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Industrially produced antibiotics are produced by fermentation, where the source microorganism is grown in large liquid growth medium.
Learning Objectives
• Describe how antibiotics are produced in industry by fermentation
Key Points
• Antibiotics are the secondary metabolites of microorganisms.
• During processing, the antibiotic must be extracted and purified to a crystalline product.
• Useful antibiotics are often discovered using a screening process or a rational design process.
Key Terms
• antibiotic: Any substance that can destroy or inhibit the growth of bacteria and similar microorganisms.
• fermentation: Any of many anaerobic biochemical reactions in which an enzyme (or several enzymes produced by a microorganism) catalyses the conversion of one substance into another; especially the conversion (using yeast) of sugars to alcohol or acetic acid with the evolution of carbon dioxide.
• metabolite: Any substance produced by, or taking part in, a metabolic reaction.
Antibiotics are produced industrially by a process of fermentation, where the source microorganism is grown in large containers (100,000 – 150,000 liters or more) containing a liquid growth medium.
Oxygen concentration, temperature, pH, and nutrient levels must be optimal and are closely monitored and adjusted if necessary. As antibiotics are secondary metabolites, the population size must be controlled very carefully to ensure that maximum yield is obtained before the cells die. Once the process is complete, the antibiotic must be extracted and purified to a crystalline product. This is simpler to achieve if the antibiotic is soluble in organic solvent. Otherwise it must first be removed by ion exchange, adsorption, or chemical precipitation.
Microorganisms used in fermentation are rarely identical to their counterparts in the wild. This is because species are often genetically modified to yield the maximum amounts of antibiotics. Mutation is often used and is encouraged by introducing mutagens such as ultraviolet radiation, x-rays, or certain chemicals. Selection and further reproduction of the higher yielding strains over many generations can raise yields by 20-fold or more. Another technique used to increase yields is gene amplification, where copies of genes coding for enzymes involved in the antibiotic production can be inserted back into a cell, via vectors such as plasmids. This process must be closely linked with retesting of antibiotic production and effectiveness.
Despite the wide variety of known antibiotics, less than 1% of antimicrobial agents have medical or commercial value. For example, whereas penicillin has a high therapeutic index as it does not generally affect human cells, this is not the case for many antibiotics. Other antibiotics simply lack advantage over those already in use or have no other practical applications.
Useful antibiotics are often discovered using a screening process. To conduct such a screen, isolates of many different microorganisms are cultured and then tested for production of diffusible products that inhibit the growth of test organisms. Most antibiotics identified in such a screen are already known and must therefore be disregarded. The remainder must be tested for their selective toxicities and therapeutic activities, and the best candidates can be examined and possibly modified.
A more modern version of this approach is a rational design program. This involves screening directed towards finding new natural products that inhibit a specific target, such as an enzyme only found in the target pathogen, rather than tests to show general inhibition of a culture. | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.02%3A_Microbial_Products_in_the_Health_Industry/17.2A%3A_Industrial_Production_of_Antibiotics.txt |
Microorganisms and plants can synthesize many uncommon amino acids and vitamins.
Learning Objectives
• Describe how microorganisms and plants can synthesize many uncommon amino acids and vitamins
Key Points
• Amino acids are organic compounds made from amine (-NH2) and carboxylic acid (-COOH) functional groups, and a specific side-chain.
• Vitamin K is a group of structurally similar, fat-soluble vitamins that are needed for the posttranslational modification of certain proteins.
• Bacteria in the colon (large intestine) can produce a range of vitamin K2 forms, and can also convert vitamin K1 into vitamin K2.
Key Terms
• amino acid: Any organic compound containing both an amino and a carboxylic acid functional group.
• vitamin: Any of a specific group of organic compounds essential in small quantities for healthy human growth, metabolism, development, and body function; found in minute amounts in plant and animal foods or sometimes produced synthetically; deficiencies of specific vitamins produce specific disorders.
• synthesize: To combine two or more things to produce a new, more complex product.
• dichotomous key: a tool for identification of plants and animals, written as a sequence of paired questions, the choice of which determines the next pair of questions until a name or identification is reached
Amino Acids
Amino acids are biologically important organic compounds made from amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen. About 500 amino acids are known which can be classified in many ways.
Microorganisms and plants can synthesize many uncommon amino acids. For example, some microbes make 2-aminoisobutyric acid and lanthionine, which is a sulfide-bridged derivative of alanine. Both of these amino acids are found in peptidic lantibiotics such as alamethicin. While in plants, 1-aminocyclopropane-1-carboxylic acid is a small disubstituted cyclic amino acid that is a key intermediate in the production of the plant hormone ethylene.
Vitamins
Vitamin K is a group of structurally similar, fat-soluble vitamins that are needed for the posttranslational modification of certain proteins required for blood coagulation and in metabolic pathways in bone and other tissue. They are 2-methyl-1,4-naphthoquinone (3-) derivatives. This group of vitamins includes two natural vitamers: vitamin K1 and vitamin K2.
Vitamin K1, also known as phylloquinone or phytomenadione (also called phytonadione), is synthesized by plants, and is found in highest amounts in green leafy vegetables because it is directly involved in photosynthesis.
Vitamin K2 has several subtypes, one of which is involved in bone metabolism. Vitamin K2 homologs (menaquinones) are characterized by the number of isoprenoid residues in their side chain. Menaquinones are abbreviated MK-n, where n represents the number of isoprenoid side chain residues. For example, menaquinone-4 (abbreviated MK-4), has four isoprene residues in its side chain. Bacteria in the colon (large intestine) can produce a range of vitamin K2 forms, and can also convert K1 into K2 (MK-7 homolog). No known toxicity exists for vitamins K1 or K2.
Three synthetic types of vitamin K are known: vitamins K3, K4, and K5. Although the natural K1 and K2 forms are nontoxic, the synthetic form K3 (menadione) has shown toxicity. | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.02%3A_Microbial_Products_in_the_Health_Industry/17.2B%3A_Vitamins_and_Amino_Acids.txt |
Learning Objectives
• Describe steroid biosynthesis
A steroid is a type of organic compound that contains a characteristic arrangement of four cycloalkane rings that are joined to each other. Examples of steroids include the dietary fat cholesterol, the sex hormones estradiol and testosterone, and the anti-inflammatory drug dexamethasone. The core of steroids is composed of twenty carbon atoms bonded together that take the form of four fused rings: three cyclohexane rings (designated as rings A, B, and C in the figure to the right) and one cyclopentane ring (the D ring). The steroids vary by the functional groups attached to this four-ring core and by the oxidation state of the rings. Sterols are special forms of steroids, with a hydroxyl group at position-3 and a skeleton derived from cholestane.
Hundreds of distinct steroids are found in plants, animals, and fungi. All steroids are made in cells either from the cycloartenol (plants) or sterols lanosterol (animals and fungi). Both lanosterol and cycloartenol are derived from the cyclization of the triterpene squalene.
Steroid biosynthesis is an anabolic metabolic pathway that produces steroids from simple precursors. A unique biosynthetic pathway is followed in animals compared to many other organisms, making the pathway a common target for antibiotics and other anti-infective drugs. In humans and other animals, the biosynthesis of steroids follows the mevalonate pathway that uses acetyl-CoA as building blocks to form dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). In plants and bacteria, the non-mevalonate pathway uses pyruvate and glyceraldehyde 3-phosphate as substrates.
The non-mevalonate pathway or 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway (MEP/DOXP pathway) of isoprenoid biosynthesis is an alternative metabolic pathway leading to the formation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).
The classical mevalonate pathway or HMG-CoA reductase pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. It is important for the production of IPP and DMAPP that serve as the basis for the biosynthesis of molecules used in processes as diverse as protein prenylation, cell membrane maintenance, hormones, protein anchoring, and N-glycosylation.
In contrast to the classical mevalonate pathway of isoprenoid biosynthesis, plants and apicomplexan protozoa such as malaria parasites have the ability to produce their isoprenoids (terpenoids) using an alternative pathway, the non-mevalonate pathway, which takes place in their plastids. In addition, most bacteria including important pathogens such as Mycobacterium tuberculosis synthesize IPP and DMAPP via the non-mevalonate pathway.
Key Points
• Steroid biosynthesis is an anabolic metabolic pathway that produces steroids from simple precursors.
• In plants and bacteria, the non-mevalonate pathway uses pyruvate and glyceraldehyde 3-phosphate as substrates.
• The classical mevalonate pathway or HMG-CoA reductase pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria.
Key Terms
• steroid: A class of organic compounds having a structure of 17 carbon atoms arranged in four rings; they are lipids, and occur naturally as sterols, bile acids, adrenal and sex hormones, and some vitamins; many drugs are synthetic steroids.
• anabolic: Anabolism is the set of metabolic pathways that construct molecules from smaller units.
• biosynthesis: The synthesis of organic compounds within a living organism, especially the synthesis of large compounds from small ones. | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.02%3A_Microbial_Products_in_the_Health_Industry/17.2C%3A_Steroids.txt |
Enzymes are biological molecules that catalyze (increase the rates of) chemical reactions.
Learning Objectives
• Describe the use of enzymes in industry
Key Points
• Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.
• Synthetic molecules called artificial enzymes also display enzyme-like catalysis.
• Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required.
Key Terms
• chemical reactions: Processes that lead to the transformation of one set of chemical substances to another.
• rennin: a proteolytic enzyme, obtained the gastric juice of the abomasum of calves, used to coagulate milk and make cheese
• enzymes: Biological molecules that catalyze (i.e., increase the rates of) chemical reactions.
• Synthetic: The combination of two or more parts, whether by design or by natural processes. It may imply being prepared or made artificially, in contrast to naturally.
Enzymes are biological molecules that catalyze (increase the rates of) chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates sufficient for life. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.
Like all catalysts, enzymes work by lowering the activation energy for a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions.
As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts in that they are highly specific for their substrates. A few RNA molecules called ribozymes also catalyze reactions, with an important example being some parts of the ribosome. Synthetic molecules, called artificial enzymes, also display enzyme-like catalysis.
Enzyme activity can be affected by other molecules. Inhibitors can decrease enzyme activity; activators can increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, pressure, chemical environment (e.g., pH), and substrate concentration. Some enzymes are used commercially; for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins into smaller molecules, making the meat easier to chew).
Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. However, enzymes in general are limited in the number of reactions they have evolved to catalyze, and by their lack of stability in organic solvents and at high temperatures. As a consequence, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been designed “from scratch” to catalyze reactions that do not occur in nature.
In food processing, the enzymes used include amylases from fungi and plants. These enzymes are used in the production of sugars from starch, such as in making high-fructose corn syrup. In baking, they catalyze the breakdown of starch in the flour to sugar. Yeast fermentation of sugar produces the carbon dioxide that raises the dough. Proteases are used by biscuit manufacturers to lower the protein level of flour. Trypsin is used to predigest baby foods. For the processing of fruit juices, cellulases and pectinases are used to clarify fruit juices. Papain is used to tenderize meat for cooking.
In the dairy industry, rennin, derived from the stomachs of young ruminant animals (like calves and lambs) is used to manufacture of cheese, used to hydrolyze protein. Lipases are implemented during the production of Roquefort cheese to enhance the ripening of the blue-mold cheese. Lactases are used to break down lactose to glucose and galactose.
In the brewing industry, enzymes from barley are released during the mashing stage of beer production. They degrade starch and proteins to produce simple sugar, amino acids, and peptides that are used by yeast for fermentation. Industrially-produced barley enzymes are widely used in the brewing process to substitute for the natural enzymes found in barley. Amylase, glucanases, and proteases are used to split polysaccharides and proteins in the malt. Betaglucanases and arabinoxylanases are used to improve the wort and beer filtration characteristics. Amyloglucosidase and pullulanases are used for low-calorie beer and adjustment of fermentability. Proteases are used to remove cloudiness produced during storage of beers.
In the starch industry, amylases, amyloglucosideases, and glucoamylases convert starch into glucose and various syrups. Glucose isomerase converts glucose into fructose in production of high-fructose syrups from starchy materials.
In the paper industry, amylases, xylanases, cellulases, and ligninases are used to degrade starch to lower viscosity, aiding sizing and coating paper.
In the biofuel industry, cellulases used to break down cellulose into sugars that can be fermented (see cellulosic ethanol).
In the production of biological detergents, proteases, produced in an extracellular form from bacteria, are used in pre-soak conditions and direct liquid applications, helping with the removal of protein stains from clothes.
In molecular biology, restriction enzymes, DNA ligase, and polymerases are used to manipulate DNA in genetic engineering, important in pharmacology, agriculture and medicine, and are essential for restriction digestion and the polymerase chain reaction. Molecular biology is also important in forensic science.
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Microorganisms from sewage can cause human disease, but can also negatively affect important ecosystems on which humans rely.
Learning Objectives
• Explain the relationship between microorganisms and water quality
Key Points
• Scientists typically measure water quality by testing for the presence of “indicator species ” of bacteria, harmless microorganisms that are found in the human gut alongside pathogenic species.
• Typical indicator species include coliform bacteria (related to the pathogenic E. coli) and Pseudomonas aeruginosa. When levels of these bacteria are high, scientists begin testing for disease causing bacteria.
• Ecosystems may also suffer from contaminated water. In aquatic ecosystems, sewage bacteria may cause “dead zones” when they use up the oxygen in the water while decomposing nutrients. Coral reefs may also become infected with sewage bacteria and die.
Key Terms
• hypoxic: Of, pertaining to, or suffering from hypoxia (lack of oxygen)
Water Quality and Human Health
Waterborne diseases are a infections transmitted by contaminated drinking water. Although there are many pathogens which can be transmitted through water, bacteria and protozoa are some of the most common organisms that cause disease. Monitoring for waterborne disease can be difficult because humans often shed very low numbers of pathogenic bacteria when they are infected. To test whether disease causing bacteria might be present, researchers measure the presence of indicator species, such as coliform bacteria (which are the group to which the pathogenic E. coli belongs) orPseudomonas aeruginosa. Although most coliform bacteria do not cause disease, they are commonly found in the human gut and in sewage, and their presence implies that human waste has reached the water supply. Researchers usually test water quality by sampling water and measuring the concentration of all bacteria in a sample. If the number of bacteria exceeds the limits set by water quality standards, the next step is to test for the presence of specific pathogens. Scientists can use genetic probes, or specific culture techniques to check whether harmful pathogens are present. Standards for testing may differ depending on the water source: drinking water is held to very high standards, while water quality in lakes and rivers may be held to more lax standards because recreational swimmers typically ingest very little water.
Water Quality and The Environment
Microrganisms can also have important impacts on the environment. All healthy ecosystems have their own communities of bacteria that decompose biological matter. However, contamination by sewage and human waste can disrupt the natural balance of bacteria and affect aquatic ecosystems. An influx of human pathogens can cause problems for ecosystems in several ways. First, sewage bacteria can cause hypoxic “dead zones” in aquatic ecosystems. The foreign bacteria rapidly reproduce and consume debris and nutrients in the sewage, but use up all the oxygen in the water in doing so. The de-oxygenated water is harmful to fish and other aquatic life. Coral reefs are also affected by sewage contaminated water. Coral can become infected by human gut bacteria, and this can cause “coral bleaching disease” where coral lose their normal bacterial and algae communities and die. Water quality is not just important for human health, it is important for the human communities that depend on aquatic and marine ecosystems. | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.03%3A_Wastewater_Treatment_and_Water_Purification/17.3A%3A_Microorganisms_and_Water_Quality.txt |
Wastewater is treated in 3 phases: primary (solid removal), secondary (bacterial decomposition), and tertiary (extra filtration).
Learning Objectives
• List the steps of wastewater/sewage treatment
Key Points
• Primary treatment is the first phase of sewage treatment: wastewater is placed in a holding tank and solids settle to the bottom where they are collected and lighter substances like fats and oils are scraped off the top.
• Secondary treatment is where waste is broken down by aerobic bacteria incorporated into the wastewater treatment system.
• Tertiary treatment is designed to filter out nutrients and waste particles that might damage sensitive ecosystems; wastewater is passed through additional filtering lagoons or tanks to remove extra nutrients.
Key Terms
• Effluent: Sewage water that has been partially treated and is released into a natural body of water; a flow of any liquid waste.
• zooplankton: Small protozoa, crustaceans (such as krill), and the eggs and larvae from larger animals.
• aerobic: Living or occurring only in the presence of oxygen.
Sewage is generated by residential and industrial establishments. It includes household waste liquid from toilets, baths, showers, kitchens, sinks, and so forth that is disposed of via sewers. In many areas, sewage also includes liquid waste from industry and commerce. The separation and draining of household waste into greywater and blackwater is becoming more common in the developed world. Greywater is water generated from domestic activities such as laundry, dishwashing, and bathing, and can be reused more readily. Blackwater comes from toilets and contains human waste.
Sewage treatment is done in three stages: primary, secondary and tertiary treatment.
Primary Treatment
In primary treatment, sewage is stored in a basin where solids (sludge) can settle to the bottom and oil and lighter substances can rise to the top. These layers are then removed and then the remaining liquid can be sent to secondary treatment. Sewage sludge is treated in a separate process called sludge digestion.
Secondary Treatment
Secondary treatment removes dissolved and suspended biological matter, often using microorganisms in a controlled environment. Most secondary treatment systems use aerobic bacteria, which consume the organic components of the sewage (sugar, fat, and so on). Some systems use fixed film systems, where the bacteria grow on filters, and the water passes through them. Suspended growth systems use “activated” sludge, where decomposing bacteria are mixed directly into the sewage. Because oxygen is critical to bacterial growth, the sewage is often mixed with air to facilitate decomposition.
Tertiary Treatment
Tertiary treatment (sometimes called “effluent polishing”) is used to further clean water when it is being discharged into a sensitive ecosystem. Several methods can be used to further disinfect sewage beyond primary and secondary treatment. Sand filtration, where water is passed through a sand filter, can be used to remove particulate matter. Wastewater may still have high levels of nutrients such as nitrogen and phosphorus. These can disrupt the nutrient balance of aquatic ecosystems and cause algae blooms and excessive weed growth. Phosphorus can be removed biologically in a process called enhanced biological phosphorus removal. In this process, specific bacteria, called polyphosphate accumulate organisms that store phosphate in their tissue. When the biomass accumulated in these bacteria is separated from the treated water, these biosolids have a high fertilizer value. Nitrogen can also be removed using nitrifying bacteria. Lagooning is another method for removing nutrients and waste from sewage. Water is stored in a lagoon and native plants, bacteria, algae, and small zooplankton filter nutrients and small particles from the water.
Sludge Digestion
Sewage sludge scraped off the bottom of the settling tank during primary treatment is treated separately from wastewater. Sludge can be disposed of in several ways. First, it can be digested using bacteria; bacterial digestion can sometimes produce methane biogas, which can be used to generate electricity. Sludge can also be incinerated, or condensed, heated to disinfect it, and reused as fertilizer. | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.03%3A_Wastewater_Treatment_and_Water_Purification/17.3B%3A_Wastewater_and_Sewage_Treatment.txt |
Water is purified with filters to remove larger protozoans, and by chemical or UV disinfection to kill bacteria and other small pathogens.
Learning Objectives
• Illustrate the steps of drinking water purification
Key Points
• Water is first passed through a system of filters and a coagulating agent is added to remove particulate matter.
• Water is then passed through a membrane filter to remove large pathogens such as cryptosporidum and giardia.
• To finalize the purification process, chemical disinfection (usually with chlorine or ozone ) or UV light is applied to the water to kill bacteria, viruses, and the hardy cysts produced by cryptosporidium and giardia.
Key Terms
• ozone: A triatomic molecule, consisting of three oxygen atoms. It is an allotrope of oxygen that is much less stable than the diatomic allotrope (O2), breaking down with a half life of about half an hour in the lower atmosphere, to normal dioxygen. Ozone is formed from dioxygen by the action of ultraviolet light and also atmospheric electrical discharges, and is present in low concentrations throughout the Earth’s atmosphere. In total, ozone makes up only 0.6 parts per million of the atmosphere.
• coagulation: The precipitation of suspended particles as they increase in size (by any of several physical or chemical processes).
• protozoa: Protozoa are a diverse group of unicellular eukaryotic organisms, many of which are motile. Originally, protozoa had been defined as unicellular protists with animal-like behavior, e.g., movement. Protozoa were regarded as the partner group of protists to protophyta, which have plant-like behaviour, e.g., photosynthesis.
Drinking Water Purification
In order to purify drinking water from a source (such as a lake, river, reservoir or groundwater), the water must go through several steps to remove large particles and different types of pathogens.
1. Pumping and Containment: Water is pumped from the source into holding tanks.
2. Screening: Water is passed through a screen filter to remove large debris.
3. Storage: Water is stored in reservoirs, tanks, and water towers in preparation for purification. Sometimes water is “pre-cholrinated” in this system to prevent bacterial growth while it is in storage.
4. Coagulation and Sedimentation: Although there are many processes by which large particles are removed from drinking water, most water purification systems implement some kind of coagulation system. A chemical that causes particle aggregation is added to the water, and clumps of particles form and settle to the bottom of the reservoir. This is called sedimentation.
5. Membrane Filtration: Membrane filters are able to remove all particles larger than 0.2 um. Larger pathogens such as giardia lamblia and cryptosporidium are trapped in these filters, but the cysts they produce are small enough to pass through.
6. Disinfection: Before water is considered potable, it must be disinfected to remove any pathogens that passed through the membrane filter.
Methods of Disinfection
• Chlorination is the most common form of disinfection. Chlorine is a strong oxidant, and rapidly kills many microorganisms, especially bacteria. Because chlorine is a toxic gas, it can also be dangerous to sanitation workers. Chlorine based compounds like choloramine are often used. Although chlorine is very effective against bacteria, it is not as effective against the cysts formed by protozoans (like giardia lamblia and cryptosporidium). Chlorine can sometimes leave residual byproducts in water.
• Ozone is an unstable molecule that readily gives up one atom of oxygen providing a powerful oxidizing agent. This agent is toxic to most waterborne organisms. Ozone is widely used in Europe, and is an effective method to kill cysts formed by protozoans. It also works well against almost all other pathogens.
• Ultraviolet Light is very effective at inactivating protozoan cysts, and will also kill bacteria and viruses. However, it is not as effective in cloudy water. It is sometimes used in concert with chlorination.
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The production of alcohol beverages is a process that involves the active participation of microorganisms, most often yeasts.
Learning Objectives
• Explain why microorganisms are used for beer, wine, and sake production.
Key Points
• Yeasts are the main fermentor and alcohol producer in the production of wine, beer and other alcohol drinks.
• The main yeast species used is Saccharomyces cerevisiae. It ferments the sugars, coming from different sources, e.g., grapes for wine, barley for beer, to alcohol and carbon dioxide.
• Both wild and cultivated strains are used. The species or strains used in the fermentation play an important role in giving the final taste properties of the drink.
Key Terms
• must: The unfermented grape juice of crushed grapes that contains fruit, seeds and skins.
Humans have been producing alcoholic beverages for thousands of years. The production of alcohol in these drinks is based primarily on yeast fermentation. Yeasts are eukaryotic microorganisms that ferment variety of sugars from different sources into the final products of carbon dioxide and alcohol.
Wine production
Wine is made from grapes or other fruit. The grapes are first cleaned of leaves and stems and the fruit is crushed into must that is ready for fermentation. The yeasts used for the fermentation grow a film on the fruit or in the environment. These wild strains play an important role in the final properties of the drink. However, cultivated strains of Saccharomyces cerevisiae are often added to improve the consistency of the final product. There are hundreds of commercially available yeast strains for wine fermentation.
In the fermentation process, energy that is converted to heat is produced as well. It is important to keep the temperature in the fermentation vessel lower than 40ºC to keep the yeasts alive. To improve yeast growth, additional nutrients, like diammonium phosphate, are sometimes added in the fermentation step.
When making red wine, there is an additional fermentation step after alcoholic fermentation. Malic acid, naturally present in grape juice, can be converted to lactic acid by lactic acid bacteria naturally found in wineries or added artificially.
Beer production
Beer is the most consumed alcoholic beverage in the world. It is made most often of malted barley and malted wheat. Sometimes a mixture of starch sources can be used, such as rice. Unmalted maize can be added to the barley or wheat to lower cost. Potatoes, millet and other foods high in starch are used in different places in the world as the primary carbohydrate source.
The process of making beer is called brewing. It includes breaking the starch in the grains into a sugary liquid, called wort, and fermenting the sugars in the wort into alcohol and carbon dioxide by yeasts. Two main species are used in the fermentation process: Saccharomyces cerevisiae (top-fermenting, since it forms foam on top of the wort) and Saccharomyces uvarum (bottom-fermenting). Top-fermenting yeasts are used to produce ale, while bottom-fermenting produce lagers. The temperature used for top-fermenting (15-24ºC) leads to the production of a lot of esters and flavor products that give beer a fruity taste. Hops are added to introduce a bitter taste and to serve as a preservative.
Brewer’s yeasts are very rich in essential minerals and B vitamins, with the exception of vitamin B12. Beer brewing in modern days is performed by added pure cultures of the desired yeast species to the wort. Additional yeasts species that are used in making beer are Dekkera/Brettanomyces. After the fermentation is finished, the beer is cleared of the yeasts by precipitation or with the use of clearing additives.
Other types of alcohol beverages are made by the fermentation activity of microorganisms as well. A few examples are sake (uses the fungus Aspergillus oryzae to facilitate starch fermentation from rice), brandy, whiskey (both are distilled alcohol), and other alcohol beverages with higher percentage of alcohol compared to wine and beer. | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.04%3A_The_Microbiology_of_Food/17.4A%3A_Wine_Beer_and_Alcohol.txt |
Vinegar is a food product made by acetic acid bacteria that can ferment the alcohol in alcoholic liquids to acetic acid.
Learning Objectives
• Describe how vinegar is made and the common uses of vinegar
Key Points
• The fermentation of alcohol requires oxygen and its availability can determine the rate of vinegar production.
• The main genus used for vinegar fermentation is Acetobacter sp. since the final product of its fermentation can contain acetic acid as high as 20%.
• Some of the most common uses of vinegar are in food preparation, as a cleaning agent, and as a medicine.
Key Terms
• starter culture: Starter culture consists of live microorganisms with some medium used to start fermentation or growth in a fresh new medium (substrate).
Vinegar has been used for cooking and in the household and different industries due to its mildly acidic nature for many centuries. It is one of the foods together with beer, wine, bread and fermented dairy products, that is the result of fermentation by microorganisms and has been around for thousands of years. It is a mixture of acetic acid (most often 5%) and water.
The fermentation is performed usually by acetic acid bacteria, from the genus Acetobacter, from the alcohol in variety of sources (e.g., apple cider, wine, potatoes, fermented grain). Acetobacter bacteria are Gram negative aerobic rods. They are naturally present in environments where alcohol is being produced and can be isolated from damaged fruit, apple cider, etc. In these liquids, the bacteria form a film on the surface, since they are aerobic and need good oxygen supply. This film, called mother of vinegar, can be used as a starter culture of acetic fermentation in fresh alcohol liquids. Mother of vinegar can also be found in unpasteurized store brand vinegar. Acetic acid bacteria are transmitted in nature by vectors like fruit flies and Vinegar eels.
This acetic acid fermentation needs oxygenation. If left at room temperature alcohol containing solution with Acetobacter will be converted to vinegar in months. The industrial process can be completed within hours since air is bubbled and mixed through the solution.
Vinegar can also be an undesired product in wine production. If the temperature in the fermentation vessel is too high, the Acetobacter will outgrow the yeasts and the produced alcohol will be converted to vinegar.
There are bacteria that can convert sugars straight to acetic acid in anaerobic fermentation. Such species include Clostridium and Acetobacterium but they can not tolerate acetic acid of concentrations higher than a few percent. The product made from these bacteria must be concentrated while oxidative fermentation by Acetobactercan produce up to 20% acetic acid.
Vinegar is a food product made all over the world from many different carbohydrate sources where alcohol fermentation has been performed. Some of them are more commonly used, such as apple cider and grapes, while others such as coconut water, dates, kiwifruit are used in specific regions of the world. Vinegar is used not only in food preparation but also as a cleaning agent due to its acidic nature and strong antibacterial properties. It can be used to lower the glycemic index of foods if consumed together with them. It has also been shown to reduce the risk of fatal ischemic heart disease when consumed frequently with oil in salad dressings.
17.4C: Citric Acid and Other Organic Compounds
Many organic compounds, like citric acid, are produced industrially by microorganisms.
Learning Objectives
• Explain how citric acid and other organic compounds are produced by the mold Aspergillus niger
Key Points
• The major industrial producer of citric acid is the mold Aspergillus niger.
• It has the ability to produce citric acid in high quantities and exports it outside the cells.
• Citric acid is used in the food, chemical and pharmaceutical industries.
Key Terms
• molasses: Molasses is viscous syrup produced from a variety of sources, such as sugar beets, sugarcane and grapes.
Citric acid (citrate) is an important substance in the Krebs cycle. It is produced from acetyl coenzyme A and oxaloacetate in the presence of the enzyme citrate synthase. The Krebs cycle is key in the oxidation of sugars, proteins and fats to carbon dioxide and water. Many of the cycle compounds are also needed for the synthesis of the cells’ own proteins, carbohydrates, and fats.
Citrate has been used for centuries in different industries and in the households. It is used as a food additive to give a sour taste to foods or to preserve certain qualities of food products (e.g., prevents separating of the fats in ice cream). It has natural antibacterial properties and is used as a preservative as well. Its buffering property is used in cosmetics and pharmaceuticals to adjust the pH of products.
For centuries, the source of citric acid were citrus fruits. After World War I, the ability of some microorganisms to produce citric acid was further explored and the technology for industrial production was developed. Penicillium mold was the first described organism to produce citric acid but industrially another mold, Aspergillus niger, became the microorganism of choice. The mold is grown in a medium with sucrose or glucose as the main carbon source. The sugar source is usually an inexpensive solution like molasses or corn steep liquor. The microorganism makes more citric acid in the Krebs cycle than needed for the cell’s metabolism and exports it outside the cell. The citric acid is then precipitated out of solution and regenerated.
Microorganisms replaced the industrial chemical production of many different organic compounds, like enzymes and amino acids. Enzymes, such as glucoamylase (used to make high-fructose corn syrup) and pectinase (clearing agent for apple cider and wines) are produced industrially by Aspergillus. The food additivemonosodium glutamate (MSG) is produced in the form of glutamic acid by Corynebacterium glutamicum. | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.04%3A_The_Microbiology_of_Food/17.4B%3A_Vinegar.txt |
Fungi are used as food or as producers of a variety of food products (bread, wine, beer, etc. ) or compounds used in different industries.
Learning Objectives
• Describe how yeast, molds and mushrooms are used in the food industry
Key Points
• The single cell yeast species, Saccharomyces cerevisiae, has been used as the major leavening agent in making bread for thousands of years.
• Different species of the mold Penicillium are added to milk or curd when making soft cheese to produce blue cheese.
• Mushrooms have fleshy fruit body with certain aroma and flavors as well as good nutritional properties and are used mostly as food.
Key Terms
• leavening agent: An organism or compound that can make dough rise and produce soft bread.
• curd: Curd is the solid coagulated fraction of milk after it has been digested with enzymes or treated with sour substances.
• gangrene: The death of tissue due to reduced blood supply as a result of infection or a blocked blood vessel.
Fungi are eukaryotic organisms that are separated taxonomically in the Fungi kingdom. The kingdom includes yeasts and molds (both microorganisms) and mushrooms. These organisms are ubiquitous all over the world. They have been used by people as food or as producers of a huge variety of food products or compounds used in different industries.
Yeasts
The yeast species Saccharomyces cerevisiae has been used as leavening agentfor the production of bread since ancient times. The yeasts ferment the carbohydrates in the dough and produce carbon dioxide that causes the dough to rise and the bread to be softer after baking. Different sources provided the starter cultures. Dough could be left exposed to the air before cooking. Beer foam or grape juice paste were alos used as yeasts sources. Nowadays, the common used starters are pure cultures of Saccharomyces cerevisiae produced and sold as baker’s yeasts, although some artisan bakers maintain their own starter cultures. Other yeasts and some bacteria can be used as leavening agents too. For example, sourdough is made with Saccharomyces exiguus and Lactobacillus cultures that give it its sour taste.
Molds
Molds are fungi which cells grow in long chains of filamentous hyphae. The first antibiotic used in modern medicine, penicillin, was isolated form Penicillium mold. Different species of the mold Penicillium are added to milk or curd when making soft cheese to produce blue cheese. The mold adds specific smell and flavor to the cheese. Some bacteria, such as Brevibacterium linens, are also used to give blue cheese its characteristic odor.
Each mold species is usually found in the environment of the local region where the production of specific brand started. To enhance the mold growth in cheese, different techniques are applied to improve the access of air. The cheese is ripen for weeks to months in dark cold places. Even before the discovery of penicillin, people used blue cheese to prevent gangrene in wounds.
Mushrooms
Edible mushrooms are macrofungi since they are visible with a naked eye. Mushrooms have fleshy fruit body with certain aroma and flavors as well as good nutritional properties and are used mostly as food. A few species of mushrooms have been cultivated but wild mushrooms are harvested as well. However, some mushrooms produce toxic compounds that can be life-threatening. Proper identification is key since quite often poisonous mushrooms mimic edible ones in appearance. Even if not dangerous, mushrooms in general are great absorbants of chemicals from the environment and sometimes they can make them toxic, e.g., pesticides, insecticides, heavy metals. Certain mushrooms have been used for their medicinal properties in some cultures.
17.4E: Edible Algae
Edible algae have been used as food for centuries in many coastal regions all over the world.
Learning Objectives
• Describe the nutritional value of algae
Key Points
• Algae are a very diverse group of generally simple unicellular or multicellular eukaryotic organisms.
• Algae are of excellent nutritional value since they contain complete protein, fiber, and sometimes high levels of omega-3 fatty acids, many vitamins and minerals.
• Some compounds that are used as additives in the food industry are isolated from algae.
Key Terms
• complete protein: Complete protein (whole protein) is a protein that contains all of the nine essential amino acids.
Algae are a very diverse group of generally simple unicellular or multicellular eukaryotic organisms. Most of them are autotrophic which means that they can harvest carbon dioxide from the atmosphere and convert it to organic matter. They inherited their photosynthetic apparatus from cyanobacteria. Cyanobacteria are sometimes called blue-green algae but they are prokaryotic organisms and are not true algae. Some cyanobacterial species are used as food as well.
Seaweeds are edible algae that have been used for centuries as food in many coastal regions all over the world. They may belong to one of three groups of multicellular algae: red, green or brown. In countries such as China, Japan, Korea and to some extent Iceland, Ireland, Chile and New Zealand algae are part of people’s regular diet. They are usually of marine origin since freshwater algae are often poisonous.
Algae are of excellent nutritional value since they contain complete protein (in contrast to plant food harvested on land), fiber, and sometimes high levels of omega-3 fatty acids. In fact, the omega-3 acids in fish comes from the microalgae consumed at the bottom of the food pyramide and gradually passed up to the fish at the top. Algae are also rich in many vitamins, such as A, C, B1, B2, B3 and B6, as well as minerals, such as iodine, calcium, potassium, magnesium and iron. They are consumed both cooked, dried and raw.
Cultivated microalgae and cyanobacteria such as Spirulina and Chlorella are sold as nutritional supplements. Hydrocolloids such as agar, alginate and carrageenan are isolated from wild and cultivated algae and used as additives in the food industry for their emulsifying and thickening properties. Some of the complex polysaccharides found in algae may be digested by bacteria in the gut since the needed enzymes for digestion are abundantly present in Japanese people but absent in people from North America. | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.04%3A_The_Microbiology_of_Food/17.4D%3A_Edible_Fungi.txt |
Fermented dairy products have been prepared and consumed by people for centuries due to their high nutritional values.
Learning Objectives
• Describe how fermented milk and dairy products are produced and explain their nutritional value
Key Points
• The fermentation is usually performed by lactic acid bacteria which ferment the lactose in milk and convert it to lactic acid leading to precipitation of the proteins.
• There is a tremendous variety of fermented dairy products in many regions in the world. The properties of each product depend on the local strains used for the fermentation.
• Many lactic acid bacteria have also been investigated for medicinal health benefits in the past few decades but so far the results are inconclusive.
Key Terms
• rennet: Enzymes derived from mammalian stomachs that contain proteases and lipase.
• whey: The remaining liquid after milk curdling.
Fermented milk or dairy products have been part of human diet since ancient times. Various fermented products are made by different strains. Lactic acid fermentation is performed most often by lactic acid bacteria. Due to their abundance in nature, including mucosal surfaces of the human body, and their use in fermented foods they are labeled as GRAS (generally recognized as safe). The main genera that belong to the lactic acid bacteria group are: Lactobacillus, Leuconostoc, Lactococcus, Pediococcusand Streptococcus. These bacteria ferment the carbohydrates in milk, the major one being lactose, to lactic acid and some other products. The acid precipitates the proteins in the milk and that is why fermented products are usually of thicker consistency than milk. The high acidity and low pH hinders the growth of other bacteria, including pathogens. Some lactic acid bacteria can produce agents with antimicrobial properties. Since milk is rich in many nutrients such as protein, calcium, phosphorus, and B vitamins dairy products are an excellent food.
Some of the most popular and widespread cultured dairy products are yogurt and cheese.
Records of yogurt preparation as food date back to centuries BCE. Classic yogurt is the result of the fermentation of two main bacterial species: Lactobacillus bulgaricus and Streptococcus thermophilus. Sometimes other lactic acid bacteria are added as well. Yogurt is most often made of cow’s milk although milk from sheep, goat, water buffalo, camels and yaks is used as well depending on the region of cultivation.
To make yogurt, the milk is first heated to 80ºC or boiled to kill any pathogenic bacteria and to denature the milk proteins to prevent the formation of curds. After it is cooled down to about 45ºC, the starter culture of the two species is mixed well with the milk and incubated at the same temperature for a few hours. In many countries, the traditional food is yogurt without any sweeteners which could be consumed plain or used to prepare a variety of dishes usually with vegetables. Yogurt has been traditionally consumed in Eastern cultures as a cold drink after mixing with water (e.g., lassi, ayran, doogh). After the industrialization of yogurt production in the twentieth century, yogurt with added sweetener and fruit or fruit jam has become popular in the Western world.
Cheese is another popular and ancient dairy product. It consists of milk proteins and fat together with lactic acid bacteria. It has longer shelf life than uncultured milk. Currently there are a few hundred varieties of cheese produced all over the world. Making cheese is similar to yogurt but after acidification usually with lactic acid bacteria (Lactococci, Lactobacilli, Streptococci), the solids are separated from the whey by coagulation with rennet and processed further to yield the final product. Depending on the type of cheese, the solids could go straight to packaging or other bacteria or mold could be added (e.g., Penicillium mold for blue cheese) for additional fermentation.
Other fermented and widely consumed cultured dairy products include kefir (lactic acid bacteria and yeasts are used for the fermentation), sour cream (fermented cream), cultured buttermilk (fermented cow’s milk with Streptococcus lactis or Lactobacillus bulgaricus only).
Lactic acid bacteria have been researched for medicinal health benefits. In the early twentieth century, the Nobel laureate in medicine, Elie Metchnikoff, believed that the longevity of peasants in Bulgaria and the Russian steppes was due to their high consumption of milk-fermented products. He hypothesized that the lactic acid bacteria would inhabit the gut after consumption, create and acidic environment as they grow and multiply, and hence prevent the growth of proteolytic. After it was discovered that Lactobacillus bulgaricus can not live in the human gut, the idea was abandoned. Years later, strains of Lactobacillus acidophilus were found to thrive in the gut after implantation and the research started again. The term “probiotics” was introduced and defined as live microorganisms that provide beneficial effects for their host when administered in adequate concentration. Most of the researched species were isolated from different fermented dairy products. The research has been focused on curing or preventing a number of diseases like diarrhea, intestinal inflammations, urogenital infections, allergies, etc. Some species have been prepared and sold as nutritious supplements. However, so far there has not been enough evidence to establish a definite cause and effect relationship about any of the marketed products.
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Fermentation is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using microorganisms.
Learning Objectives
• Describe the process of fermentation and its use in the food industry
Key Points
• Fermentation usually implies that the action of microorganisms is desirable.
• Fermentation produces alcoholic beverages such as wine, beer, and cider.
• Fermentation is also employed in the leavening of bread and the production of dairy products.
Key Terms
• fermentation: Fermentation in food processing typically is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts, bacteria, or a combination thereof, under anaerobic conditions.
• carbohydrates: A major class of foods that includes sugars and starches.
• microorganisms: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.
• oligodynamic action: that is active in small quantities; used especially to describe the sterilizing effect of some heavy metals against bacteria
Fermentation in food processing typically is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts, bacteria, or a combination thereof, under anaerobic conditions. Fermentation in simple terms is the chemical conversion of sugars into ethanol. The science of fermentation is also known as zymology or zymurgy.
Historically, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that the fermentation was catalyzed by a vital force, called “ferments,” within the yeast cells. The “ferments” were thought to function only within living organisms. “Alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells,” he wrote.
Fermentation usually implies that the action of microorganisms is desirable. This process is used to produce alcoholic beverages such as wine, beer, and cider. Fermentation is also employed in the leavening of bread (CO2 produced by yeast activity); in preservation techniques to produce lactic acid in sour foods such as sauerkraut, dry sausages, kimchi, and yogurt; and in the pickling of foods with vinegar (acetic acid).
Food fermentation has been said to serve five main purposes:
1. Enrichment of the diet through development of a diversity of flavors, aromas, and textures in food substrates.
2. Preservation of substantial amounts of food through lactic acid, alcohol, acetic acid, and alkaline fermentations.
3. Biological enrichment of food substrates with protein, essential amino acids, essential fatty acids, and vitamins.
4. Elimination of antinutrients.
5. A decrease in cooking time and fuel requirement.
17.5B: Food Spoilage by Microbes
Food spoilage is the process in which food deteriorates to the point it is not edible to humans or its quality of edibility becomes reduced.
Learning Objectives
• Describe the process of food spoilage
Key Points
• Bacteria can cause food spoilage by breaking down the food, producing acids or other waste products during this process.
• Harvested crops decompose from the moment they are harvested due to attacks from microorganisms.
• Signs of food spoilage may include an appearance different from the food in its fresh form, such as a change in color, a change in texture, an unpleasant odor, or an undesirable taste.
Key Terms
• food spoilage: Food spoilage is the process in which food deteriorates to the point that it is not edible to humans or its quality of edibility becomes reduced.
• microorganisms: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.
• bacteria: A type, species, or strain of bacterium.
Food spoilage is the process in which food deteriorates to the point that it is not edible to humans or its quality of edibility becomes reduced. Various external forces are responsible for the spoilage of food. Food that is capable of spoiling is referred to as perishable food.
Harvested crops decompose from the moment they are harvested due to attacks from microorganisms.
Various bacteria can be responsible for the spoilage of food. When bacteria breaks down the food, acids and other waste products are created in the process. While the bacteria itself may or may not be harmful, the waste products may be unpleasant to taste or may even be harmful to one’s health.
Yeasts can be responsible for the decomposition of food with a high sugar content. The same effect is useful in the production of various types of food and beverages, such as bread, yogurt, cider, and alcoholic beverages.
Signs of food spoilage may include an appearance different from the food in its fresh form, such as a change in color, a change in texture, an unpleasant odor, or an undesirable taste. The item may become softer than normal. If mold occurs, it is often visible externally on the item.
Some spoiled foods are harmless to eat, and may simply be diminished in quality. But foods exhibiting certain types of spoilage may be harmful to consume. Uncooked or under-cooked animal flesh that spoils is typically quite toxic, and consumption can result in serious illness or death. The toxic effects from consuming spoiled food are known colloquially as “food poisoning”, and more properly as “foodborne illness. ” | textbooks/bio/Microbiology/Microbiology_(Boundless)/17%3A_Industrial_Microbiology/17.05%3A_Food_Preservation/17.5A%3A_Fermented_Foods.txt |
Food preservation is the process of treating food to stop or slow down spoilage, loss of quality, edibility, or nutritional value.
Learning Objectives
• Describe how food preservation processes stop or slow down food spoilage thus allowing for longer food storage
Key Points
• Preservation usually involves preventing the growth of bacteria, fungi (such as yeasts), and other microorganisms, as well as retarding the oxidation of fats which cause rancidity.
• A number of methods of prevention can be used that can either totally prevent, delay, or otherwise reduce food spoilage.
• Maintaining or creating nutritional value, texture, and flavor is an important aspect of food preservation.
Key Terms
• Preservation: Food preservation is the process of treating and handling food to stop or slow down food spoilage, loss of quality, edibility, or nutritional value and thus allow for longer food storage.
• microorganisms: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.
• food spoilage: Spoilage is the process in which food deteriorates to the point that it is not edible to humans or its quality of edibility becomes reduced.
Food Preservation
Food preservation is the process of treating and handling food to stop or slow down food spoilage, loss of quality, edibility, or nutritional value and thus allow for longer food storage.
Preservation usually involves preventing the growth of bacteria, fungi (such as yeasts), and other microorganisms, as well as retarding the oxidation of fats which cause rancidity.
Methods of Food Preservation
A number of methods of prevention can be used that can either totally prevent, delay, or otherwise reduce food spoilage. Preservatives can expand the shelf life of food and can lengthen the time long enough for it to be harvested, processed, sold, and kept in the consumer’s home for a reasonable length of time.
Maintaining or creating nutritional value, texture and flavor is an important aspect of food preservation, although, historically, some methods drastically altered the character of the food being preserved. In many cases these changes have now come to be seen as desirable qualities, as with cheese, yogurt, and pickled onions.
Drying is one of the most ancient food preservation techniques, which reduces water activity sufficiently to prevent bacterial growth.
Refrigeration preserves food by slowing down the growth and reproduction of microorganisms and the action of enzymes which cause food to rot.
Freezing is also one of the most commonly used processes for preserving a very wide range of food including prepared foodstuffs which would not have required freezing in their unprepared state.
Vacuum-packing stores food in a vacuum environment, usually in an air-tight bag or bottle. The vacuum environment strips bacteria of oxygen needed for survival, thereby slowing spoiling. Vacuum-packing is commonly used for storing nuts to reduce the loss of flavor from oxidation.
Salting or curing draws moisture from the meat through a process of osmosis. Meat is cured with salt or sugar, or a combination of the two. Nitrates and nitrites are also often used to cure meat and contribute to the characteristic pink color, as well as inhibition of Clostridium botulinum.
Sugar is used to preserve fruits, either in syrup with fruit such as apples, pears, peaches, apricots, plums, or in crystallized form where the preserved material is cooked in sugar to the point of crystallisation and the resultant product is then stored dry. This method is used for the skins of citrus fruit (candied peel), angelica, and ginger. A modification of this process produces glacé fruit such as glacé cherries where the fruit is preserved in sugar but is then extracted from the syrup and sold, the preservation being maintained by the sugar content of the fruit and the superficial coating of syrup. The use of sugar is often combined with alcohol for preservation of luxury products such as fruit in brandy or other spirits. These should not be confused with fruit flavored spirits such as cherry brandy.
Smoking is used to lengthen the shelf life of perishable food items. This effect is achieved by exposing the food to smoke from burning plant materials such as wood. Most commonly subjected to this method of food preservation are meats and fish that have undergone curing. Fruits and vegetables like paprika, cheeses, spices, and ingredients for making drinks such as malt and tea leaves are also smoked, but mainly for cooking or flavoring them. It is one of the oldest food preservation methods, which probably arose after the development of cooking with fire.
Preservative food additives can be antimicrobial. These inhibit the growth of bacteria or fungi, including mold, or antioxidant, such as oxygen absorbers, which inhibit the oxidation of food constituents. Common antimicrobial preservatives include calcium propionate, sodium nitrate, sodium nitrite, sulfites (sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc.), and disodium EDTA. Antioxidants include BHA and BHT. Other preservatives include formaldehyde (usually in solution), glutaraldehyde (kills insects), ethanol, and methylchloroisothiazolinone.
Pickling is a method of preserving food in an edible anti-microbial liquid. Pickling can be broadly categorized into two categories: chemical pickling and fermentation pickling.
Canning involves cooking food, sealing it in sterile cans or jars, and boiling the containers to kill or weaken any remaining bacteria as a form of sterilization. Foods have varying degrees of natural protection against spoilage and may require that the final step occur in a pressure cooker. High-acid fruits like strawberries require no preservatives to can and only a short boiling cycle, whereas marginal fruits such as tomatoes require longer boiling and addition of other acidic elements. Low acid foods, such as vegetables and meats require pressure canning. Food preserved by canning or bottling is at immediate risk of spoilage once the can or bottle has been opened.
Other forms of preservation can include: jellying, jugging, irradiation, pulsed electric field processing, modified atmosphere, high pressure, burial in the ground, and biopreservation.
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What is microbiology? If we break the word down it translates to “the study of small life,” where the small life refers to microorganisms or microbes. But who are the microbes? And how small are they? Generally microbes can be divided into two categories: the cellular microbes (or organisms) and the acellular microbes (or agents). In the cellular camp we have the bacteria, the archaea, the fungi, and the protists (a bit of a grab bag composed of algae, protozoa, slime molds, and water molds). Cellular microbes can be either unicellular, where one cell is the entire organism, or multicellular, where hundreds, thousands or even billions of cells can make up the entire organism. In the acellular camp we have the viruses and other infectious agents, such as prions and viroids. In this textbook the focus will be on the bacteria and archaea (traditionally known as the “prokaryotes,”) and the viruses and other acellular agents.
• 1: Introduction to Microbiology
Generally microbes can be divided into two categories: the cellular microbes (or organisms) and the acellular microbes (or agents). Cellular microbes include bacteria, the archaea, the fungi, and the protists ( algae, protozoa, slime molds, and water molds). Cellular microbes can be either unicellular or multicellular. Acellular microbes include viruses and other infectious agents, such as prions and viroids.
• 2: Microscopes
With the advent of molecular biology there’s a lot of microbiology nowadays that happens without a microscope. But if you want to actually visualize microbes, you’ll need the ability to magnify – you’ll need a microscope of some type. And, since “seeing is believing,” it was the visualization of microbes that got people interested in them in the first place.
• 3: Cell Structure I
Cellular organisms are divided into two broad categories, based on their cell type: prokaryotic or eukaryotic. Generally, prokaryotes are smaller, simpler, with a lot less stuff, and eukaryotes are larger, more complex. The crux of their key difference can be deduced from their names: “karyose” is a Greek word meaning “nut” or “center,” a reference to the nucleus. “Pro” means “before,” while “Eu” means “true,” indicating that prokaryotes lack a nucleus while eukaryotes have a true nucleus.
• 4: Bacteria - Cell Walls
It is important to note that not all bacteria have a cell wall. Having said that though, it is also important to note that most bacteria (about 90%) have a cell wall and they typically have one of two types: a gram positive cell wall or a gram negative cell wall.
• 5: Bacteria - Internal Components
We have already covered the main internal components found in all bacteria, namely, cytoplasm, the nucleoid, and ribosomes. Remember that bacteria are generally thought to lack organelles, those bilipid membrane-bound compartments so prevalent in eukaryotic cells (although some scientists argue that bacteria possess structures that could be thought of as simple organelles). But bacteria can be more complex, with a variety of additional internal components to be found.
• 6: Bacteria - Surface Structures
What have we learned so far, in terms of cell layers? All cells have a cell membrane. Most bacteria have a cell wall. But there are a couple of additional layers that bacteria may, or may not, have. These would be found outside of both the cell membrane and the cell wall, if present.
• 7: Archaea
The Archaea are a group of organisms that were originally thought to be bacteria (which explains the initial name of “archaeabacteria”), due to their physical similarities. More reliable genetic analysis revealed that the Archaea are distinct from both Bacteria and Eukaryotes, earning them their own domain in the Three Domain Classification originally proposed by Woese in 1977, alongside the Eukarya and the Bacteria.
• 8: Introduction to Viruses
Viruses are typically described as obligate intracellular parasites, acellular infectious agents that require the presence of a host cell in order to multiply. Viruses that have been found to infect all types of cells – humans, animals, plants, bacteria, yeast, archaea, protozoa…some scientists even claim they have found a virus that infects other viruses! But that is not going to happen without some cellular help.
• 9: Microbial Growth
Provided with the right conditions (food, correct temperature, etc) microbes can grow very quickly. It’s important to have knowledge of their growth, so we can predict or control their growth under particular conditions. While growth for muticelluar organisms is typically measured in terms of the increase in size of a single organism, microbial growth is measured by the increase in population, either by measuring the increase in cell number or the increase in overall mass.
• 10: Environmental Factors
What environmental conditions can affect microbial growth? Temperature, oxygen, pH, water activity, pressure, radiation, lack of nutrients…these are the primary ones. We will cover more about metabolism (i.e. what type of food can they eat?) later, so let us focus now on the physical characteristics of the environment and the adaptations of microbes.
• 11: Microbial Nutrition
All microbes have a need for three things: carbon, energy, and electrons. There are specific terms associated with the source of each of these items, to help define organisms.
• 12: Energetics & Redox Reactions
Metabolism refers to the sum of chemical reactions that occur within a cell. Catabolism is the breakdown of organic and inorganic molecules, used to release energy and derive molecules that could be used for other reactions. Anabolism is the synthesis of more complex molecules from simpler organic and inorganic molecules, which requires energy.
• 13: Chemoorganotrophy
Chemoorganotrophy is a term used to denote the oxidation of organic chemicals to yield energy. In other words, an organic chemical serves as the initial electron donor. The process can be performed in the presence or absence of oxygen, depending upon what is available to a cell and whether or not they have the enzymes to deal with toxic oxygen by-products.
• 14: Chemolithotrophy & Nitrogen Metabolism
Chemolithotrophy is the oxidation of inorganic chemicals for the generation of energy. The process can use oxidative phosphorylation, just like aerobic and anaerobic respiration, but now the substance being oxidized (the electron donor) is an inorganic compound. The nitrogen cycle depicts the different ways in which nitrogen, an essential element for life, is used and converted by organisms for various purposes.
• 15: Phototrophy
Phototrophy (or “light eating”) refers to the process by which energy from the sun is captured and converted into chemical energy, in the form of ATP. The term photosynthesis is more precisely used to describe organisms that both convert sunlight into ATP (the “light reaction”) but then also proceed to use the ATP to fix carbon dioxide into organic compounds (the Calvin cycle). These organisms are the photoautotrophs. In the microbial world, there are also photoheterotrophs.
• 16: Taxonomy & Evolution
It is believed that the Earth is 4.6 billion year old, with the first cells appearing approximately 3.8 billion years ago. Those cells were undoubtedly microbes, eventually giving rise to all the life forms that we envision today, as well as the life forms that went extinct before we got here. How did this progression occur?
• 17: Microbial Genetics
Bacteria do not have sex, which presents a real problem for bacteria (and archaea, too); how do they get the genetic variability that they need? They might need a new gene to break down an unusual nutrient source or degrade an antibiotic threatening to destroy them – acquiring the gene could mean the difference between life and death. We are going to explore the processes that bacteria use to acquire new genes, the mechanisms known as Horizontal Gene Transfer (HGT).
• 18: Genetic Engineering
Genetic engineering is the deliberate manipulation of DNA, using techniques in the laboratory to alter genes in organisms. Even if the organisms being altered are not microbes, the substances and techniques used are often taken from microbes and adapted for use in more complex organisms.
• 19: Genomics
Genomics is a field that studies the entire collection of an organism’s DNA or genome. It involves sequencing, analyzing, and comparing the information contained within genomes. Since sequencing has become much less expensive and more efficient, vast amounts of genomic information is now available about a wide variety of organisms, but particularly microbes, with their smaller genome size. In fact, the biggest bottleneck currently is not the lack of information but the lack of computing power.
• 20: Microbial Symbioses
Symbiosis, strictly defined, refers to an intimate relationship between two organisms. The relationship could be good, bad, or neutral for either partner. A mutualistic relationship is one in which both partners benefit, while a commensalistic relationship benefits one partner but not the other. In a pathogenic relationship, one partner benefits at the expense of the other. This chapter looks at a few examples of symbiosis, where microbes are one of the partners.
• 21: Bacterial Pathogenicity
A microbe that is capable of causing disease is referred to as a pathogen, while the organism being infected is called a host. The ability to cause disease is referred to as pathogenicity, with pathogens varying in their ability. An opportunistic pathogen is a microbe that typically infects a host that is compromised in some way, either by a weakened immune system or breach to the body’s natural defenses, such as a wound. The measurement of pathogenicity is called virulence.
• 22: The Viruses
Since viruses lack ribosomes (and thus rRNA), they cannot be classified within the Three Domain Classification scheme with cellular organisms. Alternatively, Dr. David Baltimore derived a viral classification scheme, one that focuses on the relationship between a viral genome to how it produces its mRNA. The Baltimore Scheme recognizes seven classes of viruses.
Microbiology (Bruslind)
Obviously microbes are small. The traditional definition describes microbes as organisms or agents that are invisible to the naked eye, indicating that one needs assistance in order to see them. That assistance is typically in the form of a microscope of some type. The only problem with that definition is that there are microbes that you can see without a microscope. Not well, but you can see them. It would be easy to dismiss these organisms as non-microbes, but in all other respects they look/act/perform like other well-studied microbes (who follow the size restriction).
So, the traditional definition is modified to describe microbes as fairly simple agents/organisms that are not highly differentiated, meaning even the multicellular microbes are composed of cells that can act independently– there is no set division of labor. If you take a giant fungus and chop half the cells off, the remaining cells will continue to function unimpeded. Versus if you chopped half my cells off, well, that would be a problem. Multicellular microbes, even if composed of billions of cells, are relatively simple in design, usually composed of branching filaments.
It is also acknowledged that research in the field of microbiology will require certain common techniques, largely related to the size of the quarry. Because microbes are so small and there are so many around, it is important to be able to isolate the one type that you are interested in. This involves methods of sterilization, to prevent unwanted contamination, and observation, to confirm that you have fully isolated the microbe that you want to study.
Microbe Size
Since size is a bit of theme in microbiology, let us talk about actual measurements. How small is small? The cellular microbes are typically measured in micrometers (µm). A typical bacterial cell (let us say E. coli) is about 1 µm wide by 4 µm long. A typical protozoal cell (let us say Paramecium) is about 25 µm wide by 100 µm long. There are 1000 µm in every millimeter, so that shows why it is difficult to see most microbes without assistance. (An exception would be a multicellular microbe, such as a fungus. If you get enough cells together in one place, you can definitely see them without a microscope!)
When we talk about the acellular microbes we have to use an entirely different scale. A typical virus (let us say influenza virus) has a diameter of about 100 nanometers (nm). There are 1000 nanometers in every micrometer, so that shows why you need a more powerful microscope to see a virus. If a typical bacterium (let us pick on E.coli again) were inflated to be the size of the Statue of Liberty, a typical virus (again, influenza virus works) would be the size of an adult human, if we keep the correct proportions.
http://learn.genetics.utah.edu/content/cells/scale/
The Discovery of Microbes
The small size of microbes definitely hindered their discovery. It is hard to get people to believe that their skin is covered with billions of small creatures, if you cannot show it to them. “Seeing is believing,” that is what I always say. Or someone says that.
In microbiology, there are two people that are given the credit for the discovery of microbes. Or at least providing the proof of their discovery, both around the same time period:
Robert Hooke (1635-1703)
Robert Hooke was a scientist who used a compound microscope, or microscope with two lenses in tandem, to observe many different objects. He made detailed drawings of his observations, publishing them in the scientific literature of the day, and is credited with publishing the first drawings of microorganisms. In 1665 he published a book by the name of Micrographia, with drawing of microbes such as fungi, as well as other organisms and cell structures. His microscopes were restricted in their resolution, or clarity, which appeared to limit what microbes he was able to observe.
Antony van Leeuwenhoek (1632-1723)
Antony van Leeuwenhoek was a Dutch cloth merchant, who also happened to dabble in microscopes. He constructed a simple microscope (which has a single lens), where the lens was held between two silver plates. Apparently he relished viewing microbes from many different sample types – pond water, fecal material, teeth scrapings, etc. He made detailed drawings and notes about his observations and discoveries, sending them off to the Royal Society of London, the scientific organization of that time. This invaluable record clearly indicates that he saw both bacteria and a wide variety of protists. Some microbiologists refer to van Leeuwenhoek as the “Father of Microbiology,” because of his contributions to the field.
Microbial Groups
Classification of organisms, or the determination of how to group them, continually changes as we acquire new information and new tools of assessing the characteristics of an organism. Currently all organisms are grouped into one of three categories or domains: Bacteria, Archaea, and Eukarya. The Three Domain Classification, first proposed by Carl Woese in the 1970s, is based on ribosomal RNA (rRNA) sequences and widely accepted by scientists today as the most accurate current portrayal of organism relatedness.
Tree of Life.
Bacteria
The Bacteria domain contains some of the best known microbial examples (E. coli, anyone?). Most of the members are unicellular, cells lack a nucleus or any other organelle, most members have a cell wall with a particular substance known as peptidoglycan (not found anywhere else but in bacteria!), and humans are intimately familiar with many members, since they are common in soil, water, our foods, and our own bodies.
Archaea
Archaea is a relatively new domain, since these organisms used to be grouped with the bacteria. There are some obvious similarities, since they are mostly unicellular and cells lack a nucleus or any other organelle. But they have completely different cell walls that vary markedly in composition (but notably lack peptidoglycan) and their rRNA sequences have shown that they are not closely related to the Bacteria at all. In fact, they appear to be more closely related to the eukaryotes! These organisms are found in soil, water, even sometimes in the human body, but they are also found in some very extreme environments on Earth – very cold, very hot, very salty, very pressurized, very acidic, earning them the commonly used name “the extremophiles,” or extreme-loving organisms.
Eukarya
The Eukarya Domain includes many non-microbes, such as animals and plants, but there are numerous microbial examples as well, such as fungi, protists, slime molds, and water molds. The eukaryotic cell type has a nucleus, as well as many organelles, such as mitochondria or endoplasmic reticulum.
Viruses
Viruses are not part of the Three Domain Classification, since they lack ribosomes and therefore lack rRNA sequences for comparison. They are classified separately, using characteristics specific to viruses. Viruses are typically described as “obligate intracellular parasites,” a reference to their strict requirement for a host cell in order to replicate or increase in number. These acellular entities are often agents of disease, a result of their cell invasion.
Taxonomic Ranks
Taxonomic ranks are a way for scientists to organize information about organisms, by determining relatedness. Domains are the largest grouping used, followed by numerous smaller groupings, where each smaller grouping consists of organisms that share specific features in common. Each level becomes more and more restrictive as to whom can be a member. Eventually we get down to genus and species, the groupings used for formation of a scientific name. This is the binomial nomenclature devised by Carl Linnaeus in the 1750s.
Taxonomic Ranks. By Annina Breen (Own work) [CC BY-SA 4.0], via Wikimedia Commons
Binomial Nomenclature
When referring to the actual scientific name assigned to an organism, it is important to follow convention, so it is clear to everyone that you are referring to the scientific name. There are rules in science (just like in English class, where you would never refer to “mr. robert louis stevenson,” or at least not without expecting to get your paper back with red all over it).
A scientific name is composed of a genus and a species, where the genus is a generic name and the species is specific. The species name, once assigned, is permanent for the organism, while the genus can change if new information becomes available. For example, the bacterium previously known as Streptococcus faecalis is now Enterococcus faecalis because sequencing information indicates that it is more closely related to the members of the Enterococcus genus. It is important to note that it is inappropriate to refer to an organism by the species alone (i.e. you should never refer to E. coli as “coli” alone. Other bacteria can have the species “coli” as well.)
Now for the rules: The genus is always capitalized. The species is always lowercase. And both the genus and the species are italicized (common if typewritten) or underlined (common if handwritten). The genus may be shortened to its starting letter, but only if the name has been referred to in the text in its entirety at least once first (the exception to this is E. coli, due to its commonality, where hardly anyone spells out the Escherichia genus anymore).
Key Words
microbiology, microorganisms, microbes, unicellular, multicellular, differentiation, sterilization, observation, micrometers (µm), nanometers (nm), Robert Hooke, compound microscope, Antony van Leeuwenhoek, simple microscope, Royal Society of London, Father of Microbiology, Three Domain Classification, ribosomal RNA (rRNA), Bacteria, Archaea, Eukarya, obligate intracellular parasites, taxonomic ranks, genus, species, binomial nomenclature
Study Questions
1. Who are the members of the microbial world?
2. What is the complete definition of microbiology? What characteristics are relevant?
3. What size are different groups of microbes?
4. What were the contributions of Hooke and Van Leeuwenhoek to the field of microbiology? How did they make these contributions?
5. What is the basis for Woese’s classification and what are the three domains?
6. What are the basic characteristics of members of the three domains? Where do microbes fit in?
7. What are the basic characteristics of viruses? Why are they not classified in one of the three domains?
8. What are taxonomic ranks? What is the system of binomial nomenclature? What are the basic rules? How are bacteria named? What is a genus and species? Be able to write a bacterial name correctly. | textbooks/bio/Microbiology/Microbiology_(Bruslind)/01%3A_Introduction_to_Microbiology.txt |
You have probably figured out that microbes (AKA microorganisms) are pretty small, right? Yeah, well, size isn’t everything. But numbers, that is something. If you were to take one gram of soil and start counting the microbes in it at a rate of 1 microbe/second, it would take you over 33 years to complete your counting. Then most of you would be in your 50s and having a mid-life crisis, so let’s not go there…but the small size of microbes certainly has made it difficult to study them, particularly in the beginning. (If you want a visual idea of scale, check out the Cell Size and Scale tool, which allows you to zoom in from a coffee bean to a carbon atom. Be sure to pay careful attention to the microbes in between!)
O.k., if you want to see something really, really, really small, who ya gonna call? Not GhostbustersTM, that’s for sure. I would try someone with a microscope. (Microscope Man? Maybe not.) Now I will admit, with the advent of molecular biology there’s a lot of microbiology nowadays that happens without a microscope. But if you want to actually visualize microbes, you’ll need the ability to magnify – you’ll need a microscope of some type. And, since “seeing is believing,” it was the visualization of microbes that got people interested in them in the first place.
Microscopy in the 1600s
It is believed that Robert Hooke was one of the first scientists to actually observe microbes, in 1665. His illustrations and observations from a variety of objects viewed under a microscope were published in the book Micrographia. Hooke used a compound microscope, meaning it contained two sets of lenses for magnification: the ocular lens next to the eye and the objective lens, next to the specimen or object. The magnification of a compound microscope is a product of the ocular lens magnification and the objective lens magnification. Thus a microscope with an ocular magnification of 10x and an objective magnification of 50x would have a total magnification of 500x. You can see a drawing of Hooke’s microscope.
Antonie van Leeuwenhoek, often called the “Father of Microbiology,” wasn’t a scientist by profession. He was a cloth merchant from Holland who was believed to be inspired by Mr. Hooke’s work, probably with the original intention of examining textiles to determine quality. Very quickly van Leeuwenhoek started examining just about everything under the microscope and we know this because he kept detailed notes about both his samples and his observations. Van Leeuwenhoek was using what is called a simple microscope, a microscope with just a single lens. Essentially, it is a magnifying glass. But the lenses that he produced were of such high quality that he is given credit for the discovery of single-celled life forms. You can learn more about van Leeuwenhoek’s observations.
Modern Microscopy: Light Microscopes
Let us face it, a modern microscope is a pretty technical tool, even one of the cheaper versions. If you want to understand the limitations of a light microscope you have to understand concepts like resolution, wavelength, and numerical aperture, where their relationship to one another is summed up by the Abbé equation:
In microscopy the definition of resolution is typically the ability of a lens to distinguish two objects that are close together. So, in the Abbé equation d becomes the minimal distance where two objects next to one another can be resolved or distinguished as individual objects. Resolution is dependent upon the wavelengthof illumination being used, where a shorter wavelength will result in a smaller d. Lastly, we have the effect of the numerical aperture, which is a function of the objective lens and its ability to gather light. The numerical aperture value is actually defined by two components: n, which is the refractive index of the medium the lens is working in, and sin θ, which is a measurement of the cone of light that enters the objective. A lens can typically work in two media: air, with a refractive index of 1.00, or oil, with a refractive index of 1.25. Oil will allow more of the light to be collected, by directing more of the light rays into the objective lens. The maximum total magnification for a microscope using visual light for illumination is around 1500X, where the microscope might have 15x oculars and a 100x oil immersion objective. The highest resolution possible is around 0.2 μm. If objects or cells are closer together than this, they can’t be distinguished as separate entities.
Here’s a nice description from Nikon, including an interactive tutorial on numerical aperture and image resolution. And then there are so many microscopes, so little time! The type that you need depends upon the specific type of microbes that you want to visualize.
For light microscopes there are six different types of microscopes, all using light as the source of illumination: bright-field microscope, dark-field microscope, phase contrast microscope, differential interference contrast (DIC), fluorescence microscope, and confocal scanning laser microscope (CSLM). Let us look at the details of each type:
Bright-field Microscope
The bright-field microscope is your standard microscope that you could purchase for your niece or nephew at any toy store. Here is a website on the basic parts of a bright-field compound microscope, in case you are not enrolled in the general microbiology lab. The specimen is illuminated by a light source at the base of the microscope and then initially magnified by the objective lens, before being magnified again by the ocular lens. Remember that the total magnification achieved is a product of the magnification of both lenses.
The specimen is typically visualized because of differences in contrast between the specimen itself and its surrounding environment. But that does not apply to unstained bacteria, which have very little contrast with their environment, unless the cells are naturally pigmented. That is why staining (see section below) is such an important concept in microscopy. A bright-field microscope will work reasonably well to view the larger eukaryotic microbes (i.e. protozoa, algae) without stain, but unstained bacteria will be almost invisible. Stained bacteria will appear dark against a bright background (ah, I knew that there was a reason for the term “bright-field.”)
Bright-Field Microscope
Dark-field Microscope
The dark-field microscope is really just a slightly modified bright-field microscope. In fact, you could make this modification to the microscope you have at home! It makes use of what is known as a dark-field stop, an opaque disk that blocks light directly underneath the specimen so that light reaches it from the sides. The result is that only light that has been reflected or refracted by the specimen will be collected by the objective lens, resulting in cells that appear bright against a dark background (thus the term “dark-field.” Yes, it’s all making sense now). This allows for observation of living, unstained cells which is particularly nice if you want to observe motility or eukaryotic organelles.
Phase Contrast Microscope
The phase-contrast microscope is also a modified bright-field microscope, although the modifications are getting more complex, as well as more expensive. This microscope also uses an opaque ring or annular stop, but this one has a transparent ring that only releases light in a hollow cone. The principle of this microscope gets back to the idea of refractive index and the fact that cells have a different refractive index than their surroundings, resulting in light that differs slightly in phase. The difference is amplified by a phase ring found in a special phase objective. The phase differences can be translated into differences in brightness, resulting in a dark image amidst a bright background. This allows for the observation of living, unstained cells, once again useful to observe motility or eukaryotic organelles.
Phase Contrast Microscope Mechanics.
Differential Interference Contrast (DIC) Microscope
The differential interference contrast microscope operates on much the same principle as the phase-contrast microscope, by taking advantage of the differences in refractive index of a specimen and its surroundings. But it uses polarized light that is then split into two beams by a prism. One beam of light passes through the specimen, the other passes through the surrounding area. When the beams are combined via a second prism they “interfere” with one another, due to being out of phase. The resulting images have an almost 3D effect, useful for observing living, unstained cells.
Differential Contrast Microscope Mechanism.
Fluorescence Microscope
A fluorescence microscope utilizes light that has been emitted from a specimen, rather than passing through it. A mercury-arc lamp is used to generate an intense beam of light that is filtered to produce a specific wavelength of light directed at the specimen by use of dichromatic mirror, which reflects short wavelengths and transmits longer wavelengths. Naturally fluorescent organisms will absorb the short-wavelengths and emit fluorescent light with a longer wavelength that will pass through the dichromatic mirror and can be visualized. There are a variety of microbes with natural fluorescence but there are certainly far more organisms that lack this quality. Visualization of the latter organisms depends upon the use of fluorochromes, fluorescent dyes that bind to specific cell components. The fluorochromes can also be attached to antibodies, to highlight specific structures or areas of the cell, or even different organisms.
Fluorescence Microscope. By Masur (Own work) [GFDL, CC-BY-SA-3.0 or CC BY-SA 2.5-2.0-1.0], via Wikimedia Commons
Fluorescence Microscope Mechanism.
Confocal Scanning Laser Microscope (CSLM)
In order to understand how a confocal scanning laser microscope works, it is helpful to understand how a fluorescence microscope works, so hopefully you already read the previous section. A CSLM uses a laser for illumination, due to the high intensity. The light is directed at dichromatic mirrors that move, “scanning” the specimen. The longer wavelengths emitted by the fluorescently stained specimen pass back through the mirrors, through a pinhole, and are measured by a detector. The pinhole serves to conjugate the focal point of the lens (ah, that’s where the term confocal came from!), which means it allows for complete focus of a given point. Since the entire specimen is scanned in the x-z planes (all three axes), the information acquired by the detector can be compiled by a computer to create a single 3D image entirely in focus. This is a particularly useful tool for viewing complex structures such as biofilms.
Confocal Scanning Laser Microscope Mechanism.
Staining
Most of the microbes, particularly unicellular microbes, would not be apparent without the help of staining. It helps to make something so small a bit easier to see, by providing contrast between the microorganism and its background. A simple stain makes use of a single dye, either to stain the cells directly (direct stain) or to stain the background surrounding the cells (negative stain). From this a researcher can gather basic information about a cell’s size, morphology (shape), and cell arrangement.
There are also more complex stains, known as differential stains, that combine stains to allow for differentiation of organisms based on their characteristics. The Gram stain, developed in 1884, is the most common differential stain used in microbiology, where bacterial cells are separated based on their cell wall type: gram positive bacteria which stain purple and gram negative bacteria which stain pink. Some bacteria have a specialized cell wall that must be stained with the acid-fast stain, where acid-fast bacteria stain red and non-acid-fast bacteria stain blue. Other differential stains target specific bacterial structures, such as endospores, capsules, and flagella, to be talked about later.
Even More Modern Microscopy: Electron Microscopes
Light microscopes are great if you are observing eukaryotic microbes and they might work for observing bacteria and archaea, but they are not going to work at all to observe viruses. Remember that the limit of resolution for a light microscope is 0.2 μm or 200 nm and most viruses are smaller than that. So, we need something more powerful. Enter the electron microscopes, which replace light with electrons for visualization. Since electrons have a wavelength of 1.23 nm (as opposed to the 530 nm wavelength of blue-green light), resolution increases to around 0.5 nm, with magnifications over 150,000x. The drawback of using electrons is that they must be contained in a vacuum, eliminating the possibility of working with live cells. There is also some concern that the extensive sample preparation might distort the specimen’s characteristics or cause artifacts to form.
There are two different types of electron microscope, the transmission electron microscope (TEM) and the scanning electron microscope (SEM):
Transmission Electron Microscope (TEM)
The transmission electron microscope utilizes an electron beam directed at the specimen with the use of electromagnets. Dense areas scatter the electrons, resulting in a dark area on the image, while electrons can pass (or “transmit”) through the less dense areas, resulting in a brighter section. The image is generated on a fluorescent screen and can then be captured.
Since the electrons are easily scattered by extremely thick specimens, samples must be sliced down to 20-100 nm in thickness, typically by being embedded in some type of plastic and then being cut with a diamond knife into extremely thin sections. The resulting pictures represent one slice or plane of the specimen.
Transmission Electron Microscope.
Transmission Electron Microscope. By kallerna (Own work) [Public domain], via Wikimedia Commons
Scanning Electron Microscope (SEM)
The scanning electron microscope also utilizes an electron beam but the image is formed from secondary electrons that were released from the surface of the specimen and then collected by a detector. More electrons are released from raised areas of the specimen, while less secondary electrons will be collected from sunken areas. In addition, the electron beam is scanned over the specimen surface, producing a 3D image of the external features.
If you want to see some beautiful TEM and SEM photomicrographs, check out Dennis Kunkel’s site. Most have been colorized, but they are quite stunning. On the other end of the spectrum, here are pictures taken with the Intel Play QX3, a plastic microscope for children. Be careful, you could get lost in this website. But it is great to see what an inexpensive microscope can produce in the hands of someone who knows what they’re doing! These pictures are stunning as well.
Scanning Electron Microscope Mechanism.
Scanning Electron Microscope. By en:User:Olaboy [CC BY-SA 2.5], Via Wikipedia Commons
21st Century Microscopy: Scanning Probe Microscopes
As technology has advanced, even more powerful microscopes have been invented, ones that can even allow for visualization at the atomic level. These microscopes can be used in microbiology but more often they are used in other fields, to allow visualization of chemicals, metals, magnetic samples, and nanoparticles, wherever the 0.1 nm resolution and 100,000,000x magnification might be needed.
The scanning probe microscopes are thus named because they move some type of probe over a specimen’s surface in the x-z planes, allowing computers to generate an extremely detailed 3D image of the specimen. Resolution is so high because the probe size is much smaller than the wavelength of either visible light or electrons. Both microscopes can be used to study objects in liquid, allowing for the examination of biological molecules. There are two different types of scanning probe microscopes, the scanning tunneling microscope (STM) and the atomic force microscope (AFM):
Scanning Tunneling Microscope (STM)
The scanning tunneling microscope has an extremely sharp probe, 1 atom thick, that maintains a constant voltage with the specimen surface allowing electrons to travel between them. This tunneling current is maintained by raising and lowering the probe to sustain a constant height above the sample. The resulting motion is tracked by a computer, which generates the final image.
Scanning Tunneling Microscope Mechanism.
Atomic Force Microscope (AFM)
The atomic force microscope was developed as an alternative to the STM, for use with samples that do not conduct electricity well. The microscope utilizes a cantilever with an extremely sharp probe tip that maintains a constant height above the specimen, typically by direct contact with the sample. Movement of the cantilever to maintain this contact deflects a laser beam, translating into an image of the object. Once again, computers are used to generate the image.
Atomic Force Microscope Mechanism.
Key Words
magnification, Robert Hooke, compound microscope, ocular lens, objective lens, total magnification, van Leeuwenhoek, simple microscope, Abbé equation, resolution, wavelength, numerical aperture, refractive index, oil immersion objective, light microscope, bright-field microscope, dark-field microscope, phase contrast microscope, differential interference contrast (DIC) microscope, fluorescence microscope, fluorochrome, confocal scanning laser microscope (CSLM), simple stain, direct stain, negative stain, differential stain, Gram stain, acid-fast stain, electron microscope (EM), transmission electron microscope (TEM), scanning electron microscope (SEM), scanning probe microscopes, scanning tunneling microscopes (STM), atomic force microscope (AFM).
Essential Questions/Objectives
1. What roles did Hooke and van Leeuwenhoek play in the development of microscopy? How did their contributions differ?
2. How do magnification & resolution differ? How is total magnification determined?
3. How does the Abbé equation explain the resolution of a microscope? What components impact resolution? What is the function of immersion oil?
4. Know the main uses and understand the principle mechanics of the following light microscopes: brightfield, dark-field, phase contrast, fluorescence, and differential interference contrast.
5. How does the confocal scanning laser microscope work to form a 3 dimensional image? How does it improve resolution compared to other light microscopes?
6. How is staining used in microscopy? What are the general categories of stains and how are they used?
7. What are the advantages and problems with electron microscopes? What are the effective magnification, resolution and main uses of electron microscopes?
8. How does the TEM differ from the SEM in terms of function and final product?
9. How do scanning probe microscopes work and what do they allow us to see? Why are they useful for studying biological molecules? What is the difference in the scanning tunneling and the atomic force microscope?
Exploratory Questions (OPTIONAL)
1. How have microscopes improved our understanding of microbes? What are the limitations of microscopes and the information that we get from them? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/02%3A_Microscopes.txt |
Traditionally, cellular organisms have been divided into two broad categories, based on their cell type. They are either prokaryotic or eukaryotic. In general, prokaryotes are smaller, simpler, with a lot less stuff, which would make eukaryotes larger, more complex, and more cluttered. The crux of their key difference can be deduced from their names: “karyose” is a Greek word meaning “nut” or “center,” a reference to the nucleus of a cell. “Pro” means “before,” while “Eu” means “true,” indicating that prokaryotes lack a nucleus (“before a nucleus”) while eukaryotes have a true nucleus. More recently, microbiologists have been rebelling against the term prokaryote because it lumps both bacteria and the more recently discovered archaea in the same category. Both cells are prokaryotic because they lack a nucleus and other organelles (such as mitochondria, Golgi apparatus, endoplasmic reticulum, etc), but they aren’t closely related genetically. So, to honor these differences this text will refer to the groups as the archaea, the bacteria, and the eukaryotes and try to leave the prokaryotic reference out of it.
Cell Morphology
Cell morphology is a reference to the shape of a cell. It might seem like a trivial concept but to a cell it is not. The shape dictates how that cell will grow, reproduce, obtain nutrients, move, and it’s important to the cell to maintain that shape to function properly. Cell morphology can be used as a characteristic to assist in identifying particular microbes but it’s important to note that cells with the same morphology are not necessarily related. Bacteria tend to display the most representative cell morphologies, with the most common examples listed here:
Bacterial Cell Morphologies.
• Coccus (pl. cocci) – a coccus is a spherically shaped cell.
• Bacillus (pl. bacilli) – a bacillus is a rod-shaped cell.
• Curved rods – obviously this is a rod with some type of curvature. There are three sub-categories: the vibrio, which are rods with a single curve and the spirilla/spirochetes, which are rods that form spiral shapes. Spirilla and spirochetes are differentiated by the type of motility that they exhibit, which means it is hard to separate them unless you are looking at a wet mount.
• Pleomorphicpleomorphic organisms exhibit variability in their shape.
There are additional shapes seen for bacteria, and an even wider array for the archaea, which have even been found as star or square shapes. Eukaryotic microbes also tend to exhibit a wide array of shapes, particularly the ones that lack a cell wall such as the protozoa.
Cell Size
Cell size, just like cell morphology, is not a trivial matter either, to a cell. There are reasons why most archaeal/bacterial cells are much smaller than eukaryotic cells. Much of it has to do with the advantages derived from being small. These advantages relate back to the surface-to-volume ratio of the cell, a ratio of the external cellular layer in contact with the environment compared to the liquid inside. This ratio changes as a cell increases in size. Let us look at a 2 μm cell in comparison with a cell that is twice as large at 4 μm.
r = 1 μm
surface area = 12.6 μm2
volume = 4.2 μm3
r = 2 μm
surface area = 50.3 μm2
volume = 33.5 μm3
The surface-to-volume ratio of the smaller cell is 3, while the surface-to-volume ratio of the larger cell decreases to 1.5. Think of the cell surface as the ability of the cell to bring in nutrients and let out waste products. The larger the surface area, the more possibilities exist for engaging in these activities. Based on this, the larger cell would have an advantage. Now think of the volume as representing what the cell has to support. As the surface-to-volume ratio goes down, it indicates that the cell has less of an opportunity to bring in the nutrients needed to support the cell’s activities – activities such as growth and reproduction. So, small cells grow and reproduce faster. This also means that they evolve faster over time, giving them more opportunities to adapt to environments.
Keep in mind that the size difference (bacterial/archaeal cells = smaller, eukaryotic cells = larger) is on average. A typical bacterial/archaeal cell is a few micrometers in size, while a typical eukarytotic cell is about 10x larger. There are a few monster bacteria that fall outside the norm in size and still manage to grow and reproduce very quickly. One such example is Thiomargarita namibiensis, which can measure from 100-750 μm in length, compared to the more typical 4 μm length of E. coli. T. namibiensis manages to maintain its rapid reproductive rate by producing very large vacuoles or bubbles that occupy a large portion of the cell. These vacuoles reduce the volume of the cell, increasing the surface-to-volume ratio. Other very large bacteria utilize a ruffled membrane for their outer surface layer. This increases the surface area, which also increase the surface-to-volume ratio, allowing the cell to maintain its rapid reproductive rate.
Cell Components
All cells (bacterial, archaeal, eukaryotic) share four common components:
• Cytoplasmcytoplasm is the gel-like fluid that fills each cell, providing an aqueous environment for the chemical reactions that take place in a cell. It is composed of mostly water, with some salts and proteins.
• DNA – deoxyribonucleic acid or DNA is the genetic material of the cell, the instructions for the cell’s abilities and characteristics. This complete set of genes, referred to as a genome, is localized in an irregularly-shaped region known as the nucleoid in bacterial and archaeal cells, and enclosed into a membrane-bound nucleus in eukaryotic cells.
• Ribosomes – the protein-making factories of the cell are the ribosomes. Composed of both RNA and protein, there are some distinct differences between the ones found in bacteria/archaea and the ones found in eukaryotes, particularly in terms of size and location. The ribosomes of bacteria and archaea are found floating in the cytoplasm, while many of the eukaryotic ribosomes are organized along the endoplasmic reticulum, a eukaryotic organelle. Ribosomes are measured using the Svedberg unit, which corresponds to the rate of sedimentation when centrifuged. Bacterial/archaeal ribosomes have a measurement of 70S as a sedimentation value, while eukaryotic ribosomes have a measurement of 80S, an indication of both their larger size and mass.
• Plasma Membrane – one of the outer boundaries of every cell is the plasma membrane or cell membrane. (A plasma membrane can be found elsewhere as well, such as the membrane that bounds the eukaryotic nucleus, while the term cell membrane refers specifically to this boundary of the cell proper). The plasma membrane separates the cell’s inner contents from the surrounding environment. While not a strong layer, the plasma membrane participates in several crucial processes for the cell, particularly for bacteria and archaea, which typically only have the one membrane:
• Acts as a semi-permeable barrier to allow for the entrance and exit of select molecules. It functions to let in nutrients, excrete waste products, and possibly keep out dangerous substances such as toxins or antibiotics.
• Performs metabolic processes by participating in the conversion of light or chemical energy into a readily useable form known as ATP. This energy conservation involves the development of a proton motive force (PMF), based on the separation of charges across the membrane, much like a battery.
• “Communicates” with the environment by binding or taking in small molecules that act as signals and provide information important to the cell. The information might relate to nutrients or toxin in the area, as well as information about other organisms.
Typical Prokaryotic Cell.
Typical Eukaryotic Cell. By Mediran (Own work) [CC BY-SA 3.0], via Wikimedia Commons
Eukaryotes have numerous additional components called organelles, such as the nucleus, the mitochrondria, the endoplasmic reticulum, the Golgi apparatus, etc. These are all membrane-bound compartments that house different activities for the cell. Because each structure is bounded by its very own plasma membrane, it provides the cell with multiple locations for membranous functions to occur.
Plasma Membrane Structure
When talking about the details of the plasma membrane it gets a little bit complicated, since bacteria and eukaryotes share the same basic structure, while archaea have marked differences. For now let us cover the basic structure, while the archaeal modifications and variations will be covered in the chapter on archaea.
The plasma membrane is often described by the fluid-mosaic model, which accounts for the movement of various components within the membrane itself. The general structure is explained by the separation of individual substances based on their attraction or repulsion of water. The membrane is typically composed of two layers (a bilayer) of phospholipids, which form the basic structure. Each phospholipid is composed of a polar region that is hydrophilic (“water loving”) and a non-polar region that is hydrophobic (“water fearing”). The phospholipids will spontaneously assemble in such a way as to keep the polar regions in contact with the aqueous environment outside of the cell and the cytoplasm inside, while the non-polar regions are sequestered in the middle, much like the jelly in a sandwich.
The phospholipids themselves are composed of a negatively-charged polar head which is a phosphate group, connected by a glycerol linkage to two fatty acid tails. The phosphate group is hydrophilic while the fatty acid tails are hydrophobic. While the membrane is not considered to be particularly strong, it is strengthened somewhat by the presence of additional lipid components, such as the steroids in eukaryotes and the sterol-like hopanoids in bacteria. Embedded and associated with the phospholipid bilayer are various proteins, with myriad functions. Proteins that are embedded within the bilayer itself are called integral proteins while proteins that associate on the outside of the membrane are called peripheral proteins. Some of the peripheral proteins are anchored to the membrane via a lipid tail, and many associate with specific integral proteins to fulfill cellular functions. Integral proteins are the dominant type, representing about 70-80% of the proteins associated with a plasma membrane, while the peripheral proteins represent the remaining 20-30%.
Plasma Membrane Structure.
The amount of protein composing a plasma membrane, in comparison to phospholipid, differs by organism. Bacteria have a very high protein to phospholipid ratio, around 2.5:1, while eukaryotes exhibit a ratio of 1:1, at least in their cell membrane. But remember that eukaryotes have multiple plasma membranes, one for every organelle. The protein to phospholipid ratio for their mitochondrial membrane is 2.5:1, just like the bacterial plasma membrane, providing additional evidence for the idea that eukaryotes evolved from a bacterial ancestor.
Key Words
prokaryote, eukaryote, morphology, coccus, bacillus, vibrio, spirilla, spirochete, pleomorphic, surface-to-volume (S/V) ratio, cytoplasm, DNA, genome, nucleoid, nucleus, ribosome, Svedberg unit, plasma membrane, cell membrane, proton motive force (PMF), fluid-mosaic model, phospholipid, hydrophilic, hydrophobic, polar head, phosphate group, glycerol linkage, fatty acid tail, steroids, hopanoids, phospholipid bilayer, integral protein, peripheral protein.
Essential Questions/Objectives
1. Why are microbiologists questioning the traditional ways of thinking about “prokaryotes”?
2. What are the 3 basic shapes of Bacteria?
3. How do microbes belonging to the categories of Eukaryotes vs. Bacteria/Archaea typically differ in terms of size? How does size impact a cell? What role does the surface:volume ratio play? How can cells get around limitations imposed by the surface:volume ratio?
4. What are two ways that bacteria can adapt to being large? Give specific examples.
5. What are the basic components of any cell?
6. What are the roles of the plasma membrane?
7. What is the fluid mosaic model?
8. Understand the basic structure of the phospholipids of the plasma membrane and the role that it plays in membrane design.
9. What are other lipids found in the plasma membrane?
10. What are the 2 categories of proteins found in the plasma membrane and how do they differ?
11. How do the phospholipids and the protein come together to form a working plasma membrane?
12. What is the importance of the protein to phospholipid ratio in terms of evolution?
Exploratory Questions (OPTIONAL)
1. What is the largest bacterium or archaean ever discovered? What is the smallest eukaryote ever discovered? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/03%3A_Cell_Structure_I.txt |
It is important to note that not all bacteria have a cell wall. Having said that though, it is also important to note that most bacteria (about 90%) have a cell wall and they typically have one of two types: a gram positive cell wall or a gram negative cell wall. The two different cell wall types can be identified in the lab by a differential stain known as the Gram stain. Developed in 1884, it’s been in use ever since. Originally, it was not known why the Gram stain allowed for such reliable separation of bacterial into two groups. Once the electron microscope was invented in the 1940s, it was found that the staining difference correlated with differences in the cell walls. Here is a website that shows the actual steps of the Gram stain. After this stain technique is applied the gram positive bacteria will stain purple, while the gram negative bacteria will stain pink.
Overview of Bacterial Cell Walls
A cell wall, not just of bacteria but for all organisms, is found outside of the cell membrane. It’s an additional layer that typically provides some strength that the cell membrane lacks, by having a semi-rigid structure.
Both gram positive and gram negative cell walls contain an ingredient known as peptidoglycan (also known as murein). This particular substance hasn’t been found anywhere else on Earth, other than the cell walls of bacteria. But both bacterial cell wall types contain additional ingredients as well, making the bacterial cell wall a complex structure overall, particularly when compared with the cell walls of eukaryotic microbes. The cell walls of eukaryotic microbes are typically composed of a single ingredient, like the cellulose found in algal cell walls or the chitin in fungal cell walls.
The bacterial cell wall performs several functions as well, in addition to providing overall strength to the cell. It also helps maintain the cell shape, which is important for how the cell will grow, reproduce, obtain nutrients, and move. It protects the cell from osmotic lysis, as the cell moves from one environment to another or transports in nutrients from its surroundings. Since water can freely move across both the cell membrane and the cell wall, the cell is at risk for an osmotic imbalance, which could put pressure on the relatively weak plasma membrane. Studies have actually shown that the internal pressure of a cell is similar to the pressure found inside a fully inflated car tire. That is a lot of pressure for the plasma membrane to withstand! The cell wall can keep out certain molecules, such as toxins, particularly for gram negative bacteria. And lastly, the bacterial cell wall can contribute to the pathogenicity or disease –causing ability of the cell for certain bacterial pathogens.
Structure of Peptidoglycan
Let us start with peptidoglycan, since it is an ingredient that both bacterial cell walls have in common. Peptidoglycan is a polysaccharide made of two glucose derivatives, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), alternated in long chains. The chains are cross-linked to one another by a tetrapeptide that extends off the NAM sugar unit, allowing a lattice-like structure to form. The four amino acids that compose the tetrapeptide are: L-alanine, D-glutamine, L-lysine or meso-diaminopimelic acid (DPA), and D-alanine. Typically only the L-isomeric form of amino acids are utilized by cells but the use of the mirror image D-amino acids provides protection from proteases that might compromise the integrity of the cell wall by attacking the peptidoglycan. The tetrapeptides can be directly cross-linked to one another, with the D-alanine on one tetrapeptide binding to the L-lysine/ DPA on another tetrapeptide. In many gram positive bacteria there is a cross-bridge of five amino acids such as glycine (peptide interbridge) that serves to connect one tetrapeptide to another. In either case the cross-linking serves to increase the strength of the overall structure, with more strength derived from complete cross-linking, where every tetrapeptide is bound in some way to a tetrapeptide on another NAG-NAM chain.
While much is still unknown about peptidoglycan, research in the past ten years suggests that peptidoglycan is synthesized as a cylinder with a coiled substructure, where each coil is cross-linked to the coil next to it, creating an even stronger structure overall.
Peptidoglycan Structure.
Gram Positive Cell walls
The cell walls of gram positive bacteria are composed predominantly of peptidoglycan. In fact, peptidoglycan can represent up to 90% of the cell wall, with layer after layer forming around the cell membrane. The NAM tetrapeptides are typically cross-linked with a peptide interbridge and complete cross-linking is common. All of this combines together to create an incredibly strong cell wall.
The additional component in a gram positive cell wall is teichoic acid, a glycopolymer, which is embedded within the peptidoglycan layers. Teichoic acid is believed to play several important roles for the cell, such as generation of the net negative charge of the cell, which is essential for development of a proton motive force. Teichoic acid contributes to the overall rigidity of the cell wall, which is important for the maintenance of the cell shape, particularly in rod-shaped organisms. There is also evidence that teichoic acids participate in cell division, by interacting with the peptidoglycan biosynthesis machinery. Lastly, teichoic acids appear to play a role in resistance to adverse conditions such as high temperatures and high salt concentrations, as well as to β-lactam antibiotics. Teichoic acids can either be covalently linked to peptidoglycan (wall teichoic acids or WTA) or connected to the cell membrane via a lipid anchor, in which case it is referred to as lipoteichoic acid.
Since peptidoglycan is relatively porous, most substances can pass through the gram positive cell wall with little difficulty. But some nutrients are too large, requiring the cell to rely on the use of exoenzymes. These extracellular enzymes are made within the cell’s cytoplasm and then secreted past the cell membrane, through the cell wall, where they function outside of the cell to break down large macromolecules into smaller components.
Gram Negative Cell Walls
The cell walls of gram negative bacteria are more complex than that of gram positive bacteria, with more ingredients overall. They do contain peptidoglycan as well, although only a couple of layers, representing 5-10% of the total cell wall. What is most notable about the gram negative cell wall is the presence of a plasma membrane located outside of the peptidoglycan layers, known as the outer membrane. This makes up the bulk of the gram negative cell wall. The outer membrane is composed of a lipid bilayer, very similar in composition to the cell membrane with polar heads, fatty acid tails, and integral proteins. It differs from the cell membrane by the presence of large molecules known as lipopolysaccharide (LPS), which are anchored into the outer membrane and project from the cell into the environment. LPS is made up of three different components: 1) the O-antigen or O-polysaccharide, which represents the outermost part of the structure , 2) the core polysaccharide, and 3) lipid A, which anchors the LPS into the outer membrane. LPS is known to serve many different functions for the cell, such as contributing to the net negative charge for the cell, helping to stabilize the outer membrane, and providing protection from certain chemical substances by physically blocking access to other parts of the cell wall. In addition, LPS plays a role in the host response to pathogenic gram negative bacteria. The O-antigen triggers an immune response in an infected host, causing the generation of antibiotics specific to that part of LPS (think of E. coli O157). Lipid A acts as a toxin, specifically an endotoxin, causing general symptoms of illness such as fever and diarrhea. A large amount of lipid A released into the bloodstream can trigger endotoxic shock, a body-wide inflammatory response which can be life-threatening.
The outer membrane does present an obstacle for the cell. While there are certain molecules it would like to keep out, such as antibiotics and toxic chemicals, there are nutrients that it would like to let in and the additional lipid bilayer presents a formidable barrier. Large molecules are broken down by enzymes, in order to allow them to get past the LPS. Instead of exoenzymes (like the gram positive bacteria), the gram negative bacteria utilize periplasmic enzymes that are stored in the periplasm. Where is the periplasm, you ask? It is the space located between the outer surface of the cell membrane and the inner surface of the outer membrane, and it contains the gram negative peptidoglycan. Once the periplasmic enzymes have broken nutrients down to smaller molecules that can get past the LPS, they still need to be transported across the outer membrane, specifically the lipid bilayer. Gram negative cells utilize porins, which are transmembrane proteins composed of a trimer of three subunits, which form a pore across the membrane. Some porins are non-specific and transport any molecule that fits, while some porins are specific and only transport substances that they recognize by use of a binding site. Once across the outer membrane and in the periplasm, molecules work their way through the porous peptidoglycan layers before being transported by integral proteins across the cell membrane.
The peptidoglycan layers are linked to the outer membrane by the use of a lipoprotein known as Braun’s lipoprotein (good ol’ Dr. Braun). At one end the lipoprotein is covalently bound to the peptidoglycan while the other end is embedded into the outer membrane via its polar head. This linkage between the two layers provides additional structural integrity and strength.
Unusual and Wall-less Bacteria
Having emphasized the important of a cell wall and the ingredient peptidoglycan to both the gram positive and the gram negative bacteria, it does seem important to point out a few exceptions as well. Bacteria belonging to the phylum Chlamydiae appear to lack peptidoglycan, although their cell walls have a gram negative structure in all other regards (i.e. outer membrane, LPS, porin, etc). It has been suggested that they might be using a protein layer that functions in much the same way as peptidoglycan. This has an advantage to the cell in providing resistance to β-lactam antibiotics (such as penicillin), which attack peptidoglycan.
Bacteria belonging to the phylum Tenericutes lack a cell wall altogether, which makes them extremely susceptible to osmotic changes. They often strengthen their cell membrane somewhat by the addition of sterols, a substance usually associated with eukaryotic cell membranes. Many members of this phylum are pathogens, choosing to hide out within the protective environment of a host.
Key Words
cell wall, gram positive bacteria, gram negative bacteria, Gram stain, peptidoglycan, murein, osmotic lysis, N-acetylglucosamine (NAG), N-acetylmuramic acid (NAM), tetrapeptide, L-alanine, D-glutamine, L-lysine, meso-diaminopimelic acid (DPA), D-alanine, direct cross-link, peptide interbridge, complete cross-linking, teichoic acid, wall teichoic acid (WTA), lipoteichoic acid, exoenzymes, outer membrane, lipopolysaccharide (LPS), O-antigen or O-polysaccharide, core polysaccharide, lipid A, endotoxin, periplasmic enzymes, periplasm, porins, Braun’s lipoprotein, Chlamydiae, Tenericutes, sterols.
Essential Questions/Objectives
1. What are the basic characteristics and functions of the cell wall in Bacteria?
2. What is the Gram stain and how does it relate to the different cell wall types of Bacteria?
3. What is the basic unit structure of peptidoglycan? What components are present and how do they interact? Be able to diagram peptidoglycan and its’ components.
4. What is cross linking and why does this play such an important role in the cell wall? What different types of cross-linking are there?
5. Why are D-amino acids unusual and how does having D-amino acids in the peptidoglycan keep this macromolecule stable?
6. What are the differences between gram positive and negative organisms in terms of thickness of peptidoglycan, different constituents of PG and variations in cross linkage and strength, and other molecules associated with cell wall?
7. What is teichoic acid and what are its’ proposed roles and functions? What are lipteichoic acids?
8. What is the periplasm of gram negative bacteria? What purpose can it serve? What alternatives are available for cells?
9. What is the general composition of the outer membrane of gram-negative microorganisms, its function and toxic properties? How is it linked to the cell? What is a porin and what are their functions?
