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Learning Objectives
• Outline the various mechanisms utilized by nitrogen-fixing bacteria to protect nitrogenases from oxygen
Central to nitrogen fixation (N2 to NH3) are the enzymes that do the actual fixation, these are known as nitrogenases. Due to the oxidation carried out by oxygen, most nitrogenases, which are essential large reduction complexes are irreversibly inhibited by O2, which degradatively oxidizes the Fe-S cofactors. In essence, O2binds to the iron (Fe) found in nitrogenases and blocks their ability to bind to N2. To protect nitrogenases, there are mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo. One known exception is the nitrogenase of Streptomyces thermoautotrophicus, which is unaffected by the presence of oxygen. This is complicated by the fact the bacteria still need the presence of oxygen for proper respiration.
Some microbes have a proteoglycan rich extra cellular matrix which traps a layer of water, often referred to as a slime layer. This slime layer acts as a barrier for oxygen. The ability of some nitrogen fixers such as azotobacteraceae to employ an oxygen-amendable nitrogenase under aerobic conditions has been attributed to a high metabolic rate, allowing oxygen reduction at the cell membrane; however, the effectiveness of this mechanism is in question.
Many rhizobia, nitrogen fixing bacteria, live in a symbiotic relationship with plants known as legumes. They have an interesting strategy to deal with O2. In plants infected with Rhizobium, (legumes such as alfalfa or soybeans), the presence of oxygen in the root nodules would reduce the activity of the oxygen-sensitive nitrogenase. In these situations, the roots of such plants produce a protein known as leghemoglobin (also leghaemoglobin or legoglobin). Leghemoglobin buffers the concentration of free oxygen in the cytoplasm of infected plant cells to ensure the proper function of root nodules. Leghemoglobin is a nitrogen or oxygen carrier; naturally occurring oxygen and nitrogen interact similarly with this protein. Leghemoglobin buffers the concentration of free oxygen in the cytoplasm of infected plant cells to ensure the proper function of root nodules. It has close chemical and structural similarities to hemoglobin, and, like hemoglobin, is red in colour. Leghemoglobin has a high affinity for oxygen, about ten times higher than of human hemoglobin. This allows an oxygen concentration that is low enough to allow nitrogenase to function but not so high as to bind all the O2 in the bacteria, providing the bacteria with oxygen for respiration.
Leghemoglobin is produced by legumes in response to the roots being infected by rhizobia, as part of the symbiotic interaction between the plant and these nitrogen-fixing bacterium. Interestingly, it is widely believed that leghemoglobin is the product of both the plant and the bacterium in which a protein precursor is produced by the plant and the heme (an iron atom bound in a porphyrin ring, which binds O2) is produced by the bacterium. The protein and heme come together to function, allowing the bacteria to fix-nitrogen, giving the plant usable nitrogen and thus the plant provides the rhizobia a home.
Key Points
• The iron (Fe) found in nitrogenases is very sensitive to oxygen, if there is too much oxygen this will in the end disrupt nitrogenase function.
• Some bacteria produce barriers which protect themselves from oxygen, while others use proteins such as leghemoglobin to bind up oxygen which may interfere with nitrogenases.
• Portions of leghemoglobin are thought to be produced by rhizobia residing in plant nodules, while other parts are produced by the plant, an elegant example of symbiosis.
Key Terms
• oxidation: A reaction in which the atoms of an element lose electrons and the valence of the element increases.
• reduction: A reaction in which electrons are gained and valence is reduced; often by the removal of oxygen or the addition of hydrogen.
• proteoglycan: Any of many glycoproteins that have heteropolysaccharide side chains | textbooks/bio/Microbiology/Microbiology_(Boundless)/05%3A_Microbial_Metabolism/5.15%3A_Nitrogen_Fixation/5.15D%3A_Anaerobiosis_and_N_Fixation.txt |
Learning Objectives
• Discuss the role of the nif genes in controlling nitrogen fixation
The fixation of atmospheric nitrogen (N2) is a very energy intensive endeavor. If there is no need for N2 fixation, the production of proteins needed for fixation are tightly controlled. The nif genes are responsible for the coding of proteins related and associated with the fixation of atmospheric nitrogen into a form of nitrogen available to plants. These genes are found in nitrogen fixing bacteria and cyanobacteria. The nif genes are found in both free living nitrogen fixing bacteria and in symbiotic bacteria in various plants.
The nif genes are genes encoding enzymes involved in the fixation of atmospheric nitrogen. The primary enzyme encoded by the nif genes is the nitrogenase complex which is in charge of converting atmospheric nitrogen to other nitrogen forms such as ammonia, which plants can use for various purposes. Besides the nitrogenase enzyme, the nif genes also encode a number of regulatory proteins involved in nitrogen fixation. The expression of the nif genes is induced as a response to low concentrations of fixed nitrogen and oxygen concentrations (the low oxygen concentrations are actively maintained in the root environment). Nitrogen fixation is regulated by nif regulon, which is a set of seven operons which includes 17 nif genes. Nif genes have both positive and negative regulators. Some of nif genes are: Nif A, D, L,K, F,H S,U,Y,W,Z.
Activation of nif genes transcription is done by the nitrogen sensitive NifA protein. When there isn’t enough fixed nitrogen factor available for the plant’s use, NtrC, which is a RNA polymerase, triggers NifA’s expression. NifA then activates the rest of the transcription for the nif genes. If there is a sufficient amount of reduced nitrogen or oxygen is present, another protein is activated, NifL. In turn, NifL inhibits NifA activity, which results in the inhibition of nitrogenase formation. NifL is then regulated by other proteins that are sensors for the levels of O2 and ammonium in the surrounding environment.
The nif genes can be found on bacteria’s chromosomes, but many times they are found on bacteria’s plasmids with other genes related to nitrogen fixation, such as the genes needed for the bacteria to communicate with the plant host.
Key Points
• Nitrogen fixation takes a great deal of energy. If the conditions are not favorable for nitrogen fixation or there is enough ammonia around, nitrogen-fixing bacteria turn off the production of proteins needed for nitrogen fixation.
• Nitrogen fixing protein production is regulated by the nif regulon.
• The nif regulon contains factors which both turn on and off the production of proteins needed for nitrogen-fixation.
Key Terms
• regulon: A group of genes that is regulated by the same regulatory molecule. The genes of a regulon share a common regulatory element binding site or promoter. The genes comprising a regulon may be located non-contiguously in the genome.
• operon: A unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together.
LICENSES AND ATTRIBUTIONS
CC LICENSED CONTENT, SPECIFIC ATTRIBUTION
• Nitrogenase. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogenase. License: CC BY-SA: Attribution-ShareAlike
• Nitrogenase. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogenase. License: CC BY-SA: Attribution-ShareAlike
• Nitrogenase. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogenase. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen fixation. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogen_fixation. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen fixation. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogen_fixation. License: CC BY-SA: Attribution-ShareAlike
• cofactor. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/cofactor. License: CC BY-SA: Attribution-ShareAlike
• fixation. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/fixation. License: CC BY-SA: Attribution-ShareAlike
• heterometal. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/heterometal. License: CC BY-SA: Attribution-ShareAlike
• nitrogen fixation. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/nitrogen_fixation. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen Cycle. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...ogen_Cycle.svg. License: CC BY-SA: Attribution-ShareAlike
• Hermann Hellriegel. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Hermann_Hellriegel. License: CC BY-SA: Attribution-ShareAlike
• Martinus Beijerinck. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Martinus_Beijerinck. License: CC BY-SA: Attribution-ShareAlike
• Crop rotation. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Crop_rotation. License: CC BY-SA: Attribution-ShareAlike
• Hermann Hellriegel. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Hermann_Hellriegel. License: CC BY-SA: Attribution-ShareAlike
• Martinus Beijerinck. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Martinus_Beijerinck. License: CC BY-SA: Attribution-ShareAlike
• legume. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/legume. License: CC BY-SA: Attribution-ShareAlike
• symbiosis. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/symbiosis. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen Cycle. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...ogen_Cycle.svg. License: CC BY-SA: Attribution-ShareAlike
• Martinus Beijerinck. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...Beijerinck.png. License: Public Domain: No Known Copyright
• Nitrogenase. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogenase. License: CC BY-SA: Attribution-ShareAlike
• Nif regulon. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nif_regulon. License: CC BY-SA: Attribution-ShareAlike
• Nitrogenase general catalytic scheme. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...tic_scheme.svg. License: CC BY-SA: Attribution-ShareAlike
• Nitrogenase. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogenase. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen fixation. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogen_fixation. License: CC BY-SA: Attribution-ShareAlike
• Nitrogenase. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogenase. License: CC BY-SA: Attribution-ShareAlike
• sulfide. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/sulfide. License: CC BY-SA: Attribution-ShareAlike
• reduction. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/reduction. License: CC BY-SA: Attribution-ShareAlike
• enthalpy. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/enthalpy. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen Cycle. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...ogen_Cycle.svg. License: CC BY-SA: Attribution-ShareAlike
• Martinus Beijerinck. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...Beijerinck.png. License: Public Domain: No Known Copyright
• Nitrogenase general catalytic scheme. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...tic_scheme.svg. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen fixation. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nitrogen_fixation. License: CC BY-SA: Attribution-ShareAlike
• Leghemoglobin. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Leghemoglobin. License: CC BY-SA: Attribution-ShareAlike
• proteoglycan. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/proteoglycan. License: CC BY-SA: Attribution-ShareAlike
• oxidation. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/oxidation. License: CC BY-SA: Attribution-ShareAlike
• reduction. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/reduction. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen Cycle. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...ogen_Cycle.svg. License: CC BY-SA: Attribution-ShareAlike
• Martinus Beijerinck. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...Beijerinck.png. License: Public Domain: No Known Copyright
• Nitrogenase general catalytic scheme. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...tic_scheme.svg. License: CC BY-SA: Attribution-ShareAlike
• Leghemoglobin 1FSL. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...lobin_1FSL.png. License: Public Domain: No Known Copyright
• Nif regulon. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nif_regulon. License: CC BY-SA: Attribution-ShareAlike
• Nif gene. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nif_gene. License: CC BY-SA: Attribution-ShareAlike
• Nif gene. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Nif_gene. License: CC BY-SA: Attribution-ShareAlike
• regulon. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/regulon. License: CC BY-SA: Attribution-ShareAlike
• operon. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/operon. License: CC BY-SA: Attribution-ShareAlike
• Nitrogen Cycle. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/File:Nitrogen_Cycle.svg. License: CC BY-SA: Attribution-ShareAlike
• Martinus Beijerinck. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/File:Martinus_Beijerinck.png. License: Public Domain: No Known Copyright
• Nitrogenase general catalytic scheme. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/File:Nitrogenase_general_catalytic_scheme.svg. License: CC BY-SA: Attribution-ShareAlike
• Leghemoglobin 1FSL. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/File:Leghemoglobin_1FSL.png. License: Public Domain: No Known Copyright
• NIF REGULON. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...IF_REGULON.png. License: CC BY-SA: Attribution-ShareAlike | textbooks/bio/Microbiology/Microbiology_(Boundless)/05%3A_Microbial_Metabolism/5.15%3A_Nitrogen_Fixation/5.15E%3A_Genetics_and_Regulation_of_N_Fixation.txt |
Thumbnail: A Petri dish with bacterial colonies on an agar-based growth medium. (Public Domain; U.S. National Oceanic and Atmospheric Administration).