10. What group of bacteria lack peptidoglycan in their cell wall? What advantage does this confer?
11. What group of bacteria normally does not have cell walls and how do they maintain themselves?
Exploratory Questions (OPTIONAL)
1. How does the mechanism of the Gram stain relate to specific components of the bacterial cell wall? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/04%3A_Bacteria%3A_Cell_Walls.txt |
We have already covered the main internal components found in all bacteria, namely, cytoplasm, the nucleoid, and ribosomes. Remember that bacteria are generally thought to lack organelles, those bilipid membrane-bound compartments so prevalent in eukaryotic cells (although some scientists argue that bacteria possess structures that could be thought of as simple organelles). But bacteria can be more complex, with a variety of additional internal components to be found that can contribute to their capabilities. Most of these components are cytoplasmic but some of them are periplasmic, located in the space between the cytoplasmic and outer membrane in gram negative bacteria.
Cytoskeleton
It was originally thought that bacteria lacked a cytoskeleton, a significant component of eukaryotic cells. In the last 20 years, however, scientists have discovered bacterial filaments made of proteins that are analogues to the cytoskeletal proteins found in eukaryotes. It has also been determined that the bacterial cytoskeleton plays important roles in cell shape, cell division, and integrity of the cell wall.
FtsZ
FtsZ, homologous to the eukaryotic protein tubulin, forms a ring structure in the middle of the cell during cell division, attracting other proteins to the area in order to construct a septum that will eventually separate the two resulting daughter cells.
MreB
MreB, homologous to the eukaryotic protein actin, is found in bacillus and spiral-shaped bacteria and plays an essential role in cell shape formation. MreB assumes a helical configuration running the length of the cell and dictates the activities of the peptidoglycan-synthesis machinery, assuring a non-spherical shape.
Crescentin
Crescentin, homologous to the eukaryotic proteins lamin and keratin, is found in spiral-shaped bacteria with a single curve. The protein assembles lengthwise in the inner curvature of the cell, bending the cell into its final shape.
Cytoskeleton Structures.
Inclusions
Bacterial inclusions are generally defined as a distinct structure located either within the cytoplasm or periplasm of the cell. They can range in complexity, from a simple compilation of chemicals such as crystals, to fairly complex structures that start to rival that of the eukaryotic organelles, complete with a membranous external layer. Their role is often to store components as metabolic reserves for the cell when a substance is found in excess, but they can also play a role in motility and metabolic functions as well.
Carbon storage
Carbon is the most common substance to be stored by a cell, since all cells are carbon based. In addition, carbon compounds can often be broken down quickly by the cell, so they can serve as energy sources as well. One of the simplest and most common inclusions for carbon storage is glycogen, in which glucose units are linked together in a multi-branching polysaccharide structure.
Another common way for bacteria to store carbon is in the form of poly-β-hydroxybutyrate (PHB), a granule that forms when β-hydroxybutyric acid units aggregate together. This lipid is very plastic-like in composition, leading some scientists to investigate the possibility of using them as a biodegradeable plastic. The PHB granules actually have a shell composed of both protein and a small amount of phospholipid. Both glycogen and PHB are formed when there is an excess of carbon and then broken down by the cell later for both carbon and energy.
Inorganic storage
Often bacteria need something other than carbon, either for synthesis of cell components or as an alternate energy reserve. Polyphosphate granules allow for the accumulation of inorganic phosphate (PO43-), where the phosphate can be used to make nucleic acid (remember the sugar-phosphate backbone?) or ATP (adenosine triphosphate, of course).
Other cells need sulfur as a source of electrons for their metabolism and will store excess sulfur in the form of sulfur globules, which result when the cell oxidizes hydrogen sulfide (H2S) to elemental sulfur (S0), resulting in the formation of refractile inclusions.
Non-storage functions
There are times when a bacterium needs to do something beyond simple storage of organic or inorganic compounds for use in metabolism and there are inclusions to help with these non-storage functions. One such example is gas vacuoles, which are used by the cell to control buoyancy in a water column, providing the cell with some control over where it is in the environment. It is a limited form of motility, on the vertical axis only. Gas vacuoles are composed of conglomerations of gas vesicles, cylindrical structures that are both hollow and rigid. The gas vesicles are freely permeable to all types of gases by passive diffusion and can be quickly constructed or collapsed, as needed by the cell to ascend or descend.
Magnetosomes are inclusions that contain long chains of magnetite (Fe3O4), which are used by the cell as a compass in geomagnetic fields, for orientation within their environment. Magnetotactic bacteria are typically microaerophilic, preferring an environment with a lower level of oxygen than the atmosphere. The magenetosome allows the cells to locate the optimum depth for their growth. Magenetosomes have a true lipid bilayer, reminiscent of eukaryotic organelles, but it is actually an invagination of the cell’s plasma membrane that has been modified with specific proteins.
Microcompartments
Bacterial microcompartments (BMCs) are unique from other inclusions by virtue of their structure and functionality. They are icosahedral in shape and composed of a protein shell made up of various proteins in the BMC family. While their exact role varies, they all participate in functions beyond simple storage of substances. These compartments provide both a location and the substances (usually enzymes) necessary for particular metabolic activities.
The best studied example of a BMC is the carboxysome, which are found in many CO2-fixing bacteria. Carboxysomes contain the enzyme ribulose-1,5-bisphosphate carboxylase (luckily it is also known as RubisCO), which plays a crucial role in converting CO2 into sugar. The carboxysome also plays a role in the concentration of CO2, thus ensuring that the components necessary for CO2-fixation are all in the same place at the same time.
Anammoxosome
The anammoxosome is a large membrane-bound compartment found in bacterial cells capable of carrying out the annamox reaction (anaerobic ammonium oxidation), where ammonium (NH4+) and nitrite (NO2-) are converted to dinitrogen gas (N2). The process is performed as a way for the cell to get energy, using ammonium as an electron donor and nitrite as an electron accept, with the resulting production of nitrogen gas. This chemical conversion of nitrogen is important for the nitrogen cycle.
Nitrogen Cycle. By Shou-Qing Ni and Jian Zhang [CC BY 3.0], via Wikimedia Commons
Chlorosome
Found in some phototrophic bacteria, a chlorosome is a highly efficient structure for capturing low light intensities. Lining the inside perimeter of the cell membrane, each chlorosome can contain up to 250,000 bacteriochlorophyll molecules, arranged in dense arrays. Harvested light is transferred to reaction centers in the cell membrane, allowing the conversion from light energy to chemical energy in the form of ATP. The chlorosome is bounded by a lipid monolayer.
Plasmid
A plasmid is an extrachromosomal piece of DNA that some bacteria have, in addition to the genetic material found in the nucleoid. It is composed of double-stranded DNA and is typically circular, although linear plasmids have been found. Plasmids are described as being “non-essential” to the cell, where the cell can function normally in their absence. But while plasmids have only a few genes, they can confer important capabilities for the cell, such as antibiotic resistance. Plasmids replicate independently of the cell and can be lost (known as curing), either spontaneously or due to exposure to adverse conditions, such as UV light, thymine starvation, or growth above optimal conditions. Some plasmids, known as episomes, can be integrated into the cell chromosome where the genes will be replicated during cell division.
Endospore
Then there is the endospore, a marvel of bacterial engineering. This is located under the heading “bacterial internal components,” but it is important to note that an endospore isn’t an internal or external structure so much as a conversion of the cell into an alternate form. Cells start out as a vegetative cell, doing all the things a cell is supposed to do (metabolizing, reproducing, mowing the lawn…). If they get exposed to hostile conditions (dessication, high heat, an angry neighbor…) and they have the ability, they might convert from a vegetative cell into an endospore. The endospore is actually formed within the vegetative cell (doesn’t that make it an internal structure?) and then the vegetative cell lyses, releasing the endospore (does that make it an external structure?).
Endospore Layers.
Endospores are only formed by a few gram positive genera and provide the cell with resistance to a wide variety of harsh conditions, such as starvation, extremes in temperature, exposure to drying, UV light, chemicals, enzymes, and radiation. While the vegetative cell is the active form for bacterial cells (growing, metabolizing, etc), the endospore can be thought of as a dormant form of the cell. It allows for survival of adverse conditions, but it does not allow the cell to grow or reproduce.
Structure
In order to be so incredibly resistant to so many different substances and environmental conditions, many different layers are necessary. The bacterial endospore has many different layers, starting with a core in the center. The core is the location of the nucleoid, ribosomes, and cytoplasm of the cell, in an extremely dehydrated form. It typically contains only 25% of the water found in a vegetative cell, increasing heat resistance. The DNA is further protected by the presence of small acid-soluble proteins (SASPs), which stabilize the DNA and protect it from degradation. DNA stabilization is increased by the presence of dipicolinic acid complexed with calcium (Ca-DPA), which inserts between the DNA bases. The core is wrapped in an inner membrane that provides a permeability barrier to chemicals, which is then surrounded by the cortex, a thick layer consisting of peptidoglycan with less cross-linking than is found in the vegetative cell. The cortex is wrapped in an outer membrane.Lastly are several spore coats made of protein, which provide protection from environmental stress such as chemicals and enzymes.
Sporulation: conversion from vegetative cell to endospore
Sporulation, the conversion of vegetative cell into the highly protective endospore, typically occurs when the cell’s survival is threatened in some way. The actual process is very complex and typically takes several hours until completion. Initially the sporulating cells replicates its DNA, as if it were about to undergo cell division. A septum forms asymmetrically, sequestering one copy of the chromosome at one end of the cell (called the forespore). Synthesis of endospore-specific substances occurs, altering the forespore and leading to the development of the layers specific for an endospore, as well as dehydration. Eventually the “mother cell” is lyses, allowing for the release of the mature endospore into the environment.
Sporulation.
Conversion from endospore to vegetative cell
The endospore remains dormant until environmental conditions improve, causing a chemical change that initiates gene expression. There are three distinct stages in the conversion from an endospore to the metabolically active vegetative cells: 1) activation, a preparation step that can be initiated by the application of heat; 2) germination, when the endospore becomes metabolically active and begins to take on water; 3) outgrowth, when the vegetative cell fully emerges from the endospore shell.
Key Words
cytoskeleton, FtsZ, tubulin, MreB, actin, crescentin, lamin, keratin, inclusion, glycogen, poly-β-hydroxybutyrate (PHB), polyphosphate granule, sulfur globule, gas vacuole, gas vesicle, magnetosome, microaerophilic, microcompartment, bacterial microcompartments (BMCs), carboxysome, ribulose-1,5-bisphosphate carboxylase, RubisCO, anammoxosome, annamox reaction, chlorosome, plasmid, curing, episome, endospore, vegetative cell, core, small acid-soluble proteins (SASPs), dipicolinic acid, Ca-DPA, inner membrane, cortex, outer membrane, spore coat, sporulation, forespore, activation, germination, outgrowth.
Essential Questions/Objectives
1. What are the roles and composition of the bacterial cytoskeleton? How does it differ from the eukaryotic cytoskeleton? What are the specific bacterial cytoskeleton proteins and what details are known about each?
2. What is the purpose of inclusions found in bacteria? What are their characteristics?
3. What are the specific examples of storage inclusions found in bacteria? Be able to describe each type in terms of structure and purpose.
4. What are other inclusions found in bacteria? Be able to describe each type in terms of structure and purpose.
5. How do microcompartments differ from inclusions? What are specific examples? What is the composition and purpose?
6. What are anammoxosomes? What is their composition and purpose?
7. What are plasmids and what characteristics do they have? What are episomes? What is curing and what causes it?
8. What are bacterial endospores? What is their purpose? What characteristics do they have? What are the various layers of an endospore and what role does each layer play?
Exploratory Questions (OPTIONAL)
1. What bacterial structures could be useful to scientists in addressing societal problems? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/05%3A_Bacteria%3A_Internal_Components.txt |
Layers Outside the Cell Wall
What have we learned so far, in terms of cell layers? All cells have a cell membrane. Most bacteria have a cell wall. But there are a couple of additional layers that bacteria may, or may not, have. These would be found outside of both the cell membrane and the cell wall, if present.
Capsule
A bacterial capsule is a polysaccharide layer that completely envelopes the cell. It is well organized and tightly packed, which explains its resistance to staining under the microscope. The capsule offers protection from a variety of different threats to the cell, such as desiccation, hydrophobic toxic materials (i.e. detergents), and bacterial viruses. The capsule can enhance the ability of bacterial pathogens to cause disease and can provide protection from phagocytosis (engulfment by white blood cells known as phagocytes). Lastly, it can help in attachment to surfaces.
Slime Layer
A bacterial slime layer is similar to the capsule in that it is typically composed of polysaccharides and it completely surrounds the cell. It also offers protection from various threats, such as desiccation and antibiotics. It can also allow for adherence to surfaces. So, how does it differ from the capsule? A slime layer is a loose, unorganized layer that is easily stripped from the cell that made it, as opposed to a capsule which integrates firmly around the bacterial cell wall.
S-Layer
Some bacteria have a highly organized layer made of secreted proteins or glycoproteins that self-assemble into a matrix on the outer part of the cell wall. This regularly structured S-layer is anchored into the cell wall, although it is not considered to be officially part of the cell wall in bacteria. S-layers have very important roles for the bacteria that have them, particularly in the areas of growth and survival, and cell integrity.
S layers help maintain overall rigidity of the cell wall and surface layers, as well as cell shape, which are important for reproduction. S layers protect the cell from ion/pH changes, osmotic stress, detrimental enzymes, bacterial viruses, and predator bacteria. They can provide cell adhesion to other cells or surfaces. For pathogenic bacteria they can provide protection from phagocytosis.
Structures Outside the Cell Wall
Bacteria can also have structures outside of the cell wall, often bound to the cell wall and/or cell membrane. The building blocks for these structures are typically made within the cell and then secreted past the cell membrane and cell wall, to be assembled on the outside of the cell.
Fimbriae (sing. fimbria)
Fimbriae are thin filamentous appendages that extend from the cell, often in the tens or hundreds. They are composed of pilin proteins and are used by the cell to attach to surfaces. They can be particularly important for pathogenic bacteria, which use them to attach to host tissues.
Pili (sing. pilin)
Pili are very similar to fimbriae (some textbooks use the terms interchangeably) in that they are thin filamentous appendages that extend from the cell and are made of pilin proteins. Pili can be used for attachment as well, to both surfaces and host cells, such as the Neisseria gonorrhea cells that use their pili to grab onto sperm cells, for passage to the next human host. So, why would some researchers bother differentiating between fimbriae and pili?
Pili are typically longer than fimbriae, with only 1-2 present on each cell, but that hardly seems enough to set the two structures apart. It really boils down to the fact that a few specific pili participate in functions beyond attachment. The conjugative pili participate in the process known as conjugation, which allows for the transfer of a small piece of DNA from a donor cell to a recipient cell. The type IV pili play a role in an unusual type of motility known as twitching motility, where a pilus attaches to a solid surface and then contracts, pulling the bacterium forward in a jerky motion.
Flagella (sing. flagellum)
Bacterial motility is typically provided by structures known as flagella. The bacterial flagellum differs in composition, structure, and function from the eukaryotic flagellum, which operates as a flexible whip-like tail utilizing microtubules. The bacterial flagellum is rigid in nature and operates more like the propeller on a boat.
There are three main components to the bacterial flagellum:
1. the filament – a long thin appendage that extends from the cell surface. The filament is composed of the protein flagellin and is hollow. Flagellin proteins are transcribed in the cell cytoplasm and then transported across the cell membrane and cell wall. A bacterial flagellar filament grows from its tip (unlike the hair on your head), adding more and more flagellin units to extend the length until the correct size is reached. The flagellin units are guided into place by a protein cap.
2. the hook – this is a curved coupler that attaches the filament to the flagellar motor.
3. the motor – a rotary motor that spans both the cell membrane and the cell wall, with additional components for the gram negative outer membrane. The motor has two components: the basal body, which provides the rotation, and the stator, which provides the torque necessary for rotation to occur. The basal body consists of a central shaft surrounded by protein rings, two in the gram positive bacteria and four in the gram negative bacteria. The stator consists of Mot proteins that surround the ring(s) embedded within the cell membrane.
Flagellum base diagram. By LadyofHats (Own work) [Public Domain], Via Wikipedia Commons
Bacterial Movement
Bacterial movement typically involves the use of flagella, although there are a few other possibilities as well (such as the use of type IV pili for twitching motility). But certainly the most common type of bacterial movement is swimming, which is accomplished with the use of a flagellum or flagella.
Swimming
Rotation of the flagellar basal body occurs due to the proton motive force, where protons that accumulate on the outside of the cell membrane are driven through pores in the Mot proteins, interacting with charges in the ring proteins as they pass across the membrane. The interaction causes the basal body to rotate and turns the filament extending from the cell. Rotation can occur at 200-1000 rpm and result in speeds of 60 cell lengths/second (for comparison, a cheetah moves at a maximum rate of 25 body lengths/second).
Rotation can occur in a clockwise (CW) or a counterclockwise (CCW)direction, with different results to the cell. A bacterium will move forward, called a “run,” when there is a CCW rotation, and reorient randomly, called a “tumble,” when there is a CW rotation.
Corkscrew Motility
Some spiral-shaped bacteria, known as the Spirochetes, utilize a corkscrew-motility due to their unusual morphology and flagellar conformation. These gram negative bacteria have specialized flagella that attach to one end of the cell, extend back through the periplasm and then attach to the other end of the cell. When these endoflagella rotate they put torsion on the entire cell, resulting in a flexing motion that is particularly effective for burrowing through viscous liquids.
Gliding Motility
Gliding motility is just like it sounds, a slower and more graceful movement than the other forms covered so far. Gliding motility is exhibited by certain filamentous or bacillus bacteria and does not require the use of flagella. It does require that the cells be in contact with a solid surface, although more than one mechanism has been identified. Some cells rely on slime propulsion, where secreted slime propels the cell forward, where other cells rely on surface layer proteins to pull the cell forward.
Chemotaxis
Now that we have covered the basics of the bacterial flagellar motor and mechanics of bacterial swimming, let us combine the two topics to talk about chemotaxis or any other type of taxes (just not my taxes). Chemotaxis refers to the movement of an organism towards or away from a chemical. You can also have phototaxis, where an organism is responding to light. In chemotaxis, a favorable substance (such as a nutrient) is referred to as an attractant, while a substance with an adverse effect on the cell (such as a toxin) is referred to as a repellant. In the absence of either an attractant or a repellant a cell will engage in a “random walk,” where it alternates between tumbles and runs, in the end getting nowhere in particular. In the presence of a gradient of some type, the movements of the cell will become biased, resulting over time in the movement of the bacterium towards an attractant and away from any repellants. How does this happen?
First, let us cover how a bacterium knows which direction to go. Bacteria rely on protein receptors embedded within their membrane, called chemoreceptors, which bind specific molecules. Binding typically results in methylation or phosphorylation of the chemoreceptor, which triggers an elaborate protein pathway that eventually impacts the rotation of the flagellar motor. The bacteria engage in temporal sensing, where they compare the concentration of a substance with the concentration obtained just a few seconds (or microseconds) earlier. In this way they gather information about the orientation of the concentration gradient of the substance. As a bacterium moves closer to the higher concentrations of an attractant, runs (dictated by CCW flagellar rotation) become longer, while tumbling (dictated by CW flagellar rotation) decreases. There will still be times that the bacterium will head off in the wrong direction away from an attractant since tumbling results in a random reorientation of the cell, but it won’t head in the wrong direction for very long. The resulting “biased random walk” allows the cell to quickly move up the gradient of an attractant (or move down the gradient of a repellant).
Bacterial Movement. By Brudersohn (Own work (Original text: selbst erstellt)) [CC BY-SA 2.0 de], via Wikimedia Commons
Key Words
capsule, slime layer, S-layer, fimbriae/fimbria, pilin, pili/pilus, conjugative pili, conjugation, type IV pili, twitching motility, flagella/flagellum, filament, flagellin, hook, motor, basal body, stator, Mot proteins, swimming, clockwise (CW), counterclockwise (CCW), run, tumble, Spirochetes, corkscrew-motility, endoflagella, gliding motility, chemotaxis, phototaxis, attractant, repellant, random walk, chemoreceptors, temporal sensing, biased random walk.
Essential Questions/Objectives
1. What are the compositions and functions of capsules and slime layers? When are they produced? How do capsules or slime layers increase the survival chances of bacteria in different environments?
2. What are fimbriae and pili; what are their compositions and functions?What is the size of bacterial flagella and how can they be arranged on a bacterial cell? How common are flagella in bacteria?
3. What is the basic composition of a bacterial flagellum and how does this differ from flagella found in eukaryotes? How do bacterial flagella grow and how are proteins transported across the membrane? How do they cause movement? How is the movement different from Eukaryotic flagella?
4. How are bacterial flagella attached to the body? How do the 2 inner rings work to cause movement and what powers the movement? What is the purpose of the 2 outer rings found in the basal body of gram-bacteria? What do gram + have instead?
5. How do endoflagella differ from flagella and in what type of bacteria are they found? Where do they work better than flagella?
6. What is chemotaxis? How does the direction of rotation of the flagella affect the way a bacteria moves? What do we know about the mechanism of chemotaxis in terms of membrane binding-proteins and chemotactic mediator? How long do stimuli last in chemotaxis and why is this important to the phenomenon?
Exploratory Questions (OPTIONAL)
1. How could chemotaxis in microbes be used to address environmental pollution problems? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/06%3A_Bacteria_-_Surface_Structures.txt |
The Archaea are a group of organisms that were originally thought to be bacteria (which explains the initial name of “archaeabacteria”), due to their physical similarities. More reliable genetic analysis revealed that the Archaea are distinct from both Bacteria and Eukaryotes, earning them their own domain in the Three Domain Classification originally proposed by Woese in 1977, alongside the Eukarya and the Bacteria.
Phylogenetic Tree of Life.
Similarities to Bacteria
So, why were the archaea originally thought to be bacteria? Perhaps most importantly, they lack a nucleus or other membrane-bound organelles, putting them into the prokaryotic category (if you are using the traditional classification scheme). Most of them are unicellular, they have 70S sized ribosomes, they are typically a few micrometers in size, and they reproduce asexually only. They are known to have many of the same structures that bacteria can have, such as plasmids, inclusions, flagella, and pili. Capsules and slime layers have been found but appear to be rare in archaea.
While archaea were originally isolated from extreme environments, such as places high in acid, salt, or heat, earning them the name “extremophiles,” they have more recently been isolated from all the places rich with bacteria: surface water, the ocean, human skin, soil, etc.
Key Differences
Plasma Membrane
There are several characteristics of the plasma membrane that are unique to Archaea, setting them apart from other domains. One such characteristic is chirality of the glycerol linkage between the phopholipid head and the side chain. In archaea it is in the L-isomeric form, while bacteria and eukaryotes have the D-isomeric form. A second difference is the presence of an ether-linkage between the glycerol and the side chain, as opposed to the ester-linked lipids found in bacteria and eukaryotes. The ether-linkage provides more chemical stability to the membrane. A third and fourth difference are associated with the side chains themselves, unbranched fatty acids in bacteria and eukaryotes, while isoprenoid chains are found in archaea. These isoprenoid chains can have branching side chains.
Comparison of Plasma Membrane Lipid Between Bacteria and Archaea. OpenStax, Structure of Prokaryotes. OpenStax CNX. Mar 28, 2014 http://cnx.org/contents/9e7c7540-5794-4c31-917d-fce7e50ea6dd@11.
Lastly, the plasma membrane of Archaea can be found as monolayers, where the isoprene chains of one phospholipid connect with the isoprene chains of a phospholipid on the opposite side of the membrane. Bacteria and eukaryotes only have lipid bilayers, where the two sides of the membrane remain separated.
Cell Wall
Like bacteria, the archaeal cell wall is a semi-rigid structure designed to provide protection to the cell from the environment and from the internal cellular pressure. While the cell walls of bacteria typically contain peptidoglycan, that particular chemical is lacking in archaea. Instead, archaea display a wide variety of cell wall types, adapted for the environment of the organism. Some archaea lack a cell wall altogether.
While it is not universal, a large number of Archaea have a proteinaceous S-layer that is considered to be part of the cell wall itself (unlike in Bacteria, where an S-layer is a structure in addition to the cell wall). For some Archaea the S-layer is the only cell wall component, while in others it is joined by additional ingredients (see below). The archaeal S-layer can be made of either protein or glycoprotein, often anchored into the plasma membrane of the cell. The proteins form a two-dimensional crystalline array with a smooth outer surface. A few S-layers are composed of two different S-layer proteins.
While archaea lack peptidoglycan, a few contain a substance with a similar chemical structure, known as pseudomurein. Instead of NAM, it contains N-acetylalosaminuronic acid (NAT) linked to NAG, with peptide interbridges to increase strength.
Methanochondroitin is a cell wall polymer found in some archaeal cells, similar in composition to the connective tissue component chondroitin, found in vertebrates.
Some archaea have a protein sheath composed of a lattice structure similar to an S-layer. These cells are often found in filamentous chains, however, and the protein sheath encloses the entire chain, as opposed to individual cells.
Cell Wall Structural Diversity.
Ribosomes
While archaea have ribosomes that are 70S in size, the same as bacteria, it was the rRNA nucleotide differences that provided scientists with the conclusive evidence to argue that archaea deserved a domain separate from the bacteria. In addition, archaeal ribosomes have a different shape than bacterial ribosomes, with proteins that are unique to archaea. This provides them with resistance to antibiotics that inhibit ribosomal function in bacteria.
Structures
Many of the structures found in bacteria have been discovered in archaea as well, although sometimes it is obvious that each structure was evolved independently, based on differences in substance and construction.
Cannulae
Cannulae, a structure unique to archaea, have been discovered in some marine archaeal strains. These hollow tube-like structures appear to connect cells after division, eventually leading to a dense network composed of numerous cells and tubes. This could serve as a means of anchoring a community of cells to a surface.
Hamus (pl. hami)
Another structure unique to archaea is the hamus, a long helical tube with three hooks at the far end. Hami appear to allow cells to attach both to one another and to surfaces, encouraging the formation of a community.
Pilus (pl. pili)
Pili have been observed in archaea, composed of proteins most likely modified from the bacterial pilin. The resulting tube-like structures have been shown to be used for attachment to surfaces.
Flagellum (pl. flagella)
The archaeal flagellum, while used for motility, differs so markedly from the bacterial flagellum that it has been proposed to call it an “archaellum,” to differentiate it from its bacterial counterpart.
What is similar between the bacterial flagellum and the archaeal flagellum? Both are used for movement, where the cell is propelled by rotation of a rigid filament extending from the cell. After that the similarities end.
What are the differences? The rotation of an archaeal flagellum is powered by ATP, as opposed to the proton motive force used in bacteria. The proteins making up the archaeal flagellum are similar to the proteins found in bacterial pili, rather than the bacterial flagellum. The archaeal flagellum filament is not hollow so growth occurs when flagellin proteins are inserted into the base of the filament, rather than being added to the end. The filament is made up of several different types of flagellin, while just one type is used for the bacterial flagellum filament. Clockwise rotation pushes an archaeal cells forward, while counterclockwise rotation pulls an archaeal cell backwards. An alternation of runs and tumbles is not observed.
Classification
Currently there are two recognized phyla of archaea: Euryarchaeota and Proteoarchaeota. Several additional phyla have been proposed (Nanoarchaeota, Korarchaeota, Aigarchaeota, Lokiarchaeota), but have yet to be officially recognized, largely due to the fact that the evidence comes from environmental sequences only.
Key Words
Archaea, L-isomeric form, D-isomeric form, ether-linkages, ester-linkages, isoprenoid chains, branching side chains, lipid monolayer, lipid bilayer, S-layer, pseudomurein, N-acetylalosaminuronic acid (NAT), methanochondroitin, protein sheath, cannulae, hamus/hami, pilus/pili, flagellum/flagella, archaellum, Euryarchaeota, Proteoarchaeota.
Essential Questions/Objectives
1. How are the archaea similar to bacteria?
2. Describe the differences between the plasma membranes of archaea, compared to bacteria & eukaryotes. Explain the differences.
3. What types of cell walls exist in Archaea and what are they composed of?
4. How are archaeal ribosomes both similar and different from bacterial ribosomes?
5. How do the pili of archaea differ from those of bacteria?
6. What are cannulae and hami? What role could they play for archaea?
7. How does archaeal flagella differ from bacterial flagella, in terms of composition, assembly, and function?
8. Understand the commonalities and differences between archaea and bacteria, in terms of physical characteristics.
Exploratory Questions (OPTIONAL)
1. What explains the fact that archaea appear to be more closely related to eukaryotes, despite their physical similarities to bacteria? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/07%3A_Archaea.txt |
Viruses are typically described as obligate intracellular parasites, acellular infectious agents that require the presence of a host cell in order to multiply. Viruses that have been found to infect all types of cells – humans, animals, plants, bacteria, yeast, archaea, protozoa…some scientists even claim they have found a virus that infects other viruses! But that is not going to happen without some cellular help.
Virus Characteristics
Viruses can be extremely simple in design, consisting of nucleic acid surrounded by a protein coat known as a capsid. The capsid is composed of smaller protein components referred to as capsomers. The capsid+genome combination is called a nucleocapsid.
Viruses can also possess additional components, with the most common being an additional membranous layer that surrounds the nucleocapsid, called an envelope. The envelope is actually acquired from the nuclear or plasma membrane of the infected host cell, and then modified with viral proteins called peplomers. Some viruses contain viral enzymes that are necessary for infection of a host cell and coded for within the viral genome. A complete virus, with all the components needed for host cell infection, is referred to as a virion.
Virus Characteristics, Image created by Ben Taylor, Public Domain, Via Wikipedia commons
Virus Genome
While cells contain double-stranded DNA for their genome, viruses are not limited to this form. While there are dsDNA viruses, there are also viruses with single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), and single-stranded RNA (ssRNA). In this last category, the ssRNA can either positive-sense (+ssRNA, meaning it can transcribe a message, like mRNA) or it can be negative-sense (-ssRNA, indicating that it is complementary to mRNA). Some viruses even start with one form of nucleic acid in the nucleocapsid and then convert it to a different form during replication.
Virus Structure
Viral nucleocapsids come in two basic shapes, although the overall appearance of a virus can be altered by the presence of an envelope, if present. Helical viruses have an elongated tube-like structure, with the capsomers arranged helically around the coiled genome. Icosahedral viruses have a spherical shape, with icosahedral symmetry consisting of 20 triangular faces. The simplest icosahedral capsid has 3 capsomers per triangular face, resulting in 60 capsomers for the entire virus. Some viruses do not neatly fit into either of the two previous categories because they are so unusual in design or components, so there is a third category known as complex viruses. Examples include the poxvirus with a brick-shaped exterior and a complicated internal structure, as well as bacteriophage with tail fibers attached to an icosahedral head.
Virus Replication Cycle
While the replication cycle of viruses can vary from virus to virus, there is a general pattern that can be described, consisting of five steps:
1. Attachment – the virion attaches to the correct host cell.
2. Penetration or Viral Entry – the virus or viral nucleic acid gains entrance into the cell.
3. Synthesis – the viral proteins and nucleic acid copies are manufactured by the cells’ machinery.
4. Assembly – viruses are produced from the viral components.
5. Release – newly formed virions are released from the cell.
Attachment
Outside of their host cell, viruses are inert or metabolically inactive. Therefore, the encounter of a virion to an appropriate host cell is a random event. The attachment itself is highly specific, between molecules on the outside of the virus and receptors on the host cell surface. This accounts for the specificity of viruses to only infect particular cell types or particular hosts.
Penetration or Viral Entry
Many unenveloped (or naked) viruses inject their nucleic acid into the host cell, leaving an empty capsid on the outside. This process is termed penetration and is common with bacteriophage, the viruses that infect bacteria. With the eukaryotic viruses, it is more likely for the entire capsid to gain entrance into the cell, with the capsid being removed in the cytoplasm. An unenveloped eukaryotic virus often gains entry via endocytosis, where the host cell is compelled to engulf the capsid resulting in an endocytic vesicle. An enveloped eukaryotic virus gains entrance for its nucleocapsid when the viral envelope fuses with the host cell membrane, pushing the nucleocapsid past the cell membrane. If the entire nucleocapsid is brought into the cell then there is an uncoating process to strip away the capsid and release the viral genome.
Synthesis
The synthesis stage is largely dictated by the type of viral genome, since genomes that differ from the cell’s dsDNA genome can involve intricate viral strategies for genome replication and protein synthesis. Viral specific enzymes, such as RNA-dependent RNA polymerases, might be necessary for the replication process to proceed. Protein production is tightly controlled, to insure that components are made at the right time in viral development.
Assembly
The complexity of viral assembly depends upon the virus being made. The simplest virus has a capsid composed of 3 different types of proteins, which self-assembles with little difficulty. The most complex virus is composed of over 60 different proteins, which must all come together in a specific order. These viruses often employ multiple assembly lines to create the different viral structures and then utilize scaffolding proteins to put all the viral components together in an organized fashion.
Release
The majority of viruses lyse their host cell at the end of replication, allowing all the newly formed virions to be released to the environment. Another possibility, common for enveloped viruses, is budding, where one virus is released from the cell at a time. The cell membrane is modified by the insertion of viral proteins, with the nucleocapsid pushing out through this modified portion of the membrane, allowing it to acquire an envelope.
Active Virus Life Cycle by John Kellogg Via OER at Oregon State University
Bacteriophage
Viruses that infect bacteria are known as bacteriophage or phage. A virulent phage is one that always lyses the host cell at the end of replication, after following the five steps of replication described above. This is called the lytic cycle of replication.
There are also temperate phage, viruses that have two options regarding their replication. Option 1 is to mimic a virulent phage, following the five steps of replication and lysing the host cell at the end, referred to as the lytic cycle. But temperate phage differ from virulent phage in that they have another choice: Option 2, where they remain within the host cell without destroying it. This process is known as lysogeny or the lysogenic cycle of replication.
A phage employing lysogeny still undergoes the first two steps of a typical replication cycle, attachment and penetration. Once the viral DNA has been inserted into the cell it integrates with the host DNA, forming a prophage. The infected bacterium is referred to as a lysogenor lysogenic bacterium. In this state, the virus enjoys a stable relationship with its host, where it does not interfere with host cell metabolism or reproduction. The host cell enjoys immunity from reinfection from the same virus.
Exposure of the host cell to stressful conditions (i.e. UV irradiation) causes induction, where the viral DNA excises from the host cell DNA. This event triggers the remaining steps of the lytic cycle, synthesis, maturation, and release, leading to lysis of the host cell and release of newly formed virions.
Lytic Cycle Versus Lysogenic Cycle of Replication. OpenStax, Virus Infections and Hosts. OpenStax CNX. Apr 11, 2013 http://cnx.org/contents/7cbd15ad-5bff-4678-a99f-85fd579e070c@3.
So, what dictates the replication type that will be used by a temperate phage? If there are plenty of host cells around, it is likely that a temperate phage will engage in the lytic cycle of replication, leading to a large increase in viral production. If host cells are scarce, a temperate phage is more likely to enter lysogeny, allowing for viral survival until host cell numbers increase. The same is true if the number of phage in an environment greatly outnumber the host cells, since lysogeny would allow for host cells numbers to rebound, ensuring long term viral survival.
Lysogens can experience a benefit from lysogeny as well, since it can result in lysogenic conversion, a situation where the development of a prophage leads to a change in the host’s phenotype. One of the best examples of this is for the bacterium Corynebacterium diphtheriae, the causative agent of diphtheria. The diphtheria toxin that causes the disease is encoded within the phage genome, so only C. diphtheriaelysogens cause diphtheria.
Eukaryotic Viruses
Eukaryotic viruses can cause one of four different outcomes for their host cell. The most common outcome is host cell lysis, resulting from a virulent infection (essentially the lytic cycle of replication seen in phage). Some viruses can cause a latent infection, co-existing peacefully with their host cells for years (much like a temperate phage during lysogeny). Some enveloped eukaryotic viruses can also be released one at a time from an infected host cell, in a type of budding process, causing a persistent infection. Lastly, certain eukaryotic viruses can cause the host cell to transform into a malignant or cancerous cell, a mechanism known as transformation.
Viruses and Cancer
There are many different causes of cancer, or unregulated cell growth and reproduction. Some known causes include exposure to certain chemicals or UV light. There are also certain viruses that have a known associated with the development of cancer. Such viruses are referred to as oncoviruses. Oncoviruses can cause cancer by producing proteins that bind to host proteins known as tumor suppressor proteins, which function to regulate cell growth and to initiate programmed cell death, if needed. If the tumor suppressor proteins are inactivated by viral proteins then cells grow out of control, leading to the development of tumors and metastasis, where the cells spread throughout the body.
Key Words
virus, obligate intracellular parasite, capsid, bacteriophage, capsomere, nucleocapsid, envelope, peplomer, virion, dsDNA, ssDNA, dsRNA, +ssRNA, -ssRNA, helical viruses, icosahedral viruses, complex viruses, attachment, penetration, viral entry, synthesis, assembly, release, naked virus, endocytosis, budding, bacteriophage, phage, virulent phage, lytic cycle, temperate phage, lysogeny, lysogenic cycle, prophage, lysogen, lysogenic bacterium, induction, lysogenic conversion, virulent infection, latent infection, persistent infection, transformation, oncovirus, tumor suppressor proteins.
Essential Questions/Objectives
1. What are the general properties of a virus?
2. What is the size range of viruses? How do they compare, size-wise, to bacteria?
3. What is the general structure of viruses? What are the different components?
4. What viral shapes exist?
5. How do envelopes and enzymes relate to viruses?
6. What types of viral genomes exist?
7. What are the steps of viral multiplication? What is happening at each step? How do Bacterial/Archaeal viruses differ from eukaryotic viruses, in regards to multiplication?
8. What are the 2 types of viral infection found in Bacteria/Archaea? What are the specific terms associated with viral infection of bacterial/archaeal cells?
9. What are the 4 types of viral infection found in eukaryotes?
10. How do some viruses cause cancer?
Exploratory Questions (OPTIONAL)
1. What is the largest bacterium or archaean ever discovered? What is the smallest eukaryote ever discovered? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/08%3A_Introduction_to_Viruses.txt |
Provided with the right conditions (food, correct temperature, etc) microbes can grow very quickly. Depending on the situation, this could be a good thing for humans (yeast growing in wort to make beer) or a bad thing (bacteria growing in your throat causing strep throat). It’s important to have knowledge of their growth, so we can predict or control their growth under particular conditions. While growth for muticelluar organisms is typically measured in terms of the increase in size of a single organism, microbial growth is measured by the increase in population, either by measuring the increase in cell number or the increase in overall mass.
Bacterial Division
Bacteria and archaea reproduce asexually only, while eukartyotic microbes can engage in either sexual or asexual reproduction. Bacteria and archaea most commonly engage in a process known as binary fission, where a single cell splits into two equally sized cells. Other, less common processes can include multiple fission, budding, and the production of spores.
The process begins with cell elongation, which requires careful enlargement of the cell membrane and the cell wall, in addition to an increase in cell volume. The cell starts to replicate its DNA, in preparation for having two copies of its chromosome, one for each newly formed cell. The protein FtsZ is essential for the formation of a septum, which initially manifests as a ring in the middle of the elongated cell. After the nucleoids are segregated to each end of the elongated cell, septum formation is completed, dividing the elongated cell into two equally sized daughter cells. The entire process or cell cyclecan take as little as 20 minutes for an active culture of E. coli bacteria.
Growth Curve
Since bacteria are easy to grow in the lab, their growth has been studied extensively. It has been determined that in a closed system or batch culture (no food added, no wastes removed) bacteria will grow in a predictable pattern, resulting in a growth curve composed of four distinct phases of growth: the lag phase, the exponential or log phase, the stationary phase, and the death or decline phase. Additionally, this growth curve can yield generation time for a particular organism – the amount of time it takes for the population to double.
Bacterial Growth Curve. By Michał Komorniczak. If you use on your website or in your publication my images (either original or modified), you are requested to give me details: Michał Komorniczak (Poland) or Michal Komorniczak (Poland). For more information, write to my e-mail address: [email protected] [CC BY-SA 3.0], via Wikimedia Commons
The details associated with each growth curve (number of cells, length of each phase, rapidness of growth or death, overall amount of time) will vary from organism to organism or even with different conditions for the same organism. But the pattern of four distinct phases of growth will typically remain.
Lag phase
The lag phase is an adaptation period, where the bacteria are adjusting to their new conditions. The length of the lag phase can vary considerably, based on how different the conditions are from the conditions that the bacteria came from, as well as the condition of the bacterial cells themselves. Actively growing cells transferred from one type of media into the same type of media, with the same environmental conditions, will have the shortest lag period. Damaged cells will have a long lag period, since they must repair themselves before they can engage in reproduction.
Typically cells in the lag period are synthesizing RNA, enzymes, and essential metabolites that might be missing from their new environment (such as growth factors or macromolecules), as well as adjusting to environmental changes such as changes in temperature, pH, or oxygen availability. They can also be undertaking any necessary repair of injured cells.
Exponential or Log phase
Once cells have accumulated all that they need for growth, they proceed into cell division. The exponential or log phase of growth is marked by predictable doublings of the population, where 1 cell become 2 cells, becomes 4, becomes 8 etc. Conditions that are optimal for the cells will result in very rapid growth (and a steeper slope on the growth curve), while less than ideal conditions will result in slower growth. Cells in the exponential phase of growth are the healthiest and most uniform, which explains why most experiments utilize cells from this phase.
Bacterial Growth Rates.
Due to the predictability of growth in this phase, this phase can be used to mathematically calculate the time it takes for the bacterial population to double in number, known as the generation time (g). This information is used by microbiologists in basic research, as well as in industry. In order to determine generation time, the natural logarithm of cell number can be plotted against time (where the units can vary, depending upon speed of growth for the particular population), using a semilogarithmic graph to generate a line with a predictable slope.
The slope of the line is equal to 0.301/g. Alternatively one can rely on the fixed relationship between the initial number of cells at the start of the exponential phase and the number of cells after some period of time, which can be expressed by:
\[N = N_02^{n}\]
where \(N\) is the final cell concentration, \(N_0\) is the initial cell concentration, and \(n\) is the number of generations that occurred between the specified period of time.
Generation time (g) can be represented by t/n, with t being the specified period of time in minutes, hours, days, or months. Thus, if one knows the cell concentration at the start of the exponential phase of growth and the cell concentration after some period of time of exponential growth, the number of generations can be calculated. Then, using the amount of time that growth was allowed to proceed (t), one can calculate g.
Stationary Phase
All good things must come to an end (otherwise bacteria would equal the mass of the Earth in 7 days!). At some point the bacterial population runs out of an essential nutrient/chemical or its growth is inhibited by its own waste products (it is a closed container, remember?) or lack of physical space, causing the cells to enter into the stationary phase. At this point the number of new cells being produced is equal to the number of cells dying off or growth has entirely ceased, resulting in a flattening out of growth on the growth curve.
Physiologically the cells become quite different at this stage, as they try to adapt to their new starvation conditions. The few new cells that are produced are smaller in size, with bacilli becoming almost spherical in shape. Their plasma membrane becomes less fluid and permeable, with more hydrophobic molecules on the surface that promote cell adhesion and aggregation. The nucleoid condenses and the DNA becomes bound with DNA-binding proteins from starved cells (DPS), to protect the DNA from damage. The changes are designed to allow the cell to survive for a longer period of time in adverse conditions, while waiting for more optimal conditions (such as an infusion of nutrients) to occur. These same strategies are used by cells in oligotrophic or low-nutrient environments. It has been hypothesized that cells in the natural world (i.e. outside of the laboratory) typically exist for long periods of time in oligotrophic environments, with only sporadic infusions of nutrients that return them to exponential growth for very brief periods of time.
During the stationary phase cells are also prone to producing secondary metabolites, or metabolites produced after active growth, such as antibiotics. Cells that are capable of making an endospore will activate the necessary genes during this stage, in order to initiate the sporulation process.
Death or Decline phase
In the last phase of the growth curve, the death or decline phase, the number of viable cells decreases in a predictable (or exponential) fashion. The steepness of the slope corresponds to how fast cells are losing viability. It is thought that the culture conditions have deteriorated to a point where the cells are irreparably harmed, since cells collected from this phase fail to show growth when transferred to fresh medium. It is important to note that if the turbidity of a culture is being measured as a way to determine cell density, measurements might not decrease during this phase, since cells could still be intact.
It has been suggested that the cells thought to be dead might be revived under specific conditions, a condition described as viable but nonculturable (VBNC). This state might be of importance for pathogens, where they enter a state of very low metabolism and lack of cellular division, only to resume growth at a later time, when conditions improve.
It has also been shown that 100% cell death is unlikely, for any cell population, as the cells mutate to adapt to their environmental conditions, however harsh. Often there is a tailing effect observed, where a small population of the cells cannot be killed off. In addition, these cells might benefit from their death of their fellow cells, which provide nutrients to the environment as they lyse and release their cellular contents.
Key Words
binary fission, multiple fission, budding, spores, cell cycle, closed system, batch culture, growth curve, lag phase, exponential or log phase, generation time (g), N, N0, n, t, stationary phase, DNA-binding proteins from starved cells (DPS), oligotrophic, secondary metabolites, death or decline phase, viable but nonculturable (VBNC).
Essential Questions/Objectives
1. How is growth measured in microbial populations?
2. How do eukaryotes and bacteria/archaea differ in their reproductive methods?
3. What are the steps of binary fission? What is happening at each step?
4. Know what the growth curve of an organism grown in a closed system looks like. Know the various stages and what is occurring at each stage, physiologically. What can influence lag phase? What are the 2 differing explanations for cell loss in the death or senescence phase?
5. Understand generation time and how can it be determined on a log number of cells vs. time graph. Know the advantage of plotting the log number of cells vs. time instead of the number of cells vs. time. What factors affect the generation time of an organism?
6. Practice problem: Six Staphylococcus aureus are inoculated into a cream pie by the hands of a pastry chef. The generation time of S. aureus in cream pie at room temperature is 30 minutes. a) How many S. aureus are in the pie after 4 hours at RT? b) After 24 hours?
Exploratory Questions (OPTIONAL)
1. In what situation would the VBNC occurrence benefit cells? How could this pose a public health threat? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/09%3A_Microbial_Growth.txt |
Competition is fierce out in the microbial world (non-microbial world, too!) and resources can be scarce. For those microbes that are willing and able to adapt to what might be considered a harsh environment, it can certainly mean less competition. So what environmental conditions can affect microbial growth? Temperature, oxygen, pH, water activity, pressure, radiation, lack of nutrients…these are the primary ones. We will cover more about metabolism (i.e. what type of food can they eat?) later, so let us focus now on the physical characteristics of the environment and the adaptations of microbes.
Osmolarity
Cells are subject to changes in osmotic pressure, due to the fact that the plasma membrane is freely permeable to water (a process known as passive diffusion). Water will generally move in the direction necessary to try and equilibrate the cell’s solute concentration to the solute concentration of the surrounding environment. If the solute concentration of the environment is lower than the solute concentration found inside the cell, the environment is said to be hypotonic. In this situation water will pass into the cell, causing the cell to swell and increasing internal pressure. If the situation is not rectified then the cell will eventually burst from lysis of the plasma membrane. Conversely, if the solute concentration of the environment is higher than the solute concentration found inside the cell, the environment is said to be hypertonic. In this situation water will leave the cell, causing the cell to dehydrate. Extended periods of dehydration will cause permanent damage to the plasma membrane.
Hypertonic vs. Hypotonic Solutions.
Cells in a hypotonic solution need to reduce the osmotic concentration of their cytoplasm. Sometimes cells can use inclusions to chemically change their solutes, reducing molarity. In a real pinch they can utilize what are known as mechanosensitive (MS) channels, located in their plasma membrane. MS channels open as the plasma membrane stretches due to the increased pressure, allowing solutes to leave the cell and thus lowering the osmotic pressure.
Cells in a hypertonic solution needing to increase the osmotic concentration of their cytoplasm can take up additional solutes from the environment. However, cells have to be careful about what solutes they take up, since some solutes can interfere with cellular function and metabolism. Cells need to take up compatible solutes, such as sugars or amino acids, which typically will not interfere with cellular processes.
There are some microbes that have evolved to extreme hypertonic environments, specifically high salt concentrations, to the point where they now require the presence of high levels of sodium chloride to grow. Halophiles, which require a NaCl concentration above 0.2 M, take in both potassium and chloride ions as a way to offset the effects of the hypertonic environment that they live in. Their evolution has been so complete that their cellular components (ribosomes, enzymes, transport proteins, cell wall, plasma membrane) now require the presence of high concentrations of both potassium and chloride to function.
pH
pH is defined as the negative logarithm of the hydrogen ion concentration of a solution, expressed in molarity. The pH scale ranges from 0 to 14, with 0 representing an extremely acidic solution (1.0 M H+) and 14 representing an extremely alkaline solution (1.0 x 10-14 M H+). Each pH units represents a tenfold change in hydrogen ion concentration, meaning a solution with a pH of 3 is 10x more acidic than a solution with a pH of 4.
Typically cells would prefer a pH that is similar to their internal environment, with cytoplasm having a pH of 7.2. That means that most microbes are neutrophiles (“neutral lovers”), preferring a pH in the range of 5.5 to 8.0. There are some microbes, however, that have evolved to live in the extreme pH environments.
Acidophiles (“acid lovers”), preferring an environmental pH range of 0 to 5.5, must use a variety of mechanisms to maintain their internal pH in an acceptable range and preserve the stability of their plasma membrane. These organisms transport cations (such as potassium ions) into the cell, thus decreasing H+ movement into the cell. They also utilize proton pumps that actively pump H+ out.
Alkaliphiles (“alkaline lovers”), preferring an environmental pH range of 8.0 to 11.5, must pump protons in, in order to maintain the pH of their cytoplasm. They typically employ antiporters, which pump protons in and sodium ions out.
pH Scale. OpenStax, Inorganic Compounds Essential to Human Functioning. OpenStax CNX. Jun 18, 2013 http://cnx.org/contents/e4e45509-bfc0-4aee-b73e-17b7582bf7e1@3.
Temperature
Microbes have no way to regulate their internal temperature so they must evolve adaptations for the environment they would like to live in. Changes in temperature have the biggest effect on enzymes and their activity, with an optimal temperature that leads to the fastest metabolism and resulting growth rate. Temperatures below optimal will lead to a decrease in enzyme activity and slower metabolism, while higher temperatures can actually denature proteins such as enzymes and carrier proteins, leading to cell death. As a result, microbes have a growth curve in relation to temperature with an optimal temperature at which growth rate peaks, as well as minimum and maximum temperatures where growth continues but is not as robust. For a bacterium the growth range is typically around 30 degrees.
The psychrophiles are the cold lovers, with an optimum of 15oC or lower and a growth range of -20oC to 20oC. Most of these microbes are found in the oceans, where the temperature is often 5oC or colder. They can also be found in the Arctic and the Antarctic, living in ice wherever they can find pockets of liquid water. Adaptation to the cold required evolution of specific proteins, particularly enzymes, that can still function in low temperatures. In addition, it also required modification to the plasma membrane to keep it semifluid. Psychrophiles have an increased amount of unsaturated and shorter-chain fatty acids. Lastly, psychrophiles produce cryoprotectants, special proteins or sugars that prevent the development of ice crystals that might damage the cell. Psychrotophs or cold tolerant microbes have a range of 0-35oC, with an optimum of 16oC or higher.
Humans are best acquainted with the mesophiles, microbes with a growth optima of 37oC and a range of 20-45oC. Almost all of the human microflora fall into this category, as well as almost all human pathogens. The mesophiles occupy the same environments that humans do, in terms of foods that we eat, surfaces that we touch, and water that we drink and swim in.
On the warmer end of the spectrum is where we find the thermophiles(“heat lovers”), the microbes that like high temperatures. Thermophiles typically have a range of 45-80oC, and a growth optimum of 60oC. There are also the hyperthermophiles, those microbes that like things extra spicy. These microbes have a growth optima of 88-106oC, a minimum of 65oC and a maximum of 120oC. Both the thermophiles and the hyperthermophiles require specialized heat-stable enzymes that are resistant to denaturation and unfolding, partly due to the presence of protective proteins known as chaperone proteins. The plasma membrane of these organisms contains more saturated fatty acids, with increased melting points.
Growth Curves.
Oxygen Concentration
The oxygen requirement of an organism relates to the type of metabolism that it is using. Energy generation is tied to the movement of electrons through the electron transport chain (ETC), where the final electron acceptor can be oxygen or a non-oxygen molecule.
Organisms that use oxygen as the final electron acceptor are engaging in aerobic respiration for their metabolism. If they require the presence of atmospheric oxygen (20%) for their metabolism then they are referred to as obligate aerobes. Microaerophiles require oxygen, but at a lower level than normal atmospheric levels – they only grow at levels of 2-10%.
Organisms that can grow in the absence of oxygen are referred to as anaerobes, with several different categories existing. The facultative anaerobes are the most versatile, being able to grow in the presence or absence of oxygen by switching their metabolism to match their environment. They would prefer to grow in the presence of oxygen, however, since aerobic respiration generates the largest amount of energy and allows for faster growth. Aerotolerant anaerobes can also grow in the presence or absence of oxygen, exhibiting no preference. Obligate anaerobes can only grow in the absence of oxygen and find an oxygenated environment to be toxic.
While the use of oxygen is dictated by the organism’s metabolism, the ability to live in an oxygenated environment (regardless of whether it is used by the organism or not) is dictated by the presence/absence of several enzymes. Oxygen by-products (called reactive oxygen species or ROS) can be toxic to cells, even to the cells using aerobic respiration. Enzymes that can offer some protection from ROS include superoxide dismutase (SOD), which breaks down superoxide radicals, and catalase, which breaks down hydrogen peroxide. Obligate anaerobes lack both enzymes, leaving them little or no protection against ROS.
Oxygen and Bacterial Growth.
Pressure
The vast majority of microbes, living on land or water surface, are exposed to a pressure of approximately 1 atmosphere. But there are microbes that live on the bottom of the ocean, where the hydrostatic pressure can reach 600-1,000 atm. These microbes are the barophiles(“pressure lovers”), microbes that have adapted to prefer and even require the high pressures. These microbes have increased unsaturated fatty acids in their plasma membrane, as well as shortened fatty acid tails.
Radiation
All cells are susceptible to the damage cause by radiation, which adversely affects DNA with its short wavelength and high energy. Ionizing radiation, such as x-rays and gamma rays, causes mutations and destruction of the cell’s DNA. While bacterial endospores are extremely resistant to the harmful effects of ionizing radiation, vegetative cells were thought to be quite susceptible to its impact. That is, until the discovery of Deinococcus radiodurans, a bacterium capable of completely reassembling its DNA after exposure to massive doses of radiation.
Ultraviolet (UV) radiation also causes damage to DNA, by attaching thymine bases that are next to one another on the DNA strand. These thymine dimers inhibit DNA replication and transcription. Microbes typically have DNA repair mechanisms that allow them to repair limited damage, such as the enzyme photolyase that splits apart thymine dimers.
Key Words
osmotic pressure, passive diffusion, solute, hypotonic, hypertonic, mechanosensitive (MS) channel, compatible solute, halophile, pH, neutrophile, acidophile, alkaliphile, optimum temperature, minimum temperature, maximum temperature, psychrophile, psychrotroph, mesophile, thermophile, hyperthermophile, chaperone protein, electron transport chain (ETC), aerobic respiration, obligate aerobe, microaerophile, anaerobe, facultative anaerobe, aerotolerant anaerobe, obligate anaerobe, reactive oxygen species (ROS), superoxide dismutase (SOD), catalase, barophile, ionizing radiation, Deinococcus radiodurans, ultraviolet (UV) radiation, thymine dimmers, photolyase.
Essential Questions/Objectives
1. What are all the descriptive terms used for microbes that live in different environments or the terms used for the environments that they live in? What does each term mean? In what types of environments are each microbial group found?
2. What effect does solute concentration have on microbes? How can cells adapt when going from a low solute to a high solute environment and vice versa? What is a compatible solute? What microbial groups have a requirement for high solute concentrations? How do microbes differ in their response to water activity?
3. How do microbes differ in their response to pH? What does pH affect in a cell and what do cells that live at high or low pH have to do to survive these conditions?
4. How do microbes differ in their response to temperatures? What terms are used for these responses? If a cell is to grow at low or high temperatures, what adaptations does it need to make?
5. How do microbes differ in their response to oxygen levels? Why would they differ? What enzymes are needed to adapt to environments containing differing amounts of oxygen?
6. How do microbes respond to high pressure? To ionizing radiation? To UV light? What populations are resistant to these conditions?
Exploratory Questions (OPTIONAL)
1. What is the largest bacterium or archaean ever discovered? What is the smallest eukaryote ever discovered? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/10%3A_Environmental_Factors.txt |
All microbes have a need for three things: carbon, energy, and electrons. There are specific terms associated with the source of each of these items, to help define organisms.
Let us focus on carbon first. All organisms are carbon-based with macromolecules – proteins, carbohydrates, lipids, nucleic acid – having a fundamental core of carbon. On one hand, organisms can use reduced, preformed organic substances as a carbon source. These are the heterotrophs or “other eaters.” Alternatively, they can rely on carbon dioxide (CO2) as a carbon source, reducing or “fixing” it this inorganic form of carbon into an organic molecule. These are the autotrophs or “self feeders.”
For energy, there are two possibilities as well: light energy or chemical energy. Light energy comes from the sun, while chemical energy can come from either organic or inorganic chemicals. Those organisms that use light energy are called phototrophs (“light eaters”), while those that use chemical energy are called chemotrophs (“chemical eaters”). Chemical energy can come from inorganic sources or organic sources. An organism that uses inorganic sources is known as a lithotroph (“rock eater”), while an organism that uses organic sources is called an organotroph (“organic eater”).
These terms can all be combined, to derive a single term that gives you an idea of what an organism is using to meet its basic needs for energy, electrons, and carbon.
Macronutrients
In addition to carbon, hydrogen and oxygen, cells need a few other elements in sufficient quantity. In particular, cells need nitrogen for the formation of proteins, nucleic acids, and a few other cell components. Cells also need phosphorous, which is a crucial component of nucleic acids (think sugar-phosphate backbone!), phospholipids, and adenosine triphosphate or ATP. Sulfur is necessary for a few amino acids, as well as several vitamins, while potassium is needed for enzymes, and magnesium is used to stabilize ribosomes and membrane. Collectively these elements (including C, H, and O) are referred to as the macronutrients.
Growth Factors
Some microbes can synthesize certain organic molecules that they need from scratch, as long as they are provided with carbon source and inorganic salts. Other microbes require that certain organic compounds exist within their environment. These organic molecules essential for growth are called growth factors and fall in three categories: 1) amino acids (building blocks of protein), 2) purines and pyrimidines (building blocks of nucleic acid), and 3) vitamins (enzyme cofactors).
Uptake of Nutrients
In order to support its’ activities, a cell must bring in nutrients from the external environment across the cell membrane. In bacteria and archaea, several different transport mechanisms exist.
Passive Diffusion
Passive or simple diffusion allows for the passage across the cell membrane of simple molecules and gases, such as CO2, O2, and H2O. In this case, a concentration gradient must exist, where there is higher concentration of the substance outside of the cell than there is inside the cell. As more of the substance is transported into the cell the concentration gradient decreases, slowing the rate of diffusion.
Facilitated Diffusion
Facilitated diffusion also involves the use of a concentration gradient, where the concentration of the substance is higher outside the cell, but differs with the use of carrier proteins (sometimes called permeases). These proteins are embedded within the cell membrane and provide a channel or pore across the membrane barrier, allowing for the passage of larger molecules. If the concentration gradient dissipates, the passage of molecules into the cell stops. Each carrier protein typically exhibits specificity, only transporting in a particular type of molecule or closely related molecules.
Active Transport
Many types of nutrient uptake require that a cell be able to transport substances against a concentration gradient (i.e. with a higher concentration inside the cell than outside). In order to do this, a cell must utilize metabolic energy for the transport of the substance through carrier proteins embedded in the membrane. This is known as active transport. All types of active transport utilize carrier proteins.
Active Transport Versus Facilitated Diffusion.
Primary active transport
Primary active transport involves the use of chemical energy, such as ATP, to drive the transport. One example is the ABC system, which utilizes ATP-Binding Cassette transporters. Each ABC transporter is composed of three different components: 1) membrane-spanning proteins that form a pore across the cell membrane (i.e. carrier protein), 2) an ATP binding region that hydrolyzes ATP, providing the energy for the passage across the membrane, and 3) a substrate-binding protein, a peripheral protein that binds to the appropriate substance to be transporter and ferries it to the membrane-spanning proteins. In gram negative bacteria the substrate-binding protein is located in the cell’s periplasm, while in gram positive bacteria the substrate-binding protein is attached to the outside of the cell membrane.
ABC Transporter Structure.
Secondary active transport
Secondary active transport utilizes energy from a proton motive force (PMF). A PMF is an ion gradient that develops when the cell transports electrons during energy-conserving processes. Positively charged protons accumulate along the outside of the negatively charged cell, creating a proton gradient between the outside of the cell and the inside.
There are three different types of transport events for simple transport: uniport, symport, and antiport and each mechanism utilizes a different protein porter. Uniporters transport a single substance across the membrane, either in or out. Symporters transport two substances across the membrane at the same time, typically a proton paired with another molecule. Antiporters transport two substances across the membrane as well, but in opposite directions. As one substance enters the cell, the other substance is transported out.
Uniport Synport Antiport. By Lupask (Own work) [Public domain], via Wikimedia Commons
Group Translocation
Group translocation is a distinct type of active transport, using energy from an energy-rich organic compound that is not ATP. Group translocation also differs from both simple transport and ABC transporters in that the substance being transported is chemically modified in the process.
One of the best studied examples of group translocation is the phosphoenolpyruvate: sugar phosphotransferase system (PTS), which uses energy from the high-energy molecule phosphoenolpyruvate (PEP) to transport sugars into the cell. A phosphate is transferred from the PEP to the incoming sugar during the process of transportation.
Group Translocation via PTS.
Iron Uptake
Iron is required by microbes for the function of their cytochromes and enzymes, resulting in it being a growth-limiting micronutrient. However, little free iron is available in environments, due to its insolubility. Many bacteria have evolved siderophores, organic molecules that chelate or bind ferric iron with high affinity. Siderophores are released by the organism to the surrounding environment, whereby they bind any available ferric iron. The iron-siderophore complex is then bound by a specific receptor on the outside of the cell, allowing the iron to be transported into the cell.
Siderophores and Receptor Sites.
Key Words
heterotroph, autotroph, phototroph, chemotroph, lithotroph, organotroph, photolithoautotroph, photoorganoheterotroph, chemoorganoheterotroph, chemolithoautotroph, chemolithoheterotroph, macronutrients, growth factors, passive/simple diffusion, facilitated diffusion, carrier protein/permease, active transport, primary active transport, ABC system, ATP-binding cassette transporter, ABC transporter, secondary active transport, proton motive force (PMF), uniport, symport, antiport, porter, uniporter, symporter, antiporter, group translocation, phosphoenolpyruvate: sugar phosphotransferase system (PTS), phosphoenolpyruvate (PEP), siderophore.
Study Questions
1. What are the different terms associated with microbial nutritional types? How can these terms be combined to define the nutritional types of microbes in terms of their sources of carbon, electrons, and energy?
2. What are macroelements and why are they important to a cell? What are growth factors and what is their significance to a cell?
3. What is the importance of nutrient uptake for a cell? What are the common features of nutrient uptake by bacteria?
4. What is transported into a bacteria cell by passive diffusion and how does this affect a bacterial cell?
5. Explain diffusion (passive and facilitated) and active transport.
6. What are the 3 types of active transport? Be able to diagram each processes. What is required for each of these processes? How are they similar, how are they different?
7. Why is iron uptake important for a cell? What is used to accomplish this?
Exploratory Questions (OPTIONAL)
1. What is the largest bacterium or archaean ever discovered? What is the smallest eukaryote ever discovered? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/11%3A_Microbial_Nutrition.txt |
Metabolism refers to the sum of chemical reactions that occur within a cell. Catabolism is the breakdown of organic and inorganic molecules, used to release energy and derive molecules that could be used for other reactions. Anabolism is the synthesis of more complex molecules from simpler organic and inorganic molecules, which requires energy.
Energetics
While some energy is lost as heat in chemical reactions, the measurement of interest for cells is the amount of free energy (G), or the energy available to do work. Cells perform three different types of work: chemical work (such as anabolism), transport work (such as nutrient uptake), and mechanical work (such as the rotation of a flagellum).
The change in free energy is typically denoted as ΔG°’, which indicates the change in free energy under standard conditions of pH 7, 25oC, 1 atmosphere pressure (also known as the standard free energy change). A reaction that generates a positive ΔG°’ indicates that the reaction requires energy and is endergonic in nature. A reaction that generates a negative ΔG°’ indicates that the reaction releases energy and is exergonic in nature. Reactions that are exergonic release energy that can be conserved by the cell to do work.
Adenosine triphosphate (ATP)
Adenosine triphosphate or ATP is a high-energy molecule used by all cells for energy currency, partly because it readily donates a phosphoryl group to other molecules. An exergonic reaction will release energy, driving the synthesis of ATP from the addition of a phosphate molecule (orthophosphate or Pi) to adenosine diphosphate or ADP. An endergonic reaction, which requires energy, will couple with the hydrolysis of ATP to ADP + Pi, using the released energy to drive the reaction.
Enzymes
In order for a chemical reaction to proceed, chemical bonds must be broken. The energy required to break bonds is called activation energy. The amount of activation energy required by a cell can be lowered with the help of a catalyst, substances which assist the reaction to proceed without being changed themselves by the reaction. Cells use protein catalysts known as enzymes.
Activation energy. By Originally uploaded by Jerry Crimson Mann, vectorized by Tutmosis, corrected by Fvasconcellos (en:Image:Activation2.png) [GFDL or CC-BY-SA-3.0], via Wikimedia Commons
Redox Reactions
Cells conserve energy in the form of ATP by coupling its synthesis to the release of energy via oxidation-reduction (redox) reactions, where electrons are passed from an electron donor to an electron acceptor. The oxidation of a molecule refers to the loss of its electrons, while the reduction of a molecule refers to its gain of electrons. Organic chemists often refer to the process by the mnemonic OIL RIG: Oxidation Is Loss, Reduction Is Gain. A molecule being oxidized is acting as an electron donor, while the molecule being reduced is acting as an electron acceptor. Since electrons represent energy, a substance with many electrons to donate can be thought of as energy-rich.
Conjugate Redox Pair
Electrons do not exist freely in solution, they must be coupled with atoms or molecules. Every redox reaction consists of two half reaction, where one substance donates electrons and thus becomes an oxidized product while another substance accepts the electrons and thus becomes a reduced product. Conjugate redox pair refers to the acceptor and donor of a half reaction.
A substance can be either an electron donor or an electron acceptor, dependent upon the other substances in the reaction. A redox couple represents both forms of a substance in a half reaction, with the oxidized form (the electron acceptor) always placed on the left and the reduced form (the electron donor) on the right. An example would be ½ O2/H2O, where H2O could serve as an electron donor and O2 could serve as an electron acceptor. Each half reaction is given a standard reduction potential (E’0) in volts or millivolts, which is a measurement of the tendency of the donor in the reaction to give up electrons. A substance with greater tendency to donate electrons in the reduced form has a more negative E’0, while a substance with a weak tendency to donate electrons in the reduced form has a less negative or even positive E’0. A substance with a negative E’0 makes a very good electron donor, in the reduced form.
Redox Tower
The information regarding standard reduction potentials for various redox couples is displayed in the form of a redox tower, which lists the couples in a vertical form based on their E’0. Redox couples with the most negative E’0 on listed at the top while those with the most positive E’0 are listed on the bottom. The reduced substance with the greatest tendency to donate electrons would be found at the top of the tower on the right, while the oxidized substance with the greatest tendency to accept electrons would be found at the bottom of the tower on the left. Redox couples in the middle can serve as either electron donors or acceptors, depending upon what substance they partner with for a reaction. The difference between reduction potentials of a donor and an acceptor (ΔE’0) is measured as acceptor E’0 minus donor E’0. The larger the value for ΔE’0, the more potential energy for a cell. Larger values are derived when there is the biggest distance between the donor and the acceptor (or a bigger fall down the tower).
Electron Tower.
While ΔE’0 is proportional to ΔG°’, the number of electrons that a substance has to donate is important too. The actual formula is:
$\Delta \mathrm{G}^{\circ\prime} = -nF \cdot \Delta {\mathrm{E}^{\prime}}_{0}$
where n is the number of electrons being transferred and F is the Faraday constant (23,062 cal/mole-volt, 96, 480 J/mole-volt).
Electron Carriers
The transference of electrons from donor to acceptor does not occur directly, since chemically dissimilar electron donors and acceptors might never interact with one another. Instead, many cellular intermediates participate in the process, with the possibility for energy capture occurring along the way. These intermediates are called electron carriersand they go back and forth between a reduced form (when they are carrying an electron) and an oxidized form (after they have passed the electron on), without being consumed in the reaction themselves.
In order for the reaction to be energetically favorable for the cell, the carriers must be arranged in order of their standard reduction potential (i.e. going down the redox tower), with an electron being passed from a carrier with the most negative E’0 to a carrier with a less negative E’0. It is important to note that some carriers accept both electrons and protons, while other carriers accept electrons only. This fact will become of crucial importance later, in the discussion of how energy is generated.
While there are many different electron carriers, some unique to specific organisms or groups of organisms, let us cover some of the more common ones:
• Nicotinamide adenine dinucleotide (NAD+/NADH) – a co-enzyme that carriers both electrons (e-) and protons (H+), two of each. A closely related molecule is nicotinamide adenine dinucleotide phosphate (NADP+/ NADPH), which accepts 2 electrons and 1 proton.
• Flavin adenine dinucleotide (FAD/FADH) and flavin mononucleotide (FMN/FMNH) – carry 2 electrons and 2 protons each. Proteins with these molecules are called flavoproteins.
• Coenzyme Q (CoQ)/ubiquinone – carries 2 electrons and 2 protons.
• Cytochromes – use iron atoms as part of a heme group to carry 1 electron at a time.
• Iron-sulfur (Fe-S) proteins, such as ferredoxin – use iron atoms not part of heme group to carry 1 electron at a time.
Electron Transport Chain
The process starts with an initial electron donor, a substance from outside of the cell, and ends with a final electron acceptor, another substance from outside of the cell. In the middle the electrons are passed from carrier to carrier, as the electrons work their way down the electron tower. In order to make the process more efficient, most of the electron carriers are embedded within a membrane of the cell, in the order that they are arranged on a redox tower. These electron transport chains are found within the cell membrane of bacteria and archaea, and within the mitochondrial membrane of eukaryotes.
Electron Transport Chain.
Key Words
metabolism, catabolism, anabolism, free energy (G), chemical work, transport work, mechanical work, ΔG°’, standard free energy change, exergonic, endergonic, adenosine triphosphate (ATP), orthophosphate (Pi), activation energy, catalyst, enzyme, oxidation-reduction (redox) reaction, electron donor, electron acceptor, OIL RIG, conjugate redox pair, redox couple, standard reduction potential (E’0), redox tower, ΔE’0, electron carriers, nicotinamide adenine dinucleotide (NAD+/NADH), nicotinamide adenine dinucleotide phosphate (NADP+/ NADPH), flavin adenine dinucleotide (FAD/FADH), flavin mononucleotide (FMN/FMNH), coenzyme Q (CoQ)/ubiquinone, cytochrome, iron-sulfur (Fe-S) proteins, ferredoxin, electron transport chain (ETC).
Study Questions
1. How are metabolism, catabolism, and anabolism defined?
2. What are the 3 major types of work carried out by cells? What’s an example of each type?
3. What is free energy? What is standard free energy?
4. What are the characteristics of an endergonic and an exergonic reaction? How can cells conserve the energy given off by reactions?
5. What is the role of ATP in the cell and why it is a good compound for this role?
6. What are enzymes? What role do enzymes play in energy conservation?
7. What is oxidation and reduction? What does standard reduction potential represent, such as 2H+/H2 = -0.42V? Indicate what each term in this equation represents. In redox pairs with a more negative O-R potential the reduced form is more likely to be an electron ____________________ and has __________________________ potential energy. What is a conjugate redox pair?
8. What is an electron tower and how does this concept help to explain energy exchange in a cell?
9. What is ΔG0’? What does it represent and how is it calculated?
10. What is an electron carrier, what role do they play, what are the most common electron carriers in the cell and why must they constantly be recycled?
11. What is an electron transport chain and how does it function to conserve energy for the cell? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/12%3A_Energetics_and_Redox_Reactions.txt |
Chemoorganotrophy is a term used to denote the oxidation of organic chemicals to yield energy. In other words, an organic chemical serves as the initial electron donor. The process can be performed in the presence or absence of oxygen, depending upon what is available to a cell and whether or not they have the enzymes to deal with toxic oxygen by-products.
Aerobic Respiration
To start, let us focus on the catabolism of organic compounds when it occurs in the presence of oxygen. In other words, oxygen is being used as the final electron acceptor. When the process utilizes glycolysis and the tricarboxylic acid (TCA) cycle to completely oxidize an organic compound down to CO2, it is known as aerobic respiration. This generates the most ATP for a cell, given the large amount of distance between the initial electron donor (glucose) and the final electron acceptor (oxygen), as well as the large number of electrons that glucose has to donate.
Organic Energy Sources
In chemoorganotrophy, energy is derived from the oxidation of an organic compound. There are many different organic compounds available to a cell, such as proteins, polysaccharides, and lipids. But cellular pathways are arranged in such a way to increase metabolic efficiency. Thus, the cell funnels reactions into a few common pathways. By convention, glucose is used as the starting molecule to describe each process.
Glycolysis
Glycolysis is a nearly universal pathway for the catabolism of glucose to pyruvate. The pathway is divided into two parts: part I, which focuses on modifications to the 6-carbon sugar glucose, and part II, where the 6-carbon compound is split into two 3-carbon molecules, yielding a bifurcated pathway. Part I actually requires energy in the form of 2 molecules of ATP, in order to phosphorylate or activate the sugar. Part II is the energy conserving phase of the reaction, where 4 molecules of ATP are generated by substrate-level phosphorylation, where a high-energy molecule directly transfers a Pi to ADP.
The net yield of energy from glycolysis is 2 molecules of ATP for every molecule of glucose. In addition, 2 molecules of the carrier NAD+ are reduced, forming NADH. In aerobic respiration, these electrons will ultimately be transferred by NADH to an electron transport chain, allowing the cell to capture more energy. Lastly, 2 molecules of the 3-carbon compound pyruvate are produced, which can be further oxidized to capture more energy for the cell.
Glycolysis.
Tricarboxylic acid (TCA) cycle
The tricarboxylic acid (TCA) cycle picks up at the end of glycolysis, in order to fully oxidize each molecule of pyruvate down to 3 molecules of CO2, as occurs in aerobic respiration. It begins with a type of connecting reaction before the molecules can enter the cycle proper. The connecting reaction reduces 1 molecule of NAD+ to NADH for every molecule of pyruvate, in the process of making citrate.
The citrate enters the actual cycle part of the process, undergoing a series of oxidations that yield many different products, many of them important precursor metabolites for other pathways. As electrons are released, carriers are reduced, yielding 3 molecules of NADH and 1 molecule of FADH2 for every molecule of pyruvate. In addition, 1 molecule of GTP (which can be thought of as an ATP-equivalent molecule) is generated by substrate-level phosphorylation.
Taking into account that there were two molecules of pyruvate generated from glycolysis, the net yield of the TCA cycle and its connecting reaction are: 2 molecules of GTP, 8 molecules of NADH, and 2 molecules of FADH2. But where does the ATP come from? So far we only have the net yield of 2 molecules from glycolysis and the 2 molecules of ATP-equivalents (i.e. GTP) from the TCA cycle. This is where the electron transport chain comes into play.
TCA at the End of Glycolysis.
Oxidative Phosphorylation
The synthesis of ATP from electron transport generated from oxidizing a chemical energy source is known oxidative phosphorylation. We have already established that electrons get passed from carrier to carrier, in order of their standard reduction potential. We have also established that some carriers accept electrons and protons, while others accept electrons only. What happens to the unaccepted protons? And how does this generate ATP for the cell? Welcome to the wonderful world of the proton motive force (PMF) and ATP synthase!
Proton Motive Force
Protons that are not accepted by electron carriers migrate outward, to line the outer part of the membrane. For bacteria and archaea, this means lining the cell membrane and explains the importance for the negative charge of the cell.
As the positively charged protons accumulate, a concentration gradient of protons develops. This results in the cytoplasm of the cell being more alkaline and more negative, leading to both a chemical and electrical potential difference. This proton motive force (PMF) can be used to do work for the cell, such as in the rotation of the bacterial flagellum or the uptake of nutrients.
ATP synthase
The PMF can also be used to synthesize ATP, with the help of an enzyme known as ATP synthase (or ATPase). This large enzyme has two components, one that spans the membrane and one that sticks into the cytoplasm and synthesizes the ATP. Protons are driven through the membrane-spanning component, generating torque that drives the rotation of the cytoplasmic portion. When the cytoplasmic component returns to its original configuration it binds Pi to ADP, generating a molecule of ATP.
Aerobic Respiration Summary
After all that, what did the cell end up with, from using aerobic respiration? Using substrate-level phosphorylation the cell generated 2 net molecules of ATP during glycolysis, in addition to 2 molecules of ATP-equivalents from the TCA cycle. For reduced carriers, there were 2 molecules of NADH generated during glycolysis, in addition to 8 molecules from the TCA cycle or its connecting reaction. There were also 2 molecules of FADH2 from the TCA cycle. All of those electrons were passed on to the ETC (and eventually to oxygen), in order to develop a PMF, so that ATP synthase could generate ATP. How much ATP is generated?
Research indicates that the process is not completely efficient and there is some “leakage” that occurs. Current estimates are that 2.5 ATP are generated for every molecule of NADH, while 1.5 ATP are generated for every molecule of FADH2. Using these values would allow the cell to synthesize 25 molecules of ATP from all the NAD+ that was reduced in the process, in addition to 3 molecules of ATP from the FAD+ that was reduced. This would bring the grand total of maximum ATP produced to 32 (counting the GTP in that figure).
ATP Generation.
Anaerobic Chemoorganotrophy
Certainly oxygen is a wonderful final electron acceptor, particularly when paired with glucose as an initial electron donor. It is part of the lowest redox couple on an electron tower, with an extremely positive standard electron potential. But what does a microbe do, if oxygen is not available or it lacks the protections necessary from toxic oxygen by-products? Let us focus on the generation of energy in the absence of oxygen, using a different electron acceptor, when an organic chemical is still being used as the initial electron donor. Examples of anaerobic chemoorganotrophy include anaerobic respiration and fermentation.
Anaerobic Respiration
Anaerobic respiration starts with glycolysis as well and the pyruvate can be shunted off to the TCA cycle, just like in aerobic respiration. In fact, oxidative phosphorylation is used to generate most of the ATP, which means the use of an ETC and ATP synthase. The key difference is that the final electron acceptor will not be oxygen.
There are a variety of possible final electron acceptors that can be used in anaerobic respiration, allowing microbes to live in a wide variety of locations. The best electron acceptor will be the one that is lowest down on the electron tower, in an oxidized form (i.e. on the left-hand side of the redox couple). Some common electron acceptors include nitrate (NO3-), ferric iron (Fe3+), sulfate (SO42-), carbonate (CO32-) or even certain organic compounds, like fumarate.
How much ATP is generated by anaerobic respiration? That will depend upon the final electron acceptor being used. It will not be as much as is generated during aerobic respiration, since we know that oxygen in the best possible electron acceptor. Selection of an electron acceptor other than oxygen pushes an organism up the electron tower, shortening the distance between the electron donor and the acceptor, reducing the amount of ATP produced.
Fermentation
No matter what they might teach you in a biochemical class, fermentation and anaerobic respiration are not the same thing, at least not to a microbiologist.
Fermentation is catabolism of glucose in the absence of oxygen as well and it does have some similarities to anaerobic respiration. Most obviously, it does not use oxygen as the final electron acceptor. It actually uses pyruvate, an organic compound. Fermentation starts with glycolysis, a process which we have already covered, that also starts off both aerobic respiration and anaerobic respiration. What does it yield? Two net molecules of ATP by substrate-level phosphorylation and 2 molecules of NADH. Organisms doing either aerobic or anaerobic respiration would then utilize oxidative phosphorylation in order to increase their ATP yield. Fermenters, however, lack an ETC or repress synthesis of their ETC when oxygen is not available, so they do not use the TCA cycle at all.
Without the use of an ETC (or a PMF or ATP synthase), no further ATP is generated beyond what was synthesized during glycolysis. But organisms using fermentation cannot just stop with glycolysis, since eventually all their molecules of NAD+ would become reduced. In order to re-oxidize this electron carrier they use pyruvate as a final electron acceptor, yielding a variety of fermentation products such as ethanol, CO2, and various acids.
Lactate Fermentation. By Sjantoni (Own work) [CC BY-SA 3.0], via Wikimedia Commons
Fermentation products, although considered waste products for the cell, are vitally important for humans. We rely on the process of fermentation to produce a variety of fermented foods (beer, wine, bread, cheese, tofu), in addition to using the products for a variety of industrial processes.
Key Words
chemoorganotrophy, aerobic respiration, glycolysis, substrate-level phosphorylation, tricarboxylic acid (TCA) cycle, GTP, oxidative phosphorylation, proton motive force (PMF), ATP synthase/ATPase, anaerobic respiration, fermentation.
Study Questions
1. What is chemoorganotrophy?
2. In glycolysis, what’s the starting compound? How many molecules of ATP (total and net) are produced? How molecules of NADH are reduced?
3. What is substrate level phosphorylation?
4. How do organisms reoxidize NADH, after the breakdown of glucose to pyruvate? Why is it important for them to reoxidize the NADH?
5. During the TCA cycle and connecting reaction, what is glucose broken down to? How many molecules of ATP/ATP equivalents are formed by substrate phosphorylation? How many molecules of NAD and how many molecules of FAD are reduced?
6. What does the cell get from the TCA cycle, in terms of energy & intermediates?
7. In aerobic respiration, how is NADH reoxidized? What is the maximum ATP’s per NADH or FADH formed during this reoxidation? What is the final electron acceptor?
8. What components are involved in electron transport? What is a proton motive force and what role does it play in energy generation?
9. What is oxidative phosphorylation? Where specifically is energy given off in electron transport and how is that energy conserved?
10. How does ATP synthase work to harvest the conserved energy?
11. How many ATPs are formed when glucose is completely broken down in bacterial aerobic respiration and where do they come from? What other products are formed?
12. How is anaerobic respiration similar and different from aerobic respiration? How does the energy yield compare? Why?
13. How is fermentation similar and different to aerobic & anaerobic respiration? How does the energy yield compare? Why? What are the end products of fermentation?
14. For each type of metabolism in this chapter, what is the initial electron donor? What is the final electron acceptor? What processes are used to generate energy? What is the energy yield?
Lactate Fermentation. By Sjantoni (Own work) [CC BY-SA 3.0], via Wikimedia Commons
Fermentation products, although considered waste products for the cell, are vitally important for humans. We rely on the process of fermentation to produce a variety of fermented foods (beer, wine, bread, cheese, tofu), in addition to using the products for a variety of industrial processes.
Key Words
chemoorganotrophy, aerobic respiration, glycolysis, substrate-level phosphorylation, tricarboxylic acid (TCA) cycle, GTP, oxidative phosphorylation, proton motive force (PMF), ATP synthase/ATPase, anaerobic respiration, fermentation.
Study Questions
1. What is chemoorganotrophy?
2. In glycolysis, what’s the starting compound? How many molecules of ATP (total and net) are produced? How molecules of NADH are reduced?
3. What is substrate level phosphorylation?
4. How do organisms reoxidize NADH, after the breakdown of glucose to pyruvate? Why is it important for them to reoxidize the NADH?
5. During the TCA cycle and connecting reaction, what is glucose broken down to? How many molecules of ATP/ATP equivalents are formed by substrate phosphorylation? How many molecules of NAD and how many molecules of FAD are reduced?
6. What does the cell get from the TCA cycle, in terms of energy & intermediates?
7. In aerobic respiration, how is NADH reoxidized? What is the maximum ATP’s per NADH or FADH formed during this reoxidation? What is the final electron acceptor?
8. What components are involved in electron transport? What is a proton motive force and what role does it play in energy generation?
9. What is oxidative phosphorylation? Where specifically is energy given off in electron transport and how is that energy conserved?
10. How does ATP synthase work to harvest the conserved energy?
11. How many ATPs are formed when glucose is completely broken down in bacterial aerobic respiration and where do they come from? What other products are formed?
12. How is anaerobic respiration similar and different from aerobic respiration? How does the energy yield compare? Why?
13. How is fermentation similar and different to aerobic & anaerobic respiration? How does the energy yield compare? Why? What are the end products of fermentation?
14. For each type of metabolism in this chapter, what is the initial electron donor? What is the final electron acceptor? What processes are used to generate energy? What is the energy yield? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/13%3A_Chemoorganotrophy.txt |
Chemolithotrophy
Chemolithotrophy is the oxidation of inorganic chemicals for the generation of energy. The process can use oxidative phosphorylation, just like aerobic and anaerobic respiration, but now the substance being oxidized (the electron donor) is an inorganic compound. The electrons are passed off to carriers within the electron transport chain, generating a proton motive force that is used to generate ATP with the help of ATP synthase.
Chemolithotrophy Pathways.
Electrons donors
Chemolithotrophs use a variety of inorganic compounds as electron donors, with the most common substances being hydrogen gas, sulfur compounds (such as sulfide and sulfur), nitrogen compounds (such as ammonium and nitrite), and ferrous iron.
• Hydrogen oxidizers – these organisms oxidize hydrogen gas (H2) with the use of a hydrogenase enzyme. Both aerobic and anaerobic hydrogen oxidizers exist, with the aerobic organisms eventually reducing oxygen to water.
• Sulfur oxidizers – as a group these organisms are capable of oxidizing a wide variety of reduced and partially reduced sulfur compounds such as hydrogen sulfide (H2S), elemental sulfur (S0), thiosulfate (S2O32-), and sulfite (SO32-). Sulfate (SO42-) is frequently a by-product of the oxidation. Often the oxidation occurs in a stepwise fashion with the help of the sulfite oxidase enzyme.
• Nitrogen oxidizers – the oxidation of ammonia (NH3) is performed as a two-step process by nitrifying microbes, where one group oxidizes ammonia to nitrite (NO2-) and the second group oxidizes the nitrite to nitrate (NO3-). The entire process is known as nitrification and is performed by small groups of aerobic bacteria and archaea, often found living together in soil or in water systems.
• Iron oxidizers – these organisms oxidize ferrous iron (Fe2+) to ferric iron (Fe3+). Since Fe2+ has such a positive standard reduction potential, the bioenergetics are not extremely favorable, even using oxygen as a final electron acceptor. The situation is made more difficult for these organisms by the fact that Fe2+ spontaneously oxidizes to Fe3+ in the presence of oxygen; the organisms must use it for their own purposes before that happens.
Electron acceptors
Chemolithotrophy can occur aerobically or anaerobically. Just as with either type of respiration, the best electron acceptor is oxygen, to create the biggest distance between the electron donor and the electron acceptor. Using a non-oxygen acceptor allows chemolithotrophs to have greater diversity and the ability to live in a wider variety of environments, although they sacrifice energy production.
Amount of ATP generated
Just as both the electron donors and acceptors can vary widely for this group of organisms, the amount of ATP generated for their efforts will vary widely as well. They will not make as much ATP as an organism using aerobic respiration, since the largest ΔE0’ is found using glucose as an electron donor and oxygen as an electron acceptor. But how much less than 32 molecules of ATP greatly depends upon the actual donor and acceptor being used. The smaller the distance between the two, the less ATP that will be formed.
Chemolithoautotrophs vs chemolithoheterotrophs
Most chemolithotrophs are autotrophs (chemolithoautotrophs), where they fix atmospheric carbon dioxide to assemble the organic compounds that they need. These organisms require both ATP and reducing power (i.e. NADH/NADPH) in order to ultimately convert the oxidized molecule CO2 into a greatly reduced organic compound, like glucose. If a chemolithoautotroph is using an electron donor with a higher redox potential than NAD+/NADP, they must use reverse electron flow to push electrons back up the electron tower. This is energetically unfavorable to the cell, consuming energy from the proton motive force to drive electrons in a reverse direction back through the ETC.
Some microbes are chemolithoheterotrophs, using an inorganic chemical for their energy and electron needs, but relying on organic chemicals in the environment for their carbon needs. These organisms are also called mixotrophs, since they require both inorganic and chemical compounds for their growth and reproduction.
Nitrogen Metabolism
The nitrogen cycle depicts the different ways in which nitrogen, an essential element for life, is used and converted by organisms for various purposes. Much of the chemical conversions are performed by microbes as part of their metabolism, performing a valuable service in the process for other organisms in providing them with an alternate chemical form of the element.
Nitrogen Cycle.
Nitrogen Fixation
Nitrogen fixation describes the conversion of the relatively inert dinitrogen gas (N2) into ammonia (NH3), a much more useable form of nitrogen for most life forms. The process is performed by diazotrophs, a limited number of bacteria and archaea that can grow without an external source of fixed nitrogen, because of their abilities. Nitrogen fixation is an essential process for Earth’s organisms, since nitrogen is a required component of various organic molecules, such as amino acids and nucleotides. Plants, animals, and other organisms rely on bacteria and archaea to provide nitrogen in a fixed form, since no eukaryote is known that can fix nitrogen.
Nitrogen fixation is an extremely energy and electron intensive process, in order to break the triple bond in N2 and reduce it to NH3. It requires a particular enzyme known as nitrogenase, which is inactivated by O2. Thus, nitrogen fixation must take place in an anaerobic environment. Aerobic nitrogen-fixing organisms must devise special conditions or arrangements in order to protect their enzyme. Nitrogen-fixing organisms can either exist independently or pair up with a plant host:
1. Symbiotic nitrogen-fixing organisms: these bacteria partner up with a plant, to provide them with an environment appropriate for the functioning of their nitrogenase enzyme. The bacteria live in the plant’s tissue, often in root nodules, fixing nitrogen and sharing the results. The plant provides both the location to fix nitrogen, as well as additional nutrients to support the energy-taxing process of nitrogen fixation. It has been shown that the bacteria and the host exchange chemical recognition signals that facilitate the relationship. One of the best known bacteria in this category is Rhizobium, which partners up with plants of the legume family (clover, soybeans, alfalfa, etc).
2. Free-living nitrogen-fixing organisms: these organisms, both bacteria and archaea, fix nitrogen for their own use that ends up being shared when the organisms dies or is ingested. Free-living nitrogen-fixing organisms that grow anaerobically do not have to worry about special adaptations for their nitrogenase enzyme. Aerobic organisms must make adaptations. Cyanobacteria, a multicellular bacterium, make specialized cells known as heterocystsin which nitrogen fixation occurs. Since Cyanobacteria produce oxygen as part of their photosynthesis, an anoxygenic version occurs within the heterocyst, allowing the nitrogenase to remain active. The heterocysts share the fixed nitrogen with surrounding cells, while the surrounding cells provide additional nutrients to the heterocysts.
Assimilation
Assimilation is a reductive process by which an inorganic form of nitrogen is reduced to organic nitrogen compounds such as amino acids and nucleotides, allowing for cellular growth and reproduction. Only the amount needed by the cell is reduced. Ammonia assimilation occurs when the ammonia (NH3)/ammonium ion (NH4+) formed during nitrogen fixation is incorporated into cellular nitrogen. Assimilative nitrate reduction is a reduction of nitrate to cellular nitrogen, in a multi-step process where nitrate is reduced to nitrite then ammonia and finally into organic nitrogen.
Nitrification
As mentioned above, nitrification is performed by chemolithotrophs using a reduced or partially reduced form of nitrogen as an electron donor to obtain energy. ATP is gained by the process of oxidative phosphorylation, using a ETC, PMF, and ATP synthase.
Denitrification
Denitrification refers to the reduction of NO3- to gaseous nitrogen compounds, such as N2. Denitrifying microbes perform anaerobic respiration, using NO3- as an alternate final electron acceptor to O2. This is a type of dissimilatory nitrate reduction where the nitrate is being reduced during energy conservation, not for the purposes of making organic compounds. This produces large amounts of excess byproducts, resulting in the loss of nitrogen from the local environment to the atmosphere.
Anammox
Anammox or anaerobic ammonia oxidation is performed by marine bacteria, relatively recently discovered, that utilize nitrogen compounds as both electron acceptor and electron donor. Ammonia is oxidized anaerobically as the electron donor while nitrite is utilized as the electron acceptor, with dinitrogen gas produced as a byproduct. The reactions occur within the anammoxosome, a specialized cytoplasmic structure which constitutes 50-70% of the total cell volume. Just like denitrification, the anammox reaction removes fixed nitrogen from a local environment, releasing it to the atmosphere.
Key Words
chemolithotrophy, hydrogen oxidizers, hydrogenase, sulfur oxidizers, sulfite oxidase, nitrogen oxidizers, nitrification, iron oxidizers, chemolithoautotroph, reverse electron flow, chemolithoheterotroph, mixotroph, nitrogen fixation, diazotroph, nitrogenase, symbiotic nitrogen-fixing organisms, Rhizobium, legume, free-living nitrogen-fixing organisms, Cyanobacteria, heterocyst, assimilation, ammonia assimilation, assimilative nitrate reduction, denitrification, dissimilatory nitrate reduction, anammox, anaerobic ammonia oxidation, anammoxosome.
Study Questions
1. What is chemolithotrophy?
2. What are the most common electron donors and acceptors for chemolithotrophs? How does their amount of ATP produced compare to chemoorganotrophs?
3. How do chemolithoautotrophs and chemolithoheterotrophs differ? What is the reverse electron flow and how/why is it used by some chemolithoautotrophs?
4. What roles do bacteria/archaea play in the nitrogen cycle? How are different nitrogen compounds used in their metabolism?
5. What is required for nitrogen fixation? How do free living nitrogen fixers and plant associated nitrogen fixers differ? How do Rhizobium and Cyanobacteria protect their nitrogenase from oxygen?
6. What are the different mechanisms of nitrogen metabolism? What conversion is occurring for each? What is the purpose of each and how does it relate to the organism’s metabolism?
Nitrogen Cycle.
Nitrogen Fixation
Nitrogen fixation describes the conversion of the relatively inert dinitrogen gas (N2) into ammonia (NH3), a much more useable form of nitrogen for most life forms. The process is performed by diazotrophs, a limited number of bacteria and archaea that can grow without an external source of fixed nitrogen, because of their abilities. Nitrogen fixation is an essential process for Earth’s organisms, since nitrogen is a required component of various organic molecules, such as amino acids and nucleotides. Plants, animals, and other organisms rely on bacteria and archaea to provide nitrogen in a fixed form, since no eukaryote is known that can fix nitrogen.
Nitrogen fixation is an extremely energy and electron intensive process, in order to break the triple bond in N2 and reduce it to NH3. It requires a particular enzyme known as nitrogenase, which is inactivated by O2. Thus, nitrogen fixation must take place in an anaerobic environment. Aerobic nitrogen-fixing organisms must devise special conditions or arrangements in order to protect their enzyme. Nitrogen-fixing organisms can either exist independently or pair up with a plant host:
1. Symbiotic nitrogen-fixing organisms: these bacteria partner up with a plant, to provide them with an environment appropriate for the functioning of their nitrogenase enzyme. The bacteria live in the plant’s tissue, often in root nodules, fixing nitrogen and sharing the results. The plant provides both the location to fix nitrogen, as well as additional nutrients to support the energy-taxing process of nitrogen fixation. It has been shown that the bacteria and the host exchange chemical recognition signals that facilitate the relationship. One of the best known bacteria in this category is Rhizobium, which partners up with plants of the legume family (clover, soybeans, alfalfa, etc).
2. Free-living nitrogen-fixing organisms: these organisms, both bacteria and archaea, fix nitrogen for their own use that ends up being shared when the organisms dies or is ingested. Free-living nitrogen-fixing organisms that grow anaerobically do not have to worry about special adaptations for their nitrogenase enzyme. Aerobic organisms must make adaptations. Cyanobacteria, a multicellular bacterium, make specialized cells known as heterocystsin which nitrogen fixation occurs. Since Cyanobacteria produce oxygen as part of their photosynthesis, an anoxygenic version occurs within the heterocyst, allowing the nitrogenase to remain active. The heterocysts share the fixed nitrogen with surrounding cells, while the surrounding cells provide additional nutrients to the heterocysts.
Assimilation
Assimilation is a reductive process by which an inorganic form of nitrogen is reduced to organic nitrogen compounds such as amino acids and nucleotides, allowing for cellular growth and reproduction. Only the amount needed by the cell is reduced. Ammonia assimilation occurs when the ammonia (NH3)/ammonium ion (NH4+) formed during nitrogen fixation is incorporated into cellular nitrogen. Assimilative nitrate reduction is a reduction of nitrate to cellular nitrogen, in a multi-step process where nitrate is reduced to nitrite then ammonia and finally into organic nitrogen.
Nitrification
As mentioned above, nitrification is performed by chemolithotrophs using a reduced or partially reduced form of nitrogen as an electron donor to obtain energy. ATP is gained by the process of oxidative phosphorylation, using a ETC, PMF, and ATP synthase.
Denitrification
Denitrification refers to the reduction of NO3- to gaseous nitrogen compounds, such as N2. Denitrifying microbes perform anaerobic respiration, using NO3- as an alternate final electron acceptor to O2. This is a type of dissimilatory nitrate reduction where the nitrate is being reduced during energy conservation, not for the purposes of making organic compounds. This produces large amounts of excess byproducts, resulting in the loss of nitrogen from the local environment to the atmosphere.
Anammox
Anammox or anaerobic ammonia oxidation is performed by marine bacteria, relatively recently discovered, that utilize nitrogen compounds as both electron acceptor and electron donor. Ammonia is oxidized anaerobically as the electron donor while nitrite is utilized as the electron acceptor, with dinitrogen gas produced as a byproduct. The reactions occur within the anammoxosome, a specialized cytoplasmic structure which constitutes 50-70% of the total cell volume. Just like denitrification, the anammox reaction removes fixed nitrogen from a local environment, releasing it to the atmosphere.
Key Words
chemolithotrophy, hydrogen oxidizers, hydrogenase, sulfur oxidizers, sulfite oxidase, nitrogen oxidizers, nitrification, iron oxidizers, chemolithoautotroph, reverse electron flow, chemolithoheterotroph, mixotroph, nitrogen fixation, diazotroph, nitrogenase, symbiotic nitrogen-fixing organisms, Rhizobium, legume, free-living nitrogen-fixing organisms, Cyanobacteria, heterocyst, assimilation, ammonia assimilation, assimilative nitrate reduction, denitrification, dissimilatory nitrate reduction, anammox, anaerobic ammonia oxidation, anammoxosome.
Study Questions
1. What is chemolithotrophy?
2. What are the most common electron donors and acceptors for chemolithotrophs? How does their amount of ATP produced compare to chemoorganotrophs?
3. How do chemolithoautotrophs and chemolithoheterotrophs differ? What is the reverse electron flow and how/why is it used by some chemolithoautotrophs?
4. What roles do bacteria/archaea play in the nitrogen cycle? How are different nitrogen compounds used in their metabolism?
5. What is required for nitrogen fixation? How do free living nitrogen fixers and plant associated nitrogen fixers differ? How do Rhizobium and Cyanobacteria protect their nitrogenase from oxygen?
6. What are the different mechanisms of nitrogen metabolism? What conversion is occurring for each? What is the purpose of each and how does it relate to the organism’s metabolism? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/14%3A_Chemolithotrophy_and_Nitrogen_Metabolism.txt |
Of course, getting energy from sunlight, that’s pretty cool. Or is it hot? Bad jokes aside, hopefully you remember the basic process of photosynthesis from previous classes, where they might have talked about what happens in plants. The Z pathway? Anything? Do not panic – we will talk about it. Along with the other types of phototrophy that microbes use.
Photoautotrophs vs Photoheterotrophs
Phototrophy (or “light eating”) refers to the process by which energy from the sun is captured and converted into chemical energy, in the form of ATP. The term photosynthesis is more precisely used to describe organisms that both convert sunlight into ATP (the “light reaction”) but then also proceed to use the ATP to fix carbon dioxide into organic compounds (the Calvin cycle). These organisms are the photoautotrophs. In the microbial world, there are also photoheterotrophs, organisms that convert sunlight into ATP but utilize pre-made organic compounds available in the environment. The ATP could then be used for other purposes.
Pigments
In order to convert energy from sunlight into ATP, organisms use light-sensitive pigments. Plants and algae utilize chlorophylls, which are used by cyanobacteria as well. Chlorophylls are green in color, due to the fact that they absorb red and blue wavelengths (≈675 nm and 430 nm) and transmit green light. The purple and green bacteria have bacteriochlorophylls, which absorb higher wavelengths (≈870 nm) than the chlorophylls, allowing different phototrophs to occupy the same environment without competing with one another.