06: Culturing Microorganisms
Learning Objectives
• Describe the ultrastructure of the microbial cytoplasm
The bacterial cytoplasmic membrane plays a role in permeability and energy conservation in microbial cell structure. The cytoplasmic membranes in bacteria are composed of a phospholipid bilayer; this layer differs from those found in eukaryotes in their lack of sterols. The membranes however, may contain compounds called hopanoids and various fatty acids as well.
The fatty acids present within the cytoplasmic membrane are categorized into classes based on the addition of functional groups; methyl or hydroxyl groups are two examples of such groups. The cytoplasm itself is enclosed within the membrane. In bacteria it lacks the structures seen in eukaryotic cells, including a mitochondria, nucleus, or chloroplasts. The components of the microbial cytoplasm include macromolecules, smaller molecules, various inorganic ions, and cytoplasmic inclusions. The macromolecules included in a bacterial cytoplasm include proteins, DNA, RNA molecules. Smaller molecules include precursors to macromolecules and vitamins. Below is an overview of the components found in microbial cytoplasm based on chemical analysis.
The cytosol is a major component of the cytoplasm; it is the liquid portion of the cytoplasm that is not enclosed within a membrane-bound component. The cytosol is typically composed of water, salts, and organic molecules. Elements found within the cytosol include carbon, hydrogen, nitrogen, oxygen, phosphorous, and sulfur. These elements are critical to metabolic processes used by microbes.
Macromolecules found within bacterial cytoplasm include the nucleoid region, ribosomes, proteins, and enzymes. The nucleoid region is the area within the cell that houses the genetic material. Prokaryotes may sometimes contain an extra chromosomal piece of DNA referred to as the plasmid. The ribosomes, similar to ribosomes in eukaryotes, are responsible for protein synthesis. Unlike eukaryotes, prokaryotes, specifically bacteria, typically contain one cytosol-specific ribosome. Eukaryotes have multiple types of ribosomes, including the mitochondria and cytosol).
Another group of substances found within the cytoplasm include small particles referred to as inclusions. These inclusions are characterized by their granular appearance and insolubility. They are suspended in the cytosol. Inclusions vary, based on cell types. Typically, inclusions function as reserve materials. In prokaryotes, for example, lipid droplets are plentiful in cells which require lipid storage mechanisms. These lipid droplets store molecules such as fatty acids which are present in the cytoplasmic membrane of prokaryotes. E. coli offers another example of bacterial inclusions. These E. coli inclusions are composed of protein aggregates. In addition, inclusions can contain phosphate reserves, sulfur reserves, or photosynthetic pigments.
Key Points
• The cytosol contains the liquid portion of the cytoplasm and is not contained within a membrane -bound component.
• Macromolecules within the cytoplasm include DNA, ribosomes and proteins.
• Cytoplasmic inclusions within the cytoplasm are characterized by a granular appearance and vary based on cell type.
Key Terms
• hopanoids: a group of pentacyclic compounds that exhibit various functions in prokaryotes
6.1B: Sources of Essential Nutrients
Learning Objectives
• Describe the types of nutrients that are used by microorganisms for growth and metabolism
Nutrients are materials that are acquired from the environment and are used for growth and metabolism. Microorganisms (or microbes) vary significantly in terms of the source, chemical form, and amount of essential elements they need. Some examples of these essential nutrients are carbon, oxygen, hydrogen, phosphorus, and sulfur. There are two categories of essential nutrients: macro-nutrients (which are needed in large amounts) and micro-nutrients (which are needed in trace or small amounts). Macro-nutrients usually help maintain the cell structure and metabolism. Micro-nutrients help enzyme function and maintain protein structure.
Organic and Inorganic Nutrients
Organic nutrients contain some combination of carbon and hydrogen atoms. Inorganic nutrients are elements or simply molecules that are made of elements other than carbon and hydrogen.
Essential Nutrients
The sources of common essential nutrients are carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. Organisms usually absorb carbon when it is in its organic form. Carbon in its organic form is usually a product of living things. Another essential nutrient, nitrogen, is part of the structure of protein, DNA, RNA, and ATP. Nitrogen is important for heterotroph survival, but it must first be degraded into basic building blocks, such as amino acids, in order to be used. Oxygen is an important component of both organic and inorganic compounds. It is essential to the metabolism of many organisms. Hydrogen has many important jobs including maintaining the pH of solutions and providing free energy in reactions of respiration. Phosphate is an important player in making nucleic acids and cellular energy transfers. Without sufficient phosphate, an organism will cease to grow. Lastly, sulfur is found in rocks and sediments and is found widely in mineral form.
Key Points
• Nutrients are materials that are acquired from the environment and are used for growth and metabolism. Microorganisms (or microbes) vary significantly in the source, chemical form, and amount they will need of these essential elements.
• Macro-nutrients are needed in large amounts and micro-nutrients are needed in trace or small amounts.
• Organic nutrients contain some combination of carbon and hydrogen atoms. Inorganic nutrients are elements or simply molecules that are made of elements other than carbon and hydrogen.
Key Terms
• organic: relating to the compounds of carbon, and relating to natural products
• inorganic: relating to a compound that does not contain carbon.
• metabolism: the complete set of chemical reactions that occur in living cells.
6.1C: Limitation of Microbial Growth by Nutrient Supply
Learning Objectives
• Describe the role of nutrients in microbial growth and their culture in the lab
Nutrients are necessary for microbial growth and play a vital role in the proper cultivation of microorganisms in the laboratory and for proper growth in their natural environments. The types of nutrients that are required include those that supply energy, carbon and additional necessary materials. The nutrients used to propagate growth are organism -specific, based on their cellular and metabolic processes..
The common nutrients which are found to be required in all living things include carbon, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium, oxygen, iron and additional trace elements. Essential nutrients are nutrients absolutely required by an organism. Two categories of essential nutrients are macro- and micro-nutrients. Macronutrients are necessary in large amounts; micronutrients tend to be needed in smaller amounts and are often trace elements.
Nutrients as Limiting Factors
In regard to required nutrients for proper growth, there are often limiting factors involved. The limiting factor or limiting nutrient affects and controls growth. The availability of specific nutrients dictates organismal growth by controlling and limiting activation of cellular and metabolic pathways necessary for progress. When all nutrients and parameters are ideal and constant during the growth phase, this is regarded as a steady state: all requirements are present and microorganisms thrive. In circumstances where there are less than ideal parameters, such as a lack of specific requirements, the growth process is affected.
In industrial microbiology this concept is critical, as microbial growth and production is dictated by proper cellular growth and metabolism. The production of necessary components if often controlled by the presence and concentration of a limiting nutrient. Hence, it is critical to identify the required nutrients and ensure these are supplied in the culturing of microorganisms.
Key Points
• The common nutrients which are found to be required in all living things include carbon, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium, oxygen, iron and additional trace elements.
• Both and macro- and micro-nutrients are critical in proper organismal growth as they play important roles in cellular and metabolic processes.
• The limiting nutrient is essential for growth and based on its concentration and presence or absence can control growth.
Key Terms
• micronutrient: an element or nutrient required in small quantities.
• macronutrients: any element or nutrient required in large amounts.
LICENSES AND ATTRIBUTIONS
CC LICENSED CONTENT, SPECIFIC ATTRIBUTION | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.01%3A_Microbial_Nutrition/6.1A%3A_Chemical_Analysis_of_Microbial_Cytoplasm.txt |
Stringent response is a stress response that occurs in bacteria and plant chloroplasts in reaction to stress conditions.
Learning Objectives
• Discuss how starvation activates survival genes
Key Points
• The stringent response is signaled by the alarmone (p)ppGpp.
• In Escherichia coli (p)ppGpp, production is mediated by the ribosomal protein L11 and the ribosome-associated protein RelA with the A-site bound deacylated tRNA being the ultimate inducer.
• In other bacteria, stringent response is mediated by a variety of RelA/SpoT Homologue (RSH) proteins, some of which activate synthetically, hydrolytically, or both (Rel).
Key Terms
• stringent response: a stress response in bacteria in reaction to amino-acid starvation, fatty acid limitation, and other stress conditions.
Stringent Response, also called stringent control, is a stress response that occurs in bacteria and plant chloroplasts in reaction to amino-acid starvation, fatty acid limitation, iron limitation, heat shock, and other stress conditions. The stringent response is signaled by the alarmone (p)ppGpp and modulating transcription of up to 1/3 of all genes in the cell. This in turn causes the cell to divert resources away from growth and division and toward amino acid synthesis in order to promote survival until nutrient conditions improve.
In Escherichia coli, (p)ppGpp production is mediated by the ribosomal protein L11 and the ribosome-associated protein RelA with the A-site bound deacylated tRNA being the ultimate inducer. RelA converts GTP and ATP into pppGpp by adding the pyrophosphate from ATP onto the 3′ carbon of the ribose in GTP, releasing AMP. pppGpp is converted to ppGpp by the gpp gene product, releasing Pi. ppGpp is converted to GDP by the spoT gene product, releasing pyrophosphate ( PPi ). GDP is converted to GTP by the ndk gene product. Nucleoside triphosphate (NTP) provides the Pi, and is converted to Nucleoside diphosphate (NDP).
In other bacteria, stringent response is mediated by a variety of RelA/SpoT Homologue (RSH) proteins. Some of these proteins activate synthetically, hydrolytically, or both (Rel). The disabling of the stringent response by distruption of relA and spoT in Pseudomonas aeruginosa, produced in infectious cells and biofilms characterized by nutrient limitation, causes greater susceptibility to antibiotics.