Phototrophs can contain accessory pigments as well, such as the carotenoids and phycobiliproteins. Carotenoids, which absorb blue light (400-550 nm), are typically yellow, orange, or red in color. The phycobiliproteins can be split in two groups: phycoerythrin, which transmits a red color, and phycocyanin, which transmits a blue color. The accessory pigments can serve to expand the wavelength range of light being absorbed, allowing better utilization of light available. In addition, these pigments can serve a protective function for the organism by acting as an antioxidant.
Phototrophic Pigment.
In bacteria and archaea, the phototrophic pigments are housed within invaginations of the cell membrane or within a chlorosome. Light-harvesting pigments form antennae, which funnel the light to other molecules in reaction centers, which actually perform the conversion of light energy into ATP.
Photophosphorylation in general
For any organism, the general process of phototrophy is going to be the same. A photosystem antennae absorbs light and funnels the energy to a reaction center, specifically to a special pair of chlorophyll/bacteriochlorophyll molecules. The molecules become excited, changing to a more negative reduction potential (i.e. jumping up the electron tower). The electrons can then be passed through an electron transport chain of carriers, such as ferredoxin and cytochromes, allowing for the development of a proton motive force. The protons are brought back across the plasma membrane through ATPase, generating ATP in the process. Since the original energy from the process came from sunlight, as opposed to a chemical, the process is called photophosphorylation. If the electrons are returned to the special pair of chlorophyll/bacteriochlorophyll molecules (cyclic photophosphorylation), the process can be repeated over and over again. If the electrons are diverted elsewhere, such as for the reduction of NAD(P) (non-cyclic photophosphorylation), then an external electron source must be used to replenish the system.
Anoxygenic Phototrophy
Purple phototrophic bacteria
Purple phototrophic bacteria engage in anoxygenic phototrophy, indicating that they do not generate oxygen during the process. They have a single photosystem with bacteriochlorophyll, allowing them to use cyclic photophosphorylation as described above for the formation of ATP. But if a purple bacterium wants to grow as a photoautotroph, it will also need reducing power in the form of NAD(P)H.
The reaction center of purple bacteria (known as P870) has an E0’ of +0.5V. After being hit by a photon of light, the potential changes to -1.0V, which is insufficient to reduce NAD(P) with its E0’ of -0.32V. Thus, autotrophic purple bacteria must engage in a process known as reverse electron flow, using energy from the proton motive force to drive electrons up the electron tower. Additionally, they must find an external electron donor to replenish the electrons now diverted to NAD(P). Typically the electrons come from H2S or elemental sulfur, with various sulfur byproducts produced.
Photophosphorylation in Purple Bacteria.
In the presence of organic compounds, the purple bacteria often exist as photoheterotrophs, utilizing cyclic photophosphorylation to generate ATP and getting their organic compounds from the environment. This eliminates the need for using reverse electron flow, an energetically unfavorable process, as well as the need for external electron donors.
Green phototrophic bacteria
Green phototrophic bacteria also engage in anoxygenic phototrophy, utilizing a single photosystem with bacteriochlorophyll for cyclic photophosphorylation in the production of ATP. However, they also use this same photosystem for generation of reducing power, by periodically drawing off electrons to NAD+. The use of reverse electron flow is unnecessary, however, since the initial carrier, ferredoxin (Fd) has a E0’ with a more negative reduction potential than NAD(P). An external electron donor is required, typically by using H2S or thiosulfate. Thus, the green bacteria operate as photoautotrophs, by alternating the use of their photosystem for ATP or NAD(P)H.
Green and Purple Phototrophic Bacteria.
Oxygenic Phototrophy
Oxygenic phototrophy is used by cyanobacteria containing chlorophyll a, with two distinct photosystems, each containing separate reaction centers. This allows for the generation of both ATP and reducing power in one process, facilitating photoautotrophic growth through the fixation of CO2. This can appropriately be referred to as photosynthesis and it is the same process used by plants, commonly referred to as the “Z pathway.”
The process starts when light energy decreases the reduction potential of P680 chlorophyll a molecules contained in photosystem II (PSII). The electrons are then passed through an electron transport chain, generating ATP via a proton motive force. Electrons are then passed to photosystem I (PSI), where they get hit by another photon of light, decreasing their reduction potential even more. The electrons are then passed through a different electron transport chain, eventually being passed off to NADP+ for the formation of NADPH.
Overview of Oxygenic Photosynthesis in Cyanobacteria.
The process is an example of noncyclic photophosphorylation, since the electrons are not returned to the original photosystem. Thus, an external electron donor is required in order to allow the process to repeat. Water, found on the right side of the redox couple O2/H2O, is normally a poor electron donor, due to its extremely positive reduction potential. But the reduction potential of P680 chlorophyll a is even more positive when not excited, allowing for water to serve as an electron donor. The hydrolysis of water leads to the evolution of oxygen, a welcome byproduct for all organisms that use aerobic respiration. It is thought that cyanobacteria are responsible for the oxygenation of Earth, allowing for the development of aerobic respiration as a form of metabolism.
There are some conditions under which cyanobacteria only use PSI, essentially performing a form of anoxygenic phototrophy, despite their possession of chlorophyll a. This occurs within the heterocysts of cyanobacteria, where oxygen inactivates the nitrogenase enzymes. Heterocysts degrade PSII, ensuring that oxygen will not be produced as a byproduct, while still allowing for the production of ATP with the remaining photosystem.
Oxygen-free Phototrophy in Cyanobacterial Heterocysts.
Rhodopsin-Based Phototrophy
One unusual form of phototrophy is used by archaea, without the use of chlorophyll or bacteriochlorophyll. Instead these organisms use a bacteriorhodopsin (more appropriately called an archaearhodopsin), a retinal molecule related to the one found in vertebrate eyes. When the rhodopsin absorbs light it undergoes a conformational change, pumping a proton across the cell membrane and leading to the development of a proton motive force, without the participation of an electron transport chain.
Rhodopsin-Based Phototrophy. By Darekk2 (Own work) [CC BY-SA 3.0], via Wikimedia Commons
Key Words
phototrophy, photosynthesis, photoautotroph, photoheterotroph, chlorophylls, bacteriochlorophylls, carotenoid, phycobiliprotein, phycoerythrin, phycocyanin, chlorosome, antennae, reaction centers, photophosphorylation, cyclic photophosphorylation, non-cyclic photophosphorylation, purple phototrophic bacteria, anoxygenic phototrophy, reverse electron flow, green phototrophic bacteria, oxygenic phototrophy, Z pathway, photosystem II (PSII), photosystem I (PSI), rhodopsin-based phototrophy, bacteriorhodopsin/archaeorhodopsin.
Essential Questions/Objectives
1. How do phototrophy and photosynthesis differ? How do photoautotrophs and photoheterotrophs fit with each term?
2. What is the difference among chlorophyll, bacteriochlorophyll, and accessory pigments of phototrophs?
3. How is ATP formed during photophosphorylation? What mechanisms are used?
4. What is anoxygenic phototrophy? How does the process differ between the purple and the green phototrophic bacteria? How does each process differ between photoautotrophs and photohetertrophs? What is reverse electron flow?
5. Be able to diagram and explain cyclic or anoxygenic photosynthesis in Purple and Green photosynthetic bacteria – include pigments used, products formed, source of electrons, final electron acceptor, and how ATP is formed.
6. Be able to diagram and explain the Z pathway of photosynthesis in Cyanobacterium including pigment used, products formed, source of electrons, final electron acceptor and how ATP is formed.
7. What is cyclic photosynthesis in Cyanobacteria? When is this type of process used by Cyanobacteria?
8. What is rhodopsin-based phototrophy? What does it involve and what organisms use it? Under what conditions is it used?
Exploratory Questions (OPTIONAL)
1. Under what conditions would it be advantageous for a microbe to operate as a photoheterotroph, as opposed to a photoautotroph? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/15%3A_Phototrophy.txt |
Evolution
It is believed that the Earth is 4.6 billion year old, with the first cells appearing approximately 3.8 billion years ago. Those cells were undoubtably microbes, eventually giving rise to all the life forms that we envision today, as well as the life forms that went extinct before we got here. How did this progression occur?
Early Earth
Conditions on early Earth were most likely extremely hot, anoxic (lacking oxygen), with reduced inorganic chemicals in abundance. While no one knows exactly how cells came about, it is likely that they were initially suited to these harsh conditions.
RNA World
RNA, in its many forms, plays a crucial role in cellular activities. It has been hypothesized that RNA played an even more central role in primitive cells, with self-replicating RNA containing a cell’s information as well as having catalytic activity to synthesize proteins. Eventually this RNA world evolved to one in which proteins took over the catalytic responsibilities and DNA became the common form of information storage.
The “RNA World” and the Modern World.
Metabolic Diversity
Initial cells probably had a relatively primitive electron transference system, perhaps through just one carrier, that still allowed for the development of a proton motive force to conserve energy. As chemolithoautotrophs proliferated, organic material started to accumulate in the environment, providing the conditions needed for the development of chemoorganotrophic organisms. These new cells oxidized organic compounds, with their more negative redox potential and increased number of electrons. This most likely lengthened electron transport chains, resulting in faster growth, and speeding up diversity even more.
Phototrophy & Photosynthesis
At about 3.5 billion year ago some cells evolved phototrophic pigments, allowing for the conversion of light energy into chemical energy. Initially phototrophs utilized anoxygenic phototrophy, using sulfur products as an electron donor when performing CO2 fixation.
Stromatolites are layered rocks that form when minerals are incorporated into thick mats of microbes, growing on water surfaces. Ancient stromatolites contain fossilized microbial mats made up of cyanobacteria-like cells, indicating their presence relatively early in Earth’s history.
Approximately 2.5-3.3 billion year ago the cyanobacterial ancestors developed oxygenic photosynthesis by acquiring two photosystems and the pigment chlorophyll a. This led to the use of water as an electron donor, causing oxygen to accumulate in Earth’s atmosphere. This Great Oxidation Event substantially changed the types of metabolism possible, allowing for the use of oxygen as a final electron acceptor.
Ozone Shield Formation
The development of an ozone shield around the Earth occurred around 2 billion years ago. Ozone (O3) serves to block out much of the ultraviolet (UV) radiation coming from the sun, which can cause significant damage to DNA. As oxygen accumulated in the environment, the O2 was converted to O3 when exposed to UV light, causing an ozone layer to form around Earth. This allowed organisms to start inhabiting the surface of the planet, as opposed to just the ocean depths or soil layers.
Endosymbiosis
Evolution supports the idea of more primitive molecules or organisms being generated first, followed by the more complex components or organisms over time. Endosymbiosis offers an explanation for the development of eukaryotic cells, a more complex cell type with organelles or membrane-bound enclosures.
It is generally accepted that eukaryotic ancestors arose when a cell ingested another cell, a free-living bacterium, but did not digest it. This endosymbiont had capabilities that the proto-eukaryotic cell lacked, such as the ability for phototrophy (i.e. chloroplasts) or oxidative phosphorylation (i.e. mitochondria). Eventually the two became mutually dependent upon one another with the endosymbiont becoming an organelle, with the chloroplast being derived from a cyanobacterial ancestor and the mitochondrion being derived from a gram negative bacillus ancestor.
Endosymbiosis. By Signbrowser (Own work) [CC0], via Wikimedia Commons
Evidence to support this idea includes the fact that mitochondria and chloroplasts: have a single, circular chromosome; undergo binary fission separate from the eukaryotic cell; have 70S sized ribosomes; have a lipid bilayer with a 2:1 ratio of protein to lipid; and, perhaps most importantly, have rRNA sequences that place them phylogenetically with the bacteria.
Phylogeny
Molecular Phylogeny
Phylogeny is a reference to the development of an organism evolutionarily. Molecular techniques allow for the evolutionary assessment of organisms using genomes or ribosomal RNA (rRNA) nucleotide sequences, generally believed to provide the most accurate information about the relatedness of microbes.
Nucleic acid hybridization or DNA-DNA hybridization is a commonly used tool for molecular phylogeny, comparing the similarities between genomes. The genomes of two organisms are heated up or “melted” to separate the complementary strand and then allowed to cool down. Strands that have complementary base sequences will re-anneal, while strands without complementation will remain upaired. Typically one source of DNA is labeled, usually with radioactivity, to allow for identification of each DNA source.
Nucleic acid sequencing, typically using the rRNAs from small ribosomal subunits, allows for direct comparison of sequences. The ribosomal sequence is seen as ideal because the genes encoding it do not change very much over time, nor does it appear to be strongly influenced by horizontal gene transfer. This makes it an excellent “molecular chronometer,” or way to track genetic changes over a long period of time, even between closely related organisms.
Phylogenetic Trees
Phylogenetic trees serve to show a pictorial example of how organisms are believed to be related evolutionarily. The root of the tree is the last common ancestor for the organisms being compared (Last Universal Common Ancestor or LUCA, if we are doing a comparison of all living cells on Earth). Each node (or branchpoint) represents an occurrence where the organisms diverged, based on a genetic change in one organism. The length of each branch indicates the amount of molecular changes over time. The external nodes represent specific taxa or organisms (although they can also represent specific genes). A cladeindicates a group of organisms that all have a particular ancestor in common.
Taxonomy
Taxonomy refers to the organization of organisms, based on their relatedness. Typically it involves some type of classification scheme, the identification of isolates, and the naming or nomenclature of included organisms. Many different classification schemes exist, although many have not been appropriate for comparison of microorganisms.
Classification Systems
A phenetic classification system relies upon the phenotypes or physical appearances of organisms. Phylogenetic classication uses evolutionary relationships of organisms. A genotypic classification compares genes or genomes between organisms. The most popular approach is to use a polyphasic approach, which combines aspects of all three previous systems.
Microbial Species
Currently there is no widely accepted “species definition” for microbes. The definition most commonly used is one that relies upon both genetic and phenotypic information (a polyphasic approach), with a threshold of 70% DNA-DNA hybridization and 97% 16S DNA sequence identity in order for two organisms to be deemed as belonging to the same species.
Key Words
evolution, RNA world, stromatolites, Great Oxidation Event, ozone shield, endosymbiosis, chloroplast, mitochondria, phylogeny, ribosomal RNA/rRNA, molecular phylogeny, nucleic acid hybridization, DNA-DNA hybridization, nucleic acid sequencing, molecular chronometer, phylogenetic tree, Last Universal Common Ancestor/LUCA, node, branch, external node, clade, taxonomy, phonetic classification, phylogenetic classification, genotypic classification, polyphasic classiciation, species definition.
Study Questions
1. What is the approximate age of earth? What is the age of the oldest microbial fossils?
2. What are thought to be the conditions of early earth? How would this influence microbial selection?
3. What is the premise of the “RNA world”?
4. What are the important steps in the evolution of metabolism? How does each step influence microbial growth/life on earth?
5. What is the endosymbiotic theory and what evidence do we have for it?
6. What is phylogeny? What is molecular phylogeny?
7. What is DNA-DNA hybridization? What is nucleic acid sequencing? How is each performed? What information is gained?
8. What is a molecular chronometer? Which molecule has been most useful and why?
9. What is a phylogenetic tree? What is the difference among a node, external node, branch, and a clade? What does the length of a branch indicate? What is LUCA?
10. What is taxonomy and what is its purpose? What is the difference between classification, nonmenclature, and identification in taxonomy?
11. What are differences among the following classification systems: phenetic, phylogenetic, genotypic, polyphasic. What characteristics are used for each? Where do they overlap?
12. How are a microbial species currently defined? What criteria are applied? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/16%3A_Taxonomy_and_Evolution.txt |
Let’s talk about sex. Bacterial sex. Ha! That is going to be difficult, since bacteria do not have sex. Which presents a real problem for bacteria (and archaea, too) – how do they get the genetic variability that they need? They might need a new gene to break down an unusual nutrient source or degrade an antibiotic threatening to destroy them – acquiring the gene could mean the difference between life and death. But where would these genes come from? How would the bacteria get a hold of them? We are going to explore the processes that bacteria use to acquire new genes, the mechanisms known as Horizontal Gene Transfer (HGT).
Conjugation
Conjugation is the process by which a donor bacterium transfers a copy of a plasmid to a recipient bacterium, through a pilus. The process requires cell-to-cell contact. The donor cell (F+) has a conjugative plasmid, an extrachromosomal piece of dsDNA that codes for the proteins necessary to make a threadlike filament known as a pilus. The pilus is used to bind to the recipient (F-) cell, bringing it in close proximity to the donor cell. It is believed that a channel is then opened between the two cells, allowing for a ssDNA copy of the plasmid to enter the recipient cells. Both cells then make the complementary copy to the ssDNA, resulting in two F+ cells capable of conjugation.
Conjugation. By Adenosine (Own work) [CC BY-SA 3.0], via Wikimedia Commons
Transformation
The process of transformation also allows a bacterial cell to acquire new genes, but it does not require cell-to-cell contact. In this process the new genes are acquired directly from the environment. Typically the process requires a donor cell that at some point lysed and released naked DNA to the environment. The recipient cell is one that is capable of taking up the DNA from the environment and incorporating it into its own genome, where the cell is described as being competent.
There are mechanical and chemical means of encouraging a cell to pick up DNA from the environment, but natural competence is determined genetically. The process typically occurs at the end of exponential phase of growth or beginning of the stationary phase, in the presence of high cell density and limited nutrients. Under these conditions specific proteins are manufactured including DNA-binding proteins (DNA translocase), endonucleases, and transmembrane channel proteins. Gram negative cells also make a cell wall autolysin, to transport the DNA across the outer membrane.
Gene Acquisition via Transformation.
Random pieces of DNA bind to receptors on the outside of the cell and are then transported into the cell by the DNA translocase, through the transmembrane channel, a large structure often involving numerous different proteins. An endonuclease can be used to degrade one strand of dsDNA, if only ssDNA may pass into the cell, or to cleave the DNA fragment into smaller sizes .Once inside the cell, the DNA must be incorporated into the bacterial chromosome by RecA (see Molecular Recombination below), for the genes to be expressed.
Transduction
Transduction involves the use of a virus, a bacteriophage, to act as a conduit for shuttling bacteria genes from one cell to another, thus negating the necessity for cell-to-cell contact. There are two different types of transduction: generalized transduction and specialized transduction.
Generalized Transduction
In generalized transduction, a bacterial host cell is infected with either a virulent or a temperate bacteriophage engaging in the lytic cycle of replication. After the first three steps of replication (absorption, penetration, and synthesis), the virus enters into the assembly stage, during which fully formed virions are made. During this stage, random pieces of bacterial DNA are mistakenly packaged into a phage head, resulting in the production of a transducing particle. While these particles are not capable of infecting a cell in the conventional sense, they can bind to a new bacterial host cell and inject their DNA inside. If the DNA (from the first bacterial host cell) is incorporated into the recipient’s chromosome, the genes can be expressed.
Generalized Transduction.
Specialized Transduction
Specialized transduction can only occur with temperate bacteriophage, since it involves the lysogenic cycle of replication. The bacteriophage randomly attaches to a bacterial host cell, injecting viral DNA inside. The DNA integrates into the chromosome of the host cell, forming a prophage. At some point induction occurs, where the prophage is excised from the bacterial chrosomsome. In specialized transduction, the excision is incorrectly performed and a portion of bacterial genes immediately adjacent to the viral genes are excised too. Since this DNA is used as the template for the synthesis stage, all copies will be a hybrid of viral and bacterial DNA, and all resulting virions will contain both viral and bacterial DNA.
Once the cell is lysed, the virions are released to infect other bacterial host cells. Each virion will attach to the host cell and inject in the DNA hybrid, which could be incorporated into the host chromosome, if a prophage is formed. At this point the second bacterial host cell can contain its own DNA, DNA from the previous bacterial host cell, and viral DNA.
Molecular Recombination
In each of the cases of HGT, the process is only successful if the genes can be expressed by the altered cell. In conjugation, the genes are located on a plasmid, under the control of promoters on the plasmid. In transformation and transduction, where naked DNA is gaining access to the cell, the DNA could easily be broken down by the cell with no genetic expression occurring. In order for the genes to be expressed, the DNA must be recombined with the recipient’s chromosome.
The most common mechanism of molecular recombination is homologous recombination, involving the RecA protein. In this process DNA from two sources are paired, based on similar nucleotide sequence in one area. An endonuclease nicks one strand, allowing RecA to pair up bases from different strands, a process known as strand invasion. The cross-over between DNA molecules is resolved with resolvase, which cuts and rejoins the DNA into two separate dsDNA molecules.
Recombination can also occur using site-specific recombination, a process often used by viruses to insert their genome into the chromosome of their host. This type of recombination is also used by transposable elements (see next section).
Transposable Elements
Finally, we shouldn’t leave the topic of microbial genetics without at least exploring the role of transposable elements or “jumping genes.” While these can play a very big role in the activation and inactivation of bacterial genes, the best explanation derives from the work of Barbara McClintock in corn, who won the Nobel Prize for her research in 1983. She demonstrated that transposable elements can be responsible for the activation or inactivation of genes within an organism.
Transposable elements are relatively simple in structure, designed to move from one location to another within a DNA molecule by a process known as transposition. All transposable elements code for the enzyme transposase, the enzyme responsible allowing transposition to occur, and have short inverted repeats (IRs) at each end.
Transposition.
The simplest transposable element is an insertion sequence (IS), which contains the transposase and IRs of varying lengths. A transposontypically contains additional genes, with the exact type varying widely from transposon to transposon. A transposon can be removed from one location and relocated to another (the cut-and-paste model), a process known as conservative transposition. Alternatively, it can be copied, with the copy being inserted at a second site, in a process known as replicative transposition.
Key Words
Horizontal Gene Transfer (HGT), conjugation, donor, recipient, conjugative plasmid, F-, F, transformation, naked DNA, competence, competent cell, DNA translocase, endonuclease, autolysin, RecA, transduction, generalized transduction, transducing particle, specialized transduction, molecular recombination, homologous recombination, resolvase, site-specific recombination, transposable elements, transposition, transposase, inverted repeats (IR), insertion sequence (IS), transposon, conservative transposition, replicative transposition.
Essential Questions/Objectives
1. What is horizontal gene transfer? What are the three mechanisms for this to occur in bacteria?
2. What are the components needed for the processes of transformation, conjugation, and transduction? How does each process occur? What genes are involved in each process?
3. How do generalized and specialized transduction differ? What is the end result of each?
4. What is recombination? What is the importance to bacteria & archaea?
5. What are the two types of recombination? What are the details of each type? What components are needed for each type?
6. What is transposition? What is required for the process to occur? What is a transposable element?
Exploratory Questions (OPTIONAL)
1. How could transposons be used in the study of bacterial genetics? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/17%3A_Microbial_Genetics.txt |
Genetic engineering is the deliberate manipulation of DNA, using techniques in the laboratory to alter genes in organisms. Even if the organisms being altered are not microbes, the substances and techniques used are often taken from microbes and adapted for use in more complex organisms.
Steps in Cloning a Gene
Let us walk through the basic steps for cloning a gene, a process by which a gene of interest can be replicated many times over. Let us pretend that we are going to genetically engineer E. coli cells to glow in the dark, a characteristic that they do not naturally possess.
1. Isolate DNA of interest – first we need to identify the genes or genes that we are interested in, the target DNA. If we want our E. coli cells to glow in the dark, we need to find an organism that possesses this trait and identify the gene or genes responsible for the trait. The green fluorescent protein (GFP) commonly used as an expression marker in molecular techniques was originally isolated from jellyfish.In cloning a gene it is helpful to use a cloning vector, typically a plasmid or virus, capable of independent replication that will stably carry the target DNA from one location to another. Plasmid vectors are available from both bacteria and yeast.
2. Cut DNA with restriction endonucleases – once the target and vector DNA have been identified, both types of DNA are cut using restriction endonucleases. These enzymes recognize short sequences of DNA that are 4-8 bp long. The enzymes are widespread in both bacteria and archaea, with each enzyme recognizing a specific inverted repeat sequence that is palindromic (reads the same on each DNA strand, in the 5’ to 3’ direction).
Restriction Endonucleases.
While some restriction endonucleases cut straight across the DNA (i.e. blunt cut), many make staggered cuts, producing a very short region of single-stranded DNA on each strand. These single-stranded regions are referred to as “sticky ends,” and are invaluable in molecular cloning since the unpaired bases will recombine with any DNA having the complementary base sequence.
3. Combine target and vector DNA – after both types of DNA have been cleaved by the same restriction endonuclease, the two types of DNA are combined together with the addition of DNA ligase, an enzyme that repairs the covalent bonds on the sugar-phosphate backbone of the DNA. This results in the creation of recombinant DNA, DNA molecules that contain the DNA from two or more sources, also known as chimeras.
Ligation.
4. Introduce recombined molecule into host cell – once the target DNA has been stably combined with vector DNA, the recombinant DNA must be introduced into a host cell, in order for the genes to be replicated or expressed. There are different methods for introducing the recombinant DNA, largely depending upon the complexity of the host organism. In the case of bacteria, transformation is often the easiest method, using competent cells to pick up the recombinant DNA molecules. Alternatively, electroporation can be used, where the cells are exposed to a brief pulse of high –voltage electricity causing the plasma membrane to become temporarily permeable to DNA passage.While some cells will acquire recombinant DNA with the appropriate configuration (i.e. target DNA combined with vector DNA), the method also will yield cells carrying recombinant DNA with alternate DNA combinations (i.e. plasmid DNA combining with another plasmid DNA molecule or target DNA attached to more target DNA). The mixture is referred to as a genomic library and must be screened to select the appropriate clone. If random fragments of DNA were originally used (instead of isolation of the appropriate target DNA genes), the process is referred to as shotgun cloning and can yield thousands or tens of thousands of clones to be screened.
Introducing recombinant DNA into cells other than bacteria
Agrobacterium tumefaciens and the Ti plasmid
Tumor-Inducing Plasmid.
Agrobacterium tumefaciens is a plant pathogen that causes tumor formation called crown gall disease. The bacterium contains a plasmid known as the Ti (tumor inducing) plasmid, which inserts bacterial DNA into the host plant genome. Scientists utilize this natural process to do genetic engineering of plants by inserting foreign DNA into the Ti plasmid and removing the genes necessary for disease, allowing for the production of transgenic plants.
Gene gun
A gene gun uses very small metal particles (microprojectiles) coated with the recombinant DNA, which are blasted at plant or animal tissue at a high velocity. If the DNA is transformed or taken up by the cell’s DNA, the genes are expressed.
Viral vectors
For a viral vector, virulence genes from a virus can be removed and foreign DNA inserted, allowing the virus capsid to be used as a mechanism for shuttling genetic material into a plant or animal cell. Marker genes are typically added that allow for identification of the cells that took up the genes.
DNA techniques
Gel Electrophoresis
Gel electrophoresis is a technique commonly used to separate nucleic acid fragments based on size. It can be used to identify particular fragments or to verify that a technique was successful.
A porous gel is prepared made of agarose, with the concentration adjusted based on expected size. Nucleic acid samples are deposited into wells in the gel and an electrical current is applied. Nucleic acid, with its negative charge, will move towards the positive electrode, which should be placed at the bottom of the gel. The nucleic acid will move through the gel, with the smallest pieces encountering the least resistance and thus moving through the fastest. The length of passage of each nucleic acid fragment can be compared to a DNA ladder, with fragments of known size.
Polymerase Chain Reaction (PCR)
The polymerase chain reaction or PCR is a method used to copy or amplify DNA in vitro. The process can yield a billionfold copies of a single gene within a short period of time. The template DNA is mixed with all the ingredients necessary to make DNA copies: primers (small oligonucleotides that flank the gene or genes of interest by recognizing sequences on either side of it), nucleotides (the building blocks of DNA), and DNA polymerase. The steps involve heating the template DNA in order to denature or separate the strands, dropping the temperature to allow the primers to anneal, and then heating the mixture up to allow the DNA polymerase to extend the primers, using the original DNA as an initial template. The cycle is repeated 20-30 times, exponentially increasing the amount of target DNA in a few hours.
Polymerase Chain Reaction (PCR). By Enzoklop (Own work) [CC BY-SA 3.0], via Wikimedia Commons
Uses of Genetically Engineered Organisms
There can be numerous reasons to create a genetically modified organism (GMO) or transgenic organism, defined as a genetically modified organism that contains a gene from a different organism. Typically the hope is that the GMO will provide needed information or a product of value to society.
Source of DNA
Genetically engineered organisms can be made so that a piece of DNA can be easily replicated, providing a large source of that DNA. For example, a gene associated with breast cancer can be spliced into the genome of E. coli, allowing for the rapid production of the gene so that it may be sequenced, studied, and manipulated, without requiring repeated tissue donations from human volunteers.
Source of RNA
Antisense RNA is ssRNA that is complementary to the mRNA that will code for a protein. In cells it is made as a way to control target genes. There has been increasing interest in the use of antisense RNA as a way to prevent diseases that are caused by the production of a particular protein.
Antisense RNA. By Robinson R [CC BY 2.5], via Wikimedia Commons
Source of Protein
Since microbes replicate so rapidly, it can be extremely advantageous to use them to manufacture proteins of interest or value. Given the right promoters, bacteria will express genes for proteins that are not naturally found in bacteria, such as cytokine. Genetically engineered cells have been used to make a wide variety of proteins of use to humans, such as insulin or human growth hormone.
Key Words
genetic engineering, cloning, target DNA, green fluorescent protein (GFP), cloning vector, restriction endonuclease, sticky ends, DNA ligase, recombinant DNA, chimera, transformation, electroporation, genomic library, shotgun cloning, Agrobacterium tumefaciens, Ti plasmid, gene gun, microprojectiles, viral vector, gel electrophoresis, DNA ladder, polymerase chain reaction (PCR), template DNA, primer, nucleotide, DNA polymerase, denaturing, annealing, extending, genetically modified organism (GMO), transgenic organisms, antisense RNA.
Study Questions
1. Define genetic engineering.
2. Identify and describe the basic steps used in the genetic engineering of a bacterial cell. What components are needed and why?
3. Summarize the different ways that recombinant DNA can be inserted into a cell or organism. Be able to provide specific examples.
4. Describe techniques used in the manipulation of DNA. What are the essential components of each process?
5. Explain the different applications of genetic engineering. | textbooks/bio/Microbiology/Microbiology_(Bruslind)/18%3A_Genetic_Engineering.txt |
Genomics is a field that studies the entire collection of an organism’s DNA or genome. It involves sequencing, analyzing, and comparing the information contained within genomes. Since sequencing has become much less expensive and more efficient, vast amounts of genomic information is now available about a wide variety of organisms, but particularly microbes, with their smaller genome size. In fact, the biggest bottleneck currently is not the lack of information but the lack of computing power to process the information!
Sequencing
Sequencing, or determining the base order of an organism’s DNA or RNA, is often one of the first steps to finding out detailed information about an organism. A bacterial genome can range from 130 kilobase pairs (kbp) to over 14 Megabase pairs (Mbp), while a viral genome ranges from 0.859 to 2473 kbp. For comparison, the human genome contains about 3 billion base pairs.
Shotgun sequencing
Shotgun sequencing initially involves construction of a genomic library, where the genome is broken into randomly sized fragments that are inserted into vectors to produce a library of clones. The fragments are sequenced and then analyzed by a computer, which searches for overlapping regions to form a longer stretch of sequence. Eventually all the sequences are aligned to give the complete genome sequence. Errors are reduced because many of the clones contain identical or near identical sequences, resulting in good “coverage” of the genome.
Shotgun Sequencing. By Commins, J., Toft, C., Fares, M. A. [CC BY-SA 2.5], via Wikimedia Commons
Second generation DNA sequencing
Second-generation DNA sequencing uses massively parallel methods, where multiple samples are sequenced side-by-side. DNA fragments of a few hundred bases each are amplified by PCR and then attached to small bead, so that each bead carries several copies of the same section of DNA. The beads are put into a plate containing more than a million wells, each with one bead, and the DNA fragments are sequenced.
Third- and fourth-generation DNA sequencing
Third-generation DNA sequencing involves the sequencing of single molecules of DNA. Fourth-generation DNA sequencing, also known as “post light sequencing,” utilizes methods other than optical detection for sequencing.
Bioinformatics
After sequencing, it is time to make sense of the information. The field of bioinformatics combines many fields together (i.e. biology, computer science, statistics) to use the power of computers to analyze information contained in the genomic sequence. Locating specific genes within a genome is referred to as genome annotation.
Open Reading Frames (ORFS)
An open reading frame or ORF denotes a possible protein-coding gene. For double-stranded DNA, there are six reading frames to be analyzed, since the DNA is read in sets of three bases at a time and there are two strands of DNA. An ORF typically has at least 100 codons before a stop codon, with 3’ terminator sequences. A functional ORF is one that is actually used by the organism to encode a protein. Computers are used to search the DNA sequence looking for ORFs, with those presumed to encode protein further analyzed by a bioinformaticist.
It is often helpful for the sequence to be compared against a database of sequences coding for known proteins. GenBank is a database of over 200 billion base pairs of sequences that scientists can access, to try and find matches to the sequence of interest. The database search tool BLAST (basic local alignment search tool) has programs for comparing both nucleotide sequences and amino acid sequences, providing a ranking of results in order of decreasing similarity.
BLAST Results.
Comparative Genomics
Once the sequences of organisms have been obtained, meaningful information can be gathered using comparative genomics. For this genomes are assessed for information regarding size, organization, and gene content.
Comparison of the genome of microbial strains has given scientists a better picture regarding the genes that organisms pick up. A group of multiple strains share a core genome, genes coding for essential cellular functions that they all have in common. The pan genome represents all the genes found in all the members of species, so provides a good idea of the diversity of a group. Most of these “extra” genes are probably picked up by horizontal gene transfer.
Comparative genomics also shows that many genes are derived as a result of gene duplication. Genes within a single organism that likely came about because of gene duplication are referred to as paralogs. In many cases one of the genes might be altered to take on a new function. It is also possible for gene duplication to be found in different organisms, as a result of acquiring the original gene from a common ancestor. These genes are called orthologs.
Functional Genomics
The sequence of a genome and the location of genes provide part of the picture, but in order to fully understand an organism we need an idea of what the cell is doing with its genes. In other words, what happens when the genes are expressed? This is where functional genomics comes in – placing the genomic information in context.
The first step in gene expression is transcription or the manufacture of RNA. Transcriptome refers to the entire complement of RNA that a cell can make from its genome, while proteome refers to all the proteins encoded by an organisms’ genome, in the final step of gene expression.
Microarrays
Microarrays or gene chips are solid supports upon which multiple spots of DNA are placed, in a grid-like fashion. Each spot of DNA represents a single gene or ORF. Known fragments of nucleic acid are labeled and used as probes, with a signal produced if binding occurs. Microarrays can be used to determine what genes might be turned on or off under particular conditions, such as comparing the growth of a bacterial pathogen inside the host versus outside of the host.
Proteomics
The study of the proteins of an organism (or the proteome) is referred to as proteomics. Much of the interest focuses on functional proteomics, which examines the functions of the cellular proteins and the ways in which they interact with one another.
One common technique used in the study of proteins is two-dimensional gel electrophoresis, which first separates proteins based on their isoelectric points. This is accomplished by using a pH gradient, which separates the proteins based on their amino acid content. The separated proteins are then run through a polyacrylamide gel, providing the second dimension as proteins are separated by size.
Structural proteomics focuses on the three-dimensional structure of proteins, which is often determined by protein modeling, using computer algorithms to predict the most likely folding of the protein based on amino acid information and known protein patterns.
Metabolomics
Metabolomics strives to identify the complete set of metabolic intermediates produced by an organism. This can be extremely complicated, since many metabolites are used by cells in multiple pathways.
Metagenomics
Metagenomics or environmental genomics refers to the extraction of pooled DNA directly from a specific environment, without the initial isolation and identification of organisms within that environment. Since many microbial species are difficult to culture in the laboratory, studying the metagenome of an environment allows scientists to consider all organisms that might be present. Taxa can even be identified in the absence of organism isolation using nucleic acid sequences alone, where the taxon is known as phylotype.
Key Words
genomics, sequencing, shotgun sequencing, genomic library, second generation DNA sequencing, massively parallel methods, third- and fourth-generation DNA sequencing, bioinformatics, genome annotation, open reading frame/ORF, functional ORF, GenBank, BLAST/basic local alignment search tool, comparative genomics, core genome, pan genome, paralog, ortholog, functional genomics, transcriptome, proteome, microarray/gene chips, probe, proteomics, functional proteomics, two-dimensional gel electrophoresis, structural proteomics, metabolomics, metagenomics/environmental genomics, metagenome, phylotype.
Study Questions
1. What does the field of genomics encompass?
2. What is shotgun sequencing and how does this allow for the complete sequencing of an organism’s genome?
3. What are the basic differences among 2nd, 3rd, and 4th generation sequencing?
4. What is an open reading frame and how can scientists use it to determine information about a genome and its products?
5. How does functional genomics differ from comparative genomics? What are the tools used in functional genomics and what information can be obtained from each? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/19%3A_Genomics.txt |
Symbiosis, strictly defined, refers to an intimate relationship between two organisms. Although many people use the term to describe a relationship beneficial to both participants, the term itself is not that specific. The relationship could be good, bad, or neutral for either partner. A mutualistic relationship is one in which both partners benefit, while a commensalistic relationship benefits one partner but not the other. In a pathogenic relationship, one partner benefits at the expense of the other. This chapter looks at a few examples of symbiosis, where microbes are one of the partners.
The Human Microbiome
The human microbiome describes the genes associated with all the microbes that live in and on a human. All 10^14 of them! The microbes are mostly bacteria but can include archaea, fungi, and eukartyotic microbes The locations include skin, upper respiratory tract, stomach, intestines, and urogenital tracts. Colonization occurs soon after birth, as infants acquire microbes from people, surfaces and objects that they come in contact with.
Gut microbes and human metabolism
Most of the microbes associated with the human body are found in the gut, particularly about 1-4 hours after eating a meal when the microbial population dramatically increases. The gut microbiota is extremely diverse and it has been estimated that from 500-1000 species of bacteria live in the human gastrointestinal tract (typically described as from 5-8 pounds of bacteria!).
The gut microbes are essential for host digestion and nutrition, aiding in digestion by breaking down carbohydrates that humans could not break down on their own, by liberating short chain fatty acids from indigestible dietary fibers. In addition, they produce vitamins such as biotin and vitamin K.
Gut microbes and obesity
There has been increased interest in the microbial gut population, due to the possibility that it might play a role in obesity. Although currently hypothetical, research has shown that obese mice have a microbial gut community that differs from the microbes found in the gut of non-obese mice, with more Firmicutes bacteria and methanogenic Archaea. It has been suggested that these microbes are more efficient at absorbing nutrients.
Human microbiome and disease
It has been shown that microbiota changes are associated with diseased states or dysbiosis. Preliminary research has shown that the microbiota might be associated rheumatoid arthritis, colorectal cancer, diabetes, in addition to obesity.
Research
The Human Microbiome Project (HMP) was an international research program based in the U.S. that was focused on the functions of gut microbiota. Some 200 researchers used advanced DNA-sequencing techniques to determine what microbes are present and in what populations.
Many current research projects are focused on determining the role of the human microbiome in both health and the diseased state. There is no doubt that our knowledge will continue to grow as we find out more about the vast populations of microbes that live in and on us.
Biofilms
Biofilms are a complex aggregation of cells that are encased within an excellular matrix and attached to a surface. Bioforms can form on just about any surface and are common in nature and industry, being found on the surfaces of rocks, caves, pipes, boat hulls, cooking vessels, and medical implants, just to name a few. They have also been around a long time, since the fossil record shows evidence for biofilms going back 3.4 billion years!
The microbial community of a biofilm can be composed of one or two species, but more commonly contains many different species of bacteria, each influencing the others gene expression and growth.
Biofilm development
The basic steps for biofilm formation can be broken down into four steps:
1. Cell disposition and attachment – in order for biofilm development to occur, free-floating or planktonic cells must collide with a suitable surface. Typically the surface has been preconditioned with the deposits of environmental proteins and other molecules.
2. Colonization – cell-to-cell signaling occurs, leading to the expression of biofilm specific genes. These genes are associated with the communal production of extracellular polymeric DNA released by some cells can be taken up by others, stimulating the expression of new genes.
3. Maturation – the EPS matrix fully encases all the cells, as the biofilm continues to thicken and grow, forming a complex, dynamic community. Water channels form throughout the structure.
4. Detachment and sloughing – individual cells or pieces of the biofilm are released to the environment, as a form of active dispersal. This release can be trigger by environmental factors, such as the concentration of nutrients or oxygen.
Biofilm Development. Each stage of development in the diagram is paired with a photomicrograph of a developing Pseudomonas aeruginosa biofilm. All photomicrographs are shown to same scale. By D. Davis [CC BY 2.5], via Wikimedia Commons
Cellular advantages of biofilms
Why do bioforms develop? There are certain advantages that cells enjoy while in a biofilm, over their planktonic growth. Perhaps most importantly, biofilms offer cells increased protection from harmful conditions or substances, such as UV light, physical agitation, antimicrobial agents, and phagocytosis. It has been shown that bacteria within a biofilm are up to a thousand times more resistant to antibiotics than free-floating cells!
A biofilm also allows a cell population to “put down roots,” so to speak, so that they can stay in close proximity to a nutrient-rich area. For example, a biofilm that develops on a conduit pipe at a dairy plant will have continual access to fresh food, which is much better than being swept away with the final product.
Lastly, biofilms allow for cells to grow in microbial populations, where they can easily benefit from cell-to-cell communication and genetic exchange.
Biofilm impacts
Biofilms have huge impacts throughout many different types of industry. Medical implants ranging from catheters to artificial joints are particularly susceptible to biofilm formation, leading to huge problems for the medical industry. Biofilms are responsible for many chronic infections, due to their increased resistance to antimicrobial compounds and antibiotics. A type of biofilm that affects almost everyone is the formation of dental plaque, which can lead to cavity formation.
Outside of medicine, biofilms affect just about any industry relying on pipes to convey water, food, oil, or other liquids, where their resistance makes its particularly difficult to completely eliminate the biofilm.
Quorum Sensing
The word quorum refers to having a minimum number of members needed for an organization to conduct business, such as hold a vote. Quorum sensing refers to the ability of some bacteria to communicate in a density-dependent fashion, allowing them to delay the activation of specific genes until it is the most advantageous for the population.
Quorum sensing involves cell-to-cell communication, using small diffusible substances known as autoinducers. An autoinducer is produced by a cell, diffusing across the plasma membrane to be released into the environment. As the cell population increases in the environment the concentration of autoinducer increases as well, causing the molecule to diffuse back into individual cells where it triggers the activation of specific genes.
Quorum sensing in Bioluminescent Bacteria.
Quorum sensing example
One of the best studied examples of quorum sensing is the mutualistic relationship between the bioluminescent bacterium Aliivibrio fischeriand the bobtail squid. The bobtail squid actually has a light organ that evolved to house the bacterium, relying on its luminescence to provide a camouflage effect against predators. At low cell density the luminescence would not provide the desired effect, representing a waste in energy by the bacterial population. Therefore, quorum sensing is used so that the lux gene that codes for the luciferase enzyme necessary for luminescence is only activated when the bacterial population is at sufficient density.
Key Words
symbiosis, mutualistic, commensalistic, pathogenic relationship, human microbiome, dysbiosis, Human Microbiome Project (HMP), biofilm, planktonic, extracellular polymeric substances/EPS, quorum sensing, autoinducer.
Essential Questions/Objectives
1. What is a symbiosis? What are different specific examples of symbioses?
2. How is the human microbiome defined? What microbes does it include?
3. What impact does or can the human microbiome have on its host?
4. What are biofilms? Where do they form?
5. What are the steps leading to biofilm formation?
6. How do cells benefit from existing in a biofilm? What are the impacts of biofilms to humans?
7. What is quorum sensing? How does the mechanism work?
8. Describe the example of Aliivibrio fischeri and the bobtail squid. What role does quorum sensing play in the relationship between these two organisms? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/20%3A_Microbial_Symbioses.txt |
A microbe that is capable of causing disease is referred to as a pathogen, while the organism being infected is called a host. The ability to cause disease is referred to as pathogenicity, with pathogens varying in their ability. An opportunistic pathogen is a microbe that typically infects a host that is compromised in some way, either by a weakened immune system or breach to the body’s natural defenses, such as a wound. The measurement of pathogenicity is called virulence, with highly virulent pathogens being more likely to cause disease in a host.
It is important to remember that there are many variables to take into account in a host-pathogen interaction, which is a dynamic relationship that is constantly changing. The virulence of the pathogen is important, but so is the number of microbes that gained entry to the host, the location of entry, the overall health of the host, and the state of the host’s defenses. Exposure to a pathogen does not ensure that disease will occur, since a host might be able to fight off the infection before disease signs/symptoms develop.
Host-Pathogen Interactions.
Pathogen Transmission
An infection starts with exposure to a pathogen. The natural site or home for a pathogen is known as a reservoir and can either be animate (human or animal) or inanimate (water, soil, food). A pathogen can be picked up from its reservoir and then spread from one infected host to another. Carriers play an important role in the spread of disease, since they carry the pathogen but show no obvious symptoms of disease. A disease that primarily occurs within animal populations but can be spread to humans is called a zoonosis, while a hospital-acquired infection is known as a nosocomial infection.
The mechanism by which a pathogen is picked up by a host is referred to as mode of transmission, with the main mechanisms listed below:
Direct contact
Direct contact includes host-to-host contact, such as through kissing or sexual intercourse, where one person might come in contact with another person’s skin or body fluids. An expectant mother may transmit a pathogen to her infant by vertical contact while pregnant, or during the act of giving birth.
Droplet transmission
Droplet transmission is often considered to be a form of direct contact as well. It involves transmission by respiratory droplets, where an infected host expels the pathogen in tiny droplets by coughing or sneezing, which are then inhaled by a host nearby. These droplets are not transmitted through the air over long distances, nor do they remain infectious for very long.
Indirect contact
Indirect contact involves the transfer of the infectious agent through some type of intermediary, such as a contaminated object or person. The pathogen might be deposited on an inanimate object, called a fomite, which is then used by another person. This could include a shared toy or commonly-touched surface, like a doorknob or computer keyboard. Alternatively, a healthcare worked might transmit a pathogen from one patient to another, if they did not change their gloves between patients.
Airborne transmission
Airborne transmission occurs due to pathogens that are in small particles or droplets in the environment, which can remain infectious over time and distance. An example might be fungal spores that are inhaled during a dust storm.
Fecal-oral transmission
Fecal-oral transmission occurs when an infected host is shedding the pathogen in their feces which contaminate food or water that is consumed by the next host.
Vectorborne transmission
Vectorborne transmission occurs when an arthropod vector, such as mosquitoes, flies, ticks, are involves in the transmission. Sometimes the vector just picks up the infectious agents on their external body parts and carries it to another host, but typically the vector picks up the infectious agent when biting an infected host. The agent is picked up in the blood, and then spread to the next host when the vector moves on to bite someone else.
Pathogen Transmission.
Virulence Factors
In order for a bacterium to be virulent, it must have capabilities that allow it to infect a host. These capabilities arise from physical structures that the bacterium has or chemical substances that the bacterium can produce. Collectively the characteristics that contribute to virulence are called virulence factors. The genes that code for virulence factors are commonly found clustered on the pathogen’s chromosome or plasmid DNA, called pathogenicity islands. These pathogenicity islands can be distinguished by a G+C content that differs from the rest of the genome and the presence of insertion-like sequences flanking the gene cluster. Pathogenicity islands facilitate the sharing of virulence factors between bacteria due to horizontal gene transfer, leading to the development of new pathogens over time.
Often the genes for virulence factors are controlled by quorum sensing, to ensure gene activation when the pathogen population is at an optimal density. Triggering the genes too soon could alert the host’s immune system to the invader, cutting short the bacterial infection.
Adherence and Colonization
Bacterial pathogens must be able to grab onto host cells or tissue, and resist removal by physical means (such as sneezing) or mechanical means (such as movement of the cilated cells that line our airway). Adherence can involve polysaccharide layers made by the bacteria, such as a capsule or slime layer, which provide adhesion to host cells as well as resistance from phagocytosis. Adherence can also be accomplished by physical structures such as a pilus or flagellum.
Once cells are successfully adhering to a surface, they increase in number, utilizing resources available at the site. This colonization is important for pathogen survival and invasion to other sites, which will yield increased nutrients and space for the growing population.
Invasion
Invasion refers to the ability of the pathogen to spread to other locations in the host, by invading host cells or tissue. It is typically at this point when disease or obvious signs/symptoms of illness will occur. While physical structures can still play a role in invasion, most bacterial pathogens produce a wide array of chemicals, specifically enzymes that effect the host’s cells and tissue. Enzymes such as collagenase, which allows the pathogen to spread by breaking down the collagen found in connective tissue. Or leukocidins, which destroy the host’s white blood cells, decreasing resistance. Hemolysins lyse the host’s red blood cells, releasing iron, a growth-limiting factor for bacteria.
Bacteria in the bloodstream, a condition known as bacteremia, can quickly spread to locations throughout the host. This can result in a massive, systemic infection known as septicemia, which can result in septic shock and death, as the host becomes overwhelmed by the bacterial pathogen and its products.
Toxins
Toxins are a very specific virulence factor produced by some bacterial pathogens, in the form of substances that are poisonous to the host. Toxigenicity refers to an organism’s ability to make toxins. For bacteria, there are two categories of toxins, the exotoxins and the endotoxins.
Exotoxins
Exotoxins are heat-sensitive soluble proteins that are released into the surrounding environment by a living organism. These incredibly potent substances can spread throughout the host’s body, causing damage distant from the original site of infection. Exotoxins are associated with specific diseases, with the toxin genes often carried on plasmids or by prophages. There are many different bacteria that produce exotoxins, causing diseases such as botulism, tetanus, and diphtheria. There are three categories of exotoxins:
1. Type I: cell surface-active – these toxins bind to cell receptors and stimulate cell responses. One example is superantigen, that stimulates the host’s T cells, an important component of the immune system. The stimulated T cells produce an excessive amount of the signaling molecule cytokine, causing massive inflammation and tissue damage.
2. Type II: membrane-damaging – these toxins exert their effect on the host cell membrane, often by forming pores in the membrane of the target cell. This can lead to cell lysis as cytoplasmic contents rush out and water rushes in, disrupting the osmotic balance of the cell.
3. Type III: intracellular – these toxins gain access to a particular host cell and stimulate a reaction within the target cell. One example is the AB-toxin – these toxins are composed of two subunits, an A portion and a B portion. The B subunit is the binding portion of the toxin, responsible for recognizing and binding to the correct cell type. The A subunit is the portion with enzymatic activity. Once delivered into the correct cell by the B subunit, the A subunit enacts some mechanism on the cell, leading to decreased cell function and/or cell death. An example is the tetanus toxin produced by the bacterium Clostridrium tetani. Once delivered to a neuron, the A subunit will cleave the cellular synaptobreven, resulting in a decrease in neurotransmitter release. This results in spastic paralysis of the host. Each AB-toxin is associated with a different disease.
AB-toxin: Host Cell Binding.
Endotoxins
Endotoxins are made by gram negative bacteria, as a component of the outer membrane of their cell wall. The outer membrane contains lipopolysaccharide or LPS, with the toxic component being the lipid part known as lipid A. Lipid A is heat-stable and is only released when the bacterial cell is lysed. The effect on the host is the same, regardless of what bacterium made the lipid A – fever, diarrhea, weakness, and blood coagulation. A massive release of endotoxin in a host can cause endotoxin shock, which can be deadly.
Key Words
pathogen, host, pathogenicity, opportunistic pathogen, virulence, reservoir, carrier, nosocomial infection, mode of transmission, direct contact, vertical contact, droplet transmission, indirect contact, fomite, airborne transmission, fecal-oral transmission, vectorborne transmission, virulence factor, pathogenicity island, adherence, colonization, invasion, bacteremia, septicemia, toxin, toxigenicity, exotoxin, Type I/cell surface-active toxin, superantigen, T cell, cytokine, Type II/membrane-damaging toxin, Type III/intracellular toxin, AB toxin, endotoxin, lipid A, endotoxin shock.
Study Questions
1. What are the different terms associated with bacterial pathogenesis? How do they differ and what to they refer to?
2. What are the components that play a role in the host-pathogen interaction?
3. What terms are associated with pathogen transmission? What are the different ways in which pathogens can be transmitted? What is involved for each mode of transmission?
4. What are virulence factors? What role does pathogenicity islands play in the dispersal of virulence factors?
5. Why are adherence and colonization important to a bacterial pathogen? How does invasion differ?
6. What types of toxins are made by bacterial pathogens? What characteristics do they have? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/21%3A_Bacterial_Pathogenicity.txt |
Viral Classification
Since viruses lack ribosomes (and thus rRNA), they cannot be classified within the Three Domain Classification scheme with cellular organisms. Alternatively, Dr. David Baltimore derived a viral classification scheme, one that focuses on the relationship between a viral genome to how it produces its mRNA. The Baltimore Scheme recognizes seven classes of viruses.
DNA viruses
Class I: dsDNA
DNA viruses with a dsDNA genome, like bacteriophages T4 and lambda, have a genome exactly the same as the host cell that they are infecting. For this reason, many host enzymes can be utilized for replication and/or protein production. The flow of information follows a conventional pathway: dsDNA → mRNA → protein, with a DNA-dependent RNA-polymerase producing the mRNA and the host ribosome producing the protein. The genome replication, dsDNA → dsDNA, requires a DNA-dependent DNA-polymerase from either the virus or the host cell.
dsDNA.
The virus often employs strategies for control of gene expression, to insure that particular viral products are made at specific times in the virus replication. In the case of T4, the host RNA polymerase binds to the viral DNA and begins transcribing early genes immediately after the DNA is injected into the cell. One of the early viral proteins modifies the host RNA polymerase so that it will no longer recognize host promoters at all, in addition to moving on to transcribe genes for middle-stage viral proteins. A further modification (catalyzed by middle-stage viral proteins) further modified the RNA polymerase so it will recognize viral genes coding for late-stage proteins. This insures an orderly production of viral proteins.
The replication of several dsDNA viruses results in the production of concatemers, where several viral genomes are linked together due to short single-stranded regions with terminal repeats. As the genome is packaged into the capsid a viral endonuclease cuts the concatemer to an appropriate length.
There are several animal viruses with dsDNA genomes, such as the pox viruses and the adenoviruses. The herpesviruses have several notable features, such as the link of several members with cancer and the ability of the viruses to remain in a latent form within their host. A productive infection results in an explosive viral population, cell death, and development of disease signs, during which neurons are infected. A latent infection develops in the neurons, allowing the virus to remain undetected in the host. If the viral genome is reactivated, a productive infection results, leading to viral replication and disease signs again.
Class II: ssDNA
The flow of information for ssDNA viruses, such as the parvoviruses, will still follow the conventional pathway, to a certain extent: DNA → mRNA → protein. But the viral genome can either have the same base sequence as the mRNA (plus-strand DNA) or be complementary to the mRNA (minus-strand DNA). In the former case, a DNA strand that is complementary to the viral genome must be manufactured first, forming a double-stranded replicative form (RF). This can be used to both manufacture viral proteins and as a template for viral genome copies. For the minus-strand DNA viruses, the genome can be used directly to produce mRNA but a complementary copy will still need to be made, to serve as a template for viral genome copies.
ssDNA.
The replicative form can be used for rolling-circle replication, where one strand is nicked and replication enzymes are used to extend the free 3’ end. As a complementary strand is synthesized around the circular DNA, the 5’ end is peeled off, leading to a displaced strand that continues to grow in length.
Rolling-Circle Replication.
Class VII: DNA viruses that use reverse transcriptase
The hepadnaviruses contain a DNA genome that is partially double-stranded, but contains a single-stranded region. After gaining entrance into the cell’s nucleus, host cell enzymes are used to fill in the gap with complementary bases to form a dsDNA closed loop. Gene transcription yields a plus-strand RNA known as the pregenome, as well as the viral enzyme reverse transcriptase, an RNA-dependent DNA-polymerase. The pregenome is used as a template for the reverse transcriptase to produced minus-strand DNA genomes, with a small piece of pregenome used as a primer to produce the double-stranded region of the genomes.
RNA viruses
Class III: dsRNA
Double-stranded RNA viruses infect bacteria, fungi, plants, and animals, such as the rotavirus that causes diarrheal illness in humans. But cells do not utilize dsRNA in any of their processes and have systems in place to destroy any dsRNA found in the cell. Thus the viral genome, in its dsRNA form, must be hidden or protected from the cell enzymes. Cells also lack RNA-dependent RNA-polymerases, necessary for replication of the viral genome so the virus must provide this enzyme itself. The viral RNA-dependent RNA polymerase acts as both a transcriptase to transcribe mRNA, as well as a replicase to replicate the RNA genome.
dsRNA.
For the rotavirus, the viral nucleocapsid remains intact in the cytoplasm with replication events occurring inside, allowing the dsRNA to remain protected. Messenger RNA is transcribed from the minus-strand of the RNA genome and then translated by the host ribosome in the cytoplasm. Viral proteins aggregate to form new nucleocapsids around RNA replicase and plus-strand RNA. The minus-strand RNA is then synthesized by the RNA replicase within the nucleocapsid, once again insuring protection of the dsRNA genome.
Class IV: +ssRNA
Viruses with plus-strand RNA, such as poliovirus, can use their genome directly as mRNA with translation by the host ribosome occurring as soon as the unsegmented viral genome gains entry into the cell. One of the viral genes expressed yields an RNA-dependent RNA-polymerase (or RNA replicase), which creates minus-strand RNA from the plus-strand genome. The minus-strand RNA can be used as a template for more plus-strand RNA, which can be used as mRNA or as genomes for the newly forming viruses.
+ssRNA.
Translation of the poliovirus genome yields a polyprotein, a large protein with protease activity that cleaves itself into three smaller proteins. Additional cleavage activity eventually yields all the proteins needed for capsid formation, as well as an RNA-dependent RNA-polymerase.
The formation of a polyprotein that is cut into several smaller proteins illustrates one possible strategy to an issue faced by many +ssRNA viruses – how to generate multiple proteins from an unsegmented +ssRNA genome? Other possibilities include:
• subgenomic mRNA – during translation, portions of the viral RNA may be skipped, resulting in different proteins than what is made from the viral RNA in its entirety.
• ribosomal frame-shifting – the ribosome “reads” the mRNA in groups of three nucleotides or codon, which translate to one amino acid. If the ribosome starts with nucleotide #1, that is one open reading frame (ORF), resulting in one set of amino acids. If the ribosome were to move forward where nucleotide 2 is the starting nucleotide that would be ORF #2, resulting in a completely different set of amino acids. If the ribosome were to move forward again where nucleotide 3 is the starting nucleotide that would be ORF#3, resulting in an entirely different set of amino acids. Some viruses have viral genes that deliberately overlap within different ORFs, leading to the production of different proteins from a single mRNA.
• readthrough mechanism – a viral genome can have stop codons embedded throughout the sequence. When the ribosome comes to a stop codon it can either stop, ending the amino acid sequence, or it can ignore the stop codon, continuing on to make a longer string of amino acids. For viruses with the readthrough mechanism, they acquire a variety of proteins by having stop codons that are periodically ignored. Sometimes this function is combined with the ribosomal frame-shifting to produce an even greater variety of viral proteins.
Class V: -ssRNA
Minus-strand RNA viruses include many members notable for humans, such as influenza virus, rabies virus, and Ebola virus. Since the genome of minus-strand RNA viruses cannot be used directly as mRNA, the virus must carry an RNA-dependent RNA-polymerase within its capsid. Upon entrance into the host cell, the plus-strand RNAs generated by the polymerase are used as mRNA for protein production. When viral genomes are needed the plus-strand RNAs are used as templates to make minus-strand RNA.
-ssRNA.
Class VI: +ssRNA, retroviruses
Despite the fact that the retroviral genome is composed of +ssRNA, it is not used as mRNA. Instead, the virus uses its reverse transcriptase to synthesize a piece of ssDNA complementary to the viral genome. The reverse transcriptase also possesses ribonuclease activity, which is used to degrade the RNA strand of the RNA-DNA hybrid. Lastly, the reverse transcriptase is used as a DNA polymerase to make a complementary copy to the ssDNA, yielding a dsDNA molecule. This allows the virus to insert its genome, in a dsDNA form, into the host chromosome, forming a provirus. Unlike a prophage, a provirus can remain latent indefinitely or cause the expression of viral genes, leading to the production of new viruses. Excision of the provirus does not occur for gene expression.
+ssRNA, retroviruses.
Other Infectious Agents
Viroids
Viroids are small, circular ssRNA molecules that lack protein. These infectious molecules are associated with a number of plant diseases. Since ssRNA is highly susceptible to enzymatic degradation, the viroid RNA has extensive complementary base pairing, causing the viroid to take on a hairpin configuration that is resistant to enzymes. For replication viroids rely on a plant RNA polymerase with RNA replicase activity.
Prions
Prions are infectious agents that completely lack nucleic acid of any kind, being made entirely of protein. They are associated with a variety of diseases, primarily in animals, although a prion has been found that infects yeast (!). Diseases include bovine spongiform encephalopathy (BSE or “mad cow disease”), Creutzfeld-Jakob disease in humans, and scrapie in sheep.
The prion protein is found in the neurons of healthy animals (PrPC or Prion Protein Cellular), with a particular secondary structure. The pathogenic form (PrPSC or Prion Protein Scrapie) has a different secondary structure and is capable of converting the PrPC into the pathogenic form. Accumulation of the pathogenic form causes destruction of brain and nervous tissue, leading to disease symptoms such as memory loss, lack of coordination, and eventually death.
Prions. Joannamasel at English Wikipedia [CC BY-SA 3.0], via Wikimedia Commons
Key Words
Baltimore Scheme, Class I, Class II, Class III, Class IV, Class V, Class VI, Class VII, DNA-dependent RNA polymerase, DNA-dependent DNA-polymerase, concatemer, productive infection, latent infection, plus-strand DNA/+DNA, minus-strand DNA/-DNA, dsDNA, ssDNA, replicative form (RF), rolling-circle replication, pregenome, reverse transcriptase, RNA-dependent DNA-polymerase, dsRNA, RNA-dependent RNA-polymerase, transcriptase, replicase, plus-strand RNA/+ssRNA, minus-strand RNA/-ssRNA, polyprotein, subgenomic mRNA, ribosomal frame-shifting, open reading frame (ORF), readthrough mechanism, stop codon, retrovirus, ribonuclease, provirus, viroid, prion, PrPC/Prion Protein Cellular, PrPSC/Prion Protein Scrapie.
Essential Questions/Objectives
1. What is the Baltimore system of classification? What viral characteristics does it use?How does each viral group make proteins and replicate their genome? Where do the necessary components come from? (virus or host cell) What modifications are necessary, for viruses with a genome different from the host cell?
2. What strategy do dsDNA viruses use for control of gene expression? What are concatemers? What are productive and latent infections?
3. What is a replicative form? What is rolling circle replication? What is the advantage of these viral mechanisms?
4. What is a pregenome? What is reverse transcriptase? What role does it play for the Class VII viruses?
5. What issues do dsRNA viruses face? How do they overcome these issues? What is a transcriptase? What is a replicase?
6. How is the genome used by Class IV +ssRNA viruses? What are the strategies used by these viruses to generate multiple proteins from an unsegmented genome?
7. What steps are necessary for the –ssRNA viruses?
8. How do the retroviruses, as +ssRNA viruses, differ from the Class IV viruses? What is a ribonuclease? What is a provirus?
9. What is a viroid? What is a prion? How do these agents cause disease? How do they replicate?
Exploratory Questions (OPTIONAL)
1. Why were scientists initially so resistant to the idea of prions lacking any type of nucleic acid? | textbooks/bio/Microbiology/Microbiology_(Bruslind)/22%3A_The_Viruses.txt |
Organism
• Clostridium tetani is a moderately-sized Gram-positive, endospore-producing bacillus.
• Motile with a peritrichous arrangement of flagella.
• Produce round, terminal endospores that give the bacterium a "tennis-racquet" appearance.
• An obligate anaerobe(def).
Habitat
• Colonizes the intestinal tract in humans and animals.
Source
• Endospores found in fertile soil or feces.
Epidemiology
• Endospores are found in most soils and in the intestinal tract of many animals and humans.
• Although exposure to endospores is commom, disease is uncommon except in countries with poor medical care and vaccination compliance.
• Fewer than 50 cases per year in the U.S.; most in elderly individuals with waning immunity.
• It is estimated that there is more than one million cases a year worldwide, with a mortality rate of 20% to 50%.
• Most deaths occur in neonates and originates from infection of umbilical stumps in mothers that have no immunity.
Clinical Disease
• Generalized tetanus is most common. Typical presenting symptoms include lockjaw and sardonic smile, arrising as a result of spastic paralysis of the masseter muscles and other facial muscles. Difficulty in swallowing, drooling, irritability, and persistent back spasms are other early symptoms. When the autonomic nervous system is involved, symptoms include perfuse sweating, hyperthermia , cardiac arrhythmias , and fluctuations in blood pressure.
• Cephalic infection primarily infects the head and involves cranial nerves.
• Localized infection involves the muscles in the area of primary injury.
• Neonatal tetanus is in newborns and originates from infection of umbilical stumps in mothers that have no immunity.
• The infection begins when endospores of C. tetani enter an anaerobic wound . Since the bacterium is an obligate anaerobe, an anaerobic environment is needed for the endospores to germinate and the vegetative bacteria to grow. Vegetative bacteria eventually produce tetanospasmin, the toxin responsible for symptoms of tetanus.
** CDC Recommendations for tetanus prophylaxis.
From Tetanus, by Daniel J Dire, MD, FACEP, FAAEM, Associate Professor, Department of Emergency Medicine, University of Alabama at Birmingham and Daniel J Dire, MD, FACEP, FAAEM, is a member of the following medical societies: American Academy of Clinical Toxicology, American Academy of Emergency Medicine, Association of Military Surgeons of the US, and Society for Academic Emergency Medicine
Escherichia coli
Gram Stain of Escherichia coli. Note gram-negative (pink) bacilli.
Haemophilus influenzae
Organism
• Haemophilus influenzae is a small Gram-negative bacillus.
• It is nonmotile.
• Facultative anaerobe (def).
• Fastideous growth needs. Requires enrichments for growth.
Habitat
• Mucous membranes of the respiratory tract in humans.
Source
• The patient's own mucous membranes or transmitted patient-to-patient.
Epidemiology
• Haemophilus parainfluenzae and nonencapsulated H. influenzae typically colonize the upper respiratory tract in humans within the first few months of life. These bacteria typically cause sinusitis, otitis media (def), bronchitis(def), and pneumonia (def).
• Encapsulated H. influenzae, primarily H. influenzae type b, is uncommon as normal flora of the upper respiratory tract but can be a common cause of serious infection in children.
• Until immunization of children against H. influenzae type b became routine in developed countries, this bacterium was the most common cause of pneumonia, septicemia(def), meningitis (def), and epiglottitis (def) in children under the age of four. Immunization has reduced the incidence of systemic infection by this bacterium 95%.
Clinical Disease
• Haemophilus influenzae does not cause influenza. Influenza is a viral infection.
• Haemophilus parainfluenzae and nonencapsulated H. influenzae typically cause sinusitis, otitis media (def), bronchitis (def), and pneumonia (def).
• H. influenzae type b is the most common cause of pneumonia, septicemia (def), meningitis (def), epiglottitis (def), and cellulitis in children under the age of four who are not immunized.
From Haemophilus influenzae Infections, by Mark R Schleiss, MD, Associate Professor, Department of Pediatrics, Division of Infectious Diseases, University of Cincinnati and Children's Hospital Research Foundation. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Bacteria/Clostridium_tetani.txt |
Organism
(left) Structure of Helicobacter pylori. (right) Scanning electron micrograph of Helicobacter bacteria (originally classified as Flexispira rappini, now deprecated). Obtained from the CDC Public Health Image Library. Image credit: CDC/Dr. Patricia Fields, Dr. Collette Fitzgerald (PHIL #5715), 2004.
Habitat
• The human gastrointestinal tract is the primary source.
Source
• Person-to-person spread by the fecal-oral route.
Epidemiology
• In developing countries, 70%-90% of individuals are colonized by the age of 10; in developed countries, colonization is low during children but increases to around 45% in older adults.
• Between 70% and 90% of people with gastritis, peptic ulcers, or doedonal ulcers are infected with H. pylori.
Clinical Disease
• Appears as gastritis (def), peptic ulcers (def), gastric adenocarcinoma (def), and certain B-cell lymphomas (def).
• Chronic gastritis is a risk factor for gastric carcinoma.
From Helicobacter pylori Infection, by Luigi Santacroce, MD, Assistant Professor, Department of Dentistry and Surgery, Section of General Surgery, Medical and Dentistry School, State University at Bari, Italy and Giuseppe Miragliotta, MD, Chairman, Professor, Section of Microbiology, University Hospital of Bari, Italy; Manoop S Bhutani, MD, Associate Professor of Medicine, Division of Gastroenterology, University of Texas Medical Branch at Galveston
Neisseria gonorrhoeae
Positive GC smear for gonorrhea.
Note the Neisseria gonorrhoeae (gram-negative diplococci) inside the white blood cells.
Neisseria meningitidis
Organism
• Neisseria meningitidis is a Gram-negative diplococcus, typically flattened where the cocci meet.
• Aerobic (def).
• There are 13 serogroups of meningococci. Serogroups B and C commonly cause meningitis (def) and meningococcemia (def) in developed countries; serogroups Y and W135 typically cause pneumonia.
Habitat
• Humans are the only natural host.
Source
• Transmitted person-to-person by aerosolized respiratory tract secretions.
Clinical Disease
• There are between 2000 and 3000 cases of meningococcal meningitis per year in the U.S. A total of 2725 cases were reported to CDC in 1998.
• N. meningitidis infects the nasopharynx of humans causing a usually mild or subclinical upper respiratory infection. However in about 15% of these individuals, the organism invades the blood and disseminates, causing septicemia and from the there may cross the blood-brain barrier causing meningitis (def). A petechial skin rash, caused by endotoxin in the blood, appears in about 75 percent of the septic cases and fatality rates for meningococcal septicemia are as high as 30 percent as a result of the shock cascade. A fulminating form of the disease, called Waterhouse-Frederichsen syndrome, can be fatal within several hours due to massive intravascular coagulation and resulting shock, probably a result of massive endotoxin release. N. meningitidis is especially dangerous in young children.
• Typical symptoms are headache, meningeal signs, and fever.
• Mortality is close to 100% if untreated; less than 10% with prompt and appropriate antibiotic therapy.
From Meningococcal Infections, by Thomas A Hoffman, MD, Professor, Department of Internal Medicine, Division of Infectious Diseases, Jackson Memorial Hospital, University of Miami.
Streptococcus pneumoniae
Streptococcus pneumoniae, or the pneumococcus, is a gram-positive lanceolate coccus usually appearing as a diplococcus, but occasionally appearing singularly or in short chains. Pneumococci are frequently found as normal flora of the nasopharynx of healthy carriers. From 10% to 40% of adults carry the bacterium in the nasopharynx. In the U.S., they are the most common cause of community-acquired pneumonia requiring hospitalization, causing around 500,000 cases per year and usually occurring as a secondary infection in the debilitated or immunocompromised host. The pneumococci also cause over 7,000,000 cases of otitis media per year, are the leading cause of sinusitis in people of all ages, are responsible for 500,000 cases of bacteremia, and 3000 cases of meningitis, being the most common cause of meningitis in adults and children over 4 years of age. Note gram-positive encapsulated diplococci. The large cells with the dark red nuclei are while blood cells.
Encapsulated Streptococcus pneumoniae. Encapsulated Streptococcus pneumoniae. © Gloria Delisle and Lewis Tomalty, authors. Licensed for use, ASM MicrobeLibrary.
Streptococcus pyogenes
Note gram-positive (purple) cocci in chains (arrows).
Organism
• Streptococcus pyogenes, a group A beta streptococcus, is a Gram-positive coccus typically arranged in chains.
• Facultative anaerobe (def).
Habitat
• Asymptomatic colonization of the upper respiratory tract in humans.
Source
• Pharyngitis is pread person to person primarily by respiratory droplets; skin infections are spread by direct contact with an infected person or through fomites (def).
Epidemiology
• The group A beta hemolytic streptococci are responsible for most acute human streptococcal infections. Between 5% and 20% of children are asymptomatic carriers. The most common infection is pharyngitis (def) with the organism usually being limited to the mucous membranes and lymphatic tissue of the upper respiratory tract. Children are at greatest risk for infection.
Clinical Disease
• The most common infection is pharyngitis (streptococcal sore throat) with the organism usually being limited to the mucous membranes and lymphatic tissue of the upper respiratory tract. From the pharynx, however, the streptococci sometimes spread to other areas of the respiratory tract resulting in laryngitis (def), bronchitis (def), pneumonia, and otitis media (def).
• Occasionally, it may enter the lymphatic vessels or the blood and disseminate to other areas of the body, causing septicemia (def), osteomyelitis (def), endocarditis(def), septic arthritis (def), and meningitis (def).
• If it enters injured skin, it may cause pyogenic (def) cutaneous infections such as impetigo , erysipelas (def), orcellulitis (def).
• Group A beta streptococcus infections can result in two autoimmune diseases (def), rheumatic fever and acute glomerulonephritis, where antibodies made against streptococcal antigens cross react with joint membranes and heart valve tissue in the case of rheumatic fever, or glomerular cells and basement membranes of the kidneys in the case of acute glomerulonephritis.
• Certain strains of S. pyogenes cause invasive group A beta streptococcal infections. Each year in the U.S. there are between 750 and 1500 cases of necrotizing fasciitis where a streptococcal-coded protease called Exotoxin B destroys the muscle (myositis) or the muscle covering (necrotizing fasciitis). There are another 750 - 1500 cases of toxic shock-like syndrome (def) due to group A beta streptococci producing Streptococcal pyrogenic exotoxin (Spe).
From Streptococcus Group A Infections, by Sat Sharma, MD, FRCPC, FACP, FCCP, DABSM, Program Director, Associate Professor, Department of Internal Medicine, Divisions of Pulmonary and Critical Care Medicine, University of Manitoba; Site Coordinator of Respiratory Medicine, St Boniface General Hospital; and Godfrey Harding, MD, FRCPC, Program Director of Medical Microbiology, Professor, Department of Medicine, Section of Infectious Diseases and Microbiology, St Boniface Hospital, University of Manitoba, Canada.
Vibrio cholerae
Monotrichous Flagellum of Vibrio cholerae. Courtesy of the Centers for Disease Control and Prevention. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Bacteria/Helicobacter_pylori.txt |
Thumbnail: A cluster of Escherichia coli bacteria magnified 10,000 times. (Public Domain; Eric Erbe, digital colorization by Christopher Pooley, both of USDA, ARS, EMU).
1: Fundamentals of Microbiology
Learning Objectives
1. State three harmful effects and four beneficial effects associated with the activities of microorganisms.
2. Define microbiota and microbiome.
3. Briefly describe two different beneficial things the human microbiome does for the normal function of our body.
4. State several diseases associated with a change in our "normal" microbiota.
5. List and recognize a description of the each of the 5 basic groups of microbes.
Microorganisms are the dominant life forms on earth, are found in almost every conceivable environment, and are essential to sustaining life on this planet. There are five basic groups of microorganisms:
• Bacteria are typically unicellular, microscopic, prokaryotic organisms that reproduce by binary fission.
• Fungi (yeasts and molds) are typically unicellular, microscopic, eukaryotic fungi that reproduce asexually by budding. Molds are typically filamentous, eukaryotic fungi that reproduce by producing asexual reproductive spores.
• Viruses are typically submicroscopic, acellular infectious particles that can only replicate inside a living host cell. The vast majority of viruses possess either DNA or RNA, but not both.
• Protozoa are typically unicellular, microscopic, eukaryotic organisms that lack a cell wall.
• Algae are typically eukaryotic microorganisms that carry out photosynthesis.
To get us started on our introduction of microorganisms we will go through the following Think-Pair-Share Questions.
Exercise \(1\): Think-Pair-Share Questions
This tube contains 7 milliliters of a culture of Escherichia coli. The total number of bacteria in this tube is equal to:
1. The number of people in Baltimore city.
2. The number of people in Maryland.
3. The number of people in North America.
4. The number of people in the world.
Exercise \(2\): Think-Pair-Share Questions
Are microbes such as bacteria mostly beneficial or harmful? Briefly explain your answer.
Exercise \(3\): Think-Pair-Share Questions
• In what ways might microbes such as bacteria be beneficial?
• In what ways might microbes such as bacteria be harmful?
In this course we will be looking at various fundamental concepts of microbiology, with particular emphasis on their relationships to human health. The overall goal is to better understand the total picture of infectious diseases in terms of host-infectious agent interaction. We will look at various groups of microbes and learn what they might do to establish infection and harm the body, we will look at the body to see the ways in which it defends itself against these microbes, and we will learn what can be done to help the body in its defense efforts.
The Big Picture of Infectious Diseases
One of the most important things in microbiology is learining the "Big Picture of Infectious Diseases," which is the biological basis of host parasite interaction. There are four interlocking parts to this big picture:
1. The microbe's side of the story - why some microbes have more potential to be harmful: The overwhelming majority of microbes are harmless to humans and, in fact, many are beneficial, being key players in the recycling of nutrients in nature. We will look at the major groups of microbes, learn what they are composed of chemically and structurally, and see how how they carry out their metabolism and reproduce. We will learn of a variety of factors some microbes may possess that play a role in increasing their ability to cause disease. Also we will learn how, through mutation, genetic recombination, and natural selection, microbes may adapt to resist our control attempts.
2. The body's side of the story - ways in which the body is able to defend itself naturally against infectious disease agents: Here will learn about the phenomenal defenses the body has available to defend itself against infectious disease agents, as well as altered body cells such as cancer cells and infected cells. The body is able to do this through the innate immune system and the adaptive immune system. Innate immune defenses are those you are born with and include anatomical barriers, mechanical removal, cytokines, pattern-recognition receptors, phagocytosis, inflammation, the complement pathways, and fever. The adaptive immune defenses are those you develop throughout your life and include antibody production and cell-mediated immunity.
3. Ways in which we can artificially help the body defend itself by removing the microbes or enhancing body defenses: We will learn how we can artificially help ourselves to avoid or reduce the risk of infection. Also we will learn ways in which we are able to artificially remove microbes from the body and its environment using agents such as antiseptics, disinfectants, physical agents such as heat and cold, antimicrobial chemotherapeutic chemicals, and antibiotics. Finally we will learn ways we are currently able to - or potentially in the future will be able to - improve or restore the body's immune responses through such techniques as immunization, adoptive immunotherapy, or immune modulation.
4. Relationship between the Human Microbiome and Human Health: The complex mutually beneficial symbiotic relationship between humans and their natural microbes is critical to good health. It is now recognized that the millions of genes associated with the normal flora or microbiota of the human body -especially in the intestinal tract - aid in the digestion of many foods, the regulation of multiple host metabolic pathways, and the regulation the body's immune defenses.
Benefits of Microbial Activity
Most people tend to think of microorganisms as harmful because of their roles in causing infectious diseases in humans and other animals, and agricultural loss as a result of infectious diseases of plants and the spoilage of food. The fact is, however, the vast majority of microorganisms are not harmful but rather beneficial. Without them there would be no life on earth. Therefore, we will start this course by looking at a few of the many benefits from microbial activity on this planet.
1. Food production: Many food products employ microorganisms in their production. These include the microbial fermentation processes used to produce yogurt, buttermilk, cheeses, alcoholic beverages, leavened breads, sauerkraut, pickles, and kimchi.
2. Energy production and cleaning up the environment: Methane, or natural gas, is a product of methanogenic microorganisms. Many aquatic microbes capture light energy and store it in molecules used as food then used by other organisms. Animal wastes, domestic refuse, biomass, and grain can be converted to biofuels such as ethanol and methane by microorganisms. In addition, through a process called bioremediation , some pollutants such pesticides, solvents, and oil spills can be cleaned up with the aid of microbes.
3. Sustaining agriculture: Through their roles in recycling nitrogen, carbon, and sulfur, microorganism are able to convert these essential elements into forms that can be used by plants in their growth. They are also essential in enabling ruminant animals such as cows and sheep to digest cellulose from the grasses they eat.
4. Production of useful natural gene products or products from bioengineering. Examples include specific enzymes, antibiotics, vaccines, and medications such as human insulin, interferons, and growth hormones.
5. The human microbiota and microbiome: Where we be without microorganisms? While the typical human body contains an estimated 37 trillion human cells, it also contains over 100 trillion bacteria and other microbes. The human body has 3 times as many bacterial cells as it does human cells! It is estimated the the mass of the human microbiota is 2.5 pounds.
The complex mutually beneficial symbiotic relationship between humans and their natural microbes is critical to good health. It is now recognized that the millions of genes associated with the microbiota of the human body -especially in the intestinal tract - aid in the digestion of many foods, the regulation of multiple host metabolic pathways, and the regulation the body's immune defenses. These collective microbes and their genes are referred to as the human microbiome. There are currently an estimated 5,000,000 - 10,000,000 genes from over 1000 species that constitute the human microbiome compared to the approximately 20,000 - 23,000 genes that make up the human genome. There are approximately 300 non-human genes in the human body for every human gene.
1. The mutually beneficial interaction between the human host and its resident microbiota is essential to human health. Microbial genes produce metabolites essential to the host while human genes contribute to development of the microbiota. The microbiome aids in the following:
1. The digestion of many foods, especially plant polysaccharides that would normally be indigestible by humans.
2. The regulation of many host metabolic pathways. The metabolism of many substrates in the human body is carried out by a combination of genes from both the microbiome and the human genome. Within the intestinal tract there is constant chemical communication not only between microbial species but also between microbial cells and human cells. Multiple factors, including diet, antibiotic use, disease, life style, and a person's environment can alter the composition of the microbiota within the gastrointestinal tract and, as a result, influence host biochemistry and the body's susceptibility to disease.
3. Metabolic disorders such as diabetes, nonalcoholic fatty liver disease, hypertension, obesity, gastric ulcers, colon cancer, and possibly some mood and behavior changes through hormone signaling have been linked to alterations in the microbiota.
Summary
1. Microorganisms are typically too small to be seen with the naked eye.
2. Bacteria, fungi, viruses, protozoa, and algae are the major groups of microorganisms.
3. The vast majority of microorganisms are not harmful but rather beneficial.
4. Microbiota refers to all of the microorganisms that live in a particular environment.
5. A microbiome is the entire collection of genes found in all of the microbes associated with a particular host.
6. The microbiome of the human body - especially in the intestinal tract - aid in the digestion of many foods, the regulation of multiple host metabolic pathways, and the regulation the body's immune defenses. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/1%3A_Fundamentals_of_Microbiology/1.1%3A_Introduction_to_Microbiology.txt |
Learning Objectives
1. Briefly describe why, in terms of differences in cell size, a eukaryotic cell is structurally more complex and compartmentalized than a cell that is prokaryotic.
2. When given a description, determine whether a cell is prokaryotic or eukaryotic and explain why.
3. Briefly state why viruses are not considered as prokaryotic nor eukaryotic.
According to the cell theory, the cell is the basic unit of life. All living organisms are composed of one or more cells. Based on the organization of their cellular structures, all living cells can be divided into two groups: prokaryotic and eukaryotic (also spelled procaryotic and eucaryotic). Animals, plants, fungi, protozoans, and algae all possess eukaryotic cell types. Only bacteria have prokaryotic cell types.
Prokaryotic cells are generally much smaller and more simple than eukaryotic (Figure $1$). Prokaryotic cells are, in fact, able to be structurally more simple because of their small size. The smaller a cell, the greater is its surface-to-volume ratio (the surface area of a cell compared to its volume).
The surface area of a spherical object can be calculated using the following formula:
$S = 4\, \pi\, r^2$
The volume of a spherical object can be calculated using the formula:
$V = \dfrac{4}{3}\, \pi\, r^3$
For example, a spherical cell 1 micrometer (µm) in diameter - the average size of a coccus-shaped bacterium - has a surface-to-volume ratio of approximately 6:1, while a spherical cell having a diameter of 20 µm has a surface-to-volume ratio of approximately 0.3:1.
A large surface-to-volume ratio, as seen in smaller prokaryotic cells, means that nutrients can easily and rapidly reach any part of the cells interior. However, in the larger eukaryotic cell, the limited surface area when compared to its volume means nutrients cannot rapidly diffuse to all interior parts of the cell. That is why eukaryotic cells require a variety of specialized internal organelles to carry out metabolism, provide energy, and transport chemicals throughout the cell. Both, however, must carry out the same life processes. Some features distinguishing prokaryotic and eukaryotic cells are shown in Table $1$. All of these features will be discussed in detail later in Unit 1.
Table $1$: Eukaryotic Versus Prokaryotic Cells
Nuclear Body
eukaryotic cell
a. The nuclear body is bounded by a nuclear membrane having pores connecting it with the endoplasmic reticulum (see Figure $2$ and Figure $3$).
b. It contains one or more paired, linear chromosomes composed of deoxyribonucleic acid (DNA) associated with histone proteins ).
c. A nucleolus is present. Ribosomal RNA (rRNA) is transcribed and assembled in the nucleolus.
d. The nuclear body is called a nucleus.
An electron micrograph of a cell nucleus, showing the darkly stained nucleolus. (Public Domain; US National Institute of General Medical Sciences/National Institutes of Health)
prokaryotic cell
a. The nuclear body is not bounded by a nuclear membrane (see Figure $4$).
b. It usually contains one circular chromosome composed of deoxyribonucleic acid (DNA) associated with histone-like proteins.
c. There is no nucleolus.
d. The nuclear body is called a nucleoid .
Cell Division
eukaryotic cell
a. The nucleus divides by mitosis .
b. Haploid (1N) sex cells in diploid or 2N organisms are produced through meiosis .
prokaryotic cell
a. The cell usually divides by binary fission . There is no mitosis.
b. Prokaryotic cells are haploid. Meiosis is not needed.
Cytoplasmic Membrane - also known as a cell membrane or plasma membrane
eukaryotic cell
a. The cytoplasmic membrane (see Figure $2$ and Figure $3$) is a fluid phospholipid bilayer (see Figure $5$) containing sterols (see Figure $6$) .
b. The membrane is capable of endocytosis (phagocytosis and pinocytosis) and exocytosis .
prokaryotic cell
a. The cytoplasmic membrane (Figure $4$) is a fluid phospholipid bilayer (Figure $5$) that usually lacking sterols. Bacteria generally contain sterol-like molecules called hopanoids (Figure $7$).
b.The membrane is incapable of endocytosis and exocytosis.
Cytoplasmic Structures
eukaryotic cell
a. The ribosomes are composed of a 60S and a 40S subunit that come together during protein synthesis to form an 80S ribosome .
- Ribosomal subunit densities: 60S and 40S
b. Internal membrane-bound organelles such as mitochondria , endoplasmic reticulum , Golgi apparatus , vacuoles, and lysosomes are present (see Figure $2$ and Figure $3$).
c. Chloroplasts serve as organelles for photosynthesis.
d. A mitotic spindle involved in mitosis is present during cell division.
e. A cytoskeleton is present. It contains microtubules, actin micofilaments, and intermediate filaments. These collectively play a role in giving shape to cells, allowing for cell movement, movement of organelles within the cell and endocytosis, and cell division.
prokaryotic cell
a. The ribosomes are composed of a 50S and a 30S subunit that come together during protein synthesis to form a 70S ribosome . See Figure $8$.
- Ribosomal subunit densities: 50S and 30S
b. Internal membrane-bound organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, and lysosomes are absent (see Figure $4$)
c. There are no chloroplasts. Photosynthesis usually takes place in infoldings or extensions derived from the cytoplasmic membrane.
d. There is no mitosis and no mitotic spindle.
e. The various structural filaments in the cytoplasm collectively make up the prokaryotic cytoskeleton. Cytoskeletal filaments play essential roles in determining the shape of a bacterium (coccus, bacillus, or spiral) and are also critical in the process of cell division by binary fission and in determining bacterial polarity.
Respiratory Enzymes and Electron Transport Chains
eukaryotic cell
- The electron transport system is located in the inner membrane of the mitochondria. It contributes to the production of ATP molecules via chemiosmosis.
-Electron micrograph of a mitochondrion from the Biology Department at the University of New Mexico.
prokaryotic cell
- The electron transport system is located in the cytoplasmic membrane. It contributes to the production of ATP molecules via chemiosmosis.
Cell Wall
eukaryotic cell
a. Plant cells, algae, and fungi have cell walls, usually composed of cellulose or chitin. Eukaryotic cell walls are never composed of peptidoglycan (see Figure $3$).
b. Animal cells and protozoans lack cell walls (see Figure $2$).
prokaryotic cell
a. With few exceptions, members of the domain Bacteria have cell walls composed of peptidoglycan (see Figure $4$).
b. Members of the domain Archae have cell walls composed of protein, a complex carbohydrate, or unique molecules resembling but not the same as peptidoglycan.
Locomotor Organelles
eukaryotic cell
- Eukaryotic cells may have flagella or cilia. Flagella and cilia are organelles involved in locomotion and in eukaryotic cells consist of a distinct arrangement of sliding microtubules surrounded by a membrane. The microtubule arrangement is referred to as a 2X9+2 arrangement (see Figure $9$).
prokaryotic cell
- Many prokaryotes have flagella, each composed of a single, rotating fibril and usually not surrounded by a membrane (see Figure $10$). There are no cilia.
Representative Organisms
• eukaryotic cell: The domain Eukarya: animals, plants, algae, protozoans, and fungi (yeasts, molds, mushrooms).
• prokaryotic cell: The domain Bacteria and the domain Archae.
Since viruses are acellular- they contain no cellular organelles, cannot grow and divide, and carry out no independent metabolism - they are considered neither prokaryotic nor eukaryotic. Because viruses are not cells and have no cellular organelles, they can only replicate and assemble inside a living host cell. They turn the host cell into a factory for manufacturing viral parts and viral enzymes and assembling the viral components.
Viruses, which possess both living and nonliving characteristics, will be discussed in Unit 4. Recently, viruses have been declared as living entities based on the large number of protein folds encoded by viral genomes that are shared with the genomes of cells. This indicates that viruses likely arose from multiple ancient cells.
Summary
1. There are two basic types of cells in nature: prokaryotic and eukaryotic.
2. Prokaryotic cells are structurally simpler than eukaryotic cells.
3. The smaller a cell, the greater its surface to volume ratio.
4. The smaller the surface to volume ratio, the more structurally complex (compartmentalized) a cell needs to be in order to carry out life functions.
5. There are fundamental differences between prokaryotic and eukaryotic cells.
6. Bacteria are prokaryotic cells; fungi, protozoa, algae, plants, and animals are composed of eukaryotic cells.
7. Viruses are not cells so they are neither prokaryotic nor eukaryotic. They can replicate only inside a living cell. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/1%3A_Fundamentals_of_Microbiology/1.2%3A_Cellular_Organization_-_Prokaryotic_and_Eukaryotic_Cells.txt |
Learning Objectives
1. Define phylogeny.
2. Name the 3 Domains of the 3 Domain system of classification and recognize a description of each.
3. Name the four kingdoms of the Domain Eukarya and recognize a description of each.
4. Define horizontal gene transfer.
The Earth is 4.6 billion years old and microbial life is thought to have first appeared between 3.8 and 3.9 billion years ago; in fact, 80% of Earth's history was exclusively microbial life. Microbial life is still the dominant life form on Earth. It has been estimated that the total number of microbial cells on Earth on the order of 2.5 X 1030 cells, making it the major fraction of biomass on the planet.
Phylogeny refers to the evolutionary relationships between organisms. The Three Domain System, proposed by Woese and others, is an evolutionary model of phylogeny based on differences in the sequences of nucleotides in the cell's ribosomal RNAs (rRNA), as well as the cell's membrane lipid structure and its sensitivity to antibiotics. Comparing rRNA structure is especially useful. Because rRNA molecules throughout nature carry out the same function, their structure changes very little over time. Therefore similarities and dissimilarities in rRNA nucleotide sequences are a good indication of how related or unrelated different cells and organisms are.
There are various hypotheses as to the origin of prokaryotic and eukaryotic cells. Because all cells are similar in nature, it is generally thought that all cells came from a common ancestor cell termed the last universal common ancestor (LUCA). These LUCAs eventually evolved into three different cell types, each representing a domain. The three domains are the Archaea, the Bacteria, and the Eukarya.
More recently various fusion hypotheses have begun to dominate the literature. One proposes that the diploid or 2N nature of the eukaryotic genome occurred after the fusion of two haploid or 1N prokaryotic cells. Others propose that the domains Archaea and Eukarya emerged from a common archaeal-eukaryotic ancestor that itself emerged from a member of the domain Bacteria. Some of the evidence behind this hypothesis is based on a "superphylum" of bacteria called PVC, members of which share some characteristics with both archaea and eukaryotes. There is growing evidence that eukaryotes may have originated within a subset of archaea. In any event, it is accepted today that there are three distinct domains of organisms in nature: Bacteria, Archaea, and Eukarya. A description of the three domains follows.
Domains?
There is a "superphylum" of bacteria called PVC, referring to the three members of that superphylum: the Planctomycetes, the Verrucomicrobia, and the Chlamydiae. Members of the PVC, while belonging to the domain Bacteria, show some features of the domains Archaea and Eukarya.
Some of these bacteria show cell compartmentalization wherein membranes surround portions of the cell interior, such as groups of ribosomes or DNA, similar to eukaryotic cells. Some divide by budding or contain sterols in their membranes, again similar to eukaryotes. Some lack peptidoglycan, similar to eukaryotes and archaea. It has been surmised that these bacteria migh be an intermediate step between an ancestor that emerged from a bacterium (domain Bacteria) and an archael-eukaryotic ancestor prior to its split into the domains Archaea and Eukarya.
The Archaea (archaebacteria)
The Archaea possess the following characteristics:
1. Archaea are prokaryotic cells.
2. Unlike the Bacteria and the Eukarya, the Archaea have membranes composed of branched hydrocarbon chains (many also containing rings within the hydrocarbon chains) attached to glycerol by ether linkages (Figure \(3\)).
3. The cell walls of Archaea contain no peptidoglycan.
4. Archaea are not sensitive to some antibiotics that affect the Bacteria, but are sensitive to some antibiotics that affect the Eukarya.
5. Archaea contain rRNA that is unique to the Archaea as indicated by the presence molecular regions distinctly different from the rRNA of Bacteria and Eukarya.
Archaea often live in extreme environments and include methanogens, extreme halophiles, and hyperthermophiles. One reason for this is that the ether-containing linkages in the Archaea membranes is more stabile than the ester-containing linkages in the Bacteria and Eukarya and are better able to withstand higher temperatures and stronger acid concentrations.
The Bacteria (eubacteria)
Bacteria (also known as eubacteria or "true bacteria") are prokaryotic cells that are common in human daily life, encounter many more times than the archaebacteria. Eubacteria can be found almost everywhere and kill thousands upon thousands of people each year, but also serve as antibiotics producers and food digesters in our stomachs. The Bacteria possess the following characteristics:
1. Bacteria are prokaryotic cells.
2. Like the Eukarya, they have membranes composed of unbranched fatty acid chains attached to glycerol by ester linkages (Figure \(3\)).
3. The cell walls of Bacteria, unlike the Archaea and the Eukarya, contain peptidoglycan.
4. Bacteria are sensitive to traditional antibacterial antibiotics but are resistant to most antibiotics that affect Eukarya.
5. Bacteria contain rRNA that is unique to the Bacteria as indicated by the presence molecular regions distinctly different from the rRNA of Archaea and Eukarya.
Bacteria include mycoplasmas, cyanobacteria, Gram-positive bacteria, and Gram-negative bacteria.
The Eukarya (eukaryotes)
The Eukarya (also spelled Eucarya) possess the following characteristics:
1. Eukarya have eukaryotic cells.
2. Like the Bacteria, they have membranes composed of unbranched fatty acid chains attached to glycerol by ester linkages (Figure \(3\)).
3. Not all Eukarya possess cells with a cell wall, but for those Eukarya having a cell wall, that wall contains no peptidoglycan.
4. Eukarya are resistant to traditional antibacterial antibiotics but are sensitive to most antibiotics that affect eukaryotic cells.
5. Eukarya contain rRNA that is unique to the Eukarya as indicated by the presence molecular regions distinctly different from the rRNA of Archaea and Bacteria.
The Eukarya are subdivided into the following four kingdoms:
1. Protista Kingdom: Protista are simple, predominately unicellular eukaryotic organisms. Examples includes slime molds, euglenoids, algae, and protozoans.
2. Fungi Kingdom: Fungi are unicellular or multicellular organisms with eukaryotic cell types. The cells have cell walls but are not organized into tissues. They do not carry out photosynthesis and obtain nutrients through absorption. Examples include sac fungi, club fungi, yeasts, and molds.
3. Plantae Kingdom: Plants are multicellular organisms composed of eukaryotic cells. The cells are organized into tissues and have cell walls. They obtain nutrients by photosynthesis and absorption. Examples include mosses, ferns, conifers, and flowering plants.
4. Animalia Kingdom: Animals are multicellular organisms composed of eukaryotic cells. The cells are organized into tissues and lack cell walls. They do not carry out photosynthesis and obtain nutrients primarily by ingestion. Examples include sponges, worms, insects, and vertebrates.
It used to be thought that the changes that allow microorganisms to adapt to new environments or alter their virulence capabilities was a relatively slow process occurring within an organism primarily through mutations, chromosomal rearrangements, gene deletions and gene duplications. Those changes would then be passed on to that microbe's progeny and natural selection would occur. This gene transfer from a parent organism to its offspring is called vertical gene transmission.
It is now known that microbial genes are transferred not only vertically from a parent organism to its progeny, but also horizontally to relatives that are only distantly related, e.g., other species and other genera. This latter process is known as horizontal gene transfer. Through mechanisms such as transformation, transduction, and conjugation, genetic elements such as plasmids, transposons, integrons, and even chromosomal DNA can readily be spread from one microorganism to another. As a result, the old three-branched "tree of life" in regard to microorganisms (Figure \(1\)) now appears to be more of a "net of life."
Microbes are known to live in remarkably diverse environments, many of which are extremely harsh. This amazing and rapid adaptability is a result of their ability to quickly modify their repertoire of protein functions by modifying, gaining, or losing their genes. This gene expansion predominantly takes place by horizontal transfer.
Summary
1. Phylogeny refers to the evolutionary relationships between organisms.
2. Organisms can be classified into one of three domains based on differences in the sequences of nucleotides in the cell's ribosomal RNAs (rRNA), the cell's membrane lipid structure, and its sensitivity to antibiotics.
3. The three domains are the Archaea, the Bacteria, and the Eukarya.
4. Prokaryotic organisms belong either to the domain Archaea or the domain Bacteria; organisms with eukaryotic cells belong to the domain Eukarya.
5. Microorganism transfer genes to other microorganisms through horizontal gene transfer - the transfer of DNA to an organism that is not its offspring. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/1%3A_Fundamentals_of_Microbiology/1.3%3A_Classification_-_The_Three_Domain_System.txt |
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
1.1: Introduction to Microbiology
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. List 5 basic groups of microbes. (ans)
2. State 3 of the many benefits from microbial activity on this planet. (ans)
3. State 2 of the harmful effects associated with microbial activities. (ans)
4. Briefly describe two different beneficial things the human microbiome does for the normal function of our body. (ans)
1.2: Cellular Organization: Prokaryotic and Eukaryotic Cells
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. An electron micrograph of a cell shows a rigid cell wall, cytoplasmic membrane, nuclear body without a nuclear membrane, and no endoplasmic reticulum or mitochondria. Explain why it is or is not each of the following.
1. a bacterium (ans)
2. a yeast (ans)
3. a virus (ans)
4. an animal cell (ans)
2. Match the descriptions below with the best type of cellular organization.
_____ no nuclear membrane, circular chromosome of DNA, no mitosis (ans)
_____ capable of endocytosis, sterols in membrane, 80S ribosomes (ans)
_____ mitochondria, Golgi apparatus, endoplasmic reticulum (ans)
_____ cell wall contains peptidoglycan (ans)
1. eukaryotic
2. prokaryotic
3. Multiple Choice (ans)
1.3: Classification: The Three Domain System
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Matching
_____ Eukaryotic cells. They have membranes composed of straight fatty acid chains attached to glycerol by ester linkages.If they possess cell walls, those walls contain no peptidoglycan. (ans)
_____ Prokaryotic cells. They have membranes composed of branched hydrocarbon chains attached to glycerol by ether linkages and have cell walls that contain no peptidoglycan. They often live in extreme environments. (ans)
_____ Prokaryotic cells. They have membranes composed of straight fatty acid chains attached to glycerol by ester linkages and have cell walls containing peptidoglycan. (ans)
1. Archaea
2. Bacteria
3. Eukarya
2. Matching
_____ Simple, predominately unicellular eukaryotic organisms. Examples includes slime molds, euglenoids, algae, and protozoans. (ans)
_____ Multicellular organisms composed of eukaryotic cells. The cells are organized into tissues and lack cell walls. They do not carry out photosynthesis and obtain nutrients primarily by ingestion. (ans)
_____ Multicellular organisms composed of eukaryotic cells. The cells are organized into tissues and have cell walls. They obtain nutrients by photosynthesis and absorption. (ans)
1. Fungi Kingdom
2. Protista Kingdom
3. Plantae Kingdom
4. Animalia Kingdom
3. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/1%3A_Fundamentals_of_Microbiology/1.E%3A_Fundamentals_of_Microbiology_%28Exercises%29.txt |
Thumbnail: Electron micrograph of Treponema pallidum on cultures of cotton-tail rabbit epithelium cells (Sf1Ep). Treponema pallidum is the causative agent of syphilis. (Public Domain; CDC / Dr. David Cox).
2: The Prokaryotic Cell - Bacteria
Learning Objectives
1. List the three basic shapes of bacteria.
2. List and describe 5 different arrangements of cocci.
3. Define and give the abbreviation for the metric unit of length termed micrometer and state the average size of a coccus-shaped bacterium and a rod-shaped bacterium.
4. List and describe 2 different arrangements of bacilli.
5. List and describe 3 different spiral forms of bacteria.
Bacteria are prokaryotic, single-celled, microscopic organisms (Exceptions have been discovered that can reach sizes just visible to the naked eye. They include Epulopiscium fishelsoni, a bacillus-shaped bacterium that is typically 80 micrometers (µm) in diameter and 200-600 µm long, and Thiomargarita namibiensis, a spherical bacterium between 100 and 750 µm in diameter.)
1. generally much smaller than eukaryotic cells.
2. very complex despite their small size. Even though bacteria are single-celled organisms, they are able to communicate with one another through a process called quorum sensing. In this way they can function as a multicellular population rather than as individual bacteria. This will be discussed in greater detail in Unit 2.
a. Division in one plane produces either a diplococcus or streptococcus arrangement.
diplococcus: cocci arranged in pairs (see Figure \(2\))
- scanning electron micrograph of a Streptococcus pneumoniae, a diplococcus; courtesy of CDC
- scanning electron micrograph of a Neisseria, a diplococcus; courtesy of Dennis Kunkel's Microscopy
streptococcus: cocci arranged in chains (see Figure \(3\))
- scanning electron micrograph of a Streptococcus pyogenes, a streptococcus; courtesy of Dennis Kunkel's Microscopy
- transmission electron micrograph of Streptococcus from the Rockefeller University web page.
- scanning Electron Micrograph of Enterococcus
b. Division in two planes produces a tetrad arrangement.
tetrad: cocci arranged in squares of 4 (see Figure \(4\))
- scanning electron micrograph of Micrococcus luteus showing several tetrads
c. Division in three planes produces a sarcina arrangement.
sarcina: cocci in arranged cubes of 8 (see Figure \(5\))
d. Division in random planes produces a staphylococcus arrangement.
staphylococcus: cocci arranged in irregular, often grape-like clusters (see Figure \(6\))
- negative image of Staphylococcus aureus
- scanning electron micrograph of Staphylococcus aureus, a staphylococcus; courtesy of Dennis Kunkel's Microscopy
- Scanning electron micrograph of methicillin-resistant Staphylococcus aureus (MRSA); courtesy of CDC
An average coccus is about 0.5-1.0 micrometer (µm) in diameter. (A micrometer equals 1/1,000,000 of a meter.)
The rod or bacillus
Bacilli are rod-shaped bacteria. Bacilli all divide in one plane producing a bacillus, streptobacillus, or coccobacillus arrangement (see Figure \(7\)).
a. bacillus: single bacilli (see Figure \(8\))
- scanning electron micrograph of a bacillus; courtesy of CDC
- scanning electron micrograph of Escherichia coli O157H7, a bacillus; courtesy of CDC
b. streptobacillus: bacilli arranged in chains (see Figure \(9\))
c. coccobacillus: oval and similar to a coccus (see Figure \(9\)A and Figure \(9\)B)
An average bacillus is 0.5-1.0 µm wide by 1.0-4.0 µm long.
The spiral
Spirals come in one of three forms, a vibrio, a spirillum, or a spirochete. (see Figure \(10\))
a. vibrio: a curved or comma-shaped rod (see Figure \(11\))
- scanning electron micrograph of a Vibrio cholerae, a vibrio; courtesy of Dennis Kunkel's Microscopy
b. spirillum: a thick, rigid spiral (see Figure \(12\))
c. spirochete: a thin, flexible spiral (see Figure \(13\))
- scanning electron micrograph of the spirochete Leptospira
; courtesy of CDC
- scanning electron micrograph of the spirochete Treponema pallidum; courtesy of CDC
Spirals range in size from 1 µm to over 100 µm in length.
Exceptions to the above shapes
There are exceptions to the three basic shapes of coccus, bacillus, and spiral. They include sheathed, stalked, filamentous, square, star-shaped, spindle-shaped, lobed, trichome-forming, and pleomorphic bacteria.
Ultrasmall Bacteria
150 could fit in a single Escherichia coli) have been discovered in groundwater that was passed through a filter with a pore size of 0.2 micrometers µm). They showed an average length of only 323 nanometers(nm) and an average width of 242 nm. They contain DNA, an average of 42 ribosomes per bacterium, and possessed pili . It is thought that they use these pili to attach to other bacteria from which they scavenge nutrients. Because the surface to volume ratio is even greater than in more traditional sized bacteria, they might be better designed to take up scarce nutrients from more nutrient-poor environments.
Summary
1. There are three basic shapes of bacteria: coccus, bacillus, and spiral.
2. Based on planes of division, the coccus shape can appear in several distinct arrangements: diplococcus, streptococcus, tetrad, sarcina, and staphylococcus.
3. The bacillus shape can appear as a single bacillus, a streptobacillus, or a coccobacillus.
4. The spiral shape can appear in several forms: vibrio, spirillum, and spirochete.
5. The metric unit micrometer (1/1,000,000 or 10-6 of a meter) is used to measure bacterial size. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.1%3A_Sizes_Shapes_and_Arrangements_of_Bacteria.txt |
State the chemical composition and major function of the cytoplasmic membrane in bacteria. Briefly describe the fluid phospholipid bilayer arrangement of biological membranes. State the net flow of water when a cell is placed in an isotonic, hypertonic, or hypotonic environment and relate this to the solute concentration. Define the following means of transport: passive diffusion osmosis facilitated diffusion transport through channel proteins transport through uniporter active transport transport through antiporter transport through symporter the ABC transport system group translocation State how the antibiotic polymyxin and disinfectants such as orthophenylphenol, chlorhexidine, hexachlorophene, zephiran, and alcohol affect bacteria. Define binary fission and geometric progression and relate this to bacteria being able to astronomically increase their numbers in a relatively short period of time. Briefly describe the process of binary fission in bacteria, stating the functions of Par proteins, the divisome, and FtsZ proteins.
All molecules and atoms possess kinetic energy (energy of motion). If the molecules or atoms are not evenly distributed on both sides of a membrane, the difference in their concentration forms a concentration gradient that represents a form of potential energy (stored energy). The net movement of these particles will therefore be down their concentration gradient - from the area of higher concentration to the area of lower concentration. Diffusion is powered by the potential energy of a concentration gradient and does not require the expenditure of metabolic energy.
A cell can find itself in one of three environments: isotonic, hypertonic, or hypotonic (the prefixes iso-, hyper-, and hypo- refer to the solute concentration).
• In an isotonic environment (Figure \(5\)) both the water and solute concentration are the same inside and outside the cell and water goes into and out of the cell at an equal rate.
• If the environment is hypertonic ( Figure \(6\)A and Figure \(6\)B) the water concentration is greater inside the cell while the solute concentration is higher outside (the interior of the cell is hypotonic to the surrounding hypertonic environment). Water goes out of the cell.
• In an environment that is hypotonic (Figure \(7\)) the water concentration is greater outside the cell and the solute concentration is higher inside (the interior of the cell is hypertonic to the hypotonic surroundings). Water goes into the cell.
2. Channel proteins transport water or certain ions down either a concentration gradient, in the case of water, or an electric potential gradient in the case of certain ions, from an area of higher concentration to lower concentration ( Figure \(6\)B). While water molecules can directly cross the membrane by passive diffusion, as mentioned above, channel proteins called aquaporins can enhance their transport.
Active Transport
Active transport is a process whereby the cell uses both transport proteins and metabolic energy to transport substances across the membrane against the concentration gradient. In this way, active transport allows cells to accumulate needed substances even when the concentration is lower outside. Active transport enables bacteria to successfully compete with other organisms for limited nutrients in their natural habitat, and as will be seen in Unit 2, enables pathogens to compete with the body's own cells and normal flora bacteria for the same nutrients.
The energy is provided by proton motive force, the hydrolysis of ATP, or the breakdown of some other high-energy compound such as phosphoenolpyruvate (PEP). Proton motive force is an energy gradient resulting from hydrogen ions (protons) moving across the membrane from greater to lesser hydrogen ion concentration. ATP is the form of energy cells most commonly use to do cellular work. PEP is one of the intermediate high-energy phosphate compounds produced at the end of glycolysis.
Specific transport proteins (carrier proteins) are required in order to transport the majority of molecules a cell requires across its cytoplasmic membrane. This is because the concentration of nutrients in most natural environments is typically quite low. Transport proteins allow cells to accumulate nutrients from even a sparse environment. Transport proteins involved in active transport include antiporters, symporters, the proteins of the ATP-binding cassette (ABC) system, and the proteins involved in group translocation.
a. Antiporter: Antiporters are transport proteins that transport one substance across the membrane in one direction while simultaneously transporting a second substance across the membrane in the opposite direction (Figure \(9\)A). Antiporters in bacteria generally use the potential energy of electrochemical gradients from protons (H+), that is, proton motive force to co-transport ions, glucose, and amino acids against their concentration gradient (Figure \(9\)B). Sodium ions (Na+) and protons (H+), for example, are co-transported across bacterial membranes by antiporters.
b. Symporter: Symporters are transport proteins that simultaneously transport two substances across the membrane in the same direction (Figure \(10\)A). Symporters use the potential energy of electrochemical gradients from protons (H+), that is, proton motive force to co-transport ions, glucose, and amino acids against their concentration gradient (Figure \(10\)B). Sulfate (HSO4-) and protons (H+) as well as phosphate (HPO4-) and protons (H+) are co-transported across bacterial membranes by symporters.
c. ATP-binding cassette (ABC) system: An example of an ATP-dependent active transport found in various gram-negative bacteria is the ATP-binding cassette (ABC) system. This involves substrate-specific binding proteins located in the bacterial periplasm, the gel-like substance between the bacterial cell wall and cytoplasmic membrane. The periplasmic-binding protein picks up the substance to be transported and carries it to a membrane-spanning transport protein (Figure \(11\)A). Meanwhile, an ATP-hydrolyzing protein breaks ATP down into ADP, phosphate, and energy (Figure \(11\)B). It is this energy that powers the transport of the substrate, by way of the membrane-binding transporter, across the membrane (Figure 11C and Figure \(11\)D) and into the cytoplasm. Examples of active transport include the transport of certain sugars and amino acids. Over 200 different ABC transport systems have been found in bacteria.
d. Group translocation is another form of active transport that can occur in prokaryotes. In this case, a substance is chemically altered during its transport across a membrane so that once inside, the cytoplasmic membrane becomes impermeable to that substance and it remains within the cell.
An example of group translocation in bacteria is the phosphotransferase system. A high-energy phosphate group from phosphoenolpyruvate (PEP) is transferred by a series of enzymes to glucose. The final enzyme both phosphorylates the glucose and transports it across the membrane as glucose 6-phosphate (Figure \(12\)A through 12D). (This is actually the first step in glycolysis.) Other sugars that are transported by group translocation are mannose and fructose.
Functions of the cytoplasmic membrane other than selective permeability
A number of other functions are associated with the bacterial cytoplasmic membrane and associated proteins of a collection of cell division machinery known as the divisome. In fact, many of the functions associated with specialized internal membrane-bound organelles in eukaryotic cells are carried out generically in bacteria by the cytoplasmic membrane. Functions associated with the bacterial cytoplasmic membrane and/or the divisome include:
1. energy production. The electron transport system ( Fig.) for bacteria with aerobic and anaerobic respiration, as well as photosynthesis for bacteria converting light energy into chemical energy is located in the cytoplasmic membrane.
2. motility. The motor that drives rotation of bacterial flagella ( see Fig.) is located in the cytoplasmic membrane.
3. Movie of motile Rhodobacter spheroides with fluorescent labelled-flagella. Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
4. waste removal. Waste by products of metabolism within the bacterium must exit through the cytoplasmic membrane.
5. formation of endospores (discussed later in this unit; see Fig. and animation).
Binary fission
Bacteria divide by binary fission wherein one bacterium splits into two. Therefore, bacteria increase their numbers by geometric progression whereby their population doubles every generation time. In general it is thought that during DNA replication (discussed in Unit 6), each strand of the replicating bacterial DNA attaches to proteins at what will become the cell division plane. For example, Par proteins function to separate bacterial chromosomes to opposite poles of the cell during cell division. They bind to the origin of replication of the DNA and physically pull or push the chromosomes apart, similar to the mitotic apparatus of eukaryotic cells.
Figure \(8\): Bacterial Divisome.In general it is thought that during DNA replication (discussed in Unit 6), each strand of the replicating bacterial DNA attaches to proteins at what will become the cell division plane. For example, Par proteins function to separate bacterial chromosomes to opposite poles of the cell during cell division. They bind to the origin of replication of the DNA and physically pull or push the chromosomes apart, similar to the mitotic apparatus of eukaryotic cells. In the center of the bacterium, a group of proteins called Fts (filamentous temperature sensitive) proteins interact to form a ring at the cell division plane. These proteins form the cell division apparatus known as the divisome and are directly involved in bacterial cell division by binary fission. The divisome is responsible for directing the synthesis of new cytoplasmic membrane and new peptidoglycan to form the division septum.
In the center of the bacterium, a group of proteins called Fts (filamentous temperature sensitive) proteins interact to form a ring at the cell division plane. These proteins form the cell division apparatus known as the divisome and are directly involved in bacterial cell division by binary fission (Figure \(1\) and Figure \(13\)).
• electron micrograph of a divisome: see under Bacterial Cell Division, Jon Beckwith's Lab.
The divisome is responsible for directing the synthesis of new cytoplasmic membrane and new peptidoglycan to form the division septum. The function of a number of divisome proteins have been identified, including:
• MinE: Directs formation of the FtsZ ring and divisome complex at the bacterium's division plane.
• FtsZ: Similar to tubulin in eukaryotic cells, FtsZ forms a constricting ring at the division site. As FtsZ depolymerizes, it directs an inward growth of the cell wall to form the division septum. It is found in both Bacteria and Archaea, as well as in mitochondria and chloroplasts.
• ZipA: A protein that connects the FtsZ ring to the bacterial cytoplasmic membrane.
• FtsA: An ATPase that breaks down ATP to provide energy for cell division and also helps connect the FtsZ ring to the bacterial cytoplasmic membrane.
• FtsK: Helps in separating the replicated bacterial chromosome.
• FtsI: Needed for peptidoglycan synthesis.
- Scanning electron micrograph of dividing Escherichia coli; courtesy of CDC.
- Scanning electron micrograph of dividing Salmonella typhimurium; courtesy of CDC.
- To view an transmission electron micrograph of dividing streptococci, see the Rockefeller University home page.
Using Antimicrobial Agents that Alter the Cytoplasmic Membrane to Control Bacteria
As will be discussed later in Unit 2, a very few antibiotics, such as polymyxins and tyrocidins as well as many disinfectants and antiseptics, such as orthophenylphenol, chlorhexidine, hexachlorophene, zephiran, alcohol, triclosans, etc., used during disinfection alter the microbial cytoplasmic membranes and cause leakage of cellular needs.
Summary
1. The bacterial cytoplasmic membrane is a fluid phospholipid bilayer that encloses the bacterial cytoplasm.
2. The cytoplasmic membrane is semipermeable and determines what molecules enter and leave the bacterial cell.
3. Passive diffusion is the net movement of gases or small uncharged polar molecules such as water across a membrane from an area of higher concentration to an area of lower concentration.
4. Passive diffusion is powered by the potential energy of a concentration gradient and does not require the expenditure of metabolic energy or the use of transport proteins.
5. Facilitated diffusion is powered by the potential energy of a concentration gradient and does not require the expenditure of metabolic energy, but it does require the use of transport proteins.
6. A solution refers to solute dissolved in a solvent.
7. Osmosis is the movement of water across a membrane from an area of higher water (lower solute) concentration to an area of lower water (higher solute) concentration by both passive diffusion and facilitated diffusion.
8. Active transport is a process whereby the cell uses both transport proteins and metabolic energy to transport substances across the membrane against the concentration gradient.
9. Most molecules and ions that a cell needs to concentrate within the cytoplasm in order to support life require active transport for entry into the cell.
10. In order to colonize any environment, a bacterium must be able to effectively use its transport systems to compete with other bacteria, as well as the cells of other organisms – such as human cells - for limited nutrients.
11. Bacteria divide by binary fission and increase their numbers by geometric progression.
12. Some antimicrobial agents alter the microbial cytoplasmic membranes and cause leakage of cellular needs.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Match the following descriptions with the best answer.
2. Even though there is a lower concentration of a particular nutrient outside a bacterium than inside, the bacterium is still able to transport that nutrient into its cytoplasm. Explain how this might occur and what is required for this transport. (ans)
3. A bacterium is placed in a new environment and subsequently water flows out of the bacterium. Is this new environment isotonic, hypotonic, or hypertonic to the bacterium? Is the solute concentration higher inside the bacterium or outside? (ans)
4. Bacteria normally do not grow in jams and jellies. In terms of osmosis, what might explain this? (ans)
5. Define the following:
1. binary fission (ans)
2. geometric progression (ans)
6. State the functions of the following in bacterial cell division:
1. Par proteins (ans)
2. divisome (ans)
3. FtsZ proteins (ans)
7. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.2%3A_The_Cytoplasmic_Membrane.txt |
Learning Objectives
1. State the three parts of a peptidoglycan monomer and state the function of peptidoglycan in bacteria.
2. Briefly describe how bacteria synthesize peptidoglycan, indicating the roles of autolysins, bactoprenols, transglycosylases, and transpeptidases.
3. Briefly describe how antibiotics such as penicillins, cephalosporins, and vancomycin affect bacteria and relate this to their cell wall synthesis.
4. State what color Gram-positive bacteria stain after Gram staining.
5. State what color Gram-negative bacteria stain after Gram staining.
6. State what color acid-fast bacteria stain after acid-fast staining.
The mycoplasmas are the only bacteria that naturally lack a cell wall. Mycoplasmas maintain a nearly even pressure between the outside environment and the cytoplasm by actively pumping out sodium ions. Their cytoplasmic membranes also contain sterols that most likely provide added strength. The remaining bacteria in the domain Bacteria, with the exception of a few bacteria such as the Chlamydias, have a semirigid cell wall containing peptidoglycan. (While bacteria belonging to the domain Archaea also have a semirigid cell wall, it is composed of chemicals distinct from peptidoglycan such as protein or pseudomurein. We will not take up the Archaea here.)
Function of Peptidoglycan
Peptidoglycan prevents osmotic lysis. As seen earlier under the cytoplasmic membrane, bacteria concentrate dissolved nutrients (solute) through active transport. As a result, the bacterium's cytoplasm is usually hypertonic to its surrounding environment and the net flow of free water is into the bacterium. Without a strong cell wall, the bacterium would burst from the osmotic pressure of the water flowing into the cell.
Structure and Composition of Peptidoglycan
With the exceptions above, members of the domain Bacteria have a cell wall containing a semirigid, tight knit molecular complex called peptidoglycan. Peptidoglycan, also called murein, is a vast polymer consisting of interlocking chains of identical peptidoglycan monomers (Figure \(1\)). A peptidoglycan monomer consists of two joined amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a pentapeptide coming off of the NAM (Figure \(2\)). The types and the order of amino acids in the pentapeptide, while almost identical in gram-positive and gram-negative bacteria, show some slight variation among the domain Bacteria.
The peptidoglycan monomers are synthesized in the cytosol of the bacterium where they attach to a membrane carrier molecule called bactoprenol. As discussed below, The bactoprenols transport the peptidoglycan monomers across the cytoplasmic membrane and work with the enzymes discussed below to insert the monomers into existing peptidoglycan enabling bacterial growth following binary fission.
Once the new peptidoglycan monomers are inserted, glycosidic bonds then link these monomers into the growing chains of peptidoglycan. These long sugar chains are then joined to one another by means of peptide cross-links between the peptides coming off of the NAMs. By linking the rows and layers of sugars together in this manner, the peptide cross-links provide tremendous strength to the cell wall, enabling it to function similar to a molecular chain link fence around the bacterium (see Figure \(1\)).
Synthesis of Peptidoglycan
In order for bacteria to increase their size following binary fission, links in the peptidoglycan must be broken, new peptidoglycan monomers must be inserted, and the peptide cross links must be resealed. The following sequence of events occur:
Step 1. Bacterial enzymes called autolysins:
a) Break the glycosidic bonds between the peptidoglycan monomers at the point of growth along the existing peptidoglycan (see Figure \(3\), steps 1-3); and
b) Break the peptide cross-bridges that link the rows of sugars together (see Figure \(3\), steps 1-3).
Step 2. The peptidoglycan monomers are synthesized in the cytosol (see Figure \(4\), step-1 and Figure \(4\), step-2) and bind to bactoprenol. The bactoprenols transport the peptidoglycan monomers across the cytoplasmic membrane and interacts with transglycosidases to insert the monomers into existing peptidoglycan (see Figure \(4\), step-3, Figure \(4\), step-4, Figure \(4\), step-5, and Figure \(4\), step-6)
Step 3. Transglycosylase (transglycosidase) enzymes insert and link new peptidoglycan monomers into the breaks in the peptidoglycan (see Figure \(5\), step 1 and Figure \(5\), step 2).
Step 4. Finally, transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong (see Figure \(6\), step 1 and see Figure \(6\), step 2).
In Escherichia coli, the terminal D-alanine is cleaved from the pentapeptides to form a tetrapeptides. This provides the energy to bond the D-alanine of one tetrapeptide to the diaminopimelic acid of another tetrapeptide (see Figure \(1\)B). In the case of Staphylococcus aureus, the terminal D-alanine is cleaved from the pentapeptides to form a tetrapeptides. This provides the energy to bond a pentaglycine bridge (5 molecules of the amino acid glycine) from the D-alanine of one tetrapeptide to the L-lysine of another (see Figure \(1\)A).
Exercise: Think-Pair-Share Questions
1. As we will see in Unit 2, the antibiotic bacitracin binds to bactoprenol after it inserts a peptidoglycan monomer into the growing bacterial cell wall.
Explain how this can lead to the death of that bacterium.
2. As we will see in Unit 2, the penicillin antibiotics binds to the bacterial enzyme transpeptidase.
1. Explain how this can lead to the death of that bacterium.
2. Could this antibiotic be used to treat protozoan infections such as giardiasis and toxoplasmosis?
In the center of the bacterium, a group of proteins called Fts (filamentous temperature sensitive) proteins interact to form a ring at the cell division plane. These proteins form the cell division apparatus known as the divisome and are directly involved in bacterial cell division by binary fission (see Figure \(1\) and Figure \(2\)).
The divisome is responsible for directing the synthesis of new cytoplasmic membrane and new peptidoglycan to form the division septum.
Antimicrobial Agents that Inhibit Peptidoglycan Synthesis Causing Bacterial Lysis
Many antibiotics work by inhibiting normal synthesis of peptidoglycan in bacteria causing them to burst as a result of osmotic lysis. As just mentioned, in order for bacteria to increase their size following binary fission, enzymes called autolysins break the peptide cross links in the peptidoglycan, transglycosylase enzymes then insert and link new peptidoglycan monomers into the breaks in the peptidoglycan, and transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong.
Interference with this process results in a weak cell wall and lysis of the bacterium from osmotic pressure. Examples include the penicillins (penicillin G, methicillin, oxacillin, ampicillin, amoxicillin, ticarcillin, etc.), the cephalosporins (cephalothin, cefazolin, cefoxitin, cefotaxime, cefaclor, cefoperazone, cefixime, ceftriaxone, cefuroxime, etc.), the carbapenems (imipenem, metropenem), the monobactems (aztreonem), the carbacephems (loracarbef), and the glycopeptides (vancomycin, teichoplanin).
• For example, penicillins and cephalosporins bind to the transpeptidase enzymes (also called penicillin-binding proteins) responsible for resealing the cell wall as new peptidoglycan monomers are added during bacterial cell growth. This blocks the transpeptidase enzymes from cross-linking the sugar chains and results in a weak cell wall and subsequent osmotic lysis of the bacterium (see Figure \(8\)).
Flash animation showing how penicillins inhibit peptidoglycan synthesis.
© Juliet V. Spencer, Stephanie K.M. Wong, authors, Licensed for use, ASM MicrobeLibrary.
Antimicrobial chemotherapy will be discussed in greater detail later in Unit 2 under Control of Bacteria by Using Antibiotics and Disinfectants.
Gram-Positive, Gram-Negative, and Acid-Fast Bacteria
Most bacteria can be placed into one of three groups based on their color after specific staining procedures are performed: Gram-positive, Gram-negative, or acid-fast.
• Gram-positive Bacteria: These retain the initial dye crystal violet during the Gram stain procedure and appear purple when observed through the microscope. Common Gram-positive bacteria of medical importance include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, and Clostridium species.
(left) Gram Stain of Staphylococcus aureus which are gram-positive (purple) cocci in clusters. (right) Gram Stain of Escherichia coli which are Gram-negative (pink) bacilli.
• Gram-negative Bacteria: These decolorize during the Gram stain procedure, pick up the counterstain safranin, and appear pink when observed through the microscope. Common Gram-negative bacteria of medical importance include Salmonella species, Shigella species, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Proteus species, and Pseudomonas aeruginosa. Also see gram stain of a mixture of gram-positive and gram-negative bacteria.
A Gram Stain of a Mixture of Gram-Positive and Gram-Negative Bacteria. Note Gram-negative (pink) bacilli and Gram-positive (purple) cocci.
• acid-fast Bacteria: These resist decolorization with an acid-alcohol mixture during the acid-fast stain procedure, retain the initial dye carbolfuchsin and appear red when observed through the microscope. Common acid-fast bacteria of medical importance include Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium-intracellulare complex.
Acid-Fast Stain of Mycobacterium tuberculosis in Sputum. Note the reddish acid-fast bacilli among the blue normal flora and white blood cells in the sputum that are not acid-fast.
These staining reactions are due to fundamental differences in their cell wall as will be discussed in Lab 6 and Lab 16. We will now look at each of these three bacterial cell wall types.
The S-layer
1. Structure and Composition
The most common cell wall in species of Archaea is a paracrystalline surface layer (S-layer). It consists of a regularly structured layer composed of interlocking glycoprotein or protein molecules. In electron micrographs, has a pattern resembling floor tiles. Although they vary with the species, S-layers generally have a thickness between 5 and 25 nm and possess identical pores with 2-8 nm in diameter. Several species of Bacteria have also been found to have S-layers.
To view electron micrographs of S-layers see the following:
• S-Layer Proteins, the Structural Biology Homepage at Karl-Franzens University in Austria.
• Characteristic Properties of S-layer Proteins, at Foresight Nanotech Institute in Austria.
2. Functions and Significance to Bacteria Causing Infections
The S-layer has been associated with a number of possible functions. These include the following:
a. The S-layer may protect bacteria from harmful enzymes, from changes in pH, from the predatory bacterium Bdellovibrio, a parasitic bacterium that actually uses its motility to penetrate other bacteria and replicate within their cytoplasm, and from bacteriophages.
b. The S-layer can function as an adhesin, enabling the bacterium to adhere to host cells and environmental surfaces, colonize, and resist flushing.
c. The S-layer may contribute to virulence by protecting the bacterium against complement attack and phagocytosis.
d. The S-layer may act as a as a coarse molecular sieve.
Summary
1. The vast majority of the domain Bacteria have a rigid cell wall composed of peptidoglycan.
2. The peptidoglycan cell wall surrounds the cytoplasmic membrane and prevents osmotic lysis.
3. Peptidoglycan is composed of interlocking chains of building blocks called peptidoglycan monomers.
4. In order to grow following binary fission, bacteria have to synthesize new peptidoglycan monomers in the cytoplasm, transport those monomers across the cytoplasmic membrane, put breaks in the existing cell wall so the monomers can be inserted, connect the monomers to the existing peptidoglycan, and cross-link the rows and layers of peptidoglycan.
5. Many antibiotics inhibit peptidoglycan synthesis in bacteria and lead to osmotic lysis of the bacteria.
6. Most bacteria can be placed into one of three groups based on their color after specific staining procedures are performed: Gram-positive, Gram-negative, or acid-fast. These staining reactions are due to fundamental differences in the bacterial cell wall.
7. Gram-positive bacteria stain purple after Gram staining while Gram-negative bacteria stain pink.
8. Acid-fast bacteria stain red after acid-fast staining.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. A monomer of peptidoglycan consists of _____________, _____________, and _______________. (ans)
2. State the function of peptidoglycan in bacteria. (ans)
3. State the role of the following enzymes in peptidoglycan synthesis:
1. autolysins (ans)
2. bactoprenols (ans)
3. transpeptidases (ans)
4. transglycosylase (ans)
4. A penicillin is used to treat a bacterial infection. Describe the mechanism by which this antibiotic eventually kills the bacteria. (ans)
5. Gram-positive bacteria stain ____________ (ans) after Gram staining while Gram-negative bacteria stain _____________ (ans).
6. Bacteria normally live in a hypotonic environment. Since water flows into a cell in an environment that is hypotonic, why don't the bacteria burst from osmotic pressure? (ans)
7. Multiple Choice (ans)
2.3: The Peptidoglycan Cell Wall
State what color Gram-positive bacteria stain after the Gram stain procedure. Describe the composition of a Gram-positive cell wall and indicate the possible beneficial functions to the bacterium of peptidoglycan, teichoic acids, and surface proteins. Briefly describe how PAMPs of the Gram-positive cell wall can promote inflammation. State the function of bacterial adhesins, secretion systems, and invasins. Define antigen and epitope. Highlighted Bacterium Read the description of Enterococcus, andmatch the bacterium with the description of the organism and the infection it causes.
For More Information: Preview of the Gram stain from Lab 6.
Flash animation illustrating the interaction of the Gram's stain reagents at a molecular level
© Daniel Cavanaugh, Mark Keen, authors, Licensed for use, ASM MicrobeLibrary.
Common Gram-positive bacteria of medical importance include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, and Clostridium species.
Structure and Composition of the Gram-Positive Cell Wall
1. In electron micrographs, the Gram-positive cell wall appears as a broad, dense wall 20-80 nm thick and consisting of numerous interconnecting layers of peptidoglycan (see Figs. 1A and 1B). Chemically, 60 to 90% of the Gram-positive cell wall is peptidoglycan. In Gram-positive bacteria it is thought that the peptidoglycan is laid down in cables of several cross-linked glycan strands approximately 50 nm wide. These cables then themselves become cross-linked for further cell wall strength.
2. Interwoven in the cell wall of Gram-positive are teichoic acids and lipoteichoic acids. Teichoic acids extend through and beyond the rest of the cell wall and are polyalcohols composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol and are covalently bound to the peptidoglycan. Teichoic acids covalently bound to cytoplasmic membrane lipids are called lipoteichoic acids (see Figure \(1\)B).
3. The outer surface of the peptidoglycan is studded with surface proteins that differ with the strain and species of the bacterium (see Figure \(1\)B).
4. The periplasm is the gelatinous material between the peptidoglycan and the cytoplasmic membrane.
To view an electron micrograph of Streptococcus showing a Gram-positive cell wall, see the Rockefeller University web page.
Functions of the Gram-Positive Cell Wall Components
1. The peptidoglycan in the Gram-positive cell wall prevents osmotic lysis.
2. The teichoic acids probably help make the cell wall stronger (see Figure \(1\)B).
3. The surface proteins (see Figure \(1\)B) in the bacterial peptidoglycan, depending on the strain and species, carry out a variety of activities.
a. Some surface proteins function as enzymes.
b. Other proteins serve as adhesins. Adhesins enable the bacterium to adhere intimately to host calls and other surfaces in order to colonize those cells and resist flushing (See Figure \(2\) ).
c. Many bacteria involved in infection have the ability to co-opt the functions of host cells for the bacterium's own benefit. This is done by way of bacterial secretions systems that enable the bacterium to directly inject bacterial effector molecules into the cytoplasm of the host cell in order to alter its cellular machinery or cellular communication to the benefit of the bacteria. They do this by producing secretion systems such as the type 3 secretion system that produces hollow, needle-like tubes called injectisomes. Certain bacteria, for example, inject invasins into the cytoplasm of the host cell that enable the bacterium to enter that cell.
The role of these cell wall surface proteins will be discussed in greater detail later in Unit 3 under Bacterial Pathogenicity.
4. The periplasm contains enzymes for nutrient breakdown.
Significance of Gram-Positive Cell Wall Components to the Initiation of Body Defenses
The body has two immune systems: the innate immune system and the adaptive immune system.
1. Innate immunity is an antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to almost any microbe. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.
2. Adaptive (acquired) immunity refers to antigen-specific defense mechanisms that take several days to become protective and are designed to react with and remove a specific antigen. This is the immunity one develops throughout life.
Initiation of Innate Immunity
In order to protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPs. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometime referred to as microbe-associated molecular patterns or MAMPs.)
Fragments of peptidoglycan and teichoic acids are PAMPS associated with the cell wall of Gram-positive bacteria. In addition, bacteria and other microorganisms also possess mannose-rich glycans (short carbohydrate chains with the sugar mannose or fructose as the terminal sugar) that function as PAMPs. These mannose-rich glycans are common in microbial glycoproteins and glycolipids but rare in those of humans (see Figure \(3\)).
These PAMPS bind to pattern-recognition receptors or PRRs on a variety of defense cells of the body and trigger such innate immune defenses as inflammation, fever, and phagocytosis.
Inflammation is the first response to infection and injury and is critical to body defense. Basically, the inflammatory response is an attempt by the body to restore and maintain homeostasis after injury. Most of the body defense elements are located in the blood, and inflammation is the means by which body defense cells and body defense chemicals leave the blood and enter the tissue around an injured or infected site.
Body defense cells such as macrophages, and dendritic cells have pattern recognition receptors such as toll-like receptors on their surface that are specific for the peptidoglycan fragments and lipoteichoic acids in the Gram-positive cell wall and/or to NODs in their cytoplasm that are specific for peptidoglycan fragments.
The binding of these cell wall components to their corresponding pattern recognition receptors triggers the macrophages to release various defense regulatory chemicals called cytokines, including IL-1, IL-6, IL-8, TNF-alpha, and PAF. The cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate both the complement pathways and the coagulation pathway (see Figure \(4\)).
The peptidoglycan and teichoic acids also activate the alternative complement pathway and the lectin pathway, innate immune defense pathways that play a variety of roles in body defense.
Innate immunity will be discussed in greater detail in Unit 5.
Initiation of Adaptive Immunity
Proteins and polysaccharides associated with the Gram-positive cell wall function as antigens and initiate adaptive immunity. An antigen is defined as a molecular shape that reacts with antibody molecules and with antigen receptors on lymphocytes. We recognize those molecular shapes as foreign or different from our body's molecular shapes because they fit specific antigen receptors on our B-lymphocytes and T-lymphocytes, the cells that carry out adaptive immunity.
The actual portions or fragments of an antigen that react with antibodies and with receptors on B-lymphocytes and T-lymphocytes are called epitopes. An epitope is typically a group of 5-15 amino acids with a unique shape that makes up a portion of a protein antigen, or 3-4 sugar residues branching off of a polysaccharide antigen. A single microorganism has many hundreds of different shaped epitopes that our lymphocytes can recognize as foreign and mount an adaptive immune response against.
The body recognizes an antigen as foreign when epitopes of that antigen bind to B-lymphocytes and T-lymphocytes by means of epitope-specific receptor molecules having a shape complementary to that of the epitope. The epitope receptor on the surface of a B-lymphocyte is called a B-cell receptor and is actually an antibody molecule. The receptor on a T-lymphocyte is called a T-cell receptor (TCR).
There are two major branches of the adaptive immune responses: humoral immunity and cell-mediated immunity.
1. Humoral immunity : Humoral immunity involves the production of antibody molecules in response to an antigen and is mediated by B-lymphocytes. Through a variety of mechanisms, these antibodies are able to remove or neutralize microorganisms and their toxins after binding to their epitopes. For example, antibodies made against cell wall antigens can stick bacteria to phagocytes, a process called opsonization. Antibodies made against cell wall adhesins can prevent bacteria from adhering to and colonizing host cells.
2. Cell-mediated immunity : Cell-mediated immunity involves the production of cytotoxic T-lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigen and is mediated by T-lymphocytes. These defense cells help to remove infected cells and cancer cells displaying foreign epitopes.
Adaptive immunity will be discussed in greater detail in Unit 6.
Significance of Gram-Positive Cell Wall Components to Bacterial Pathogenicity
During severe systemic infections with large numbers of bacteria present, however, high levels of Gram-positive PAMPs are released resulting in excessive cytokine production by the macrophages and other cells and this, in turn, can harm the body (see Figure \(5\)).
Summary
1. Because of the nature of their cell wall, Gram-positive bacteria stain purple after Gram staining.
2. The Gram-positive cell wall consists of many interconnected layers of peptidoglycan and lacks an outer membrane.
3. Peptidoglycan prevents osmotic lysis in the hypotonic environment in which most bacteria live.
4. Teichoic acids and lipoteichoic acids are interwoven through the peptidoglycan layers.
5. Surface proteins embedded in the cell wall can function as adhesins, secretion systems, and enzymes.
6. The Gram-positive cell wall activates both the body's innate immune defenses and its adaptive immune defenses.
7. The body activates innate immunity by recognizing molecules unique to microorganisms that are not associated with human cells called pathogen-associated molecular patterns or PAMPs. PAMPs bind to Pattern-recognition receptors (PRRs) on defense cells to trigger the production of inflammatory cytokines.
8. Inflammation is the means by which the body delivers defense cells and defense molecules to an infection site,however, excessive inflammation can be harmful and even deadly to the body.
9. PAMPs associated with the Gram-positive cell wall include peptidoglycan monomers, teichoic acids, lipoteichoic acids, and mannose-rich sugar chains.
10. An antigen is a molecular shape that reacts with antigen receptors on lymphocytes to initiate an adaptive immune response.
11. Cell wall molecules can also trigger adaptive immunity such as the production of antibody molecules against bacterial cell wall antigens.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State what color Gram-positive bacteria appear after the Gram stain procedure. (ans)
2. Describe the structure and appearance of a Gram-positive cell wall. (ans)
3. State the beneficial function to the bacterium of the following components of the gram-positive cell wall:
1. peptidoglycan (ans)
2. teichoic acids (ans)
3. adhesins (ans)
4. invasins (ans)
4. Briefly describe how PAMPs of the Gram-positive cell wall can promote inflammation. (ans)
5. Define antigen. (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.3%3A_The_Peptidoglycan_Cell_Wall/2.3A%3A_The_Gram-Positive_Cell_Wall.txt |
State what color Gram-negative bacteria stain after the Gram stain procedure. Describe the composition of a Gram-negative cell wall and indicate the possible beneficial functions to the bacterium of peptidoglycan, the outer membrane, lipopolysaccharides, porins, and surface proteins. Briefly describe how LPS and other PAMPs of the Gram-negative cell wall can promote inflammation. State the function of bacterial adhesins, secretion systems, and invasins. Define periplasm. Define antigen and epitope. Highlighted Bacterium Read the description of Escherichia coli, and match the bacterium with the description of the organism and the infection it causes. Highlighted Disease: Urinary Tract Infections (UTIs) Define the following: urethritis cystitis pyelonephritis Name at least 4 risk factors for UTIs. Name the most common bacterium to cause UTIs; name at least 3 other bacteria that commonly cause UTIs. Name at least 3 common symptoms of UTIs.
We will now look at the Gram-negative bacterial cell wall. As mentioned in the previous section on peptidoglycan, Gram-negative bacteria are those that decolorize during the Gram stain procedure, pick up the counterstain safranin, and appear pink (Figure \(2\)B.1).
Common Gram-negative bacteria of medical importance include Salmonella species, Shigella species, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Proteus species, and Pseudomonas aeruginosa.
Escherichia coli
Organism
• Escherichia coli is a moderately-sized Gram-negative bacillus.
• Possess a peritrichous arrangement of flagella.
• Facultative anaerobe.
Habitat
• Normal flora of the intestinal tract in humans and animals.
Source
• Usually the patient's own fecal flora; some transmission is patient-to-patient.
Clinical Disease
• E. coli causes around 80 percent of all uncomplicated urinary tract infections (UTIs) and more than 50 percent of nosocomial UTIs. UTIs account for more than 7, 000,000 physician office visits per year in the U.S. Between 35 and 40 percent of all nosocomial infections, about 900,000 per year in the U.S., are UTIs and are usually associated with urinary catheterization.
• E. coli causes wound infections, usually a result of fecal contamination of external wounds or a result of wounds that cause trauma to the intestinal tract, such as surgical wounds, gunshot wounds, knife wounds, etc.
• E. coli is by far the most common Gram-negative bacterium causing sepsis. Septicemia is a result of bacteria getting into the blood. They are usually introduced into the blood from some other infection site, such as an infected kidney, wound, or lung. There are approximately 500,000 cases of septicemia per year in the U.S. and the mortality rate is between 20 and 50 percent. Approximately 45 percent of the cases of septicemia are due to Gram-negative bacteria. Klebsiella, Proteus, Enterobacter, Serratia, and E. coli, are all common gram-negative bacteria causing septicemia.
• E. coli, along with group B streptococci, are the leading cause of neonatal meningitis.
• While E. coli is one of the dominant normal flora in the intestinal tract of humans and animals, some strains can cause gastroenteritis, an infection of the intestinal tract.
• Enterotoxigenc E. coli (ETEC) produce enterotoxins that cause the loss of sodium ions and water from the small intestines resulting in a watery diarrhea. Over half of all travelers' diarrhea is due to ETEC; almost 80,000 cases a year in the U.S.
• Enteropathogenic E. coli (EPEC) cause an endemic diarrhea in areas of the developing world, especially in infants younger than 6 months. The bacterium disrupts the normal microvilli on the epithelial cells of the small intestines resulting in maladsorbtion and diarrhea.
• Enteroaggregative E. coli (EAEC) is a cause of persistant diarrhea in developing countries. It probably causes diarrhea by adhering to mucosal epithelial cells of the small intestines and interfering with their function.
• Enteroinvasive E. coli (EIEC) invade and kill epithelial cells of the large intestines causing a dysentery-type syndrome similar to Shigella common in underdeveloped countries.
• Enterohemorrhagic E. coli (EHEC), such as E. coli 0157:H7, produce a shiga-like toxin that kills epithelial cells of the large intestines causing hemorrhagic colitis, a bloody diarrhea. In rare cases, the shiga-toxin enters the blood and is carried to the kidneys where, usually in children, it damages vascular cells and causes hemolytic uremic syndrome. E. coli 0157:H7 is thought to cause more than 20,000 infections and up to 250 deaths per year in the U.S.
• Diffuse aggreegative E. coli(DAEC) causes watery diarrhea in infants 1-5 years of age. They stimulate elongation of the microvilli on the epithelial cells lining the small intestines.
• For More Information: The Gram Stain from Lab 6.
• Flash animation illustrating the interaction of the Gram's stain reagents at a molecular level © Daniel Cavanaugh, Mark Keen, authors, Licensed for use, ASM MicrobeLibrary.
• Highlighted Infection: Urinary Tract Infections (UTIs)
Structure and Composition of the Gram-Negative Cell Wall
The periplasm is the gelatinous material between the outer membrane, the peptidoglycan, and the cytoplasmic membrane. This periplasmic space is about 15nm wide and contains a variety of hydrolytic enzymes for nutrient breakdown, periplasmic binding proteins for transport via the ATP-binding cassette (ABC) system, and chemoreceptors for chemotaxis (discussed under Bacterial Flagella later in this Unit).
Concept map for the Gram-negative cell wall.
Functions of the Gram-Negative Cell Wall Components
c. Many bacteria involved in infection have the ability to co-opt the functions of host cells for the bacterium's own benefit. This is done by way of bacterial secretions systems that enable the bacterium to directly inject bacterial effector molecules into the cytoplasm of the host cell in order to alter its cellular machinery or cellular communication to the benefit of the bacteria. They do this by producing secretion systems such as the type 3 secretion system that produces hollow, needle-like tubes called injectisomes. Certain bacteria, for example, inject invasins into the cytoplasm of the host cell that enable the bacterium to enter that cell.
The role of these cell wall surface proteins will be discussed in greater detail later in Unit 3 under Bacterial Pathogenicity.
4. The periplasm contains enzymes for nutrient breakdown as well as periplasmic binding proteins to facilitate the transfer of nutrients across the cytoplasmic membrane.
The Role of Gram-Negative Cell Wall Components to the Initiation of Body Defenses
The body has two immune systems: the innate immune system and the adaptive immune system. Innate immunity is an antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to almost any microbe. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection. Adaptive (acquired) immunity refers to antigen-specific defense mechanisms that take several days to become protective and are designed to react with and remove a specific antigen. This is the immunity one develops throughout life.
Initiation of Innate Immunity
To protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPS. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometime referred to as microbe-associated molecular patterns or MAMPs.)
LPS, porins, and fragments of peptidoglycan are PAMPs associated with the cell wall of Gram-negative bacteria. In addition, bacteria and other microorganisms also possess mannose-rich glycans (short carbohydrate chains with the sugar mannose or fructose as the terminal sugar) that function as PAMPs. These mannose-rich glycans are common in microbial glycoproteins and glycolipids but rare in those of humans (Figure \(3\)).
These PAMPS bind to pattern-recognition receptors or PRRs on a variety of defense cells of the body and triggers innate immune defenses such as inflammation , fever, and phagocytosis.
Inflammation is the first response to infection and injury and is critical to body defense. Basically, the inflammatory response is an attempt by the body to restore and maintain homeostasis after injury. Most of the body defense elements are located in the blood, and inflammation is the means by which body defense cells and body defense chemicals leave the blood and enter the tissue around an injured or infected site.
Body defense cells called macrophages, and dendritic cells have pattern recognition receptors such as toll-like receptors on their surface that are specific for the peptidoglycan fragments and LPS in the Gram-negative cell wall and/or to NODs in their cytoplasm that are specific for peptidoglycan fragments. The binding of these cell wall components to their corresponding pattern recognition receptors triggers the macrophages to release various defense regulatory chemicals called cytokines, including IL-1, IL-6, IL-8, TNF-alpha, and PAF. The cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate both the complement pathways and the coagulation pathway (Figure \(4\)).
The LPS binds to a LPS-binding protein circulating in the blood and this complex, in turn, binds to a receptor molecule (CD14) found on the surface of body defense cells called macrophages. This is thought to promote the ability of the toll-like receptor pair TLR-4/TLR4 to respond to the LPS. The binding of these cell wall components to their corresponding pattern recognition receptors triggers macrophages to release various defense regulatory chemicals called cytokines, including IL-1, IL-6, IL-8, TNF-alpha, and PAF. The cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate both the complement pathways and the coagulation pathway (Figure \(4\)).
Explain how the body is able to recognize these bacteria and eventually send phagocytes and defense molecules to the infected site. How might this mechanism lead to the symptoms of the infection?
The LPS also activates the alternative complement pathway and the lectin pathway, innate defense pathways that play a variety of roles in body defense.
Innate immunity will be discussed in greater detail in Unit 5.
Initiation of Adaptive Immunity
Proteins and polysaccharides associated with the Gram-negative cell wall function as antigens and initiate adaptive immunity. An antigen is defined as a molecular shape that reacts with antibody molecules and with antigen receptors on lymphocytes. We recognize those molecular shapes as foreign or different from our body's molecular shapes because they fit specific antigen receptors on our B-lymphocytes and T-lymphocytes, the cells that carry out adaptive immunity.
The actual portions or fragments of an antigen that react with antibodies and with receptors on B-lymphocytes and T-lymphocytes are called epitopes. An epitope is typically a group of 5-15 amino acids with a unique shape that makes up a portion of a protein antigen, or 3-4 sugar residues branching off of a polysaccharide antigen. A single microorganism has many hundreds of different shaped epitopes that our lymphocytes can recognize as foreign and mount an adaptive immune response against.
The body recognizes an antigen as foreign when epitopes of that antigen bind to B-lymphocytes and T-lymphocytes by means of epitope-specific receptor molecules having a shape complementary to that of the epitope. The epitope receptor on the surface of a B-lymphocyte is called a B-cell receptor and is actually an antibody molecule. The receptor on a T-lymphocyte is called a T-cell receptor (TCR).
There are two major branches of the adaptive immune responses: humoral immunity and cell-mediated immunity.
1. Humoral immunity: Humoral immunity involves the production of antibody molecules in response to an antigen and is mediated by B-lymphocytes. Through a variety of mechanisms, these antibodies are able to remove or neutralize microorganisms and their toxins after binding to their epitopes. For example, antibodies made against cell wall antigens can stick bacteria to phagocytes, a process called opsonization. Antibodies made against cell wall adhesins can prevent bacteria from adhering to and colonizing host cells.
2. Cell-mediated immunity: Cell-mediated immunity involves the production of cytotoxic T-lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigen and is mediated by T-lymphocytes. These defense cells help to remove infected cells and cancer cells displaying foreign epitopes.
Adaptive immunity will be discussed in greater detail in Unit 6.
Significance of Gram-Negative Cell Wall Components to Bacterial Pathogenicity
The lipid A portion of the LPS portion in the outer membrane is also known as endotoxin. During severe systemic infections with large numbers of bacteria present, high levels of LPS are released resulting in excessive cytokine production by the macrophages and other cells and this, in turn, can harm the body (Figure \(5\)).
Summary
1. Because of the nature of their cell wall, Gram-negative bacteria stain pink after Gram staining.
2. The Gram-negative cell wall consists of 2-3 interconnected layers of peptidoglycan surrounded by an outer membrane.
3. Peptidoglycan prevents osmotic lysis in the hypotonic environment in which most bacteria live.
4. The outer membrane is a semipermeable structure that contains pore-forming proteins called porins that allow nutrients to pass through the outer membrane.
5. Surface proteins embedded in the cell wall can function as adhesins, secretion systems, and enzymes.
6. The Gram-negative cell wall activates both the body's innate immune defenses and its adaptive immune defenses.
7. The body activates innate immunity by recognizing molecules unique to microorganisms that are not associated with human cells called pathogen-associated molecular patterns or PAMPs. PAMPs bind to Pattern-recognition receptors (PRRs) on defense cells to trigger the production of inflammatory cytokines.
8. Inflammation is the means by which the body delivers defense cells and defense molecules to an infection site, however, excessive inflammation, can be harmful and even deadly to the body.
9. PAMPs associated with the Gram-negative cell wall include peptidoglycan monomers, lipopolysaccharide (LPS), porins, and mannose-rich sugar chains.
10. An antigen is a molecular shape that reacts with antigen receptors on lymphocytes to initiate an adaptive immune response.
11. Cell wall molecules can also trigger adaptive immunity such as the production of antibody molecules against bacterial cell wall antigens.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State what color Gram-negative bacteria appear after the Gram stain procedure. (ans)
2. Describe the structure and appearance of a Gram-negative cell wall. (ans)
3. State the beneficial function to the bacterium of the following components of the gram-negative cell wall:
1. peptidoglycan (ans)
2. outer membrane (ans)
3. adhesins (ans)
4. invasins (ans)
4. Briefly describe how the LPS (endotoxin) of the Gram-negative cell wall can promote inflammation. (ans)
5. Define epitope. (ans)
6. When Gram-negative bacteria enter the blood and cause septicemia, most of the harm to the body is due to a massive inflammatory response. What might explain this? (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.3%3A_The_Peptidoglycan_Cell_Wall/2.3B%3A_The_Gram-Negative_Cell_Wall.txt |
Fundamental Statements for this Learning Object:
In this section on Prokaryotic Cell Anatomy we are looking at the various anatomical parts that make up a bacterium. As mentioned in the introduction to this section, a typical bacterium usually consists of:
• a cytoplasmic membrane surrounded by a peptidoglycan cell wall and maybe an outer membrane;
• a fluid cytoplasm containing a nuclear region (nucleoid) and numerous ribosomes; and
• often various external structures such as a glycocalyx, flagella, and pili.
There are three primary types of bacterial cell wall: Gram-positive, Gram-negative, and acid-fast. We will now look at the acid-fast cell wall.
Acid-fast bacteria stain poorly with the Gram stain procedure, appearing weakly Gram-positive or Gram-variable. They are usually characterized using the acid-fast staining procedure. As mentioned in the previous section on peptidoglycan, bacteria with an acid-fast cell wall resist decolorization with an acid-alcohol mixture during the acid-fast staining procedure , retain the initial dye carbol fuchsin and appear red (Figure \(\PageIndex{1; left}\)). Common acid-fast bacteria of medical importance include Mycobacterium tuberculosis, Mycobacterium leprae,Mycobacterium avium-intracellulare complex, and Nocardia species.
Structure and Composition of the Acid-Fast Cell Wall
Acid-fast bacteria are gram-positive, but in addition to peptidoglycan, the outer membrane or envelope of the acid-fast cell wall of contains large amounts of glycolipids, especially mycolic acids that in the genus Mycobacterium, make up approximately 60% of the acid-fast cell wall (Figure \(2\)).
• Layer 1: The acid-fast cell wall of Mycobacterium has a thin, inner layer of peptidoglycan.
• Layer 2: The peptidoglycan layer is, in turn, linked to arabinogalactan (D-arabinose and D-galactose).
• Layer 3: The arabinogalactan is then linked to an outer membrane containing high-molecular weight mycolic acids. The arabinogalactan/mycolic acid layer is overlaid with a layer of polypeptides and mycolic acids consisting of free lipids, glycolipids, and peptidoglycolipids. Other glycolipids include lipoarabinomannan and phosphatidyinositol mannosides (PIM). Like the outer membrane of the gram-negative cell wall, porins are required to transport small hydrophilic molecules through the outer membrane of the acid-fast cell wall.
• Layer 4: The outer surface of the acid-fast cell wall is studded with surface proteins that differ with the strain and species of the bacterium.
• Layer 5:The periplasm is the gelatinous material between the peptidoglycan and the cytoplasmic membrane.
Functions of the Acid-Fast Cell Wall Components
• Layer 1: The peptidoglycan prevents osmotic lysis.
• Layer 2: The arabinogalactan layer is linked to both the peptidoglycan and to the mycolic acid outer membrane and probably provides additional strength to the cell wall.
• Layer 3: The mycolic acids and other glycolipids also impede the entry of chemicals causing the organisms to grow slowly and be more resistant to chemical agents and lysosomal components of phagocytes than most bacteria (Figure \(2\)). There are far fewer porins in the acid-fast cell wall compared to the gram-negative cell wall and the pores are much longer. This is thought to contribute significantly to the lower permeability of acid-fast bacteria.
• Layer 4:The surface proteins in the acid-fast cell wall, depending on the strain and species, carry out a variety of activities, including functioning as enzymes and serving as adhesins, which enable the bacterium to adhere intimately to host cells and other surfaces in order to colonize and resist flushing.
• Layer 15 The periplasm contains enzymes for nutrient breakdown.
Exercise: Think-Pair-Share Questions
Mycobacterium tuberculosis is a very slow growing bacterium with a generation time often measured in days to weeks. It is also resistant to the vast majority of antibiotics that are commonly effective against other bacteria and treatment is typically with a combination of drugs for up to 9 months.
Based on what we just learned, explain what might account for these two characteristics.
Significance of Acid-Fast Cell Wall Components to the Initiation of Body Defenses
The body has two immune systems: the innate immune system and the adaptive immune system.
1. Innate immunity is an antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to almost any microbe. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.
2. Adaptive (acquired) immunity refers to antigen-specific defense mechanisms that take several days to become protective and are designed to react with and remove a specific antigen. This is the immunity one develops throughout life.
Initiation of Innate Immunity
To protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPs. Pathogenic Mycobacterium species such as Mycobacterium tuberculosis and Mycobacterium leprae release mycolic acid, arabinogalactan, and peptidoglycan fragments from their acid-fast cell wall. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometime referred to as microbe-associated molecular patterns or MAMPs.)
These PAMPS bind to pattern-recognition receptors or PRRs on a variety of defense cells of the body causing them to synthesize and secrete a variety of proteins called cytokines. These cytokines can, in turn promote innate immune defenses such as inflammation , phagocytosis, activation of the complement pathways , and activation of the coagulation pathway .
Inflammation is the first response to infection and injury and is critical to body defense. Basically, the inflammatory response is an attempt by the body to restore and maintain homeostasis after injury. Most of the body defense elements are located in the blood, and inflammation is the means by which body defense cells and body defense chemicals leave the blood and enter the tissue around an injured or infected site.
Body defense cells called macrophages , and dendritic cells have pattern recognition receptors such as toll-like receptors on their surface that are specific for the peptidoglycan fragments and mycolic acids in the acid-fast cell wall and/or to NODs in their cytoplasm that are specific for peptidoglycan fragments. The binding of these cell wall components to their corresponding pattern recognition receptors triggers the macrophages to release various defense regulatory chemicals called cytokines, including IL-1 and TNF-alpha. The cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate both the complement pathways and the coagulation pathway.
Innate immunity will be discussed in greater detail in Unit 5.
Initiation of Adaptive Immunity
Proteins and polysaccharides associated with the acid-fast cell wall function as antigens and initiate adaptive immunity. An antigen is defined as a molecular shape that reacts with antibody molecules and with antigen receptors on lymphocytes. We recognize those molecular shapes as foreign or different from our body's molecular shapes because they fit specific antigen receptors on our B-lymphocytes and T-lymphocytes, the cells that carry out adaptive immunity.
The actual portions or fragments of an antigen that react with antibodies and with receptors on B-lymphocytes and T-lymphocytes are called epitopes . An epitope is typically a group of 5-15 amino acids with a unique shape that makes up a portion of a protein antigen, or 3-4 sugar residues branching off of a polysaccharide antigen. A single microorganism has many hundreds of different shaped epitopes that our lymphocytes can recognize as foreign and mount an adaptive immune response against.
The body recognizes an antigen as foreign when epitopes of that antigen bind to B-lymphocytes and T-lymphocytes by means of epitope-specific receptor molecules having a shape complementary to that of the epitope. The epitope receptor on the surface of a B-lymphocyte is called a B-cell receptor and is actually an antibody molecule. The receptor on a T-lymphocyte is called a T-cell receptor (TCR).
There are two major branches of the adaptive immune responses: humoral immunity and cell-mediated immunity.
1. Humoral immunity: Humoral immunity involves the production of antibody molecules in response to an antigen and is mediated by B-lymphocytes. Through a variety of mechanisms, these antibodies are able to remove or neutralize microorganisms and their toxins after binding to their epitopes.
2. Cell-mediated immunity: Cell-mediated immunity involves the production of cytotoxic T-lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigen and is mediated by T-lymphocytes. These defense cells help to remove infected cells and cancer cells displaying foreign epitopes.
Adaptive immunity will be discussed in greater detail in Unit 6.
Significance of Acid-Fast Cell Wall Components to Bacterial Pathogenicity
Most of the damage in the lungs during tuberculosis is thought to be due to the inflammatory effects from excessive TNF-alpha production, along with the release of toxic lysosomal components of the macrophages trying to kill the Mycobacterium tuberculosis.
Click on this link, read the description of Mycobacterium tuberculosis, and be able to match the bacterium with its description on an exam.
Antimicrobial Agents that Inhibit Acid-Fast Cell Wall Synthesis to Control Mycobacterium Species
INH (isoniazid) blocks the incorporation of mycolic acid into acid-fast cell walls while ethambutol interferes with the incorporation of arabinoglactan (Figure \(2\)). Both inhibit synthesis of the acid-fast cell wall. Pyrazinamide inhibits fatty acid synthesis in Mycobacterium tuberculosis.
Think-Pair-Share Questions
Look at the following transmission electron micrograph and Gram stain of the same bacterium.
(left) Transmission electron micrograph: (right) Gram stain
1. Is this organism Gram-positive, Gram-negative, or acid-fast?
2. How can you tell? State all reasons.
Summary
1. Because of the nature of their cell wall, acid-fast bacteria stain red after acid-fast staining.
2. The genus Mycobacterium and the genus Nocardia are among the few bacteria possessing an acid-fast cell wall.
3. The acid-fast cell wall consists of a thin, inner layer of peptidoglycan linked to a layer of arabinogalactin, which in turn is linked to an outer membrane containing mycolic acids and overlaid with a variety of polypeptides and glycolipids.
4. Porins are required to transport small hydrophilic molecules through the outer membrane of the acid-fast cell wall.
5. The acid-fast cell wall activates both the body's innate immune defenses and its adaptive immune defenses.
6. The body activates innate immunity by recognizing molecules unique to microorganisms that are not associated with human cells called pathogen-associated molecular patterns or PAMPs. PAMPs bind to Pattern-recognition receptors (PRRs) on defense cells to trigger the production of inflammatory cytokines.
7. Inflammation is the means by which the body delivers defense cells and defense molecules to an infection site, however, excessive inflammation, can be harmful and even deadly to the body.
8. PAMPs associated with the acid-fast cell wall include peptidoglycan monomers, arabinogalactin, and mycolic acids.
9. An antigen is a molecular shape that reacts with antigen receptors on lymphocytes to initiate an adaptive immune response.
10. Cell wall molecules can also trigger adaptive immunity such as the production of antibody molecules against bacterial cell wall antigens.
11. A few antimicrobial chemotherapeutic agents inhibit acid-fast cell wall synthesis
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State what color acid-fast bacteria appear after the acid-fast stain procedure. (ans)
2. Describe the structure and appearance of an acid-fast cell wall. (ans)
3. State the beneficial function to the bacterium of the following components of the acid-fast cell wall:
1. peptidoglycan (ans)
2. mycolic acid and other glycolipids (ans)
3. porins (ans)
4. Mycobacterium tuberculosis is much more resistant to antibiotics and disinfectants than most other bacteria. It also grows much more slowly. Why might this be? (ans)
5. Multiple Choice Cell Wall Quiz (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.3%3A_The_Peptidoglycan_Cell_Wall/2.3C%3A_The_Acid-Fast_Cell_Wall.txt |
Name the various structures that may be located within the cytoplasm of bacteria.
In this section on Prokaryotic Cell Anatomy we are looking at the various anatomical parts that make up a bacterium. As mentioned in the introduction to this section, a typical bacterium usually consists of:
• a cytoplasmic membrane surrounded by a peptidoglycan cell wall and maybe an outer membrane;
• a fluid cytoplasm containing a nuclear region (nucleoid) and numerous ribosomes; and
• often various external structures such as a glycocalyx, flagella, and pili.
2.4: Cellular Components within the Cytoplasm
Define the following: exoenzymes endoenzymes. cytosol State the primary function of the bacterial cytoplasm. Define the following: metabolism catabolic reactions anabolic reactions.
We will now look at the bacterial cytoplasm. In bacteria, the cytoplasm refers to everything enclosed by the cytoplasmic membrane. About 80% of the cytoplasm of bacteria is composed of water. Within the cytoplasm can be found nucleic acids (DNA and RNA), enzymes and amino acids, carbohydrates, lipids, inorganic ions, and many low molecular weight compounds. The liquid component of the cytoplasm is called the cytosol. Some groups of bacteria produce cytoplasmic inclusion bodies that carry out specialized cellular functions.
Functions
While bacteria secrete exoenzymes to hydrolize macromolecules into smaller molecules capable of being transported across the cytoplasmic membrane, the cytoplasm is the site of most bacterial metabolism. This includes catabolic reactions in which molecules are broken down in order to obtain building block molecules for more complex cellular molecules and macromolecules, and anabolic reactions used to synthesize cellular molecules and macromolecules. The chemical reactions occuring within the bacterium are under the control of endoenzymes.
The various structurural filaments in the cytoplasm collectively make up the prokaryotic cytoskeleton. Prokaryotic cells possess analogs for all of the cytoskeletal proteins found in eukaryotic cells, as well as cytoskeletal proteins with no eukaryotic homologues. Cytoskeletal filaments play essential roles in determining the shape of a bacterium (coccus, bacillus, or spiral) and are also critical in the process of cell division by binary fission and in determining bacterial polarity.
Summary
1. In bacteria, the cytoplasm refers to anything enclosed by the cytoplasmic membrane.
2. The liquid portion of the cytoplasm is called the cytosol.
3. The cytoplasm is the site of most bacterial metabolism.
4. During catabolic reactions larger molecules are broken down to obtain cellular building block molecules and energy; during anabolic reactions cellular molecules and macromolecules are synthesized.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. _____ Enzymes that function within the bacterium. (ans)
_____ Chemical reactions in which more complex molecules are synthesized. (ans)
_____ Chemical reactions in which more complex molecules are broken down into smaller, more simple molecules. (ans)
1. Metabolism
2. Catabolic reactions
3. Anabolic reactions
4. Exoenzymes
5. Endoenzymes
2. State the primary function of bacterial cytoplasm. (ans)
2.4B: The Bacterial Chromosome an
Learning Objectives
1. Define genome.
2. Describe the composition of the bacterial chromosome.
3. Name the enzymes that enables bacterial DNA to become circular, supercoiled, and unwind during DNA replication.
4. Briefly describe the process of DNA replication.
5. State the function of the following enzymes in bacterial DNA replication:
1. DNA polymeraseIII
2. DNA polymerase II
3. DNA helicase
4. primase
5. DNA ligase
6. State the function of DNA.
7. In terms of protein synthesis, briefly describe the process of transcription and translation.
8. Briefly state how the following antibacterial chemotherapeutic agents affect bacteria:
1. fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.)
2. trimethoprim and sulfamethoxazole
We will now look at the bacterial chromosome located in the nuclear region called the nucleoid.
A. Structure and Composition of the Bacterial Chromosome
The term genome refers to the sum of an organism's genetic material. The bacterial genome is composed of a single molecule of chromosomal deoxyribonucleic acid or DNA and is located in a region of the bacterial cytoplasm visible when viewed with an electron microscope called the nucleoid. Unlike the eukaryotic nucleus, the bacterial nucleoid has no nuclear membrane or nucleoli.
In general it is thought that during DNA replication, each strand of the replicating bacterial DNA attaches to proteins at what will become the cell division plane. For example, Par proteins function to separate bacterial chromosomes to opposite poles of the cell during cell division. They bind to the origin of replication of the DNA and physically pull or push the chromosomes apart, similar to the mitotic apparatus of eukaryotic cells (Figure \(1\)).
In the center of the bacterium, a group of proteins called Fts (filamentous temperature sensitive) proteins interact to form a ring at the cell division plane. These proteins form the cell division apparatus known as the divisome and are directly involved in bacterial cell division by binary fission. The divisome is responsible for directing the synthesis of new cytoplasmic membrane and new peptidoglycan to form the division septum.
Since bacteria are haploid, that is they have only one chromosome and only reproduce asexually, there is also no meiosis in bacteria.
The bacterial chromosome is one long, single molecule of double stranded, helical, supercoiled DNA. In most bacteria, the two ends of the double-stranded DNA covalently bond together to form both a physical and genetic circle. The chromosome is generally around 1000 µm long and frequently contains as many as 3500 genes (Figure \(2\)). E. coli, a bacterium that is 2-3 µm in length, has a chromosome approximately 1400 µm long.
To enable a macromolecule this large to fit within the bacterium, histone-like proteins bind to the DNA, segregating the DNA molecule into around 50 chromosomal domains and making it more compact. A DNA topoisomerase enzyme called DNA gyrase then supercoils each domain around itself, forming a compacted mass of DNA approximately 0.2 µm in diameter. In actively growing bacteria, projections of the nucleoid extend into the cytoplasm. Presumably, these projections contain DNA that is being transcribed into mRNA.Supercoils are both inserted and removed by topoisomerases.
DNA topoisomerases are, therefore, essential in the unwinding, replication, and rewinding of the circular, supercoiled bacterial DNA. In order for the long molecule of DNA to fit within the bacterium, the DNA must be supercoiled. However, this supercoiled DNA must be uncoiled and relaxed in order for DNA polymerase to bind for DNA replication and RNA polymerase to bind for transcription of the DNA. For example, a topoisomerase called DNA gyrase catalyzes the negative supercoiling of the circular DNA found in bacteria. Topoisomerase IV, on the other hand, is involved in the relaxation of the supercoiled circular DNA, enabling the separation of the interlinked daughter chromosomes at the end of bacterial DNA replication.
B. DNA Replication in Bacteria
In general, DNA is replicated by uncoiling of the helix, strand separation by breaking of the hydrogen bonds between the complementary strands, and synthesis of two new strands by complementary base pairing. Replication begins at a specific site in the DNA called the origin of replication (oriC).
DNA replication is bidirectional from the origin of replication. To begin DNA replication, unwinding enzymes called DNA helicases cause short segments of the two parent DNA strands to unwind and separate from one another at the origin of replication to form two "Y"-shaped replication forks. These replication forks are the actual site of DNA copying (Figure \(3\)). All the proteins involved in DNA replication aggregate at the replication forks to form a replication complex called a replisome (Figure \(4\)).
Single-strand binding proteins bind to the single-stranded regions so the two strands do not rejoin. Unwinding of the double-stranded helix generates positive supercoils ahead of the replication fork. Enzymes called topoisomerases counteract this by producing breaks in the DNA and then rejoin them to form negative supercoils in order to relieve this stress in the helical molecule during replication.
As the strands continue to unwind and separate in both directions around the entire DNA molecule, new complementary strands are produced by the hydrogen bonding of free DNA nucleotides with those on each parent strand. As the new nucleotides line up opposite each parent strand by hydrogen bonding, enzymes called DNA polymerases join the nucleotides by way of phosphodiester bonds. Actually, the nucleotides lining up by complementary base pairing are deoxynucleotide triphosphates, composed of a nitrogenous base, deoxyribose, and three phosphates. As the phosphodiester bond forms between the 5' phosphate group of the new nucleotide and the 3' OH of the last nucleotide in the DNA strand, two of the phosphates are removed providing energy for bonding (see Figure \(6\)). In the end, each parent strand serves as a template to synthesize a complementary copy of itself, resulting in the formation of two identical DNA molecules (see Figure \(7\)). In bacteria, Par proteins function to separate bacterial chromosomes to opposite poles of the cell during cell division. They bind to the origin of replication of the DNA and physically pull or push the chromosomes apart, similar to the mitotic apparatus of eukaryotic cells. Fts proteins, such as FtsK in the divisome, also help in separating the replicated bacterial chromosome.
GIF animation illustrating DNA replication by complementary base pairing
In reality, DNA replication is more complicated than this because of the nature of the DNA polymerases. DNA polymerase enzymes are only able to join the phosphate group at the 5' carbon of a new nucleotide to the hydroxyl (OH) group of the 3' carbon of a nucleotide already in the chain. As a result, DNA can only be synthesized in a 5' to 3' direction while copying a parent strand running in a 3' to 5' direction.
Each DNA strand has two ends. The 5' end of the DNA is the one with the terminal phosphate group on the 5' carbon of the deoxyribose; the 3' end is the one with a terminal hydroxyl (OH) group on the deoxyribose of the 3' carbon of the deoxyribose (see Figure \(8\)). The two strands are antiparallel, that is they run in opposite directions. Therefore, one parent strand - the one running 3' to 5' and called the leading strand - can be copied directly down its entire length (see Figure \(9\)). However, the other parent strand - the one running 5' to 3' and called the lagging strand - must be copied discontinuously in short fragments (Okazaki fragments) of around 100-1000 nucleotides each as the DNA unwinds. This occurs, as mentioned above, at the replisome. The lagging DNA strand loops out from the leading strand and this enables the replisome to move along both strands pulling the DNA through as replication occurs. It is the actual DNA, not the DNA polymerase that moves during bacterial DNA replication (see Figure \(5\)).
In addition, DNA polymerase enzymes cannot begin a new DNA chain from scratch. They can only attach new nucleotides onto 3' OH group of a nucleotide in a preexisting strand. Therefore, to start the synthesis of the leading strand and each DNA fragment of the lagging strand, an RNA polymerase complex called a primase is required. The primase, which is capable of joining RNA nucleotides without requiring a preexisting strand of nucleic acid, first adds several comlementary RNA nucleotides opposite the DNA nucleotides on the parent strand. This forms what is called an RNA primer (see Figure \(10\)).
DNA polymerase III then replaces the primase and is able to add DNA nucleotides to the RNA primer (see Figure \(11\)). Later, DNA polymerase II digests away the RNA primer and replaces the RNA nucleotides of the primer with the proper DNA nucleotides to fill the gap (see Figure \(12\)). Finally, the DNA fragments themselves are hooked together by the enzyme DNA ligase (see Figure \(9\)). Yet even with this complicated procedure, a 1000 micrometer-long macromolecule of tightly-packed, supercoiled DNA can make an exact copy of itself in only about 10 minutes time under optimum conditions, inserting nucleotides at a rate of about 1000 nucleotides per second!
YouTube movie illustrating DNA replication in prokaryotic cells, #1.
YouTube movie illustrating DNA replication in prokaryotic cells, #2.
GIF animation illustrating the replication of leading and lagging strands of DNA
Animation of DNA replication.
Courtesy of HHMI's Biointeractive.
For More Information: Review of Prokaryotic DNA Replication from Unit 7
C. Functions of the Bacterial Chromosome
The chromosome is the genetic material of the bacterium. Genes located along the DNA are transcribed into RNA molecules, primarily messenger RNA (mRNA), transfer RNA (tRNA, and ribosomal RNA (rRNA). Messenger RNA is then translated into protein at the ribosomes.
• Transcription: Ribonucleic acid (RNA) is synthesized by complementary base pairing of ribonucleotides with deoxyribonucleotides to match a portion of one strand of DNA called a gene. Although genes are present on both strands of DNA, only one strand is transcribed for any given gene. Following transcription of genes into mRNA, 30S and 50S ribosomal subunits attach to the mRNA and tRNA inserts the correct amino acids which are subsequently joined to form a polypeptide or a protein through a process called translation.
• Translation: During translation, specific tRNA molecules pick up specific amino acids, transfer those amino acids to the ribosomes, and insert them in their proper place according to the mRNA "message." This is done by the anticodon portion of the tRNA molecules complementary base pairing with the codons along the mRNA.
In general then, DNA determines what proteins and enzymes an organism can synthesize and, therefore, what chemical reactions it is able to carry out.
D. The Bacterial Epigenome
The epigenome refers to a variety of chemical compounds that modify the genome typically by adding a methyl (CH3) group to the nucleotide base adenine at specific locations along the DNA molecule. This methylation can, in turn, either repress or activate transcription of specific genes. By basically turning genes on or off, the epigenome enables the bacterial genome to interact with and respond to the bacterium's environment. The epigenome can be inherited just like the genome.
All cells, including human cells, possess an epigenome. Just as the bacterial epigenome can affect the bacterial genome, bacteria, can affect our epigenome and subsequently modify the function of our genome by causing either DNA methylation of nucleotides or by modifying our histone proteins. The resulting modification can either help activate various genes involved in immune defenses, or, in the case of some pathogens, suppress immune response genes.
E. Significance of the Chromosome to the Initiation of Body Defense
To protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPS. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometimes referred to as microbe-associated molecular patterns or MAMPs.)
Bacterial and viral genomes contain a high frequency of unmethylated cytosine-guanine (CpG) dinucleotide sequences (a cytosine lacking a methyl or CH3 group and located adjacent to a guanine). Mammalian DNA has a low frequency of cytosine-guanine dinucleotides and most are methylated. These unmethylated cytosine-guanine dinucleotide sequences in bacterial DNA are PAMPS that bind to pattern-recognition receptors on a variety of defense cells of the body and triggers innate immune defenses such as inflammation, fever, and phagocytosis.
F. Antimicrobial Agents that Inhibiting Normal Nucleic Acid Replication in Bacteria
Some antibacterial chemotherapeutic affect bacteria by inhibiting normal nucleic acid replication.
• The fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.) work by inhibiting one or more of the topoisomerases, the enzymes needed for bacterial nucleic acid synthesis.
• Co-trimoxazole, a combination of sulfamethoxazole and trimethoprim, block enzymes in the bacteria pathway required for the synthesis of tetrahydrofolic acid, a cofactor needed for bacteria to make the nucleotide bases thymine, guanine, uracil, and adenine. Without the tetrahydrofolic acid, the bacteria cannot synthesize DNA or RNA.
Antimicrobial chemotherapy will be discussed in greater detail later in Unit 2 under Control of Bacteria by Using Antibiotics and Disinfectants.
Exercise: Think-Pair-Share Questions
As we are learning, pathogen-associated molecular patterns (PAMPs) are microbial molecules many microbes share but are not found as a part of the human body and are able to initiate innate immune responses. Examples thus far include peptidoglycan fragments, lipopolysaccharide in the gram-negative cell wall, and lipoteichoic acids in the gram-positive cell wall, molecules that human cells lack. Bacterial and viral genomes also act as PAMPs.
Our cells also have DNA and RNA. How can bacterial and viral genomes initiate innate immunity when our genomes do not?
Summary
1. The genome is the sum of an organism’s genetic material.
2. Bacteria contain a single chromosome of double-stranded deoxyribonucleic acid (DNA).
3. The region of the bacterial cytoplasm where the chromosome is located and visible when viewed with an electron microscope called the nucleoid.
4. The bacterial chromosome is typically a physical and genetic circle, becomes supercoiled,and is not surrounded by a nuclear membrane.
5. Bacteria do not carry out mitosis or meiosis.
6. DNA topoisomerase enzymes are used to supercoil and relax the bacterial chromosome during DNA replication and transcription.
7. Like eukaryotic DNA, prokaryotic DNA replicates by sequential unwinding of the two DNA parent strands and the subsequent complementary base pairing of DNA nucleotides with each parent strand.
8. During DNA replication the nitrogenous base adenine forms hydrogen bonds with thymine and guanine forms hydrogen bonds with cytosine.
9. Genes located along the DNA are transcribed into RNA molecules, primarily messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Messenger RNA is then translated into protein at the ribosomes.
10. During transcription, ribonucleic acid (RNA) is synthesized by complementary base pairing of ribonucleotides with deoxyribonucleotides to match a portion of one strand of DNA called a gene.
11. During translation, specific tRNA molecules pick up specific amino acids, transfer those amino acids to the ribosomes, and insert them in their proper place according to the mRNA "message."
12. Bacterial and viral genomes act as PAMPs to stimulate innate immunity.
13. Some antibacterial chemotherapeutic agents inhibiting normal nucleic acid replication in bacteria.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. The sum of an organism's genetic material is called its____________. (ans)
2. Bacterial enzymes involved in in the unwinding, replication, and rewinding of the circular, supercoiled bacterial DNA called ______________. (ans)
3. Describe the general composition of the chromosome in most bacteria. (ans)
4. Briefly describe the process of DNA replication. (ans)
5. State what enzyme carries out the following functions during DNA replication.
1. Unwinds the helical DNA by breaking the hydrogen bonds between complementary bases. (ans)
2. Synthesizes a short RNA primer at the beginning of each origin of replication. (ans)
3. Adds DNA nucleotides to the RNA primer. (ans)
4. Digests away the RNA primer and replaces the RNA nucleotides of the primer with the proper DNA nucleotides. (ans)
5. Links the DNA fragments of the lagging strand together. (ans)
6. State the overall function of DNA. (ans)
7. Define transcription. (ans)
8. Define translation. (ans)
9. Ciprofloxacin (Cipro) is used to treat a variety of bacterial infections. How does it stop bacteria from growing? (ans)
10. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.4%3A_Cellular_Components_within_the_Cytoplasm/2.4A%3A_Cytoplasm.txt |
Learning Objectives
1. Describe plasmids and indicate their possible benefit to bacteria.
2. State the function of the following:
1. transposons
2. integrons
3. episome
4. conjugative plasmid
3. State the most common way plasmids are transmitted from one bacterium to another.
4. Define horizontal gene transfer.
In addition to the bacterial chromosome, many bacteria often contain small nonchromosomal DNA molecules called plasmids. Plasmids usually contain between 5 and 100 genes. Plasmids are not essential for normal bacterial growth and bacteria may lose or gain them without harm. They can, however, provide an advantage under certain environmental conditions. For example, under normal environmental growth conditions, bacteria are not usually exposed to antibiotics and having a plasmid coding for an enzyme capable of denaturing a particular antibiotic is of no value. However, if that bacterium finds itself in the body when the particular antibiotic that the plasmid-coded enzyme is able to degrade is being given to treat an infection, the bacterium containing the plasmid is able to survive and grow.
Structure and Composition
Plasmids are small molecules of double stranded, helical, non-chromosomal DNA. In most plasmids the two ends of the double-stranded DNA molecule that make up plasmids covalently bond together forming a physical circle. Some plasmids, however, have linear DNA. Plasmids replicate independently of the host chromosome, but some plasmids, called episomes, are able to insert or integrate into the host cell’s chromosome where their replication is then regulated by the chromosome.
Although some plasmids can be transmitted from one bacterium to another by transformation and by generalized transduction, the most common mechanism of plasmid transfer is conjugation. Plasmids that can be transmitted by cell-to-cell contact are called conjugative plasmids. They contain genes coding for proteins involved in both DNA transfer and and the formation of mating pairs.
Functions
Plasmids code for synthesis of a few proteins not coded for by the bacterial chromosome. For example, R-plasmids, found in some Gram-negative bacteria, often have genes coding for both production of a conjugation pilus (discussed later in this unit) and multiple antibiotic resistance. Through a process called conjugation, the conjugation pilus enables the bacterium to transfer a copy of the R-plasmids to other bacteria, making them also multiple antibiotic resistant and able to produce a conjugation pilus. In addition, some exotoxins, such as the tetanus exotoxin, Escherichia coli enterotoxin, and E. coli shiga toxin discussed later in Unit 2 under Bacterial Pathogenicity, are also coded for by plasmids. Thousands of different plasmids are known to exist.
Transposons
Transposons (transposable elements or "jumping genes" ) are small pieces of DNA that encode enzymes that transpose the transposon, that is, move it from one DNA location to another, either on the same molecule of DNA or on a different molecule. Transposons may be found as part of a bacterium's nucleoid (conjugative transposons) or in plasmids and are usually between one and twelve genes long. A transposon contains a number of genes, coding for antibiotic resistance or other traits, flanked at both ends by insertion sequences coding for an enzyme called transpoase. Transpoase is the enzyme that catalyzes the cutting and resealing of the DNA during transposition. Thus, such transposons are able to cut themselves out of a bacterial nucleoid or a plasmid and insert themselves into another nucleoid or plasmid and contribute in the transmission of antibiotic resistance among a population of bacteria.
Plasmids can also acquire a number of different antibiotic resistance genes by means of integrons. Integrons are transposons that can carry multiple gene clusters called gene cassettes that move as a unit from one piece of DNA to another. An enzyme called integrase enables these gene cassettes to integrate and accumulate within the integron. In this way, a number of different antibiotic resistance genes can be transferred as a unit from one bacterium to another.
Plasmids and conjugative transposons are very important in horizontal gene transfer in bacteria. Horizontal gene transfer , also known as lateral gene transfer, is a process in which an organism transfers genetic material to another organism that is not its offspring. The ability of Bacteria and Archaea to adapt to new environments as a part of bacterial evolution most frequently results from the acquisition of new genes through horizontal gene transfer rather than by the alteration of gene functions through mutations. (It is estimated that as much as 20% of the genome of Escherichia coli originated from horizontal gene transfer.)
Horizontal gene transfer is able to cause rather large-scale changes in a bacterial genome. For example, certain bacteria contain multiple virulence genes called pathogenicity islands that are located on large, unstable regions of the bacterial genome. These pathogenicity islands can be transmitted to other bacteria by horizontal gene transfer. However, if these transferred genes provide no selective advantage to the bacteria that acquire them, they are usually lost by deletion. In this way the size of the bacterium's genome can remain approximately the same size over time.
CRISPR
Because bacteria are always taking in new DNA from horizontal gene transfer or being infected by bacteriophages, bacteria have developed a system for removing viral nucleic acid or DNA from self-serving or harmful plasmids. This system represents a type of adaptive immunity in bacteria, and is carried out by clustered, regularly interspaced, short palindromic repeat (CRISPR) sequences and CRISPR-associated (Cas) proteins that possess nuclease activity. The CRISPR/Cas system targets specific foreign DNA sequences in bacteria for destruction.
Video: YouTube Movie of the CRISPER/Cas9 System in Bacteria (www.youtube.com/v/ZsxIU5-s5Ds)
Applications of CRISPR technology has now become a common tool used in molecular biology for CRISPR/nuclease mediated genome editing (genetic engineering) in a wide variety of different cell types. Molecular biologists are now beginning to use this to carry out highly efficient, targeted alterations of genome sequence and gene expression and hope to eventually use it to repair damaged or dysfunctional genes.
Exercise: Think-Pair-Share Questions
An F+ plasmid is a conjugative plasmid that codes strictly for the ability to produce a conjugation pilus and a mating pair.
State what medically significant event might occur if a transposon located in the nucleoid of a normal flora intestinal bacterium and containing genes for antibiotic resistance were to cut out of the bacterium’s nucleoid and insert into the F+ plasmid.
Summary
1. Many bacteria often contain small nonchromosomal DNA molecules called plasmids.
2. While plasmids are not essential for normal bacterial growth and bacteria may lose or gain them without harm, they can provide an advantage under certain environmental conditions.
3. Plasmids code for synthesis of a few proteins not coded for by the bacterial chromosome.
4. Transposons (jumping genes) are small pieces of DNA that encode enzymes that enable the transposon to, move from one DNA location to another.
5. Transposons may be found as part of a bacterium's chromosome or in plasmids
6. Integrons are transposons that can carry multiple gene clusters called gene cassettes that move as a unit from one piece of DNA to another
7. Horizontal gene transfer is a process in which an organism transfers genetic material to another cell that is not its offspring.
8. Horizontal gene transfer is able to cause rather large-scale changes in a bacterial genome.
9. The ability of Bacteria and Archaea to adapt to new environments as a part of bacterial evolution, most frequently results from the acquisition of new genes through horizontal gene transfer rather than by the alteration of gene functions through mutations.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Describe plasmids and indicate their possible benefit to bacteria. (ans)
2. State why R-plasmids are presenting quite a problem today in treating many Gram-negative infections. (ans)
3. _____________ are small pieces of DNA that encode enzymes that cut segments of DNA from a location in a bacterial chromosome or in a plasmid and insert it into another chromosome or plasmid. These segments of translocated DNA often contain genes for antibiotic resistance. (ans)
4. The genes coding for antibiotic resistance in bacterial plasmids frequently change over time, enabling the bacterium to resist new antibiotics. What might account for this? (ans)
5. State the most common way plasmids are transmitted from one bacterium to another. (ans)
6. Define horizontal gene transfer. (ans)
7. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.4%3A_Cellular_Components_within_the_Cytoplasm/2.4C%3A_Plasmids_and_Transposons.txt |
Learning Objectives
1. Describe the structure and chemical composition of bacterial ribosomes and state their function.
2. In terms of protein synthesis, briefly describe the process of transcription and translation.
3. State, in a general sense, how antibiotics like neomycin, tetracycline, doxycycline, erythromycin, and azithromycin affect bacterial growth.
Ribosome Structure and Composition
Ribosomes are composed of ribosomal RNA (rRNA) and protein. Prokaryotic cells have three types of rRNA: 16S rRNA, 23S rRNA, and 5S rRNA. Like transfer RNA (tRNA), rRNAs use intrastrand H-bonding between complementary nucleotide bases to form complex folded structures. Ribosomes are composed of two subunits with densities of 50S and 30S ("S" refers to a unit of density called the Svedberg unit). The 30S subunit contains 16S rRNA and 21 proteins; the 50S subunit contains 5S and 23S rRNA and 31 proteins.The two subunits combine during protein synthesis to form a complete 70S ribosome about 25nm in diameter. A typical bacterium may have as many as 15,000 ribosomes.
The Density of Ribosomal Subunits
Ribosomes are composed of two subunits that come together to translate messenger RNA (mRNA) into polypeptides and proteins during translation and are typically described in terms of their density. Density is the mass of a molecule or particle divided by its volume and is measured in Svedberg (S) units, a unit of density corresponding to the relative rate of sedimentation during ultra-high-speed centrifugation. The greater the S-value, the more dense the particle.
Ribosomal subunits are composed of ribosomal RNA (rRNA) and proteins. Ribosomal subunits with different S-values are composed of different molecules of rRNA, as well as different proteins. Remember that RNA is a polymer of ribonucleotides containing the nitrogenous base adenine, uracil, guanine, or cytosine. Different molecules of rRNA are of different lengths and have a different order of these ribonucleotide bases. Because rRNA is single stranded, many of the rRNA nucleotide bases are involved in intrastrand hydrogen bonds and this is what gives the rRNA molecule its specific shape (see Figure \(1\)). The shape, in turn, helps determine its function - much like the the interactions between amino acids in a protein determine that protein's shape and function (see Figure \(2\)).
Illustration of a 16S rRNA in Escherichia coli
Animation of a 16S rRNA
Illustration of the enzyme catalase
Prokaryotic ribosomes, for example, are composed of two subunits with densities of 50S and 30S. The 30S subunit contains 16S rRNA 1540 nucleotides long and 21 proteins; the 50S subunit contains a 5S rRNA 120 nucleotides long, a 23S rRNA 2900 nucleotides long, and 31 proteins. The two subunits combine during protein synthesis to form a complete 70S ribosome.
Eukaryotic ribosomal subunits have densities of 60S and 40S because they contain different rRNA molecules and proteins than prokaryotic ribosomal subunits. In most eukaryotes, the 40S subunit contains an 18S rRNA 1900 nucleotides long and approximately 33 proteins; the 60S subunit contains a 5S rRNA 120 nucleotides long, a 5.8S rRNA 160 nucleotides long, a 28S rRNA 4700 nucleotides long, and approximately 49 proteins. The two subunits combine during protein synthesis to form a complete 80S ribosome about 25nm in diameter.
Because of this difference in specific rRNAs and proteins the resulting "shape," there are drugs that can bind either to a 30S or 50S ribosomal subunit of a prokaryotic ribosome and subsequently block its function but are unable to bind to the equivalent 40S or 60S subunit of a eukaryotic ribosome.
Ribosome Functions
Ribosomes function as a workbench for protein synthesis, that is, they receive and translate genetic instructions for the formation of specific proteins. During protein synthesis, mRNA attaches to the 30s subunit and amino acid-carrying transfer RNAs (tRNA) attach to the 50s subunit (Figure \(1\)). Protein synthesis is discussed in detail in Unit 6.
The chromosome is the genetic material of the bacterium. Genes located along the DNA are transcribed into RNA molecules, primarily messenger RNA (mRNA), transfer RNA (tRNA, and ribosomal RNA (rRNA). Messenger RNA is then translated into protein at the ribosomes.
• Transcription: Ribonucleic acid (RNA) is synthesized by complementary base pairing of ribonucleotides with deoxyribonucleotides to match a portion of one strand of DNA called a gene. Although genes are present on both strands of DNA, only one strand is transcribed for any given gene. Following transcription of genes into mRNA, 30S and 50S ribosomal subunits attach to the mRNA and tRNA inserts the correct amino acids which are subsequently joined to form a polypeptide or a protein through a process called translation.
• Translation: During translation, specific tRNA molecules pick up specific amino acids, transfer those amino acids to the ribosomes, and insert them in their proper place according to the mRNA "message." This is done by the anticodon portion of the tRNA molecules complementary base pairing with the codons along the mRNA.
Exercise: Think-Pair-Share Questions
In order for any of the tetracycline group of antibiotics to inhibit Gram-negative bacterial growth, they must enter the cytoplasm of that bacterium and bind to the 30S subunit of its ribosomes.
Earlier we learned the composition and functions of both the Gram-negative cell wall and the cytoplasmic membrane. We have also previously learned how the order of deoxyribonucleotide bases in DNA determines the order of ribonucleotide bases in rRNA which, in turn, determines the 3-dimensional shape of that RNA. Likewise, the order of deoxyribonucleotide bases in DNA determines the order of amino acids in a protein or enzyme which determines the 3-dimensional shape of that protein.
Considering all of this and using the illustration above, think of three physical changes that could occur within the bacterium as a result of acquiring new or altered genes through mutation or horizontal gene transfer that could enable the bacterium to resist that tetracycline.
Antimicrobial Agents that Alter Prokaryotic Ribosomal Subunits and Block Translation in Bacteria
Many antibiotics alter bacterial ribosomes, interfering with translation and thereby causing faulty protein synthesis. The portion of the ribosome to which the antibiotic binds determines how translation is effected. For example:
• The tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.) bind reversibly to the 30S subunit, distorting it in such a way that the anticodons of charged tRNAs cannot align properly with the codons of the mRNA.
• The macrolides (erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, etc.) bind reversibly to the 50S subunit. They appear to inhibit elongation of the protein by preventing the enzyme peptidyltransferase from forming peptide bonds between the amino acids. They may also prevent the transfer of the peptidyl tRNA from the A-site to the P-site.
Antimicrobial chemotherapy will be discussed in greater detail later in Unit 2 under Control of Bacteria by Using Antibiotics and Disinfectants.
Summary
1. Ribosomes are composed of ribosomal RNA (rRNA) and protein.
2. Bacterial ribosomes are composed of two subunits with densities of 50S and 30S, as opposed to 60S and 40S in eukaryotic cells.
3. Ribosomes function as a workbench for protein synthesis whereby they receive and translate genetic instructions for the formation of specific proteins.
4. During translation, specific tRNA molecules pick up specific amino acids, transfer those amino acids to the ribosomes, and insert them in their proper place according to the mRNA "message."
5. Many antibiotics bind to either the 30S or the 50S subunit of bacterial ribosomes, interfering with translation and thereby causing faulty protein synthesis.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Describe bacterial ribosomes. (ans)
2. State the function of ribosomes. (ans)
3. Define translation. (ans)
4. State, in a general sense, how antibiotics like neomycin, tetracycline, doxycycline, erythromycin, and azithromycin affect bacterial growth. (ans)
5. The tetracyclines (tetracycline, doxycycline) are antibiotics that bind to the 30S subunit of bacterial ribosomes. The macrolides (erythromycin, azithromycin, clarithromycin) are antibiotics that bind to the 50S subunit of bacterial ribosomes. Why won't these antibiotics be effective for fungal, protozoal, or viral infections? (ans)
6. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.4%3A_Cellular_Components_within_the_Cytoplasm/2.4D%3A_Ribosomes.txt |
Name 2 common genera of bacteria capable of producing endospores and state which is an obligate anaerobe. Briefly discuss the function of a bacterial endospore. Describe the structure of a bacterial endospore. Define sporulation and germination. Name three infections that may be transmitted to humans by endospores. Highlighted Bacterium Read the description of Clostridium tetani and match the bacterium with the description of the organism and the infection it causes.
Endospores are dormant alternate life forms produced by the genus Bacillus, the genus Clostridium, and a number other genera of bacteria, including Desulfotomaculum, Sporosarcina, Sporolactobacillus, Oscillospira, and Thermoactinomyces. Bacillus species (see Figure \(1\)) are obligate aerobes that live in soil while Clostridium species (see Figure \(2\)) are obligate anaerobes often found as normal flora of the gastrointestinal tract in animals.
Figure \(1\): Endospore stain of Bacillus megaterium Figure \(2\): Endospore stain of Clostridium tetani
Note green endospores within pink bacilli.
Note the endospore within the rod gives the bacterium a "tennis racquet" shape (arrows).
• Scanning electron micrograph of Clostridium botulinum with endospore; courtesy of Dennis Kunkel's Microscopy.
Formation of Endospores
Under conditions of starvation, especially the lack of carbon and nitrogen sources, a single endospores form within some of the bacteria. The process is called sporulation .
First the DNA replicates (Figure \(3\), step 1)and a cytoplasmic membrane septum forms at one end of the cell (Figure \(3\). step 3). A second layer of cytoplasmic membrane then forms around one of the DNA molecules (Figure \(3\), step 4) - the one that will become part of the endospore - to form a forespore (Figure \(3\), step 5). Both of these membrane layers then synthesize peptidoglycan in the space between them to form the first protective coat, the cortex (Figure \(3\), step 6) that lies adjacent to the germ cell wall that will eventually form the cell wall of the bacterium upon germination.
Calcium dipocolinate is also incorporated into the forming endospore. A spore coat composed of a keratin-like protein then forms around the cortex (Figure \(3\), step 7). Sometimes an outer membrane composed of lipid and protein and called an exosporium is also seen (Figure \(3\), step 8).
Finally, the remainder of the bacterium is degraded and the endospore is released (Figure \(3\), step 9). Sporulation generally takes around 15 hours. The process is summarized in Figure \(3\).
YouTube animation of endospore formation by Global Institute of Medical Sciences
Endospore Structure (see Figure \(3\), step 10)
The completed endospore consists of multiple layers of resistant coats (including a cortex, a spore coat, and sometimes an exosporium) surrounding a nucleoid, some ribosomes, RNA molecules, and enzymes.
• To view an electron micrograph of an endospore of Bacillus stearothermophilus, see the Microbe Zoo web page of Michigan State University.
(Some bacteria produce spore-like structures distinct from endospores. Exospores are heat resistant spores produced by a budding process in members of the genus Metylosinus and Rhodomicrobium. Cysts are resistant to drying and are formed singly within vegetative cells by Azotobacter, Myxococcus, and Sporocytophaga. Conidia are heat-susceptible asexual reproductive spores produced by various genera of branching bacteria belonging to the group Actinomycetes.)
Function of Endospores
An endospore is not a reproductive structure but rather a resistant, dormant survival form of the organism. Endospores are quite resistant to high temperatures (including boiling), most disinfectants, low energy radiation, drying, etc. The endospore can then survive until a variety of environmental stimuli trigger germination , allowing outgrowth of a single vegetative bacterium as shown in Fig 3, step 11 and step 12 and in Figure \(4\). Viable endospores have reportedly been isolated from the GI tract of a bee embedded in amber between 25 and 40 million years ago. Viable endospores of a halophilic (salt-loving) bacterium have also reportedly been isolated from fluid inclusions in salt crystals dating back over 250 million years!
Bacterial endospores are resistant to antibiotics, most disinfectants, and physical agents such as radiation, boiling, and drying. The impermeability of the spore coat is thought to be responsible for the endospore's resistance to chemicals. The heat resistance of endospores is due to a variety of factors:
• Calcium-dipicolinate, abundant within the endospore, may stabilize and protect the endospore's DNA.
• Small acid-soluble proteins (SASPs) saturate the endospore's DNA and protect it from heat, drying, chemicals, and radiation. They also function as a carbon and energy source for the development of a vegetative bacterium during germination.
• The cortex may osmotically remove water from the interior of the endospore and the dehydration that results is thought to be very important in the endospore's resistance to heat and radiation.
• Finally, DNA repair enzymes contained within the endospore are able to repair damaged DNA during germination.
Endospores and Infectious Disease
Although harmless themselves until they germinate, they are involved in the transmission of some diseases to humans. Infections transmitted to humans by endospores include:
• Anthrax, caused by Bacillus anthracis; endospores can be inhaled, ingested, or enter wounds where they germinate and the vegetative bacteria subsequently replicate.
• Tetanus, caused by Clostridium tetani; endospores enter anaerobic wounds where they germinate and the vegetative bacteria subsequently replicate.
• Botulism, caused by Clostridium botulinum; endospores enter the anaerobic environment of improperly canned food where they germinate and subsequently replicate.
• Gas gangrene, caused by Clostridium perfringens); endospores enter anaerobic wounds where they germinate and the vegetative bacteria subsequently replicate.
• Pseudomembranous colitis (Clostridium difficile); antibiotics destroy the normal microbiota of the intestines that keep the growth of C. difficile in check while the endospores of C. difficile survive and subsequently germinate and replicate before the microbiota is restored.
Summary
1. Endospores are dormant alternate life forms produced by a few genera of bacteria.
2. The genus Bacillus (an obligate aerobe often living in the soil) and the genus Clostridium (an obligate anaerobe living in the gastrointestinal tract of animals) produce endospores.
3. Under conditions of starvation, a single endospore forms within a bacterium through a process called sporulation, after which the remainder of the bacterium is degraded.
4. The completed endospore consists of multiple layers of resistant coats (including a cortex, a spore coat, and sometimes an exosporium) surrounding a nucleoid, some ribosomes, RNA molecules, and enzymes.
5. Endospores are quite resistant to high temperatures (including boiling), most disinfectants, low energy radiation, and drying.