During the stringent response, (p)ppGpp accumulation affects the resource-consuming cell processes replication, transcription, and translation. (p)ppGpp is thought to bind RNA polymerase and alter the transcriptional profile, decreasing the synthesis of translational machinery (such as rRNA and tRNA), and increasing the transcription of biosynthetic genes. Additionally, the initiation of new rounds of replication is inhibited and the cell cycle arrests until nutrient conditions improve. Translational GTPases involved in protein biosynthesis are also affected by ppGpp, with Initiation Factor 2 (IF2) being the main target.
6.2B: Oligotrophs
Learning Objectives
• Examine oligotrophs and their adaptation to nutrient poor environments
An oligotroph is an organism that thrives in an environment that offers very low levels of nutrients. They may be contrasted with copiotrophs, which prefer nutritionally rich environments. Oligotrophs are characterized by slow growth, low rates of metabolism, and generally low population density. Oligotrophic environments include deep oceanic sediments, caves, glacial and polar ice, deep subsurface soil, aquifers, ocean waters, and leached soils. An ecosystem or environment is said to be oligotrophic if it offers little to sustain life. The term is commonly utilized to describe environments of water, ice, air, rock or soil with very low nutrient levels. Oligotrophic environments are of special interest for alternative energy sources and survival strategies upon which life could rely. An example of oligotrophic bacterium are Caulobacter crescentus.
Caulobacter crescentus is a Gram-negative, oligotrophic bacterium widely distributed in fresh water lakes and streams. The control circuitry that directs and paces Caulobacter cell cycle progression involves the entire cell operating as an integrated system. The control circuitry monitors the environment and the internal state of the cell, including the cell topology, as it orchestrates activation of cell cycle subsystems and Caulobacter crescentus asymmetric cell division. The proteins of the Caulobacter cell cycle control system and its internal organization are co-conserved across many alphaproteobacteria species, but there are great differences in the regulatory apparatus’ functionality and peripheral connectivity to other cellular subsystems from species to species. The Caulobacter cell cycle control system has been exquisitely optimized by evolutionary selection as a total system for robust operation in the face of internal stochastic noise and environmental uncertainty.
The bacterial cell’s control system has a hierarchical organization. The signaling and the control subsystem interfaces with the environment by means of sensory modules largely located on the cell surface. The genetic network logic responds to signals received from the environment and from internal cell status sensors to adapt the cell to current conditions.
Key Points
• Oligotrophic environments are of special interest for alternative energy sources and survival strategies upon which life could rely.
Key Terms
• oligotroph: An organism capable of living in an environment that offers very low levels of nutrients. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.02%3A_Cell_Differentiation_and_Starvation/6.2A%3A_Activation_of_Starvation_by_Survival_Genes.txt |
Starvation-induced fruiting bodies can aggregate up to 500 micrometres long and contain approximately 100,000 bacterial cells.
Learning Objectives
• Explain starvation induced fruit bodies
Key Points
• In fruiting bodies, the bacteria perform separate tasks; this type of cooperation is a simple type of multicellular organisation.
• Myxococcus xanthus colonies exist as a self-organized, predatory single- species biofilm called a swarm.
• The fruiting process is thought to benefit myxobacteria by ensuring that cell growth is resumed with a group (swarm) of myxobacteria, rather than as isolated cells.
Key Terms
• quorum sensing: A proposed method of communication between bacterial cells by the release and sensing of small diffusible signal molecules.
• stigmergy: A mechanism of spontaneous, indirect coordination between agents or actions, where the trace left in the environment by an action stimulates the performance of a subsequent action.
• saprotrophic: Extra-cellular digestion involved in the processing of dead or decayed organic matter
Starvation-Induced Fruiting Bodies
When starved of amino acids, myxobacteria, or slime bacteria, detect surrounding cells in a process known as quorum sensing . Migrating towards each other, they aggregate to form fruiting bodies up to 500 micrometers long containing approximately 100,000 bacterial cells. In these fruiting bodies, the bacteria perform separate tasks; this type of cooperation is a simple type of multicellular organisation. About one in 10 cells migrate to the top of these fruiting bodies and differentiate into a specialized dormant state called myxospore, which is more resistant to drying and other adverse environmental conditions than ordinary cells.
The myxobacteria are a group of bacteria that predominantly live in the soil and feed on insoluble organic substances. The myxobacteria have very large genomes, relative to other bacteria e.g., 9–10 million nucleotides. Sorangium cellulosum has the largest known (as of 2008) bacterial genome, at 13.0 million nucleotides.
Myxobacteria are included among the delta group of proteobacteria, a large taxon of Gram-negative forms. They can move actively by gliding and typically travel in swarms (also known as wolf packs), containing many cells kept together by intercellular molecular signals. Individuals benefit from aggregation as it allows accumulation of extracellular enzymes which are used to digest food that increases feeding efficiency.
Myxobacteria produce a number of biomedically and industrially-useful chemicals, such as antibiotics, and export those chemicals outside of the cell. When nutrients are scarce, myxobacterial cells aggregate into fruiting bodies, a process long-thought to be mediated by chemotaxis but now considered to be a function of a form of contact-mediated signaling. These fruiting bodies can take different shapes and colors, depending on the species.
Within the fruiting bodies, cells begin as rod-shaped vegetative cells and develop into rounded myxospores with thick cell walls. These myxospores, analogous to spores in other organisms, are more likely to survive until nutrients are more plentiful. The fruiting process is thought to benefit myxobacteria by ensuring that cell growth is resumed with a group (swarm) of myxobacteria, rather than as isolated cells. At a molecular level, initiation of fruiting body development is regulated by Pxr sRNA.
Myxococcus xanthus colonies exist as a self-organized, predatory, saprotrophic, single-species biofilm called a swarm. Myxococcus xanthus, which can be found almost ubiquitously in soil, are thin rod-shaped, gram-negative cells that exhibit self-organizing behavior as a response to environmental cues. The swarm modifies its environment through stigmergy. This behavior facilitates predatory feeding, as the concentration of extracellular digestive enzymes secreted by the bacteria increases.
M. xanthus is a model organism for studying development, the behavior in which starving bacteria self-organize to form fruiting bodies: dome shaped structures of approximately 100,000 cells. These swarms differentiate into metabolically quiescent and environmentally resistant myxospores over the course of several days. During this process of self-organizing, dense ridges of cells move in traveling waves (ripples) that grow and shrink over several hours. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.02%3A_Cell_Differentiation_and_Starvation/6.2C%3A_Starvation-Induced_Fruiting_Bodies.txt |
Several bacteria alter their morphology in response to the types and concentrations of external compounds.
Learning Objectives
• Explain bacterial differentiation to eukaryotic-like sturctures
Key Points
• Bacterial morphology changes help to optimize interactions with cells and the surfaces to which they attach.
• Oxidative stress, nutrient limitation, DNA damage and antibiotics exposure are some of stress conditions to which bacteria respond, altering their DNA replication and cell division.
• The most frequent shape alteration may be filamentation triggered by a limitation in the availability of one or more nutrients.
Key Terms
• cytoskeleton: A cellular structure like a skeleton, contained within the cytoplasm.
• septum: a partition that separates the cells of a (septated) fungus
• segrosomes: multiprotein complexes that partition chromosomes/plasmids in bacteria.
• cell division: a process by which a cell divides into two cells.
Bacterial morphological plasticity refers to evolutionary changes in the shape and size of bacterial cells. As bacteria evolve, morphological changes occur to maintain the consistency of the cell. However, this consistency could be affected in some circumstances (such as environmental stress) and changes in bacterial shape and size. In bacteria, the transformation into filamentous organisms have been recently demonstrated. These are survival strategies that affect the normal physiology of the bacteria in response to factors such as innate immune response, predator sensing, quorum sensing and antimicrobial signs.
Normally, bacteria have different shapes and sizes which include coccus, rod and helical/spiral (among others less common). This forms the basis for their classification. For instance, rod shapes may allow bacteria to attach more readily in environments with shear stress (e.g., in flowing water). Cocci may have access to small pores, creating more attachment sites per cell and hiding themselves from external shear forces. Spiral bacteria combine some of the characteristics of cocci (small footprints) and of filaments (more surface area on which shear forces can act) and the ability to form an unbroken set of cells to build biofilms. Several bacteria alter their morphology in response to the types and concentrations of external compounds. Bacterial morphology changes help to optimize interactions with cells and the surfaces to which they attach. This mechanism has been described in bacteria such as Escherichia coli and Helicobacter pylori.
Oxidative stress, nutrient limitation, DNA damage and antibiotics exposure are some stress conditions to which bacteria respond, altering their DNA replication and cell division. Filamentous bacteria have been considered to be over-stressed, sick and dying members of the population. However, the filamentous members of some communities have vital roles in the population’s continued existence, since the filamentous phenotype can confer protection against lethal environments.Filamentous E. coli can be up to 70 µm in length and has been identified as playing an important role in pathogenesis in human cystitis. There are different mechanisms identified in some bacteria that are attributable to the development of filamentous forms.
Nutritional stress can change bacterial morphology. The most frequent shape alteration may be filamentation triggered by a limitation in the availability of one or more nutrients. Since the filament can increase a cell’s uptake–proficiency surface without changing its surface-to-volume ratio appreciably, this may be enough reason for cells to be filament. Moreover, the filamentation benefits bacterial cells attaching to a surface because it increases specific surface area in direct contact with the solid medium. In addition, the filamentation may allow bacterial cells to access nutrients by enhancing the possibility that the filament will be exposed to a nutrient-rich zone and pass compounds to the rest of the cell’s biomass. For example, Actinomyces israelii grows as filamentous rods or branched in the absence of phosphate, cysteine, or glutathione. However, it returns to a regular rod-like morphology when adding back these nutrients.
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Learning Objectives
• Classify culture media
Culture medium or growth medium is a liquid or gel designed to support the growth of microorganisms. There are different types of media suitable for growing different types of cells. Here, we will discuss microbiological cultures used for growing microbes, such as bacteria or yeast.
NUTRIENT BROTHS AND AGAR PLATES
These are the most common growth media, although specialized media are sometimes required for microorganism and cell culture growth. Some organisms, termed fastidious organisms, need specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells. Many human microbial pathogens also require the use of human cells or cell lysates to grow on a media.
The most common growth media nutrient broths (liquid nutrient medium) or LB medium (Lysogeny Broth) are liquid. These are often mixed with agar and poured into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar. Many microbes can also be grown in liquid cultures comprised of liquid nutrient media without agar.
DEFINED VS UNDEFINED MEDIA
This is an important distinction between growth media types. A defined medium will have known quantities of all ingredients. For microorganisms, it provides trace elements and vitamins required by the microbe and especially a defined carbon and nitrogen source. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract, which consists of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.
There are many different types of media that can be used to grow specific microbes, and even promote certain cellular processes; such as wort, the medium which is the growth media for the yeast that makes beer. Without wort in certain conditions, fermentation cannot occur and the beer will not contain alcohol or be carbonated (bubbly).
COMMON BROADLY-DEFINED CULTURE MEDIA
Nutrient media – A source of amino acids and nitrogen (e.g., beef, yeast extract). This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown. These media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory-culture collections.
Minimal media – Media that contains the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow “wild type” microorganisms. These media can also be used to select for or against the growth of specific microbes. Usually a fair amount of information must be known about the microbe to determine its minimal media requirements.
Selective media – Used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing.
Differential media – Also known as indicator media, are used to distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection and identification of microorganisms.
These few examples of general media types provide some indication only; there are a myriad of different types of media that can be used to grow and control microbes.
Key Points
• Culture media contains the nutrients needed to sustain a microbe.
• Culture media can vary in many ingredients allowing the media to select for or against microbes.
• Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources in culture media.
Key Terms
• culture: The process of growing a bacterial or other biological entity in an artificial medium.
• lysogeny broth: Lysogeny broth (LB) is a nutritionally-rich medium; primarily used for the growth of bacteria. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.03%3A_Culturing_Bacteria/6.3A%3A_Culture_Media.txt |
Learning Objectives
• Differentiate complex and synthetic medias
There are many types of culture media, which is food that microbes can live on. Two major sub types of media are complex and synthetic medias, known as undefined and defined media.
An undefined medium has some complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.A defined medium (also known as chemically defined medium or synthetic medium) is a medium in which all the chemicals used are known, no yeast, animal, or plant tissue is present. A chemically defined medium is a growth medium suitable for the culture of microbes or animal cells (including human) of which all of the chemical components are known. The term chemically defined medium was defined by Jayme and Smith as a ‘Basal formulation which may also be protein-free and is comprised solely of biochemically-defined low molecular weight constituents.
A chemically defined medium is entirely free of animal-derived components (including microbial derived components such as yeast extract) and represents the purest and most consistent cell culture environment. By definition chemically defined media cannot contain either fetal bovine serum, bovine serum albumin, or human serum albumin as these products are derived from bovine or human sources and contain complex mixes of albumins and lipids. The term ‘chemically defined media’ is often misused in the literature to refer to serum albumin-containing media. Animal serum or albumin is routinely added to culture media as a source of nutrients and other ill-defined factors, despite technical disadvantages to its inclusion and its high cost. Technical disadvantages to using serum include the undefined nature of serum, batch-to-batch variability in composition, and the risk of contamination. There are increasing concerns about animal suffering inflicted during serum collection that add an ethical imperative to move away from the use of serum wherever possible. Chemically defined media differ from serum-free media in that bovine serum albumin or human serum albumin with either a chemically defined recombinant version (which lacks the albumin associated lipids) or synthetic chemical such as the polymer polyvinyl alcohol which can reproduce some of the functions of serums.
Key Points
• Defined media is made from constituents that are completely understood.
• Undefined media has some part of which is not entirely defined.
• The presence of extracts from animals or other microbes makes a media undefined as the entire chemical composition of extracts are not completely known.
Key Terms
• recombinant: This term refers to something formed by combining existing elements in a new combination. Thus, the phrase recombinant DNA refers to an organism created in the lab by adding DNA from another species.
• serum: The clear yellowish fluid obtained upon separating whole blood into its solid and liquid components after it has been allowed to clot. Also called blood serum.
6.3C: Selective and Differential Media
Learning Objectives
• Compare selective and differential media
There are many types of media used in the studies of microbes. Two types of media with similar implying names but very different functions, referred to as selective and differential media, are defined as follows.
Selective media are used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. Media lacking an amino acid such as proline in conjunction with E. coli unable to synthesize it were commonly used by geneticists before the emergence of genomics to map bacterial chromosomes. Selective growth media are also used in cell culture to ensure the survival or proliferation of cells with certain properties, such as antibiotic resistance or the ability to synthesize a certain metabolite. Normally, the presence of a specific gene or an allele of a gene confers upon the cell the ability to grow in the selective medium. In such cases, the gene is termed a marker. Selective growth media for eukaryotic cells commonly contain neomycin to select cells that have been successfully transfected with a plasmid carrying the neomycin resistance gene as a marker. Gancyclovir is an exception to the rule as it is used to specifically kill cells that carry its respective marker, the Herpes simplex virus thymidine kinase (HSV TK). Some examples of selective media include:
• Eosin methylene blue (EMB) that contains methylene blue – toxic to Gram-positive bacteria, allowing only the growth of Gram negative bacteria.
• YM (yeast and mold) which has a low pH, deterring bacterial growth.
• MacConkey agar for Gram-negative bacteria.
• Hektoen enteric agar (HE) which is selective for Gram-negative bacteria.
• Mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol. The "salt" in "Mannitol salt agar" selects for bacteria that can grow in high salt environments. Staphylococci thrive on the medium, largely because of their adaptation to salty habitats such as human skin.
• Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of Escherichia coli.
• Xylose lysine desoxyscholate (XLD), which is selective for Gram-negative bacteria buffered charcoal yeast extract agar, which is selective for certain gram-negative bacteria, especially Legionella pneumophila.
Differential media or indicator media distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria. Examples of differential media include:
• Blood agar (used in strep tests), which contains bovine heart blood that becomes transparent in the presence of hemolytic.
• Streptococcuseosin methylene blue (EMB), which is differential for lactose and sucrose fermentation.
• MacConkey (MCK), which is differential for lactose fermentationmannitol salt agar (MSA), which is differential for mannitol fermentation.
• X-gal plates, which are differential for lac operon mutants.
Key Points
• Selective media generally selects for the growth of a desired organism, stopping the growth of or altogether killing non-desired organisms.
• Differential media takes advantage of biochemical properties of target organisms, often leading to a visible change when growth of target organisms are present.
• Differential media, unlike selective media, does not kill organisms. It indicates if a target organism is present.
Key Terms
• recombinant: This term refers to something formed by combining existing elements in a new combination. Thus, the phrase recombinant DNA refers to an organism created in the lab by adding DNA from another species.
• gene: A unit of heredity; a segment of DNA or RNA that is transmitted from one generation to the next. It carries genetic information such as the sequence of amino acids for a protein.
• allele: One of a number of alternative forms of the same gene occupying a given position on a chromosome. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.03%3A_Culturing_Bacteria/6.3B%3A_Complex_and_Synthetic_Media.txt |
Microbiologists rely on aseptic technique, dilution, colony streaking and spread plates for day-to-day experiments.
Learning Objectives
• Recall aseptic technique, dilution series, streaking and spreading plates
Key Points
• Aseptic technique is basically the mindset of keeping things free of contamination, as the world we live in has so many microbes that can interfere with experiments.
• Colony streaking leads to to the isolation of individual colonies, which are a group of microbes that came from one single progenitor mircrobe.
• Spread plates allow for the even spreading of bacteria onto a petri dish; allowing for the isolation of individual colonies, for counting or further experiments.
Key Terms
• colony: A bacterial colony is defined as a visible cluster of bacteria growing on the surface of or within a solid medium, presumably cultured from a single cell.
• bunsen burner: A small laboratory gas burner whose air supply may be controlled with an adjustable hole.
Microbiologists have many tools, but four relatively simple techniques are used by microbiologists daily, these are outlined here.
Aseptic technique or sterile technique is used to avoid contamination of sterile media and equipment during cell culture. Sterile technique should always be employed when working with live cell cultures and reagents/media that will be used for such cultures. This technique involves using flame to kill contaminating organisms, and a general mode of operation that minimizes exposure of sterile media and equipment to contaminants.
When working with cultures of living organisms, it is extremely important to maintain the environments in which cells are cultured and manipulated as free of other organisms as possible. This requires that exposure of containers of sterilized culture media to outside air should be minimized, and that flame is used to “re-sterilize” container lids and rims. This means passing rims and lids through the flame produced by a Bunsen burner in order to kill microorganisms coming in contact with those surfaces.
Sterile technique, in general, is a learned state-of-being, or mantra, where every utilization of any sterile material comes with the caveat of taking every precaution to ensure it remains as free of contaminants as possible for as long as possible. Heat is an excellent means of killing microorganisms, and the Bunsen burner is the sterile technician’s best friend.
A serial dilution is the step-wise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M… Serial dilutions are used to accurately create highly-diluted solutions as well. A culture of microbes can be diluted in the same fashion. For a ten-fold dilution on a 1 mL scale, vials are filled with 900 microliters of water or media, and 100 microliters of the stock microbial solution are serially transferred, with thorough mixing after every dilution step. The dilution of microbes is very important to get to microbes diluted enough to count on a spread plate (described later).
In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. This is dipped in an inoculum such as a broth or patient specimen containing many species of bacteria.The sample is spread across one quadrant of a petri dish containing a growth medium, usually an agar plate which has been sterilized in an autoclave. Choice of which growth medium is used depends on which microorganism is being cultured, or selected for. Growth media are usually forms of agar, a gelatinous substance derived from seaweed.
Spread plates are simply microbes spread on a media plate. Microbes are in a solution, and can be diluted. They are then transferred to a petri dish with media specific for the growth of the microbe of interest. The solution is then spread uniformly through a number of possible means, the most popular is the use of sterile glass beads that are shook on top of the media, spreading the microbe-containing liquid evenly on the plate. Also common is the use of a bent-glass rod, often referred to as a hockey stick, due to its similar shape. The glass rod is sterilized and used to spread the microbe-containing liquid uniformly on the plate. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.03%3A_Culturing_Bacteria/6.3D%3A_Aseptic_Technique_Dilution_Streaking_and_Spread_Plates.txt |
Learning Objectives
• Evaluate special culture techniques
Microbiologists would prefer to use well-defined media to grow a microbe, making the microbe easier to control. However, microbes are incredibly varied in what they use as a food source, the environments they live in, and the danger levels they may have for humans and other organisms they may compete with. Therefore they need special nutrient and growth environments. To grow these difficult microbes, microbiologists often turn to undefined media which is chosen based on price and more-so in this case by necessity as some microorganisms have never been cultured on defined media. Some special culture conditions are relatively simple as demonstrated by microaerophile.