6. The endospore survives until a variety of environmental stimuli trigger germination, allowing outgrowth of a single vegetative bacterium.
7. Infectious diseases such as anthrax, tetanus, gas gangrene, botulism, and pseudomembranous colitis are transmitted to humans by endospores.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Name 2 common genera of bacteria capable of producing endospores and state which is an obligate anaerobe. (ans)
2. Briefly discuss the function of a bacterial endospore. (ans)
3. The emergence of a vegetative bacterium from an endospore is called ________________. (ans)
4. Name three infections transmitted to humans by bacterial endospores. (ans)
5. Botulism is caused by Clostridium botulinum, a bacterium that is normal flora of the intestinal tract of grazing animals. A person home-canned some green beans by boiling the beans and placing them in jars and screwing on lids. The lids popped down indicating a vacuum had formed within the jar. Upon ingesting these beans the person contracted botulism. Based on what was learned about Clostridium, explain. (ans)
6. Multiple Choice (ans)
2.4F: Inclusion Bodies and Organe
Name three major types of photosynthetic bacteria and briefly describe where its photosynthetic system is located. State the function of the following inclusion bodies: cyanophycin granules carboxysomes gas vacuoles polyhydroxybutyrate and glycogen granules magnetosomes volutin granules and sulfur granules
There are several major groups of photosynthetic bacteria: cyanobacteria, purple bacteria, green sulfur bacteria, green nonsulfur bacteria, heliobacteria, and acidobacteria. Comparing the cyanobacteria, the purple bacteria, and the green bacteria:
The cyanobacteria carry out oxygenic photosynthesis, that is, they use water as an electron donor and generate oxygen during photosynthesis. The photosynthetic system is located in an extensive thylakoid membrane system that is lined with particles called phycobilisomes that contain light-harvesting phycobiliproteins.
• Photograph of the cyanobacteria Anabaena. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.4%3A_Cellular_Components_within_the_Cytoplasm/2.4E%3A_Endospores.txt |
The overall purpose of this Learning Object is to list the various cellular components that are often found external to the bacterial cell wall.
In this section on Prokaryotic Cell Anatomy we are looking at the various anatomical parts that make up a bacterium. We will now look at the following structures located outside the cell wall of many bacteria: (1) glycocalyx (capsule) and S-layer, (2) flagella, and (3) pili.
2.5: Structures Outside the Cell Wall
State the chemical composition and 2 common functions of a bacterial glycocalyx. Briefly describe the following steps in phagocytosis: unenhanced attachment enhanced attachment engulfment destruction Briefly describe how a capsule might initially enable some bacteria to resist being phagocytosed by white blood cells. Define biofilm and state at least 3 advantages of biofilm formation to bacteria. Highlighted Bacterium Read the description of Strepococcus pneumoniae and match the bacterium with the description of the organism and the infection it causes.
All bacteria secrete some sort of glycocalyx (Capsules and Slime Layers), an outer viscous covering of fibers extending from the bacterium (see Figure \(1\), Figure \(2\), and Figure \(3\)). If it appears as an extensive, tightly bound accumulation of gelatinous material adhering to the cell wall, it is called a capsule as shown in the photomicrograph in Figure \(2\). If the glycocalyx appears unorganized and more loosely attached, it is referred to as a slime layer.
Structure and Composition
The glycocalyx is usually a viscous polysaccharide or polypeptide slime. Actual production of a glycocalyx often depends on environmental conditions.
• A capsule stain of Streptococcus lactis.
Functionsand Significance to Bacterial Pathogenicity
Although a number of functions have been associated with the glycocalyx, such as protecting bacteria against drying, trap nutrients, etc., for our purposes there are two very important functions. The glycocalyx enables certain bacteria to resist phagocytic engulfment by white blood cells in the body or protozoans in soil and water. The glycocalyx also enables some bacteria to adhere to environmental surfaces (rocks, root hairs, teeth, etc.), colonize, and resist flushing.
1. Preview of the Steps in Phagocytosis
As will be seen in Unit 5, there are several steps involved in phagocytosis.
a. Attachment
First the surface of the microbe must be attached to the cytoplasmic membrane of the phagocyte. Attachment of microorganisms is necessary for ingestion and may be unenhanced or enhanced.
• Unenhanced attachment is a general recognition of what are called pathogen-associated molecular patterns or PAMPs - components of common molecules such as peptidoglycan, teichoic acids, lipopolysaccharide, mannans, and glucans common in microbial cell walls but not found on human cells - by means of glycoprotein known as endocytic pattern-recognition receptors on the surface of the phagocytes (see Figure \(4\)).
Flash animation illustrating the function of endocytic pattern-recognition receptors on phagocytes.
html5 version of animation for iPad illustrating the function of endocytic pattern-recognition receptors on phagocytes.
For More Information: Pathogen-Associated Molecular Patterns (PAMPs) from Unit 5
• Enhanced attachment is the attachment of microbes to phagocytes by way of an antibody molecule called IgG or proteins produced during the complement pathways called C3b and C4b (see Figure \(5\)). Molecules such as IgG and C3b that promote enhanced attachment are called opsonins and the process is called opsonization. Enhanced attachment is much more specific and efficient than unenhanced.
b. Engulfment
Following attachment, polymerization and then depolymerization of actin filaments send pseudopods out to engulf the microbe (see Figure \(6\)) and place it in a vesicle called a phagosome (see Figure \(7\)).
c. Destruction
Finally, lysosomes, containing digestive enzymes and microbicidal chemicals, fuse with the phagosome containing the ingested microbe and the microbe is destroyed (see Figure \(8\)).
Role of the Glycocalyx in Resisting Phagocytosis
Capsules enable bacteria to resist phagocytosis. For example, capsules can resist unenhanced attachment by preventing the glycoprotein receptors on phagocytes from recognizing the bacterial cell wall components and mannose-containing carbohydrates (see Figure \(10\)). Also, some capsules simply cover the C3b that does bind to the bacterial surface and prevent the C3b receptor on phagocytes from making contact with the C3b (see Figure \(9\)). This will be discussed in greater detail later in Unit 3 under Bacterial Pathogenesis.
Examples of bacteria that use their capsule to resist phagocytic engulfment include Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitidis, Bacillus anthracis , and Bordetella pertussis.
For More Information: The Ability to Resist Phagocytic Engulfment from Unit 3
The body's immune defenses, however, can eventually get around the capsule by producing opsonizing antibodies (IgG) against the capsule. The antibody then sticks the capsule to the phagocyte. In vaccines against pneumococccal pneumonia and Haemophilus influenzae type b, it is capsular polysaccharide that is given as the antigen in order to stimulate the body to make opsonizing antibodies against the encapsulated bacterium.
Flash animation showing phagocytosis of an encapsulated bacterium through opsonization.
html5 version of animation for iPad showing phagocytosis of an encapsulated bacterium through opsonization.
• Movie of an encapsulated bacterium resisting engulfment by a neutrophil. Phagocytosis. © James Sullivan, author. Licensed for use, ASM MicrobeLibrary.
3. Role of the Glycocalyx in Adhering to and Colonizing Environmental Surfaces
The glycocalyx also enables some bacteria to adhere to environmental surfaces (rocks, root hairs, teeth, etc.), colonize, and resist flushing. For example, many normal flora bacteria produce a capsular polysaccharide matrix or glycocalyx to form a biofilm on host tissue (see Figure \(3\)) as discussed below.
Significance of the glycocalyx in the Initiation of Body Defense
Initiation of Adaptive Immunity
Polysaccharides or polypeptides associated with the bacterial glycocalyx or capsule function as antigens and initiate adaptive immunity. An antigen is defined as a molecular shape that reacts with antibody molecules and with antigen receptors on lymphocytes. We recognize those molecular shapes as foreign or different from our body's molecular shapes because they fit specific antigen receptors on our B-lymphocytes and T-lymphocytes, the cells that carry out adaptive immunity.
The actual portions or fragments of an antigen that react with antibodies and with receptors on B-lymphocytes and T-lymphocytes are called epitopes. An epitope is typically a group of 5-15 amino acids with a unique shape that makes up a portion of a protein antigen, or 3-4 sugar residues branching off of a polysaccharide antigen. A single microorganism has many hundreds of different shaped epitopes that our lymphocytes can recognize as foreign and mount an adaptive immune response against.
The body recognizes an antigen as foreign when epitopes of that antigen bind to B-lymphocytes and T-lymphocytes by means of epitope-specific receptor molecules having a shape complementary to that of the epitope. The epitope receptor on the surface of a B-lymphocyte is called a B-cell receptor and is actually an antibody molecule. The receptor on a T-lymphocyte is called a T-cell receptor (TCR).
There are two major branches of the adaptive immune responses: humoral immunity and cell-mediated immunity.
1. Humoral immunity: Humoral immunity involves the production of antibody molecules in response to an antigen and is mediated by B-lymphocytes. Through a variety of mechanisms, these antibodies are able to remove or neutralize microorganisms and their toxins after binding to their epitopes. For example, antibodies made against capsular antigens can stick bacteria to phagocytes, a process called opsonization.
2. Cell-mediated immunity: Cell-mediated immunity involves the production of cytotoxic T-lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigen and is mediated by T-lymphocytes. These defense cells help to remove infected cells and cancer cells displaying foreign epitopes.
Adaptive immunity will be discussed in greater detail in Unit 6.
Biofilms
Many pathogenic bacteria, as well as normal flora and many environmental bacteria, form complex bacterial communities as biofilms. Biofilms are groups of bacteria attached to a surface and enclosed in a common secreted adhesive matrix, typically polysaccharide in nature. Bacteria in biofilms are often able to communicate with one another by a process called quorum sensing (discussed later in Unit 2) and are able to interact with and adapt to their environment as a population of bacteria rather than as individual bacteria. By living as a community of bacteria as a biofilm, these bacteria are better able to:
• resist attack by antibiotics;
• trap nutrients for bacterial growth and remain in a favorable niche;
• adhere to environmental surfaces and resist flushing;
• live in close association and communicate with other bacteria in the biofilm; and
• resist phagocytosis and attack by the body's complement pathways.
Biofilms are, therefore, functional, interacting, and growing bacterial communities. Biofilms even contain their own water channels for delivering water and nutrients throughout the biofilm community.
• Electron micrograph of a biofilm of Haemophilus influenzae from Biomedcentral.com
• Photomicrograph of a biofilm with water channels from Centers for Disease Control and Prevention Rodney M. Donlan: "Biofilms: Microbial Life on Surfaces"
• Biofilm of Pseudomonas aeruginosa from the Ausubel Lab, Department of Molecular Biology, Massachusetts General Hospital
To initiate biofilm formation, planktonic bacteria (free individual bacteria not in a biofilm) contact an environmental surface through their motility or by random collision. These planktonic bacteria then attach to that surface using pili or cell wall adhesins. This attachment then signals the expression of genes involved in quorum sensing and, ultimately, biofilm formation. As the biofilm matrix is secreted, motile bacteria lose their flagella and become nonmotile.
Planktonic Pseudomonas aeruginosa, for example, uses its polar flagellum to move through water or mucus and make contact with a solid surface such as the body's mucous membranes. It then can use pili and cell wall adhesins to attach to the epithelial cells of the mucous membrane. Attachment activates signaling and quorum sensing genes to eventually enable the population of P. aeruginosa to start synthesizing a polysaccharide biofilm composed of alginate. As the biofilm grows, the bacteria lose their flagella to become nonmotile and secrete a variety of enzymes that enable the population to obtain nutrients from the host cells. Eventually the biofilm mushrooms up and develops water channels to deliver water and nutrients to all the bacteria within the biofilm. As the biofilm begins to get too crowded with bacteria, quorum sensing enables some of the Pseudomonas to again produce flagella, escape the biofilm, and colonize a new location (See Figs. 11A-11G).
Streptococcus mutans, and Streptococcus sobrinus, two bacteria implicated in initiating dental caries, break down sucrose into glucose and fructose. Streptococcus mutans can uses an enzyme called dextransucrase to convert sucrose into a sticky polysaccharide called dextran that forms a biofilm enabling the bacteria to adhere to the enamel of the tooth and form plaque. This will be discussed in greater detail later in Unit 2 under Bacterial Pathogenicity. S. mutans and S. sobrinus also ferment glucose in order to produce energy. The fermentation of glucose results in the production of lactic acid that is released onto the surface of the tooth and initiates decay.
• Scanning electron micrograph of Streptococcus growing in the enamel of a tooth.© Lloyd Simonson, author. Licensed for use, ASM MicrobeLibrary.
• Scanning electron micrograph of dental plaque.© H. Busscher, H. van der Mei, W. Jongebloed, R Bos, authors. Licensed for use, ASM MicrobeLibrary.
• Scanning electron micrograph of Staphylococcus aureus forming a biofilm in an indwelling catheter courtesy of CDC.
• Biofilm of Staphylococcus aureus from Montana State University
A number of biofilm-forming bacteria, such as uropathogenic Escherichia coli (UPEC), enterohemorrhagic E. coli (EHEC), Citrobacter species, Salmonella species, and Mycobacterium tuberculosis are able to produce amyloid fibers that can play a role in such processes as attachment to host cells, invasion of host cells, and biofilm formation. Curli is an example of such an amyloid fiber produced by UPEC and Salmonella.
Many chronic and difficult-to-treat infections are caused by bacteria in biofilms. Within biofilms, bacteria grow more slowly, exhibit different gene expression than free planktonic bacteria, and are more resistant to antimicrobial agents such as antibiotics because of the reduced ability of these chemicals to penetrate the dense biofilms matrix. Biofilms have been implicated in tuberculosis, kidney stones, Staphylococcus infections, Legionnaires' disease, and periodontal disease. It is further estimated that as many as 10 million people a year in the US may develop biofilm-associated infections as a result of invasive medical procedures and surgical implants.
You Tube movie and animation: What are Biofilms?
Summary
1. All bacteria secrete some sort of glycocalyx, an outer viscous covering of fibers extending from the bacterium.
2. An extensive, tightly bound glycocalyx adhering to the cell wall is called a capsule.
3. Phagocytosis involves several distinct steps including attachment of the microbe to the phagocyte through unenhanced or enhanced attachment, ingestion of the microbe and its placement into a phagosome, and the destruction of the microbe after fusion of lysosomes with the phagosome.
4. Capsules enable bacteria to resist unenhanced attachment by covering up bacterial PAMPs so they are unable to bind to endocytic pattern-recognition receptors.
5. The glycocalyx also enables some bacteria to adhere to environmental surfaces, colonize, and resist flushing.
6. The body's adaptive immune defenses can eventually overcome bacterial capsules by producing opsonizing antibodies (IgG) against the capsule that are able to stick the capsule to the phagocyte.
7. Biofilms are groups of bacteria attached to a surface and enclosed in a common secreted adhesive matrix and are functional, interacting, and growing bacterial communities.
8. Most bacteria in nature exist as biofilm populations.
9. Many chronic and difficult-to-treat infections are caused by bacteria in biofilms.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State two common functions associated with the bacterial glycocalyx. (ans)
2. Briefly describe how a bacterial capsule might block phagocytosis. (ans)
3. State three possible functions associated with a bacterial biofilm. (ans)
4. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.5%3A_Structures_Outside_the_Cell_Wall/2.5A%3A_Glycocalyx_%28Capsules%29_and_Biofi.txt |
Structure and Composition of Flagella
1. The filament is the rigid, helical structure that extends from the cell surface. It is composed of the protein flagellin arranged in helical chains so as to form a hollow core. During synthesis of the flagellar filament, flagellin molecules coming off of the ribosomes are transported through the hollow core of the filament where they attach to the growing tip of the filament causing it to lengthen. With the exception of a few bacteria, such as Bdellovibrio and Vibrio cholerae, the flagellar filament is not surrounded by a sheath (see Figure \(1\)).
2. The hook is a flexible coupling between the filament and the basal body (see Figure \(1\)).
3. The basal body consists of a rod and a series of rings that anchor the flagellum to the cell wall and the cytoplasmic membrane (see Figure \(1\)). Unlike eukaryotic flagella, the bacterial flagellum has no internal fibrils and does not flex. Instead, the basal body acts as a rotary molecular motor, enabling the flagellum to rotate and propel the bacterium through the surrounding fluid. In fact, the flagellar motor rotates very rapidly. (Some flagella can rotate up to 300 revolutions per second!)
The MotA and MotB proteins form the stator of the flagellar motor and function to generate torque for rotation of the flagellum. The MS and C rings function as the rotor. (See Figure \(1\)). Energy for rotation comes from the proton motive force provided by protons moving through the Mot proteins along a concentration gradient from the peptidoglycan and periplasm towards the cytoplasm.
• Electron micrograph and illustration of the basal body of bacterial flagella; Cover photo of Molecular Biology of the Cell, May 1, 2000.
• Animation of a rotating bacterial flagellum from the ARN Molecular Museum
• YouTube movie of the assembly and rotation of a bacterial flagellum
Bacteria flagella (see Figure \(2\) and Figure \(3\)) are 10-20 µm long and between 0.01 and 0.02 µm in diameter.
Flagellar Arrangements (see Figure \(4\))
1. monotrichous: a single flagellum, usually at one pole
2. amphitrichous: a single flagellum at both ends of the organism
3. lophotrichous: two or more flagella at one or both poles
• Scanning electron micrograph of Helicobacter pylori showing lophotrichous arrangement of flagella ; from Science Photolab.com
4. peritrichous: flagella over the entire surface
• Scanning electron micrograph of Proteus vulgaris showing peritrichous arrangement of flagella and pili; from fineartamerica.com
5. axial filaments: internal flagella found only in the spirochetes. Axial filaments are composed of from two to over a hundred axial fibrils (or endoflagella) that extend from both ends of the bacterium between the outer membrane and the cell wall, often overlapping in the center of the cell. (see Figure \(5\) and Figure \(6\)). A popular theory as to the mechanism behind spirochete motility presumes that as the endoflagella rotate in the periplasmic space between the outer membrane and the cell wall, this could cause the corkscrew-shaped outer membrane of the spirochete to rotate and propel the bacterium through the surrounding fluid.
• Axial filaments of the spirochete Leptospira; Midlands Technical College, Bio 255 course site
Concept map for Bacterial Flagella
Functions
Flagella are the organelles of locomotion for most of the bacteria that are capable of motility. Two proteins in the flagellar motor, called MotA and MotB, form a proton channel through the cytoplasmic membrane and rotation of the flagellum is driven by a proton gradient. This driving proton motive force occurs as protons accumulating in the space between the cytoplasmic membrane and the cell wall as a result of the electron transport system travel through the channel back into the bacterium's cytoplasm. Most bacterial flagella can rotate both counterclockwise and clockwise and this rotation contributes to the bacterium's ability to change direction as it swims. A protein switch in the molecular motor of the basal body controls the direction of rotation.
1. A bacterium with peritrichous flagella:
If a bacterium has a peritrichous arrangement of flagella, counterclockwise rotation of the flagella causes them to form a single bundle that propels the bacterium in long, straight or curved runs without a change in direction. Counterclockwise rotation causes the flagellum to exhibit a left-handed helix. During a run, that lasts about one second, the bacterium moves 10 - 20 times its length before it stops. This occurs when some of the the flagella rotate clockwise, disengage from the bundle, and trigger a tumbling motion. Clockwise rotation causes the flagellum to assume a right-handed helix. A tumble only lasts about one-tenth of a second and no real forward progress is made. After a “tumble”, the direction of the next bacterial run is random because every time the bacterium stops swimming, Brownian motion and fluid currents cause the bacterium to reorient in a new direction.
Movie of swimming Escherichia coli as seen with phase contrast microscopy.
Flagella are not visible with under phase contrast microscopy. Note runs and tumbles.
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
Movie of motile Escherichia coli with fluorescent labelled-flagella #1.
This technique allows the the flagella to be seen as the bacteria swim. Note some flagella leaving the flagellar bundle to initiate tumbling.
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
Movie of motile Escherichia coli with fluorescent labelled-flagella #2.
This technique allows the the flagella to be seen as the bacteria swim. Note some flagella leaving the flagellar bundle to initiate tumbling.
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
Movie of tethered Escherichia coli showing that the bacterial flagella rotate.
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
When bacteria with a peritrichous arrangement grow on a nutrient-rich solid surface, they can exhibit a swarming motility wherein the bacteria elongate, synthesize additional flagella, secrete wetting agents, and move across the surface in coordinated manner.
Movie of swarming motility of Escherichia coli.
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
2. A bacterium with polar flagella:
Most bacteria with polar flagella, like the peritrichous above, can rotate their flagella both clockwise and counterclockwise. If the flagellum is rotating counterclockwise, it pushes the bacterium forward. When it rotates clockwise, it pulls the bacterium backward. These bacteria change direction by changing the rotation of their flagella.
Video \(4\)B.1: Phase contrast movie of motile Pseudomonas. Pseudomonas has a single polar flagellum that can rotate both counterclockwise and clockwise but is not visible under phase contrast microscopy (http://www.youtube.com/embed/EWj2TGsTQEI).
Movie of Spirillum volutans, a spiral-shaped bacterium with a bundle of flagella at either end.
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
Some bacteria with polar flagella can only rotate their flagellum clockwise. In this case, clockwise rotation pushes the bacterium forward. Every time the bacterium stops, Brownian motion and fluid currents cause the bacterium to reorient in a new direction.
Movie of Rhodobacter spheroides with fluorescent-labelled flagella.
The flagellum can only rotate clockwise.
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
Taxis
Around half of all known bacteria are motile. Motility serves to keep bacteria in an optimum environment via taxis. Taxis is a motile response to an environmental stimulus. Bacteria can respond to chemicals (chemotaxis), light (phototaxis), osmotic pressure (osmotaxis), oxygen (aerotaxis), and temperature (thermotaxis). Chemotaxis is a response to a chemical gradient of attractant or repellent molecules in the bacterium's environment.
• In an environment that lacks a gradient of attractant or repellent, the bacterium moves randomly. In this way the bacterium keeps searching for a gradient.
• In an environment that has a gradient of attractant or repellent, the net movement of the bacterium is towards the attractant or away from the repellent.
For More Information: Chemotaxis in Escherichia coli
Chemotaxis is regulated by chemoreceptors located in the cytoplasmic membrane or periplasm of the bacterium bind chemical attractants or repellents. In most cases, this leads to either the methylation or demethylation of methyl-accepting chemotaxis proteins (MCPs) that in turn, eventually trigger either a counterclockwise or clockwise rotation of the flagellum. An increasing concentration of attractant or decreasing concentration of repellent (both conditions beneficial) causes less tumbling and longer runs; a decreasing concentration of attractant or increasing concentration of repellent (both conditions harmful) causes normal tumbling and a greater chance of reorienting in a "better" direction. As a result, the organism's net movement is toward the optimum environment..
Significance of Flagella in the Initiation of Body Defense
Initiation of Innate Immunity
To protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPs. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometimes referred to as microbe-associated molecular patterns or MAMPs.)
The protein flagellin in bacterial flagella is a PAMP that binds to pattern-recognition receptors or PRRs on a variety of defense cells of the body and triggers innate immune defenses such as inflammation, fever, and phagocytosis.
For More Information: Pathogen-Associated Molecular Patterns (PAMPs) from Unit 5
For More Information: Pattern-Recognition Receptors from Unit 5
Initiation of Adaptive Immunity
Proteins associated with bacterial flagella function as antigens and initiate adaptive immunity. An antigen is defined as a molecular shape that reacts with antibody molecules and with antigen receptors on lymphocytes. We recognize those molecular shapes as foreign or different from our body's molecular shapes because they fit specific antigen receptors on our B-lymphocytes and T-lymphocytes, the cells that carry out adaptive immunity.
The actual portions or fragments of an antigen that react with antibodies and with receptors on B-lymphocytes and T-lymphocytes are called epitopes. An epitope is typically a group of 5-15 amino acids with a unique shape that makes up a portion of a protein antigen, or 3-4 sugar residues branching off of a polysaccharide antigen. A single microorganism has many hundreds of different shaped epitopes that our lymphocytes can recognize as foreign and mount an adaptive immune response against.
The body recognizes an antigen as foreign when epitopes of that antigen bind to B-lymphocytes and T-lymphocytes by means of epitope-specific receptor molecules having a shape complementary to that of the epitope. The epitope receptor on the surface of a B-lymphocyte is called a B-cell receptor and is actually an antibody molecule. The receptor on a T-lymphocyte is called a T-cell receptor (TCR).
There are two major branches of the adaptive immune responses: humoral immunity and cell-mediated immunity.
1. Humoral immunity: Humoral immunity involves the production of antibody molecules in response to an antigen and is mediated by B-lymphocytes. Through a variety of mechanisms, these antibodies are able to remove or neutralize microorganisms and their toxins after binding to their epitopes. For example, antibodies made against flagellar antigens can stick bacteria to phagocytes, a process called opsonization. They can also interfere with bacterial motility.
2. Cell-mediated immunity: Cell-mediated immunity involves the production of cytotoxic T-lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigen and is mediated by T-lymphocytes. These defense cells help to remove infected cells and cancer cells displaying foreign epitopes.
Adaptive immunity will be discussed in greater detail in Unit 6.
For More Information: Review of antigens and epitopes from Unit 6
Significance of Motility to Bacterial Pathogenicity
Motility and chemotaxis probably help some intestinal pathogens to move through the mucous layer so they can attach to the epithelial cells of the mucous membranes. In fact, many bacteria that can colonize the mucous membranes of the bladder and the intestines are motile. Motility probably helps these bacteria move through the mucus in places where it is less viscous.
Flash animation showing a motile bacterium contacting a host cell by swimming through the mucus.
html5 version of animation for iPad showing a motile bacterium contacting a host cell by swimming through the mucus.
Motility and chemotaxis also enable spirochetes to move through viscous environments and penetrate cell membranes. Examples include Treponema pallidum (inf), Leptospira (inf), and Borrelia burgdorferi ) (inf). Because of their thinness, their internal flagella (axial filaments), and their motility, spirochetes are more readily able to penetrate host mucous membranes, skin abrasions, etc., and enter the body. Motility and invasins may also enable the spirochetes to penetrate deeper in tissue and enter the lymphatics and bloodstream and disseminate to other body sites.
Flash animation showing spirochetes using motility to enter a blood vessel.
html5 version of animation for iPad showing spirochetes using motility to enter a blood vessel.
Movie of motile Borrelia bergdorferi, the spirochete that causes Lyme disease. Note corkscrewing motility.
From You Tube, courtesy of CytoVivo.
Electron micrograph of Treponema pallidum invading a host cell.
This will be discussed in more detail under Bacterial Pathogenesis in Unit 3.
Summary
1. Many bacteria are motile and use flagella to swim through liquid environments.
2. The basal body of a bacterial flagellum functions as a rotary molecular motor, enabling the flagellum to rotate and propel the bacterium through the surrounding fluid.
3. Bacterial flagella appear in several arrangements, each unique to a particular organism.
4. Motility serves to keep bacteria in an optimum environment via taxis.
5. Taxis refers to a motile response to an environmental stimulus enabling the net movement of bacteria towards some beneficial attractant or away from some harmful repellent.
6. Most bacterial flagella can rotate both clockwise and counterclockwise enabling to stop and change direction.
7. The protein flagellin that forms the filament of bacterial flagella functions as a pathogen-associated molecular pattern or PAMP that binds to pattern-recognition receptors or PRRs on a variety of defense cells of the body to trigger innate immune defenses.
8. Motility and chemotaxis probably help some intestinal pathogens to move through the mucous layer so they can attach to the epithelial cells of the mucous membranes and colonize the intestines.
9. Motility enables some spirochetes to penetrate deeper in tissue and enter the lymphatics and bloodstream and disseminate to other body sites.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. _____ a single flagellum at both ends (ans)
1. monotrichous
2. amphitrichous
3. lophotrichous
4. peritrichous
5. axial filaments
2. State how bacterial flagella may play a role in the initiation of innate immune defenses. (ans)
3. Briefly describe how bacterial flagella and chemotaxis may play a role in the pathogenocity of some bacteria. (ans)
4. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.5%3A_Structures_Outside_the_Cell_Wall/2.5B%3A_Flagella.txt |
Learning Objectives
1. State the chemical composition, structure, and function of the short adhesion pili of bacteria.
2. State the function of a bacterial conjugation (sex) pilus.
3. Define bacterial conjugation.
4. State how the ability to change the shape of the adhesive tip of its pili could be an advantage to a bacterium.
5. Briefly describe twitching motility induced by type IV pili.
Highlighted Bacterium
1. Read the description of Neisseria gonorrhoeae and match the bacterium with the description of the organism and the infection it causes.
Structure and Composition
Fimbriae and pili are thin, protein tubes originating from the cytoplasmic membrane of many bacteria. Both are able to stick bacteria to surfaces, but pili are typically longer and fewer in number than fimbriae. They are found in virtually all Gram-negative bacteria but not in many Gram-positive bacteria. The fimbriae and pili have a shaft composed of a protein called pilin. At the end of the shaft is the adhesive tip structure having a shape corresponding to that of specific glycoprotein or glycolipid receptors on a host cell (Figure \(1\)). There are two basic types of pili: short attachment pili and long conjugation pili.
Short attachment pili, also known as fimbriae, are usually short and quite numerous (Figure \(1\)) and enable bacteria to colonize environmental surfaces or cells and resist flushing.
Long conjugation pili, also called "F" or sex pili (Figure \(4\)), that are longer and very few in number. The conjugation pilus enables conjugation. As will be seen later in this unit, conjugation is the transfer of DNA from one bacterium to another by cell-to-cell contact. In gram-negative bacteria it is typically the transfer of DNA from a donor or "male bacterium" with a sex pilus to a recipient or "female bacterium" to enable genetic recombination.
Significance of Pili to Bacterial Pathogenicity
The short attachment pili or fimbriae are organelles of adhesion allowing bacteria to colonize environmental surfaces or cells and resist flushing. The pilus has a shaft composed of a protein called pilin. At the end of the shaft is the adhesive tip structure having a shape corresponding to that of specific glycoprotein or glycolipid receptors on a host cell (Figure \(1\)). Because both the bacteria and the host cells have a negative charge, pili may enable the bacteria to bind to host cells without initially having to get close enough to be pushed away by electrostatic repulsion. Once attached to the host cell, the pili can depolymerize and enable adhesions in the bacterial cell wall to make more intimate contact.
Bacteria are constantly losing and reforming pili as they grow in the body and the same bacterium may switch the adhesive tips of the pili in order to adhere to different types of cells and evade immune defenses (Figure \(6\)). This will be discussed in detail later in Unit 3 under Bacterial Pathogenesis. Bacteria that use pili to initially colonize host cells include Neisseria gonorrhoeae, Neisseria meningitidis (inf), uropathogenic strains of Escherichia coli, and Pseudomonas aeruginosa (inf).
Highlighted Bacterium: Neisseria gonorrhoeae
Click on this link, read the description of Neisseria gonorrhoeae, and be able to match the bacterium with its description on an exam.
One class of pili, known as type IV pili , not only allow for attachment but also enable a twitching motility. They are located at the poles of bacilli and allow for a gliding motility along a solid surface such as a host cell. Extension and retraction of these pili allows the bacterium to drag itself along the solid surface (see Figure \(5\)). In addition, bacteria can use their type IV pili to "slingshot" the bacterium over a cellular surface. In this case, as the pili contract they are thought to become taut like a stretched rubber band. When an anchoring pilus detaches, the taut pili "slingshot" the bacterium in the opposite direction (see Figure \(6\)). This motion typically alternates with the twitching motility and enables a more rapid motion and direction change than with the twitching motility because the rapid slingshotting motion reduces the viscosity of the surrounding biofilm.
This enables bacteria with these types of pili within a biofilm to move around a cellular surface and find an optimum area on that cell for attachment and growth once they have initially bound. Bacteria with type IV pili include Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, and Vibrio cholerae.
• Electron micrograph of type IV pili of Neisseria gonorrhoeae from Magdalene So, University of Arizona
You Tube movie showing Pseudomonas using type IV pili to "walk" on end following binary fission.
Courtesy of Gerard Wong, UCLA Bioengineering, CNSI
Movie of twitching motility of Pseudomonas
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
Retraction of pili of Pseudomonas used in twitching motility
Courtesy of Dr. Howard C. Berg from the Roland Institute at Harvard.
Exercise: Think-Pair-Share Questions
Neisseria gonorrhoeae is a gram-negative diplococcus that has multiple alleles coding for different and distinct pili adhesive tips as well as different and distinct cell wall adhesins called Opa proteins.
The gonococcus is able to colonize and infect a numerous sites in the body, including the urethra, the rectum, the throat, the conjunctiva of the eye, and the fallopian tubes. It can also colonize sperm.
1. Considering the locations in the body where it colonizes, why doesn't the body simply flush the bacterium out of the body?
2. Why is N. gonorrhoeae able to colonize so many different sites in the body?
3. We recognize pili adhesive tips and cell wall adhesins as foreign and, during adaptive immunity, make antibodies that bind to these microbial molecules. State how this might help to protect the body.
Significance of Fimbriae and Pili in the Initiation of Body Defense
Initiation of Adaptive Immunity
Proteins associated with bacterial fimbriae and pili function as antigens and initiate adaptive immunity. An antigen is defined as a molecular shape that reacts with antibody molecules and with antigen receptors on lymphocytes. We recognize those molecular shapes as foreign or different from our body's molecular shapes because they fit specific antigen receptors on our B-lymphocytes and T-lymphocytes, the cells that carry out adaptive immunity.
Epitopes of an Antigen (Polysaccharide). Proteins have many epitopes of different specificities. During humoral immunity, antibodies are made to fit each epitope of each antigen.
The actual portions or fragments of an antigen that react with antibodies and with receptors on B-lymphocytes and T-lymphocytes are called epitopes . An epitope is typically a group of 5-15 amino acids with a unique shape that makes up a portion of a protein antigen, or 3-4 sugar residues branching off of a polysaccharide antigen. A single microorganism has many hundreds of different shaped epitopes that our lymphocytes can recognize as foreign and mount an adaptive immune response against.
Epitopes of an Antigen (Polysaccharide)
The body recognizes an antigen as foreign when epitopes of that antigen bind to B-lymphocytes and T-lymphocytes by means of epitope-specific receptor molecules having a shape complementary to that of the epitope. The epitope receptor on the surface of a B-lymphocyte is called a B-cell receptor and is actually an antibody molecule. The receptor on a T-lymphocyte is called a T-cell receptor (TCR).
There are two major branches of the adaptive immune responses: humoral immunity and cell-mediated immunity.
1. Humoral immunity: Humoral immunity involves the production of antibody molecules in response to an antigen and is mediated by B-lymphocytes. Through a variety of mechanisms, these antibodies are able to remove or neutralize microorganisms and their toxins after binding to their epitopes. For example, antibodies made against pili antigens can stick bacteria to phagocytes, a process called opsonization. Antibodies made against the adhesive tips of pili can prevent bacteria from adhering to and colonizing host cells.
2. Cell-mediated immunity: Cell-mediated immunity involves the production of cytotoxic T-lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigen and is mediated by T-lymphocytes. These defense cells help to remove infected cells and cancer cells displaying foreign epitopes.
Adaptive immunity will be discussed in greater detail in Unit 6.
Medscape article on infections associated with organisms mentioned in this Learning Object. Registration to access this website is free.
• Neisseria gonorrhoeae
• Neisseria meningitidis
• Escherichia coli
• Pseudomonas aeruginosa
• Vibrio cholerae
Summary
1. Fimbriae and pili are thin, protein tubes originating from the cytoplasmic membrane found in virtually all Gram-negative bacteria but not in many Gram-positive bacteria. Pili are typically longer and fewer in number than fimbriae.
2. The short attachment pili or fimbriae are organelles of adhesion allowing bacteria to colonize environmental surfaces or cells and resist flushing.
3. The long conjugation pilus enables conjugation in Gram-negative bacteria.
4. The pilus has a shaft composed of a protein called pilin with an adhesive tip structure at the end having a shape corresponding to that of specific receptors on a host cell.
5. The same bacterium may switch the adhesive tips of the pili in order to adhere to different types of cells and evade immune defenses.
6. Type IV pili not only allow for attachment but also enable a twitching motility that enables bacteria to “crawl” or “walk” over the surfaces to which they have attached by extending and retracting their type IV pili.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State the function of the short adhesion pili of bacteria. (ans)
2. Define bacterial conjugation. (ans)
3. State how the ability to change the shape of the adhesive tip of its pili could be an advantage to a bacterium. (ans)
4. Multiple Choice (ans)
2.E: The Prokaryotic Cell: Bacteria (Exercises)
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
Fundamental Statements for this Learning Object:
1. Physical control includes such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration.
2. Chemical control refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial chemicals.
3. Sterilization is the process of destroying all living organisms and viruses.
4. Disinfection is the elimination of microorganisms, but not necessarily endospores, from inanimate objects or surfaces.
5. Decontamination is the treatment of an object or inanimate surface to make it safe to handle.
6. A disinfectant is an agents used to disinfect inanimate objects but generally to toxic to use on human tissues.
7. An antiseptic is an agent that kills or inhibits growth of microbes but is safe to use on human tissue.
8. A sanitizer is an agent that reduces microbial numbers to a safe level.
9. An antibiotic is a metabolic product produced by one microorganism that inhibits or kills other microorganisms.
10. Synthetic chemicals that can be used therapeutically.
11. An agent that is cidal in action kills microorganisms.
12. An agent that is static in action inhibits the growth of microorganisms.
13. Selective toxicity means that the chemical being used should inhibit or kill the intended pathogen without seriously harming the host.
14. A broad spectrum agent is one generally effective against a variety of Gram-positive and Gram-negative bacteria.
15. A narrow spectrum agent generally works against just Gram-positives, Gram-negatives, or only a few bacteria.
2.1: Sizes, Shapes, and Arrangements of Bacteria
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Match the following descriptions with the best answer.
_____ Division in one plane; cocci arranged in pairs (ans)
_____ Division in one plane; cocci arranged in chains (ans)
_____ Division in two planes; cocci arranged in a square of four (ans)
_____ Division in one plane; rods completely separate after division. (ans)
_____ Division in one plane; rods arranged in chains. (ans)
_____ A comma shaped bacterium. (ans)
_____ A thin, flexible spiral. (ans)
_____ A thick, rigid spiral. (ans)
1. bacillus
2. streptobacillus
3. spirochete
4. spirillum
5. vibrio
6. streptococcus
7. staphylococcus
8. diplococcus
9. tetrad
10. sarcina
2. A Gram stain of discharge from an abcess shows cocci in irregular, grape-like clusters. What is the most likely genus of this bacterium? (ans)
3. State the diameter of an average-sized coccus-shaped bacterium. (ans)
4. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/2%3A_The_Prokaryotic_Cell_-_Bacteria/2.5%3A_Structures_Outside_the_Cell_Wall/2.5C%3A_Fimbriae_and_Pili.txt |
Bacterial genetics is the subfield of genetics devoted to the study of bacteria. Bacterial genetics are subtly different from eukaryotic genetics, however bacteria still serve as a good model for animal genetic studies. One of the major distinctions between bacterial and eukaryotic genetics stems from the bacteria's lack of membrane-bound organelles (this is true of all prokaryotes. While it is a fact that there are prokaryotic organelles, they are never bound by a lipid membrane, but by a shell of proteins), necessitating protein synthesis occur in the cytoplasm.
• 3.1: Horizontal Gene Transfer in Bacteria
Horizontal gene transfer enables bacteria to respond and adapt to their environment much more rapidly by acquiring large DNA sequences from another bacterium in a single transfer. Horizontal gene transfer is a process in which an organism transfers genetic material to another organism that is not its offspring. Mechanisms of bacterial horizontal gene transfer include transformation, transduction, and conjugation.
• 3.2: Bacterial Quorum Sensing, Pathogenicity Islands, and Secretion Systems (Injectosomes)
Pathogenicity is the ability of a microbe to cause disease and inflict damage upon its host; virulence is the degree of pathogenicity within a group or species of microbes. The pathogenicity of an organism is determined by its virulence factors. Virulence factors enable that bacterium to colonize the host, resist body defenses, and harm the body. Most of the virulence factors are the products of quorum sensing genes.
• 3.3: Enzyme Regulation
In living cells there are hundreds of different enzymes working together in a coordinated manner, and since cells neither synthesize nor break down more material than is required for normal metabolism and growth, precise enzyme regulation is required for turning metabolic reactions on and off. There is tremendous diversity in the mechanisms bacteria use to regulate enzyme synthesis and enzyme activity.
• 3.E: Bacterial Genetics (Exercises)
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
3: Bacterial Genetics
Learning Objectives
After completing this section you should be able to perform the following objectives.
1. Compare and contrast mutation and horizontal gene transfer as methods of enabling bacteria to respond to selective pressures and adapt to new environments.
2. Define horizontal gene transfer and state the most common form of horizontal gene transfer in bacteria.
3. Briefly describe the mechanisms for transformation in bacteria.
4. Briefly describe the following mechanisms of horizontal gene transfer in bacteria:
1. generalized transduction
2. specialized transduction
5. Briefly describe the following mechanisms of horizontal gene transfer in bacteria:
1. Transfer of conjugative plasmids, conjugative transposons, and mobilizable plasmids in Gram-negative bacteria
2. F+ conjugation
3. Hfr conjugation
6. Describe R-plasmids and the significance of R-plasmids to medical microbiology.
Bacteria are able to respond to selective pressures and adapt to new environments by acquiring new genetic traits as a result of mutation, a modification of gene function within a bacterium, and as a result of horizontal gene transfer, the acquisition of new genes from other bacteria. Mutation occurs relatively slowly. The normal mutation rate in nature is in the range of 10-6 to 10-9 per nucleotide per bacterial generation, although when bacterial populations are under stress, they can greatly increase their mutation rate. Furthermore, most mutations are harmful to the bacterium. Horizontal gene transfer, on the other hand, enables bacteria to respond and adapt to their environment much more rapidly by acquiring large DNA sequences from another bacterium in a single transfer.
Horizontal gene transfer, also known as lateral gene transfer, is a process in which an organism transfers genetic material to another organism that is not its offspring. The ability of Bacteria and Archaea to adapt to new environments as a part of bacterial evolution most frequently results from the acquisition of new genes through horizontal gene transfer rather than by the alteration of gene functions through mutations. (It is estimated that as much as 20% of the genome of Escherichia coli originated from horizontal gene transfer.)
Horizontal gene transfer is able to cause rather large-scale changes in a bacterial genome. For example, certain bacteria contain multiple virulence genes called pathogenicity islands that are located on large, unstable regions of the bacterial genome. These pathogenicity islands can be transmitted to other bacteria by horizontal gene transfer. However, if these transferred genes provide no selective advantage to the bacteria that acquire them, they are usually lost by deletion. In this way the size of the bacterium's genome can remain approximately the same size over time.
There are three mechanisms of horizontal gene transfer in bacteria: transformation, transduction, and conjugation. The most common mechanism for horizontal gene transmission among bacteria, especially from a donor bacterial species to different recipient species, is conjugation. Although bacteria can acquire new genes through transformation and transduction, this is usually a more rare transfer among bacteria of the same species or closely related species.
Transformation
Transformation is a form of genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and is exchanged for a piece of DNA of the recipient. Transformation usually involves only homologous recombination, a recombination of homologous DNA regions having nearly the same nucleotide sequences. Typically this involves similar bacterial strains or strains of the same bacterial species.
A few bacteria, such as Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneomophila, Streptococcus pneumoniae, and Helicobacter pylori tend to be naturally competent and transformable. Competent bacteria are able to bind much more DNA than noncompetent bacteria. Some of these genera also undergo autolysis that then provides DNA for homologous recombination. In addition, some competent bacteria kill noncompetent cells to release DNA for transformation.
During transformation, DNA fragments (usually about 10 genes long) are released from a dead degraded bacterium and bind to DNA binding proteins on the surface of a competent living recipient bacterium. Depending on the bacterium, either both strands of DNA penetrate the recipient, or a nuclease degrades one strand of the fragment and the remaining DNA strand enters the recipient. This DNA fragment from the donor is then exchanged for a piece of the recipient's DNA by means of RecA proteins and other molecules and involves breakage and reunion of the paired DNA segments as seen in (Figure \(1\)). Transformation is summarized in Figure \(2\).
Figure \(2\): Transformation: Step 1: A donor bacterium dies and is degraded.Step 2: DNA fragments, typically around 10 genes long, from the dead donor bacterium bind to transformasomes on the cell wall of a competent, living recipient bacterium.Step 3: In this example, a nuclease degrades one strand of the donor fragment and the remaining DNA strand enters the recipient. Competence-specific single-stranded DNA-binding proteins bind to the donor DNA strand to prevent it from being degraded in the cytoplasm. Step 4: RecA proteins promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA (see Figure \(1\) for the functions of RecA proteins). This involves breakage and reunion of paired DNA segments. Step 5: Transformation is complete.
Transduction
Transduction involves the transfer of a DNA fragment from one bacterium to another by a bacteriophage. There are two forms of transduction: generalized transduction and specialized transduction.
During the replication of lytic bacteriophages and temperate bacteriophages, occasionally the phage capsid accidently assembles around a small fragment of bacterial DNA. When this bacteriophage, called a transducing particle, infects another bacterium, it injects the fragment of donor bacterial DNA it is carrying into the recipient where it can subsequently be exchanged for a piece of the recipient's DNA by homologous recombination. Generalized transduction is summarized in Figure \(3\).
• Step 1: A bacteriophage adsorbs to a susceptible bacterium.
• Step 2: The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes. Bacteriophage-coded enzymes will also breakup the bacterial chromosome.
• Step 3: Occasionally, a bacteriophage capsid mistakenly assembles around either a fragment of the donor bacterium's chromosome or around a plasmid instead of around a phage genome.
• Step 4: The bacteriophages are released as the bacterium is lysed. Note that one bacteriophage is carrying a fragment of the donor bacterium's DNA rather than a bacteriophage genome.
• Step 5: The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium.
• Step 6: The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium.
• Step 7: Homologous recombination occurs and the donor bacterium's DNA is exchanged for some of the recipient's DNA. (Figure \(1\) shows the functions of the RecA proteins involved in homologous recombination.)
Generalized transduction occurs in a variety of bacteria, including Staphylococcus, Escherichia, Salmonella, and Pseudomonas.
Plasmids, such as the penicillinase plasmid of Staphylococcus aureus, may also be carried from one bacterium to another by generalized transduction.
Specialized transduction: This may occur occasionally during the lysogenic life cycle of a temperate bacteriophage. During spontaneous induction, a small piece of bacterial DNA may sometimes be exchanged for a piece of the bacteriophage genome, which remains in the bacterial nucleoid. This piece of bacterial DNA replicates as a part of the bacteriophage genome and is put into each phage capsid. The bacteriophages are released, adsorb to recipient bacteria, and inject the donor bacterium DNA/phage DNA complex into the recipient bacterium where it inserts into the bacterial chromosome (Figure \(4\)).
Conjugation
Genetic recombination in which there is a transfer of DNA from a living donor bacterium to a living recipient bacterium by cell-to-cell contact. In Gram-negative bacteria it typically involves a conjugation or sex pilus.
Conjugation is encoded by plasmids or transposons. It involves a donor bacterium that contains a conjugative plasmid and a recipient cell that does not. A conjugative plasmid is self-transmissible, in that it possesses all the necessary genes for that plasmid to transmit itself to another bacterium by conjugation. Conjugation genes known as tra genes enable the bacterium to form a mating pair with another organism, while oriT (origin of transfer) sequences determine where on the plasmid DNA transfer is initiated by serving as the replication start site where DNA replication enzymes will nick the DNA to initiate DNA replication and transfer. In addition, mobilizable plasmids that lack the tra genes for self-transmissibility but possess the oriT sequences for initiation of DNA transfer may also be transferred by conjugation if the bacterium containing them also possesses a conjugative plasmid. The tra genes of the conjugative plasmid enable a mating pair to form, while the oriT of the mobilizable plasmid enable the DNA to moves through the conjugative bridge (Figure \(5\)).
Transposons ("jumping genes") are small pieces of DNA that encode enzymes that enable the transposon to move from one DNA location to another, either on the same molecule of DNA or on a different molecule. Transposons may be found as part of a bacterium's chromosome (conjugative transposons) or in plasmids and are usually between one and twelve genes long. A transposon contains a number of genes, such as those coding for antibiotic resistance or other traits, flanked at both ends by insertion sequences coding for an enzyme called transpoase. Transpoase is the enzyme that catalyzes the cutting and resealing of the DNA during transposition.
Conjugative transposons, like conjugative plasmids, carry the genes that enable mating pairs to form for conjugation. Therefore, conjugative transposons also enable mobilizable plasmids and nonconjugative transposons to be transferred to a recipient bacterium during conjugation.
Many conjugative plasmids and conjugative transposons possess rather promiscuous transfer systems that enables them to transfer DNA not only to like species, but also to unrelated species. The ability of bacteria to adapt to new environments as a part of bacterial evolution most frequently results from the acquisition of large DNA sequences from another bacterium by conjugation.
a. General mechanism of transfer of conjugative plasmids by conjugation in Gram-negative bacteria
In Gram-negative bacteria, the first step in conjugation involves a conjugation pilus (sex pilus or F pilus) on the donor bacterium binding to a recipient bacterium lacking a conjugation pilus. Typically the conjugation pilus retracts or depolymerizes pulling the two bacteria together. A series of membrane proteins coded for by the conjugative plasmid then forms a bridge and an opening between the two bacteria, now called a mating pair.
Using the rolling circle model of DNA replication, a nuclease breaks one strand of the plasmid DNA at the origin of transfer site (oriT) of the plasmid and that nicked strand enters the recipient bacterium. The other strand remains behind in the donor cell. Both the donor and the recipient plasmid strands then make a complementary copy of themselves. Both bacteria now possess the conjugative plasmid. This process is summarized in Figure \(6\)).
This is the mechanism by which resistance plasmids (R-plasmids), coding for multiple antibiotic resistance and conjugation pilus formation, are transferred from a donor bacterium to a recipient. This is a big problem in treating opportunistic Gram-negative infections such as urinary tract infections, wound infections, pneumonia, and septicemia by such organisms as E. coli, Proteus, Klebsiella, Enterobacter, Serratia, and Pseudomonas, as well as with intestinal infections by organisms like Salmonella and Shigella.
There is also evidence that the conjugation pilus may also serve as a direct channel through which single-stranded DNA may be transferred during conjugation.
b. F+ conjugation
This results in the transfer of an F+ plasmid possessing tra genes coding only for a conjugation pilus and mating pair formation from a donor bacterium to a recipient bacterium. One strand of the F+ plasmid is broken with a nuclease at the origin of transfer (oriT) sequence that determines where on the plasmid DNA transfer is initiated by serving as the replication start site where DNA replication enzymes will nick the DNA to initiate DNA replication and transfer. The nicked strand enters the recipient bacterium while the other plasmid strand remains in the donor. Each strand then makes a complementary copy. The recipient then becomes an F+ male and can make a sex pilus (see 7A through 7D).
In addition, mobilizable plasmids that lack the tra genes for self-transmissibility but possess the oriT sequences for initiation of DNA transfer, may also be transferred by conjugation. The tra genes of the F+ plasmid enable a mating pair to form and the oriT sequences of the mobilizable plasmid enable the DNA to moves through the conjugative bridge (Figure \(5\)).
c. Hfr (high frequency recombinant) conjugation
Hfr conjugation begins when an F+ plasmid with tra genes coding for mating pair formation inserts or integrates into the chromosome to form an Hfr bacterium. (A plasmid that is able to integrate into the host nucleoid is called an episome.) A nuclease then breaks one strand of the donor's DNA at the origin of transfer (oriT) location of the inserted F+ plasmid and the nicked strand of the donor DNA begins to enter the recipient bacterium. The remaining non-nicked DNA strand remains in the donor and makes a complementary copy of itself.
The bacterial connection usually breaks before the transfer of the entire chromosome is completed so the remainder of the F+ plasmid seldom enters the recipient. As a result, there is a transfer of some chromosomal DNA, which may be exchanged for a piece of the recipient's DNA through homologous recombination, but not the ability to form a conjugation pilus and mating pairs (see Figure \(8\)A through 8E).
Exercise: Think-Pair-Share Questions
1. A strain of living Streptococcus pneumoniae that cannot make a capsule is injected into mice and has no adverse effect. This strain is then mixed with a culture of heat-killed Streptococcus pneumoniae that when alive was able to make a capsule and kill mice. After a period of time, this mixture is injected into mice and kills them. In terms of horizontal gene transfer, describe what might account for this.
2. A gram-negative bacterium that was susceptible to most common antibiotics suddenly becomes resistant to several of them. It also appears to be spreading this resistance to others of its kind. Describe the mechanism that most likely accounts for this.
Summary
1. Mutation is a modification of gene function within a bacterium and while it enables bacteria to adapt to new environments, it occurs relatively slowly.
2. Horizontal gene transfer enables bacteria to respond and adapt to their environment much more rapidly by acquiring large DNA sequences from another bacterium in a single transfer.
3. Horizontal gene transfer is a process in which an organism transfers genetic material to another organism that is not its offspring.
4. Mechanisms of bacterial horizontal gene transfer include transformation, transduction, and conjugation.
5. During transformation, a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and is exchanged for a piece of DNA of the recipient. Typically this involves similar bacterial strains or strains of the same bacterial species.
6. Transduction involves the transfer of either a chromosomal DNA fragment or a plasmid from one bacterium to another by a bacteriophage.
7. Conjugation is a transfer of DNA from a living donor bacterium to a living recipient bacterium by cell-to-cell contact. In Gram-negative bacteria it involves a conjugation pilus.
8. A conjugative plasmid is self-transmissible, that is, it possesses conjugation genes known as tra genes enable the bacterium to form a mating pair with another organism, and oriT (origin of transfer) sequences that determine where on the plasmid DNA transfer is initiated.
9. Mobilizable plasmids that lack the tra genes for self-transmissibility can be co-transfered in a bacterium possessing a conjugative plasmid.
10. Transposons ("jumping genes") are small pieces of DNA that encode enzymes that enable the transposon to move from one DNA location to another, either on the same molecule of DNA or on a different molecule.
11. Conjugative transposons carry the genes that enable mating pairs to form for conjugation.
12. F+ conjugation is the transfer of an F+ plasmid possessing tra genes coding only for a conjugation pilus and mating pair formation from a donor bacterium to a recipient bacterium. Mobilizable plasmids may be co-transfered during F+ conjugation.
13. During Hfr conjugation, an F+ plasmid with tra genes coding for mating pair formation inserts into the bacterial chromosome to form an Hfr bacterium. This results in a transfer of some chromosomal DNA from the donor to the recipient which may be exchanged for a piece of the recipient's DNA through homologous recombination. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/3%3A_Bacterial_Genetics/3.1%3A_Horizontal_Gene_Transfer_in_Bacteria.txt |
Learning Objectives
1. Define the following:
1. pathogenicity
2. virulence
2. Even though a microorganism may be considered pathogenic, it still may not be able to cause disease upon entering the body. Discuss why.
3. Define and briefly describe the overall process of quorum sensing in bacteria and how it may enable bacteria to behave as a multicellular population.
4. State at least two possible advantages of individual bacterial behavior.
5. State at least two possible advantages of multicellular bacterial behavior.
6. State what is meant by intraspecies, interspecies, and interkingdom communication.
7. State the function of bacterial secretions systems (injectisomes) such as the type 3 and type 6 secretion systems in bacterial pathogenicity.
In this Learning Object we are going to look at several aspects of bacterial genetics that are directly related to bacterial pathogenicity, namely, quorum sensing, pathogenicity islands, and secretion systems. Pathogenicity and virulence are terms that refer to an organism's ability to cause disease. Pathogenicity is the ability of a microbe to cause disease and inflict damage upon its host, whereas virulence is the degree of pathogenicity within a group or species of microbes as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenicity of an organism, that is its ability to cause disease, is determined by its virulence factors .
Many of the virulence factors that enable bacteria to colonize the body and/or harm the body are the products of quorum sensing genes. Many bacteria are able to sense their own population density, communicate with each other by way of secreted chemical factors, and behave as a population rather than as individual bacteria . This plays an important role in pathogenicity and survival for many bacteria.
Bacterial Quorum Sensing
Bacteria can behave either as individual single-celled organisms or as multicellular populations. Bacteria exhibit these behaviors by chemically "talking" to one another through a process called quorum sensing. Quorum sensing involves the production, release, and community-wide sensing of molecules called autoinducers that modulate gene expression, and ultimately bacterial behavior, in response to the density of a bacterial population.
To initiate the process of quorum sensing, bacterial genes code for the production of signaling molecules called autoinducers that are released into the bacterium's surrounding environment. These signaling molecules then bind to signaling receptors either on the bacterial surface or in the cytoplasm. When these autoinducers reach a critical, threshold level, they activate bacterial quorum sensing genes that enable the bacteria to behave as a multicellular population rather than as individual single-celled organisms (Figure \(3\).2.2). The autoinducer/receptor complex is able to bind to DNA promoters and activate the transcription of quorum sensing-controlled genes in the bacterium. In this way, individual bacteria within a group are able to benefit from the activity of the entire group.
1. In Gram-negative bacteria, the autoinducers are typically molecules called acyl-homoserine lactones or AHL. AHLs diffuse readily out of and into bacterial cells where they bind to AHL receptors in the cytoplasm of the bacteria. When a critical level of AHL is reached, the cytoplasmic autoinducer/receptor complex functions as a DNA-binding transcriptional activator.
2. In Gram-positive bacteria, the autoinducers are oligopeptides, short peptides typically 8-10 amino acids long. Oligopeptides cannot diffuse in and out of bacteria like AHLs, but rather leave bacteria via specific exporters. They then bind to autoinducer receptors on the surface of the bacterium. When a critical level of oligopeptide is reached, the binding of the oligopeptide to its receptor starts a phosphorylation cascade that activates DNA-binding transcriptional regulatory proteins called response regulators.
The outcomes of bacteria-host interaction are often related to bacterial population density. Bacterial virulence, that is its ability to cause disease, is largely based on the bacterium's ability to produce gene products called virulence factors that enable that bacterium to colonize the host, resist body defenses, and harm the body.
At a low density of bacteria, the autoinducers diffuse away from the bacteria (Figure \(3\).2.2). Sufficient quantities of these molecules are unable to bind to the signaling receptors on the bacterial surface and the quorum sensing genes that enable the bacteria to act as a population are not activated. This enables the bacteria to behave as individual, single-celled organisms.
Possible advantages of individual bacterial behavior seen at low bacterial density
If a relatively small number of a specific bacterium were to enter the body and immediately start producing their virulence factors, chances are the body's immune systems would have sufficient time to recognize and counter those virulence factors and remove the bacteria before there was sufficient quantity to cause harm. The bacterium instead utilizes genes that enable it to act as an individual organism rather than as part of a multicellular population.
Acting as individual organisms may better enable that low density of bacteria to gain a better foothold in their new environment in the following ways:
1. Many bacteria are capable of motility and motility serves to keep bacteria in an optimum environment via taxis .
Motility and chemotaxis probably help some intestinal and urinary pathogens to move through the mucous layer so they can attach to the epithelial cells of the mucous membranes. In fact, many bacteria that can colonize the mucous membranes of the bladder and the intestines are motile. Motility probably helps these bacteria move through the mucus in places where it is less viscous.
2. One of the body's innate defenses is the ability to physically remove bacteria from the body through such means as the constant shedding of surface epithelial cells from the skin and mucous membranes, the removal of bacteria by such means as coughing, sneezing, vomiting, and diarrhea, and bacterial removal by bodily fluids such as saliva, blood, mucous, and urine. Bacteria may resist this physical removal by producing pili (see Figure \(3\)), cell wall adhesin proteins (Figure \(3\).2.4), and/or biofilm-producing capsules . Some pili, called type IV pili also allow some bacteria to "walk" or "crawl" along surfaces to spread out and eventually form microcolonies.
Figure \(3\).2.3: Adhesive Tip of Bacterial Pili Binding to Host Cell Receptors
3. Many bacteria secrete an extracellular polysaccharide or polypeptide matrix called a capsule or glycocalyx that enables the bacteria to adhere to host cells, resist phagocytosis, and form microcolonies. As the bacteria geometrically increase in number by binary fission, so does the amount of their secreted autoinducers, and production of high levels of autoinducers then enables the population of bacteria to communicate with one another by quorum sensing. At a high density of bacteria, large quantities of autoinducers are produced (Figure \(3\).2.5) and are able to bind to the signaling receptors on the bacterial surface in sufficient quantity so as to activate the quorum sensing genes that enable the bacteria to behave as a multicellular population (Figure \(3\).2.1).
Advantages of Multicellular Behavior seen at High Bacterial Density
1. By behaving as a multicellular population, individual bacteria within a group are able to benefit from the activity of the entire group. As the entire population of bacteria simultaneously turn on their virulence genes, the body's immune systems are much less likely to have enough time to counter those virulence factors before harm is done.
2. This triggers production of an extracellular adhesive matrix (glycocalyx) enabling the bacteria to form microcolonies and irreversibly attachment to the mucous membranes. Biofilm formation begins.
3. Virulence factors such as exoenzymes and toxins can damage host cells enabling the bacteria in the biofilm to obtain nutrients. The biofilm continues to develop and mature.
4. As the area becomes over-populated with bacteria, quorum sensing enables some of the bacteria to escape the biofilm, often by again producing flagella, and return to individual single-celled organism behavior in order to find a new sight to colonize.
Pseudomonas aeruginosa is an example of a quorum sensing bacterium. P. aeruginosa causes severe hospital-acquired infections, chronic infections in people with cystic fibrosis, and potentially fatal infections in those who are immunocompromised.
1. When P. aeruginosa first enters the body, they are at a low density of bacteria. The autoinducers diffuse away from the bacteria (Figure \(3\).2.2), sufficient quantities of these molecules are unable to bind to the signaling receptors, and the quorum sensing genes that enable the bacteria to act as a population are not activated. The P. aeruginosa continue to function as individual bacteria. Motility genes (coding for flagella) and adhesin genes (coding for pili and cell wall adhesins) are expressed. The flagella enable the initial bacteria to swim through mucus towards host tissues such as mucous membranes. Pili then enable the bacteria to reversibly attach to host cells in order to resist flushing and begin colonization (Figure \(3\).2.6; left). Type IV pili, which enable a twitching motility in some bacteria, then enable the bacteria as they replicate to crawl along and spread out over the mucous membranes (Figure \(3\).2.6; middle). The pili subsequently retract and bacterial cell wall adhesins enable a more intimate attachment of the bacterium to the mucous membranes (Figure \(3\).2.6; right).
2. Once P. aeruginosa has colonized, it is able to replicate geometrically and achieve a high population density. Quorum sensing genes are activated and the bacteria function as a population. This triggers production of an extracellular polysaccharide called alginate to form microcolonies and enables irreversible attachment to the mucous membranes (Figure 3.2.7; left). Biofilm formation begins.
3. Quorum sensing genes coding for enzymes and toxins that damage host cells are produced. These are injected into the host cells by way of an injectosome. This releases nutrients for the bacteria in the biofilm. The bacteria continue to replicate as the biofilm continues to develop, mushroom up, and mature (Figure 3.2.7; middle).
4. As the bacteria replicate, the biofilm continues to mature (Figure 3.2.7; right). Water channels form within the biofilm to deliver water, oxygen, and nutrients to the growing population of P. aeruginosa. The high density of bacteria bacteria are now acting as a multicellular population rather than as individual bacteria.
The biofilm enables bacteria to:
• resist attack by antibiotics;
• trap nutrients for bacterial growth and remain in a favorable niche;
• adhere to environmental surfaces and resist flushing;
• live in close association and communicate with other bacteria in the biofilm; and
• resist phagocytosis and attack by the body's complement pathways.
5. When the population of P. aeruginosa begins to outgrow their local environment, quorum sensing enables them to turn off adhesin genes and turn on flagella genes that allow some of the bacteria to spread out of the biofilm to new location within that environment via motility (Figure \(3\).2.8).
It turns out that bacteria are multilingual. They use quorum sensing not only to "talk" to members their own species (intraspecies communication), but also to "talk" to bacteria that are not of their genus and species (interspecies communication). Intraspecies autoinducers and receptors enable bacteria to communicate with others of their own species while interspecies autoinducers and receptors enable bacteria to communicate with bacteria of a different species or genus (Figure \(3\).2.9). The autoinducers for interspecies communications are referred to as AI-2 family autoinducers and are different from the intraspecies (AI-1) autoinducers. In some cases bacteria use interspeciecies communication to work cooperatively with various other bacteria in their biofilm to the benefit all involved; in other cases, bacteria may use interspecies communication in such a way that one group benefits at the expense of another.
Figure \(3\).2.9: Intraspecies and Interspecies Communication. Intraspecies autoinducers and receptors enable bacteria to communicate with others of their own species while interspecies autoinducers and receptors enable bacteria to communicate with bacteria of a different species or genus.
Furthermore, bacteria are capable of interkingdom communication, communication between bacteria and their animal or plant host. Increasing numbers of bacteria are being found that have signaling receptors that recognize human hormones. For example, a number of bacteria that are pathogens of the human intestinal tract have a sensing molecule called QseC that binds the human hormones adrenaline and noradrenaline. This, in turn, activates various virulence genes of the bacteria. On the other hand, some bacterial autoinducers can enter human host cells and regulate human cellular function. For example, at low concentration some bacterial autoinducers suppress host immune responses thus better enabling those bacteria to better establish themselves in the body. At high concentrations, however, they stimulate an inflammatory response in the host to help the bacteria to spread from the initial infection site. One bacterial autoinducer has been found to initiate apoptosis (cell suicide) in phagocytes such as neutrophils and macrophages.
Bacterial Pathogenicity Islands
The genomes of pathogenic bacteria, when compared with those of similar nonpathogenic species or strains, often show extra genes coding for virulence factors , that is, molecules expressed and secreted by the bacterium that enable them to colonize the host, evade or inhibit the immune responses of the host, enter into or out of a host cell, and/or obtain nutrition from the host. These include virulence factors such as capsules, adhesins, type 3 secretion systems, invasins, and toxins.
Most genes coding for virulence factors in bacteria are located in pathogenicity islands or PAIs and are usually acquired by horizontal gene transfer . These PAIs may be located in the bacterial chromosome, in plasmids, or even in bacteriophage genomes that have entered the bacterium. The genomes of most pathogenic bacteria typically contain multiple PAIs that can account for up to 10 transpoases ,- 20% of the bacterium's genome. PAIs carry genes such as integrases , or insertion sequences that enable them to insert into host bacterial DNA. Transfer RNA (tRNA) genes are often the target site for integration of PAIs. Conjugative plasmids are the most frequent means of transfer of PAIs from one bacterium to another and the transfer of PAIs can then confer virulence to a previously nonpathogenic bacterium.
Type 3 Secretion Systems (T3SS or Injectisomes) and Type 6 Secretion Systems (T6SS)
Many bacteria involved in infection have the ability to co-opt the functions of host cells for the bacterium’s own benefit. This is done by way of bacterial secretions systems that enable the bacterium to directly inject bacterial effector molecules into the cytoplasm of the host cell in order to alter its cellular machinery or cellular communication to the benefit of the bacteria.
The most common type is the type 3 secretion system or T3SS (Figure \(3\).2.10). A secretion apparatus in the cytoplasmic membrane and cell wall of the bacterium polymerizes a hollow needle that is lowered to the cytoplasmic membrane of the host cell and a translocon protein is then delivered to anchor the needle to the host cell. Effector proteins in the bacterium can now be injected into the cytoplasm of the host cell. The delivery system is sometimes called an injectisome. (A type 4 secretion system can transfer effector proteins and/or DNA into the host cell because it is similar to the conjugation transfer system initiated by tra genes discussed under horizontal gene transfer.)
Electron micrograph of an injectisome. A transmission electron-microscope image of isolated T3SS needle complexes from Salmonella typhimurium. (CC BY-SA 2.5; Schraidt O, Lefebre MD, Brunner MJ, Schmied WH, Schmidt A, Radics J, Mechtler K, Galán JE, Marlovits TC - Cropped image from Schraidt et al. (2010), Topology and Organization of the Salmonella typhimurium Type III Secretion Needle Complex Components. PLoS Pathog 6(4): e1000824.doi:10.1371/journal.ppat.1000824)
Some bacteria, such as Pseudomonas aeruginosa and Vibrio cholerae, produce a type 6 secretion system, or T6SS, that consists of a protein tube surrounded by a contractile sheath, similar to the tail of T4-bacteriophages (a bacteriophage is a virus that only infects bacteria.) The type 6 secretion system not only injects effector molecules into eukaryotic cells, but also is able to inject antibacterial effector molecules into other bacteria in order to kill those bacteria. Predator bacteria can use their T6SS to kill prey bacteria. In fact, V. cholerae and P. aeruginosa have been shown to "duel" with one another via their respective T6SSs.
V. cholerae also uses its T6SS to promote horizontal gene transfer by way of transformation. Individual V. cholerae cells also use their T6SS to attack one another upon cell-to-cell contact. Most members of the population, however, produce immunity proteins that protect them from being killed by the effector molecules that are injected. Not all strains of V. cholerae in the population, however, produce these immunity proteins and these non-immune cells are subsequently lysed, releasing their DNA into the environment. This DNA can then be taken up by neighboring competent V. cholerae via transformation.
Exercise: Think-Pair-Share Questions
1. Briefly describe how bacterial quorum sensing may play a role in pathogenicity by:
1. Promoting colonization of a new host by bacteria that have just entered the body.
2. Enabling the bacterium to persist within that host once they have colonized.
3. Allowing some of the bacteria to spread to a new location within a host or to a new host.
2. Briefly describe how the ability to produce a type 3 secretion system might play a role in a pathogen colonizing the body and causing an infection.
Summary
1. Pathogenicity is the ability of a microbe to cause disease and inflict damage upon its host; virulence is the degree of pathogenicity within a group or species of microbes.
2. The pathogenicity of an organism is determined by its virulence factors.
3. Virulence factors enable that bacterium to colonize the host, resist body defenses, and harm the body.
4. Most of the virulence factors are the products of quorum sensing genes.
5. Quorum sensing involves the production, release, and community-wide sensing of molecules called autoinducers that modulate gene expression, and ultimately bacterial behavior, in response to the density of a bacterial population.
6. The outcomes of bacteria-host interaction are often related to bacterial population density.
7. At a low density of bacteria, the autoinducers diffuse away from the bacteria and there are insufficient quantities of these molecules to activate the quorum sensing genes that enable the bacteria to act as a population. As a result the bacteria behave as individual, single-celled organisms.
8. Acting as individual organisms may enable a low density of bacteria to gain a better foothold in their new environment by enabling bacteria to use motility and taxis to contact host cells, use pili to initially adhere to and crawl over host cell surfaces, use adhesins to adhere to host cells and resist flushing, and secrete a glycocalyx to form microcolonies.
9. As the bacteria increase in numbers geometrically as a result of binary fission and reach high density, large quantities of autoinducers are produced and are able to bind to the signaling receptors on the bacterial surface in sufficient quantity so as to activate the quorum sensing genes that enable the bacteria to now behave as a multicellular population.
10. By behaving as a multicellular population, individual bacteria within a group are able to benefit from the activity of the entire group.
11. As the entire population of bacteria simultaneously turn on their virulence genes, the body's immune systems are much less likely to have enough time to counter those virulence factors before harm is done. Virulence factors such as exoenzymes and toxins can damage host cells enabling the bacteria in the biofilm to obtain nutrients.
12. As the area becomes over-populated with bacteria, quorum sensing enables some of the bacteria to escape the biofilm and return to individual single-celled organism behavior in order to find a new sight to colonize.
13. Quorum sensing enables bacteria to communicate with members of their own species, with other species of bacteria, and with their eukaryotic host cells.
14. Most genes coding for virulence factors in bacteria are located in pathogenicity islands or PAIs and are usually acquired by horizontal gene transfer.
15. Many bacteria involved in infection have the ability to co-opt the functions of the host cell for the bacterium’s own benefit by producing secretions systems that enable the bacterium to directly inject bacterial effector molecules into the cytoplasm of the host cell in order to alter the host cell’s cellular machinery, cellular function, or cellular communication. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/3%3A_Bacterial_Genetics/3.2%3A_Bacterial_Quorum_Sensing_Pathogenicity_Islands_and_Secretion_Systems_%28Injectosomes%29.txt |
null Learning Objectives
1. Compare and contrast the genetic control of enzyme activity (enzyme synthesis) in bacteria with the control of enzyme activity through feedback inhibition.
2. Compare and contrast an inducible operon with a repressible operon and give an example of each.
3. Compare how the presense or absence of tryptophan affects the trp operon.
4. Compare how the presense or absence of lactose affects the lac operon.
5. Compare how the presense or absence of an inducer affects activators.
6. Briefly describe how small RNAs can regulate enzyme activity.
7. Define the following:
1. repressor
2. inducer
3. activator
4. enhancer
5. small RNAs
8. Compare and contrast competitive inhibition with noncompetitive inhibition.
In living cells, there are hundreds of different enzymes working together in a coordinated manner. Living cells neither synthesize nor break down more material than is required for normal metabolism and growth. All of this necessitates precise control mechanisms for turning metabolic reactions on and off. There is tremendous diversity in the mechanisms bacteria use to regulate enzyme synthesis and enzyme activity. For pretty much every step between the activation of a gene and the final enzyme reaction from that gene product there is some bacterial mechanism for regulation that step. Here we will look at several well studied examples.
Genetic Control of Enzyme Synthesis through Repression, Induction, or Enhancement of Transcription
Genetic control of enzyme activity refers to controlling transcription of the mRNA needed for an enzyme's synthesis. In prokaryotic cells, this involves the induction, repression, or enhancement of enzyme synthesis by regulatory proteins that can bind to DNA and either induce, block, or enhance the function of RNA polymerase , the enzyme required for transcription. The regulatory proteins are often part of either an operon or a regulon. An operon is a set of genes transcribed as a polycistronic message that is collectively controlled by a regulatory protein. A regulon is a set of related genes controlled by the same regulatory protein but transcribed as monocistronic units. Regulatory proteins may function either as repressors, activators, or enhancers.
a. Repressors
Repressors are regulatory proteins that block transcription of mRNA. They do this by binding to a portion of DNA called the operator (operators are often called boxes now) that lies downstream of a promoter. The binding of the regulatory protein to the operator prevents RNA polymerase from binding to the promoter and transcribing the coding sequence for the enzymes. This is called negative control and is mostly n in biosynthetic reactions where a bacterium only makes a molecule like a particular amino acid when that amino acid is not present in the cell.
Repressors are allosteric proteins that have a binding site for a specific molecule. Binding of that molecule to the allosteric site of the repressor can alter the repressor's shape that, in turn affects its ability to bind to DNA. This can work in one of two ways:
1. Some repressors are synthesized in a form that cannot by itself bind to the operator. This is referred to as a repressible system. The binding of a molecule called a corepressor, however, alters the shape of the regulatory protein to a form that can bind to the operator and subsequently block transcription. An example of this type of repressible system is the trp operon in Escherichia coli that encodes the five enzymes in the pathway for the biosynthesis of the amino acid tryptophan. In this case, the repressor protein coded for by the trp regulatory gene, normally does not bind to the operator region of the trp operon and the five enzymes needed to synthesize the amino acid tryptophan are made (Figure \(1\)A and Figure \(1\)B).
Tryptophan, the end product of these enzyme reactions, however, functions as a corepressor. Once sufficient tryptophan has been synthesized, the cell needs to terminate its synthesis. The tryptophan is able to bind to a site on the allosteric repressor protein, changing its shape and enabling it to interact with the trp operator region. Once the repressor binds to the operator, RNA polymerase is unable to bind to the promoter and transcribe the genes for tryptophan biosynthesis. Therefore, when sufficient tryptophan is present, transcription of the enzymes that allows for its biosynthesis are turned off ( Figure \(2\)A and Figure \(2\)B).
In addition to repression, the expression of the trp operon is also regulated by attenuation. The trpL gene codes for a mRNA leader sequence that controls operon expression through attenuation. This leader sequence mRNA consists of domains 1, 2, 3, and 4. Domain 3 can base pair with either domain 2 or domain 4.
At high tryptophan concentrations, domains 3 and 4 pair in such a way as to form stem and loop structures that block the transcription of the remainder of the leader sequence mRNA and subsequently, the transcription of the structural genes for tryptophan biosynthesis ( Figure \(3\)A). However, at low concentrations of tryptophan, domains 3 and 2 pair. This pairing allows for the full transcription of the leader sequence mRNA, as well as that of the structural genes for tryptophan biosynthesis ( Figure \(3\)B).
2. Other repressors are synthesized in a form that readily binds to the operator and blocks transcription. However, the binding of a molecule called an inducer alters the shape of the regulatory protein in a way that now blocks its binding to the operator and thus permits transcription. This is referred to as an inducible system.
An example of an inducible system is the lac operon that encodes for the three enzymes needed for the degradation of lactose by E. coli. E. coli will only synthesize the enzymes it requires to utilize lactose if that sugar is present in the surrounding environment. In this case, lactose functions as an inducer . In the absence of lactose, the active repressor protein binds to the operator and RNA polymerase is unable to bind to the promoter and transcribe the genes for utilization of lactose. As a result, the enzymes needed for the utilization of lactose are not synthesized (Figure \(4\)A and Figure \(4\)B). When lactose, the inducer, is present, a metabolite of lactose called allolactose binds to the allosteric repressor protein and causes it to change shape in such a way that it is no longer able to bind to the operator. Now RNA polymerase is able to transcribe the three lac operon structural genes and the bacterium is able to synthesize the enzymes required for the utilization of lactose (Figure \(5\)A and Figure \(5\)B).
b. Activators
Activators are regulatory proteins that promote transcription of mRNA. Activators control genes that have a promotor to which RNA polymerase cannot bind. The promotor lies adjacent to a segment of DNA called the activator-binding site. The activator is an allosteric protein synthesized in a form that cannot normally bind to the activator-binding site. As a result, RNA polymerase is unable to bind to the promoter and transcribe the genes ( Figure \(6\)). However, binding of a molecule called an inducer to the activator alters the shape of the activator in a way that now allows it to bind to the activator-binding site. The binding of the activator to the activator-binding site, in turn, enables RNA polymerase to bind to the promotor and initiate transcription ( Figure \(7\)A and Figure \(7\)B). This is called positive control and is mostly n in catabolic reactions where a bacterium only makes enzymes for the catabolism of a substrate when that substrate is available to the cell.
c. Enhancers
Enhancers are regulatory proteins that bind to DNA located some distance from the operon they control by working with DNA-bending proteins. The DNA-binding proteins bend the DNA in a way that now allows the enhancer to interact with the promoter in such a way that RNA polymerase can now bind and initiate transcription ( Figure \(8\)).
2. Genetic Control of Enzyme Synthesis through Promoter Recognition and through DNA Supercoiling
a. Promoter Recognition: The specific sigma factors that bind to RNA polymerase determine which operon will be transcribed.
b. DNA Supercoiling: DNA supercoiling can change the tertiary shape of a DNA molecule from its normal form to one that has a left-handed twist called Z-DNA. The activities of some promoters are decreased with Z-DNA while others are increased.
3. Genetic Control of Enzyme Synthesis through the Translational Control of Enzyme Synthesis
a. RNA interference (RNAi)
RNA interference (RNAi) is a process whereby small non-coding regulatory RNAs (ncRNAs) such as microRNAs (miRNAs) regulate gene expression. These ncRNAs are regulatory molecules that are complementary to an early portion of the 5' end of the mRNA coding for the enzyme. When the small RNA binds to the mRNA by complementary base pairing , ribosomes cannot attach to the mRNA blocking its translation. As a result, the enzyme is not made ( Figure \(9\)). In bacteria these ncRNAs are often called small RNAs (sRNAs); in animal cells, plant cells, and viruses they are often called microRNAs (miRNA).
b. Ribosomal Proteins (r-proteins)
Ribosomal proteins bind to rRNA to form ribosomal subunits. Because the nucleotide base sequence for the mRNA coding for the r-proteins has similarities to that of the rRNA to which that r-protein binds during subunit formation, r-proteins not yet incorporated into ribosomal subunits can bind to that mRNA and block translation
4. Controlling the Enzyme's Activity (Feedback Inhibition).
Enzyme activity can be controlled by competitive inhibition and non-competitive inhibition.
a. With what is termed non-competitive inhibition , the inhibitor is the end product of a metabolic pathway that is able to bind to a second site (the allosteric site) on the enzyme. Binding of the inhibitor to the allosteric site alters the shape of the enzyme's active site thus preventing binding of the first substrate in the metabolic pathway. In this way, the pathway is turned off ( Figure \(10\)).
b. In the case of what is called competitive inhibition , the inhibitor is the end product of an enzymatic reaction. That end product is also capable of reacting with the enzyme's active site and prevents the enzyme from binding its normal substrate. As a result, the end product is no longer synthesized ( Figure \(11\)).
Summary
1. In living cells there are hundreds of different enzymes working together in a coordinated manner, and since cells neither synthesize nor break down more material than is required for normal metabolism and growth, precise enzyme regulation is required for turning metabolic reactions on and off.
2. There is tremendous diversity in the mechanisms bacteria use to regulate enzyme synthesis and enzyme activity.
3. Ways in which enzymes can be controlled or regulated include controlling the synthesis of the enzyme (genetic control) and controlling the activity of the enzyme (feedback inhibition).
4. In prokaryotes, genetic control of enzyme activity includes the induction or repression of enzyme synthesis by regulatory proteins that can bind to DNA and either block or enhance the function of RNA polymerase, the enzyme required for transcription.
5. An operon is a set of genes collectively controlled by a regulatory protein.
6. Regulatory proteins may function either as repressors or activators.
7. Repressors are regulatory proteins that block transcription of mRNA by preventing RNA polymerase from transcribing the coding sequence for the enzymes.
8. Some repressors, as in the case of the trp operon, are synthesized in a form that cannot by itself bind to the operator. This is referred to as a repressible system. The binding of a molecule called a corepressor, however, alters the shape of the regulatory protein to a form that can bind to the operator and subsequently block transcription.
9. Some repressors, as in the case of the lac operon, are synthesized in a form that readily binds to the operator and blocks transcription. However, the binding of a molecule called an inducer alters the shape of the regulatory protein in a way that now blocks its binding to the operator and thus permits transcription. This is referred to as an inducible system.
10. Activators are regulatory proteins that promote transcription of mRNA by enabling RNA polymerase to transcribing the coding sequence for the enzymes.
11. Enhancers are regulatory proteins that bind to DNA located some distance from the operon they control by working with DNA-bending proteins. The DNA-bending proteins bend the DNA in a way that now allows the enhancer to interact with the promoter in such a way that RNA polymerase can now bind and initiate transcription
12. Bacteria also use translational control of enzyme synthesis. One method is for the bacteria to produce noncoding RNA (ncRNA) molecules that are complementary to the mRNA coding for the enzyme, and when the small RNA binds to the mRNA by complementary base pairing, ribosomes cannot attach to the mRNA, the mRNA is not transcribed and translated into protein, and the enzyme is not made. In bacteria, these ncRNAs are often called small RNAs (sRNAs).
13. Feedback inhibition controls the activity of the enzyme rather than its synthesis and can be noncompetitive or competitive.
14. In the case of non-competitive inhibition, the inhibitor is the end product of a metabolic pathway that is able to bind the allosteric site on the enzyme. Binding of the inhibitor to the allosteric site alters the shape of the enzyme's active site thus preventing binding of the first substrate in the metabolic pathway. In this way, the pathway is turned off.
15. In the case of what is called competitive inhibition, the inhibitor is the end product of an enzymatic reaction. That end product is also capable of reacting with the enzyme's active site and prevents the enzyme from binding its normal substrate. As a result, the end product is no longer synthesized. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/3%3A_Bacterial_Genetics/3.3%3A_Enzyme_Regulation.txt |
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
3.1: Horizontal Gene Transfer in Bacteria
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Define horizontal gene transfer. (ans)
2. State three mechanisms of horizontal gene transfer in bacteria. (ans)
3. Briefly describe the mechanisms for transformation in bacteria. (ans)
4. Briefly describe the mechanism of generalized transduction in bacteria. (ans)
5. Briefly describe the following mechanisms of horizontal gene transfer in bacteria:
1. Transfer of conjugative plasmids in gram-negative bacteria (ans)
2. F+ conjugation (ans)
6. Describe R-plasmids, R-plasmid conjugation, and the significance of R-plasmids to medical microbiology. (ans)
7. Multiple Choice (ans)
3.2: Bacterial Quorum Sensing, Pathogenicity Islands, and Secretion Systems (Injectosomes)
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Define pathogenicity. (ans)
2. Define virulence. (ans)
3. Even though a microorganism may be considered pathogenic, it still may not be able to cause disease upon entering the body. Discuss why. (ans)
4. Define and briefly describe the overall process of quorum sensing in bacteria and how it may enable bacteria to behave as a multicellular population. (ans)
5. Multiple Choice (ans)
3.3: Enzyme Regulation
Study the material in this section and then write out the answers to these question. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Matching
_____ Regulatory proteins that block transcription of mRNA by binding to a portion of DNA called the operator that lies downstream of a promoter. (ans)
_____ A molecule that alters the shape of the regulatory protein in a way that blocks its binding to the operator and thus permits transcription. (ans)
_____ Regulatory proteins that promote transcription of mRNA. (ans)
_____ A molecule that alters the shape of the regulatory protein to a form that can bind to the operator and block transcription. (ans)
_____ Producing antisense RNA that is complementary to the mRNA coding for the enzyme. When the antisense RNA binds to the mRNA by complementary base pairing, the mRNA cannot be translated into protein and the enzyme is not made. (ans)
_____ The induction or repression of enzyme synthesis by regulatory proteins that can bind to DNA and either block or enhance the function of RNA polymerase. (ans)
_____ The inhibitor is the end product of a metabolic pathway that is able to bind to a second site (the allosteric site) on an enzyme. Binding of the inhibitor to the allosteric site alters the shape of the enzyme's active site thus preventing binding of the first substrate in the metabolic pathway. (ans)
_____ The inhibitor is the end product of an enzymatic reaction. That end product is also capable of reacting with the enzyme's active site and prevents the enzyme from binding its normal substrate. (ans)
_____Regulatory proteins that bind to DNA located some distance from the operon they control by working with DNA-bending proteins that enable RNA polymerase can to bind to a promoter and initiate transcription. (ans)
1. activators
2. competitive inhibition
3. corepressors
4. genetic control
5. inducer
6. non-competitive inhibition
7. repressors
8. translational control
9. enhancers
2. Describe how the lac operon in E. coli functions as an inducible operon. (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/3%3A_Bacterial_Genetics/3.E%3A_Bacterial_Genetics_%28Exercises%29.txt |
Thumbnail: Staphylococcus aureus - Antibiotics Test plate. (Public Domain; CDC / Provider: Don Stalons).
4: Using Antibiotics and Chemical Agents to Control Bacteria
selective toxicity broad spectrum antibiotic narrow spectrum antibiotic antibiotic chemotherapeutic synthetic drug cidal static sterilization disinfection disinfectant antiseptic physical agent
Control of microorganisms is essential in order to prevent the transmission of diseases and infection, stop decomposition and spoilage, and prevent unwanted microbial contamination. Microorganisms are controlled by means of physical agents and chemical agents. Physical agents include such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration. Control by chemical agents refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial chemicals.
In this unit we will concentrate on the chemical control of microbial growth with a special emphasis on the antibiotics and chemotherapeutic antimicrobial chemicals used in treating bacterial infections. Control of microorganisms by means of physical agents will be covered in Lab 18 and control by means of disinfectants, antiseptics, and sanitizers will be discussed in Lab 19.
The basis of chemotherapeutic control of bacteria is selective toxicity. Selective toxicity means that the chemical being used should inhibit or kill the intended pathogen without seriously harming the host. A broad spectrum agent is one generally effective against a variety of Gram-positive and Gram-negative bacteria; a narrow spectrum agent generally works against just Gram-positives, Gram-negatives, or only a few bacteria. As mentioned above, such agents may be cidal or static in their action. A cidal agent kills the organism while a static agent inhibits the organism's growth long enough for body defenses to remove it.
There are two categories of antimicrobial chemotherapeutic agents: antibiotics and synthetic drugs. Antibiotics are metabolic products of one microorganism that inhibit or kill other microorganisms. Chemotherapeutic synthetic drugs are antimicrobial drugs synthesized by chemical procedures in the laboratory. Many of today's antibiotics are now actually semi-synthetic and some are even made synthetically.
Antibiotics are metabolic products of one microorganism that inhibit or kill other microorganisms. Why then do bacteria produce antibiotics? There is growing support for multiple actions for microbial antibiotic production:
• If produced in large enough amounts, antibiotics may be used as a weapon to inhibit or kill other microbes in the vicinity to reduce competition for food.
• Antibiotics produced in sublethal quantities may function as interspecies quorum sensing molecules enabling a number of different bacteria to form within a common biofilm where metabolic end products of one organism may serve as a substrate for another. All the organisms are protected within the same biofilm.
• Antibiotics produced in sublethal quantities may function as interspecies quorum sensing molecules enabling some bacteria to manipulate others to become motile and swim away thus reducing the competition for food.
• Antibiotics action may result in the degradation of bacterial cell walls or DNA and these products can act as cues that trigger other bacteria to produce a protective biofilm.
• Antibiotics produced in sublethal quantities may trigger intraspecies quorum sensing. Exposure to low concentrations of an antibiotic may trigger bacteria to produce quorum sensing molecules that trigger the population to produce a protective biofilm. The biofilm then protects the population from greater concentrations of the antibiotic.
Summary
1. Physical control includes such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration.
2. Chemical control refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial chemicals.
3. Sterilization is the process of destroying all living organisms and viruses.
4. Disinfection is the elimination of microorganisms, but not necessarily endospores, from inanimate objects or surfaces.
5. Decontamination is the treatment of an object or inanimate surface to make it safe to handle.
6. A disinfectant is an agents used to disinfect inanimate objects but generally to toxic to use on human tissues.
7. An antiseptic is an agent that kills or inhibits growth of microbes but is safe to use on human tissue.
8. A sanitizer is an agent that reduces microbial numbers to a safe level.
9. An antibiotic is a metabolic product produced by one microorganism that inhibits or kills other microorganisms.
10. Synthetic chemicals that can be used therapeutically.
11. An agent that is cidal in action kills microorganisms.
12. An agent that is static in action inhibits the growth of microorganisms.
13. Selective toxicity means that the chemical being used should inhibit or kill the intended pathogen without seriously harming the host.
14. A broad spectrum agent is one generally effective against a variety of Gram-positive and Gram-negative bacteria.
15. A narrow spectrum agent generally works against just Gram-positives, Gram-negatives, or only a few bacteria.
Glossary
Basic terms used in discussing the control of microorganisms include:
1. Sterilization
Sterilization is the process of destroying all living organisms and viruses. A sterile object is one free of all life forms, including bacterial endospores, as well as viruses.
2. Disinfection
Disinfection is the elimination of microorganisms, but not necessarily endospores, from inanimate objects or surfaces.
3. Decontamination
Decontamination is the treatment of an object or inanimate surface to make it safe to handle.
4. Disinfectant
A disinfectant is an agents used to disinfect inanimate objects but generally to toxic to use on human tissues.
5. Antiseptic
An antiseptic is an agent that kills or inhibits growth of microbes but is safe to use on human tissue.
6. Sanitizer
A sanitizer is an agent that reduces microbial numbers to a safe level.
7. Antibiotic
An antibiotic is a metabolic product produced by one microorganism that inhibits or kills other microorganisms.
8. Chemotherapeutic synthetic drugs
Synthetic chemicals that can be used therapeutically.
9. Cidal
An agent that is cidal in action will kill microorganisms and viruses.
10. Static
An agent that is static in action will inhibit the growth of microorganisms. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/4%3A_Using_Antibiotics_and_Chemical_Agents_to_Control_Bacteria/4.1%3A_An_Overview_to_Control_of_Microorganisms.txt |
Describe six different ways antibiotics or disinfectants may affect bacterial structures or macromolecules and state how each ultimately causes harm to the cell. State which of the following groups of antibiotics: 1) inhibit peptidoglycan synthesis; 2) inhibit nucleic acid synthesis; 3) alter bacterial 30S ribosomal subunits blocking translation; or 4) alter bacterial 50S ribosomal subunits blocking translation. macrolides(erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, etc.), oxazolidinones (linezolid), and streptogramins penicillins, monobactams, carbapenems, cephalosporins, and vancomycin fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.), sulfonamides and trimethoprim, and metronidazole aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc.) and tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.) State two modes of action for disinfectants, antiseptics, and sanitizers.
The basis of chemotherapeutic control of bacteria is selective toxicity. Selective toxicity means that the chemical being used should inhibit or kill the intended pathogen without seriously harming the host. A broad spectrum agent is one generally effective against a variety of Gram-positive and Gram-negative bacteria; a narrow spectrum agent generally works against just Gram-positives, Gram-negatives, or only a few bacteria. Such agents may be cidal or static in their action. A cidal agent kills the organism while a static agent inhibits the organism's growth long enough for body defenses to remove it. There are two categories of antimicrobial chemotherapeutic agents: antibiotics and synthetic drugs. Antibiotics are metabolic products of one microorganism that inhibit or kill other microorganisms. Synthetic drugs are antimicrobial drugs synthesized by chemical procedures in the laboratory. Many of today's antibiotics are now actually semisynthetic and some are even made synthetically. We will now look at the various ways in which our control agents affect bacteria altering their structures or interfering with their cellular functions.
Many Antibiotics inhibit Synthesis of Peptidoglycan and cause Osmotic Lysis
Interference with this process results in the formation of a weak cell wall and osmotic lysis of the bacterium. Agents that inhibit peptidoglycan synthesis include the penicillins (penicillin G, methicillin, oxacillin, ampicillin, amoxicillin, ticarcillin, etc.), the cephalosporins (cephalothin, cefazolin, cefoxitin, cefotaxime, cefaclor, cefoperazone, cefixime, ceftriaxone, cefuroxime, etc.), the carbapenems (imipenem, metropenem), the monobactems (aztreonem), and the carbacephems (loracarbef). Penicillins, monobactams, carbapenems, and cephalosporins are known chemically as beta-lactam antibiotics because they all share a molecular structure called a beta-lactam ring (see Figure \(5\)). The glycopeptides (vancomycin, teichoplanin) and lipopeptides (daptomycin) also inhibit peptidoglycan synthesis.
a. Beta lactam antibiotics such as penicillins and cephalosporins
Penicillins, cephalosporins, as well as other beta-lactam antibiotics (see Common Antibiotics), bind to the transpeptidase enzymes (also called penicillin-binding proteins) responsible for reforming the peptide cross-links between rows and layers of peptidoglycan of the cell wall as new peptidoglycan monomers are added during bacterial cell growth. This binding blocks the transpeptidase enzymes from cross-linking the sugar chains and results in a weak cell wall. In addition, these antibiotics appear to interfere with the bacterial controls that keep autolysins in check, with resulting degradation of the peptidoglycan and osmotic lysis of the bacterium (see Figure \(6\)).
Flash animation showing how penicillins inhibit peptidoglycan synthesis.
© Juliet V. Spencer, Stephanie K.M. Wong, authors, Licensed for use, ASM MicrobeLibrary.
YouTube movie showing lysis of E. coli after exposure to a penicillin #1
YouTube movie showing lysis of E. coli after exposure to a penicillin #2
b. Glycopeptides
Glycopeptides such as vancomycin (see Common Antibiotics) and the lipoglycopeptide teichoplanin bind to the D-Ala-D-Ala portion of the pentapeptides of the peptidoglycan monomers and block the formation of gycosidic bonds between the sugars by the transgycosidase enzymes, as well as the formation of the peptide cross-links by the transpeptidase enzymes. This results in a weak cell wall and subsequent osmotic lysis of the bacterium (see Figure \(7\)).
Flash animation showing how vancomycin inhibit peptidoglycan synthesis.
© Juliet V. Spencer, Stephanie K.M. Wong, authors, Licensed for use, ASM MicrobeLibrary.
c. Bacitracin
Bacitracin (see Common Antibiotics) binds to the transport protein bactoprenol after it inserts a peptidoglycan monomer into the growing cell wall. It subsequently prevents the dephosphorylation of the bactoprenol after it releases the monomer it has transported across the membrane. Bactoprenol molecules that have not lost the second phosphate group cannot assemble new monomers and transport them across the cytoplasmic membrane. As a result, no new monomers are inserted into the growing cell wall. As the autolysins continue to break the peptide cross-links and new cross-links fail to form, the bacterium bursts from osmotic lysis (see Figure \(8\)).
Flash animation showing how bacitracin inhibit peptidoglycan synthesis.
© Juliet V. Spencer, Stephanie K.M. Wong, authors, Licensed for use, ASM MicrobeLibrary.
A few antimicrobial chemotherapeutic agents inhibit normal synthesis of the acid-fast cell wall
A few antimicrobial chemotherapeutic agents inhibit normal synthesis of the acid-fast cell wall of the genus Mycobacterium (see Common Antibiotics).. INH(isoniazid) appears to block the synthesis of mycolic acid, a key component of the acid-fast cell wall of mycobacteria (see Figure \(9\)). Ethambutol interferes with the synthesis of the outer membrane of acid-fast cell walls (see Figure \(9\)).
A very few antibiotics alter the bacterial cytoplasmic membrane causing leakage of molecules and enzymes needed for normal bacterial metabolism.
A very few antibiotics, such as polymyxins, colistins, and daptomycin (Common Antibiotics), as well as many disinfectants and antiseptics, such as orthophenylphenol, chlorhexidine, hexachlorophene, zephiran, alcohol, and triclosans, alter the bacterial cytoplasmic membrane causing leakage of molecules and enzymes needed for normal bacterial metabolism.
1. Polymyxins and colistins act as detergents and alter membrane permeability in Gram-negative bacteria. They cannot effectively diffuse through the thick peptidoglycan layer in gram-positives.
2. Daptomycin disrupts the bacteria cytoplasmic membrane function by apparently binding to the membrane and causing rapid depolarization. This results on a loss of membrane potential and leads to inhibition of protein, DNA and RNA synthesis, resulting in bacterial cell death.
3. Pyrazinamide inhibits fatty acid synthesis in the membranes of Mycobacterium tuberculosis.
Some antimicrobial chemotherapeutic agents inhibit normal nucleic acid replication in bacteria (see Common Antibiotics).
a. Fluoroquinolones
Fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, gatifloxacin, etc., (Common Antibiotics))) work by inhibiting one or more of a group of enzymes called topoisomerase, enzymes needed for supercoiling, replication, and separation of circular bacterial DNA (see Figure \(10\)). For example, DNA gyrase (topoisomerase II) catalyzes the negative supercoiling of the circular DNA found in bacteria. It is critical in bacterial DNA replication, DNA repair, transcription of DNA into RNA, and genetic recombination. Topoisomerase IV, on the other hand, is involved in the relaxation of the supercoiled circular DNA, enabling the separation of the interlinked daughter chromosomes at the end of bacterial DNA replication.
In Gram-negative bacteria, the main target for fluoroquinolones is DNA gyrase (topoisomerase II), an enzyme responsible for supercoiling of bacterial DNA during DNA replication; in Gram-positive bacteria, the primary target is topoisomerase IV, an enzyme responsible for relaxation of supercoiled circular DNA and separation of the inter-linked daughter chromosomes.
b. Sulfonamides
Sulfonamides (sulfamethoxazole, sulfanilamide) and diaminopyrimidines (trimethoprim) (see Common Antibiotics) block enzymes in the bacteria pathway required for the synthesis of tetrahydrofolic acid, a cofactor needed for bacteria to make the nucleotide bases thymine, guanine, uracil, and adenine (see Figure \(11\)).
This is done through a process called competitive antagonism whereby a drug chemically resembles a substrate in a metabolic pathway. Because of their similarity, either the drug or the substrate can bind to the substrate's enzyme. While the enzyme is bound to the drug, it is unable to bind to its natural substrate and that blocks that step in the metabolic pathway (see Figure \(12\)). Typically, a sulfonamide and a diaminopyrimidine are combined. Co-trimoxazole, for example, is a combination of sulfamethoxazole and trimethoprim.
Sulfonamides such as sulfamethoxazole tie up the first enzyme in the pathway, the conversion of para-aminobenzoic acid to dihydropteroic acid (see Figure \(11\)). Trimethoprim binds to the third enzyme in the pathway, an enzyme that is responsible for converting dihydrofolic acid to tetrahydrofolic acid (see Figure \(11\)). Without the tetrahydrofolic acid, the bacteria cannot synthesize DNA or RNA.
c. Metronidazole
Metronidazole (see Common Antibiotics) is a drug that is activated by the microbial proteins flavodoxin and feredoxin found in microaerophilc and anaerobic bacteria and certain protozoans. Once activated, the metronidazole puts nicks in the microbial DNA strands.
d. Rifampin
Rifampin (rifamycin) (see Common Antibiotics) blocks transcription by inhibiting bacterial RNA polymerase, the enzyme responsible for transcription of DNA to mRNA.