A microaerophile is a microorganism that requires oxygen to survive, but requires environments containing lower levels of oxygen than are present in the atmosphere (~20% concentration). Many microphiles are also capnophiles, as they require an elevated concentration of carbon dioxide. In the laboratory they can be easily cultivated in a candle jar. A candle jar is a container into which a lit candle is introduced before sealing the container’s airtight lid. The candle’s flame burns until extinguished by oxygen deprivation, which creates a carbon dioxide-rich, oxygen-poor atmosphere in the jar. Many labs also have access directly to carbon dioxide and can add the desired carbon dioxide levels directly to incubators where they want to grow microaerophiles.
Animals can often be used to culture microbes. For example, armadillos are often used in the study of leprosy. They are particularly susceptible due to their unusually low body temperature, which is hospitable to the leprosy bacterium, Mycobacterium leprae. The leprosy bacterium is difficult to culture and armadillos have a body temperature of 34°C, similar to human skin. Likewise, humans can acquire a leprosy infection from armadillos by handling them or consuming armadillo meat. Additionally, Syphillis which is caused by the bacteria Treponema pallidum is difficult to grow with defined media, so rabbits are used to culture Treponema pallidum. Treponema pallidum belongs to the Spirochaetesphylum of bacteria.
To date Spirochaetes are very difficult if not impossible to rear in a controlled laboratory environment. This also includes other human pathogens like the bacterium that causes Lyme disease. Using animals to culture human-pathogens has problems. First, the use of animals is always difficult for technical and ethical reasons. Also, a microbe growing on animal other than a human may behave very differently from how that same microbe will behave on a human. Some human pathogens are grown directly on cells cultured from humans. Exemplified by the bacteria Chlamydia trachomatis, the bacteria responsible for the sexually transmitted infection (STI) in humans known as Chlamydia. As Chlamydia trachomatis only grows in humans. The human cell culture known as McCoy cell culture is used to culture this bacteria.
A large concern of microbiology is trying to find ways in which humans can avoid or get rid of microbrial infections. As typified by some of the above examples, some microbes have to be grown in the lab, and some of them can infect humans. To deal with this, microbiologists use a classification of biosafety levels. A biosafety level is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed facility. The levels of containment range from the lowest biosafety level 1 (BSL-1) to the highest at level 4 (BSL-4). In the United States, the Centers for Disease Control and Prevention (CDC) have specified these levels.
• Biosafety Level 1: This level is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, with minimal potential hazard to laboratory personnel and the environment.
• Biosafety Level 2: This level is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It includes various bacteria and viruses that cause only mild disease to humans or are difficult to contract via aerosol in a lab setting such as chlamydia.
• Biosafety Level 3: This level is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents that may cause serious or potentially lethal disease after inhalation. It includes various bacteria, parasites, and viruses that can cause severe to fatal disease in humans, but for which treatments exist (eg. yellow fever).
• Biosafety Level 4: This level is reserved for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, agents that cause severe to fatal disease in humans for which vaccines or other treatments are not available, such as Bolivian and Argentine hemorrhagic fevers, Marburg virus, and the Ebola virus. Very few laboratories are biosafety level 4.
Key Points
• Microbes, often those that we know little about, have to be cultured with undefined media or growth conditions.
• The use of animals to culture animals is sometimes necessary as no simple media can be used, this presents technical and ethical issues.
• As human pathogens are often studied by microbiologists, special safety conditions know as biosafety levels are used to keep researches free of infection from the pathogens they study.
Key Terms
• yellow fever: An acute febrile illness of tropical regions, caused by a flavivirus and spread by mosquitoes, characterized by jaundice, black vomit, and the absence of urination.
• Lyme disease: Infection by a bacterium of the genus Borrelia which is transmitted by ticks. Symptoms include a rash followed by fever, joint pain, and headaches.
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Learning Objectives
• List the growth phases of microrganisms and the different types of growth media available to culture them
The most common growth media for microorganisms are nutrient broths and agar plates; specialized media are required for some microorganisms. Some, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.
Growth media: defined vs. undefined
An important distinction between growth media types is that of defined versus undefined media.
A defined medium will have known quantities of all ingredients. For microorganisms, this consists of providing trace elements and vitamins required by the microbe, and especially, a defined source of both carbon and nitrogen. Glucose or glycerol is often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources.
An undefined medium has some complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.
Types of media
Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally-rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45°C), which turns brown and gives the medium the color for which it is named.
Selective media are used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillinor tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. Media lacking an amino acid, such as proline in conjunction with E. coli unable to synthesize it, were commonly used by geneticists before the emergence of genomics to map bacterial chromosomes.
Differential/indicator media distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria. The agar triple-sugar iron (TSI) is one of the culture media used for the differentiation of most enterobacteria.
Growth in closed culture systems, such as a batch culture in LB broth, where no additional nutrients are added and waste products are not removed, the bacterial growth will follow a predicted growth curve and can be modeled.
Growth phases
During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
Exponential phase (sometimes called the log or logarithmic phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population.Under controlled conditions, cyanobacteria can double their population four times a day. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with wastes.
The stationary phase is due to a growth-limiting factor; this is mostly depletion of a nutrient, and/or the formation of inhibitory products such as organic acids.
At death phase, bacteria run out of nutrients and die.
Culture
Batch culture is the most common laboratory-growth method in which bacterial growth is studied, but it is only one of many. The bacterial culture is incubated in a closed vessel with a single batch of medium.
In some experimental regimes, some of the bacterial culture is periodically removed and added to fresh sterile medium. In the extreme case, this leads to the continual renewal of the nutrients. This is a chemostat, also known as an open or continuous culture: a steady state defined by the rates of nutrient supply and bacterial growth. In comparison to batch culture, bacteria are maintained in exponential growth phase, and the growth rate of the bacteria is known. Related devices include turbidostats and auxostats. Bacterial growth can be suppressed with bacteriostats, without necessarily killing the bacteria.
In a synecological culture, a true-to-nature situation in which more than one bacterial species is present, the growth of microbes is more dynamic and continual.
Key Points
• The most common growth media for microorganisms are nutrient broths and agar plates.
• Open cultures allow for a replenishment of nutrients and a reduction of waste buildup in the media.
• Selective media are used for the growth of only selected microorganisms.
• Differential media or indicator media distinguish one microorganism type from another growing on the same media.
Key Terms
• closed culture: A closed culture has no additional nutrients added to the system, and waste products are not removed. Cultures in a closed system will follow a predicted growth curve.
• Enriched media: Contains nutrients required to support the growth of a wide variety of organisms.
• open culture: A continuous culture where periodically some of the bacterial culture is removed and added to fresh sterile medium. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.04%3A_Microbial_Culture_Methods/6.4A%3A_Enrichment_and_Isolation.txt |
Learning Objectives
• Describe how pure microbial cultures can be grown in agar-based growth medium
Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology. It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another. For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used. Agar is a gelatinous substance derived from seaweed. A cheap substitute for agar is guar gum, which can be used for the isolation and maintenance of thermophiles.
Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the best temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius for cultures from humans or animals or lower for environmental cultures). Another method of bacterial culture is liquid culture, in which the desired bacteria are suspended in liquid broth, a nutrient medium. These are ideal for preparation of an antimicrobial assay. The experimenter would inoculate liquid broth with bacteria and let it grow overnight (they may use a shaker for uniform growth). Then they would take aliquots of the sample to test for the antimicrobial activity of a specific drug or protein (antimicrobial peptides). As an alternative, the microbiologist may decide to use static liquid cultures. These cultures are not shaken and they provide the microbes with an oxygen gradient.
Key Points
• A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another.
• Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology.
• The most common form of microbial cultures are liquid or solid ( agar ).
Key Terms
• agar: A gelatinous material obtained from the marine algae, used as a bacterial culture medium, in electrophoresis and as a food additive.
6.4C: Preserving Bacterial Cultures
Learning Objectives
• Describe how bacterial cultures can be stored for a long time at -80 °C in glycerol
Three species of bacteria, Carnobacterium pleistocenium, Chryseobacterium greenlandensis, and Herminiimonas glaciei, have reportedly been revived after surviving for thousands of years frozen in ice. As a practical matter, as a researcher, you will want to preserve your selected bacteria so you can go back to it if something goes wrong. Whenever you successfully transform a bacterial culture with a plasmid or whenever you obtain a new bacterial strain, you will want to make a long term stock of that bacteria. Bacteria can be stored for months and years if they are stored at -80 °C and in a high percentage of glycerol.
In order to ensure a pure culture is being preserved, pick a single colony of the bacteria off a plate, grow it overnight in the appropriate liquid media, and with shaking. Take the overnight culture and and mix an aliquot with 40% glycerol in sterile water and place in a cryogenic vial. It is important to label the vial with all the relevant information (e.g. strain, vector, date, researcher, etc.). Freeze the glycerol stock and store at -80 °C. At this point you should also record the strain information and record the location.
While it is possible to make a long term stock from cells in the stationary phase, ideally your culture should be in logarithmic growth phase. Certain antibiotics in the medium should be removed first as they are supposedly toxic over time, e.g. Tetracycline. To do this, spin the culture down and resuspend it in the same volume of straight LB medium.
Key Points
• Preserve your selected bacteria so you always have something to go back to if something goes wrong.
• While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.
• Ensure a pure culture is being preserved by picking a single colony of the bacteria off a plate for cryopreservation.
Key Terms
• cryogenic: of, relating to, or performed at low temperatures
• logarithmic growth phase: exponential phase (sometimes called the log phase or the logarithmic phase) is a period characterized by cell doubling. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.04%3A_Microbial_Culture_Methods/6.4B%3A_Pure_Culture.txt |
Learning Objectives
• Describe how fluorescent in situ hybridization (FISH) is used in clinical and biomedical studies to detect and localize the presence or absence of specific DNA sequences and to identify pathogens
FISH (fluorescence in situ hybridization) is a cytogenetic technique developed by biomedical researchers in the early 1980s. It is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes bind to those targets that show a high degree of sequence complementarity. FISH can be used to detect RNA or DNA sequences of interest. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets, including mRNAs, in cells. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
Central to FISH are the use of probes. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process. They are anti-sense to the target mRNA or DNA of interest, thus they hybridize to targets. The probe can be tagged directly with fluorophores, or with targets for flourescently labelled antibodies or other substrates. Different types of tags can be used, therefore different targets can be detected in the same sample simultaneously (multi-colour FISH). Tagging can be done in various ways, such as nick translation, or PCR using tagged nucleotides. Probes can vary in length from 20 to 30 nucleotides to much longer sequences.