Many antibiotics alter bacterial ribosomes, interfering with translation of mRNA into proteins and thereby causing faulty protein synthesis (see Common Antibiotics).
To learn more detail about the specific steps involved in translation during bacterial protein synthesis, see the animation that follows. Protein synthesis is discussed in greater detail in Unit 6.
a. Aminoglycosides
The aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc. (see Common Antibiotics)) bind irreversibly to the 16S rRNA in the 30S subunit of bacterial ribosomes. Although the exact mechanism of action is still uncertain, there is evidence that some prevent the transfer of the peptidyl tRNA from the A-site to the P-site, thus preventing the elongation of the polypeptide chain. Some aminoglycosides also appear to interfere with the proofreading process that helps assure the accuracy of translation (see Figure \(13\)). Possibly the antibiotics reduce the rejection rate for tRNAs that are near matches for the codon. This leads to misreading of the codons or premature termination of protein synthesis (see Figure \(14\)). Aminoglycosides may also interfere directly or indirectly with the function of the bacterial cytoplasmic membrane. Because of their toxicity, aminoglycosides are generally used only when other first line antibiotics are not effective.
b. Tetracyclines
The tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc. (see Common Antibiotics)) bind reversibly to the 16S rRNA in the 30S ribosomal subunit, distorting it in such a way that the anticodons of charged tRNAs cannot align properly with the codons of the mRNA (see Figure \(15\)).
c. Macrolides
The macrolides (erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, etc. (see Common Antibiotics)) bind reversibly to the 23S rRNA in the 50S subunit of bacterial ribosomes. They appear to inhibit elongation of the protein by preventing the enzyme peptidyltransferase from forming peptide bonds between the amino acids (see Figure \(16\)). They may also prevent the transfer of the peptidyl tRNA from the A-site to the P-site (see Figure \(17\)) as the beginning peptide chain on the peptidyl tRNA adheres to the ribosome, creates friction, and blocks the exit tunnel of the 50S ribosomal subunit.
d. Oxazolidinones
The oxazolidinones (linezolid, sivextro) (see Common Antibiotics), following the first cycle of protein synthesis, interfere with translation sometime before the initiation phases. They appear to bind to the 50S ribosomal subunit and interfere with its binding to the initiation complex (see Figure \(18\)).
e. Streptogramins
The streptogramins (synercid, a combination of quinupristin and dalfopristin (see Common Antibiotics)) bind to two different locations on the 23S rRNA in the 50S ribosomal subunit and work synergistically to block translation. There are reports that the streptogramins may inhibit the attachment of the charged tRNA to the A-site or may block the peptide exit tunnel of the 50S ribosomal subunit.
For a more detailed description of any specific antimicrobial agent, see the website of RxList - The Internet Drug Index.
Modes of action for disinfectants, antiseptics, and sanitizers
Disinfection is the elimination of microorganisms, but not necessarily endospores, from inanimate objects or surfaces, whereas decontamination is the treatment of an object or inanimate surface to make it safe to handle. Sterilization is the process of destroying all living organisms and viruses. A sterile object is one free of all life forms, including bacterial endospores, as well as viruses.
The term disinfectant is used for an agent used to disinfect inanimate objects or surfaces but is generally too toxic to use on human tissues. An antiseptic refers to an agent that kills or inhibits growth of microbes but is safe to use on human tissue. A sanitizer describes an agent that reduces microbial numbers to a safe level. Because disinfectants and antiseptics often work slowly on some viruses - such as the hepatitis viruses, bacteria with an acid-fast cell wall such as Mycobacterium tuberculosis, and especially bacterial endospores, produced by the genus Bacillus and the genus Clostridium, they are usually unreliable for sterilization - the destruction of all life forms.
There are a number of factors which influence the antimicrobial action of disinfectants and antiseptics, including:
1. The concentration of the chemical agent.
2. The temperature at which the agent is being used. Generally, the lower the temperature, the longer it takes to disinfect or decontaminate.
3. The kinds of microorganisms present. Endospore producers such as Bacillus species, Clostridium species, and acid-fast bacteria like Mycobacterium tuberculosis are harder to eliminate.
4. The number of microorganisms present. The more microorganisms present, the harder it is to disinfect or decontaminate.
5. The nature of the material bearing the microorganisms. Organic material such as dirt and excreta interferes with some agents.
The best results are generally obtained when the initial microbial numbers are low and when the surface to be disinfected is clean and free of possible interfering substances.
There are 2 common antimicrobial modes of action for disinfectants, antiseptics, and sanitizers:
1. They may damage the lipids and/or proteins of the semipermeable cytoplasmic membrane of microorganisms resulting in leakage of cellular materials needed to sustain life.
2. They may denature microbial enzymes and other proteins, usually by disrupting the hydrogen and disulfide bonds that give the protein its three-dimensional functional shape. This blocks metabolism.
A large number of such chemical agents are in common use. Some of the more common groups are listed below:
1. Phenol and phenol derivatives: Phenol (5-10%) was the first disinfectant commonly used. However, because of its toxicity and odor, phenol derivatives (phenolics) are now generally used. The most common phenolic is orthophenylphenol, the agent found in O-syl®, Staphene®, and Amphyl®. Bisphenols contain two phenolic groups and typically have chlorine as a part of their structure. They include hexachlorophene and triclosan. Hexachlorophene in a 3% solution is combined with detergent and is found in PhisoHex®. Triclosan is an antiseptic very common in antimicrobial soaps and other products. Biguanides include chlorhexadine and alexidine. A 4% solution of chlorhexidine in isopropyl alcohol and combined with detergent (Hibiclens® and Hibitane®) is a common hand washing agent and surgical handscrub. These agents kill most bacteria, most fungi, and some viruses, but are usually ineffective against endospores. Chloroxylenol (4-chloro-3,5-dimethylphenol) is a broad spectrum antimicrobial chemical compound used to control bacteria, algae, fungi and virus and is often used in antimicrobial soaps and antiseptics. Phenol and phenolics alter membrane permeability and denature proteins. Bisphenols, biguanides, and chloroxylenol alter membrane permeability.
2. Soaps and detergents: Soaps are only mildly microbicidal. Their use aids in the mechanical removal of microorganisms by breaking up the oily film on the skin (emulsification) and reducing the surface tension of water so it spreads and penetrates more readily. Some cosmetic soaps contain added antiseptics to increase antimicrobial activity.
Detergents may be anionic or cationic. Anionic (negatively charged) detergents, such as laundry powders, mechanically remove microorganisms and other materials but are not very microbicidal. Cationic (positively charged) detergents alter membrane permeability and denature proteins. They are effective against many vegetative bacteria, some fungi, and some viruses. However, bacterial endospores and certain bacteria such as Mycobacterium tuberculosis and Pseudomonas species are usually resistant. Soaps and organic materials like excreta also inactivate them. Cationic detergents include the quaternary ammonium compounds such as benzalkonium chloride, zephiran®, diaprene, roccal, ceepryn, and phemerol. Household Lysol® contains alkyl dimethyl benzyl ammonium chloride and alcohols.
3. Alcohols
70% solutions of ethyl or isopropyl alcohol are effective in killing vegetative bacteria, enveloped viruses, and fungi. However, they are usually ineffective against endospores and non-enveloped viruses. Once they evaporate, their cidal activity will cease. Alcohols denature membranes and proteins and are often combined with other disinfectants, such as iodine, mercurials, and cationic detergents for increased effectiveness.
4. Acids and alkalies
Acids and alkalies alter membrane permeability and denature proteins and other molecules. Salts of organic acids, such as calcium propionate, potassium sorbate, and methylparaben, are commonly used as food preservatives. Undecylenic acid (Desenex®) is used for dermatophyte infections of the skin. An example of an alkali is lye (sodium hydroxide).
5. Heavy metals
Heavy metals, such as mercury, silver, and copper, denature proteins. Mercury compounds (mercurochrome, metaphen, merthiolate) are only bacteriostatic and are not effective against endospores. Silver nitrate (1%) is sometimes put in the eyes of newborns to prevent gonococcal ophthalmia. Copper sulfate is used to combat fungal diseases of plants and is also a common algicide. Selinium sulfide kills fungi and their spores.
6. Chlorine
Chlorine gas reacts with water to form hypochlorite ions, which in turn denature microbial enzymes. Chlorine is used in the chlorination of drinking water, swimming pools, and sewage. Sodium hypochlorite is the active agent in household bleach. Calcium hypochlorite, sodium hypochlorite, and chloramines (chlorine plus ammonia) are used to sanitize glassware, eating utensils, dairy and food processing equipment, hemodialysis systems, and treating water supplies.
7. Iodine and iodophores
Iodine also denatures microbial proteins. Iodine tincture contains a 2% solution of iodine and sodium iodide in 70% alcohol. Aqueous iodine solutions containing 2% iodine and 2.4% sodium iodide are commonly used as a topical antiseptic. Iodophores are a combination of iodine and an inert polymer such as polyvinylpyrrolidone that reduces surface tension and slowly releases the iodine. Iodophores are less irritating than iodine and do not stain. They are generally effective against vegetative bacteria, Mycobacterium tuberculosis, fungi, some viruses, and some endospores. Examples include Wescodyne®, Ioprep®, Ioclide®, Betadine®, and Isodine®.
8. Aldehydes
Aldehydes, such as formaldehyde and glutaraldehyde, denature microbial proteins. Formalin (37% aqueous solution of formaldehyde gas) is extremely active and kills most forms of microbial life. It is used in embalming, preserving biological specimens, and in preparing vaccines. Alkaline glutaraldehyde (Cidex®), acid glutaraldehyde (Sonacide®), and glutaraldehyde phenate solutions (Sporocidin®) kill vegetative bacteria in 10-30 minutes and endospores in about 4 hours. A 10 hour exposure to a 2% glutaraldehyde solution can be used for cold sterilization of materials. Ortho-phthalaldehyde (OPA) is dialdehyde used as a high-level disinfectant for medical instruments.
9. Peroxygens
Peroxygens are oxidizing agents that include hydrogen peroxide and peracetic acid. Hydrogen peroxide is broken down into water and oxygen by the enzyme catalase in human cells and is not that good of an antiseptic for open wounds but is useful for disinfecting inanimate objects. The high concentrations of hydrogen peroxide overwhelm the catalase found in microbes. Peracetic acid is a disinfectant that kills microorganisms by oxidation and subsequent disruption of their cytoplasmic membrane. It is widely used in health care, food processing, and water treatment.
10. Ethylene oxide gas
Ethylene oxide is one of the very few chemicals that can be relied upon for sterilization (after 4-12 hours exposure). Since it is explosive, it is usually mixed with inert gases such as freon or carbon dioxide. Gaseous chemosterilizers, using ethylene oxide, are commonly used to sterilize heat-sensitive items such as plastic syringes, petri plates, textiles, sutures, artificial heart valves, heart-lung machines, and mattresses. Ethylene oxide has very high penetrating power and denatures microbial proteins. Vapors are toxic to the skin, eyes, and mucous membranes and are also carcinogenic. Another gas that is used as a sterilant is chlorine dioxide which denatures proteins in vegetative bacteria, bacterial endospores, viruses, and fungi.
Summary
1. Many antibiotics (penicillins, cephalosporins, vancomycin, bacitracin) inhibit normal synthesis of peptidoglycan by bacteria and cause osmotic lysis. They do this by inactivating the enzymes or the transporters involved in peptidoglycan synthesis.
2. A few antimicrobial chemotherapeutic agents (INH, ethambutol) inhibit normal synthesis of the acid-fast cell wall.
3. A very few antibiotics (polymyxin, colistin, daptomycin) alter the bacterial cytoplasmic membrane causing leakage of molecules and enzymes needed for normal bacterial metabolism.
4. Some antimicrobial chemotherapeutic agents (fluoroquinolones, sulfonamides, trimethoprim) inhibit normal nucleic acid replication in bacteria.
5. Many antibiotics (tetracyclines, macrolides, oxazolidinones, streptogramins) alter bacterial ribosomes, interfering with translation of mRNA into proteins and thereby causing faulty protein synthesis.
6. There are 2 common antimicrobial modes of action for disinfectants, antiseptics, and sanitizers: damaging the lipids and/or proteins of the semipermeable cytoplasmic membrane of microorganisms resulting in leakage of cellular materials; and denaturing microbial enzymes and other proteins.
7. A number of factors which influence the antimicrobial action of disinfectants and antiseptics, including the concentration of the chemical agent, the temperature at which the agent is being used, the kinds of microorganisms present, the number of microorganisms present, and the nature of the material bearing the microorganisms.
8. Endospore producers such as Bacillus species, Clostridium species, and acid-fast bacteria like Mycobacterium tuberculosis are harder to eliminate. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/4%3A_Using_Antibiotics_and_Chemical_Agents_to_Control_Bacteria/4.2%3A_Ways_in_which_Chemical_Control_Agents_Affect_Bacteri.txt |
Name two bacteria that have low-permeability membrane barriers and are thereby intrinsically resistant to many antibiotics. Briefly describe 4 different mechanisms as a result of genetic changes in a bacterium that may enable that bacterium to resist an antibiotic. Describe R (Resistance) plasmids and state their significance to medical microbiology. State what the following stand for: MRSA, VRE,CRE, and XDR TB. Define antibiotic tolerance.
The basis of chemotherapeutic control of bacteria is selective toxicity. Selective toxicity means that the chemical being used should inhibit or kill the intended pathogen without seriously harming the host. A broad spectrum agent is one generally effective against a variety of gram-positive and gram-negative bacteria; a narrow spectrum agent generally works against just gram-positives, gram-negatives, or only a few bacteria. Such agents may be cidal or static in their action. A cidal agent kills the organism while a static agent inhibits the organism's growth long enough for body defenses to remove it. There are two categories of antimicrobial chemotherapeutic agents: antibiotics and synthetic drugs. Antibiotics are metabolic products of one microorganism that inhibit or kill other microorganisms. Synthetic drugs are antimicrobial drugs synthesized by chemical procedures in the laboratory. Many of today's antibiotics are now actually semisynthetic and some are even made synthetically.
We will now look at the two sides of the story with regards to controlling bacteria by means of chemicals:
1. Ways in which Our Control Agents Affect Bacterial Structures or Function
2. Ways in which Bacteria May Resist Our Control Agents
We will now look at the various ways in which bacteria become resistant to our control agents.
Some opportunistic pathogens, such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Enterococcus species, have low-permeability membrane barriers and are thereby intrinsically resistant to many antibiotics. Most bacteria, however, become resistant to antibiotics as a result of mutation or horizontal gene transfer. Mutation in bacterial DNA can alter the order of nucleotide bases in a gene and alter that gene product. Horizontal gene transfer can alter or add bacterial genes, again altering the bacterium's gene products. See function of DNA.
Most bacteria, become resistant to antibiotics by way of one or more of the following mechanisms that are coded for by genes in the bacterial chromosomeor in plasmids:
1. Producing an enzyme capable of inactivating the antibiotic;
2. Altering the target site receptor for the antibiotic to reduce or block its binding;
3. Preventing the entry of the antibiotic into the bacterium and/or using an efflux pump to transport the antibiotic out of the bacterium; and/or
4. Modulating gene expression to produce more of the bacterial enzyme that is being tied up or altered by the antibiotic.
Nice summary of antibiotic resistant cases and associated deaths; from the CDC.
Improving antibiotic use among hospitalized patients; from CDC.
Estimates of Healthcare-Associated Infections (HCIs) 2011; from CDC.
Getting Smart About Antibiotics; from CDC.
We will now look at each of these mechanisms of resistance.
Producing to inactivate the antibiotic
Bacteria may acquire new genes that code for an enzyme that inactivates a particular antibiotic or group of antibiotics. For example:
a. Bacteria typically become resistant to penicillins, monobactams, carbapenems, and cephalosporins are known chemically as beta-lactam antibiotics (see Figure \(2\)) and many bacteria become resistant to these antibiotics by producing various beta-lactamases that are able to inactivate some forms of these drugs. Beta-lactamases break the beta-lactam ring of the antibiotic, thus inactivating the drug. (Penicillinase is a beta-lactamase that inactivates certain penicillins.) To overcome this mechanism of resistance, sometimes beta-lactam antibiotics such as amoxicillin, ticarcillin, imipenem, or ampicillin are combined with beta-lactamase inhibitors such as clavulanate, tazobactam, or sulbactam (see Common Antibiotics) - chemicals that resemble beta-lactam antibiotic (see Figure \(2\)). These agents bind to the bacterial beta-lactamases and neutralize them.
b. Bacteria may become resistant to aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc.) and streptogramins by enzymatically adding new chemical groups to these antibiotics, thus inactivating the drug.
Altering the target site receptor for the antibiotic in the bacterium to reduce or block its binding.
Antibiotics work by binding to some bacterial target site, such as a 50S ribosomal subunit, a 30S ribosomal subunit, or a particular bacterial enzyme such as a transpeptidases or a DNA topoisomerase. Bacteria may acquire new genes that alter the molecular shape of the portion of the ribosomal subunit or the enzyme to which the drug normally binds. For example:
a. Bacteria may become resistant to to macrolides (erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, etc.) by producing a slightly altered 50S ribosomal subunit that still functions but to which the antibiotic can no longer bind (see Figure \(3\)).
b. Bacteria may become resistant to beta-lactam antibiotics (penicillins, monobactams, carbapenems, and cephalosporins) by producing altered transpeptidases (penicillin-binding proteins) with greatly reduced affinity for the binding of beta-lactam antibiotics.
c. Bacteria may become resistant to vancomycin by producing altered cross-linking peptides in the peptidoglycan to which the antibiotic no longer bonds.
d. Bacteria may become resistant to fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.) by producing altered DNA gyrase or other topoisomerases to which the drug no longer binds (see Figure \(4\)).
Altering the membranes and transport systems to prevent the entry of the antibiotic into the bacterium and/or using an efflux pump to transport the antibiotic out of the bacterium.
Antibiotics that target ribosomes or enzymes within the bacterium must first pass through the porins in the outer membrane of gram-negative and acid-fast bacterial cell walls, and then the cytoplasmic membrane in the case of all bacteria. Subsequently, the antibiotic has to accumulate to a high enough concentration within the bacterium to inhibit or kill the organism.
a. A Gram-negative or an acid-fast bacterium may block the entry of an antmicrobial drug by acquiring genes that alter the porins in the cell wall's outer membrane (see Figure \(5\)).
b. A bacterium may block the entry of an antmicrobial drug by acquiring genes that alter the carrier (transport) proteins used to transport the drug through the bacterium's cytoplasmic membrane (see Figure \(6\)). This is generally not a common mechanism of antibiotic resistance.
c. A bacterium may acquire genes coding for an energy-driven efflux pump in its the cytoplasmic membrane that is able to to pump the antibiotic out of the bacterium and preventing it from accumulating to a high enough concentration to inhibit or kill the organism (see Figure \(7\)). This is the most common method bacteria use to prevent toxic levels of antimicrobial drugs from accumulating within the cytoplasm.
Modulating gene expression to produce more of the bacterial enzyme that is being tied up or altered by the antibiotic.
Remember that enzymes function as catalysts and are present in cells in small amounts because they are not altered as they carry out their specific biochemical reactions. As mentioned in the previous section, numerous antimicrobial drugs work by inactivating bacterial enzymes and blocking metabolic reactions. Making a particular enzyme and the amount of enzyme that is made is under genetic control.
Genetic control of enzyme activity refers to controlling transcription of the mRNA needed for an enzyme's synthesis. In prokaryotic cells, this involves the induction or repression of enzyme synthesis by regulatory proteins that can bind to DNA and either block or enhance the function of RNA polymerase, the enzyme required for transcription.
Bacteria also use translational control of enzyme synthesis. In this case, the bacteria produce noncoding RNAs (ncRNAs) or antisense RNAa such as microRNAs (miRNAs) that are complementary to an early portion of the mRNA coding for the enzyme. When the noncoding RNA binds to the mRNA by complementary base pairing, ribosomes cannot attach, the mRNA cannot be translated into protein, and the enzyme is not made (See Figure \(8\)).
Mutations or horizontal gene transfer may result in a modulation of gene expression or translational events that favor increased production of the enzyme being tied up or altered by the antimicrobial agent (see Figure \(9\)). Since enzymes are normally produced in limited amounts, production of excessive amounts of enzyme may allow for the metabolic activity being blocked by the agent to still occur.
Many pathogenic bacteria, as well as normal flora, form complex bacterial communities as biofilms.
Bacteria in biofilms are often able to communicate with one another by a process called quorum sensing and are able to interact with and adapt to their environment as a population of bacteria rather than as individual bacteria. By living as a community of bacteria as a biofilm, these bacteria are:
• better able to resist attack by antibiotics, and
• are better able to resist the host immune system.
Why bacterium within a biofilm are more antibiotic resistant isn't completely understood but various mechanisms have been preposed. The extracellular polysaccharide may make it more difficult for the antibiotic to reach all of the bacteria. Bacteria within a biofilm are generally in a metabolically more inert state and this could slow down antibacterial action of the drug. Many antibiotics are static, not cidal in action; the body depends on phagocytes to remove the inhibited bacteria. The biofilm structure makes engulfment by phagocytes pretty much impossible.
Exposure to antibiotics doesn't "cause" bacteria to become drug resistant. The above changes in the bacterium that enable it to resist the antibiotic occur naturally as a result of mutation or as a result of horizontal gene transfer. For example, when under stress from antibiotics, some bacteria switch on genes whose protein products can increase the mutation rate within the bacterium 10,000 times as fast as the mutation rate that occurs during normal binary fission. This causes a sort of hyperevolution where mutation acts as a self defense mechanism for the bacterial population by increasing the chance of forming an antibiotic-resistant mutant that is able to survive at the expense of the majority of the population. (Remember that most mutations are harmful to a cell.)
In addition, horizontal gene transfer as a result of transformation, transduction, and conjugation can transfer antibiotic resistance from one bacterium to another. Horizontal gene transfer enables bacteria to respond and adapt to their environment much more rapidly than mutation by acquiring large DNA sequences from another bacterium in a single transfer.
Briefly describe 3 different mechanisms, as a result of mutation or horizontal gene transfer in a bacterium, that may enable that bacterium to resist an antibiotic State at least 4 medical dangers associated with the improper use of antibiotics and list 3 common examples of antibiotic misuse.
Exposure to the antibiotic typically selects for strains of the organism that have become resistant through these natural processes. Misuse of antibiotics, such as prescribing them for non-bacterial infections (colds, influenza, most upper respiratory infections, etc.) or prescribing the "newest" antibiotic on the market when older brands may still be as effective simply inceases the rate at which this natural selection for resistance occurs. According to the Centers for Disease Control and Prevention, as many as one-third (50 million out of 150 million) of antibiotic prescriptions given on an outpatient basis are unneeded. Patient noncompliance with antimicrobial therapy, namely, not taking the prescribed amount of the antibiotic at the proper intervals for the appropriate length of time, also plays a role in selecting for resistant strains of bacteria.
The spread of antibiotic resistance in pathogenic bacteria is due to both direct selection and indirect selection.
• Direct selection refers to the selection of antibiotic resistant pathogens at the site of infection.
• Indirect selection is the selection of antibiotic-resistant normal floras within an individual anytime an antibiotic is given. At a later date, these resistant normal flora may transfer resistance genes to pathogens that enter the body. In addition, these resistant normal flora may be transmitted from person to person through such means as the fecal-oral route or through respiratory secretions.
As an example, many Gram-negative bacteria possess R (Resistance) plasmids that have genes coding for multiple antibiotic resistance through the mechanisms stated above, as well as transfer genes coding for a conjugation (sex) pilus (see Figs. 10A-10F). It is possible for R-plasmids to accumulate transposons to increase bacterial resistance. Such an organism can conjugate with other bacteria and transfer to them an R plasmid. E. coli, Proteus, Serratia, Enterobacter, Salmonella, Shigella, and Pseudomonas are bacteria that frequently have R-factor plasmids.
In addition to plasmids, conjugative transposons also frequently transmit antibiotic resistance from one bacterium to another. Conjugative transposons, like conjugative plasmids, carry the genes that enable mating pairs to form for conjugation. Therefore, conjugative transposons also enable mobilizable plasmids and nonconjugative transposons to be transferred to a recipient bacterium during conjugation.
Examples of Antibilotic Resistant Bacteria
Examples of resistant strains of bacteria of ever increasing medical importance include:
• Penicillinase-Producing Neisseria gonorrhoeae (PPNG): Most strains of Neisseria gonorrhoeae have penicillinase plasmids and are known as PPNG (penicillinase-producing Neisseria gonorrhoeae). As a result, penicillin is no longer the drug of choice for gonorrhea.
• Carbapenem-Resistant Enterobacteriaceae (CRE): More recently, carbapenemase-producing Klebsiella pneumoniae (KPC) strains are frequently being identified among nosocomial pathogens globally. Carbapenemase is a broad-spectrum beta-lactamase enzyme first found in K. pneumoniae isolates that results in resistance to all penicillins, cephalosporins, carbapenems (i.e., imipenem, ertapenem, metropenem), and monobactams (i.e., aztreonam). These broad-spectrum beta-lactamases are also known as extended spectrum beta-lactamases or ESBLs. These ESBLs are now being seen in a variety Enterobacteriaceae including Enterobacter spp., E. coli, Serratia spp., and Salmonella enterica. These ESBL-producing Enterobacteriaceae are known as carbapenem-resistant Enterobacteriaceae, or CRE.
• Methicillin-Resistant Staphylococcus aureus (MRSA): Staphylococcus aureus resistance to methicillin confers resistance to all penicillins and cephalosporins.
• Vancomycin-Resistant Enterococcus (VRE): Vancomycin-resistant Enterococcus (VRE) are intrinsically resistant to most antibiotics and have acquired resistance to the first line drug of choice, vancomycin.
• XDR TB: Extensively drug-resistant tuberculosis (XDR TB), a relatively rare type of multidrug-resistant Mycobacterium tuberculosis that is resistant to almost all drugs used to treat TB, including the two best first-line drugs: isoniazid and rifampin. XDR TB is also resistant to the best second-line medications: fluoroquinolones and at least one of three injectable drugs i.e., amikacin, kanamycin, or capreomycin.
Dormant persisters: Another mechanism that protects some bacteria from antibiotics is antibiotic tolerance. In the case of antibiotic tolerance, the tolerant bacterium is not killed but simply stops growing when the antibiotic is present. It then is able to recover once the antibiotic is no longer in the host. For example, Streptococcus pneumoniae tolerant to vancomycin appear to repress their autolysins in the presence of the drug and don't undergo osmotic lysis. Antibiotic tolerance is especially significant in terms of bacteria that form biofilms associated with catheters, heart valves, orthopedic devices, and people with cystic fibrosis. These biofilms often contain a small percentage of dormant persisters that, because they are not dividing, tolerate the antibiotics.
Its been found that bacteria simultaneously produce toxins that inhibit their own growth and antitoxins that bind to the toxin and cause its neutralizion. Small numbers of bacteria in the population, however, become persisters because they produce lower levels of antitoxin or the antitoxin is degraded by stress. As a result, the free toxin arrests bacterial growth enabling a persistent state that is able to survive stressors such as antibiotics and starvation.
Bacteria such as E. coli, Proteus, Enterobacter, Serratia, Pseudomonas, Staphylococcus aureus, and Enterococcus mentioned above, are the leading cause of health care-associated infections. According to the Centers for Disease Control and Prevention (CDC) Healthcare-associated infection's website, "In American hospitals alone, healthcare-associated infections account for an estimated 1.7 million infections and 99,000 associated deaths each year" in the U.S. The CDC also estimates that “more than two million people in the United States get infections that are resistant to antibiotics and at least 23,000 people die as a result.”
Finally, Bacterial endospores, such as those produced by Bacillus and Clostridium, are also resistant to antibiotics, most disinfectants, and physical agents such as boiling and drying. Although harmless themselves, they are involved in the transmission of some diseases to humans. Examples include anthrax (Bacillus anthracis), tetanus (Clostridium tetani), botulism (Clostridium botulinum), gas gangrene (Clostridium perfringens), and pseudomembranous colitis (Clostridium difficile).
Summary
1. Most bacteria become resistant to antibiotics by way of one or more mechanisms that are coded for by genes in the bacterial chromosome and/or in bacterial plasmids.
2. Bacterial genes may code for production of an enzyme that inactivates the antibiotic.
3. Bacterial genes may code for an altered target site receptor (ribosomal subunit, enzyme, etc.) for the antibiotic to reduce or block its binding.
4. Bacterial genes may code for altered membrane components that prevent the entry of the antibiotic into the bacterium and/or using an efflux pump to transport the antibiotic out of the bacterium.
5. Bacterial genes may code for modulated gene expression to produce more of the bacterial enzyme that is being tied up or altered by the antibiotic.
6. When under stress from antibiotics, some bacteria switch on genes whose protein products can increase the mutation rate within the bacterium causing a hyperevolution to increase the chance of forming an antibiotic-resistant mutant that is able to survive.
7. Horizontal gene transfer as a result of transformation, transduction, and conjugation can transfer antibiotic resistance from one bacterium to another. Horizontal gene transfer enables bacteria to respond and adapt to their environment much more rapidly than mutation by acquiring large DNA sequences from another bacterium in a single transfer.
8. Another mechanism that protects some bacteria from antibiotics is antibiotic tolerance whereby the tolerant bacterium, called a dormant persister, is not killed but simply stops growing when the antibiotic is present.
9. CDC estimates that “more than two million people in the United States get infections that are resistant to antibiotics and at least 23,000 people die as a result.” | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/4%3A_Using_Antibiotics_and_Chemical_Agents_to_Control_Bacteria/4.3%3A_Ways_in_which_Bacteria_May_Resist_Chemical_Control_A.txt |
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
4.1: An Overview to Control of Microorganisms
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Matching:
_____ An agent that kills the organism. (ans)
_____ An agent that inhibits the organism's growth long enough for body defenses to remove it. (ans)
_____The chemical agent being used should inhibit or kill the intended pathogen without seriously harming the host. (ans)
_____ A chemical agent that generally works against just gram-positives, gram-negatives, or only a few bacteria. (ans)
_____ A chemical agent that is generally effective against a variety of gram-positive and gram-negative bacteria. (ans)
_____ Antimicrobial drugs synthesized by chemical procedures in the laboratory. (ans)
_____ Metabolic products of one microorganism that inhibit or kill other microorganisms. (ans)
_____ The process of destroying all living organisms and viruses. (ans)
_____ The elimination of microorganisms, but not necessarily endospores, from inanimate objects or surfaces. (ans)
_____ An agent that kills or inhibits growth of microbes but is safe to use on human tissue. (ans)
1. selective toxicity
2. broad spectrum agent
3. narrow spectrum agent
4. cidal
5. static
6. sterilization
7. antibiotic
8. chemotherapeutic synthetic drug
9. antiseptic
10. disinfection
11. disinfectant
4.2: Ways in which Chemical Control Agents Affect Bacteria
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Matching:
_____ Alter bacterial 30S ribosomal subunits blocking translation. (ans)
_____ Inhibit peptidoglycan synthesis causing osmotic lysis. (ans)
_____ Alter bacterial 50S ribosomal subunits blocking translation. (ans)
_____ Inhibit nucleic acid synthesis. (ans)
1. macrolides(erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, etc.), oxazolidinones (linezolid), and streptogramins
2. penicillins, monobactams, carbapenems, cephalosporins, and vancomycin
3. fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.), sulfonamides and trimethoprim, and metronidazole
4. aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc.) and tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.)
2. Describe 4 different ways antibiotics or disinfectants may affect bacterial structures or macromolecules and state how this ultimately causes harm to the cell.
3. Multiple Choice (ans)
4.3: Ways in which Bacteria May Resist Chemical Control Agents
Study the material in this section and then write out the answers to these question. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Name 2 bacteria that have low-permeability membrane barriers and are thereby intrinsically resistant to many antibiotics. (ans)
2. Briefly describe 3 different mechanisms as a result of genetic changes in a bacterium that may enable that bacterium to resist an antibiotic.
3. State what the following stand for:
1. MRSA (ans)
2. VRE (ans)
3. CRE (ans)
4. Briefly describe R plasmids and state their significance in our attempts to treat infections with antibiotics. (ans)
5. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_2%3A_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/4%3A_Using_Antibiotics_and_Chemical_Agents_to_Control_Bacteria/4.E%3A_Using_Antibiotics_and_Chemical_Agents_to_Control_Bac.txt |
Thumbnail: The biohazard symbol was developed by the Dow Chemical Company in 1966 for their containment products. It is used in the labeling of biological materials that carry a significant health risk. (Public Domain; Silsor).
Unit 3: Bacterial Pathogenesis
Learning Objectives
After completing this section you should be able to perform the following objectives.
1. Define the following:
1. pathogenicity
2. virulence
3. virulence factors
4. infection
5. disease
6. etiologic agent
7. reservoir
8. zoonosis
9. vector
10. portal of entry and portal of exit
2. Compare and contrast sign and symptom.
3. List four requirements for a microorganism to cause infectious disease.
4. Contrast and give examples of direct and indirect transmission of microorganisms.
5. Even though a microorganism may be considered pathogenic, it still may not be able to cause disease upon entering the body. Discuss why.
In this course we are looking at various fundamental concepts of microbiology, with particular emphasis on their relationships to human health. The overall goal is to better understand the total picture of infectious diseases in terms of host-infectious agent interaction. Bacteria are found in almost every environment. Only a relatively few bacteria cause human disease and many benefit humans. For example, many are important decomposers that assure the flow and recycling of nutrients through ecosystems. Others have important industrial and pharmaceutical uses.
While the typical human body contains an estimated 10 trillion human cells, it also contains over 100 trillion bacteria and other microbes. The complex mutually beneficial symbiotic relationship between humans and their natural microbes is critical to good health. It is now recognized that the millions of genes associated with the normal flora or microbiota of the human body -especially in the intestinal tract - aid in the digestion of many foods, the regulation of multiple host metabolic pathways, and the regulation the body's immune defenses. These collective microbial genes are referred to as the human microbiome. There are currently an estimated 3, 000,000 - 5,000,000 genes from over 1000 species that constitute the human microbiome compared to the approximately 23,000 genes that make up the human genome. Some of these same normal microbiota, however, can also cause opportunistic infections when they get into parts of the body where they do not normally live or when the body becomes immunosuppressed. However, in this section we are going to concentrate on bacteria that are potentially harmful to humans and try to understand what factors influence their ability to cause disease.
The Good, the Bad, and the Ugly
Most bacteria are not harmful. In fact, only 10% of bacteria are “bad” or pathogenic, while the other 90% "good" or neutral and are necessary components for human life.
Infection versus Disease
Pathogenicity and virulence are terms that refer to an organism's ability to cause disease. Pathogenicity is the ability of a microbe to cause disease and inflict damage upon its host, whereas virulence is the degree of pathogenicity within a group or species of microbes as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenicity of an organism, that is its ability to cause disease, is determined by its virulence factors.
As learned earlier under Bacterial Genetics, most of the virulence factors that enable bacteria to colonize the body and/or harm the body are the products of quorum sensing genes. Many bacteria use quorum sensing to sense their own population density, communicate with each other by way of secreted chemical factors, and behave as a population rather than as individual bacteria. This plays an important role in pathogenicity and survival for many bacteria.
The genomes of pathogenic bacteria, when compared with those of similar nonpathogenic species or strains, often show extra genes coding for virulence factors, that is, molecules expressed and secreted by the bacterium that enable them to colonize the host, evade or inhibit the immune responses of the host, enter into or out of a host cell, and/or obtain nutrition from the host. These include virulence factors such as capsules, adhesins, type 3 secretion systems, invasins, and toxins.
We also learned that most genes coding for virulence factors in bacteria are located in pathogenicity islands or PAIs and are usually acquired by horizontal gene transfer. These PAIs may be located in the bacterial chromosome, in plasmids, or even in bacteriophage genomes that have entered the bacterium. The genomes of most pathogenic bacteria typically contain multiple PAIs that can account for up to 10 - 20% of the bacterium's genome. PAIs carry genes such as transpoases, integrases, or insertion sequences that enable them to insert into host bacterial DNA. Transfer RNA (tRNA) genes are often the target site for integration of PAIs. Conjugative plasmids are the most frequent means of transfer of PAIs from one bacterium to another and the transfer of PAIs can then confer virulence to a previously nonpathogenic bacterium.
An infection is when a microorganism has established itself in a host - has colonized that host - whether not it causing harm or imparting damage. A disease, on the other hand, is where there is impairment to host function as a result of damage or injury. For example, the microbes that constitute the body's normal flora or microbiota have infected the body, but they seldom cause disease unless they invade a part of the body where they do not normally reside and/or the host becomes immunocompromised. In medicine, the term etiology refers to the causes of diseases or pathologies. In terms of infectious disease, the etiologic agent is the microorganism causing that disease.
The terms signs and symptoms are often used when diagnosing disease. A sign is an objective indication of some medical fact or characteristic that may be detected by a health care professional during a physical examination. They include such objective indications as blood pressure, respiration, rate, pulse, and temperature. A symptom is a condition experienced and reported by the patient.
To cause disease, a microorganism must maintain a reservoir before and after infection
The reservoir of an infectious agent is the habitat in which that microbe normally lives, grows, and multiplies. Reservoirs can include humans, animals, and the environment. Many common human infectious diseases have human reservoirs and are transferred person-to-person without intermediaries. Examples include sexually transmitted diseases, measles, most respiratory pathogens, and strep throat. Some infections are transmitted from an animal to a human in which case the infection is called a zoonosis. Examples include rabies, plague, and much salmonellosis. Plants, soil, and water in the environment are also reservoirs for some infectious agents such as histoplasmosis, coccidioidomycosis, and Legionnaires disease.
To cause disease, a microorganism must leave the reservoir and gain access to the new host
The microorganism must leave its reservoir or host through what is called a portal of exit and be transmitted to a new host. For example, the portal of exit for respiratory infections is typically the mouth or nose; for gastrointestinal infections, the feces. Modes of transmission include:
1. Direct contact, as through skin-to-skin contact, kissing, and sexual intercourse. Examples include some Staphylococcus aureus infections, infectious mononucleosis, and gonorrhea.
2. Direct droplet contact, as in the case of aerosols produced by sneezing and coughing. Examples include meningococcal infections and pertussis (whooping cough).
3. Indirect transmission of an infectious agent from a reservoir to a host by suspended air particles, inanimate objects, or vectors.
4. Airborne transmission occurs when infectious agents are carried by dust or droplets suspended in air. Some respiratory infections can be transmitted this way although most are transmitted by contact with infectious mucus.
5. Inanimate objects include water, food, blood, and fomites (inanimate objects such as toys, handkerchiefs, bedding, or clothing). Examples include cholera, salmonellosis, listeriosis, viral hepatitis).
6. Vectors such as ticks, mosquitoes, and fleas. Examples include Lyme's disease, malaria, and typhus fever.
The manner in which a pathogen enters a susceptible host is referred to as its portal of entry. For example, the portal of entry for most respiratory infections is the mouth or nose; for gastrointestinal infections, the mouth. The portal of entry must provide access to tissues with the correct physical and chemical environment (an environment with the proper oxygen content, pH, nutrients, temperature, etc.) in which the pathogen can multiply.
To cause disease, a microorganism must Adhere to cells of the skin or mucosa of its new host and colonize the body
Almost every part of the body has a mechanism for flushing microbes out of or off of the body, including the shedding of epithelial cells from the skin and mucous membranes, urination, defecation, coughing, and sneezing. Unless the microorganisms can replicate fast enough to replace those being flushed out, as in the case of much of the normal microbiota that colonize the lumen of the intestines, they need to adhere to the epithelial cells of the skin and mucous membranes. Also, this body environment must have the correct nutrients, the proper amount of oxygen or lack of oxygen, the right pH, and the right temperature to support the growth of that microorganism. Furthermore, since the body has excellent immune defense mechanisms, anything the microorganism can do to resist body defenses to some degree will also promote colonization.
To cause disease, a microorganism must Harm or damage the body
As stated above, an infection is simply when a microorganism has established itself in a host. To cause disease, that microorganism (or toxin) must inflict damage to the host.
Summary
1. Only a relatively few bacteria cause human disease.
2. The complex mutually beneficial symbiotic relationship between humans and their natural microbes is critical to good health.
3. An infection is when a microorganism has established itself in a host - has colonized that host - whether not it causing harm or imparting damage.
4. A disease is where there is impairment to host function as a result of damage or injury.
5. Etiology refers to the causes of diseases or pathologies; in terms of infectious disease, the etiologic agent is the microorganism causing that disease.
6. A sign is an objective indication of some medical fact or characteristic that may be detected by a health care professional during a physical examination; a symptom is a condition experienced and reported by the patient.
7. The reservoir of an infectious agent is the habitat in which that microbe normally lives, grows, and multiplies.
8. Transmission of microorganisms by direct contact refers to transfer by such means as skin-to-skin contact, kissing, and sexual intercourse.
9. Transmission of microorganisms by direct droplet contact refers to transfer by aerosols produced by sneezing and coughing.
10. Transmission of microorganisms by indirect contact refers to transfer by suspended air particles, inanimate objects, or vectors (ticks, mosquitoes, fleas).
11. The manner in which a pathogen enters a susceptible host is referred to as its portal of entry; the manner in which it leaves its host is its portal of exit.
12. If relatively few bacteria enter the body then the body's natural defenses against infection have a much better chance of removing them before they can colonize, multiply, and cause harm; if a large number of bacteria enter then the body's defenses may not be as successful.
13. A person with good innate and adaptive immune defenses will be much more successful in removing potentially harmful bacteria than a person that is immunocompromised.
14. Bacterial virulence factors influence a bacterium’s ability to cause infectious disease. These include virulence factors that enable bacteria to colonize the host as well as those that harm or damage the host.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Define pathogenicity. (ans)
2. Define virulence. (ans)
3. Define infection. (ans)
4. Define disease. (ans)
5. Define vector. (ans)
6. Define medical sign. (ans)
7. Even though a microorganism may be considered pathogenic, it still may not be able to cause disease upon entering the body. Discuss why. (ans)
8. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/1%3A_Overview_of_Microbial_Pathogenesis.txt |
Virulence factors are molecules expressed and secreted by that enable them to colonize the host, evade or inhibit the immune responses of the host, enter into or out of a host cell, and/or obtain nutrition from the host. The following are virulence factors that promote bacterial colonization of the host .
1. The ability to use motility and other means to contact host cells and disseminate within a host.
2. The ability to adhere to host cells and resist physical removal.
3. The ability to invade host cells.
4. The ability to compete for iron and other nutrients.
5. The ability to resist innate immune defenses such as phagocytosis and complement.
6. The ability to evade adaptive immune defenses.
• 5.0: Prelude to Virulence Factors that Promote Bacterial Colonization
Virulence factors are molecules expressed on or secreted by microorganisms that enable them to colonize the host, evade or inhibit the immune responses of the host, enter into or out of a host cell, and/or obtain nutrition from the host. To cause infectious disease, a bacterium must produce virulence factors that promote bacterial colonization of the host, as well as virulence factors that impair or damage the host.
• 5.1: The Ability to Use Motility and Other Means to Contact Host Cells
Bacteria have to make physical contact with host cells before they can adhere to those cells and resist being flushed out of the body. Motile bacteria can use their flagella and chemotaxis to swim through mucus towards mucosal epithelial cells. Because of their thinness, their internal flagella (axial filaments), their corkscrew shape, and their motility, certain spirochetes are more readily able enter lymph vessels and blood vessels and spread to other body sites.
• 5.2: The Ability to Adhere to Host Cells and Resist Physical Removal
One of the body's innate immune defenses is the ability to physically remove bacteria from the body. Bacteria may resist physical removal by producing pili, cell wall adhesin proteins, and/or biofilm-producing capsules that enable bacteria to adhere to host cells. At the end of the shaft of a bacterial pilus is an adhesive tip structure having a shape corresponding to that of specific receptor on a host cell for initial attachment. Bacteria can typically make a variety of different adhesive tips
• 5.3: The Ability to Invade Host Cells
Some bacteria produce molecules called invasins that activate the host cell's cytoskeletal machinery enabling bacterial entry into the cell by phagocytosis. Entering a non-defense host cell can provide the bacterium with a ready supply of nutrients, as well as protect the bacterium from complement, antibodies, and other body defense molecules. Some bacteria invade phagocytic cells, neutralize their killing ability, and turn them into a safe haven for bacterial replication.
• 5.4: The Ability to Compete for Nutrients
The ability to be pathogenic is directly related to the bacterium's ability to compete successfully with host tissue and normal flora for limited nutrients. They compete for nutrients by synthesizing specific transport systems or cell wall components capable of binding limiting substrates and transporting them into the cell. Iron is an essential nutrient for both bacterial growth and human cell growth. Both bacteria and their host synthesize compounds capable of binding iron for their use.
• 5.5: The Ability to Resist Innate Immune Defenses
Some bacteria are able to resist innate immune defenses such as phagocytosis and the body's complement pathways. We will break this down into two categories: (1) The ability to resist phagocytic engulfment (attachment and ingestion) and (2) the ability to resist phagocytic destruction and complement serum lysis.
• 5.6: The Ability to Evade Adaptive Immune Defenses
There are various ways that the antibodies the body makes during adaptive immunity protect the body against bacteria. Some antibodies such as IgG and IgE function as opsonins and stick bacteria to phagocytes (opsonization or enhanced attachment). Antibodies, such as IgG, IgA, and IgM, can bind to bacterial adhesins, pili, and capsules and in this way block their attachment to host cells.
• 5.E: Virulence Factors that Promote Colonization (Exercises)
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
5: Virulence Factors that Promote Colonization
List six virulence factors that promote bacterial colonization of the host.
In this section on Bacterial Pathogenesis, we are looking at bacterial virulence factors that can influence its ability to cause infectious disease. Virulence factors are molecules expressed and secreted by that enable them to colonize the host, evade or inhibit the immune responses of the host, enter into or out of a host cell, and/or obtain nutrition from the host. These virulence factors will be divided into two categories:
• Virulence factors that promote bacterial colonization of the host.
• Virulence factors that damage the host.
In this section we will look at virulence factors that promote bacterial colonization of the host.
Virulence Factors that Promote Bacterial Colonization of the Host
The following are virulence factors that promote bacterial colonization of the host .
1. The ability to use motility and other means to contact host cells and disseminate within a host.
2. The ability to adhere to host cells and resist physical removal.
3. The ability to invade host cells.
4. The ability to compete for iron and other nutrients.
5. The ability to resist innate immune defenses such as phagocytosis and complement.
6. The ability to evade adaptive immune defenses.
As mentioned in the previous section, most of the virulence factors that better enable bacteria to colonize the body are the products of quorum sensing genes. It will also be seen that bacteria often carry out these abilities by co-opting the host cell’s machinery and communication ability. Many bacteria are able to produce specialized secretion machinery that enables the bacterium to inject proteins into the host cell that reprogram various aspects of the host cell’s machinery to benefit the bacterium.
Summary
Virulence factors are molecules expressed on or secreted by microorganisms that enable them to colonize the host, evade or inhibit the immune responses of the host, enter into or out of a host cell, and/or obtain nutrition from the host. To cause infectious disease, a bacterium must produce virulence factors that promote bacterial colonization of the host, as well as virulence factors that impair or damage the host. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.0%3A_Prelude_to_Virulence_Factors_that_Promote_Bacterial_Colonization.txt |
Learning Objectives
1. State why it might be of an advantage for a bacterium trying to colonize the bladder or the intestines to be motile.
2. Describe specifically how certain bacteria are able to use motility to contact host cells and state how this can promote colonization.
3. Briefly describe why being extremely thin and being motile by means of axial filaments may be an advantage to pathogenic spirochetes.
4. Give one example of how a nonmotile bacterium may be able to better disseminate within a host.
5. Give a brief description of how a bacterium may use toxins to better disseminate from one host to another.
Highlighted Bacterium
1. Read the description of Helicobacter pylori and match the bacterium with the description of the organism and the infection it causes.
The mucosal surfaces of the respiratory tract, the intestinal tract, and the genitourinary tract constantly flush bacteria away in order to prevent colonization of host mucous membranes. Motile bacteria can use their motility and chemotaxis to swim through mucus towards mucosal epithelial cells. Many bacteria that can colonize the mucous membranes of the bladder and the intestines, in fact, are motile. Motility probably helps these bacteria move through the mucus between the mucin strands or in places where the mucus is less viscous. Examples of motile opportunists and pathogens include Helicobacter pylori, Salmonella species, Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae. Once bacteria contact host cells they can subsequently attach, and colonize. (Attachment will be discussed in the next section.)
For example, Helicobacter pylori , the bacterium that causes most gastric and duodenal ulcers, produces urease, an enzyme that breaks down urea into ammonia and carbon dioxide. The ammonia neutralizes the hydrochloric acid in the stomach. In addition, the urease is thought to alter the proteins in the mucus changing it from a solid gel to a thinner fluid that the bacteria are able to swim through by way of their flagella, and subsequently use adhesins to adhere to the epithelial cells of the mucous membranes. To further help protect the bacterium from the acid, H. pylori produces an acid-inhibitory protein that blocks acid secretion by surrounding parietal cells in the stomach. Bacterial toxins then lead to excessive production of cytokines and chemokines , as well as mucinase and phospholipase that damage the gastric mucosa. The cytokines and chemokines, in turn, result in a massive inflammatory response. Neutrophils leave the capillaries, accumulate at the area of infection, and discharge their lysosomes for extracellular killing. This not only kills the bacteria, it also destroys the mucus-secreting mucous membranes of the stomach. Without this protective layer, gastric acid causes ulceration of the stomach. This, in turn, leads to either gastritis or gastric and duodenal ulcers.
YouTube movie of a video endoscopy exam showing duodenal ulcers caused by Helicobacter pylori.
Click on this link, read the description of Helicobacter pylori, and be able to match the bacterium with its description on an exam.
Planktonic Pseudomonas aeruginosa uses its polar flagellum to move through water or mucus and make contact with a solid surface such as the body's mucous membranes (Figure \(5\).1.1). It then can use pili and cell wall adhesins to attach to the epithelial cells of the mucous membrane. Attachment activates signaling and quorum sensing genes to eventually enable the population of P. aeruginosa to start synthesizing a polysaccharide biofilm composed of alginate. As the biofilm grows, the bacteria lose their flagella to become nonmotile and secrete a variety of enzymes that enable the population to obtain nutrients from the host cells. Eventually the biofilm mushrooms up and develops water channels to deliver water and nutrients to all the bacteria within the biofilm. As the biofilm begins to get too crowded with bacteria, quorum sensing enables some of the Pseudomonas to again produce flagella, escape the biofilm, and colonize a new location.
Because of their thinness, their internal flagella (axial filaments), their corkscrew shape, and their motility (Figure \(5\).1.2), spirochetes are more readily able to penetrate host mucous membranes, skin abrasions, etc., and enter the body. Motility and penetration may also enable the spirochetes to penetrate deeper in tissue and enter the lymphatics and bloodstream and disseminate to other body sites. Spirochetes that infect humans include Treponema pallidum , Leptospira , and Borrelia burgdorferi ).
Along a different line, many bacteria produce enzymes such as elastases and proteases that degrade the extracellular matrix proteins that surround cells and tissues and make it easier for those bacteria to disseminate within the body. For example, Streptococcus pyogenes produces streptokinase that lyses the fibrin clots produced by the body in order to localize the infection. It also produces DNase that degrades cell-free DNA found in pus and reduces the viscosity of the pus. Both of these enzymes facilitate spread of the bacterium from the localized site to new tissue.
Staphylococcus aureus, on the other hand, produces surface adhesins that bind to extracellular matrix proteins and polysaccharides surrounding host cell tissue, including fibronectin, collagen, laminin, hyaluronic acid, and elastin. S. aureus proteases and hyaluronidase then dissolve these components of the extracellular matrix providing food for the bacteria and enabling the bacteria to spread.
Finally, as will be seen later in this unit under toxins, some bacteria produce toxins that induce diarrhea in the host. Diarrhea is also a part of our innate immunity to flush harmful microbes and toxins out of the intestines. On one hand, diarrhea is an advantage to the body because it flushes out harmful microbes and toxins. On the other hand, it is beneficial for the bacterium inducing the diarrhea because it also flushes out a good deal of the normal flora of the intestines and this reduces the competition for nutrients between normal flora and pathogens. In addition, diarrhea enables the pathogen to more readily leave one host and enter new hosts through the fecal-oral route.
Summary
Bacteria have to make physical contact with host cells before they can adhere to those cells and resist being flushed out of the body. Motile bacteria can use their flagella and chemotaxis to swim through mucus towards mucosal epithelial cells. Because of their thinness, their internal flagella (axial filaments), their corkscrew shape, and their motility, certain spirochetes are more readily able enter lymph vessels and blood vessels and spread to other body sites. Many bacteria produce enzymes that degrade the extracellular matrix proteins that surround cells and tissues and help to localize infection, making it easier for those bacteria to spread within the body. Some bacteria produce toxins that induce diarrhea in the host enabling the pathogen to more readily leave one host and enter new hosts through the fecal-oral route. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.1%3A_The_Ability_to_Use_Motility_and_Other_Means_to_Contact_Host_Cells.txt |
Learning Objectives
1. Briefly describe 3 different mechanisms by which bacteria can adhere to host cells and colonize and state how this can promote colonization.
2. State an advantage for bacteria in being able to switch the adhesive tips of their pili.
3. Define biofilm and state at least 3 benefits associated with bacteria living as a community within a biofilm.
Highlighted Bacterium
1. Read the description of Neisseria memingitidis andmatch the bacterium with the description of the organism and the infection it causes.
One of the body's innate immune defenses is the ability to physically remove bacteria from the body through such means as the constant shedding of surface epithelial cells from the skin and mucous membranes, the removal of bacteria by such means as coughing, sneezing, vomiting, and diarrhea, and bacterial removal by bodily fluids such as saliva, blood, mucous, and urine. Bacteria may resist this physical removal by producing pili, cell wall adhesin proteins, and/or biofilm-producing capsules. In addition, the physical attachment of bacteria to host cells can also serve as a signal for the activation of genes involved in bacterial virulence. This process is known as signal transduction.
Using Pili (fimbriae) to Adhere to Host Cells
As seen in Unit 1, pili enable some organisms to adhere to receptors on target host cells (Figure \(5\).2.1) and thus colonize and resist flushing by the body. Pili are thin, protein tubes originating from the cytoplasmic membrane and are found in virtually all Gram-negative bacteria, but not in many Gram-positive bacteria.
The pilus has a shaft composed of a protein called pilin. At the end of the shaft is the adhesive tip structure having a shape corresponding to that of specific glycoprotein or glycolipid receptors on a host cell (Figure \(5\).2.3). Because both the bacteria and the host cells have a negative charge, pili may enable the bacteria to bind to host cells without initially having to get close enough to be pushed away by electrostatic repulsion. Once attached to the host cell, the pili can depolymerize and enable adhesions in the bacterial cell wall to make more intimate contact. There is also evidence that the binding of pili to host cell receptors can serve as a trigger for activating the synthesis of some cell wall adhesins.
Bacteria are constantly losing and reforming pili as they grow in the body and the same bacterium may switch the adhesive tips of the pili in order to adhere to different types of cells and evade immune defenses (Figure \(2\).2.3). E. coli, for example, is able to make over 30 different types of pili.
One class of pili, known as type IV pili, not only allows for attachment but also enable a twitching motility. They are located at the poles of bacilli and allow for a gliding motility along a solid surface such as a host cell. Extension and retraction of these pili allows the bacterium to drag itself along the solid surface (Figure \(4\)). In addition, bacteria can use their type IV pili to "slingshot" the bacterium over a cellular surface. In this case, as the pili contract they are thought to become taut like a stretched rubber band. When an anchoring pilus detaches, the taut pili "slingshot" the bacterium in the opposite direction (Figure \(5\)). This motion typically alternates with the twitching motility and enables a more rapid motion and direction change than with the twitching motility because the rapid slingshotting motion reduces the viscosity of the surrounding biofilm.
This enables bacteria with these types of pili within a biofilm to move around a cellular surface and find an optimum area on that cell for attachment and growth once they have initially bound. Bacteria with type IV pili include Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, and Vibrio cholerae.
Examples of bacteria using pili to colonize:
1. To cause infection, Neisseria gonorrhoeae must first colonize a mucosal surface composed of columnar epithelial cells. Pili allow for this initial binding and, in fact, N. gonorrhoeae is able to rapidly lose pili and synthesize new ones with a different adhesive tip, enabling the bacterium to adhere to a variety of tissues and cells including sperm, the epithelial cells of the mucous membranes lining the throat, genitourinary tract, rectum, and the conjunctiva of the eye. Subsequently, the bacterium is able to make more intimate contact with the host cell surface by way of a cell wall adhesin called Opa (see below).
2. The pili of Neisseria meningitidis allow it to adhere to mucosal epithelial cells in the nasopharynx where it is often asymptomatic. From there, however, it sometimes enters the blood and meninges and causes septicemia and meningitis. Type IV pili are thought to help the bacterium cross the blood brain barrier.
Click on this link, read the description of Neisseria meningitidis, and be able to match the bacterium with its description on an exam.
3. Uropathogenic strains of Escherichia coli can produce pili that enable the bacterium to adhere to the urinary epithelium and cause urinary tract infections. They also produce afimbrial adhesins (see below) for attachment to epithelial cells. Enteropathogenic E. coli (EPEC) use pili to adhere to intestinal mucosal cells.
• To view an electron micrograph E. coli with pili, see Dennis Kunkel's Microscopy at the University of Hawaii-Manoa.
• To view electron micrographs of enteropathogenic E. coli (EPEC) adhering to intestinal cells, see Donnenberg Lab Images at the University of Maryland Medical School.
4. Pili of Vibrio cholerae allow it to adhere to cells of the intestinal mucosa and resist the flushing action of diarrhea.
5. Pili of Pseudomonas aeruginosa allow it to initially colonize wounds or the lung.
Using Adhesins to Adhere to Host Cells
Adhesins are surface proteins found in the cell wall of various bacteria that bind to specific receptor molecules on the surface of host cells and enable the bacterium to adhere intimately to that cell in order to colonize and resist physical removal (Figure \(6\)). Many, if not most bacteria probably use one or more adhesins to colonize host cells.
For example:
1. Streptococcus pyogenes (see electron micrograph) (group A beta streptococci) produce a number of adhesins
a. Protein F that binds to fibronectin , a common protein on epithelial cells. In this way it is able to adhere to the lymphatics and mucous membranes of the upper respiratory tract and cause streptococcal pharyngitis (strep throat).
b. Lipoteichoic acid binds to fibronectin on epithelial cells.
c. M-protein also functions as an adhesin.
2. The tip of the spirochete Treponema pallidum contains adhesins that are able to bind to fibronectin on epithelial cells.
Scanning electron Micrograph of T. pallidum adhering to a host cell by its tip.
3. The tip of the spirochete Borrelia burgdorferi contains adhesins that can bind to various host cells.
4. Escherichia coli O157 utilizes a type 3 secretion system to inject effector proteins into intestinal epithelial cells. Some of these cause polymerization of actin at the cell surface and this pushes the host cell cytoplasmic membrane up to form a pedestal. Another effector protein inserts into the membrane of the pedestal to serve as a receptor molecule for E. coli adhesins (Figure \(7\)).
5. Helicobacter pylori use a type 4 secretion system to inject effector proteins into stomach epithelial cells to induce these host cells to display more receptors on their surface for H. pylori adhesins.
6. Bordetella pertussis produces several adhesins (Figure \(8\)):
a. Filamentous hemagglutinin is an adhesin that allows the bacterium to adhere to galactose residues of the glycolipids on the membrane of ciliated epithelial cells of the respiratory tract.
b. Pertussis toxin also functions as an adhesin. One subunit of the pertussis toxin remains bound to the bacterial cell wall while another subunit binds to the glycolipids on the membrane of ciliated epithelial cells of the respiratory tract.
c. B. Pertussis also produces an adhesin called pertactin that further enables the bacterium to adhere to cells.
7. Neisseria gonorrhoeae produces an adhesin called Opa (protein II) that enables the bacterium to make a more intimate contact with the host cell after it first adheres with its pili. Like with adhesive tips of pili, N. gonorrhoeae has multiple alleles for Opa protein adhesins enabling the bacterium to adhere to a variety of host cell types.
8. Staphylococcus aureus uses protein A as an adhesin to adhere to various host cells. It also helps the bacterium to resist phagocytosis.
Using Biofilms to Adhere to Host Cells
Many normal flora bacteria produce a capsular polysaccharide matrix or glycocalyx to form a biofilm on host tissue. Biofilms are groups of bacteria attached to a surface and enclosed in a common secreted adhesive matrix, typically polysaccharide in nature. Many pathogenic bacteria, as well as normal flora and many environmental bacteria, form complex bacterial communities as biofilms.
Bacteria in biofilms are often able to communicate with one another by a process called quorum sensing and are able to interact with and adapt to their environment as a population of bacteria rather than as individual bacteria. By living as a community of bacteria as a biofilm, these bacteria are better able to:
• resist attack by antibiotics;
• trap nutrients for bacterial growth and remain in a favorable niche;
• adhere to environmental surfaces and resist flushing;
• live in close association and communicate with other bacteria in the biofilm; and
• resist phagocytosis and attack by the body's complement pathways.
Biofilms are, therefore, functional, interacting, and growing bacterial communities. Biofilms even contain their own water channels for delivering water and nutrients throughout the biofilm community.
For example:
1. Streptococcus mutans, and Streptococcus sobrinus , two bacteria implicated in initiating dental caries, break down sucrose into glucose and fructose. Streptococcus mutans can uses an enzyme called dextransucrase to convert sucrose into a sticky polysaccharide called dextran that forms a biofilm enabling the bacteria to adhere to the enamel of the tooth and initiate plaque formation.
This dextran mesh traps the S. mutans and S. sobrinus, along with other bacteria and debris, and forms plaque. S. mutans and S. sobrinus also ferment glucose in order to produce energy. The fermentation of glucose results in the production of lactic acid that is released onto the surface of the tooth and initiates decay.
2. Most children suffering from chronic ear infection (otitis media) have a biofilm of bacteria in their middle ear. This biofilm contains bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis and enables the bacteria to chronically colonize the middle ear as well as resist body defenses and antibiotics.
3. Planktonic Pseudomonas aeruginosa uses its polar flagellum to move through water or mucus and make contact with a solid surface such as the body's mucous membranes. It then can use pili and cell wall adhesins to attach to the epithelial cells of the mucous membrane. Attachment activates signaling and quorum sensing genes to eventually enable the population of P. aeruginosa to start synthesizing a polysaccharide biofilm composed of alginate. As the biofilm grows, the bacteria lose their flagella to become nonmotile and secrete a variety of enzymes that enable the population to obtain nutrients from the host cells. Eventually the biofilm mushrooms up and develops water channels to deliver water and nutrients to all the bacteria within the biofilm. As the biofilm begins to get too crowded with bacteria, quorum sensing enables some of the Pseudomonas to again produce flagella, escape the biofilm, and colonize a new location (See Figs. 9A-9H).
Many chronic and difficult-to-treat infections are caused by bacteria in biofilms. Within biofilms, bacteria grow more slowly, exhibit different gene expression than free planktonic bacteria, and are more resistant to antimicrobial agents such as antibiotics because of the reduced ability of these chemicals to penetrate the dense biofilms matrix. Biofilms have been implicated in tuberculosis, kidney stones, Staphylococcus infections, Legionnaires' disease, and periodontal disease. It is further estimated that as many as 10 million people a year in the US may develop biofilm-associated infections as a result of invasive medical procedures and surgical implants.
YouTube movie and animation: What are Biofilms?
Exercise: Think-Pair-Share Questions
Pseudomonas aeruginosa, a common cause of serious respiratory infections on people with cystic fibrosis, produces a single polar flagellum, can secrete a polysaccharide slime composed of alginate, and is able to produce both pili and cell wall adhesins. How could each of these factors contribute to the bacterium's pathogenosis and in what order might they be used?
Summary
1. One of the body's innate immune defenses is the ability to physically remove bacteria from the body.
2. Bacteria may resist physical removal by producing pili, cell wall adhesin proteins, and/or biofilm-producing capsules that enable bacteria to adhere to host cells.
3. At the end of the shaft of a bacterial pilus is an adhesive tip structure having a shape corresponding to that of specific receptor on a host cell for initial attachment. Bacteria can typically make a variety of different adhesive tips enabling them to attach to different host cell receptors.
4. Cell wall adhesins are surface proteins found in the cell wall of various bacteria that bind tightly to specific receptor molecules on the surface of host cells. Bacteria can typically make a variety of different cell wall adhesins enabling them to attach to different host cell receptors.
5. Biofilms are groups of bacteria attached to a surface and enclosed in a common secreted adhesive matrix, typically polysaccharide in nature. Many pathogenic bacteria, as well as normal flora and many environmental bacteria, form complex bacterial communities as biofilms.
6. Many chronic and difficult-to-treat infections are caused by bacteria in biofilms. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.2%3A_The_Ability_to_Adhere_to_Host_Cells_and_Resist_Physical_Removal.txt |
Briefly describe the mechanism by which invasins enable certain bacteria to enter host cells and state how this can promote colonization Briefly describe how a type 3 secretion system might be used to invade and survive inside host cells. State how certain pathogenic spirochetes such as Treponema pallidum and Borrelia bergdorferi use adhesins, invasins and motility to penetrate host cells. Highlighted Bacterium Read the description of Shigella and match the bacterium with the description of the organism and the infection it causes. Read the description of Salmonella and match the bacterium with the description of the organism and the infection it causes. Read the description of Borrelia bergdorferi and match the bacterium with the description of the organism and the infection it causes.
Some bacteria produce molecules called invasins that activate the host cell's cytoskeletal machinery enabling bacterial entry into the cell by phagocytosis. Advantages of entering a human cell include (1) providing the bacterium with a ready supply of nutrients and (2) protecting the bacteria from complement, antibodies, and other body defense molecules.
When these bacteria contact the epithelial cells of the colon, the type III secretion system delivers proteins into the epithelial cells enabling them to polymerize and depolymerize actin filaments. This cytoskeletal rearrangement is a key part of the pseudopod formation in phagocytic cells and is what enables phagocytes to engulf bacteria and place them in a vacuole. Thus the bacterium with its invasins is able to trick the epithelial cell into behaving like a phagocyte and engulfing the bacterium. The bacteria then replicate within the host cell.
We will now look at several examples of bacteria that use invasions to invade host cells.
In addition, Shigella can induce the host cells to produce signaling molecules that attract phagocytic, antigen-presenting dendritic cells to the area. It enters the dendritic cells and uses them to carry the Shigella through the intestinal wall to the underside. It then uses its type 3 secretion system to inject effector proteins from the phagosome into the cytoplasm. These proteins trigger apoptosis or cell suicide of the dendritic cell. Killing the dendritic cells prevents them from presenting Shigella to T4-lymphocytes, a step required for the production of antibodies against the Shigella (see Figure \(4\)).
• For a movie showing Shigella being propelled by actin-based motility within a cell, see the Theriot Lab Website at Stanford University Medical School. Click on "Greatest Hits" and then on "Shigella flexneri associated with actin tails in PtK2 cells."
2. Salmonella use a type 3 secretion system to inject intestinal epithelial cells with effector proteins that stimulate actin re-arrangement and cause the epithelial cell's cytoplasmic to "ruffle" up and engulf the bacteria Figs. 5A - Figure \(5\)B. The Salmonella pass through the epithelial cell where they are engulfed by phagocytic macrophages.
Once in the phagosome of the macrophage the bacterium uses its type 3 secretion system to inject proteins that prevent the lysosomes from fusing with the phagosomes, thus providing a safe haven for Salmonella replication within the phagosome and protecting the bacteria from antibodies and other defense elements (see Figs. 5C-5D).
By injecting flagellin into the cytoplasm of the macrophage the Salmonella can also eventually kill the macrophage by inducing apoptosis, a programmed cell suicide.
Molecules injected into the intestinal epithelial cells also stimulate diarrhea. Advantages of inducing diarrhea include (1) flushing out normal flora bacteria so there is less competition for nutrients; and (2) better enabling Salmonella that are not attached to host cells to be transmitted to a new host via the fecal-oral route.
For a movie showing Salmonella invading a human cell, see the Theriot Lab Website at Stanford University Medical School. Click on"Greatest Hits" and then on "Salmonella typhimurium invading a fibroblast cell."
3. Listeria monocytogenes is another bacterium that enters intestinal cells via invasins and spreads to adjacent cells by actin-based motility. Its actin-based motility enables it to moves approximately 1.5 µm per second within the host cell.
For movies showing Listeria entering host cells and being propelled by actin-based motility within a cell, see the Theriot Lab Website at Stanford University Medical School. Click on "Greatest Hits" and then on "Life history of a single infecting Listeria monocytogenes" and "Listeria monocytogenes moving in PtK2 cells."
4. Although enteroinvasive Escherichia coli (EIEC) don't have actin-based motility, they invade and kill epithelial cells of the colon in a manner similar to Shigella.
5. Legionella pneumophila, after being ingested by macrophages and placed in a phagosome, uses a type 4 secretion system to inject effector proteins that prevent the lysosomes from fusing with the phagosomes and turning the macrophage into a safe haven for bacterial replication. The same mechanism allows the Legionella to survive inside amoebas in nature. These amoebas serve as a reservoir for the bacterium in the environment.
6. F protein and M-protein of Streptococcus pyogenes (Group A beta streptococci) enables the bacterium to invade epithelial cells. This is thought to help maintain persistent streptococcal infections and enable the bacterium to spread to deeper tissues.
7. The spirochete Borrelia bergdorferi probably uses a combination of invasins and motility to penetrate host cells. In this case the host cell doesn't phagocytose the bacterium. Instead, one tip of the spirochete attaches to the host cell and some form of invasin apparently causes the host cell to release digestive enzymes that enable the spirochete with its corkscrewing motility to penetrate the host cell membrane. Once in the host cell the bacteria may remain dormant for years and hide from the immune system and antibiotics.
8. Another spirochete, Treponema pallidum, is thought to enter cells in a similar fashion. Motility also helps B. bergdorferi and T. pallidum to invade and leave blood vessels by passing between and through endothelial cells, thus enabling the spirochetes to disseminate to other locations in the body. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.3%3A_The_Ability_to_Invade_Host_Cells.txt |
State why the ability to compete for iron and other nutrients is important for bacteria to cause disease and describe briefly three ways bacteria may accomplish this as part of their pathogenicity.
Often the ability to be pathogenic is directly related to the bacterium's ability to compete successfully with host tissue and normal flora for limited nutrients. One reason the generation time of bacteria growing in the body is substantially slower than in lab culture is because essential nutrients are limited. In fact this is a major reason why the overwhelming majority of bacteria found in nature are not harmful to humans.
To be pathogenic, a bacterium must be able to multiply in host tissue. The more rapid the rate of replication, the more likely infection may be established. Pathogens, therefore, are able to compete successfully for limited nutrients in the body. Generally bacteria compete for nutrients by synthesizing specific transport systems or cell wall components capable of binding limiting substrates and transporting them into the cell. A good example of this is the ability of bacteria to compete for iron.
As we will see later in Unit 5 under innate immunity, the body makes considerable metabolic adjustment during infection to deprive microorganisms of iron. Iron is essential for both bacterial growth and human cell growth. Bacteria synthesize iron chelators - compounds capable of binding iron - called siderophores. Many siderophores are excreted by the bacterium into the environment, bind free iron, and then re-enter the cell and release the iron. Other siderophores are found on the cell wall where they bind iron and transport it into the bacterium.
Meanwhile, the body produces iron chelators of its own (transferrin, lactoferrin, ferritin, and hemin) so the concentration of free iron is very low. The ability of bacterial iron chelators to compete successfully with the body's iron chelators as well as those of normal flora may be essential to pathogenic bacteria. In addition to their own siderophores, some bacteria:
1. Produce receptors for siderophores of other bacteria in this way take iron from other bacteria.
2. Are able to bind human transferrin, lactoferrin, ferritin, and hemin and use that as their iron source. For example, Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae are able to use iron bound to human transferrin and lactoferrin for their iron needs, while pathogenic Yersinia species are able to use transferrin and hemin as iron sources.
3. Produce proteases that degrade human lactoferrin, transferrin, or heme to release the bound iron for capture by bacterial siderophores.
4. Do not use iron as a cofactor. Borrelia burgdorferi instead uses manganese as a cofactor.
5. Are able to produce exotoxins that kill host cells only when iron concentrations are low. In this way the bacteria can gain access to the iron that was in those cells.
Staphylococcus aureus, on the other hand, produces surface adhesins that bind to extracellular matrix proteins and polysaccharides surrounding host cell tissue, including fibronectin, collagen, laminin, hyaluronic acid, and elastin. S. aureus proteases and hyaluronidase then dissolve these components of the extracellular matrix providing food for the bacteria and enabling the bacteria to spread. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.4%3A_The_Ability_to_Compete_for_Nutrients.txt |
Some bacteria are able to resist innate immune defenses such as phagocytosis and the body's complement pathways. We will break this down into two categories:
• The ability to resist phagocytic engulfment (attachment and ingestion)
• The ability to resist phagocytic destruction and complement serum lysis
5.5: The Ability to Resist Innate Immune Defenses
Learning Objectives
1. Describe the following as they relate to phagocytosis:
1. unenhanced attachment
2. enhanced attachment
3. ingestion
4. destruction
2. State 4 different body defense functions of the body's complement pathways.
3. State what is meant by antibacterial peptides and give an example.
An Overview of Phagocytosis
As will be seen in Unit 5, there are several steps involved in phagocytosis.
a. Attachment
First the surface of the microbe must be attached to the cytoplasmic membrane of the phagocyte. Attachment of microorganisms is necessary for ingestion and may be unenhanced or enhanced.
1. Unenhanced attachment is a general recognition of what are called pathogen-associated molecular patterns or PAMPs - components of common molecules such as peptidoglycan, teichoic acids, lipopolysaccharide, mannans, and glucans common in microbial cell walls but not found on human cells - by means of glycoprotein known as endocytic pattern-recognition receptors on the surface of the phagocytes (Figure \(1\)).
2. Enhanced attachment is the attachment of microbes to phagocytes by way of molecules such as an antibody molecule called IgG and two proteins produced during the complement pathways called C3b and C4b (Figure \(2\)). Molecules such as IgG, C3b, and C4b that promote enhanced attachment are called opsonins and the process is called opsonization. Enhanced attachment is much more specific and efficient than unenhanced.
b. Ingestion
Following attachment, polymerization and then depolymerization of actin filaments send pseudopods out to engulf the microbe (Figure \(3\)) and place it in a vesicle called a phagosome (Figure \(4\)).
During this process, an electron pump brings hydrogen ions (H+) into the phagosome. This lowers the pH within the phagosome so that when a lysosome fuses with the phagosome, the pH is correct for the acid hydrolases to effectively break down cellular proteins.
c. Destruction
1. Intracellular destruction: Finally, lysosomes, containing digestive enzymes and microbicidal chemicals, fuse with the phagosome containing the ingested microbe and the microbe is destroyed (Figure \(5\)).
2. Extracellular destruction: If the the infection site contains very large numbers of microorganisms and high levels of inflammatory cytokines and chemokines are being produced in response to PAMPs, the phagocyte will empty the contents of its lysosomes by a process called degranulation to kill the microorganisms or cell extracellularly.
To view a scanning electron micrograph of a macrophage with pseudopods and phagocytosis of E. coli by a macrophage on a blood vessel, see Dennis Kunkel's Microscopy, University of Hawaii-Manoa.
An Overview of the Body's Complement Pathways
Some bacteria are able to interfere with the body's complement pathways. The complement pathways will be discussed in detail later in Unit 4, but a brief summary is relevant here. There are three complement pathways: the classical complement pathway, the alternative complement pathway, and the lectin pathway. While the three pathways differ in the way they are activated, once activated they all produce the same beneficial complement proteins. Basically the complement proteins are a series of serum proteins that when activated participate in four important body defense functions. These include:
a. Inflammation
Inflammation is the means by which body defense cells and defense chemicals leave the blood and enter the tissue around an injured or infected site. Complement proteins known as C5a, C3a, and C4a lead to vasodilation, increased capillary permeability, and the expression of the adhesion molecules on leukocytes and the vascular endothelium. This enables leukocytes to adhere to the inner wall of the capillaries, pass between the endothelial cells, and enter the surrounding tissue. Vasodilation also enables a variety of defense chemicals in the plasma of the blood to enter the tissue. These defense chemicals include antibodies and complement proteins. C5a also causes neutrophils to release proteases and toxic oxygen radicals for extracellular killing of microbes.
b. Phagocyte Chemotaxis
Complement proteins C3a and C4a are chemoattractants for leukocytes. Chemotaxis enables the phagocytes to move toward the infected area in order to remove microorganisms.
c. Opsonization (Enhanced Attachment)
The complement proteins C3b and C4b are known as opsonins because they bind microbes to phagocytes (Figure \(2\)). One portion of the molecule binds to microbial proteins while the other portion binds to receptors on phagocytes. In this way, microbes can be engulfed by phagocytes more effectively.
d. MAC Lysis of Biological Membranes
A series of complement proteins known as the membrane attack complex or MAC put pores in cellular membranes resulting in lysis. This is used to kill such things as Gram-negative bacteria, virus-infected cells, and tumor cells.
These processes will be discussed in greater detail in Unit 5.
Exercise: Think-Pair-Share Questions
1. Capsules often enable bacteria to resist phagocytosis by unenhanced attachment. Based on what we just learned, explain how.
2. Some bacteria are able to inhibits the C3 convertase enzyme, the enzyme that splits complement protein C3 into C3a and C3b. Explain how this might make it harder for that bacterium to be phagocytosed.
Antibacterial Peptides
The body produces a number of antibacterial peptides such as human defensins and cathelicidins that are directly toxic by forming pores in the cytoplasmic membrane of a variety of microorganisms causing leakage of cellular needs. They also activate cells for an inflammatory response. Defensins are produced by leukocytes, epithelial cells, and other cells. They are also found in blood plasma and mucus.
Some bacteria are able to resist phagocytosis and interfere with the body's complement pathways. In the next two sections we will look at the following virulence factors:
1. The ability to resist phagocytic engulfment (attachment and ingestion)
2. The ability to resist phagocytic destruction and serum lysis
Summary
1. For phagocytosis to occur, the surface of the microbe must be attached to the cytoplasmic membrane of the phagocyte through unenhanced or enhanced attachment.
2. Following attachment, the microbe must be engulfed and placed on a membrane-bound vesicle called a phagosome. The phagosome then becomes acidified to provide the correct pH for killing by lysosomal enzymes.
3. Lysosomes, containing digestive enzymes and microbicidal chemicals, fuse with the phagosome containing the ingested microbe and the microbe is destroyed. This is referred to as intracellular killing by phagocytes and happens when microbial numbers are relatively low.
4. If the the infection site contains very large numbers of microorganisms and high levels of inflammatory cytokines and chemokines are being produced, the phagocyte will empty the contents of its lysosomes by a process called degranulation in order to kill the microorganisms extracellularly. This is referred to as extracellular killing.
5. The body’s complement pathways consist of a variety of complement proteins that when activated participate in four important body defense functions: promoting inflammation, phagocyte chemotaxis, opsonization (enhanced attachment), and lysis of membrane-bound cells.
6. The body produces a number of antibacterial peptides that are directly toxic by forming pores in the cytoplasmic membrane of a variety of microorganisms causing leakage of cellular needs. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.5%3A_The_Ability_to_Resist_Innate_Immune_Defenses/5.5A%3A_An_Overview_to_Resisting_Innate_Immune_Defense.txt |
Learning Objectives
1. Briefly describe at least 3 ways capsules may enable bacteria to resist phagocytic engulfment and state how this can promote colonization.
2. State at least 2 mechanisms other than capsules that certain bacteria might use to resist phagocytic engulfment.
3. State 3 ways bacteria might resist antibacterial peptides like defensins.
Highlighted Bacterium
1. Read the description of Haemophilus influenzae and match the bacterium with the description of the organism and the infection it causes.
We will now look virulence factors that enable bacteria to resist phagocytic engulfment (attachment and ingestion) and antibacterial peptides. As we learned in Unit 1, capsule enable many organisms to resist phagocytic engulfment. For example, Streptococcus pneumoniae is able to initially evade phagocytosis and cause infections such as pneumococcal pneumonia, sinusitis, otitis media, and meningitis because of its capsule. Encapsulated strains of Haemophilus influenzae type b can causes severe respiratory infections, septicemia, epiglottitis, and meningitis in children (other non-encapsulated strains of H. influenzae usually cause mild respiratory infections such as sinusitis and otitis media). Other encapsulated bacteria include Neisseria meningitidis, Bacillus anthracis, and Bordetella pertussis.
Click on this link, read the description of Haemophilus influenzae, and be able to match the bacterium with its description on an exam.
Capsules that resist Unenhanced Attachment
Capsules can resist unenhanced attachment by preventing pathogen-associated molecular patterns or PAMPs - components of common molecules such as peptidoglycan, teichoic acids, lipopolysaccharide, mannans, and glucans common in microbial cell walls but not found on human cells - from binding to endocytic pattern-recognition receptors on the surface of the phagocytes (Figure \(1\)).
Capsules that Interfere with Complement Pathways
The capsules of some bacteria interfere with the host's complement pathways and do so in a number of ways:
The capsules of some bacteria prevent the formation of C3 convertase, an early enzyme in the complement pathways. Without this enzyme, the opsonins C3b and C4b involved in enhanced attachment, as well as the other beneficial complement proteins like C5a, are not produced.
Some capsules are rich in sialic acid, a common component of host cell glycoprotein. Sialic acid has an affinity for serum protein H, a complement regulatory protein that leads to the degradation of the opsonin C3b and the formation of C3 convertase. (Our body uses serum protein H to degrade any C3b that binds to host cell glycoproteins so that we don't stick our own phagocytes to our own cells with C3b.) Some Neisseria meningitidis strains synthesize their own sialic acid capsule. While Neisseria gonorrhoeae and Hemophilus influenzae type b do not have a sialic acid capsule, they are able to scavenge sialic acid from host cells and enzymatically transfer it to their surface where it subsequently binds protein H.
Some capsules simply cover the C3b that does bind to the bacterial surface and prevent the C3b receptor on phagocytes from making contact with the C3b (Figure \(2\)). This is seen with the capsule of Streptococcus pneumoniae.
Staphylococcus aureus produces a protein called Staphylococcal complement inhibitor that binds and inhibits the C3 convertase enzyme needed for all three complement pathways.
The body's immune defenses, however, can eventually get around these capsule by producing opsonizing antibodies (IgG) that stick capsules to the phagocyte. In vaccines against pneumococccal pneumonia and Haemophilus influenzae type b, it is capsular polysaccharide that is given as the antigen to stimulate the body to make opsonizing antibodies against the encapsulated bacterium.
Biofilms
Many pathogenic bacteria, as well as normal flora, form complex bacterial communities as biofilms. Bacteria in biofilms are often able to communicate with one another by a process called quorum sensing and are able to interact with and adapt to their environment as a population of bacteria rather than as individual bacteria. By living as a community of bacteria as a biofilm, these bacteria are better able to:
• resist attack by antibiotics;
• trap nutrients for bacterial growth and remain in a favorable niche;
• adhere to environmental surfaces and resist flushing;
• live in close association and communicate with other bacteria in the biofilm; and
• resist phagocytosis and attack by the body's complement pathways.
Biofilms are, therefore, functional, interacting, and growing bacterial communities. Biofilms even contain their own water channels for delivering water and nutrients throughout the biofilm community. For example, Pseudomonas aeruginosa produces a glycocalyx composed of alginate. This enables strains producing the glycocalyx to block neutrophil chemotaxis, scavenge the hypochlorite molecules produced by neutrophils to kill bacteria, decrease phagocytosis, and inhibit activation of the complement pathways.
Other Mechanisms
The M-protein of Streptococcus pyogenes allows these bacteria to be more resistant to phagocytic engulfment. The M-protein of S. pyogenes binds factor H, a complement regulatory protein that leads to the degradation of the opsonin C3b and the formation of C3 convertase. (Our body uses serum protein H to degrade any C3b that binds to host cell glycoproteins so that we don't stick our own phagocytes to our own cells with C3b.) S. pyogenes also produces a protease that cleaves the complement protein C5a.
Coagulase, produced by Staphylococcus aureus. Coagulase causes fibrin clots to form around the organism that help enable it to resist phagocytosis. Our adaptive immune system has difficulty in recognizing the S. aureus as foreign when it is coated with a human protein.
Pathogenic Yersinia, such as the species that causes plague, Y. pestis, contact phagocytes and, by means of a type III secretion system (Figure \(3\)), deliver proteins that depolymerize the actin microfilaments needed for phagocytic engulfment into the phagocytes (Figure \(4\)).
Blocking Phagosome Formation by Depolymerizing Actin. Molecules of some bacteria, through a type III secretion system, deliver proteins that depolymerize the phagocyte's actin microfilaments used for phagocytic engulfment.
The pili (fimbriae) of Streptococcus pyogenes both blocks the activation of the complement pathways on the bacterial cell wall and helps to resist phagocytic engulfment.
Exercise: Think-Pair-Share Questions
The vaccine for Haemophilus influenzae type b contains capsular material from this bacterium. The body recognizes this capsular material as foreign and produces antibodies against it. Describe how this might this protect the person from infection with this bacterium compared to a person who is not immunized.
Certain bacteria can resist antibacterial peptides
Human defensins are short cationic peptides 29-34 amino acids long that are directly toxic by forming pores in the cytoplasmic membrane of a variety of microorganisms causing leakage of cellular needs. They also activate cells for an inflammatory response. Defensins are produced by leukocytes, epithelial cells, and other cells. They are also found in blood plasma and mucus. Cathelicidinsare proteins produced by skin and mucosal epithelial cells. The two peptides produced upon cleavage of the cathelicidin are directly toxic to a variety of microorganisms. One pepitide also can bind to and neutralize LPS from Gram-negative cell walls to reduce inflammation.
1. Capsules help prevent antibacterial peptides from reaching the cytoplasmic membrane of some bacteria.
2. The lipopolysaccharide (LPS) of the gram-negative cell wall binds cationic antibacterial peptides and prevents them from reaching the cytoplasmic membrane.
3. Some bacteria secrete peptidases that break down antibacterial peptides.
Summary
1. Capsules can resist unenhanced attachment by by preventing pathogen-associated molecular patterns or from binding to endocytic pattern-recognition receptors on the surface of the phagocytes.
2. The capsules of some bacteria interfere with the body's complement pathway defenses.
3. The body's immune defenses can eventually get around the capsule by producing opsonizing antibodies (IgG) against the capsule that stick the capsule to the phagocyte. This is the principle behind some vaccines.
4. Biofilms enable bacteria to: resist attack by antibiotics; trap nutrients for bacterial growth and remain in a favorable niche; adhere to environmental surfaces and resist flushing; live in close association and communicate with other bacteria in the biofilm; and resist phagocytosis and attack by the body's complement pathways.
5. Certain bacteria can resist antibacterial peptides.
5.5C: The Ability to Resist Phagocytic Destruction
State at least 4 different ways bacteria might be able to resist phagocytic destruction once engulfed.
Legionella pneumophila, after being ingested by macrophages and placed in a phagosome, uses a type 4 secretion system to inject effector proteins that prevent the lysosomes from fusing with the phagosomes and turning the macrophage into a safe haven for bacterial replication. Neisseria gonorrhoeae produces Por protein (protein I) that prevents phagosomes from fusing with lysosomes enabling the bacteria to survive inside phagocytes.
Cell wall lipids of Mycobacterium tuberculosis, such as lipoarabinomannan, arrest the maturation of phagosomes preventing delivery of the bacteria to lysosomes. Some bacteria, such as species of Salmonella, Mycobacterium tuberculosis, Legionella pneumophila, and Chlamydia trachomatis, block the vesicular transport machinery that enables the lysosome to move to the phagosome for fusion.
Preventing Acidification of the Phagosome
Some bacteria, such as pathogenic Mycobacterium and Legionella pneumophilia, prevent the acidification of the phagosome that is needed for effective killing of microbes by lysosomal enzymes. (Normally after the phagosome forms, the contents become acidified because the lysosomal enzymes used for killing (acid hydrolases) function much more effectively at an acidic pH.)
Resisting killing by Lysosomal Chemicals
Some bacteria, such as Salmonella, are more resistant to toxic forms of oxygen and to defensins, the toxic peptides that kill bacteria by damaging their cytoplasmic membranes. The carotenoid pigments that give Staphylococcus aureus species its golden color and group B streptococci (GBS) its orange tint shield the bacteria from the toxic oxidants that neutrophils use to kill bacteria.
Resisting phagocytic destruction: killing the phagocyte
Some bacteria are able to kill phagocytes. Bacteria such as Staphylococcus aureus and Streptococcus pyogenes produce the exotoxin leukocidin that damages either the cytoplasmic membrane of the phagocyte or the membranes of the lysosomes, resulting in the phagocyte being killed by its own enzymes. Shigella and Salmonella, induce macrophage apoptosis, a programmed cell death.
Summary
1. Some bacteria resist phagocytic destruction by preventing fusion of the lysosome with the phagosome.
2. Some bacteria resist phagocytic destruction by escaping from the phagosome before the lysosome fuses.
3. Some bacteria resist phagocytic destruction by preventing acidification of the phagosome.
4. Some bacteria resist phagocytic destruction by resisting killing by lysosomal chemicals.
5. Some bacteria resist phagocytic destruction by killing phagocytes. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.5%3A_The_Ability_to_Resist_Innate_Immune_Defenses/5.5B%3A_The_Ability_to_Resist_Phagocytic_Engulfment_%2.txt |
Learning Objectives
1. State four ways the antibody molecules made during adaptive immunity protect us against bacteria.
2. Briefly describe at least three ways a bacterium might evade our adaptive immune defenses and name a bacterium that does each.
Overview of Adaptive Immune Defenses
One of the major defenses against bacteria is the immune defenses' production of antibody molecules against the organism. The "tips" of the antibody, called the Fab portion (Figure \(1\)) have shapes that are complementary to portions of bacterial proteins and polysaccharides called epitopes. The "bottom" of the antibody, called the Fc portion (Figure \(1\)) binds to receptors on phagocytes and NK cells) and can activate the classical complement pathway.
There are various ways that the antibodies the body makes during adaptive immunity protect the body against bacteria:
a. As mentioned above under phagocytosis, some antibodies such as IgG and IgE function as opsonins and stick bacteria to phagocytes (Figure \(2\)).
b. Antibodies, such as IgG, IgA, and IgM, can bind to bacterial adhesins, pili, and capsules and in this way block their attachment to host cells.
c. IgG and IgM can also activate the classical complement pathway providing all of its associated benefits.
d. IgA and IgM can clump bacteria together enabling them to be more readily removed by phagocytes (Figure \(3\)).
These mechanisms will be discussed in greater detail in Unit 6.
Exercise: Think-Pair-Share Questions
1. Staphylococcus aureus produces protein A, a protein that binds to the Fc portion of antibodies.
How might this enable S. aureus to resist adaptive immunity?
1. Many bacteria that colonize the mucous membranes produce immunoglobulin protease, an enzyme that hydrolizes antibodies of the IgA class.
How might this enable these bacteria to resist adaptive immunity?
Resisting Adaptive Immune Defenses
Bacteria utilize a variety of mechanisms to resist antibodies made during adaptive immunity. These include the following:
a. Certain bacteria can evade antibodies is by changing the adhesive tips of their pili as mentioned above with Escherichia coli and Neisseria gonorrhoeae (Figure \(4\)).
Bacteria can also vary other surface proteins so that antibodies previously made against those proteins will no longer "fit." (Figure \(5\)). For example, N. gonorrhoeae produces Rmp protein (protein III) that protects against antibody attack by antibodies made against other surface proteins (such as adhesins) and the lipooligosaccharide (LOS) of the bacterium.
b. Strains of Neisseria meningitidis have a capsule composed of sialic acid while strains of Streptococcus pyogenes (group A beta streptococci) have a capsule made of hyaluronic acid. Both of these polysaccharides closely resemble carbohydrates found in human tissue and because they are not recognized as foreign by the lymphocytes that carry out the adaptive immune responses, antibodies are not made against those capsules. Likewise, some bacteria are able to coat themselves with host proteins such as fibronectin, lactoferrin, or transferrin and in this way avoid having antibodies being made against them because they are unable to be recognized as foreign by lymphocytes.
c. Staphylococcus aureus produces protein A while Streptococcus pyogenes produces protein G. Both of these proteins bind to the Fc portion of the antibody IgG, the portion that is supposed to bind the bacterium to phagocytes during enhanced attachment (Figure \(1\)). The bacteria become coated with antibodies in a way that does not result in opsonization (Figure \(6\)).
d. Salmonella species can undergo phase variation of their capsular (K) and flagellar (H) antigens, that is, they can change the molecular shape of their capsular and flagellar antigens so that antibodies made against the previous form no longer fit the new form (Figure \(5\)).
e. Bacteria such as Haemophilus influenzae, Streptococcus pneumoniae, Helicobacter pylori, Shigella flexneri, Neisseria meningitidis, Neisseria gonorrhoeae and enteropathogenic E. coli produce immunoglobulin proteases. Immunoglobulin proteases degrade the body's protective antibodies (immunoglobulins) that are found in body secretions, a class of antibodies known as IgA.
f. Many pathogenic bacteria, as well as normal flora, form complex bacterial communities as biofilms. Bacteria in biofilms are often able to communicate with one another by a process called quorum sensing (discussed later in this unit) and are able to interact with and adapt to their environment as a population of bacteria rather than as individual bacteria. By living as a community of bacteria as a biofilm, these bacteria are better able to resist attack by antibiotics and are better able to resist the host immune system.
Summary
1. There are various ways that the antibodies the body makes during adaptive immunity protect the body against bacteria.
2. Some antibodies such as IgG and IgE function as opsonins and stick bacteria to phagocytes (opsonization or enhanced attachment).
3. Antibodies, such as IgG, IgA, and IgM, can bind to bacterial adhesins, pili, and capsules and in this way block their attachment to host cells.
4. IgG and IgM can activate the classical complement pathway providing all of its associated benefits.
5. IgA and IgM can clump bacteria together enabling them to be more readily removed by phagocytes.
6. Antitoxin antibodies, mainly IgG, are made against bacterial exotoxins. They combine with the exotoxin molecules before they can interact with host target cells and thus neutralize the toxin.
7. Bacteria utilize a variety of mechanisms to resist antibodies made during adaptive immunity.
8. Some bacteria can vary their surface proteins or polysaccharides so that antibodies previously made against those proteins will no longer "fit."
9. Some bacteria are able to coat themselves with host proteins and in this way avoid having antibodies being made against them because they are unable to be recognized as foreign
10. Some bacteria produce immunoglobulin proteases that degrade the body's protective antibodies (immunoglobulins) that are found in body secretions. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.6%3A_The_Ability_to_Evade_Adaptive_Immune_Defenses.txt |
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
5.0: virulence factors that promote bacterial colonization of the host
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. List 6 virulence factors that promote bacterial colonization of the host.
5.1: The Ability to Use Motility and Other Means to Contact Host Cells
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State why it might be of an advantage for a bacterium trying to colonize the bladder or the intestines to be motile. (ans)
2. Briefly describe how the spirochete Treponema pallidum that causes syphilis uses its motility to disseminate from the initial infection site to other parts of the body. (ans)
3. Give a brief description of how a bacterium may use toxins to better disseminate from one host to another. (ans)
4. Multiple Choice (ans)
5.2: The Ability to Adhere to Host Cells and Resist Physical Removal
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Briefly describe 3 different mechanisms by which bacteria can adhere to host cells and colonize. Name 2 bacteria that utilize each mechanism and name an infection that each bacterium causes.
2. Define biofilm and state 5 benefits associated with bacteria living as a community within a biofilm. (ans)
3. By activating different genes, Neisseria gonorrhoeae is able to rapidly alter the amino acid sequence of the adhesive tip of its pili. Why might this be an advantage? (ans)
4. Multiple Choice (ans)
5.3: The Ability to Invade Host Cells
Questions
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1. Briefly describe a mechanism by which invasins enable certain bacteria to enter host cells. (ans)
2. Briefly describe how a type 3 secretion system might be used to invade and survive inside host cells. (ans)
3. Multiple Choice (ans)
5.4: The Ability to Compete for Nutrients
Questions
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1. State why the ability to compete for iron is important for bacteria to cause disease. (ans)
2. Multiple Choice (ans)
5.5: The Ability to Resist Innate Immune Defenses
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Describe unenhanced attachment as it relates to phagocytosis. (ans)
2. Describe enhanced attachment as it relates to phagocytosis. (ans)
3. Describe ingestion as it relates to phagocytosis. (ans)
4. Describe destruction as it relates to phagocytosis. (ans)
5. State 4 different body defense functions of the body's complement pathways. (ans)
6. Multiple Choice (ans)
5.5B
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Briefly describe 3 ways capsules may enable bacteria to resist phagocytic engulfment. (ans)
2. State 2 mechanisms other than capsules that certain bacteria might use to resist phagocytic engulfment. (ans)
3. The vaccine for Haemophilus influenzae type b contains capsular material from this bacterium. The body recognizes this capsular material as foreign and produces antibodies against it. One part of the antibody is able to bind to the capsular material while another part has a shape that fits a receptor on phagocytic cells. Why might this protect the person from infection with this bacterium? (ans)
4. Multiple Choice (ans)
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State 4 different ways bacteria might be able to resist phagocytic destruction once engulfed. (ans)
5.6: The Ability to Evade Adaptive Immune Defenses
Study the material in this section and then write out the answers to these question. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State 4 four ways the antibody molecules made during adaptive immunity protect us against bacteria. (ans)
2. Briefly describe 3 ways a bacterium might evade our immune defenses and name a bacterium that does each.
3. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/5%3A_Virulence_Factors_that_Promote_Colonization/5.E%3A_Virulence_Factors_that_Promote_Colonization_%28Exercises%29.txt |
Virulence factors that damage the host include: (1) the ability of PAMPs to trigger the production of inflammatory cytokines that result in an excessive inflammatory response; (2) the ability to produce harmful exotoxins; (3) and the ability to induce autoimmune responses. Most of the virulence factors we will discuss in this section that enable bacteria to harm the body are the products of quorum sensing genes. We will now look at each of these factors in greater detail.
6: Virulence Factors that Damage the Host
Define exotoxin and list three types of exotoxins. State the major way the body defends itself against exotoxins.
Exotoxins (def) are toxins, often proteins in nature, secreted from a living bacterium but also released upon bacterial lysis. In addition, some bacteria use various secretion systems such as the type 3 secretion system to inject toxins directly into human cells. (As learned earlier, the lipopolysaccharide or LPS portion of the Gram-negative bacterial cell wall is known as endotoxin (def), a PAMP that can initiate an excessive inflammatory response in the host. It was originally called endotoxin because it was located within the Gram-negative cell wall as opposed to being secreted from bacteria as in the case of exotoxins.)
Not all exotoxins are necessarily produced to harm humans. Some may be designed to play a role in bacterial physiology, such as resisting bacteriophages, regulating cellular function, or quorum sensing. Other toxins may be produced primarily to target protozoa, insects, and smaller animals and harming human cells becomes an accidental side effect.
There are three main types of exotoxins:
1. superantigens (Type I toxins);
2. exotoxins that damage host cell membranes (Type II toxins); and
3. A-B toxins and other toxin that interfere with host cell function (Type III toxins).
The body's major defense against exotoxins is the production of antitoxin antibodies. Once the antibody binds to the exotoxin, the toxin can no longer bind to the receptors on the host cell membrane.
We will now look at each of these three types of exotoxins.
Summary
1. Exotoxins are toxins, often protein in nature, secreted from a living bacterium.
2. Some bacteria use various secretion systems to inject toxins directly into human cells.
3. There are three main types of exotoxins: superantigens (type I toxins); exotoxins that damage host cell membranes (type II toxins); and A-B toxins and other toxin that interfere with host cell function (type III toxins).
4. The body's major defense against exotoxins is the production of antitoxin antibodies. Once the antibody binds to the exotoxin, the toxin can no longer bind to the receptors on the host cell membrane.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. List three types of exotoxins.
1. (ans)
2. (ans)
3. (ans)
2. Define exotoxin. (ans)
3. The body's major defense against exotoxins is _______________________________________________. (ans)
6.2: The Ability to Produce Harmful Exotoxins: An Overview
Learning Objectives
1. Define superantigen.
2. Briefly describe the mechanism by which superantigens cause harm to the body.
3. Name 2 superantigens and give an example of a bacterium that produces each.
Highlighted Bacterium
1. Read the description of Streptococcus pyogenes and match the bacterium with the description of the organism and the infection it causes.
Superantigens are unusual bacterial toxins that interact with exceedingly large numbers of T4-lymphocytes. They bind to the surface of the target cell but do not enter the cell.