FISH is often used in clinical studies. If a patient is infected with a suspected pathogen, bacteria from the patient’s tissues or fluids, are typically grown on agar to determine the identity of the pathogen. Many bacteria, however, even well-known species, do not grow well under laboratory conditions. FISH can be used to directly detect the presence of the suspect on small samples of the patient’s tissue. FISH can also be used to compare the genomes of two biological species, to deduce evolutionary relationships. A similar hybridization technique is called a zoo blot. Bacterial FISH probes are often primers for the 16s rRNA region. FISH is widely used in the field of microbial ecology, to identify microorganisms. Biofilms, for example, are composed of complex (often) multi-species bacterial organizations. Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species within the biofilm. Preparing probes (in two different colors) for two species allows to visualize/study co-localization of these two species in the biofilm, and can be useful in determining the fine architecture of the biofilm.
Key Points
• FISH can be used in a clinical setting to identify pathogens or DNA / RNA targets of interest.
• FISH is used to detect and localize the presence or absence of specific DNA or RNA sequences in tissue or cells.
• FISH can also be used to compare the genomes of two biological species, such as in ecological studies, where a bacteria may not be culturable, it can be identified using FISH. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.04%3A_Microbial_Culture_Methods/6.4D%3A_The_FISH_Technique.txt |
Learning Objectives
• Describe how polymerase chain reaction (PCR) allows for the amplification and mutation of DNA and enables researchers to study very small samples
Polymerase chain reaction (PCR) is a useful technique for scientists, because it allows for the amplification and mutation of DNA. Through PCR, small quantities of DNA can be replicated by orders of magnitude, not only essentially preserving the sample if successful, but allowing for study on a much larger scale.. Without PCR, the studies we perform would be limited by the amount of DNA we were able to isolate from samples. Through PCR, the original DNA is essentially limitless, allowing scientists to induce various mutations in different genes for further study.
Through site-directed mutagenesis or customized primers, individual mutations in DNA can be made. By changing the amino acids transcribed from DNA through individual mutations, the importance of those amino acids with respect to gene function can be analyzed. However, this process can be difficult, particularly when genes act in concert (with varying expression with respect to gene activity). The length of time it takes to run a successful PCR and perform other techniques before additional studies can be done (protein expression, isolation, and purification, for example), makes biochemical research time-consuming and difficult. However, PCR, coupled with other biochemical techniques, allows us to analyze the very core of organisms and the processes by which they function. Common PCR protocols in labs today include knockout genotyping, fluorescence genotyping and mutant genotyping. Researchers can use PCR as a method of searching for genes by using primers that flank the target sequence of the gene along with all other necessary components for PCR. If the gene is present, the primers will bind and amplify the DNA, giving a band of amplified DNA on the agarose gel that will be run. If the gene is not present, the primers will not anneal and no amplification will occur.
The ability to identify specific genes to specific organisms has increased the use of PCR and has allowed it to be more specific and eliminate the possibility of cross contaminants. The identification of specific genes to specific organisms has important medical diagnostic value.
PCR is a reliable method to detect the presence of unwanted genetic materials, such as infections and bacteria in the clinical setting. It can even allow identification of an infectious agent without culturing. For example, in diagnosis of diseases like AIDS, PCR can be used to detect the small percentage of cells that are infected with HIV by utilizing primers that are specific for genes specialized to the HIV virus. PCR can reveal the presence of HIV in people who have not mounted an immune response to this pathogen, which may otherwise be missed with an antibody assay). Additionally, PCR is used for identifying bacterial species, such as Mycobacterium tuberculosis in tissue specimens. With the use of PCR, as few as 10 bacilli per million human cells can be readily detected. The bacilli are identified by using Mycobacterium tuberculosis specific genes.
Key Points
• PCR allows for identification of an infectious agent without the need for culturing.
• Researchers can use PCR as a method of searching for specific genes and/or mutations.
• PCR, coupled with other biochemical techniques, allows us to analyze the very core of organisms and the processes by which they function.
Key Terms
• polymerase chain reaction: A technique in molecular biology for creating multiple copies of DNA from a sample; used in genetic fingerprinting etc.
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There are numerous tests and assays available that are utilized to aid in bacterial identification in a variety of settings.
Learning Objectives
• Contrast the different tests that can be used in studies of microbes
Key Points
• Chemical assays used for bacterial identification utilize various components of the microorganisms including structural, cellular and metabolic indicators.
• Radioisotopic methods include the use of radioisotopes to help identify specific metabolic pathways utilized by bacteria by tracking uptake and breakdown of specific nutrients labeled with radioactivity.
• Micro-electrodes are commonly being used in bacterial identification, specifically for pathogenic bacteria, as a means to identify biological components in a variety of environments by combining with bio-sensors.
Key Terms
• peptidoglycan: A polymer of glycan and peptides found in bacterial cell walls.
Within the field of microbiology, there are specific tests or assays utilized to quantitatively and qualitatively measure microorganism components. These assays are often utilized to aid in bacterial identification. Three major types used for this purpose include chemical assays, radio isotopic methods and the use of micro electrodes. The following is an overview of these methodologies.
Chemical Assays
Chemical assays are utilized to identify and determine chemical components within a microorganism. Many of these assays test for specific cellular components and may have overlap with chemical analysis, which focuses on exact chemical composition.
Gram Staining
Examples of chemical assays include the classic test for Gram-positive or Gram-negative bacteria via Gram staining. Gram staining is utilized to differentiate bacteria into either of these Gram groups. The Gram staining technique is based on both chemical and physical properties of bacterial cell walls and tests for the presence of peptidoglycan.
Oxidative-Fermentation Glucose Test
The O-F test is utilized to determine the way in which a bacteria is capable of metabolizing carbohydrates such as glucose. The two major mechanisms from which bacteria can obtain energy include oxidation of glucose and lactose fermentation. This specific assay identifies which method bacteria use by cultivating bacteria in various conditions.
Hydrolysis Tests
The process of hydrolysis is characterized by the ability to chemically split a molecule by the addition of water. There are numerous tests utilized in bacterial identification which involve testing for hydrolysis of specific substances. These tests include hydrolysis of starch, lipids, casein and gelatin. The basis of these tests is to identify and determine if a microbe has the proper enzymes and molecules to breakdown and use these specific molecules as sources of energy for cellular growth.
Radioisotopic Methods
Radioisotopes are specific types of isotopes that emit radioactivity. Isotopes of an element vary in the number of neutrons within their nuclei. In the field of microbiology, radioisotopes have been used
Micro-electrodes
Electrodes are characterized by a system of electrical conductors that are used to make contact with a non-metallic portion of a circuit. In regards to microbiology and bacterial identification, micro-electrodes are commonly being utilized to identify pathogenic bacteria in numerous settings. The micro-electrodes have the capability to function as bio-sensors and detect specific biological components of microbes.
6.5B: Stable Isotopes
Learning Objectives
• Demonstrate how isotopes are used in bacterial identification
The term isotope refers to the number of neutrons a certain element contains. Elements of the same name (for example, oxygen) must always have the same number of protons, but the number of neutrons can change. Adding or subtracting neutrons from an atom does not change the elemental properties, but it can alter some of its features (like making it more radioactive).
Stable Isotopes
While the number of neutrons in a particular atom can change, there is a certain threshold where the atom is given more neutrons that its nuclear force can hold. At this point, the neutrons start to be released. The release is also known as decay. During this time, the atom is deemed “unstable. ” The atom will continue to lose neutrons until it become stable again. A stable isotope is a chemical isotope that is not radioactive. There are some cases where atoms have no stable isotopes so they continue to lose neutrons, and later protons and electrons, until they become another element entirely. Research has shown that there are 80 elements with one or more stable isotopes. Of these, 26 have only one stable isotope which is also known as being monoisotopic. The element with the most stable isotopes is tin which as 10 stable isotopes.
Key Points
• The term isotope refers to the number of neutrons a certain element contains.
• Elements of the same name must always have the same number of protons, but the number of neutrons can change.
• Adding or subtracting neutrons from an atom does not change the elemental properties, but it can alter some of its features (like making it more radioactive ).
• While the number of neutrons in a particular atom can change, there is a certain threshold where the atom is given more neutrons that its nuclear force can hold. At this point, the atom is deemed unstable.
• The atom will continue to lose neutrons, or decay, until it become stable again. A stable isotope is a chemical isotope that is not radioactive.
Key Terms
• isotope: Any of two or more forms of an element where the atoms have the same number of protons, but a different number of neutrons within their nuclei. As a consequence, atoms for the same isotope will have the same atomic number but a different mass number (atomic weight).
• atom: The smallest possible amount of matter which still retains its identity as a chemical element, now known to consist of a nucleus surrounded by electrons.
• Radioactive: A particle that has spontaneous emission of ionizing radiation as a consequence of a nuclear reaction, or directly from the breakdown of an unstable nucleus.
Extensions
Knowledge about stable isotopes is important in a variety of fields. Scientists have used information on these topics in botanical and plant biological investigations as well as ecological and biological studies. Additionally, some scientists have used oxygen isotope ratios to reconstruct historical atmospheric temperatures. This work is especially important due to our current concern with climate change/global warming.
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Learning Objectives
• Describe the process of binary fission in prokaryotes
Prokaryotes, such as bacteria, propagate by binary fission. For unicellular organisms, cell division is the only method used to produce new individuals. In both prokaryotic and eukaryotic cells, the outcome of cell reproduction is a pair of daughter cells that are genetically identical to the parent cell. In unicellular organisms, daughter cells are individuals.
Due to the relative simplicity of the prokaryotes, the cell division process, or binary fission, is a less complicated and much more rapid process than cell division in eukaryotes. The single, circular DNA chromosome of bacteria is not enclosed in a nucleus, but instead occupies a specific location, the nucleoid, within the cell. Although the DNA of the nucleoid is associated with proteins that aid in packaging the molecule into a compact size, there are no histone proteins and thus, no nucleosomes in prokaryotes. The packing proteins of bacteria are, however, related to the cohesin and condensin proteins involved in the chromosome compaction of eukaryotes.
The bacterial chromosome is attached to the plasma membrane at about the midpoint of the cell. The starting point of replication, the origin, is close to the binding site of the chromosome at the plasma membrane. Replication of the DNA is bidirectional, moving away from the origin on both strands of the loop simultaneously. As the new double strands are formed, each origin point moves away from the cell wall attachment toward the opposite ends of the cell. As the cell elongates, the growing membrane aids in the transport of the chromosomes. After the chromosomes have cleared the midpoint of the elongated cell, cytoplasmic separation begins. The formation of a ring composed of repeating units of a protein, FtsZ, directs the partition between the nucleoids. Formation of the FtsZ ring triggers the accumulation of other proteins that work together to recruit new membrane and cell wall materials to the site. A septum is formed between the nucleoids, extending gradually from the periphery toward the center of the cell. When the new cell walls are in place, the daughter cells separate.