Conventional antigens are engulfed by antigen presenting cells (APCs), degraded into epitopes, bind to the peptide groove of MHC-II molecules, and are put on the surface of the APC (Figure \(1\)). Here they are recognized by specific T4-lymphocytes having a TCR with a corresponding shape (Figure \(2\)).
Superantigens, however, bind directly to the outside of MHC-II molecules and activate large numbers of T4-lymphocytes (Figure \(3\)). This activation of very large numbers of T4-lymphocytes results in the secretion of excessive amounts of a cytokine called interleukin-2 (IL-2) as well as the activation of self-reactive T-lymphocytes. The normal response to a conventional antigen results in the activation of maybe 1 in 10,000 T-lymphocytes; superantigens can activate as many as 1 in 5 T-lymphocytes.
Production of high levels of IL-2 can result in circulation of IL-2 in the blood leading to symptoms such as fever, nausea, vomiting, diarrhea, and malaise. However, excess stimulation of IL-2 secretion can also lead to production of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), inflammatory chemokines such as IL-8, and platelet-activating factor (PAF), and can lead to the same endothelial damage, acute respiratory distress syndrome, disseminated intravascular coagulation, shock, and multiple organ system failure seen above with LPS and other bacterial cell wall factors. Activation of self-reactive T-lymphocytes can also lead to autoimmune attack.
The following are examples of superantigens.
1. Toxic shock syndrome toxin-1 (TSST-1), produced by some strains of Staphylococcus aureus. This exotoxin causes toxic shock syndrome (TSS). Excessive cytokine production leads to fever, rash, and shock.
2. Streptococcal pyrogenic exotoxin (Spe), produced by rare invasive strains and scarlet fever strains of Streptococcus pyogenes (the group A beta streptococci). S pyogenes produces a number of SPEs that are cytotoxic, pyrogenic, enhance the lethal effects of endotoxins, and contribute to cytokine-induced inflammatory damage. SPEs are responsible for causing streptococcal toxic shock syndrome (STSS) whereby excessive cytokine production leads to fever, rash, and triggering the shock cascade. The SPEs also appear to be responsible for inducing necrotizing fasciitis, a disease that can destroy the skin, fat, and tissue covering the muscle (the fascia). SPE B is also a precursor for a cysteine protease that can destroy muscles tissue.
Read the description of Streptococcus pyogenes, and be able to match the bacterium with its description on an exam.
1. Staphylococcal enterotoxins (SE), producedby many strains of Staphylococcus aureus. These exotoxins cause staphylococcal food poisoning. Excessive Il-2 production results in fever, nausea, vomiting,and diarrhea. The vomiting may also be due to these toxins stimulating the vagus nerve in the stomach lining that controls vomiting.
2. ETEC enterotoxin, produced by enterotoxogenic E. coli (ETEC), one of the most common causes of traveler's diarrhea.
Exercise: Think-Pair-Share Questions
What is the mechanism by which superantigens ultimately lead to SIRS?
Summary
1. Conventional antigens are only recognized by specific T4-cells having a TCR with a corresponding shape.
2. Superantigens are unusual bacterial toxins that interact with exceedingly large numbers of T4-lymphocytes.
3. Activation of very large numbers of T4-lymphocytes results in the secretion of excessive amounts of a cytokine called interleukin-2 (IL-2).
4. Excess stimulation of IL-2 secretion can also lead to production of inflammatory and can lead to the same endothelial damage, acute respiratory distress syndrome, disseminated intravascular coagulation, shock, and multiple organ system failure seen with PAMP-induced inflammation.
5. Examples of superantigens include toxic shock syndrome toxin-1 (TSST-1), Streptococcal pyrogenic exotoxins (SPE), Staphylococcal enterotoxins (SE), and enterotoxogenic E. coli (ETEC) enterotoxin.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Define superantigen (ans).
2. Briefly describe the mechanism by which superantigens cause harm to the body. (ans)
3. Name 2 superantigens and give an example of a bacterium that produces each.
1. (ans)
2. (ans)
4. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/6%3A_Virulence_Factors_that_Damage_the_Host/6.2%3A_The_Ability_to_Produce_Harmful_Exotoxins%3A_An_Overview/6.2A%3A_Type_I_Toxins%3A_Superantigens.txt |
Briefly describe the roles of alpha toxin, kappa toxin, and mu toxin, and fermentation by Clostridium perfringens in the pathogenesis of gas gangrene. State how the following toxins cause harm and name a bacterium producing each: leukotoxins such as leukocydin Bordetella tracheal cytotoxin State how Toxin A and Toxin B of Clostridium difficile lead to diarrhea and damage to the colon. Highlighted Bacterium Read the description of Clostridium difficile andmatch the bacterium with the description of the organism and the infection it causes.
In this section on Bacterial Pathogenesis we are looking at virulence factors that damage the host. Virulence factors that damage the host include:
1. The ability to produce Pathogen-Associated Molecular Patterns or PAMPs that bind to host cells causing them to synthesize and secrete inflammatory cytokines and chemokines;
2. The ability to produce harmful exotoxins.
3. The ability to induce autoimmune responses.
We are currently looking at the ability of bacteria to produce harmful exotoxins.
Exotoxins (def) are toxins, often proteins in nature, secreted from a living bacterium but also released upon bacterial lysis. In addition, some bacteria use a type 3 secretion system or a type 4 secretion system to inject toxins directly into human cells. There are three main types of exotoxins:
1. superantigens (Type I toxins),
2. exotoxins that damage host cell membranes (Type II toxins)
3. A-B toxins and other toxin that interfere with host cell function (Type II I toxins).
We will now look at exotoxins that damage host cell membranes.
The Ability to Produce Harmful Exotoxins
b. Type II Toxins: Toxins that Damage Host Cell Membranes
Type II toxins are typically phospholipases or pore-forming cytotoxins that disrupt the integrity of eukaryotic cell membranes. Damages host cells release danger-associated molecular patterns (DAMPs) (def) that bind to pattern-recognition receptors (PRRs) causing the release of inflammatory cytokines. This inflammatory response can also further contribute to tissue damage.
1. The exotoxins of Clostridium perfringens (inf). This bacterium produces at least 20 exotoxins that play a role in the pathogenesis of gas gangrene and producing expanding zones of dead tissue (necrosis) surrounding the bacteria. Toxins include:
• alpha toxin (lecithinase): increases the permeability of capillaries and muscle cells by breaking down lecithin in cytoplasmic membranes. This results in the gross edema (def) of gas gangrene. If the alpha toxin enters the blood it can damage organs. Alpha toxin is also necrotizing (def), hemolytic, and cardiotoxic.
• kappa toxin (collagenase): breaks down supportive connective tissue (def) resulting in the mushy lesions of gas gangrene. It is also necrotizing (def).
• mu toxin (hyaluronidase): breaks down the tissue cement that holds cells together in tissue.
• epsilon toxin: Increases vascular permeability and causes edema and congestion in various organs including lungs and kidneys.
• Additional necrotizing toxins (def) include beta toxin, iota toxin, and nu toxin.
A major characteristic of gas gangrene is the ability of C. perfringens to very rapidly spread from the initial wound site leaving behind an expanding zone of dead tissue. This organism spreads as a result of the pressure from fluid accumulation (due to increased capillary permeability from alpha toxin) and gas production (anaerobic fermentation of glucose by the organisms produces hydrogen and carbon dioxide), coupled with the breakdown of surrounding connective tissue (kappa toxin) and tissue cement (mu toxin).
2. Leukotoxins. Leukotoxins, such as leukocidin, are pore-forming toxins that cause lysis of white blood cells and other cells involved in immunity by binding to chemokine receptors on these cells and damaging the cell membrane. Leukotoxins is produced by various pyogenic (def) bacteria including Staphylococcus aureus (inf) and Streptococcus pyogenes (inf), (group A beta streptococci).
3. Pseudomonas aeruginosa produces a variety of toxins that lead to cell lysis and tissue damage in the host. Type II toxins include:
• Exotoxin U (Exo U): Degrades the plasma membrane of eukaryotic cells, leading to lysis.
• Phospholipase C (PLC): Damages cellular phospholipids causing tissue damage; stimulates inflammation. Delivered by a type 3 secretion system.
• Alkaline protease: leads to tissue damage.
• Cytotoxin: Damages cell membranes of leukocytes causes microvascular damage.
• Elastase: Destroys elastin, a protein that is a component of lung tissue.
• Pyocyanin: a green to blue water-soluble pigment that catalyzes the formation of tissue-damaging toxic oxygen radicles (def); impairs ciliary function, stimulates inflammation.
You Tube animation showing Pseudomonas using motility, pili, and exotoxins to cause an infection.
3D Medical Animations Library and Downloads, www.rufusrajadurai. wetpaint.com
4. Toxin A and Toxin B, produced by Clostridium difficile (inf). Toxin A damages the membranes of intestinal mucosal cells causing hypersecretion of fluids. In addition, it triggers the production of inflammatory cytokines. Finally, it also attracts and destroys neutrophils, causing them to release their lysosomal enzymes for further tissue damage leading to hemorrhagic necrosis (def). Toxin B depolymerizes actin damaging mucosal cells cytoskeleton. Clostridium difficile causes severe antibiotic-associated colitis and is an opportunistic Gram-positive, endospore-producing bacillus transmitted by the fecal-oral route. C. difficile is a common health care-associated infection (HAIs) and is the most frequent cause of health-care-associated diarrhea.
5. Streptococcus pyogenes (inf) produces a number of enzymes and toxins that damage cells and tissues and causes inflammation:
• Streptolysin S : Causes lysis of red blood cell membranes.
• Streptolysin O: Lytic to cells that contain cholesterol in their plasma membrane.
• Proteases: Degrade cellular proteins;helps organism spread.
• DNases: Degrade cellular DNA; helps organism spread.
• Streptokinase: Breaks down fibrin in clots; helps organism spread.
• Streptococcal pyrogenic exotoxin B (SPE B): A protease that facilitates bacterial spreading and survival; induces inflammation during S. pyogenes infections.
For More Information: Inflammation from Unit 5
6. Urease and phospholipase, produced by Helicobacter pylori (inf). Urease contributes to acid resistance and epithelial cell damage while phospholipase damages the membrane of gastric or intestinal mucosal cells.
Flash animation showing induction of stomach and intestinal ulcers by Helicobacter pylori.
html5 version of animation for iPad showing induction of stomach and intestinal ulcers by Helicobacter pylori.
YouTube movie of a video endoscopy exam showing duodenal ulcers caused by Helicobacter pylori.
7. Bordetella tracheal cytotoxin, produced by Bordetella pertussis (inf),causes the respiratory cell damage during whooping cough. Cell death, inhibition of ciliary movement by ciliated epithelial cells, and release of the inflammatory cytokine IL-1 triggers the violent coughing episodes, the only way the body can now remove inflammatory debris, bacteria, and mucus.
As mentioned earlier in this unit, many bacteria are able to sense their own population density, communicate with each other by way of secreted chemical factors, and behave as a population rather than as individual bacteria . This is referred to as cell-to-cell signaling or quorum sensing and plays an important role in pathogenicity and survival for many bacteria.
Quorum sensing involves the production, release, and community-wide sensing of molecules called autoinducers that modulate gene expression in response to the density of a bacterial population. When autoinducers produced by one bacterium cross the membrane of another, they bind to receptors in the cytoplasm. This autoinducer/receptor complex is then able to bind to DNA promoters and activate the transcription of quorum sensing-controlled genes. In this way, individual bacteria within a group are able to benefit from the activity of the entire group.
The outcomes of bacteria-host interaction are often related to bacterial population density. Bacterial virulence, that is its ability to cause disease, is largely based on the bacterium's ability to produce gene products called virulence factors that enable that bacterium to colonize the host, resist body defenses, and harm the body. If a relatively small number of a specific bacteria were to enter the body and immediately start producing their virulence factors, chances are the body's immune systems would have sufficient time to recognize and counter those virulence factors and remove the bacteria before there was sufficient quantity to cause harm. Many bacteria are able to delay production of those virulence factors by not expressing the genes for those factors until there is a sufficiently large enough population of that bacterium (a quorum). As the bacteria geometrically increase in number, so does the amount of their secreted autoinducers.
When a critical level of autoinducer is reached, the entire population of bacteria is able to simultaneously activate the transcription of their quorum-sensing genes and the body's immune systems are much less likely to have enough time to counter those virulence factors before harm is done.
Summary
1. Type II toxins are typically phospholipases or pore-forming cytotoxins that disrupt the integrity of eukaryotic cell membranes.
2. Damages host cells release danger-associated molecular patterns (DAMPs) that bind to pattern-recognition receptors (PRRs) causing the release of inflammatory cytokines. This inflammatory response can also further contribute to tissue damage.
3. Examples include the exotoxins of Clostridium perfringens that cause gas gangrene, exotoxins of Pseudomonas aeruginosa that causes a variety of opportunistic infections, exotoxins of Streptococcus pyogenes that causes strep throat, the exotoxins of Clostridium difficile that causes antibiotic-associated colitis, and leukotoxins, pore-forming toxins that causes lysis of white blood cells.
Questions
______ Causes the respiratory damage and violent coughing episodes seen during whooping cough. (ans)
______ Damages the membranes of intestinal mucosal cells causing hypersecretion of fluids; triggers the production of inflammatory cytokines; attracts and destroys neutrophils causing them to release their lysosomal enzymes for further tissue damage leading to hemorrhagic necrosis.
1. leukotoxins
2. Toxin A
3. Toxin B
4. Bordetella tracheal cytotoxin | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/6%3A_Virulence_Factors_that_Damage_the_Host/6.2%3A_The_Ability_to_Produce_Harmful_Exotoxins%3A_An_Overview/6.2B%3A_Type_II_Toxins%3A_Toxins_that_Damage_Hos.txt |
Define A-B toxins and state the functions of the A component and the B component. State how the following exotoxins cause harm and name a bacterium producing each: diphtheria exotoxin cholera exotoxin enterotoxins shiga toxin anthrax lethal toxin and edema toxin botulism exotoxin tetanus exotoxin Highlighted Bacterium Read the description of Corynebacterium diphtheriae andmatch the bacterium with the description of the organism and the infection it causes. Read the description of Bacillus anthracis andmatch the bacterium with the description of the organism and the infection it causes.
The classic type III toxins are A-B toxins that consist of two parts (see Figure \(1\)):
1. An "A" or active component that enzymatically inactivates some host cell intracellular target or signalling pathway to interfere with a host cell function; and
2. a "B" or binding component (see Figure \(2\)) that binds the exotoxin to a receptor molecule on the surface of the host cell membrane and determines the type of host cell to which the toxin is able to affect.
Once the exotoxin binds, it is translocated across the host cell membrane. Some A-B toxins enter by endocytosis (see Figure \(3\)), after which the A-component of the toxin separates from the B-component and enters the host cell's cytoplasm. Other A-B toxins bind to the host cell and the A component subsequently passes directly through the host cell's membrane and enters the cytoplasm (see Figure \(4\)).
The A components of most A-B toxins then catalyze a reaction by which they remove the ADP-ribosyl group from the coenzyme NAD and covalently attach it to some host cell protein, a process called ADP- ribosylation (see Figure \(5\)). This interferes with the normal function of that particular host cell protein that, in turn, determines the type of damage that is caused. Some A-B toxins work differently.
The body's major defense against exotoxins is the production of antitoxin antibodies. Once the antibody binds to the exotoxin, the toxin can no longer bind to the receptors on the host cell membrane.
Examples of A-B toxins include:
1. Diphtheria exotoxin, produced by Corynebacterium diphtheriae (inf). This toxin interferes with host cell protein synthesis by catalyzing the ADP-ribosylation of host cell elongation factor 2 (EF-2), necessary in order for tRNA to insert new amino acids into the growing protein chain. This results in cell death. Initially cells of the throat are killed by the toxin. The toxin is also released into the blood where it damages internal organs and can lead to organ failure. The "D" portion of the DTP vaccine contains diphtheria toxoid to stimulate the body to make neutralizing antibodies against the binding component of the diphtheria exotoxin. Once the antibody binds to the exotoxin, the toxin can no longer bind to the receptors on the host cell membrane.
, and be able to match the bacterium with its description on an exam.
1. Cholera exotoxin (choleragen), produced by Vibrio cholerae (inf). This exotoxin catalyzes the ADP-ribosylation of a host cell protein called Gs that turns the synthesis of a metabolic regulator molecule called cyclic AMP (cAMP) on and off. In this case, synthesis stays turned on. High levels of cAMP block intestinal epithelial cells from taking in sodium from the lumen of the intestines and stimulates them to secrete large quantities of chloride. Water and other electrolytes osmotically follow. This causes loss of fluids, diarrhea, and severe dehydration. For a movie of showing the effect of cholera exotoxin on human cells, see the Theriot Lab Website at Stanford University Medical School. Click on "Vibrio cholerae colonizing human cells."
2. Enterotoxins. A number of bacteria produce exotoxins that bind to the cells of the small intestines. Most of these toxins catalyze the ADP-ribosylation of host cell proteins that turn the synthesis of the metabolic regulator molecules cyclic AMP (cAMP) or cyclic GMP on and off in intestinal mucosal cells. High levels of cAMP and cGMP cause loss of electrolytes and water that results in diarrhea. Organisms producing enterotoxins include Clostridium perfringens (inf),and Bacillus cereus (inf). (As mentioned under Type I toxins, the enterotoxins of Staphylococcus aureus (inf) and enterotoxogenic E. coli (inf) work differently, functioning as superantigens.)
3. Pertussis exotoxin, produced by Bordetella pertussis (inf). The pertussis exotoxin catalyzes the ADP-ribosylation of a host cell protein called Gi leading to high intracellular levels of cAMP. This disrupts cellular function. In the respiratory epithelium, the high levels of cAMP results in increased respiratory secretions and mucous production and contribute to coughing. In the case of phagocytes, excessive cAMP decreases phagocytic activities such as chemotaxis, engulfment, killing. In the blood, the toxin results in increased sensitivity to histamine. This can result in increased capillary permeability, hypotension and shock. It may also act on neurons resulting in encephalopathy.
4. Pseudomonas aeruginosa produces a variety of toxins that lead to tissue damage in the host. Type II toxins include:
1. Exotoxin A: interferes with host cell protein synthesis by catalyzing the ADP-ribosylation of host cell elongation factor 2 (EF-2), necessary in order for tRNA to insert new amino acids into the growing protein chain; is also immunosuppressive.
2. Exotoxin S: inhibits host cell protein synthesis causing tissue damage; is immunosuppressive.
5. Shiga toxin, produced by species of Shigella (inf) and enterohemorrhagic Escherichia coli (EHEC) such as such as E. coli O157:H7. This toxin is an A-B toxin that cleaves host cell rRNA and prevents the attachment of charged tRNAs thus stopping host cell protein synthesis. The shiga toxin also enhances the LPS-mediated release of cytokines such as Il-1 and TNF-alpha and appears to be responsible for a complication of shigellosis and E. coli O157:H7 infection called hemolytic uremic syndrome (HUS), probably by causing blood vessel damage.
6. Anthrax toxins, produced by Bacillus anthracis. In the case of the two anthrax exotoxins, two different A-components known as lethal factor (LF) and edema factor (EF) share a common B-component known as protective antigen (PA). Protective antigen, the B-component, first binds to receptors on host cells and is cleaved by a protease creating a binding site for either lethal factor or edema factor.
1. Lethal factor is a protease that inhibits mitogen-activated kinase-kinase. At low levels, LF inhibits the release of proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-alpha, (TNF-alpha), and NO. This may initially reduce immune responses against the organism and its toxins. But at high levels, LF is cytolytic for macrophages, causing release of high levels of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and NO. Excessive release of these cytokines can lead to a massive inflammatory response and the shock cascade, similar to septic shock.
2. Edema factor is an adenylate cyclase that generates cyclic AMP in host cells. It impairs phagocytosis, and inhibits production of TNF and interleukin-6 (IL-6) by monocytes. This most likely impairs host defenses.
, and be able to match the bacterium with its description on an exam.
There are a number of other bacterial exotoxins that cause damage by interfering with host cell function. They include the following.
1. Botulinal exotoxin, produced by Clostridium botulinum (inf). This is a neurotoxin that acts peripherally on the autonomic nervous system. For muscle stimulation, acetylcholine must be released from the neural motor end plate of the neuron at the synapse between the neuron and the muscle to be stimulated. The acetylcholine then induces contraction of the muscle fibers. The botulism exotoxin binds to and enters the presynaptic neuron and blocks its release of acetylcholine. This causes a flaccid paralysis , a weakening of the involved muscles. Death is usually from respiratory failure. While two exotoxins of C. botulinum catalyze ADP-ribosylation of host cell proteins, the botulinal toxin that affects neurons does not. Since the botulinal toxin is able to cause a weakening of muscles, it is now being used therapeutically to treat certain neurologic disorders such as dystonia and achalasia that result in abnormal sustained muscle contractions, as well as a treatment to remove facial lines.
1. Tetanus exotoxin (tetanospasmin), produced by Clostridium tetani (inf). This is a neurotoxin that binds to inhibitory interneurons of the spinal cord and blocks their release of inhibitor molecules. It is these inhibitor molecules from the inhibitory interneurons that eventually allow contracted muscles to relax by stopping excitatory neurons from releasing the acetylcholine that is responsible for muscle contraction. The toxin, by blocking the release of inhibitors, keeps the involved muscles in a state of contraction and leads to spastic paralysis , a condition where opposing flexor and extensor muscles simultaneously contract. Death is usually from respiratory failure. The "T" portion of the DTP vaccine contains tetanus toxoid to stimulate the body to make neutralizing antibodies against the binding component of the diphtheria exotoxin. Once the antibody binds to the exotoxin, the toxin can no longer bind to the receptors on the host cell membrane.
1. Neutrophil activating protein, produced by Helicobacter pylori (inf). Neutrophil activating protein promotes the adhesion of human neutrophils to endothelial cells and the production of reactive oxygen radicals. The toxin induces a moderate inflammation that promote H. pylori growth by the release of nutrients factors from the inflamed tissue.
Summary
The classic type III toxins are A-B toxins that consist of two parts: an “A” or active component that enzymatically inactivates some host cell protein or signalling pathway to interfere with a host cell function; and a “B” or binding component that binds the exotoxin to a receptor molecule on the surface of the host cell membrane and determines the type of host cell to which the toxin is able to affect.
Examples include the diphtheria exotoxin produced by Corynebacterium diphtheria, the cholera exotoxin produced by Vibrio cholerae, certain enterotoxins that cause loss of electrolytes and water resulting in diarrhea, the pertussis exotoxin produced by Bordetella pertussis, shiga toxin, produced by species of Shigella and enterohemorrhagic Escherichia coli (EHEC), the anthrax toxins produced by Bacillus anthracis, the tetanus exotoxin of Clostridium tetani, and the botulism exotoxin of Clostridium botulinum.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. _____ Produced by certain strains of Escherichia coli such as E. coli O157:H7. These toxins kill intestinal epithelial cells of the colon and cause bloody diarrhea. Less commonly, the toxins enter the blood and are carried to the kidneys where they damage endothelial cells of the blood vessels and cause hemolytic uremic syndrome (HUS). (ans)
_____ Produced by a species of Clostridium. This is a neurotoxin that binds to inhibitory interneurons of the spinal cord and blocks their release of inhibitor molecules.The toxin, by blocking the release of inhibitors, keeps the involved muscles in a state of contraction and leads to spastic paralysis, a condition where opposing flexor and extensor muscles simultaneously contract. (ans)
_____ At low levels, this toxin inhibits the release of proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-alpha, (TNF-alpha), and NO. This may initially reduce immune responses against the organism and its toxins. But at high levels, it is cytolytic for macrophages, causing release of high levels of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and NO. Excessive release of these cytokines can lead to a massive inflammatory response and the shock cascade, similar to septic shock. (ans)
1. diphtheria exotoxin
2. cholera exotoxin
3. enterotoxins
4. pertussis exotoxin
5. shiga toxin
6. anthrax lethal toxin
7. botulism exotoxin
8. tetanus exotoxin
2. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/6%3A_Virulence_Factors_that_Damage_the_Host/6.2%3A_The_Ability_to_Produce_Harmful_Exotoxins%3A_An_Overview/6.2C%3A_Type_III_Toxins%3A_A-B_Toxins_and_other_.txt |
The Ability to Induce Autoimmune Responses
Autoimmunity (def) is when the body's immune defenses mistakenly attack the body. In certain cases, bacteria can serve as a trigger for this response.
One way bacteria can do this is by inducing the production of cross-reacting antibodies (def) and possibly auto-reactive cytotoxic T-lymphocytes or CTLs (def). These are antibodies and CTLs made in response to bacterial antigens (def) that accidently cross react with epitopes (def) on host cells. As a result, the antibodies and CTLs wind up destroying the host cells to which they have bound. Furthermore, when the antibodies activate the classical complement pathway (def), this further stimulates the inflammatory response resulting in more tissue damage. Rheumatic fever triggered by rheumatogenic strains of Streptococcus pyogenes(inf) is an example. Antibodies and CTLs stimulated by antigens of S. pyogenes cross-react with heart and joint tissues damaging the heart and joints.
Another way autoimmunity can be triggered by certain bacteria is by stimulating the production of soluble immune complexes. When high levels of circulating antibodies react with certain bacterial antigens, they form large amounts of immune complexes (antibodies bound to antigens). These immune complexes can lodge in filtering units such as the kidneys where they activate the complement pathway (def). The resulting inflammatory response then destroys kidney tissues. An example of this is acute glomerulonephritis that sometimes following infection by Streptococcus pyogenes (inf).
Two other possible examples of bacterial induced autoimmunity are chronic Lyme disease (arthritis, neurological abnormalities, and heart damage) following infection by Borrelia burgdorferi (inf), and tertiary syphilis (heart damage, neurological abnormalities, and destructive skin lesion) following infection by Treponema pallidum (inf).
the body by causing an autoimmune response.
Autoimmunity will be discussed in greater detail under Hypersensitivities in Unit 6.
Summary
1. Autoimmunity is when the body's immune defenses mistakenly attack the body and sometimes certain bacteria can serve as a trigger for this response.
2. One way bacteria can trigger autoimmunity by stimulating the production of cross-reacting antibodies. These are antibodies made in response to bacterial antigens then accidently cross-react with and destroy host cells to which they have bound. An example is rheumatic fever following Streptococcus pyogenes infection.
3. Another way autoimmunity can be triggered by certain bacteria is by stimulating the production of soluble antigen-antibody (immune) complexes. These immune complexes can lodge in filtering units such as the kidneys where they activate the complement pathway and trigger an inflammatory response then destroys kidney tissues. An example of this is acute glomerulonephritis that sometimes following infection by Streptococcus pyogenes.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State what is meant by autoimmunity. (ans)
2. Name 3 bacterial diseases that may result from autoimmunity.
1. (ans)
2. (ans)
3. (ans)
6.E: Virulence Factors that Damage the Host (Exercises)
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. List 3 general categories of virulence factors that damage the host. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_3%3A_Bacterial_Pathogenesis/6%3A_Virulence_Factors_that_Damage_the_Host/6.3%3A_The_Ability_to_Induce_Autoimmune_Responses.txt |
Thumbnail: A 3D rendering of an animal cell cut in half. (CC -BY-SA 4.0; Zaldua I., Equisoain J.J., Zabalza A., Gonzalez E.M., Marzo A., Public University of Navarre).
07: The Eukaryotic Cell
The cell is the basic unit of life. Based on the organization of their cellular structures, all living cells can be divided into two groups: prokaryotic and eukaryotic (also spelled procaryotic and eucaryotic). Animals, plants, fungi, protozoans, and algae all possess eukaryotic cell types. Only bacteria have prokaryotic cell types.
Eukaryotic cells are generally much larger and more complex than prokaryotic. The larger a cell, the smaller is its surface-to-volume ratio (the surface area of a cell compared to its volume). For example, a spherical cell 2 micrometers (µm) in diameter has a surface-to-volume ratio of approximately 3:1, while a spherical cell having a diameter of 20 µm has a surface-to-volume ratio of around 0.3:1. A large surface-to-volume ratio, as seen in smaller prokaryotic cells, means that nutrients can easily and rapidly reach any part of the cells interior. However, in the larger eukaryotic cell, the limited surface area when compared to its volume means nutrients cannot rapidly diffuse to all interior parts of the cell. That is why eukaryotic cells require a variety of specialized internal organelles to carry out metabolism, provide energy, and transport chemicals throughout the cell. Both, however, must carry out the same life processes.
Video \(1\): The Inner Life of a Cell. To view an excellent eight-minute animation on the inner workings of a cell created in NewTek LightWave 3D and Adobe After Effects for Harvard biology students, see . (https://www.youtube.com/watch?v=FzcTgrxMzZk)
We will now look at the various components and organelles found in eukaryotic cells.
7.1: The Cytoplasmic Membrane
State the chemical composition and major function of the cytoplasmic membrane in eukaryotic cells. State the net flow of water when a cell is placed in an isotonic, hypertonic, or hypotonic environment and relate this to the solute concentration. Define the following means of transport: passive diffusion osmosis active transport endocytosis phagocytosis pinocytosis exocytosis
In addition, it contains glycolipids as well as complex lipids called sterols, such as the cholesterol molecules found in animal cell membranes, that are not found in prokaryotic membranes (except for some mycoplasmas). The sterols make the membrane less permeable to most biological molecules, help to stabilize the membrane, and probably add rigidity to the membranes aiding in the ability of eukaryotic cells lacking a cell wall to resist osmotic lysis. The proteins and glycoproteins in the cytoplasmic membrane are quite diverse and function as:
1. channel proteins to form pores for the free transport of small molecules and ions across the membrane
2. carrier proteins for facilitated diffusion and active transport of molecules and ions across the membrane
3. cell recognition proteins that identifies a particular cell
4. receptor proteins that bind specific molecules such as hormones and cytokines
5. enzymatic proteins that catalyze specific chemical reactions.
All molecules and atoms possess kinetic energy (energy of motion). If the molecules or atoms are not evenly distributed on both sides of a membrane, the difference in their concentration forms a concentration gradient that represents a form of potential energy (stored energy). The net movement of these particles will therefore be down their concentration gradient - from the area of higher concentration to the area of lower concentration. Diffusion is powered by the potential energy of a concentration gradient and does not require the expenditure of metabolic energy.
A cell can find itself in one of three environments: isotonic, hypertonic, or hypotonic. (The prefixes iso-, hyper-, and hypo- refer to the solute concentration).
• In an isotonic environment (Figure \(5\)A), both the water and solute concentration are the same inside and outside the cell and water goes into and out of the cell at an equal rate.
• If the environment is hypertonic (Figure \(5\)B), the water concentration is greater inside the cell while the solute concentration is higher outside (the interior of the cell is hypotonic to the surrounding hypertonic environment). Water goes out of the cell.
• In an environment that is hypotonic (Figure \(5\)C), the water concentration is greater outside the cell and the solute concentration is higher inside (the interior of the cell is hypertonic to the hypotonic surroundings). Water goes into the cell.
Transport of Substances Across the Membrane by Transport (Carrier) Proteins
For the majority of substances a cell needs for metabolism to cross the cytoplasmic membrane, specific transport proteins (carrier proteins) are required. Transport proteins allow cells to accumulate nutrients from even a scarce environment. Examples of transport proteins include channel proteins, uniporters, symporters, antiporters, and the ATP- powered pumps. These proteins transport specific molecules, related groups of molecules, or ions across membranes through either facilitated diffusion or active transport.
Facilitated diffusion is the transport of substances across a membrane by transport proteins, such as uniporters and channel proteins, along a concentration gradient from an area of higher concentration to lower concentration. Facilitated diffusion is powered by the potential energy of a concentration gradient and does not require the expenditure of metabolic energy.
1. Uniporter: Uniporters are transport proteins that transport a substance from one side of the membrane to the other (Figure \(6\)A1 and Figure \(6\)A2). Amino acids, sugars, nucleosides, and other small molecules can be transported through eukaryotic membranes by different uniporters.
2. Channel proteins transport water or certain ions down either a concentration gradient, in the case of water, or an electric potential gradient in the case of certain ions, from an area of higher concentration to lower concentration (Figure \(6\)B). While water molecules can directly cross the membrane by passive diffusion, as mentioned above, their transport can be enhanced by channel proteins called aquaporins.
Active transport is a process whereby the cell uses both transport proteins and metabolic energy to transport substances across the membrane against the concentration gradient. In this way, active transport allows cells to accumulate needed substances even when the concentration is lower outside. The energy is provided by either proton motive force, the hydrolysis of ATP, or by the electric potential (voltage) difference across the membrane.
Proton motive force is an energy gradient resulting from hydrogen ions (protons) moving across the membrane from greater to lesser hydrogen ion concentration. ATP is the form of energy cells most commonly use to do cellular work. Electric potential is the difference in voltage across the cytoplasmic membrane as a result of ion concentration gradients and the selective movement of ions across membranes by ion pumps or through ion channels.
A Review of Proton Motive Force from Unit 6
A Review of ATP from Unit 6
Transport proteins involved in active transport include antiporters, symporters, the proteins of the ATP-powered pumps.
Antiporters are transport proteins that transport one substance across the membrane in one direction, while simultaneously transporting a second substance across the membrane in the opposite direction (Figure \(6\)C). Antiporters use the potential energy of electrochemical gradients from Na+ or H+ to transport ions, glucose, and amino acids against their concentration gradient (Figure \(6\)E1).
Symporters are transport proteins that simultaneously transport two substances across the membrane in the same direction (Figure \(6\)D). Like antiporters, symporters use the potential energy of electrochemical gradients from Na+ or H+ to transport ions, glucose, and amino acids against their concentration gradient (Figure \(6\)E2).
ATP- powered pumps couple the energy released from the hydrolysis of ATP with the transport of substances across the cytoplasmic membrane. ATP- powered pumps are used to transport ions such as Na+, Ca2+, K+, and H+ across membranes against their concentration gradient.
An example of active transport via an ATP- powered pump is the sodium-potassium pump found in animal cells. Three sodium ions from inside the cell first bind to the transport protein (Figure \(10\)A). Then a phosphate group is transferred from ATP to the transport protein causing it to change shape (Figure \(10\)B) and release the sodium ions outside the cell (Figure \(10\)C). Two potassium ions from outside the cell then bind to the transport protein (Figure \(10\)D) and as the phosphate is removed, the protein assumes its original shape and releases the potassium ions inside the cell (Figure \(10\)E).
Summary
The cytoplasmic membrane (also called the plasma or cell membrane) of eukaryotic cells is a fluid phospholipid bilayer embedded with proteins and glycoproteins. It contains glycolipids as well as complex lipids called sterols. The cytoplasmic membrane is a semipermeable membrane that determines what goes in and out of the cell. Substances may cross the cytoplasmic membrane of eukaryotic cells by simple diffusion, osmosis, passive transport, active transport, endocytosis and exocytosis. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/07%3A_The_Eukaryotic_Cell/7.0%3A_Eukaryotic_Cell_Anatomy.txt |
Summary
1. Algae, fungi, and plant cells have a cell wall; animal cells and protozoans lack cell walls.
2. The rigid, tightknit, polysaccharide molecular structure of the cell wall helps the cell resist osmotic lysis.
7.3A: The Nucleus
The endomembrane system compartmentalizes the cell for various different but interrelated cellular functions. It consists of the nucleus, the endoplasmic reticulum, and the Golgi complex.
We will now look at the various structures that make up the endomembrane system, including the nucleus, the endoplasmic reticulum, and the Golgi complex.
7.3: The Endomembrane System
Describe the structure and the function of the nucleus in eukaryotic cells. Define the following: nuclear envelope nucleolus nucleosome
Inside the nucleus is a fluid called nucleoplasm, a nucleolus (see Figure \(31\)), and linear chromosomes composed of negatively charged DNA associated with positively charged basic proteins called histones to form structures known as nucleosomes. The nucleosomes are part of what is called chromatin , the DNA and proteins that make up the chromosomes. The nucleolus is an area within the nucleus that is involved in the assembly of ribosomal subunits. An area of DNA called the nucleolar organizer directs the synthesis of ribosomal RNA (rRNA) that subsequently combines with ribosomal proteins to form immature ribosomal subunits that mature after they leave the nucleus by way of the pores in the nuclear envelope and mature in the cytoplasm. Genes located along the DNA are transcribed into RNA molecules, primarily messenger RNA (mRNA), transfer RNA (tRNA, and ribosomal RNA (rRNA). Messenger RNA is then translated into protein at the ribosomes. In general then, DNA determines what proteins and enzymes an organism can synthesize and, therefore, what chemical reactions it is able to carry out.
The DNA in eukaryotic cells is packaged in a highly organized way. It consists of a basic unit called a nucleosome , a beadlike structure 11 nm in diameter that consists of 146 base pairs of DNA wrapped around eight histone molecules. The nucleosomes are linked to one another by a segment of DNA approximately 60 base pairs long called linker DNA (see Figure \(27\)A). Another histone associated with the linker DNA then packages adjacent nucleotides together to form a nucleosome thread 30nm in diameter. Finally, these packaged nucleosome threads form large coiled loops that are held together by nonhistone scaffolding proteins. These coiled loops on the scaffolding proteins interact to form the condensed chromatin seen in chromosomes during mitosis.
When the cell is not replicating, the DNA and proteins appear as a threadlike mass called chromatin. During mitosis , the chromatin coils into thick rodlike bodies called chromosomes (see Figure \(31\)A) and a spindle apparatus guides the separation and movement of the chromosomes for cell division so each cell winds up with a full complement of chromosomes. During sexual reproduction the nuclei of sex cells divide by meiosis producing cells with half the normal number of chromosomes (one from each homologous pair).
For More Information: DNA from Unit 6
For More Information: DNA Replication from Unit 6
For More Information: Mitosis from Unit 6
Summary
1. Eukaryotic cells contain much more DNA than do bacteria, and this DNA is organized as multiple chromosomes located within a nucleus.
2. The nucleus in eukaryotic cells is separated from the cytoplasm by a nuclear envelope.
3. The nucleolus is an area within the nucleus that is involved in the assembly of ribosomal subunits.
4. Genes located along the DNA are transcribed into RNA molecules, primarily messenger RNA (mRNA), transfer RNA (tRNA, and ribosomal RNA (rRNA). Messenger RNA is then translated into protein at the ribosomes.
5. In general then, DNA determines what proteins and enzymes an organism can synthesize and, therefore, what chemical reactions it is able to carry out.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. _____ Separates the chromosomes from the cytoplasm. (ans)
_____ A basic unit of eukaryotic DNA appearing as beadlike structures consisting of DNA wrapped around histone molecules. (ans)
1. nuclear envelope
2. nucleolus
3. nucleosome
4. chromosomes | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/07%3A_The_Eukaryotic_Cell/7.2%3A_The_Cell_Wall.txt |
Briefly describe rough endoplasmic reticulum and state its functions. Briefly describe smooth endoplasmic reticulum and state its functions.
The endoplasmic reticulum or ER is a maze of parallel membranous tubules and flattened sacs surrounding the nucleus that connects with the nuclear membrane and runs throughout the cytoplasm (Figure \(33\)). The ER functions to:
1. provide a surface area for protein and lipid synthesis;
2. form a pathway for transporting molecules within the cell; and
3. provide a storage area for molecules the cell has synthesized.
The endoplasmic reticulum connects to the pores of the nuclear envelope. The pores in the nuclear membrane allow ribosomal subunits and mRNA transcribed off genes in the DNA to leave the nucleus, enter the cytoplasm, and participate in protein synthesis. There are two distinct regions of the ER: the rough ER and the smooth ER.
Summary
1. The endoplasmic reticulum or ER is a maze of parallel membranous tubules and flattened sacs surrounding the nucleus that connects with the nuclear membrane and runs throughout the cytoplasm.
2. ER with ribosomes attached is called rough endoplasmic reticulum and is involved in protein synthesis, production of new membrane, modification of newly formed proteins, and transport of these proteins and membrane to other locations within the cell.
3. ER without ribosomes is called smooth endoplasmic reticulum and contains enzymes for lipid biosynthesis, especially the synthesis of phospholipids and steroids. The smooth endoplasmic reticulum forms transition vesicles to transfer molecules produced in the rough ER to the Golgi complex.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. _____ Coated with ribosomes.(ans)
_____ Formstransition vesicles to transfer molecules produced in the rough ER to the Golgi apparatus and other parts of the cell. (ans)
1. smooth endoplasmic reticulum
2. rough endoplasmic reticulum
7.3C: The Golgi Complex
Briefly describe the Golgi complex and state its functions. Briefly describe how the Golgi complex packages materials for secretion from the cell.
The cell is the basic unit of life. Based on the organization of their cellular structures, all living cells can be divided into two groups: prokaryotic and eukaryotic (also spelled procaryotic and eucaryotic). Animals, plants, fungi, protozoans, and algae all possess eukaryotic cell types. Only bacteria have prokaryotic cell types.
Eukaryotic cells are generally much larger and more complex than prokaryotic. Because of their larger size, they require a variety of specialized internal membrane-bound organelles to carry out metabolism, provide energy, and transport chemicals throughout the cell.
Eukaryotic cells possess what is referred to as an internal membrane system or endomembrane system that compartmentalizes the cell for various different but interrelated cellular functions. Some of these internal membrane-bound organelles, such as the nucleus and the endoplasmic reticulum, have direct connections to one another. Other organelles, such as the endoplasmic reticulum and the Golgi complex transport materials to other organelles in vesicles. A vesicle buds off of one organelle and transports materials when it fuses with another membrane.
We will now look at the Golgi complex of eukaryotic cells.
The Golgi complex or Golgi apparatus consists of 3-20 flattened and stacked saclike structures called cisternae. A complex network of tubules and vesicles is located at the edges of the cisternae. The Golgi complex functions to:
1. sort proteins and lipids received from the ER;
2. modify certain proteins and glycoproteins; and
3. sort and package these molecules into vesicles for transport to other parts of the cell or secretion from the cell.
As mentioned above, proteins that have been produced in the rough ER are placed into transition vesicles by the smooth ER. The proteins and glycoproteins within the transition vesicle are then transported to the Golgi complex as the transition vesicles fuse with the Golgi complex membrane. Here the proteins and glycoproteins may be further modified and sorted. Finally the Golgi complex will package these molecules in membrane-bound vesicles for secretion from the cell or transport to lysosomes. The vesicles involved in secretion are called secretion vesicles. These form around the molecules to be secreted as they pinch off of the Golgi complex. The secretion vesicles then fuse with the cytoplasmic membrane to release the proteins and glycoproteins from the cell (see Figure \(33\)).
Summary
1. The Golgi complex or Golgi apparatus consists of 3-20 flattened and stacked saclike structures called cisternae. A complex network of tubules and vesicles is located at the edges of the cisternae.
2. The Golgi complex functions to sort proteins and lipids received from the ER, modify certain proteins and glycoproteins, and sort and package these molecules into vesicles for transport to other parts of the cell or secretion from the cell.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Briefly describe the Golgi complex and state its functions. (ans)
2. Briefly describe how the Golgi complex packages materials for secretion from the cell. (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/07%3A_The_Eukaryotic_Cell/7.3%3A_The_Endomembrane_System/7.3B%3A_The_Endoplasmic_Reticulum.txt |
The cell is the basic unit of life. Based on the organization of their cellular structures, all living cells can be divided into two groups: prokaryotic and eukaryotic (also spelled procaryotic and eucaryotic). Animals, plants, fungi, protozoans, and algae all possess eukaryotic cell types. Only bacteria have prokaryotic cell types. Eukaryotic cells are generally much larger and more complex than prokaryotic. Because of their larger size, they require a variety of specialized internal membrane-bound organelles to carry out metabolism, provide energy, and transport chemicals throughout the cell. Eukaryotic cells contain a variety of internal membrane-bound organelles that are not a part of the endomembrane system. These include mitochondria, chloroplasts, lysosomes, peroxisomes, vacuoles, and vesicles. We will now look at the various membrane-bound organelles.
7.4: Other Internal Membrane-Bound Organelles
Briefly describe mitochondria and state their function. State where in the mitochondria the electron transport chain is located. State where in the mitochondria the enzymes for the citric acid cycle (Krebs cycle) are located.
Mitochondria function during aerobic respiration to produce ATP through oxidative phosphorylation. The respiratory enzymes and electron carriers for the electron transport system are located within the inner mitochondria membrane. The enzymes for the citric acid cycle (Krebs cycle) are located in the matrix.
• Electron micrograph of mitochondria courtesy of Dennis Kunkel's Microscopy.
• Electron micrograph of a mitochondrion from the Biology Department at the University of New Mexico.
Summary
1. Mitochondria are rod-shaped structures ranging from 2 to 8 micrometers in length surrounded by two membranes.
2. Mitochondria are located throughout the cytoplasm.
3. Mitochondria function during aerobic respiration to produce ATP through oxidative phosphorylation.
4. The respiratory enzymes and electron carriers for the electron transport system are located within the inner mitochondria membrane. The enzymes for the citric acid cycle (Krebs cycle) are located in the matrix.
5. Mitochondria replicate giving rise to new mitochondria as they grow and divide. They also have their own DNA and ribosomes.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Briefly describe mitochondria and state their function.
2. State where in the mitochondria the electron transport chain is located.
3. State where in the mitochondria the enzymes for the citric acid cycle (Krebs cycle) are located.
7.4B: Chloroplasts
Briefly describe chloroplasts and state their function. State where in the chloroplasts the pigments and the electron transport chains needed to convert light energy into ATP are located.
Chloroplasts (see Figure \(41\)) are disk-shaped structures ranging from 5 to 10 micrometers in length. Like mitochondria, chloroplasts are surrounded by an inner and an outer membrane. The inner membrane encloses a fluid-filled region called the stroma that contains enzymes for the light-independent reactions of photosynthesis. Infolding of this inner membrane forms interconnected stacks of disk-like sacs called thylakoids, often arranged in stacks called grana. The thylakoid membrane, that encloses a fluid-filled thylakoid interior space, contains chlorophyll and other photosynthetic pigments as well as electron transport chains. The light-dependent reactions of photosynthesis occur in the thylakoids. The outer membrane of the chloroplast encloses the intermembrane space between the inner and outer chloroplast membranes (see Figure \(41\)).
The thylakoid membranes contain several pigments capable of absorbing visible light. Chlorophyll is the primary pigment of photosynthesis. Chlorophyll absorbs light in the blue and red region of the visible light spectrum and reflects green light. There are two major types of chlorophyll, chlorophyll a that initiates the light-dependent reactions of photosynthesis, and chlorophyll b, an accessory pigment that also participates in photosynthesis. The thylakoid membranes also contain other accessory pigments. Carotenoids are pigments that absorb blue and green light and reflect yellow, orange, or red. Phycocyanins absorb green and yellow light and reflect blue or purple. These accessory pigments absorb light energy and transfer it to chlorophyll.
They are found in plant cells and algae. Like Mitochondria, chloroplasts are surrounded by two membranes. The outer membrane forms the exterior of the organelle while the inner membrane folds to form a system of interconnected disclike sacs called thylakoids. The thylakoids are arranged in stacks called grana. The space enclosed by the inner chloroplast membrane is called the stroma. Chloroplasts replicate giving rise to new chloroplasts as they grow and divide. They also have their own DNA and ribosomes.
The thylakoid membranes contain the pigments chlorophyll and carotenoids, as well as enzymes and the electron transport chains used in photosynthesis (def), a process that converts light energy into the chemical bond energy of carbohydrates. Energy trapped from sunlight by chlorophyll is used to excite electrons in order to produce ATP by photophosphorylation. The light-dependent reactions that trap light energy and produce the ATP and NADPH needed for photosynthesis occur in the thylakoids. The light-independent reactions of photosynthesis use this ATP and NADPH to produce carbohydrates from carbon dioxide and water, a series of reactions that occur in the stroma of the chloroplast.
For More Information: Photosynthesis from Unit 6
Summary
1. Chloroplasts are disk-shaped structures ranging from 5 to 10 micrometers in length. Like mitochondria, chloroplasts are surrounded by an inner and an outer membrane.
2. Chloroplasts carry out photosynthesis, the process of converting light energy to chemical energy stored in the bonds of sugar.
3. Chloroplasts replicate giving rise to new chloroplasts as they grow and divide. They also have their own DNA and ribosomes.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Briefly describe chloroplasts and state their function. (ans)
2. State where in the chloroplasts the pigments and the electron transport chains needed to convert light energy into ATP are located. (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/07%3A_The_Eukaryotic_Cell/7.4%3A_Other_Internal_Membrane-Bound_Organelles/7.4A%3A_Mitochondria.txt |
Summary
1. Lysosomes, synthesized by the endoplasmic reticulum and the the Golgi complex, are membrane-enclosed spheres typically about 500 nanometers in diameter that contain powerful digestive enzymes that function to digest materials that enter by endocytosis.
2. Peroxisomes are membrane-bound organelles containing an assortment of enzymes that catalyze a variety of metabolic reactions.
3. Proteasomes are cylindrical complexes that use ATP to digest proteins into peptides and play a critical role in enabling the body to kill infected cells and cancer cells during adaptive immunity.
4. Vacuoles are large membranous sacs; vesicles are smaller. Vacuoles are often used to store materials used for energy production such as starch, fat, or glycogen. Vacuoles and vesicles also transport materials within the cell and form around particles that enter by endocytosis.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. _____ Membrane-enclosed spheres that contain powerful digestive enzymes to digest materials that enter by endocytosis. (ans)
_____ Membrane-bound organelles containing an assortment of enzymes that catalyze a variety of metabolic reactions. (ans)
1. lysosomes
2. peroxisomes
3. proteasomes
4. vacuoles
5. vesicles
7.5: Ribosomes
Briefly describe and state the function of eukaryotic ribosomes.
Ribosomes are composed of rRNA and protein and consist of 2 subunits. In eukaryotic cells, the subunits have densities of 60S and 40S ("S" refers to a unit of density called the Svedberg unit) and are composed of longer rRNA molecules and more proteins than the 50S and 30S subunits found in prokaryotic ribosomes. When the two ribosomal subunits join together during translation, they form a complete ribosome having a density of 80S.
The ribosomes are both attached to the endoplasmic reticulum and free in the cytoplasm. They serve as a workbench for protein synthesis, that is, they receive and translate genetic instructions for the formation of specific proteins or polypeptides.
7.6: The Cytoskeleton
State 4 different functions associated with the cytoskeleton of eukaryotic cells.
The cytoskeleton is a network of microfilaments, intermediate filaments, and microtubules. The cytoskeleton functions to:
1. give shape to cells lacking a cell wall;
2. allow for cell movement,e.g. , the crawling movement of white blood cells and amoebas or the contraction of muscle cells;
3. movement of organelles within the cell and endocytosis;
4. cell division, i.e., the movement of chromosomes during mitosis and meiosis and the constriction of animal cells during cytokinesis.
We will now take a closer look at microtubules, microfilaments, intermediate filaments, centrioles, flagella, and cilia.
Microtubules
Microtubules are hollow tubes made of subunits of the protein tubulin. They provide structural support for the cell and play a role in cell division, cell movement, and movement of organelles within the cell. Microtubules are components of centrioles, cilia, and flagella (see below).
Microfilaments
Microfilaments are solid, rodlike structures composed of actin. They provide structural support, and play a roll in phagocytosis, cell and organelle movement, and cell division.
Intermediate filaments
Intermediate filaments are tough fibers made of polypeptides. They help to strengthen the cytoskeleton and stabilize cell shape.
Centrioles
Centrioles are located near the nucleus and appear as cylindrical structures consisting of a ring of nine evenly spaced bundles of three microtubules. Centrioles play a role in the formation of cilia and flagella. During animal cell division, the mitotic spindle forms between centrioles.
Summary
1. The cytoskeleton is a network of microfilaments, intermediate filaments, and microtubules.
2. The cytoskeleton has a variety functions including, giving shape to cells lacking a cell wall, allowing for cell movement, enabling movement of organelles within the cell, endocytosis, and cell division. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/07%3A_The_Eukaryotic_Cell/7.4%3A_Other_Internal_Membrane-Bound_Organelles/7.4C%3A_Lysosomes_Peroxisomes_Vacuoles_and_Vesicles.txt |
Flagellar arrangement schemes
Different species of bacteria have different numbers and arrangements of flagella (Figure \(7\).7.1).
• Monotrichous bacteria have a single flagellum (e.g., Vibrio cholerae).
• Lophotrichous bacteria have multiple flagella located at the same spot on the bacteria's surfaces which act in concert to drive the bacteria in a single direction. In many cases, the bases of multiple flagella are surrounded by a specialized region of the cell membrane, the so-called polar organelle.
• Amphitrichous bacteria have a single flagellum on each of two opposite ends (only one flagellum operates at a time, allowing the bacteria to reverse course rapidly by switching which flagellum is active).
• Peritrichous bacteria have flagella projecting in all directions (e.g., E. coli).
Summary
1. Flagella are long and few in number whereas cilia are short and numerous.
2. Both flagella and cilia consist of 9 fused pairs of protein microtubules with side arms of the motor molecule dynein that originate from a centriole. These form a ring around an inner central pair of microtubules that arise from a plate near the cell surface. This complex of microtubules is surrounded by a sheath continuous with the cytoplasmic membrane.
3. Flagella and cilia function in locomotion. Cilia also function to move various materials that may surround a cell.
7.8: The Endosymbiotic Theory
Learning Objectives
• Briefly describe what is meant by the endosymbiotic theory.
• Give some evidence supporting the theory that mitochondria and chloroplasts may have arisen from prokaryotic organisms.
It is thought that life arose on earth around four billion years ago. The endosymbiotic theory states that some of the organelles in today's eukaryotic cells were once prokaryotic microbes. In this theory, the first eukaryotic cell was probably an amoeba-like cell that got nutrients by phagocytosis and contained a nucleus that formed when a piece of the cytoplasmic membrane pinched off around the chromosomes. Some of these amoeba-like organisms ingested prokaryotic cells that then survived within the organism and developed a symbiotic relationship. Mitochondria formed when bacteria capable of aerobic respiration were ingested; chloroplasts formed when photosynthetic bacteria were ingested. They eventually lost their cell wall and much of their DNA because they were not of benefit within the host cell. Mitochondria and chloroplasts cannot grow outside their host cell.
Evidence for this is based on the following:
1. Chloroplasts are the same size as prokaryotic cells, divide by binary fission, and, like bacteria, have Fts proteins at their division plane. The mitochondria are the same size as prokaryotic cells, divide by binary fission, and the mitochondria of some protists have Fts homologs at their division plane.
2. Mitochondria and chloroplasts have their own DNA that is circular, not linear.
3. Mitochondria and chloroplasts have their own ribosomes that have 30S and 50S subunits, not 40S and 60S.
4. Several more primitive eukaryotic microbes, such as Giardia and Trichomonas have a nuclear membrane but no mitochondria.
Although evidence is less convincing, it is also possible that flagella and cilia may have come from spirochetes.
Example \(1\)
1. Briefly describe what is meant by the endosymbiotic theory.
2. Give three points of evidence supporting the theory that mitochondria and chloroplasts may have arisen from prokaryotic organisms.
Solutions
1. The endosymbiotic theory states that some of the organelles in eukaryotic cells were once prokaryotic microbes.
• Mitochondria and chloroplasts are the same size as prokaryotic cells and divide by binary fission.
• Mitochondria and chloroplasts have their own DNA which is circular, not linear.
• Mitochondria and chloroplasts have their own ribosomes which have 30S and 50S subunits, not 40S and 60S.
Summary
The endosymbiotic theory states that mitochondria and chlopoplasts in today's eukaryotic cells were once separate prokaryotic microbes. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/07%3A_The_Eukaryotic_Cell/7.7%3A_Flagella_and_Cilia.txt |
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
7.1: The Cytoplasmic Membrane
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Match the following descriptions with the best answer.
_____ The movement of water across a membrane from an area of higher water concentration (lower solute concentration) to lower water concentration (higher solute concentration). (ans)
_____ The net movement of gases or small uncharge polar molecules across a phospholipid bilayer membrane from an area of higher concentration to an area of lower concentration. No metabolic energy is required. (ans)
_____ A transport where the cell uses transport proteins such as antiporters or symporters and metabolic energy to transport substances across the membrane against the concentration gradient. (ans)
_____ If the net flow of water is out of a cell, the cell is in ________________ environment. (ans)
_____ If the net flow of water is into a cell, the cell is in ________________ environment. (ans)
_____ Theingestion of dissolved materials by endocytosis whereby the cytoplasmic membrane invaginates and pinches off placing small droplets of fluid in a vesicle. (ans)
_____ The process by which a cell releases waste products or specific secretion products by the fusion of a vesicle with the cytoplasmic membrane. (ans)
1. active transport
2. passive diffusion
3. osmosis
4. exocytosis
5. pinocytosis
6. phagocytosis
7. a hypotonic
8. a hypertonic
9. an isotonic
7.2: The Cell Wall
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State which eukaryotic organisms possess a cell wall and which lack a cell wall. (ans)
2. The function of the cell wall in those eukaryotic cells that possess one is to ____________________. (ans)
7.5: Ribosomes
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Briefly describe and state the function of eukaryotic ribosomes. (ans)
7.6: The Cytoskeleton
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State 3 different functions associated with the cytoskeleton of eukaryotic cells. (ans)
7.8: The Endosymbiotic Theory
1. Parallel membranous tubules and flattened sacs with ribosomes attached. Functions in protein synthesis, production of new membrane, and transport of these proteins and membrane to other locations within the cell. This best describes the:
A. the Golgi apparatus.
B. smooth endoplasmic reticulum.
C. rough endoplasmic reticulum.
D. the nucleus.
2. Consists of 3-20 flattened and stacked saclike structures called cisternae. Modifies certain proteins and lipids received from the ER and packages these molecules into vesicles for transport to other parts of the cell or secretion from the cell. This best describes:
A. the Golgi apparatus.
B. smooth endoplasmic reticulum.
C. rough endoplasmic reticulum.
D. the nucleus.
3. Surrounded by two membranes. The outer membrane forms the exterior of the organelle while the inner membrane is arranged in a series of folds called cristae . Produces ATP through oxidative phosphorylation . This describes:
A. the Golgi apparatus.
B. mitochondria.
C. chloroplasts.
D. the endoplasmic reticulum.
4. Membrane-enclosed spheres that contain powerful digestive enzymes that function to digest materials that enter by endocytosis. This best describes:
A. peroxisomes.
B. mitochondria.
C. proteasomes.
D. lysosomes.
5. A fluid phospholipid bilayer embedded with proteins and glycoproteins. Determines what goes in and out of the cell. This best describes the:
A. cell wall.
B. cytoplasmic membrane.
C. endomembranesystem.
D. cytoskeleton.
6. Long and few in number and consisting of 9 fused pairs of protein microtubuleswith side arms of the motor molecule dynein. Originate from a centrioleand function in locomotion. This best describes:
A. cilia.
B. flagella.
C. the cytoskeleton.
Solution
1=C; 2=A; 3=B; 4=D; 5=B; 6=B | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/07%3A_The_Eukaryotic_Cell/7.E%3A_The_Eukaryotic_Cell_%28Exercises%29.txt |
Yeasts are eukaryotic microorganisms classified as members of the fungus kingdom with 1,500 species currently identified and are estimated to constitute 1% of all described fungal species.
• 8.1: Overview of Fungi
Fungi include yeasts, molds, and fleshy fungi. Fungi are are eukaryotic organisms and possess a cell wall. Most fungi are saprophytes, organisms that live off of decaying matter; a few are parasites, organisms that live off of living matter. A fungal infection is called a mycosis.
• 8.2: Yeasts
Yeasts are eukaryotic unicellular fungi Some yeast are dimorphic in that they can grow as an oval, budding yeast, but under certain culture conditions, they may produce filament-like structures called hyphae similar to molds. Components of the yeast cell wall that function as pathogen-associated molecular patterns or PAMPs include lipoteichoic acids, zymosan, and mannose-rich glycans. These PAMPs bind to pattern-recognition receptors or PRRs on a variety of body defense cells.
• 8.3: Molds
Molds are multinucleated, filamentous fungi composed of hyphae. Molds reproduce primarily by means of asexual reproductive spores. The dermatophytes are a group of molds that cause superficial mycoses of the hair, skin, and nails and utilize the protein keratin that is found in hair, skin, and nails, as a nitrogen and energy source. Dimorphic fungi may exhibit two different growth forms. Outside the body they grow as a mold, producing hyphae and asexual reproductive spores.
• 8.4: Fungal Pathogenicity
Many of the same factors that enable bacteria to colonize the body also enable fungi to colonize. Many of the same factors that enable bacteria to harm the body also enable fungi to cause harm.
• 8.5: Chemotherapeutic Control of Fungi
Because fungi, like human cells, are eukaryotic, there are far fewer chemotherapeutic agents that are selectively toxic for fungi than there are for prokaryotic bacteria. Most antifungal agents bind to or interfere with the synthesis of ergosterol, the sterol in their cytoplasmic membrane, altering membrane structure and function.
• 8.E: Fungi (Exercises)
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
08: Fungi
Name 3 groups of fungi. Define mycosis.
Mycology is the study of fungi. Fungi include yeasts, molds, and fleshy fungi. They:
1. are eukaryotic;
2. have a rigid cell wall;
3. are chemoheterotrophs (organisms that require organic compounds for both carbon and energy sources);
4. obtain their nutrients by absorption;
5. obtain nutrients as saprophytes, organisms that live off of decaying matter, or as parasites, organisms that live off of living matter.
Of the over 100,000 species of fungi, only about 100 species are pathogenic for animals. They play a major role in the recycling of nutrients by their ability to cause decay and are used by industry to produce a variety of useful products. However, they also cause many undesirable economic effects such as the spoilage of fruits, grains, and vegetables, as well as the destruction of unpreserved wood and leather products. We will be concerned mainly with the yeasts and molds, especially those causing mycoses (fungal infections). | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/08%3A_Fungi/8.1%3A_Overview_of_Fungi.txt |
Yeast Morphology
1. Yeast (see Figure \(1\)) are unicellular fungi which usually appear as oval cells 1-5 µm wide by 5-30 µm long.
2. They have typical eukaryotic structures (see Figure \(2\) and Figure \(3\)).
3. They have a thick polysaccharide cell wall.
4. They are facultative anaerobes.
5. The yeast Candida is said to be dimorphicin that it can grow as an oval, budding yeast, but under certain culture conditions, the budding yeast may elongate and remain attached producing filament-like structures called pseudohyphae. C. albicans may also produce true hyphae similar to molds (see Figure \(4\)). In this case long, branching filaments lacking complete septa form. The pseudohyphae and hyphae help the yeast to invade deeper tissues after it colonizes the epithelium. Asexual spores called blastoconidia (blastospores) develop in clusters along the hyphae, often at the points of branching. Under certain growth conditions, thick-walled survival spores called chlamydoconidia (chlamydospores) may also form at the tips or as a part of the hyphae (see Figure \(5\).)
For More Information: A Comparison of Prokaryotic and Eukaryotic Cells from Unit 1
The Role of Fungal Cell Wall Components in Initiating Body Defense
To protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPs. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometimes referred to as microbe-associated molecular patterns or MAMPs.)
Components of the yeast cell wall that function as PAMPs include lipoteichoic acids, and zymosan. In addition, bacteria and other microorganisms also possess mannose-rich glycans (short carbohydrate chains with the sugar mannose or fructose as the terminal sugar) that function as PAMPs. These mannose-rich glycans are common in microbial glycoproteins and glycolipids but rare in those of humans. These PAMPs bind to pattern-recognition receptors on a variety of defense cells of the body and triggers innate immune defenses such as inflammation, fever, and phagocytosis.
Flash animation showing the release of fungal mannans from the cell walls of yeast and their subsequent binding to pattern-recognition receptors on a macrophage.
For More Information: Review of Pathogen-Associated Molecular Patterns (PAMPs) from Unit 5
For More Information: Review of Pattern-Recognition Receptors from Unit 5
For More Information: Review of Inflammation from Unit 5
Yeast cell wall components also activate the alternative complement pathway and the lectin pathway, defense pathways that play a variety of roles in body defense. Cell wall molecules can also trigger adaptive immunity such as the production of antibody molecules against bacterial cell wall antigens. An antigen is defined as a substance that reacts with antibody molecules and antigen receptors on lymphocytes. An immunogen is an antigen that is recognized by the body as non-self and stimulates an adaptive immune response.
The body recognizes an antigen as foreign when epitopes of that antigen bind to B-lymphocytes and T-lymphocytes by means of epitope-specific receptor molecules having a shape complementary to that of the epitope. The epitope receptor on the surface of a B-lymphocyte is called a B-cell receptor and is actually an antibody molecule. The receptor on a T-lymphocyte is called a T-cell receptor (TCR).
Reproduction of yeasts
1. Yeasts reproduce asexually by a process called budding (see Figure \(1\) and Figure \(6\)). A bud is formed on the outer surface of the parent cell as the nucleus divides. One nucleus migrates into the elongating bud. Cell wall material forms between the bud and the parent cell and the bud breaks away.
• Scanning electron micrograph of Saccharomyces; courtesy of Dennis Kunkel's Microscopy.
• Movie of Saccharomyces cerevisiae reproducing by budding. Movie of Growth and Division of Budding Yeast (Saccharomyces cerevisiae) . © Phillip Meaden, author. Licensed for use, ASM MicrobeLibrary.
P. jiroveci can be found in three distinct morphologic stages:
• The trophozoite (trophic form), a haploid amoeboid form 1-4 µm in diameter that replicates by mitosis and binary fission. The trophic forms are irregular shaped and often appears in clusters.
• A precystic form or early cyst. Haploid trophic forms conjugate and produce a zygote or sporocyte (early cyst).
• The cyst form, which contains several intracystic bodies or spores are 5-8 µm in diameter. It has been postulated that in formation of the cyst form (late phase cyst), the zygote undergoes meiosis and subsequent mitosis to typically produce eight haploid ascospores (sporozoites) See Figure \(7\). As the haploid ascospores are released the cysts often collapse forming crescent-shaped bodies (see Figure \(8\)). P. jiroveci is usually transmitted by inhalation of the cyst form. Released ascospores then develop into replicating trophic forms that attach to the wall of the alveoli and replicate to fill the alveoli.
In biopsies from lung tissue or in tracheobronchial aspirates, both a trophic form about 1-4 µm in diameter with a distinct nucleus and a cyst form between 5-8 µm in diameter with 6-8 intracystic bodies (ascospores) can be seen.
Malassezia globosa
Malassezia globosa is a dimorphic yeast that is the most frequent cause of a superficial skin infection called tinea versicolor that commonly appears as a hypopigmentation of the infected skin. M. globosa is also the most common cause of dandruff and seborrheic dermatitis. The yeast is naturally found on the skin. To view additional photomicrographs of Candida, Cryptococcus, and Pneumocystis, see the AIDS Pathology Tutorial at the University of Utah.
Summary
1. Yeasts are eukaryotic unicellular fungi
2. Some yeast are dimorphic in that they can grow as an oval, budding yeast, but under certain culture conditions, they may produce filament-like structures called hyphae similar to molds.
3. Components of the yeast cell wall that function as pathogen-associated molecular patterns or PAMPs include lipoteichoic acids, zymosan, and mannose-rich glycans.
4. These PAMPs bind to pattern-recognition receptors or PRRs on a variety of body defense cells and triggers innate immune defenses.
5. Cell wall molecules can also trigger adaptive immunity such as the production of antibody molecules against bacterial cell wall antigens.
6. Yeasts reproduce asexually by a process called budding.
7. Candida albicans is found as normal flora on the mucous membranes and in the gastrointestinal tract but is usually held in check by the body’s normal microbiota and normal body defenses.
8. Candida can cause a variety of opportunistic infections in people who are debilitated, immunosuppressed, or have received prolonged antibacterial therapy, and infect the lungs, blood, heart, and meninges, especially in the compromised or immunosuppressed host.
9. Cryptococcus neoformans infections are usually mild or subclinical but, when symptomatic, usually begin in the lungs after inhalation of the yeast in dried bird feces.
10. Pneumocystis jiroveci can cause a severe pneumonia called PCP (Pneumocystis pneumonia).
11. Malassezia globosa is the most frequent cause of a superficial skin infection called tinea versicolor and also the most common cause of dandruff. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/08%3A_Fungi/8.2%3A_Yeasts.txt |
Mold Morphology
Molds are multinucleated, filamentous fungi composed of hyphae. A hypha is a branching tubular structure approximately 2-10 µm in diameter which is usually divided into cell-like units by crosswalls called septa. The total mass of hyphae is termed a mycelium. The portion of the mycelium that anchors the mold and absorbs nutrients is called the vegetative mycelium , composed of vegetative hyphae; the portion that produces asexual reproductive spores is the aerial mycelium , composed of aerial hyphae (Figure \(1\)).
Molds have typical eukaryotic structures (Figure \(2\)) and have a cell wall usually composed of chitin, sometimes cellulose, and occasionally both. Furthermore, molds are obligate aerobes and grow by elongation at apical tips of their hyphae and thus are able to penetrate the surfaces on which they begin growing.
For More Information: A Comparison of Prokaryotic and Eukaryotic Cells from Unit 1
Reproduction of Molds
1. Molds reproduce primarily by means of asexual reproductive spores (Figure \(1\)). These include the following.
a. conidiospores (conidia) See Figure \(3\).
Spores borne externally on an aerial hypha called a conidiophore ; see Figure \(4\) and Figure \(5\).
• Scanning electron micrographs of the conidiospores of Penicillium and of Aspergillus; courtesy of Dennis Kunkel's Microscopy.
b. sporangiospores See Figure \(6\).
Spores borne in a sac or sporangium on an aerial hypha called a sporangiophore ; see Figure \(7\).
• Scanning electron micrograph of the conidiospores of Rhizopus; courtesy of Dennis Kunkel's Microscopy.
c. arthrospores See Figure \(8\).
spores produced by fragmentation of a vegetative hypha (Figure \(9\)).
Pathogenic Molds
Dermatophytes
The dermatophytes are a group of molds that cause superficial mycoses of the hair, skin, and nails and utilize the protein keratin, that is found in hair, skin, and nails, as a nitrogen and energy source. Infections are commonly referred to as ringworm or tinea infections and include:
• tinea capitis (infection of the skin of the scalp, eyebrows, and eyelashes)
• tinea barbae (infection of the bearded areas of the face and neck)
• tinea faciei (infection of the skin of the face)
• tinea corporis (infection of the skin regions other than the scalp, groin, palms, and soles)
• tinea cruris (infection of the groin; jock itch)
• tinea unguium (onchomycosis; infection of the fingernails and toenails)
• tinea pedis (athlete's foot; infection of the soles of the feet and between the toes).
The three most common dermatophytes are Microsporum, Trichophyton, and Epidermophyton. They produce characteristic asexual reproductive spores called macroconidia and microconidia (Figure \(10\) and Figure \(11\)).
• Scanning electron micrograph of the macroconidia of Epidermophyton; courtesy of Dennis Kunkel's Microscopy.
Another tinea infection of the skin is tinea versicolor caused by the yeast Malassezia globosa. Tinea versicolor appears as a hypopigmentation of the infected skin. M. globosa is also the most common cause of dandruff.
Dimorphic Fungi
Dimorphic fungi may exhibit two different growth forms. Outside the body they grow as a mold, producing hyphae and asexual reproductive spores, but in the body they grow in a non-mycelial yeast form. These infections appear as systemic mycoses and usually begin by inhaling spores from the mold form. After germination in the lungs, the fungus grows as a yeast. Factors such as body temperature, osmotic stress, oxidative stress, and certain human hormones activate a dimorphism-regulating histidine kinase enzyme in dimorphic molds, causing them to switch from their avirulent mold form to their more virulent yeast form.
For example:
a. Coccidioides immitis causes coccidioidomycosis (Figure \(12\)), a disease endemic to the southwestern United States. An estimated 100,000 infections occur annually in the United States, but one to two thirds of these cases are subclinical.
The mold form of the fungus grows in arid soil and produces thick-walled, barrel-shaped asexual spores called arthrospores (Figure \(8\)) by a fragmentation of its vegetative hyphae.
After inhalation, the arthrospores germinate and develop into endosporulating spherules (Figure \(13\)) in the terminal bronchioles of the lungs. The spherules reproduce by a process called endosporulation, where the spherule produces numerous endospores (yeast-like particles), ruptures, and releases viable endospores that develop into new spherules.
b. Histoplasma capsulatum (Figure \(14\))is a dimorphic fungus that causes histoplasmosis, a disease commonly found in the Great Lakes region and the Mississippi and Ohio River valleys. Approximately 250,000 people are thought to be infected annually in the US, but clinical symptoms of histoplasmosis occur in less than 5% of the population. Most individuals with histoplasmosis are asymptomatic. Those who develop clinical symptoms are typically either immunocompromised or are exposed to a large quantity of fungal spores.
The mold form of the fungus often grows in bird or bat droppings or soil contaminated with these droppings and produces large tuberculate macroconidia and small microconidia (Figure \(15\)). Although birds cannot be infected by the fungus and do not transmit the disease, bird excretions contaminate the soil and enrich it for mycelial growth. Bats, however, can become infected and transmit histoplasmosis through their droppings. After inhalation of the fungal spores and their germination in the lungs, the fungus grows as a budding, encapsulated yeast (Figure \(16\)).
c. Blastomycosis, caused by Blastomyces dermatitidis, is common around the Great Lakes region and the Mississippi and Ohio River valleys.Infection can range from an asymptomatic, self-healing pulmonary infection to widely disseminated and potentially fatal disease. Pulmonary infection may be asymptomatic in nearly 50% of patients. Blastomyces dermatitidis can also sometimes infect the skin.
Blastomyces dermatitidis produces a mycelium with small conidiospores (Figure \(17\)) and grows actively in bird droppings and contaminated soil. When spores are inhaled or enter breaks in the skin, they germinate and the fungus grows as a yeast (Figure \(18\)).having a characteristic thick cell wall. It is diagnosed by culture and by biopsy examination.
These infections usually remains localized in the lungs, but in rare cases may spread throughout the body.
As mentioned earlier, the yeast Candida albicans can also exhibit dimorphism.
• To view additional photomicrographs of Coccidioides and Histoplasma, see the AIDS Pathology Tutorial at the University of Utah.
Opportunistic Molds
Certain molds once considered as non-pathogenic have recently become a fairly common cause of opportunistic lung and wound infections in the debilitated or immunosuppressed host. These include the common molds Aspergillus (Figure \(4\)) and Rhizopus (Figure \(6\)). Although generally harmless in most healthy individuals, Aspergillus species do cause allergic bronchopulmonary aspergillosis (ABPA), chronic necrotizing Aspergillus pneumonia (or chronic necrotizing pulmonary aspergillosis [CNPA]), aspergilloma (a mycetoma or fungus ball in a body cavity such as the lung), and invasive aspergillosis. In highly immunosuppressed individuals, however, Aspergillus may disseminate beyond the lung via the blood.
Mucormycoses are infections caused by fungi belonging to the order of Mucorales. Rhizopus species are the most common causative organisms. The most common infection is a severe infection of the facial sinuses, which may extend into the brain. Other mycoses include pulmonary, cutaneous, and gastrointestinal. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/08%3A_Fungi/8.3%3A_Molds.txt |
Name at least three fungal virulence factors that promote fungal colonization. Name at least two fungal virulence factors that damage the host.
As with the bacteria, fungal virulence factors can be divided into two categories: virulence factors that promote fungal colonization of the host; and virulence factors that damage the host.
Virulence Factors that Promote Fungal Colonization
Virulence factors that promote fungal colonization of the host include the ability to:
1. adhere to host cells and resist physical removal;
2. invade host cells;
3. compete for nutrients;
4. resist innate immune defenses such as phagocytosis and complement; and
5. evade adaptive immune defenses.
Examples of virulence factors that promote fungal colonization include:
1. A compromised immune system is the primary predisposing factor for serious fungal infections. A person highly immunosuppressed, such as a person taking immunosuppressive drugs to suppress transplant rejection, or a person with advancing HIV infection, or a person with other immunosuppressive disorders, becomes very susceptible to infections by fungi generally considered not very harmful to a healthy person with normal defenses.
2. As with bacteria, the ability to adhere to host cells with cell wall adhesins seems to play a role in fungal virulence.
3. Some fungi produce capsules allowing them to resist phagocytic engulfment, such as the yeast Cryptococcus neoformans and the yeast form of Histoplasma capsulatum (Figure \(1\)).
4. Candida albicans stimulates the production of a cytokine called GM-CSF and this cytokine can suppress the production of complement by monocytes and macrophages. This may decrease the production of the opsonin C3b as well as the complement proteins that enhance chemotaxis of phagocytes.
5. C. albicans also appears to be able to acquire iron from red blood cells.
6. C. albicans produces acid proteases and phospholipases that aid in the penetration and damage of host cell membranes.
7. Some fungi are more resistant to phagocytic destruction, e.g., Candida albicans, Histoplasma capsulatum, and Coccidioides immitis.
8. There is evidence that when the yeast form of Candida enters the blood it activates genes allowing it to switch from its budding form to its hyphal form. In addition, when engulfed by macrophages, it starts producing the tubular germ tubes which penetrate the membrane of the macrophage thus causing its death.
A movie of Candida killing a macrophage from within from the Theriot Lab Website at Stanford University Medical School: Candida albicans killing macrophages from inside out.
9. Factors such as body temperature, osmotic stress, oxidative stress, and certain human hormones activate a dimorphism-regulating histidine kinase enzyme in dimorphic molds, such as Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis, causing them to switch from their avirulent mold form to their virulent yeast form. It also triggers the yeast Candida albicans to switch from its yeast form to its more virulent hyphal form.
Virulence Factors that Damage the Host
Like bacteria, fungal PAMPs binding to PRRs can trigger excessive cytokine production leading to a harmful inflammatory response that damages tissues and organs. As fungi grow in the body, they can secrete enzymes to digest cells. These include proteases, phospholipases, and elastases. In response to both the fungus and to cell injury, cytokines are released. As seen earlier under Bacterial Pathogenesis, this leads to an inflammatory response and extracellular killing by phagocytes that leads to further destruction of host tissues.
Many molds secrete mycotoxins , especially when growing on grains, nuts and beans. These toxins may cause a variety of effects in humans and animals if ingested including loss of muscle coordination, weight loss, and tremors. Some mycotoxins are mutagenic and carcinogenic. Aflatoxins, produced by certain Aspergillus species, are especially carcinogenic. A mold called Stachybotrys chartarum is a mycotoxin producer that has been implicated as a potential serious problem in homes and buildings as one of the causes of "sick building syndrome." Mycotoxin symptoms in humans include dermatitis, inflammation of mucous membranes, , cough, fever, headache, and fatigue.
Summary
Many of the same factors that enable bacteria to colonize the body also enable fungi to colonize. Many of the same factors that enable bacteria to harm the body also enable fungi to cause harm. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/08%3A_Fungi/8.4%3A_Fungal_Pathogenicity.txt |
Briefly describe 3 different ways antifungal chemotherapeutic agents may affect fungi and give an example of an antibiotic for each way.
Remember that like human cells, fungal cells are eukaryotic. Since fungal cells, unlike prokaryotic bacterial cells, are not that different from human cells, it is more difficult to find a chemotherapeutic agent that is selectively toxic for fungi, that is, will inhibit or kill fungal cells without also inhibiting or killing human cells. Some of the common antifungal chemotherapeutic agents are listed below.
1. One antibiotic, griseofulvin (Fulvicin, Grifulvin, Gris-PEG), interferes with nuclear division by preventing the aggregation of microtubules needed for mitosis in superficial mycelial fungi. It is used only for severe dermatophyte infections.
2. The antimetabolites trimethoprim + sulfomethoxazole , trimetrexate, atovaquone, and flucytosine interfere with normal nucleic acid synthesis. Trimethoprim/sulfomethoxazole (Septra, Bactrim), atovaquone (Mepron), and trimetrexate (Neutrexin) are used to treat Pneumocystis pneumonia. Flucytosine (Ancobon) is used for more serious Candida infections.
3. Polyene antibiotics such as amphotericin B, pimaricin, and nystatin are fungicidal drugs that bind to ergosterol in the fungal cytoplasmic membrane thus altering its structure and function and causing leakage of cellular needs. Nystatin (Mycostatin) is used to treat superficial Candida infections (thrush, vaginitis, cutaneous infections), amphotericin B (Abelcet, Fungizone) is used for systemic Candida infections, Cryptococcus infections, and dimorphic fungal infections.
4. The azole derivative antibiotics such as clotrimazole, miconazole, itraconazole, fluconazole, and ketoconazole, are fungistatic drugs used to treat many fungal infections. They interfere with ergosterol biosynthesis and thus alter the structure of the cytoplasmic membrane as well as the function of several membrane-bound enzymes like those involved in nutrient transport and chitin synthesis. Clotrimazole (Lotramin, Mycelex), miconazole (Monistat), and econazole (Spectazole) are used to treat superficial Candida and dermatophyte infections; oxiconazole (Oxistat) and sulconazole (Exelderm) are used for dermatophyte infections; butaconazole (Femstat-3), terconazole (Terazole), and tioconazole (Vagistat-1) are used for Candida vaginitis; ketoconazole (Nizoral) and itraconazole (Sporanox) are used for systemic Candida, Cryptococcus, and dimorphic fungal infections; and fluconazole (Diflucan) is used for Candida infections. Voriconazole (VFEND) is a triazole is used to treat Candida infections such as candidemia, disseminated infections in skin and infections in abdomen, kidney, bladder wall, and wounds. It is also used for invasive aspergillosis.
5. Echinocandins, including caspofungin (Cancidas) and micafungin (Mycamine) are intravenous antifungals that inhibits glucan synthesis in fungal cell walls. It is used in the treatment of candidemia , Candida intra-abdominal abscesses, peritonitis, esophageal candidiasis, and pleural space infections.
6. Naftifine (Naftin) and terbinafine (Lamisil) are allylamines that block synthesis of ergosterol as does the topical thiocarbonate tolnaftate. They are used to treat dermatophyte infections.
Why are there so few antifungal chemotherapeutic agents compared to the number of antibacterial agents? Most of the antifungal agents interfere with the synthesis of ergosterol in the fungal cytoplasmic membrane. How does this harm the fungus? Why don’t these agents work on bacterial and viral infections?
For a more detailed description of any specific antimicrobial agent, see the website of RxList - The Internet Drug Index.
8.E: Fungi (Exercises)
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
8.1: Overview of Fungi
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. A fungal infection is termed a _________________. (ans)
8.2: Yeasts
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Briefly describe a typical yeast and state how it reproduces asexually. (ans)
2. Match the following:
_____ Reproductive spores produced by yeast by budding. (ans)
_____ Thick walled survival spores produced by the yeast Candida. (ans)
_____Long, continuous fungal filaments produced by dimorphic yeast. (ans)
1. hyphae
2. blastoconidia (blastospores)
3. chlamydoconidia (chlamydospores)
3. Name 3 potentially pathogenic yeasts and state an infection each causes.
4. Multiple Choice (ans)
8.3: Molds
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Define mold. (ans)
2. Match the following:
_____ The hyphae that grow up in the air and produce asexual reproductive spores. (ans)
_____ Large asexual reproductive mold spores coming of of vegetative hyphae and often produced by dermatophytes. (ans)
_____ Asexual reproductive mold spores produced inside a sac or sporangium at the end of an aerial hypha. (ans)
_____ The hyphae that anchor a mold and absorb nutrients. (ans)
_____ Asexual reproductive mold spores produced in chains at the end of an aerial hypha. (ans)
_____ A branching tubular structure of a mold that is usually divided into cell-like units by crosswalls called septa. (ans)
_____ Asexual reproductive mold spores produced by fragmentation of vegetative hyphae. (ans)
1. hypha
2. macroconidia
3. vegetative mycelium
4. aerial mycelium
5. sporangiospores
6. arthrospores
7. conidiospores
3. Define dermatophyte. (ans)
4. List 2 genera of dermatophytes.
5. Name 3 dermatophytic infections. (ans)
6. Describe what is meant by the term "dimorphic fungus", name 2 systemic infections caused by dimorphic fungi, and state how they are initially contracted. (ans)
7. Multiple Choice (ans)
8.4: Fungal Pathogenicity
Exercise
1. Name at least 3 fungal virulence factors that promote fungal colonization.
2. Name 2 fungal virulence factors that damage the host.
8.5: Chemotherapeutic Control of Fungi
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Briefly describe 2 different ways antifungal chemotherapeutic agents may affect fungi and give an example of an antibiotic for each way. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/08%3A_Fungi/8.5%3A_Chemotherapeutic_Control_of_Fungi.txt |
Thumbnail: A "Giant Amoeba", Chaos carolinense. (CC BY-SA 2.5; Dr.Tsukii Yuuji).
09: Protozoa
Learning Objectives
After completing this section you should be able to perform the following objectives.
1. Briefly describe protozoa.
2. Briefly describe 3 ways protozoans may reproduce asexually.
3. Define the following:
1. trophozoite
2. protozoan cyst.
Protozoa are unicellular eukaryotic microorganisms lacking a cell wall and belonging to the Kingdom Protista. Although there are nearly 20,000 species of protozoa, relatively few cause disease; most inhabit soil and water. Protozoa reproduce asexually by the following means:
1. fission: One cell splits into two.
2. schizogony: A form of asexual reproduction characteristic of certain protozoa, including sporozoa, in which daughter cells are produced by multiple fission of the nucleus of the parasite followed by segmentation of the cytoplasm to form separate masses around each smaller nucleus.
3. budding: Buds form around a nucleus and pinch off of the parent cell.
Some protozoa also reproduce sexually by fusion of gametes (Figure \(1\)).
Exercise: Think-Pair-Share Questions
1. Protozoa that cause gastrointestinal infections are capable of producing cyst forms as well as trophozoites. State why this is essential to these pathogens.
The Role of Protozoan Cytoplasmic Membrane Components in Initiating Body Defense
Initiation of Innate Immunity
In order to protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPs. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometimes referred to as microbe-associated molecular patterns or MAMPs.)
Components of protozoa that function as PAMPs include GPI-anchored proteins (GPI = Glycosylphosphatidylinositol) and mannose-rich glycans (short carbohydrate chains with the sugar mannose or fructose as the terminal sugar) that function as PAMPs. These mannose-rich glycans are common in microbial glycoproteins and glycolipids but rare in those of humans. These PAMPs bind to pattern-recognition receptors or PRRs on a variety of defense cells of the body and triggers innate immune defenses such as inflammation, fever, and phagocytosis.
Initiation of Adaptive Immunity
Proteins associated with protozoa function as antigens and initiate adaptive immunity. An antigen is defined as a substance that reacts with antibody molecules and antigen receptors on lymphocytes. An immunogen is an antigen that is recognized by the body as non-self and stimulates an adaptive immune response. The body recognizes an antigen as foreign when epitopes of that antigen bind to B-lymphocytes and T-lymphocytes by means of epitope-specific receptor molecules having a shape complementary to that of the epitope. The epitope receptor on the surface of a B-lymphocyte is called a B-cell receptor and is actually an antibody molecule. The receptor on a T-lymphocyte is called a T-cell receptor (TCR). This will be discussed in greater detail in Unit 6.
We will now briefly look at some medically important protozoa classified into phyla based on their motility. Illustrations can be found in your Lab Manual in Lab 20.
Summary
Protozoa are unicellular eukaryotic microorganisms lacking a cell wall and belonging to the Kingdom Protista. Protozoa reproduce asexually by fission, schizogony, or budding. Some protozoa can also reproduce sexually. Relatively few protozoa cause disease. The vegetative, reproducing, feeding form of a protozoan is called a trophozoite. Under certain conditions, some protozoa produce a protective form called a cyst. Components of protozoa that function as PAMPs include GPI-anchored proteins and mannose-rich glycans. These PAMPS bind to PRRs on various defense cells and trigger innate immunity. Protozoan molecules can also trigger adaptive immunity such as the production of antibody molecules against protozoan antigens. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/09%3A_Protozoa/9.1%3A_Characteristics_of_Protozoa.txt |
The Sarcomastigophora (Amoeboflagellates)
The amoebas (subphylum Sarcodina) move by extending lobelike projections of their cytoplasm called pseudopodia .
• Photomicrograph of an amoeba.
Video YouTube movie an amoeba moving by forming pseudopodia (https://www.youtube.com/embed/7pR7TNzJ_pA).
a. Entamoeba histolytica (see photomicrograph) which causes a gastrointestinal infection called amoebic dysentery. The organism produces protective cysts which pass out of the intestines of the infected host and are ingested by the next host. It is transmitted by the fecal-oral route.
b. Acanthamoeba can cause rare, but severe infections of the eye, skin, and central nervous system. Acanthamoeba keratitis is an infection of the eye that typically occurs in healthy persons and can result in blindness or permanent visual impairment. Granulomatous amebic encephalitis (GAE) is an infection of the brain and spinal cord typically occurring in persons with a compromised immune system. Acanthamoeba is found in soil, dust, and a variety of water sources including lakes, tap water, swimming pools, and heating and air conditioning units. It typically enters the eyes and most cases are associated with contact lens use, but it can also enter cuts or wounds and be inhaled.
c. Naegleria fowleri (sometimes called the"brain-eating amoeba"), is another amoeba that can cause a rare but devastating infection of the brain called primary amebic meningoencephalitis (PAM). The amoeba is commonly found in warm freshwater rivers, lakes, rivers, and hot springs, as well as in the soil. It typically causes infections when contaminated water enters the body through the nose where it can subsequently travel to the brain.
YouTube movie of Acanthamoeba
The flagellates (subphylum Mastigophora) move by means of flagella. Some also have an undulating membrane .
a. Giardia lamblia (see photomicrograph) can cause a gastrointestinal infection called giardiasis. Cysts pass out of the intestines of the infected host and are ingested by the next host. It is transmitted by the fecal-oral route.
YouTube animation illustrating giardiasis
• Scanning electron micrograph of Giardia in the intestines; courtesy of Dennis Kunkel's Microscopy.
• Scanning electron micrograph of Giardia;courtesy of CDC.
b. Trichomonas vaginalis (see photomicrograph) infects the vagina and the male urinary tract causing an infection called genitourinary trichomoniasis. It does not produce a cysts stage and is usually transmitted by sexual contact.
YouTube movie Trichomonas vaginalis.
YouTube movie showing motility of Trichomonas vaginalis.
c. Trypanosoma brucei gambiens (see photomicrograph) causes African sleeping sickness and is transmitted by the bite of an infected Tsetse fly. The disease primarily involves the lymphatic and nervous systems of humans.
YouTube movie of Trypanosoma
The Ciliophora
The ciliates move by means of cilia.
• Scanning electron micrograph of Paramecium, a ciliated protozoan; courtesy of Dennis Kunkel's Microscopy.
YouTube movie showing motility of Balantidium coli.
a. The only pathogenic ciliate is Balantidium coli (see photomicrograph) which causes a diarrhea-type infection called balantidiasis. Cysts pass out of the intestines of the infected host and are ingested by the next host. It is transmitted by the fecal-oral route.
Balantidium coli in a Fecal Smear
The Apicomplexans
Toxoplasma gondii is another intracellular apicomplexan and causes toxoplasmosis (see the AIDS pathology tutorial at the University of Utah). It can infect most mammals and is contracted by inhaling or ingesting cysts from the feces of infected domestic cats, where the protozoa reproduce both asexually and sexually, or by ingesting raw meat of an infected animal. Toxoplasmosis is usually mild in people with normal immune responses but can infect the brain, heart, or lungs of people who are immunosuppressed. It can also be transmitted congenitally and infect the nervous system of the infected child.
Cryptosporidium is an intracellular parasite that causes a gastrointestinal infection called cryptosporidiosis, although in people who are immunosuppressed it can also cause respiratory and gallbladder infections. It is transmitted by the fecal-oral route.
Movie of motile Cryptosporidium, courtesy of the Sibly Lab, Washington University in St. Louis School of Medicine.
Movie of Cryptosporidium entering an epithelial cell, courtesy of the Sibly Lab, Washington University in St. Louis School of Medicine.
Virulence Factors that Promote Colonization of Protozoans
Virulence factors that promote protozoal colonization of the host include the ability to:
1. contact host cells;
2. adhere to host cells and resist physical removal;
3. invade host cells;
4. compete for nutrients;
5. resist innate immune defenses such as phagocytosis and complement; and
6. evade adaptive immune defenses.
Examples of virulence factors that promote protozoal colonization include:
1. Some protozoa, such as Entamoeba histolytica,Trichomonas vaginalis, Giardia lamblia, and Balantidium coli use pseudopodia, flagella or cilia to swim through mucus and contact host cells.
2. Protozoa use adhesins associated with their cytoplasmic membrane to adhere to host cells, colonize, and resist flushing.
3. Some protozoa, such as the apicomplexans (Plasmodium (inf), Toxoplasma gondii (inf), and Cryptosporidium (inf)) possess a complex of organelles called apical complexes at their apex that contain enzymes used in penetrating host tissues and cells.
4. Protozoans such as Trypanosoma brucei gambiens (inf) and Plasmodium species (inf) are able to change their surface antigens during their life cycle in the human. As the protozoa change the amino acid sequence and shape of their surface antigens, antibodies and cytotoxic T-lymphocytes made against a previous shape will no longer fit and the body has to start a new round of adaptive immunity against the new antigen shape.
5. Some protozoa, such as Entamoeba histolytica (inf) shed their surface antigens so that antibodies made by the body against these surface antigens are tied up by the shed antigens.
To view a Quicktime movie of Cryptosporidium and electron micrographs of Giardia and Entamoeba, see the Parasites section of the CELL'S ALIVE web page.
9.E: Protozoa (Exercises)
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
9.1: Characteristics of Protozoa
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Match the following:
_____ Multiple fission. The nucleus divides many times before the cell divides. The single cell then separates into numerous daughter cells. (ans)
_____ Division in which one cell splits in two. (ans)
_____ Division in which a cell pinches off of the parent cell. (ans)
_____ The vegetative, reproducing, feeding form of a protoaoan. (ans)
_____ A protective form that enables protozoa to survive harsh environments. (ans)
1. trophozoite
2. cyst
3. fission
4. schizogony
5. budding
9.2: Medically Important Protozoa
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Matching
_____ Moves by flagella; transmitted by ingesting cysts via the fecal-oral route; causes an intestinal infection. (ans)
_____ Moves by cilia; transmitted by ingesting cysts via the fecal-oral route; causes an intestinal infection. (ans)
_____ Moves by flagella; transmitted by an infected tsetse fly; causes African sleeping sickness. (ans)
_____ Nonmotile in the body; reproduces sexually and asexually; transmitted by an infecteded Anopheles mosquito; causes malaria. (ans)
_____ Moves by flagella; transmitted sexually; causes vaginitis. (ans)
_____ Nonmotile in the body; reproduces sexually and asexually; transmitted by eating infected meat or inhaling or ingesting cysts from cat feces. (ans)
1. Entamoeba histolytica
2. Acanthamoeba
3. Giardia lamblia
4. Trichomonas vaginalis
5. Trypanosoma brucei-gambiens
6. Balantidium coli
7. Plasmodium species
8. Toxoplasma gondii
9. Cryptosporidium
2. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/09%3A_Protozoa/9.2%3A_Medically_Important_Protozoa.txt |
A virus is a small infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea.
• 10.1: General Characteristics of Viruses
Viruses are infectious agents with both living and nonliving characteristics. Living characteristics of viruses include the ability to reproduce – but only in living host cells – and the ability to mutate. Nonliving characteristics include the fact that they are not cells, have no cytoplasm or cellular organelles, and carry out no metabolism on their own and therefore must replicate using the host cell's metabolic machinery.
• 10.2: Size and Shapes of Viruses
Viruses are usually much smaller than bacteria with the vast majority being submicroscopic, generally ranging in size from 5 to 300 nanometers (nm). Helical viruses consist of nucleic acid surrounded by a hollow protein cylinder or capsid and possessing a helical structure. Polyhedral viruses consist of nucleic acid surrounded by a polyhedral (many-sided) shell or capsid, usually in the form of an icosahedron.
• 10.3: Viral Structure
Since viruses are not cells, they are structurally much simpler than bacteria. An intact infectious viral particle - or virion - consists of a genome, a capsid, and maybe an envelope. Viruses possess either DNA or RNA as their genome. The genome is typically surrounded by a protein shell called a capsid composed of protein subunits called capsomeres.
• 10.4: Classification of Viruses
Viruses can store their genetic information in six different types of nucleic acid which are named based on how that nucleic acid eventually becomes transcribed to the viral mRNA. (+) and (-) strands of nucleic acid are complementary. Copying a (+) stand gives a (-) strand; copying a (-) stand gives a (+) strand. Only (+) strands of viral RNA can be translated into viral protein. The "dependent" part of the name refers to the nucleic acid is being copied.
• 10.5: Other Acellular Infectious Agents: Viroids and Prions
Viroids are small, circular, single-stranded molecules of infectious RNA that cause several plant diseases. Prions are infectious protein particles responsible for a group of transmissible and/or inherited neurodegenerative diseases as a result of prion protein misfolding. Diseases including Creutzfeldt-Jakob disease Gerstmann-Straussler-syndrome, and mad cow disease.
• 10.6: Animal Virus Life Cycles
Viruses that infect animal cells replicate by what is called the productive life cycle. The productive life cycle is also often referred to as the lytic life cycle, even though not all viruses cause lysis of their host cell during their replication. Some viruses, such as HIV and the herpes viruses are able to become latent in certain cell types. A few viruses increase the risk of certain cancers. We will now look at the life cycles of viruses that infect animal cells.
• 10.7: Bacteriophage Life Cycles: An Overview
bacteriophages are viruses that only infect bacteria (also see Fig. 1C and Fig. 2E). There are two primary types of bacteriophages: lytic bacteriophages and temperate bacteriophages. Bacteriophages that replicate through the lytic life cycle are called lytic bacteriophages, and are so named because they lyse the host bacterium as a normal part of their life cycle. Bacteriophages capable of a lysogenic life cycle are termed temperate phages.
• 10.8: Pathogenicity of Animal Viruses
Alteration of host cell function and/or death of the host cell occurs as a result of viruses using an infected host cell as a factory for manufacturing viruses. The body’s immune defenses recognize infected host cells as foreign and destroy infected cells. The body’s adaptive immune defenses produce antibodies against viruses that block viral adsorption to host cells or result in opsonization of the virus.
• 10.9: Bacteriophage-Induced Alterations of Bacteria
Lytic bacteriophages usually cause the host bacterium to lyse. The added genetic information provided by the DNA of a prophage may enable a bacterium to possess new genetic traits. Some bacteria become virulent only when infected themselves with a specific temperate bacteriophage. The added genetic information of the prophage allows for coding of protein exotoxin or other virulence factors.
• 10.10: Antiviral Agents
Relatively few antiviral chemotherapeutic agents are currently available and they are only somewhat effective against just a few limited viruses. Many antiviral agents resemble normal DNA nucleosides molecules and work by inhibiting viral DNA synthesis. Some antiviral agents are protease inhibitors that bind to a viral protease and prevent it from cleaving the long polyprotein from polycistronic genes into proteins essential to viral structure and function.
• 10.11: General Categories of Viral Infections
Acute infections are of relatively short duration with rapid recovery. Persistent infections are where the viruses are continually present in the body. In a latent viral infection the virus remains in equilibrium with the host for long periods of time before symptoms again appear, but the actual viruses cannot be detected until reactivation of the disease occurs. In a chronic virus infection, the virus can be demonstrated in the body at all times and the disease may be present or absent.
• 10.E: Viruses (Exercises)
These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.
10: Viruses
Learning Objectives
1. State 2 living and 2 nonliving characteristics of viruses.
2. List 3 criteria used to define a virus.
3. Discuss why bacteria can be cultivated on synthetic media such as nutrient broth whereas viruses cannot.
4. Define bacteriophage.
Viruses are infectious agents with both living and nonliving characteristics. They can infect animals, plants, and even other microorganisms. Viruses that infect only bacteria are called bacteriophages and those that infect only fungi are termed mycophages . There are even some viruses called virophages that infect other viruses.
Living Characteristics of Viruses Nonliving Characteristics of Viruses
1. They reproduce at a fantastic rate, but only in living host cells.
2. They can mutate.
1. They are acellular, that is, they contain no cytoplasm or cellular organelles.
2. They carry out no metabolism on their own and must replicate using the host cell's metabolic machinery. In other words, viruses don't grow and divide. Instead, new viral components are synthesized and assembled within the infected host cell.
3. The vast majority of viruses possess either DNA or RNA but not both.
Recently, viruses have been declared as living entities based on the large number of protein folds encoded by viral genomes that are shared with the genomes of cells. This indicates that viruses likely arose from multiple ancient cells.
The vast majority of viruses contain only one type of nucleic acid: DNA or RNA, but not both. Virus are totally dependent on a host cell for replication (i.e., they are strict intracellular parasites.) Furthermore, viral components must assemble into complete viruses (virions) to go from one host cell to another. Since viruses lack metabolic machinery of their own and are totally dependent on their host cell for replication, they cannot be grown in synthetic culture media. Animal viruses are normally grown in animals, embryonated eggs, or in cell cultures where in animal host cells are grown in a synthetic medium and the viruses are then grown in these cells.
Summary
1. Viruses are infectious agents with both living and nonliving characteristics.
2. Living characteristics of viruses include the ability to reproduce – but only in living host cells – and the ability to mutate.
3. Nonliving characteristics include the fact that they are not cells, have no cytoplasm or cellular organelles, and carry out no metabolism on their own and therefore must replicate using the host cell's metabolic machinery.
4. Viruses can infect animals, plants, and even other microorganisms.
5. Since viruses lack metabolic machinery of their own and are totally dependent on their host cell for replication, they cannot be grown in synthetic culture media. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.01%3A_General_Characteristics_of_Viruses.txt |
Learning Objectives
1. Compare the size of most viruses to that of bacteria.
2. List 4 shapes of viruses.
Size
Viruses are usually much smaller than bacteria with the vast majority being submicroscopic. While most viruses range in size from 5 to 300 nanometers (nm) , in recent years a number of giant viruses, including Mimiviruses and Pandoraviruses with a diameter of 0.4 micrometers (µm) , have been identified. For a comparison of the size of a virus, a bacterium, and a human cell, scroll down to how big is... on the Cell Size and Scale Resource at the University of Utah webpage (see Figure \(1\)A, Figure \(1\)B, and Figure \(1\)C),
Shapes
Figure \(1\)C: Sizes and Shapes of Viruses (Bacteriophages)
a. Helical viruses consist of nucleic acid surrounded by a hollow protein cylinder or capsid and possessing a helical structure (Figure \(2\)A).
b. Polyhedral viruses consist of nucleic acid surrounded by a polyhedral (many-sided) shell or capsid, usually in the form of an icosahedron (Figure \(2\)B).
c. Enveloped viruses consist of nucleic acid surrounded by either a helical or polyhedral core and covered by an envelope (see Figure \(2\)C and Figure \(2\)D).
d. Binal (complex) viruses have neither helical nor polyhedral forms, are pleomorphic or irregular shaped (Figure \(3\)), or have complex structures (Figure \(2\)F).
• Transmission electron micrograph of the bacteriophage coliphage T4; courtesy of Dennis Kunkel's Microscopy.
Exercise: Think-Pair-Share Questions
We just learned that most viruses are much smaller than bacteria.
1. Compare the sizes of viruses and bacteria.
2. Why are viruses able to be so much smaller than bacteria
Summary
1. Viruses are usually much smaller than bacteria with the vast majority being submicroscopic, generally ranging in size from 5 to 300 nanometers (nm).
2. Helical viruses consist of nucleic acid surrounded by a hollow protein cylinder or capsid and possessing a helical structure.
3. Polyhedral viruses consist of nucleic acid surrounded by a polyhedral (many-sided) shell or capsid, usually in the form of an icosahedron.
4. Enveloped viruses consist of nucleic acid surrounded by either a helical or polyhedral core and covered by an envelope.
5. Binal (complex) viruses have neither helical nor polyhedral forms, have irregular shapes, or have complex structures.
10.03: Viral Structure
Learning Objectives
1. Describe what an animal virus consists of structurally.
2. Define the following:
1. capsid
2. capsomere
3. nucleocapsid.
3. Describe how most animal viruses obtain their envelope.
4. State why some bacteriophages are more complex than typical polyhedral or helical viruses.
Since viruses are not cells, they are structurally much simpler than bacteria. An intact infectious viral particle is called a virion and consists of: a genome, a capsid, and often an envelope.
Viral Genome
The viral genome is a single or segmented, circular or linear molecule of nucleic acid functioning as the genetic material of the virus. It can be single-stranded or double-stranded DNA or RNA (but almost never both), and codes for the synthesis of viral components and viral enzymes for replication. It is also becoming recognized that viruses may play a critical role in evolution of life by serving as shuttles of genetic material between other organisms.
Viral Capsid
The capsid, or core, is a protein shell surrounding the genome and is usually composed of protein subunits called capsomeres. The capsid serves to protect and introduce the genome into host cells. Some viruses consist of no more than a genome surrounded by a capsid and are called nucleocapsid or nucleocapsid (Figure \(1\)). Attachment proteins project out from the capsid and bind the virus to susceptible host cells.
The Adenovirus and Poliomyelitis viruses are examples of naked viruses (Figure \(2\)); both exhibit polyhedral structures.
Viral Envelope
Most animal viruses also have an envelope surrounding a polyhedral or helical nucleocapsid, in which case they are called enveloped viruses (Figure \(3\)). The envelope may come from the host cell's nuclear membrane, vacuolar membranes (packaged by the Golgi apparatus), or outer cytoplasmic membrane.
Although the envelope is usually of host cell origin, the virus does incorporate proteins of its own, often appearing as glycoprotein spikes, into the envelope. These glycoprotein spikes function in attaching the virus to receptors on susceptible host cells.
Viral Activation of Innate Immunity
To protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPs. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometimes referred to as microbe-associated molecular patterns or MAMPs.)
For example, most viral genomes contain a high frequency of unmethylated cytosine-guanine dinucleotide sequences (a cytosine lacking a methyl or CH3 group and located adjacent to a guanine). Mammalian DNA has a low frequency of cytosine-guanine dinucleotides and most are methylated. In addition, most viruses produce unique double-stranded viral RNA, and some viruses produce uracil-rich single-stranded viral RNA during portions of their life cycle. These forms of viral nucleic acids are common PAMPs associated with viruses. These PAMPs bind to pattern-recognition receptors or PRRs called toll-like receptors or TLRs found within the endosomes of phagocytic cells. Viral RNA can also bind to cytoplasmic PRRs called RIG-1 (retinoic acid-inducible gene-1)and MDR-5 (melanoma differentiation-associated gene-5).
Most of the PRRs that bind to viral components trigger the synthesis of cytokines called Type-I interferons that block viral replication within infected host cells. The TLRs for viral components are found in the membranes of the phagosomes used to degrade viruses during phagocytosis. As viruses are engulfed by phagocytes, the viral PAMPS bind to TLRs located within the phagolysosomes (endosomes ). The TLRs for viral components include:
1. TLR-3 binds double-stranded viral RNA;
2. TLR-7 binds uracil-rich single-stranded viral RNA such as in HIV;
3. TLR-8 binds single-stranded viral RNA;
4. TLR-9 binds unmethylated cytosine-guanine dinucleotide sequences (CpG DNA) found in bacterial and viral genomes.
5. RIG-1 (retinoic acid-inducible gene-1) and MDA-5 (melanoma differentiation-associated gene-5), are cytoplasmic sensors that both viral double-stranded and single-stranded RNA molecules produced in viral-infected cells
Bacteriophages are viruses that only infect bacteria. Some bacteriophages are structurally much more complex than typical nucleocapsid or enveloped viruses and may possess a unique tail structure composed of a base plate, tail fibers, and a contractile sheath (also see Figure \(1\)C and Figure \(2\)E). Other bacteriophages, however, are simple icosahedrals or helical (see Figure \(1\)C).
Electron Micrograph of a Bacteriophage with a Contractile Sheath. A = normal bacteriophage and B = bacteriophage after contraction of sheath
• Transmission electron micrograph of the bacteriophage coliphage T4 courtesy of Dennis Kunkel's Microscopy.
Exercise: Think-Pair-Share Questions
1. Discuss why viruses can only replicate inside living cells.
2. Most of the PRRs for viral PAMPs are found in the membranes of the phagosomes, not on the surface of the cell.
1. Why do you think this is?
2. Name the primary cytokines produced in response to viral PAMPs and state how they function to protect against viruses.
Summary
1. Since viruses are not cells, they are structurally much simpler than bacteria.
2. An intact infectious viral particle - or virion - consists of a genome, a capsid, and maybe an envelope.
3. Viruses possess either DNA or RNA as their genome.
4. The genome is typically surrounded by a protein shell called a capsid composed of protein subunits called capsomeres.
5. Some viruses consist of no more than a genome surrounded by a capsid and are called nucleocapsid or naked viruses.
6. Most animal viruses also have an envelope surrounding a polyhedral or helical nucleocapsid that is typically derived from host cell membranes by a budding process and are called enveloped viruses.
7. Specific proteins or glycoproteins on the viral surface are used to attach viruses to the surface of its host cell.
8. The viral nucleic acid functions as a pathogen-associated molecular pattern (PAMP).
9. Binding of viral PAMPs to host cell pattern-recognition receptors (PRRs) triggers the synthesis and secretion of anti-viral cytokines called type-1 interferons that block viral replication within infected host cells. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.02%3A_Size_and_Shapes_of_Viruses.txt |
Learning Objectives
1. State what criteria are used in viral classification.
2. Regarding the naming of enzymes involved in the replication of viral nucleic acid, state what the "dependent" part of the name refers to and what the "polymerase" part of the name refers to.
Viruses can store their genetic information in six different types of nucleic acid which are named based on how that nucleic acid eventually becomes transcribed to the viral mRNA (Figure \(1\)) capable of binding to host cell ribosomes and being translated into viral proteins.
In the diagrams below, (+) and (-) represent complementary strands of nucleic acid. Copying of a (+) strand by complementary base pairing forms a (-) strand. Only a (+) viral mRNA strand can be translated into viral protein. Regarding the enzymes involved in nucleic acid replication, the "dependent" part of the name refers what type of nucleic acid is being copied. The "polymerase" part of the name refers what type of nucleic acid is being synthesized, e.g., DNA-dependent RNA-polymerase would synthesize a strand of RNA complementary to a strand of DNA. These six forms of viral nucleic acid are:
• (+/-) double-stranded DNA (Figure \(2\)). To replicate the viral genome, DNA-dependent DNA polymerase enzymes copy both the (+) and (-) DNA strands producing dsDNA viral genomes. To produce viral mRNA molecules, DNA-dependent RNA polymerase enzymes copy the (-) DNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Examples include most bacteriophages, Papovaviruses, Adenoviruses, and Herpesviruses.
• (+) single-stranded DNA (Figure \(2\)). To replicate the viral genome, DNA-dependent DNA polymerase enzymes copy the (+) DNA strand of the genome producing a dsDNA intermediate. DNA-dependent DNA polymerase enzymes then copy the (-) DNA strand into ss (+) DNA genomes. To produce viral mRNA molecules, DNA-dependent RNA polymerase enzymes copy the (-) DNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Examples include Phage M13 and Parvoviruses.
• (+/-) double-stranded RNA (Figure \(4\)). To replicate the viral genome, RNA-dependent RNA polymerase enzymes copy both the (+) RNA and (-) RNA strands of the genome producing a dsRNA genomes. To produce viral mRNA molecules, RNA-dependent RNA polymerase enzymes copy the (-) RNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Reoviruses are an example.
• (-) RNA (Figure \(5\)). To replicate the viral genome, RNA-dependent RNA polymerase enzymes copy the (-) RNA genome producing ss (+) RNA. RNA-dependent RNA polymerase enzymes then copy the (+) RNA strands producing ss (-) RNA viral genome. The (+) mRNA strands also function as viral mRNA and can then be translated into viral proteins by host cell ribosomes. Examples include Orthomyxoviruses, Paramyxoviruses, Rhabdoviruses.
• (+) RNA (Figure \(6\)). To replicate the viral genome, RNA-dependent RNA polymerase enzymes copy the (+) RNA genome producing ss (-) RNA. RNA-dependent RNA polymerase enzymes then copy the (-) RNA strands producing ss (+) RNA viral genome. To produce viral mRNA molecules. RNA-dependent RNA polymerase enzymes copy the (-) RNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Examples include Picornaviruses, Togaviruses, and Coronaviruses.
• (+) RNA Retroviruses (Figure \(7\)). To replicate the viral genome, reverse transcriptase enzymes (RNA-dependent DNA polymerases) copy the (+) RNA genome producing ss (-) DNA strands. DNA-dependent DNA polymerase enzymes then copy the (-) DNA strands to produce a dsDNA intermediate. DNA-dependent RNA polymerase enzymes then copy the (-) DNA strands to produce ss (+) RNA genomes. To produce viral mRNA molecules, DNA-dependent RNA polymerase enzymes copy the (-) DNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Retroviruses, such as HIV-1, HIV-2, and HTLV-1 are examples.
Exercise: Think-Pair-Share Questions
A viral enzyme that synthesizes a complementary RNA copy of an RNA would be called what?
Table \(1\) below describes some of the medically important viruses.
Table \(1\): Classification of Viruses
Properties Viral Family Size Example
single-stranded DNA; naked; polyhedral capsid Parvoviridae 18-25 nm parvoviruses (roseola, fetal death, gastroenteritis; some depend on coinfection with adenoviruses)
double-stranded, DNA; naked; polyhedral capsid Papovaviridae; circular dsDNA 40-57 nm human papilloma viruses (HPV; benign warts and genital warts; genital and rectal cancers)
Adenoviridae; dsDNA 70-90 nm adenoviruses (respiratory infections, gastroenteritis, infectious pinkeye, rashes, meningoencephalitis)
double-stranded, circular DNA; enveloped; complex Poxviridae 200-350 nm smallpox virus (smallpox), vaccinia virus (cowpox), molluscipox virus (molluscum contagiosum-wartlike skin lesions)
double-stranded DNA; enveloped; polyhedral capsid Herpesviridae 150-200 nm herpes simplex 1 virus (HSV-1; most oral herpes; herpes simplex 2 virus (HSV-2; most genital herpes), herpes simplex 6 virus (HSV-6; roseola), varicella-zoster virus (VZV; chickenpox and shingles), Epstein-Barr virus (EBV; infectious mononucleosis and lymphomas), cytomegalovirus (CMV; birth defects and infections of a variety of body systems in immunosuppressed individuals)
Hepadnaviridae 42 nm hepatitis B virus (HBV; hepatitis B and liver cancer)
(+)single-stranded RNA; naked; polyhedral capsid picornaviridae 28-30 nm enteroviruses (poliomyelitis), rhinoviruses (most frequent cause of the common cold), Noroviruses (gastroenteritis), echoviruses (meningitis), hepatitis A virus (HAV; hepatitis A)
(+)single-stranded RNA; enveloped; usually a polyhedral capsid Togaviridae 60-70 nm arboviruses (eastern equine encephalitis, western equine encephalitis), rubella virus (German measles)
Flaviviridae 40-50 nm flaviviruses (yellow fever, dengue fever, St. Louis encephalitis), hepatitis C virus (HCV; hepatitis C)
Coronaviridae 80-160 nm coronaviruses (upper respiratory infections and the common cold; SARS)
(-)single-stranded RNA; enveloped; pleomorphic Rhabdoviridae; bullet-shaped 70-189 nm rabies virus (rabies)
Filoviridae; long and filamentous 80-14,000 nm Ebola virus, Marburg virus (hemorrhagic fevers)
Paramyxoviridae; pleomorphic 150-300 nm paramyxoviruses (parainfluenza, mumps); measles virus (measles)
(-) strand; multiple strands of RNA; enveloped Orthomyxoviridae 80-200 nm influenza viruses A, B, and C (influenza)
Bunyaviridae 90-120 nm California encephalitis virus (encephalitis); hantaviruses (Hantavirus pulmonary syndrome, Korean hemorrhagic fever)
Arenaviridae 50-300 nm arenaviruses (lymphocytic choriomeningitis, hemorrhagic fevers)
produce DNA from (+) single-stranded RNA using reverse transcriptase; enveloped; bullet-shaped or polyhedral capsid Retroviridae 100-120 nm HIV-1 and HIV-2 (HIV infection/AIDS); HTLV-1 and HTLV-2 (T-cell leukemia)
dsRNA; naked; polyhedral capsid Reoviridae 60-80 nm reoviruses (mild respiratory infections, infant gastroenteritis); Colorado tick fever virus (Colorado tick fever)
Summary
1. Viruses can store their genetic information in six different types of nucleic acid which are named based on how that nucleic acid eventually becomes transcribed to the viral mRNA.
2. (+) and (-) strands of nucleic acid are complementary. Copying a (+) stand gives a (-) strand; copying a (-) stand gives a (+) strand.
3. Only (+) strands of viral RNA can be translated into viral protein.
4. Regarding the enzymes involved in nucleic acid replication, the "dependent" part of the name refers what type of nucleic acid is being copied. The "polymerase" part of the name refers what type of nucleic acid is being synthesized.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State what criteria are used in viral classification. (ans)
2. What would a DNA-dependent RNA-polymerase enzyme do? (ans)
10.05: Other Acellular Infectious Agents: Viroids and Prions
Learning Objectives
1. Define viroid and name an infection caused by a viroid.
2. Define prion and name 3 protein misfolding diseases that apprear to be initiated by prions.
Viroids and Prions
Viroids are even more simple than viruses. They are small, circular, single-stranded molecules of infectious RNA lacking even a protein coat. They are the cause of a few plant diseases such as potato spindle-tuber disease,cucumber pale fruit, citrus exocortis disease, and cadang-cadang (coconuts).
Prions are infectious protein particles responsible for a group of transmissible and/or inherited neurodegenerative diseases including Creutzfeldt-Jakob disease, kuru, and Gerstmann-Straussler-syndrome in humans, as well as scrapie in sheep and goats, and bovine spongiform encephalopathy (mad cow disease) in cattle and in humans (where it is called new variant Creutzfeldt–Jakob disease humans). The infections are often referred to as transmissible spongiform encephalopathies.
Most evidence indicates that the infectious prion proteins are modified (misfolded) forms of normal proteins coded for by a host gene in the brain. It is thought that the normal prion protein, expressed on stem cells in the bone marrow and on cells that will become neurons, plays a role in the maturation of neurons. In the case of the disease scrapie, the normal prion protein in an animal without the disease has alpha-helices in the proteins secondary structure (Figure \(1\)) while the scrapie prion protein in diseased animals has beta-sheets for the secondary structure (Figure \(2\)). When the scrapie prion protein contacts the normal protein it causes it to change its configuration to the scrapie beta-sheet form. This suggests that the conversion of a normal prion protein into an infectious prion protein may be catalyzed by the prion protein itself upon entering the brain. Inherited forms may be a result of point mutations that make the prion protein more susceptible to a change in its protein structure.
There is growing evidence that other probable protein misfolding diseases initiated by prions include Alzheimer's disease, Hunington's disease, Parkinson's disease, frontotemporal dementias, amyotrophic lateral sclerosis, and certain cancers.
Summary
1. Viroids are small, circular, single-stranded molecules of infectious RNA that cause several plant diseases.
2. Prions are infectious protein particles responsible for a group of transmissible and/or inherited neurodegenerative diseases as a result of prion protein misfolding.
3. Diseases including Creutzfeldt-Jakob disease Gerstmann-Straussler-syndrome, and mad cow disease.
4. There is growing evidence that other probable protein misfolding diseases initiated by prions include Alzheimer's disease, Hunington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and certain cancers. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.04%3A_Classification_of_Viruses.txt |
Viruses that infect animal cells replicate by what is called the productive life cycle. The productive life cycle is also often referred to as the lytic life cycle, even though not all viruses cause lysis of their host cell during their replication. Some viruses, such as HIV and the herpes viruses are able to become latent in certain cell types. A few viruses increase the risk of certain cancers. We will now look at the life cycles of viruses that infect animal cells.
10.06: Animal Virus Life Cycles: An Overview
Learning Objectives
1. When given information about a virus in terms of how it penetrates the host cell, whether it has a DNA or RNA genome, and how it is released, describe how an enveloped virus accomplishes each of the steps of the productive life cycle listed below. (Tailor the life cycle to that virus.)
1. viral attachment or adsorption to the host cell
2. viral entry into the host cell
3. viral movement to the site of replication within the host cell
4. viral replication within the host cell
5. viral assembly or maturation within the host cell
6. viral release from the host cell
2. When given information about a virus in terms of how it penetrates the host cell, whether it has a DNA or RNA genome, and how it is released, describe how a naked virus accomplishes each of the steps of the productive life cycle listed below. (Tailor the life cycle to that virus.)
1. viral attachment or adsorption to the host cell
2. viral entry into the host cell
3. viral movement to the site of replication within the host cell
4. viral replication within the host cell
5. viral assembly or maturation within the host cell
6. viral release from the host cell
For many animal viruses, the details of each step in their life cycle have not yet been fully characterized, and among the viruses that have been well studied there is great deal of variation. What follows is a generalized productive life cycle for animal viruses consisting of the following steps: adsorption, viral entry, viral movement to the site of replication and release of the viral genome from the remainder of the virus, viral replication, viral assembly, and viral release.
Viral Attachment or Adsorption to the Host Cell
Adsorption (Figures 1) involves the binding of attachment sites on the viral surface with receptor sites on the host cell cytoplasmic membrane.
For a virus to infect a host cell, that cell must have receptors for the virus on its surface and also be capable of supporting viral replication. These host cell receptors are normal surface molecules involved in routine cellular function, but since a portion of a molecule on the viral surface resembles the chemical shape of the body's molecule that would normally bind to the receptor, the virus is able to attach to the host cell's surface.
For example:
• Most human rhinoviruses that cause the common cold bind to intercellular adhesion molecules (ICAM-1) found on cells of the nasal epithelium. These ICAM-1 molecules are used normally for the recruitment of leukocytes into the respiratory tract.
• The human immunodeficiency viruses (HIV) adsorbs to first CD4 molecules and then chemokine receptors found on the surface of human T4-lymphocytes and macrophages . CD4 molecules are normally involved in immune recognition while chemokine receptors play a role in initiating inflammation and recruiting leukocytes.
• Human cytomegaloviruses (CMV) adsorb to MHC-I molecules . MHC-I molecules on human cells enable T8-lymphocytes to recognize antigens during adaptive immunity.
• The hepatitis B virus (HBV) adsorbs to IgA receptors on human cells. These receptors normally bind the antibody isotype IgA for transport across cells.
Viral Entry into the Host Cell
a. Enveloped viruses
Enveloped viruses enter the host cell in one of two ways:
1. In some cases, the viral envelope may fuse with the host cell cytoplasmic membrane and the nucleocapsid is released into the cytoplasm (see Figs. 2A, Figure \(2\)B and Figure \(2\)C).
2. Usually they enter by endocytosis , whereby the host cell cytoplasmic membrane invaginates and pinches off, placing the virus in an endocytic vesicle (see Figure \(3\)A, Figure \(3\)B, Figure \(3\)C, and Figure \(3\)D).
3D animation illustrating adsorption and penetration of the dengue fever virus.
Janet Iwasa, Gaël McGill (Digizyme) & Michael Astrachan (XVIVO). This animation takes some time to load.
b. Naked viruses
Naked viruses enter the cell in one of two ways:
1. In some cases, interaction between the viral capsid and the host cell cytoplasmic membrane causes a rearrangement of capsid proteins allowing the viral nucleic acid to pass through the membrane into the cytoplasm (see Figure \(4\)A, Figure \(4\)B, Figure \(4\)C, and Figure \(4\)D).
2. Most naked viruses enter by receptor-mediated endocytosis whereby the host cell cytoplasmic membrane invaginates and pinches off, placing the virus in an endocytic vesicle (see Figure \(5\)A, Figure \(5\)B, Figure \(5\)C, and Figure \(5\)D).
3. Viral Movement to the Site of Replication within the Host Cell and Release of the Viral Genome from the Remainder of the Virus.
In the case of viruses that enter by endocytosis, the endocytic vesicles containing the virus move within the host cell. During this process the pH of the endocytic vesicle typically decreases and this enables the virus to leave the endocytic vesicle. Viruses exit the endocytic vesicle through a variety of mechanisms, including:
a. Fusion of the viral envelope with the membrane of the endocytic vesicle enabling the viral nucleocapsid to enter the cytoplasm of the host cell (see Figure \(7\)A, Figure \(7\)B, and Figure \(7\)C).
b. Lysis of the endocytic vesicle releasing the viral nucleocapsid into the cytoplasm of the host cell (see Figure \(7\)D , and Figure \(7\)E).
c. The viral capsid undergoing conformational changes that forms pores in the endocytic vesicle enabling the virial genome to enter the cytoplasm of the host cell (see Figure \(9\)A, Figure \(9\)B, and Figure \(9\)C).
Flash animation showing viral capsid undergoing conformational changes that forms pores in the endocytic vesicle and enable the virial genome to enter the cytoplasm.
html5 version of animation for iPad showing viral capsid undergoing conformational changes that forms pores in the endocytic vesicle and enable the virial genome to enter the cytoplasm.
Before viruses can replicate within the infected host cell, the viral genome needs to released from the remainder of the virus. This process is sometimes referred to as uncoating.
In the case of most viruses with an RNA genome, the viral RNA genome is released from the capsid and enters the cytoplasm of the host cell (see Figure \(8\)A , and Figure \(8\)B) where replication generally occurs.
Flash animation showing release of the viral genome from the capsid (uncoating).
html5 version of animation for iPad showing release of the viral genome from the capsid (uncoating).
In the case of most viruses with a DNA genome, the viral genome enters the nucleus of the host cell through one the mechanisms shown below. Most larger DNA viruses use either a or b to enter the nucleus. Method c is used by some very small DNA whose capsid is small enough to be carried through the nuclear pores.
a. The viral DNA genome is released from the capsid, enters the cytoplasm of the host cell, and subsequently enters the nucleus of the host cell through the pores in the nuclear membrane (see Figure \(9\)D and Figure \(9\)E).
b. The capsid of the viruses interacts with the nuclear membrane of the host cell enabling the viral DNA genome to enter the nucleus of the host cell via the pores in the nuclear membrane (see Figure \(9\)F and Figure \(9\)G).
c. The nucleocapsid of a small DNA virus enters the nucleus of the host cell and the capsid is subsequently removed releasing the viral DNA genome into the nucleoplasm (see Figure \(9\)H and Figure \(9\)I).
This uncoating begins the eclipse period , the period during which no intact virions can be detected within the cell. After uncoating and during the replication stage the virus is not infectious.
4. Viral Replication within the Host Cell
The viral genome directs the host cell's metabolic machinery (ribosomes, tRNA, nutrients, energy, enzymes, etc.) to synthesize viral enzymes and viral parts. The viral genome has to both replicate itself and become transcribed into viral mRNA molecules. The viral mRNA can then be translated by the host cell's ribosomes into viral structural components and enzymes need for replication and assembly of the virus.
As mentioned earlier under Viral Classification, viruses can store their genetic information in six different types of nucleic acid which are named based on how that nucleic acid eventually becomes transcribed to the viral mRNA:
a. (+/-) double-stranded DNA (see Figure \(10\)A). To replicate the viral genome, DNA-dependent DNA polymerase enzymes (usually provided by the cell) copy both the (+) and (-) DNA strands producing dsDNA viral genomes. To produce viral mRNA molecules, host cell-DNA-dependent RNA polymerase enzymes copy the (-) DNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Examples include most bacteriophages, Papovaviruses, Adenoviruses, and Herpesviruses.
b. (+) single-stranded DNA (see Figure \(10\)B). To replicate the viral genome, DNA-dependent DNA polymerase enzymes (usually provided by the cell) copy the (+) DNA strand of the genome producing a dsDNA intermediate. DNA-dependent DNA polymerase enzymes (again, usually provided by the cell) then copy the (-) DNA strand into ss (+) DNA genomes. To produce viral mRNA molecules, host cell-DNA-dependent RNA polymerase enzymes copy the (-) DNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Examples include Phage M13 and Parvoviruses.
c. (+/-) double-stranded RNA (see Figure \(10\)C) . To replicate the viral genome, viral RNA-dependent RNA polymerase enzymes (replicase) copy both the (+) RNA and (-) RNA strands of the genome producing a dsRNA genomes. To produce viral mRNA molecules, viral RNA-dependent RNA polymerase enzymes (transcriptase) copy the (-) RNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Reoviruses are an example.
d. (-) RNA (see Figure \(10\)D). To replicate the viral genome, viral RNA-dependent RNA polymerase enzymes (transcriptase) copy the (-) RNA genome producing ss (+) RNA. Transcriptase must be carried into the cell with the virion. Viral RNA-dependent RNA polymerase enzymes (replicase) then copy the (+) RNA strands producing ss (-) RNA viral genome. The (+) mRNA strands also function as viral mRNA and can then be translated into viral proteins by host cell ribosomes. Examples include Orthomyxoviruses, Paramyxoviruses, Rhabdoviruses.
e. (+) RNA (see Figure \(10\)E). To replicate the viral genome, viral RNA-dependent RNA polymerase enzymes (replicase) copy the (+) RNA genome producing ss (-) RNA. Viral RNA-dependent RNA polymerase enzymes (replicase) then copy the (-) RNA strands producing ss (+) RNA viral genome. To produce viral mRNA molecules. RNA-dependent RNA polymerase enzymes (replicase) copy the (-) RNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Examples include Picornaviruses, Togaviruses, and Coronaviruses.
f. (+) RNA Retroviruses (see Figure \(10\)F). To replicate the viral genome, viral reverse transcriptase enzymes (RNA-dependent DNA polymerases) copy the (+) RNA genome producing ss (-) DNA strands. Viral reverse transcriptase can also function as a DNA-dependent DNA polymerase enzymes and will copy the (-) DNA strands to produce a dsDNA intermediate. Reverse transcriptase must be carried into the cell with the virion. The viral DNA will move to the nucleus where it integrates into the cell’s DNA using the viral enzyme integrase which also must be carried into the host cell with the virion. Once in the host cell’s DNA, host cell DNA-dependent RNA polymerase enzymes then copy the ds (-) DNA strands to produce ss (+) RNA genomes. To produce viral mRNA molecules, host cell DNA-dependent RNA polymerase enzymes copy the ds (-) DNA strand into (+) viral mRNA. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes. Retroviruses, such as HIV-1, HIV-2, and HTLV-1 are examples.
As the host cell's ribosomes attach to the viral mRNA molecules, the mRNAs are translated into viral structural proteins and viral enzymes. During the early phase of replication, proteins needed for the replication of the viral genome are made and the genome makes thousands of replicas of itself. During the late phase of replication, viral structural proteins (capsid and matrix proteins, envelope glycoproteins, etc.) and the enzymes involved in maturation are produced.
Some viral mRNAs are monocistronic, that is, they contain genetic material to translate only a single protein or polypeptide. Other viral mRNAs are polycistronic. They contain transcripts of several genes and are translated into one or more large polyproteins. These polyproteins are subsequently cut into individual functional proteins by viral enzymes called proteases.
In the case of most RNA viruses, replication and assembly occurs in the host cell's cytoplasm. With DNA viruses, most replication and assembly occurs in the nucleus of the host cell. The viral genome enters the nucleus of the host cell and here is transcribed into viral mRNA. The viral mRNA molecules then leave the nucleus through the pores in the nuclear membrane and are translated into viral proteins by the host cell's ribosomes in the cytoplasm. Most of these viral proteins then re-enter the nucleus where the virus assembles around the replicated genomes.
• Transmission electron micrograph of Herpes simplex viruses in the nucleus of an infected host cell; courtesy of CDC.
Also during replication, viral envelope proteins and glycoproteins coded by the viral genome are incorporated into the host cell's cytoplasmic membrane (see Figure \(11\)A and Figure \(11\)B) or nuclear membrane.
Flash animation showing viral replication.
Whether a virus has an RNA or a DNA genome is significant when it comes to developing antiviral agents to control viruses. In the case of RNA viruses, all of the enzymes used in genome replication and transcription are viral encoded enzymes different from those of the host cell so these enzymes can potentially be targeted. On the other hand, DNA viruses use the host cell's RNA transcription machinery and DNA replication machinery so these enzymes, shared by the virus and the host cell, cannot be targeted without killing the host cell. Since all viruses use the host cell's translation machinery regardless of genome type, translation can not be targeted in any viruses.
5. Viral Assembly or Maturation within the Host Cell
During maturation, the capsid is assembled around the viral genome .
Viral Release from the Host Cell
a. Naked viruses
Naked viruses are predominantly released by host cell lysis (see Figure \(13\) C). While some viruses are cytolytic and lyse the host cell more or less directly, in many cases it is the body's immune defenses that lyse the infected cell.
b. Enveloped viruses
With enveloped viruses, the host cell may or may not be lysed. The viruses obtain their envelopes from host cell membranes by budding. As mentioned above, prior to budding, viral proteins and glycoproteins are incorporated into the host cell's membranes. During budding the host cell membrane with incorporated viral proteins and glycoproteins evaginates and pinches off to form the viral envelope. Budding occurs either at the outer cytoplasmic membrane, the nuclear membrane, or at the membranes of the Golgi apparatus .
1. Viruses obtaining their envelope from the cytoplasmic membrane are released during the budding process (see Figure \(14\)A and Figure \(14\)B).
2. Viruses obtaining their envelopes from the membranes of the nucleus, the endoplasmic reticulum, or the Golgi apparatus are then released by exocytosis via transport vesicles (see Figure \(15\)A and Figure \(15\)B).
Some viruses, capable of causing cell fusion, may be transported from one cell to adjacent cells without being released, that is, they are transmitted by cell-to-cell contact whereby an infected cell fuses with an uninfected cell (see Figure \(16\)A, Figure \(16\)B, and Figure \(16\)C).
Reinfection
As many as 10,000 to 50,000 animal viruses may be produced by a single infected host cell.
Exercise: Think-Pair-Share Questions
1. Animal viruses adsorb to receptors on the cytoplasmic membrane of host cells.
Why would our cells possess receptors that viruses could adsorb too?
1. When we vaccinate against viral infections such as measles, mumps, rubella, poliomyelitis, and chickenpox, we inject an attenuated or inactivated form of the virus. The body responds by making antibodies that coat the surface of that virus by binding to its surface proteins or glycoproteins.
Briefly describe two ways this may prevent future infections with this virus.
Nice Animation with Simplistic Explanation of the Replication of Influenza Viruses.
created for NPR by medical animator, David Bolinsky
The dengue virus is an RNA virus that enters by endocytosis, gets its envelope by budding into the endoplasmic reticulum, and is packaged by the Golgi apparatus and released by exocytosis.
Dengue fever is a mosquito-borne viral infection found mainly in tropical areas. Often asymptomatic and self-limiting but when symptoms do appear, they can include joint and muscle pain, headache, and a rash that may become hemorrhagic. The virus replicates in macrophages.
Courtesy of HHMI's Biointeractive.
Summary
1. For a virus to infect a host cell, that cell must have receptors for the virus on its surface and also be capable of supporting viral replication.
2. Adsorption involves the binding of attachment sites on the viral surface with receptor sites on the host cell cytoplasmic membrane.
3. Once adsorbed, many viruses enter the host cell by endocytosis, whereby the host cell cytoplasmic membrane invaginates and pinches off, placing the virus in an endocytic vesicle. Some viruses enter by a fusion process whereby part of the virus fuses with the host cell enabling the remainder of the virus to enter the host cell’s cytoplasm.
4. Following entry, the virus moves to the site of replication within the host cell. Most RNA viruses replicate in the host cell’s cytoplasm; most DNA viruses replicate in the host cell’s nucleus.
5. During replication, the viral genome directs the host cell's metabolic machinery (ribosomes, tRNA, nutrients, energy, enzymes, etc.) to synthesize viral enzymes and viral parts. The viral genome has to both replicate itself and become transcribed into viral mRNA molecules. The viral mRNA can then be transcribed by the host cell into viral structural components and enzymes need for replication and assembly of the virus.
6. During maturation, the capsid is assembled around the viral genome.
7. Prior to or during release, enveloped viruses obtain their envelopes from host cell membranes by budding. Budding occurs either at the outer cytoplasmic membrane, the nuclear membrane, or at the membranes of the Golgi apparatus.
8. Viruses obtaining their envelopes from the membranes of the nucleus, the endoplasmic reticulum, or the Golgi apparatus are then released by exocytosis via transport vesicles; viruses obtaining their envelope from the cytoplasmic membrane are released during the budding process.
9. Naked viruses are predominantly released by host cell lysis.
10. As many as 10,000 to 50,000 animal viruses may be produced by a single infected host cell.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. An enveloped virus enters by fusion, has an RNA genome, and is released by budding. Describe how it accomplishes each of the following steps during its productive life cycle.
1. viral attachment or adsorption to the host cell (ans)
2. viral entry into the host cell (ans)
3. viral movement to the site of replication within the host cell (ans)
4. viral replication within the host cell (ans)
5. viral assembly or maturation within the host cell (ans)
6. viral release from the host cell (ans)
2. When a virus infects the body, the body responds by producing antibodies that coat the virion. Discuss briefly how this might offer protection to the body. (ans)
3. Why are viruses generally very specific as to the types of hosts, tissues, and cells they are able to infect? (ans)
4. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.06%3A_Animal_Virus_Life_Cycles%3A_An_Overview/10.6A%3A_The_Productive_Life_Cycle_of_Animal_Viruses.txt |
State the major difference between the productive life cycle of animal viruses and the latent life cycle. Define provirus. Name 3 herpes viruses that may have a latent cycle, state in what cell types they become latent, and name the diseases each cause.
Some animal viruses, such as the herpes viruses and a group of viruses known as the retroviruses, are able to remain latent within infected host cells for long periods of time without replicating or causing harm. Some of these viruses remain latent within the cytoplasm of the host cell while others are able to insert or integrate their DNA into the host cell's chromosomes. When the viral DNA is incorporated into the host cell's DNA, it is called a provirus.
In many instances, viral latency, as well as viral persistence, is thought to be due to a process called RNA interference (RNAi) where small non-coding regulatory RNAs (ncRNAs) such as microRNAs (miRNAs) regulate gene expression. Certain viruses that infect humans are able to establish persistent infection by using their own miRNAs and/or miRNAs produced by their human host.
For example, viral and/or host miRNAs may bind to certain viral messenger RNA (mRNA) molecules and block translation of viral proteins required for rapid viral replication, or they may bind to the mRNA of human genes that produce proteins used in viral replication. The resulting low viral levels may then minimize immune responses against that virus. In addition, these miRNAs may directly affect host immune defenses by turning off the production of antiviral cytokinesor by blocking apoptosisof infected host cells. Examples include the herpesviruses, retroviruses, and anelloviruses.
Herpes viruses, for example, are often latent in some cell types but productive in others. Herpes viruses include herpes simplex type 1 (HSV-1) which usually causes fever blisters or oral herpes, herpes simplex type 2 (HSV-2) which usually causes genital herpes, Epstein-Barr virus (EBV) which causes infectious mononucleosis and plays a role in certain cancers, varicella-zoster virus (VZV) which causes chickenpox and shingles, and cytomegalovirus (CMV) which causes a variety of infections in immunosuppressed persons and is also a leading cause of birth defects.
For more on HSV and CMV, see the AIDS Pathology Tutorial at the University of Utah.
Herpesviruses use both host and viral miRNAs to switch between the productive life cycle in infected epithelial cells whereby large numbers of viruses are produced and the infected host cells are killed (as in the case of fever blisters) and the persistent latent state in nerve cells where low levels of viruses are produced and the infected host cells are not killed by apoptosis.
With EBV, the virus is productive in epithelial cells but latent in B-lymphocytes.
In the case of HSV-1, HSV-2, and VZV, primary infection causes the virus to replicate within epithelial cells. However, some of the viruses enter and migrate down neurons where they become latent in the body of sensory neurons. Subsequent activation of the latently infected neurons by a variety of extracellular stimuli enables the viruses to migrate back up the nerve cell and replicate again in the epithelial cells. With EBV, the virus is productive in epithelial cells but latent in B-lymphocytes.
- Scanning electron micrograph of HSV; courtesy of Dennis Kunkel's Microscopy.
In the case of HIV, the viral genome eventually becomes a provirus. After integration, the HIV proviral DNA can exist in either a latent or productive state, which is determined by genetic factors of the viral strain, the type of cell infected, and the production of specific host cell proteins.
The majority of the proviral DNA is integrated into the chromosomes of activated T4-lymphocytes. These generally comprise between 93% and 95% of infected cells and are productively infected, not latently infected. However, a small percentage of HIV-infected memory T4-lymphocytes persists in a resting state because of a latent provirus. Subsequent activation of the host cell by extracellular stimuli, however, causes the needed proteins to be made and the virus again replicates via the productive life cycle. These memory T4-lymphocytes, along with infected monocytes, macrophages, and dendritic cells, provide stable reservoirs of HIV capable of escaping host defenses and antiretroviral chemotherapy.
In the next section we will now look at the life cycle of HIV.
Summary
1. For a virus to infect a host cell, that cell must have receptors for the virus on its surface and also be capable of supporting viral replication.
2. Adsorption involves the binding of attachment sites on the viral surface with receptor sites on the host cell cytoplasmic membrane.
3. Once adsorbed, many viruses enter the host cell by endocytosis, whereby the host cell cytoplasmic membrane invaginates and pinches off, placing the virus in an endocytic vesicle. Some viruses enter by a fusion process whereby part of the virus fuses with the host cell enabling the remainder of the virus to enter the host cell’s cytoplasm.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Define provirus. (ans)
2. Name 4 herpes viruses that may have a latent cycle, state in what cell types they become latent, and name the diseases each cause.
1. (ans)
2. (ans)
3. (ans)
3. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.06%3A_Animal_Virus_Life_Cycles%3A_An_Overview/10.6B%3A_Productive_Life_Cycle_with_Possible_Latency.txt |
Learning Objectives
1. Describe how the retrovirus HIV-1 accomplishes each of the following steps during its life cycle. (Include the following key words in your description: gp120, CD4, chemokine receptors, gp41, capsid, RNA genome, reverse transcriptase, double-stranded DNA intermediate, provirus, polyproteins, proteases, and budding.)
1. viral attachment or adsorption to the host cell
2. viral entry into the host cell
3. viral movement to the site of replication within the host cell and production of a provirus.
4. viral replication within the host cell
5. viral assembly or maturation within the host cell and release from the host cell
2. Name 3 types of cells HIV primarily infects and briefly explain why.
The Structure of the Human Immunodeficiency Virus (HIV)
HIV (see HIV A, HIV B and HIV C) has an envelope derived from host cell membranes during replication. Associated with the envelope are two HIV-encoded glycoproteins, gp120 and gp41. Underneath the envelope is a protein matrix composed of p17. Inside the virus is a capsid or core made of the protein p24. The nucleocapsid also contains p6, p7, reverse transcriptase (p66/p51), integrase (p32), protease (p10), and 2 molecules of single-stranded RNA, the viral genome (see Figure \(3\)).
To view further electron micrographs of HIV, see the AIDS Pathology Tutorial at the University of Utah.
The Life Cycle for the Human Immunodeficiency Virus (HIV)
1. Attachment or Adsorption to the Host Cell
Initially, HIV uses a cellular protein called cyclophilin that is a component of its envelope to bind a low affinity host cell receptor called heparin. This first interaction (not shown in the illustrations or animations) enables the virus to initially make contact with the host cell. In order to infect a human cell, however, an envelope glycoprotein on the surface of HIV called gp120 must adsorbs to both a CD4 molecule and then a chemokine receptor found on the surface of only certain types of certain human cells.
Human cells possessing CD4 molecules include:
• T4-helper lymphocytes (also called T4-cells and CD4+ cells)
• monocytes
• macrophages
• dendritic cells
Chemokines are cytokines that promote an inflammatory response by pulling white blood cells out of the blood vessels and into the tissue to fight infection. Different white blood cells have receptors on their surface for different chemokines. The chemokine receptors are now thought to determine the type of CD4+ cell HIV is able to infect. First, a portion or domain of the HIV surface glycoprotein gp120 binds to its primary receptor, a CD4 molecule on the host cell. This induces a change in shape that enables the chemokine receptor binding domains of the gp120 to interact with a host cell chemokine receptor. The chemokine receptor functions as the viral co-receptor. This interaction brings about another conformational change that exposes a previously buried portion of the transmembrane glycoprotein gp41 called the fusion peptide that enables the viral envelope to fuse with the host cell membrane (see Figure \(1\)A, Figure \(1\)B), and Figure \(1\)C).
Animation: Adsorption of HIV to a T4-Helper Lymphocyte. The HIV envelope gp120 must attach to both a CD4 molecule and a chemokine receptor on the surface of such cells as macrophages and T4-helper lymphocytes in order to enter the cell. The gp120 first binds to a CD4 molecule on the plasma membrane of the host cell. The interaction between the gp120 and the CD4 molecule on the host cell induces a change in shape that brings the chemokine receptor binding domains of the gp120 into proximity with the host cell chemokine receptor
YouTube animation illustrating adsorption and penetration of HIV.
Most strains of HIV are referred to as M-tropic or T-tropic. The gp120 of M-tropic HIV (see Figure \(2\)) is able to adsorb to the CD4 molecules and the CCR5 chemokine receptors found on CD4+ macrophages, immature dendritic cells, and memory T4-lymphocytes. (M-tropic HIV are also called R5 viruses since they adsorb to the chemokine receptor CCR5.) M-tropic HIV require only low levels of CD4 molecules expressed on the surface of the host cell for infection. M-tropic HIV are thought to spread the infection. These strains appear to be slower-replicating and less virulent than the later T-tropic strains and do not cause the formation of syncytias. HIV initially replicates to high levels within macrophages without destroying them. (The T-tropic HIV, found later in HIV infection, are faster-replicating, more virulent, and lead to syncytia formation.)
As time goes by, mutation in the gene coding for gp120 enables some of the HIV to become dual tropic and able to infect both macrophages via the CCR5 chemokine receptor found on these cells, and T4-lymphocytes via the CCR5 and CXCR4 chemokine receptors found on these cells. (Duel-tropic HIV are also called R5X4 viruses since they adsorb to both the chemokine receptors CCR5 and CXCR4.)
Later during the course of HIV infection, most of the viruses have mutated their gp120 to become T- tropic (see Figure \(2\)) and infect primarily mature dendritic cells and T4-lymphocytes by way of CD4 and the CXCR4 co-receptors found on these cells. (T-tropic HIV are also called X4 viruses since they adsorb to the chemokine receptor CXCR4.) T-tropic HIV require high levels of CD4 molecules expressed on the surface of the host cell for infection. As mentioned, these T-tropic strains of HIV are faster-replicating and more virulent, and cause formation of syncytias and begin the cycles of T4-lymphocyte destruction.
HIV infecting microglia cells in the brain appear to bind to a CD4 molecule and a chemokine receptor called CCR3 found on these macrophage-like cells.
2. Viral Entry into the Host Cell
As mentioned above under adsorption, the binding of a portion or domain of the HIV surface glycoprotein gp120 to a CD4 molecule on the host cell induces a change in shape that brings the chemokine receptor binding domains of the gp120 into proximity with the host cell chemokine receptor. This, in turn, brings about a conformational change that exposes a previously buried portion of the transmembrane glycoprotein gp41 enabling the viral envelope to fuse with the host cell membrane (see Figure \(5\) and Figure \(6\)). After fusion of the viral envelope with the host cell cytoplasmic membrane, the genome-containing protein core of the virus enters the host cell's cytoplasm. (Occasionally the virus enters by endocytosis, after which the viral envelope fuses with the endocytic vesicle releasing the genome-containing core into the cytoplasm.)
Animation: Penetration of HIV into Host Cell. The binding of a portion or domain of the HIV surface glycoprotein gp120 to a CD4 molecule on the host cell induces a change in shape that brings the chemokine receptor binding domains of the gp120 into proximity with the host cell chemokine receptor. This, in turn, brings about a conformational change that exposes a previously buried portion of the transmembrane glycoprotein gp41 enabling the viral envelope to fuse with the host cell membrane. After fusion of the viral envelope with the host cell cytoplasmic membrane, the genome-containing protein core of the virus enters the host cell's cytoplasm.
3. Viral Movement to the Site of Replication within the Host Cell and Production of a Provirus
During uncoating, the single-stranded RNA genomes within the core or capsid of the virus are released into the cytoplasm. HIV now uses the enzyme reverse transcriptase, associated with the viral RNA genome, to make a DNA copy of the RNA genome. (Normal transcription in nature is when the DNA genome is transcribed into mRNA which is then translated into protein. In HIV reverse transcription, RNA is reverse-transcribed into DNA.)
Reverse transcriptase has three enzyme activities:
1. It has RNA-dependent DNA polymerase activity that copies the viral (+) RNA into a (-) viral complementary DNA (cDNA);
2. It has ribonuclease activity that degrades the viral RNA during the synthesis of cDNA; and
3. It has DNA-dependent DNA polymerase activity that copies the (-) cDNA strand into a (+) DNA to form a double-stranded DNA intermediate.
As the cDNA is being synthesized off of the RNA template the ribonuclease activity degrades the viral RNA genome (see Figure \(7\)A, Figure \(7\)B, and Figure \(7\)C). The reverse transcriptase then makes a complementary DNA strand to form a double-stranded viral DNA intermediate (see Figure \(7\)D).
Animation: HIV Copying RNA into DNA with Reverse Transcriptase. The single-stranded RNA genomes are released from the capsid. HIV uses the enzyme reverse transcriptase to transcribe its RNA genome into single-stranded DNA. As the DNA is being made, the RNA genome is degraded by an RNase. The reverse transcriptase then synthesizes a complementary DNA strand to produce a double-stranded DNA intermediate that enters the infected host cell's nucleus.
A viral enzyme called integrase then binds to the double-stranded viral DNA intermediate, transports it through the pores of the host cell's nuclear membrane, and inserts into one of the host cell's chromosomes to form a provirus (see Figure \(8\)A and Figure \(8\)B).
Animation: Formation of a Provirus. An HIV enzyme called integrase is used to insert the HIV double-stranded DNA intermediate into the DNA of a host cell's chromosome. HIV is now a provirus.
After integration, the HIV proviral DNA can exist in either a latent or productive state, which is determined by genetic factors of the viral strain, the type of cell infected, and the production of specific host cell proteins.
The majority of the proviral DNA is integrated into the chromosomes of activated T4-lymphocytes. These generally comprise between 93% and 95% of infected cells and are productively infected, not latently infected. However, a small percentage of HIV-infected memory T4-lymphocytes persists in a resting state because of a latent provirus. These, along with infected monocytes, macrophages, and dendritic cells, provide stable reservoirs of HIV capable of escaping host defenses and antiretroviral chemotherapy.
4. Replication of HIV within the Host Cell
The vast majority of T4-lymphocytes, which are productively infected, immediately begin producing new viruses. In the case of the small percentage of infected, resting memory T4-lymphocytes, before replication can occur, the HIV provirus must become activated. This is accomplished by such means as antigenic stimulation of the infected T4-lymphocytes or their activation by factors such as cytokines, endotoxins, and superantigens.
Following activation of the provirus, molecules of (+) mRNA are transcribed off of the (-) proviral DNA strand by the enzyme RNA polymerase II. Once synthesized,HIV mRNA goes through the nuclear pores into the rough endoplasmic reticulum to the host cell's ribosomes where it is translated into HIV structural proteins, enzymes, glycoproteins, and regulatory proteins(see Figure \(3\)).
A 9 kilobase mRNA is formed that is used for three viral functions:
a. Synthesis of Gag polyproteins (p55). These polyproteins will eventually be cleaved by HIV proteases to become HIV matrix proteins (MA; p17), capsid proteins (CA; p24), and nucleocapsid proteins (NC, p7). See Figure \(9\)A and Figure \(9\)B.
b. Synthesis of Gag-Pol polyproteins (p160). These polyproteins will eventually be cleaved by HIV proteases to become HIV matrix proteins (MA; p17), capsid proteins (CA; p24), proteinase molecules (protease or PR; p10), reverse transcriptase molecules (RT; p66/p51), and integrase molecules (IN; p32). See Figure \(9\)C and Figure \(9\)D.
c. During maturation, these RNA molecules also become the genomes of new HIV virions.
The 9kb mRNA can also be spliced to form a 4kb mRNA and a 2kb mRNA.
The 4kb mRNA is used to:
a. Synthesize the Env polyproteins (gp160). These polyproteins will eventually be cleaved by proteases to become HIV envelope glycoproteins gp120 and gp41. See Figure \(9\)E and Figure \(9\)F.
b. Synthesize 3 regulatory proteins called vif, vpr, and vpu.
The 2kb mRNA is used to synthesize 3 regulatory proteins known as tat, rev, and naf.
5. Viral Assembly or Maturation within the Host Cell and Release from the Host Cell
Assembly of HIV virions begins at the plasma membrane of the host cell. Maturation occurs either during the budding of the virion from the host cell or after its release from the cell.
Prior to budding, the Env polyprotein (gp160) goes through the endoplasmic reticulum and is transported to the Golgi complex where it is cleaved by a protease (proteinase) and processed into the two HIV envelope glycoproteins gp41 and gp120. These are transported to the plasma membrane of the host cell where gp41 anchors the gp120 to the membrane of the infected cell. See Figure \(10\)A, Figure \(10\)B, Figure \(10\)C, and Figure \(10\)D.
GIF animation showing maturation of gp41 and gp120.
The Gag (p55) and Gag-Pol (p160) polyproteins also associate with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud from the host cell.
During maturation, HIV proteases (proteinases) will cleave the remaining polyproteins into individual functional HIV proteins and enzymes such as matrix proteins (MA; p17), capsid proteins (CA; p24), reverse transcriptase molecules (RT; p66/p51), and integrase molecules (IN; p32).. See Figure \(10\)E, Figure \(10\)F, Figure \(10\)G, and Figure \(10\)H.
a. The Gag polyproteins (p55) will be cleaved by HIV proteases to become HIV matrix proteins (MA; p17), capsid proteins (CA; p24), and nucleocapsid proteins (NC, p7 and p6).
b. The Gag-Pol polyproteins (p160) will be cleaved by HIV proteases to become HIV matrix proteins (MA; p17), capsid proteins (CA; p24), proteinase molecules (protease or PR; p10), reverse transcriptase molecules (RT; p66/p51), and integrase molecules (IN; p32).
The various structural components then assemble to produce a mature HIV virion.
GIF animation showing maturation of of HIV.
6. Reinfection
Free viruses now infect new susceptible body cells. HIV can also be transmitted by cell-to-cell contact. This can occur when an infected cell with gp120 on its cytoplasmic membrane attaches to CD4 molecules and chemokine receptors on the surface of an uninfected cell. The cells then fuse (see Figure \(11\) and Figure \(12\)).
Excellent Animation Summarizing the Life Cycle of HIV
Courtesy of HHMI's Biointeractive.
YouTube Animation Illustrating Reproduction of HIV.
Courtesy of 3D Medical Animations Library, Dr. Rufus Rajadurai
Exercise: Think-Pair-Share Questions
1. State the role(s) of gp120 and gp41 in the life cycle of HIV.
2. Why does HIV primarily infect T4-lymphocytes, macrophages, and dendritic cells?
3. How do antiretroviral drugs that bind to HIV-encoded protease help to reduce the number of HIV in the body.
4. If one could destroy all of the infected white blood cells in a person infected with HIV and then reconstitute the cells by giving a bone marrow transplant from a person homozygous for a deletion mutation in their gene coding for the chemokine receptor CCR5 (he or she can not make CCR5 molecules), describe how this might prevent HIV infection in the person receiving the transplant.
Medscape article on infections associated with organisms mentioned in this Learning Object. Registration to access this website is free.
• HIV Infection and AIDS
Summary
1. During adsorption, an envelope glycoprotein on the surface of HIV called gp120 must adsorbs to both a CD4 molecule and then a chemokine receptor found on the surface of only certain types of certain human cells such as T4-lymphocytes, monocytes, macrophages, and dendritic cells.
2. Following adsorption, glycoprotein gp41 enabling the viral envelope to fuse with the host cell membrane, allowing the nucleocapsid of the virus enters the host cell's cytoplasm.
3. During uncoating, the single-stranded RNA genomes within the capsid of the virus are released into the cytoplasm and HIV now uses the enzyme reverse transcriptase to make a single-stranded DNA copy of its single-stranded RNA genome. The reverse transcriptase then makes a complementary DNA strand to form a double-stranded viral DNA intermediate.
4. A viral enzyme called integrase then binds to the double-stranded viral DNA intermediate, transports it through the pores of the host cell’s nuclear membrane, and inserts into one of the host cell's chromosomes to form a provirus.
5. Following activation of the provirus, molecules of mostly polycistronic mRNA are transcribed off of the proviral DNA strand, go through the nuclear pores into the rough endoplasmic reticulum where it is translated by host cell's ribosomes HIV structural proteins, enzymes, glycoproteins, and regulatory proteins.
6. Polyproteins translated from polycistronic mRNAs must be cleaved into function proteins by HIV protease enzymes.
7. The two HIV envelope glycoproteins gp41 and gp120 are transported to the plasma membrane of the host cell where gp41 anchors the gp120 to the membrane of the infected cell. HIV obtains its envelope from the plasma membrane by budding.
8. Most maturation occurs either during the budding of the virion from the host cell or after its release from the cell.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Describe how the retrovirus HIV-1 accomplishes each of the following steps during its life cycle. (Include the following key words in your description: gp120, CD4, chemokine receptors, gp41, capsid, RNA genome, reverse transcriptase, double-stranded DNA intermediate, provirus, polyproteins, proteases, and budding.)
1. viral attachment or adsorption to the host cell (ans)
2. viral entry into the host cell (ans)
3. viral movement to the site of replication within the host cell and production of a provirus. (ans)
4. viral replication within the host cell (ans)
5. viral assembly or maturation within the host cell and release from the host cell (ans)
2. Name 3 types of cells HIV primarily infects and briefly explain why. (ans)
3. HIV possesses a genome of RNA. How then is HIV able to insert into the DNA of host cells and form a provirus? (ans)
4. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.06%3A_Animal_Virus_Life_Cycles%3A_An_Overview/10.6C%3A_The_Life_Cycle_of_HIV.txt |
State the median incubation period for AIDS and, in terms of viral load, exhaustion of the lymphopoietic system, and immune responses, briefly describe what marks the progression to AIDS. Briefly describe the following: early or acute HIV infection chronic HIV infection AIDS
According to WHO estimates from 2004, HIV has now infected 50 to 60 million people worldwide. The virus has killed over 22 million children adults and has left 14 million children orphaned. Worldwide, over 42 million people are currently living with HIV infection/AIDS - approximately 70% of these live in Africa, 20% in Asia. Around 3 million people die each year of AIDS and it is estimated that each day 14,000 people in the world become newly infected with HIV.
The median incubation period for AIDS is around 10 years. During early or acute HIV infection the virus primarily infects and destroys memory T4-lymphocytes which express the chemokine receptor CCR5 and are very abundant in mucosal lymphoid tissues. Here HIV also encounters the dendritic cells located throughout the epithelium of the skin and the mucous membranes where in their immature form called Langerhans cells they are attached by long cytoplasmic processes. The envelope glycoproteins gp41 and gp120 of HIV contain mannose-rich glycans that bind to mannan-binding proteins (pattern recognition receptors; also called lectin receptors) on the dendritic cells.
Upon capturing antigens through pinocytosis and phagocytosis and becoming activated by pro-inflammatory cytokines, the dendritic cells detach from the epithelium, enter lymph vessels, and are carried to regional lymph nodes. By the time they enter the lymph nodes, the dendritic cells have matured and are now able to present antigens of HIV to naive T-lymphocytes located in the the lymph nodes in order to induce adaptive immune responses.
At this point the infection has transitioned from the acute phase to the chronic phase. The chronic phase of HIV infection is characterized by viral dissemination, viremia, and induction of adaptive immune responses. The viremia allows the viruses to spread and infect T4-helper lymphocytes, macrophages, and dendritic cells found in peripheral lymphoid tissues.
During the chronic phase of HIV infection, the lymph nodes and the spleen become sites for continuous viral replication and host cell destruction. During most of this phase, the immune system remains active and competent and there are few clinical symptoms. A steady state-infection generally persists where T4-lymphocyte death and T4-lymphocyte replacement by the body are in equilibrium. In a person infected with HIV, somewhere between one and two billion of these T4-cells die each day as a result of HIV infection and must be replaced by the body's lymphopoietic system in the bone marrow. It is estimated that 10 billion virions are produced and cleared in an infected individual each day. However, the enormous turnover of T4-lymphocytes eventually exhausts the lymphopoietic system and it becomes unable to replace the T4-cells being destroyed. A variety of mechanisms then eventually lead to immunodeficiency.
Mechanisms of HIV-induced immunodeficiency include:
• Direct HIV-induced cytopathic effect on infected T4-lymphocytes. This can occur through:
• Increased cell permeability as a result of gp41 expression in the host cell membrane and viral release by budding;
• Inhibition of host cell protein synthesis as a result of viral replication within the infected cell; and
• Fusion of infected T4-cells with numerous uninfected T4-cells resulting in syncytia formation.
• Killing of HIV-infected T4-cells by cytotoxic T-lymphocytes or CTLs.
• Killing of HIV-infected T4-cells by antibody-dependent cytotoxicity or ADCC.
• Apoptosis of T4-cells as a result of chronic activation by HIV and by cytokines.
• Shedding of gp120 molecules by HIV. This subsequently triggers a series of events that cause the adaptive immune system to become less and less effective, primarily by altering the normal balance of immunoregulatory TH1 and TH2 cells in the body.
• Impaired function of HIV infected macrophages and dendritic cells.
These mechanisms will be discussed in greater detail in Unit 5 under secondary immunodeficiency.
Progression to AIDS is marked by a viral load that progressively increases in number while the immune system weakens as a result of the destruction of increasing numbers of T4-lymphocytes and the inability of the body to continually replace these destroyed cells. As will be seen in Unit 5, the loss of T4-helper lymphocytes leads to a marked decline in cells called cytotoxic T-lymphocytes (CTLs), the primary cells the body's immune responses use to destroy virus-infected cells. Once a person progresses to full-blown AIDS he or she becomes susceptible to a variety of opportunistic infections by:
• bacteria such as Mycobacterium avium complex (MAC), Salmonella, and Nocardia;
• protozoa such as Cryptosporidium and Toxoplasma;
• viruses such as cytomegalovirus (CMV), herpes simplex viruses types 1 and 2 (HSV-1, HSV-2), and varicella zoster virus (VZV);
• Candida, Cryptococcus, Coccidioides, Histoplasma, and Pneumocystis.
There is also an increased incidence of tumors, such Epstein-Barr virus-associated B-cell lymphomas, other lymphomas, cervical cancer, and Kaposi’s sarcoma. Wasting syndrome and encephalopathy are also common.
Why do you think the incubation period between HIV infection and AIDS has typically been 10 years or more?
Highly active anti-retroviral therapy (HAART) with a combination of reverse transcriptase inhibitors and protease inhibitors, as will be discussed later in Unit 4 under "Control of Viruses," has had relatively good success in both improving T4-lymphocyte levels and reducing the levels of HIV in the body - sometimes to undetectable levels. However, even with undetected levels of HIV, most infected persons continue to harbor relatively small amounts of replication-competent HIV, most likely in the resting T4-memory cells produced as a normal part of the immune responses. These infected T4-memory cells probably persist for years after antiretroviral therapy has reduced viral load below the limit of laboratory detection and could represent a pool that can keep HIV infection going or reactivate the infection. Macrophages and dendritic cells may also serve as a reservoir for HIV.
Summary
1. The median incubation period for AIDS is around 10 years.
2. During early or acute HIV infection the virus primarily infects and destroys memory T4-lymphocytes which express the chemokine receptor CCR5 and are very abundant in mucosal lymphoid tissues. Here HIV also encounters the dendritic cellslocated throughout the epithelium of the skin and the mucous membranes.
3. The dendritic cells detach from the epithelium, enter lymph vessels, and are carried to regional lymph nodes where they are now able to present antigens of HIV to naive T-lymphocytes in order toinduce adaptive immune responses.
4. The virus transitions from the acute phase to the chronic phase characterized by viral dissemination, viremia, and induction of adaptive immune responses.
5. The viremia allows the viruses to spread and infect T4-helper lymphocytes, macrophages, and dendritic cells found in peripheral lymphoid tissues.
6. During the chronic phase of HIV infection, the lymph nodes and the spleen become sites for continuous viral replication and host cell destruction whereby a steady state-infection generally persists where T4-lymphocyte death and T4-lymphocyte replacement by the body are in equilibrium.
7. The enormous turnover of T4-lymphocytes eventually exhausts the lymphopoietic system and it becomes unable to replace the T4-cells being destroyed eventually leading to immunodeficiency.
8. Progression to AIDS is marked by a viral load that progressively increases in number while the immune system weakens as a result of the destruction of increasing numbers of T4-lymphocytes and the inability of the body to continually replace these destroyed cells.
9. As a result of immunosuppression, the person becomes susceptible to a variety of opportunistic infections and secondary cancers.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. State the median incubation period for AIDS. (ans)
2. In terms of viral load, exhaustion of the lymphopoietic system, and immune responses, briefly describe what marks the progression to AIDS. (ans)
3. Briefly describe the following:
1. early or acute HIV infection (ans)
2. chronic HIV infection (ans)
4. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.06%3A_Animal_Virus_Life_Cycles%3A_An_Overview/10.6D%3A_Natural_History_of_a_Typical_HIV_Infection.txt |
Describe how certain viruses may contribute to the development of tumors by altering proto-oncogenes or tumor-suppressor genes. Name 3 viruses that have been implicated in human cancers.
Some viruses can also play a role in converting normal host cells into tumor cells. These viruses are capable of viral transformation, that is, they transform normal cells into malignant cells. In fact, five viruses, hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), Epstein-Barr virus (EBV), and human T-lymphotropic virus type I (HTLV-I) are thought to contribute to over 15% of the world's cancers. Up to 80% of these human viral-associated cancers are cervical cancer (associated with HPV) and liver cancer (associated with HBV and HCV).
The hepatitis B virus (HBV) is a DNA virus that may potentially cause chronic hepatitis in those infected. There is a strong link between chronic infection with HBV and hepatocellular carcinoma, which typically appears after 30-50 years of chronic liver damage and liver cell replacement. Chronic carriers of HBV have a 300 times greater risk of eventually developing liver cancer. Around 90% of individuals infected at birth and 10% of individuals infected as adults become chronic carriers of HBV. There are about one million chronic carriers of HBV in the US. Worldwide, HBV is responsible for 60% of all liver cancer cases.
The hepatitis C virus (HCV) is a RNA virus that may also cause chronic hepatitis in those infected. As with HBV, there is a strong link between chronic infection with HCV and liver cancer, typically appearing after 30-50 years of chronic liver damage and liver cell replacement. Around 85% of individuals infected with HCV become chronic carriers and there are approximately four million chronic carriers of HCV in the US. Worldwide, HCV is responsible for 22 % of all liver cancer cases.
The human papilloma viruses (HPV) are responsible for warts. While warts are generally considered as benign tumors, some sexually-transmitted strains of HPV (HPV-16 and 18 are definitely carcinogenic in humans; HPV-31 and 33 are probably carcinogenic), have been implicated in cervical and vulvar cancer, rectal cancer, and squamous cell carcinoma of the penis. In these tumor cells the viral DNA is usually found integrated in host cell chromosomes. In the US, HPVs are associated with 82% of the deaths due to cervical cancer each year, as well as a million precancerous lesions.
The Epstein-Barr virus (EBV), a herpes virus, normally causes benign proliferations such as infectious mononucleosis and hairy leukoplakia of the tongue. However, it can contribute to non-Hodgkin's lymphoma in AIDS patients and post-transplantation lymphoproliferative diseases, appears to be an essential factor for posterior nasopharyngeal cancer in some individuals, can be a co-factor for Burkitt's lymphoma, and contributes to smooth-muscle tumors in immunosuppressed children.
The retrovirus human T-lymphotropic virus type I (HTLV-I) can induce a rare adult T-lymphocyte leukemia-lymphoma.
The development of tumors is a multistep process depending on the accumulation of mutations altering a number of genes. The altered genes then function collectively to cause malignant growth.
Proliferation of normal cells is regulated by growth-promoting proto-oncogenes and counterbalanced by growth-restricting tumor suppressor genes. Mutations that increase the activities of proto-oncogenes to create oncogenes and/or decrease the activities of tumor suppressor genes can lead to growth of tumors. It is now known that many tumors require both activation of oncogenes from proto-oncogenes and inactivation of tumor suppressor genes for their development.
Viruses are thought to play a role in cancer development both indirectly and directly. Indirectly, the viruses may induce immunosuppression so that cancer cells are not removed by immune responses, as in the case of HIV/AIDS, or they may cause long term damage to tissues resulting in large scale cell regeneration which increases the chances of natural mutation in proto-oncogenes and tumor suppressor genes, as in the case of HBV and HCV. Directly, by integrating into the host cell's chromosomes, some viruses may alter the normal function of the proto-oncogenes and tumor suppressor genes, as is seen with HPV and HBV.
However, most virus-associated cancers have long latency periods of several decades and only a small percentage of the people infected with the virus actually develop the cancer. This indicates other factors promoting changes in cellular genes are also involved. For example, in the case of cervical cancer and HPV, two variants of a tumor suppressor gene known as p53 are known. One form of the p53 gene produces a suppressor protein that is much more susceptible to degradation by an oncoprotein called E6 which is produced by carcinogenic strains of HPV.
Summary
1. Viruses are responsible for about 15% of the world’s cancers.
2. Up to 80% of these human viral-associated cancers are cervical cancer (associated with human papilloma virus or HPV) and liver cancer (associated with the hepatitis B virus or HBV and the hepatitis C virus or HCV).
3. The Epstein-Barr virus (EBV) and human T-lymphotropic virus type I (HTLV-I) also increase the risk of certain cancers.
4. The development of tumors is a multistep process depending on the accumulation of mutations altering a number of genes.
5. Most virus-associated cancers have long latency periods of several decades and only a small percentage of the people infected with the virus actually develop the cancer. This indicates other factors promoting changes in cellular genes are also involved.
Questions
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
1. Describe how certain viruses may contribute to the development of tumors by altering proto-oncogenes or tumor-suppressor genes. (ans)
2. Name 3 viruses that have been implicated in human cancers.
1. (ans)
2. (ans)
3. (ans)
3. People with chronic hepatitis B have a much higher risk of developing liver cancer. This cancer, however, usually appears after decades of chronic infection. Explain the link between HBV and liver cancer and why, if it does develop, it usually takes so long. (ans)
4. Multiple Choice (ans) | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.06%3A_Animal_Virus_Life_Cycles%3A_An_Overview/10.6E%3A_The_Role_of_Viruses_in_Tumor_Production.txt |
Name the 2 types of bacteriophage life cycles and state what the bacteriophage capable of each is called.
As mentioned in an earlier section, bacteriophages are viruses that only infect bacteria (also see Figure \(1\)C and Figure \(2\)E). There are two primary types of bacteriophages: lytic bacteriophages and temperate bacteriophages. Bacteriophages that replicate through the lytic life cycle are called lytic bacteriophages, and are so named because they lyse the host bacterium as a normal part of their life cycle. Bacteriophages capable of a lysogenic life cycle are termed temperate phages. When a temperate phage infects a bacterium, it can either replicate by means of the lytic life cycleand cause lysis of the host bacterium, or, it can incorporate its DNA into the bacterium's DNAand become a noninfectious prophage. We will now look at the lytic life cycle and lysogenic life cycle of bacteriophages.
Summary
1. Bacteriophages are viruses that only infect bacteria.
2. Bacteriophages that replicate through the lytic life cycle are called lytic bacteriophages, and are so named because they lyse the host bacterium as a normal part of their life cycle.
3. Bacteriophages capable of a lysogenic life cycle are termed temperate phages. and can either replicate by means of the lytic life cycle and cause lysis of the host bacterium, or, can incorporate their DNA into the bacterium's DNA and become a non-infectious prophage.
10.07: Bacteriophage Life Cycles: An Overview
Learning Objectives
1. Describe the steps involved in the lytic life cycle of bacteriophages.
2. Define the following:
1. lytic bacteriophage
2. eclipse period
As mentioned in an earlier section, bacteriophages are viruses that only infect bacteria (see Figure \(1\)C and Figure \(2\)E). Bacteriophages that replicate through the lytic life cycle are called lytic bacteriophages. After infecting bacteria with lytic bacteriophages in the lab, plaques can be seen on the petri plates. Plaques are small clear areas on the agar surface where the host bacteria have been lysed by lytic bacteriophages. The lytic life cycle is somewhat similar to the productive life cycle of animal viruses and consists of the following steps:
Plaques on an agar surface after infecting Escherichia coli with Coliphage T-4
Step 1: Adsorption
Attachment sites on the bacteriophage adsorb to receptor sites on the host bacterium (see Figure \(1\)). Most bacteriophages adsorb to the bacterial cell wall, although some are able to adsorb to flagella or pili. Specific strains of bacteriophages can only adsorb to specific strain of host bacteria. This is known as viral specificity.
Step 2: Penetration
In the case of bacteriophages that adsorb to the bacterial cell wall, a bacteriophage enzyme "drills" a hole in the bacterial wall and the bacteriophage injects its genome into the bacterial cytoplasm (Figure \(2\)). Some bacteriophages accomplish this by contracting a sheath which drives a hollow tube into the bacterium. This begins the eclipse period. The genomes of bacteriophages which adsorb to flagella or pili enter through these hollow organelles. In either case, only the phage genome enters the bacterium so there is no uncoating stage.
Step 3: Replication
Enzymes coded by the bacteriophage genome shut down the bacterium's macromolecular (protein, RNA, DNA) synthesis. The bacteriophage replicates its genome and uses the bacterium's metabolic machinery to synthesize bacteriophage enzymes and bacteriophage structural components (Figure \(3\) and Figure \(4\)).
Step 4: Maturation
The phage parts assemble around the genomes (Figure \(5\)).
Step 5: Release
Usually, a bacteriophage-coded lysozyme breaks down the bacterial peptidoglycan causing osmotic lysis and release of the intact bacteriophages (Figure \(6\)).
Step 6: Reinfection
From 50 to 200 bacteriophages may be produced per infected bacterium.
Adsorption of a Bacteriophage to the Cell Wall of the Bacterium. Attachment sites on the virus bind to corresponding receptors on the host cell wall.
Exercise: Think-Pair-Share Questions
1. Describe how a lytic bacteriophage might possibly play a role in horizontal gene transfer in bacteria.
2. As will be seen in lab, phage typing is a technique wherein unknown strains of a bacterium are identified by using known strains of bacteriophages. How can we use a bacteriophage to identify a bacterium?
3. We saw in the previous section that a single infected animal cell may produce 10,000-50,000 viruses yet an infected bacterium can only produce 50-200 bacteriophages. Explain this.
Summary
1. Bacteriophages that replicate through the lytic life cycle are called lytic bacteriophages,
2. Adsorption is the attachment sites on the phage adsorb to receptor sites on the host bacterium.
3. Specific strains of bacteriophages can only adsorb to specific strain of host bacteria (viral specificity).
4. In the case of bacteriophages that adsorb to the bacterial cell wall, a bacteriophage enzyme "drills" a hole in the bacterial wall and the bacteriophage injects its genome into the bacterial cytoplasm.
5. The bacteriophage replicates its genome and uses the bacterium's metabolic machinery to synthesize bacteriophage enzymes and bacteriophage structural components.
6. During maturation, the bacteriophage parts assemble around the phage genomes.
7. A phage-coded lysozyme breaks down the bacterial peptidoglycan causing osmotic lysis and release of the intact bacteriophages.
10.7B: The Lysogenic Life Cycle of Bacteriophages
Describe the lysogenic life cycle of temperate phages (including spontaneous induction). Define the following: temperate phage lysogen prophage
Bacteriophages capable of a lysogenic life cycle are termed temperate bacteriophages. When a temperate bacteriophage infects a bacterium, it can either replicate by means of the lytic life cycle and cause lysis of the host bacterium, or, it can incorporate its DNA into the bacterium's DNA and become a noninfectious prophage (see Figure \(1\)). In the latter case, the cycle begins by the bacteriophage adsorbing to the host bacterium or lysogen and injecting its genome as in the lytic life cycle (see Figure \(2\) and Figure \(3\)). However, the bacteriophage does not shut down the host cell. Instead, the bacteriophage DNA inserts or integrates into the host bacterium's DNA (see Figure \(4\)). At this stage the virus is called a prophage. Expression of the bacteriophage genes controlling bacteriophage replication is blocked by a repressor protein, and the phage DNA replicates as a part of the bacterium's DNA so that every daughter bacterium now contains the prophage (see Figure \(5\)).
The number of viruses infecting the bacterium as well as the physiological state of the bacterium appear to determine whether the temperate bacteriophage enters the lytic cycle or becomes a prophage.
In about one out of every million to one out of every billion bacteria containing a prophage, spontaneous induction occurs. The bacteriophage genes are activated and new bacteriophages are produced by the lytic life cycle (see Figure \(5\)A, Figure \(6\), Figure \(7\), Figure \(8\), and Figure \(9\)). | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.07%3A_Bacteriophage_Life_Cycles%3A_An_Overview/10.7A%3A_The_Lytic_Life_Cycle_of_Bacteriophages.txt |
Briefly describe at least 4 ways viruses can damage infected host cells. Briefly describe at least 3 different ways viruses can evade host immune defenses.
Animal viruses may cause cytopathic effect or CPE that damages infected host cells in a variety of means, including:
1. Inhibiting normal host cell DNA, RNA, or protein synthesis. This can cause structural or functional defects in the infected host cell leading to cytolysis or altered cell functions.
2. Causing nicks or breaks in the host cell's chromosomes, as seen in congenital rubella syndrome.
3. Viral proteins and glycoproteins changing the antigenic surface of the host cell's cytoplasmic membrane resulting in its being recognized as foreign and destroyed by the body's immune defenses (see Figure \(9\), Figure \(10\), Figure \(11\)A and Figure \(11\)B). This will be discussed further in Unit 6.
4. Depleting the host cell of cellular materials essential for life or normal function.
5. Stimulating body cells to release inflammatory cytokines and chemokines.
6. Stimulating body cells to release inflammatory vasoactive peptides, bradykinins, histamines, etc. resulting in vasodilation and increased mucous secretion.
7. Inducing adjacent host cells to fuse together forming giant multinucleated cells or syncytias (see Figure \(1\), Figure \(2\), Figure \(3\)A, and Figure \(3\)B) as seen with cytomegalovirus (CMV), varicella-zoster virus (VZV), and HIV.
8. Playing a role in normal cells becoming malignant (cell transformation by oncogenic viruses ).
9. Causing cytolysis of the infected host cell (see Figure \(13\)C ).
Evading Host Immune Defenses
As will be seen in Unit 6, one of the major defenses against free viruses is the immune defenses' production of antibody molecules against the virus. The "tips" of the antibody (the Fab portion; see Figure \(4\)A) have shapes that have a complementary shape to portions of viral attachment proteins and glycoproteins called epitopes found on the viral surface. When antibodies react with these attachment proteins, they block viral adsorption to host cell receptors and, therefore, block viral replication.
In addition, Antibodies such as IgG function as opsonins and stick viruses to phagocytes.
• Antibodies made against the original human influenza virus can no longer bind to the new strain of virus or stick the virus to phagocytes (see Figure \(4\)A and Figure \(4\)B).
• Likewise HIV, because of its high rate of mutation and its intracellular recombination with other strains of HIV, as mentioned earlier in this unit, produces altered gp120 to which antibodies made against the earlier strains of HIV can no longer bind.
• The hepatitis C virus (HCV) frequently through mutation produces viral variants ("escape mutants") to resist antibodies.
Another major defense against viruses, as we will see in Unit 6, is the killing of virus-infected host cells by cytotoxic T-lymphocytes (CTLs). Virus-infected host cells naturally bind viral epitopes to a host molecule called MHC-I and place the MHC-1 with bound viral epitope on the surface of the infected cell (see Figure \(5\)) where they can be recognized by CTLs having a T-cell receptors on its surface with a complementary shape. In this way the CTL can kill the infected cell by apoptosis , a programmed cell suicide (see Figure \(11\)A and Figure \(11\)B).
For a preview of CTLs killing virus-infected cells from Unit 6, Cell-Mediated Immunity, see the two animations below.
• Epstein-Barr virus (EBV) and cytomegalovirus (CMV) inhibit proteasomal activity so that viral proteins are not degraded into viral peptides. (see Figure \(5\)A)
• Herpes simplex viruses (HSV) can block the TAP transport of peptides into the endoplasmic reticulum (see Figure \(5\)B).
• Numerous viruses, such as the cytomegalovirus (CMV) and adenoviruses can block the formation of MHC-I molecules by the infected cell. As a result, no viral peptide is displayed on the infected cell and the CTLs are no longer able to recognize that the cell is infected and kill it (see Figure \(5\)C).
• Epstein-Barr virus (EBV) down regulates several host proteins involved in attaching viral epitopes to MHC-I molecules and displaying them on the host cell's surface (see Figure \(5\)D).
• Adenoviruses and Epstein-Barr Virus (EBV) code for proteins that blocks apoptosis , the programmed cell suicide mechanism triggered by various defense mechanisms in order to destroy virus-infected cells.
3. Another defense cell that is able to kill virus-infected cells is the NK cell. NK cells recognize infected cells displaying stressed-induced proteins and not displaying MHC-I molecules on their surface and kill these cells (see Figure \(7\)).
MHC-I molecules are the molecules on host cells that display viral epitopes to cytotoxic T-lymphocytes (CTLs). Some viruses suppress the production of MHC molecules by host cells, preventing CTLs from recognizing the infected cell as foreign and killing it. NK cells, however, can recognize cells not displaying MHC-I and kill them anyway.
See the three animations below for a preview of NK cells from Unit 5, Innate Immunity.
• The cytomegalovirus (CMV) can also trigger its host cell to produce altered MHC-I molecules that are unable to bind viral epitopes, and, therefore, are not recognized by CTLs. However, NK cells are also unable to kill this infected cell because it is still displaying "MHC-I molecules" on its surface.
• CMV also produces microRNAs (miRNAs), small non-coding RNA molecules that down-regulates the production of stress-induced proteins that the killer-activating receptor of NK cells first recognizes. The miRNAs do this by binding to the host cell's mRNA coding for stress-induced proteins (see Figure \(14\)). Without this binding there is no kill signal by the NK cell.
4. Some viruses cause infected host cells to secrete molecules that bind and tie up cytokines , preventing them from binding to normal cytokine receptors on host cells.
• Poxviruses cause infected host cells to secrete molecules that bind interleukin-1 (IL-1) and interferon-gamma (IFN-gamma).
• Cytomegaloviruses (CMV) cause infected host cells to secrete molecules that bind chemokines.
5. Some viruses suppress immunocompetent cells.
• Epstein-Barr virus (EBV) produces a protein that is homologous to the cytokine interleukin-10 (IL-10). IL-10 inhibits the activation of dendritic cells and macrophages , antigen-presenting cells that are needed to present antigens to T-lymphocytes for their activation. EBV also produces microRNAs (miRNAs ), small non-coding RNA molecules that inhibit an interferon response by infected cells. The miRNAs do this by binding to the host cell's mRNA coding for interferon (see Figure \(14\)).
• The human immunodeficiency virus (HIV) infects immunocompetent dendritic cells and T4-lymphocytes leading to their death or disfunction.
6. Some viruses block apoptosis of infected host cells enabling the infected host cell to survive and produce new viruses.
• Cytomegalovirus (CMV) and herpes simplex type 1 virus (HSV-1) produce microRNAs (miRNAs ), small non-coding RNA molecules that block protein involved in apoptosis, a programmed cell suicide. The miRNAs do this by binding to the host cell's mRNA coding for apoptosis-inducing proteins (see Figure \(14\)).
Summary
1. Alteration of host cell function and/or death of the host cell occurs as a result of viruses using an infected host cell as a factory for manufacturing viruses.
2. The body’s immune defenses recognize infected host cells as foreign and destroy infected cells.
3. The body’s adaptive immune defenses produce antibodies against viruses that block viral adsorption to host cells or result in opsonization of the virus.
4. The body’s adaptive immune defenses produce cytotoxic T-lymphocytes (CTLs) against viruses that bind to infected host cells and induce cell suicide (apoptosis).
5. The body’s innate immune defenses produce NK cells that can induce apoptosis of stressed, virus-infected host cells.
6. Viruses can develop resistance to antibodies and cytotoxic T-lymphocytes by altering the order of the amino acids and, therefore, the shape of viral antigens so the antibodies and CTLs no longer fit.
7. Viruses can alter infected host cells in such a way that NK cells no longer kill them.
8. Some viruses block apoptosis of infected host cells enabling the infected host cell to survive and produce new viruses.
10.09: Bacteriophage-Induced Alterations of Bacteria
Learning Objectives
1. Describe the process of lysogenic conversion and give two examples of exotoxins that result from lysogenic conversion.
1. Lytic bacteriophages usually cause the host bacterium to lyse (see Figure \(1\)).
2. Lysogenic conversion by prophages
The added genetic information provided by the DNA of a prophage (Figure \(4\)) may enable a bacterium to possess new genetic traits. For example, some bacteria become virulent only when infected themselves with a specific temperate bacteriophage. The added genetic information of the prophage allows for coding of protein exotoxin or other virulence factors.
The following bacterial exotoxins are a result of lysogenic conversion by a prophage:
1. the diphtheria exotoxin of the bacterium Corynebacterium diphtheriae;
2. the Streptococcal pyrogenic exotoxin (Spe) produced by rare invasive strains and scarlet fever strains of Streptococcus pyogenes;
3. The neurotoxin produced by Clostridium botulinum;
4. exfoliatin, an exotoxin that causes scalded skin syndrome, produced by Staphylococcus aureus;
5. the cholera exotoxin produced by Vibrio cholerae; and
6. the shiga toxins produced by E. coli O157:H7.
Animation of the Lysogenic Life Cycle of a Temperate Bacteriophage
Exercise: Think-Pair-Share Questions
State why bacteriophages themselves are harmless to humans but might enable certain bacteria to be more harmful to humans.
Summary
1. Lytic bacteriophages usually cause the host bacterium to lyse.
2. The added genetic information provided by the DNA of a prophage may enable a bacterium to possess new genetic traits.
3. Some bacteria become virulent only when infected themselves with a specific temperate bacteriophage. The added genetic information of the prophage allows for coding of protein exotoxin or other virulence factors.
4. Examples include the diphtheria exotoxin, streptococcal pyrogenic exotoxin (Spe), the botulism exotoxins, the cholera exotoxin, and the shiga toxin. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.08%3A_Pathogenicity_of_Animal_Viruses.txt |
Learning Objectives
1. State why antibiotics are of no use against viruses and what we must rely on to control viruses.
2. State the viruses the following antiviral agents are used against:
1. amantadine, rimantidine, zanamivar, and oseltamivir
2. acyclovir, famciclovir, penciclovir, and valacyclovir
3. foscarnet, gancyclovir, cidofovir, valganciclovir, and fomivirsen
4. AZT (ZDV), didanosine, zalcitabine, stavudine, lamivudine, emtricitabine, tenofovir, and abacavir
5. nevirapine, delavirdine, and efavirenz
6. saquinavir, ritonavir, idinavir, nelfinavir, amprenavir, atazanavir, fosamprenavir, ritonavir
7. telaprevir, boceprevir, simeprevir, sofosbuvir
3. Compare how the following drugs exhibit their antiviral action against HIV.
1. nucleoside reverse transcriptase inhibitors
2. protease inhibitors
3. entry inhibitors
Since viruses lack the structures and metabolic processes that are altered by common antibiotics, antibiotics are virtually useless in treating viral infections. To date, relatively few antiviral chemotherapeutic agents are available and used to treat just a few limited viruses.
Most of the antiviral agents work by inhibiting viral DNA synthesis. These drugs chemically resemble normal DNA nucleosides, molecules containing deoxyribose and either adenine, guanine, cytosine, or thymine. Viral enzymes then add phosphate groups to these nucleoside analogs to form DNA nucleotide analogs. The DNA nucleotide analogs are then inserted into the growing viral DNA strand in place of a normal nucleotide. Once inserted, however, new nucleotides can't attach and DNA synthesis is stopped. They are selectively toxic because viral polymerases are more prone to incorporate nucleotide analogs into their nucleic acid than are host cell polymerases.
Table \(1\): Antivirals used for viruses other than HIV
Antiviral Brand Name Use
amantadine Symmetrel used prophylactically against influenza A ) in high-risk individuals. It prevents influenza A viruses from the uncoating step necessary for viral replication.
rimantidine Flumadine used for treatment and prophylaxis of influenza A. It prevents influenza A viruses from the uncoating step necessary for viral replication.
zanamivir: Relenza used to limit the duration of influenza A and B infections. It is an inhibitor of the influenza virus surface enzyme called neuraminidase that is needed for release of newly formed influenza viruses from the infected cell.
oseltamivir Tamiflu used limit the duration of influenza infections. It is an inhibitor of the influenza virus surface enzyme called neuraminidase that is needed for release of newly formed influenza viruses from the infected cell.
acyclovir Zovirax used against herpes simplex viruses (HSV) to treat genital herpes, mucocutaneous herpes in the immunosuppressed, HSV encephalitis, neonatal herpes, and to reduce the rate of recurrences of genital herpes. It is also used against varicella zoster viruses (VZV) ) to treat shingles. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
trifluridine Viroptic used to treat eye infection (keratitis and conjunctivitis) caused by HSV. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
famciclovir Famvir used to treat HSV and VZV infections. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
valacyclovir
Valtrex used to treat HSV and VZV infections. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
penciclovir Denavir used in treating HSV infections. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
gancyclovir Cytovene; Vitrasert used in treating severe cytomegalovirus (CMV) infections such as retinitis. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
valganciclovir Valcyte used in treating severe CMV infections such as retinitis). It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
foscarnet Foscavir used in treating severe CMV infections such as retinitis. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
cidofovir Vistide used in treating CMV retinitis. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
fomivirsen Vitravene used in treating CMV retinitis. Fomivirsen inhibits cytomegalovirus (CMV) replication through an antisense RNA (microRNA or miRNA mechanism. The nucleotide sequence of fomivirsen is complementary to a sequence in mRNA transcripts (Figure \(1\)) that encodes several proteins responsible for regulation of viral gene expression that are essential for production of infectious CMV. Binding of fomivirsen to the target mRNA results in inhibition of protein synthesis, subsequently inhibiting virus replication.
ribavirin Copegus; Rebetol; Virazole used in treating severe acute respiratory syndrome (SARS). In combination with other drugs it is used to treat hepatitis C virus (HCV). It chemically resembles a normal RNA nucleoside. Once inserted into the growing RNA chain it inhibits further viral RNA replication.
telaprevir Incivek for the treatment of chronic hepatitis C (hepatitis C virus or HCV genotype 1). It is a protease inhibitor that binds to the active site of an HCV-encoded protease and prevent it from cleaving the long polyprotein from polycistronic HCV genes into proteins essential to the structure and function of HCV.
boceprevir
Victrelis for the treatment of chronic hepatitis C (hepatitis C virus or HCV genotype 1) infection. It is used in combination with peginterferon alfa and ribavirin. Boceprevir is a protease inhibitor that binds to the active site of an HCV-encoded protease and prevent it from cleaving the long polyprotein from polycistronic HCV genes into proteins essential to the structure and function of HCV.
simeprevir Olysio use for the treatment of chronic hepatitis C (hepatitis C virus or HCV genotype 1) infection. Used in combination with peginterferon alfa and ribavirin. Simeprevir is a protease inhibitor that binds to the active site of an HCV-encoded protease and prevent it from cleaving the long polyprotein from polycistronic HCV genes into proteins essential to the structure and function of HCV.
sofosbuvir Sovaldi Use for the treatment of chronic hepatitis C infection. Used in combination with ribavirin for hepatitis C virus or HCV genotypes 2 and 4; used in combination with peginterferon alfa and ribavirin for HCV genotypes 1 and 4. The second indication is the first approval of an interferon-free regimen for the treatment of chronic HCV infection. Sofosbuvir is a nucleotide polymerase inhibitor that binds to the active site of an HCV-encoded RNA polymerase preventing the synthesis of the viral RNA genome.
lamivudine Epivir-HBV used in treating chronic hepatitis B. It chemically resembles a normal DNA nucleoside. Once inserted into the growing DNA chain it inhibits further viral DNA replication.
adefovir dipivoxil Hepsera used in treating hepatitis B.
Current anti-HIV drugs include the following (classified by their action):
HIV nucleoside-analog reverse transcriptase inhibitors
To replicate, HIV uses the enzyme reverse transcriptase to make a DNA copy of its RNA genome. A complementary copy of this DNA is then made to produce a double-stranded DNA intermediate which is able to insert into host cell chromosomes to form a provirus. Most reverse transcriptase inhibitors are nucleoside analogs. A nucleoside is part of the building block of DNA, consisting of a nitrogenous base bound to the sugar deoxyribose but no phosphate group. A nucleoside analog chemically resembles a normal nucleoside (Figure \(2\)).
Once phosphate groups are added by either viral or host cell enzymes, the drugs now chemically resemble normal DNA nucleotides, the building block molecules for DNA synthesis. The nucleotide analog binds to the active site of the reverse transcriptase which, in turn, inserts it into the growing DNA strand in place of a normal nucleotide. Once inserted, however, new DNA nucleotides are unable to attach to the drug and DNA synthesis is stopped. This results in an incomplete provirus. For example, zidovudine (AZT, ZDV, Retrovir), as shown in Figure \(1\), resembles the deoxyribonucleotide containing the base thymine. Once zidovudine is inserted into the growing DNA strand being transcribed from the viral RNA by reverse transcriptase, no further nucleotides can be attached (Figure \(3\)).
Examples of nucleoside reverse transcriptase inhibitors include:
1. zidovudine (AZT; ZDV; Retrovir)
2. didanosine (ddI; dideoxyinosine; Videx)
3. stavudine (d4T; Zerit)
4. lamivudine (3TC; Epivir)
5. abacavir (ABC; Ziagen)
6. emtricitabine (FTC; Emtriva, Coviracil)
Nucleotide Reverse Transcriptase Inhibitors (NtRTIs)
A NtRTI inhibitor is a a nucleotide analog. A nucleotide is the building block of DNA, consisting of a nitrogenous base bound to the sugar deoxyribose, and a phosphate group. A nucleotide analog chemically resembles a normal nucleotide. The nucleotide analog binds to the active site of the reverse transcriptase which, in turn, inserts it into the growing DNA strand in place of a normal nucleotide. Once inserted, however, new DNA nucleotides are unable to attach to the drug and DNA synthesis is stopped. This results in an incomplete provirus. An example of nucleoside reverse transcriptase inhibitor is tenofovir (TDF;Viread).
3. HIV Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
These drugs do not resemble regular DNA building blocks. They bind to an allosteric site that regulates reverse transcriptase activity rather than to the enzyme's active site itself as do the above nucleoside analogues (see Figure \(4\)). This also prevents HIV provirus formation.
1. nevirapine (NVP; Viramune)
2. delavirdine (DLV;Rescriptor)
3. efavirenz (EFV; Sustiva)
4. rilpivirine (Edurant)
5. etravirine (ETR, TMC125; Intelence)
Figure \(4\): Noncompetitive Inhibition with Allosteric Enzymes. When the end product (inhibitor) of a pathway combines with the allosteric site of the enzyme, this alters the active site of the enzyme so it can no longer bind to the starting substrate of the pathway. This blocks production of the end product.
HIV Protease Inhibitors (PIs)
In order for maturation of HIV to occur, a HIV enzyme termed a protease has to cleave a long HIV-encoded gag-pol polyprotein to produce reverse transcriptase and integrase (coded by the HIV pol gene) and gag polyprotein (coded by the HIV gag gene). The HIV protease then cleaves the gag polyprotein into capsid protein p17, matrix protein p24, and nucleocapsid protein p7, as well as proteins p6, p2, and p1 whose functions are not yet fully understood (see Figs. 4A, 4B, and 4C). Proteases also cleave the env-polyprotein (coded by the HIV env gene) into the envelope glycoproteins gp120 and gp41 (see Figure \(5\)). Protease inhibitors are drugs that bind to the active site of this HIV-encoded protease and prevent it from cleaving the long gag-pol polyprotein and the gag polyprotein into essential proteins essential to the structure of HIV and to RNA packaging within its nucleocapsid (see 4C). As a result, viral maturation does not occur and noninfectious viral particles are produced.
Protease inhibitors include:
1. saquinavir (SQV; Inverase)
2. ritonavir (RTV; Norvir)
3. idinavir (IDV; Crixivan)
4. nelfinavir (NFV; Viracept)
5. amprenavir (APV; Agenerase)
6. atazanavir (ATV; Reyataz)
7. fosamprenavir (FPV; Lexiva)
8. ritonavir (RTV; Norvir)
9. darunavir (DRV; TMC114; Prezista)
10. tipranavir (TPV; Aptivus)
Entry Inhibitors (EIs)
EIs are agents interfering with the entry of HIV-1 into cells. During the adsorption and penetration stages of the life cycle of HIV, a portion or domain of the HIV surface glycoprotein gp120 binds to a CD4 molecule on the host cell. This induces a change in shape that brings the chemokine receptor binding domains of the gp120 into proximity with the host cell chemokine receptor. This brings about another conformational change that exposes a previously buried portion of the transmembrane glycoprotein gp41 that enables the viral envelope to fuse with the host cell membrane. EIs interfere with various stages of this process.
a. Agents that block the binding of gp120 to host chemokine receptor 5 (CCR5).
After the gp120 on the envelope of HIV binds to a CD4 molecule on the host cell, it must then also bind to a co-receptor - a chemokine receptor. CCR5-tropic strains of HIV bind to the chemokine receptor CCR5 (see Figure \(6\)). (An estimated 50%-60% of people having previously received HIV medication have circulating CCR5-tropic HIV.)
maraviroc (MVC; Selzentry; Celsentri) is a chemokine receptor binding blocker that binds to CCR5 and blocks gp120 from binding to the co-receptor thus blocking adsorption of HIV to the host cell.
b. Agents that block the fusion of the viral envelope with the cytoplasmic membrane of the host cell.
enfuvirtide (ENF; T-20; Fuzeon) binds a gp41 subunit of the viral envelope glycoprotein and prevents the conformational changes required for the fusion of the viral envelope with the cellular cytoplasmic membrane.
5. Integrase Inhibitors
Integrase inhibitors disable HIV integrase, the enzyme that inserts the HIV double-stranded DNA intermediate into host cell DNA. It prevents production of a provirus.
raltegravir (Isentress)
6. Fixed-dose combinations
Tablets containing two or more anti-HIV medications:
1. abacivir + lamivudine (Epzicom)
2. abacivir + lamivudine + zidovudine (Trizivir)
3. efavirenz + emtricitabine + tenofovir DF (Atripla)
4. emtricitabine + tenofovir DF (Truvada)
5. lamivudine + zidovudine (Combivir)
Certain antiviral cytokines called type-1 interferons have been produced by recombinant DNA technology and several are used to treat certain severe viral infections. These include:
1. recombinant interferon alfa-2a (Roferon-A): a cytokine used to treat Kaposi's sarcoma, chronic myelogenous leukemia, and hairy cell leukemia.
2. peginterferon alfa-2a (Pegasys) : used to treat hepatitis C (HCV).
3. recombinant interferon-alpha 2b (Intron A): a cytokine produced by recombinant DNA technology and used to treat Hepatitis B; malignant melanoma, Kaposi's sarcoma, follicular lymphoma, hairy cell leukemia, warts, and Hepatitis C.
4. peginterferon alfa-2b (PEG-Intron; PEG-Intron Redipen): used to treat hepatitis C (HCV).
5. recombinant Interferon alfa-2b plus the antiviral drug ribavirin (Rebetron): used to treat hepatitis C (HCV).
6. recombinant interferon-alpha n3 (Alferon N): used to treat warts.
7. recombinant iInterferon alfacon-1 (Infergen) : used to treat hepatitis C (HCV).
Most of the current antiviral agents don't kill and eliminate the viruses, but rather inhibit their replication and decrease the severity of the disease. As with other microbes, resistant virus strains can emerge with treatment.
Since there are no antiviral drugs for the vast majority of viral infections and most drugs that are available are only partially effective against limited types of viruses, to control viruses, we must rely on the body's immune responses. As will be seen in detail in Units 5 and 6, the immune responses include innate immunity as well as adaptive immunity (antibody production and cell-mediated immunity). Adaptive immunity can be either naturally acquired or, in some cases, artificially acquired.
For a more detailed description of any specific antimicrobial agent, see the website of RxList - The Internet Drug Index.
Summary
1. Relatively few antiviral chemotherapeutic agents are currently available and they are only somewhat effective against just a few limited viruses.
2. Many antiviral agents resemble normal DNA nucleosides molecules and work by inhibiting viral DNA synthesis.
3. Some antiviral agents are protease inhibitors that bind to a viral protease and prevent it from cleaving the long polyprotein from polycistronic genes into proteins essential to viral structure and function.
4. Some antiviral agents are entry inhibitors that prevent the virus from either binding to or entering the host cell.
5. Antiviral agents are available for only a few viruses, including certain influenza viruses, herpes viruses, cytomegaloviruses, hepatitis C viruses, and HIV.
6. Certain interferon cytokines have been produced by recombinant DNA technology and several are used for certain severe viral infections. | textbooks/bio/Microbiology/Microbiology_(Kaiser)/Unit_4%3A_Eukaryotic_Microorganisms_and_Viruses/10%3A_Viruses/10.10%3A_Antiviral_Agents.txt |
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