Mitotic Spindle Apparatus
The precise timing and formation of the mitotic spindle is critical to the success of eukaryotic cell division. Prokaryotic cells, on the other hand, do not undergo karyokinesis and, therefore, have no need for a mitotic spindle. However, the FtsZ protein that plays such a vital role in prokaryotic cytokinesis is structurally and functionally very similar to tubulin, the building block of the microtubules that make up the mitotic spindle fibers that are necessary for eukaryotes. FtsZ proteins can form filaments, rings, and other three-dimensional structures that resemble the way tubulin forms microtubules, centrioles, and various cytoskeletal components. In addition, both FtsZ and tubulin employ the same energy source, GTP (guanosine triphosphate), to rapidly assemble and disassemble complex structures.
FtsZ and tubulin are homologous structures derived from common evolutionary origins. In this example, FtsZ is the ancestor protein to tubulin (a modern protein). While both proteins are found in extant organisms, tubulin function has evolved and diversified tremendously since evolving from its FtsZ prokaryotic origin. A survey of mitotic assembly components found in present-day unicellular eukaryotes reveals crucial intermediary steps to the complex membrane-enclosed genomes of multicellular eukaryotes.
Key Points
• In bacterial replication, the DNA is attached to the plasma membrane at about the midpoint of the cell.
• The origin, or starting point of bacterial replication, is close to the binding site of the DNA to the plasma membrane.
• Replication of the bacterial DNA is bidirectional, which means it moves away from the origin on both strands simultaneously.
• The formation of the FtsZ ring, a ring composed of repeating units of protein, triggers the accumulation of other proteins that work together to acquire and bring new membrane and cell wall materials to the site.
• When new cell walls are in place, due to the formation of a septum, the daughter cells separate to form individual cells.
Key Terms
• mitotic spindle: the apparatus that orchestrates the movement of DNA during mitosis
• karyokinesis: (mitosis) the first portion of mitotic phase where division of the cell nucleus takes place
• binary fission: the process whereby a cell divides asexually to produce two daughter cells | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.06%3A_Microbial_Growth/6.6A%3A_Binary_Fission.txt |
FtsZ is a protein encoded by the ftsZ gene that assembles into a ring at the future site of the septum of bacterial cell division.
LEARNING OBJECTIVES
Evaluate the role of Fts proteins in cell division
Key Points
• FtsZ has been named after “Filamenting temperature-sensitive mutant Z”.
• During cell division, FtsZ is the first protein to move to the division site, and is essential for recruiting other proteins that produce a new cell wall between the dividing cells.
• FtsZ’s role in cell division is analogous to that of actin in eukaryotic cell division, but unlike the actin-myosin ring in eukaryotes, FtsZ has no known motor protein associated with it.
Key Terms
• segrosomes: multiprotein complexes that partition chromosomes/plasmids in bacteria.
• cell division: a process by which a cell divides into two cells.
• septum: a partition that separates the cells of a (septated) fungus
• cytoskeleton: A cellular structure like a skeleton, contained within the cytoplasm.
FtsZ is a protein encoded by the ftsZ gene that assembles into a ring at the future site of the septum of bacterial cell division. This is a prokaryotic homologue to the eukaryotic protein tubulin. FtsZ has been named after “Filamenting temperature-sensitive mutant Z”. The hypothesis was that cell division mutants of E. coli would grow as filaments due to the inability of the daughter cells to separate from one another.
FtsZ was the first protein of the prokaryotic cytoskeleton to be identified. During cell division, FtsZ is the first protein to move to the division site, and is essential for recruiting other proteins that produce a new cell wall between the dividing cells. FtsZ’s role in cell division is analogous to that of actin in eukaryotic cell division, but unlike the actin-myosin ring in eukaryotes, FtsZ has no known motor protein associated with it. The origin of the cytokinetic force thus remains unclear, but it is believed that the localized synthesis of new cell wall produces at least part of this force. It is interesting to note that L-form bacteria that lack a cell wall do not require FtsZ for division, which implies that bacteria may have retained components of an ancestral mode of cell division.
Much is known about the dynamic polymerization activities of tubulin and microtubules, but little is known about these activities in FtsZ. While it is known that single-stranded tubulin protofilaments form into 13 stranded microtubules, the multistranded structure of the FtsZ-containing Z-ring is not known. It is only speculated that the structure consists of overlapping protofilaments. Recently, proteins similar to tubulin and FtsZ have been discovered in large plasmids found in Bacillus species. They are believed to function as components of segrosomes, which are multiprotein complexes that partition chromosomes/plasmids in bacteria. The plasmid homologs of tubulin/FtsZ seem to have conserved the ability to polymerize into filaments.
FtsZ has the ability to bind to GTP, and also exhibits a GTPase domain that allows it to hydrolyze GTP to GDP and a phosphate group. In vivo, FtsZ forms filaments with a repeating arrangement of subunits, all arranged head-to-tail. These filaments form a ring around the longitudinal midpoint, or septum, of the cell. This ring is called the Z-ring. The GTP hydrolyzing activity of the protein is not essential to the formation of filaments or division. Mutants lacking the GTPase domain form twisted and disordered septa. These cells with irregular septa can still divide, although abnormally. It is unclear as to whether FtsZ actually provides the physical force that results in division or serves as a marker for other proteins to execute division.
The Z-ring forms from smaller subunits of FtsZ filaments. These filaments may pull on each other and tighten to divide the cell. If FtsZ does provide force that divides the cell, it may do so through the relative movement of subunits. In this model, FtsZ scission force comes from the relative lateral movement of subunits. Lines of FtsZ would line up together parallel and pull on each other creating a “cord” of many strings that tightens itself. In other models, FtsZ does not provide the contractile force but provides the cell a spatial scaffold for other proteins to execute the division of the cell. This is akin to the creating of a temporary structure by construction workers to access hard-to-reach places of a building. The temporary structure allows unfettered access and ensures that the workers can reach all places. If the temporary structure is not correctly built, the workers will not be able to reach certain places, and the building will be deficient.
This “scaffold theory” is supported by information that shows that the formation of the ring and localization to the membrane requires the concerted action of a number of accessory proteins. ZipA or the actin homologue FtsA permit initial FtsZ localization to the membrane. Following localization to the membrane, division proteins of the Fts family are recruited for ring assembly. Many of these proteins, such as FtsW, FtsK, and FtsQ are involved in stabilization of the Z ring and may also be active participants in the scission event. The formation of the Z-ring closely coincides with cellular processes associated with replication. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.06%3A_Microbial_Growth/6.6B%3A_Fts_Proteins_and_Cell_Division.txt |
MreB is a protein found in bacteria homologous to actin.
LEARNING OBJECTIVES
Explain the role of MreB in cell morphology determination
Key Points
• MreB proteins polymerize to form filaments that are similar to actin microfilaments.
• MreB controls the width of rod-shaped bacteria, such as Escherichia coli.
• Bacteria that are naturally spherical do not have the gene encoding MreB.
Key Terms
• peptidoglycan: A polymer of glycan and peptides found in bacterial cell walls.
• cell wall: A thick, fairly rigid layer formed around individual cells of bacteria, Archaea, fungi, plants, and algae, the cell wall is external to the cell membrane and helps the cell maintain its shape and avoid damage.
• cytoskeleton: A cellular structure like a skeleton, contained within the cytoplasm.
MreB is a protein found in bacteria that has been identified as a homologue of actin, as indicated by similarities in tertiary structure and conservation of active site peptide sequence. The conservation of protein structure suggests the common ancestry of the cytoskeletal elements formed by actin and MreB, found in prokaryotes. Indeed, recent studies have found that MreB proteins polymerize to form filaments that are similar to actin microfilaments.MreB controls the width of rod-shaped bacteria, such as Escherichia coli. A mutant E. coli that creates defective MreB proteins will be spherical instead of rod-like. Also, bacteria that are naturally spherical do not have the gene encoding MreB. Prokaryotes carrying the mreB gene can also be helical in shape. MreB has long been thought to form a helical filament underneath the cytoplasmic membrane. However, this model has been brought into question by three recent publications showing that filaments cannot be seen by electron cryotomography and that GFP-MreB can be seen as patches moving around the cell circumference. It has also been shown to interact with several proteins that are proven to be involved in length growth (for instance PBP2). Therefore, MreB probably directs the synthesis and insertion of new peptidoglycan building units into the existing peptidoglycan layer to allow length growth of the bacteria.
MreB is a cytoskeleton element that assembles into filamentous structures within the bacterial cytoplasm. MreB and its homologs have been shown to interact and co-localize with cytoplasmic protein( MurB-G), membrane-imbedded proteins ( MreD, MraY and RodA), as well as other molecules with large periplasmic domain in organism. Recent research shows that peptidoglycan precursors are inserted into cell wall following helical pattern which is dependent on MreB, and it’s reported that MreB also promote the GT activity of PBPs. This ability of MreB is because of RodZ, an inner membrane protein containing an 80-residue, N-terminal cytoplasmic region, and a 200-amino acid periplasmic C-terminal tail. RodZ co-localizes with MreB helices in a manner that is strictly dependent on its cytoplasmic region. MreB- RodZ complexes act as a major stabilizing factor in bacterial cell wall and ensure the insertion of new peptidoglycan in a spiral like fashion into the cell wall.
6.6D: Peptidoglycan Synthesis and Cell Division
Learning Objectives
• Examine Peptidoglycan synthesis during cell division
Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria (but not Archaea; []), forming the cell wall. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid. Attached to the N-acetylmuramic acid is a peptide chain of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer. Some Archaea have a similar layer of pseudopeptidoglycan or pseudomurein, where the sugar residues are β-(1,3) linked N-acetylglucosamine and N-acetyltalosaminuronic acid. That is why the cell wall of Archaea is insensitive to lysozymes, which are present in human sweat and tears as part of innate immunity.
Peptidoglycan serves a structural role in the bacterial cell wall giving it strength, as well as counteracting the osmotic pressure of the cytoplasm. A common misconception is that peptidoglycan gives the cell its shape. However, it is actually the MreB protein that facilitates cell shape. Peptidoglycan is also involved in binary fission during bacterial cell reproduction.
The peptidoglycan layer is substantially thicker in Gram-positive bacteria (20 to 80 nanometers) than in Gram-negative bacteria (7 to 8 nanometers), with the attachment of the S-layer. Peptidoglycan forms around 90% of the dry weight of Gram-positive bacteria but only 10% of Gram-negative strains. Thus, presence of high levels of peptidoglycan is the primary determinant of the characterization of bacteria as gram-positive. In Gram-positive strains, it is important in attachment roles and stereotyping purposes. For both Gram-positive and Gram-negative bacteria, particles of approximately 2 nm can pass through the peptidoglycan. Gram-positive and Gram-negative bacteria are sensitive to different types of antiobiotics.
Key Points
• The sugar component of peptidoglycan consists of alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid.
• Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength but not shape, and counteracting the osmotic pressure of the cytoplasm.
• Peptidoglycan is also involved in binary fission during bacterial cell reproduction.
Key Terms
• peptidoglycan: A polymer of glycan and peptides found in bacterial cell walls.
• MreB: MreB is a protein found in bacteria that has been identified as a homologue of actin, as indicated by similarities in tertiary structure and conservation of active site peptide sequence. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.06%3A_Microbial_Growth/6.6C%3A_MreB_and_Determinants_of_Cell_Morphology.txt |
Learning Objectives
• Examine microbial generation times
Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. Providing no mutational event occurs the resulting daughter cells are genetically identical to the original cell. Therefore, “local doubling” of the bacterial population occurs. Both daughter cells from the division do not necessarily survive. The doubling time is the generation time of the bacteria. If the number surviving exceeds unity on average, the bacterial population undergoes exponential growth.
The measurement of an exponential bacterial growth curve in batch culture was traditionally a part of the training of all microbiologists. The basic means requires bacterial enumeration (cell counting) by direct and individual (microscopic, flow cytometry), direct and bulk (biomass), indirect and individual (colony counting), or indirect and bulk (most probable number, turbidity, nutrient uptake) methods. In autecological studies, bacterial growth in batch culture can be modeled with four different phases: lag phase, exponential or log phase, stationary phase, and death phase.
During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During this phase of the bacterial growth cycle, synthesis of RNA, enzymes, and other molecules occurs. The exponential phase (sometimes called the log phase or the logarithmic phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population. If growth is not limited, doubling will continue at a constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. For this type of exponential growth, plotting the natural logarithm of cell number against time produces a straight line. The slope of this line is the specific growth rate of the organism, which is a measure of the number of divisions per cell per unit time.
The actual rate of this growth (i.e. the slope of the line in the figure) depends upon the growth conditions, which affect the frequency of cell division events and the probability of both daughter cells surviving. However, exponential growth cannot continue indefinitely because the medium is soon depleted of nutrients and enriched with wastes. Finally, the stationary phase is due to a growth-limiting factor, such as depletion of a nutrient and/or the formation of inhibitory products such as organic acids. Death of cells as a function of time is rather unpredictable and very difficult to explain. At death phase, bacteria run out of nutrients and die. This basic batch culture growth model draws out and emphasizes aspects of bacterial growth which may differ from the growth of macrofauna. It emphasizes clonality, asexual binary division, the short development time relative to replication itself, the seemingly low death rate, the need to move from a dormant state to a reproductive state or to condition the media, and finally, the tendency of lab adapted strains to exhaust their nutrients. In reality, even in batch culture, the four phases are not well defined.
Key Points
• The doubling time is the generation time of the bacteria.
• The measurement of an exponential bacterial growth curve can be done by cell counting, colony counting, or determining the turbidity of bacterial cultures.
• Bacterial growth in batch culture can be modeled with four different phases: lag phase, exponential or log phase, stationary phase, and death phase.
Key Terms
• bacterium: A single celled organism with no nucleus.
• bacterial growth: Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission.
• doubling time: The doubling time is the period of time required for a quantity to double in size or value. It is applied to population growth, inflation, resource extraction, consumption of goods, compound interest, the volume of malignant tumours, and many other things which tend to grow over time.
• lag phase: the period of bacterial growth in which bacteria adapt themselves to growth conditions; the individual bacteria are maturing and not yet able to divide
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Increases in cell size are tightly linked in unicellular organisms and under optimal conditions bacteria can grow and divide rapidly.
LEARNING OBJECTIVES
Duplicate the requirements of microbial growth cycles
Key Points
• Bacteria grow to a fixed size and then reproduce through binary fission which a form of asexual reproduction. Under optimal conditions, bacteria can grow and divide extremely rapidly.
• Different kinds of bacteria need different amounts of oxygen to survive.
• For microbial growth to process, microorganisms require certain nutrients including carbon, nitrogen, phosphorus, sulfur, and metal ions.
• Various types of bacteria thrive at different temperatures.
Key Terms
• binary fission: The process whereby a cell divides asexually to produce two daughter cells.
• Anaerobe: An anaerobic organism; one that does not require oxygen to sustain its metabolic processes.
• Aerobe: Any organism (but especially a bacterium) that can tolerate the presence of oxygen or that needs oxygen to survive.
Microbial Growth Cycle
All microbial metabolisms can be arranged according to three principles: 1) How the organism obtains carbon for synthesizing cell mass. 2) How the organism obtains reducing equivalents used either in energy conservation or in biosynthetic reactions. 3) How the organism obtains energy for living and growing (for more detail on this topic see atom on Growth Terminology). Unlike in multicellular organisms, increases in cell size (cell growth and reproduction by cell division) are tightly linked in unicellular organisms. Bacteria grow to a fixed size and then reproduce through binary fission which is a form of asexual reproduction. Under optimal conditions, bacteria can grow and divide extremely rapidly. These optimal conditions are discussed below.
Oxygen Requirements
Different kinds of bacteria need different amounts of oxygen to survive, which determines which bacteria can infect which parts of the body. They are not able infect the skin because oxygen is present, and they can only grow in the presence of oxygen. Conversely, obligate anaerobes are killed by oxygen and carry out fermentation. Tetanus is an obligate anaerobe so it will infect areas where oxygen in limited. Aerotolerant anaerobes breath anaerobically (without oxygen), but they are able to survive when oxygen is present.
Nutrient Requirements
For microbial growth to process, microorganisms require certain nutrients including carbon, nitrogen, phosphorus, sulfur, and metal ions.
Temperature Requirements
Various types of bacteria thrive at different temperatures. Microorganisms that grow best at moderate temperatures are called mesophiles. Those surviving at high temperatures are thermophiles and microorganisms surviving at very low temperatures are called psychrophiles.
6.7B: Growth Terminology
The two ways that microbial organisms can be classified are as autotrophs (supply their own energy) or as heterotrophs (use the products of others).
LEARNING OBJECTIVES
Recall bacterial growth terminology
Key Points
• An autotroph, which means self-feeding or producer, is an organism that produces complex organic compounds (such as carbohydrates and proteins) from simple substances present in its surroundings. To produce these organic compounds it either uses energy from light or inorganic chemical reactions.
• Photoautotrophs are a type of autotroph that use light (sunlight if they are green plants) as their energy source.
• Chemoautotrophs are also a type of autotroph that derive energy from chemical reactions and synthesize all necessary organic compounds from carbon dioxide.
• A heterotroph is an organism that, unlike an autotroph, cannot fix carbon and uses organic carbon for growth. Heterotrophs use the products formed by autotrophs to survive.
• Photoheterotrophs are a type of heterotroph that use light for energy, but cannot use carbon dioxide as their sole carbon source.
• Chemoheterotrophs are a type of heterotroph that are unable to fix carbon and form their own organic compounds so they must use products formed by autotrophs.
Key Terms
• autotroph: an organism that can synthesize its food from inorganic substances, using heat or light as a source of energy.
• heterotroph: An organism which requires an external supply of energy in the form of food as it cannot synthesize its own.
Growth Terminology
The two ways that microbial organisms can be classified are as autotrophs (supply their own energy) or as heterotrophs (use the products of others).
Autotrophs
An autotroph, which means self-feeding or producer, is an organism that produces complex organic compounds (such as carbohydrates, fats, and proteins) from simple substances present in its surroundings. To produce these organic compounds it either uses energy from light (by photosynthesis) or inorganic chemical reactions. Autotrophs reduce carbon dioxide (CO2) by adding hydrogen atoms to it. This reduction process forms an organic compound that stores chemical energy. Most autotrophs use water as their reducing agent (to gain hydrogen atoms), but some can use other hydrogen compounds like hydrogen sulfide. Autotrophs, and their formation of organic compounds, are an important component of the food chain because they produce the food necessary for larger, more complex organisms to grow.
Photoautotrophs
Photoautotrophs are a type of autotroph. Photoautotrophs use light (sunlight if they are green plants) as their energy source. They use this energy (physical) and convert it into chemical energy in the form of reduced carbon. This process produces energy that carries out various cellular metabolic processes.
Chemoautotrophs
Chemoautotrophs are also a type of autotroph. They derive their energy from chemical reactions and synthesize all necessary organic compounds from carbon dioxide. Most chemoautotrophs are bacteria and archaea that live in hostile environments (such as deep sea vents). Chemoautotrophs are thought to be the first organisms to inhabit earth.
Heterotrophs
A heterotroph is an organism that, unlike an autotroph, cannot fix carbon and uses organic carbon for growth. Heterotrophs use the products formed by autotrophs to survive.
Photoheterotrophs
Photoheterotrophs are a type of heterotroph. These organisms use light for energy, but cannot use carbon dioxide as their sole carbon source. They use compounds formed by autotrophs (such as carbohydrates, fatty acids, and alcohols) as their food.
Chemoheterotrophs
Chemoheterotrophs are a type of heterotroph. They are unable to fix carbon and form their own organic compounds so they must use products formed by autotrophs. These organisms use inorganic energy sources or organic energy sources to sustain life.
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Learning Objectives
• Contrast ways of directly counting bacteria
Numerous procedures in biology and medicine require that cells be counted. On almost all occasions, what gets counted is actually the concentration of the cells (for example: 5,000 cells per milliliter). By counting the cells in a known volume of a culture, the concentration can be assessed. In medicine, the concentration of various blood cells, such as red blood cells or white blood cells, can give crucial information regarding someone’s health. Similarly, the concentration of bacteria, viruses, and other pathogens in blood or bodily fluids can reveal information about the progress of an infectious disease and about how a person’s immune system is dealing with the infection. Knowing the cell concentration is important in molecular biology experiments in order to adjust the amount of reagents and chemicals applied to the experiment.
Direct counting methods include microscopic counts using a hemocytometer or a counting chamber. The hemocytometer works by creating a volumetric grid divided into differently sized cubes for accurately counting the number of particles in a cube and calculating the concentration of the entire sample. One can also quantify the number of cells in a culture by plating a known volume of the cell culture on a petri dish with a growth medium, which is also known as a streak plate. If the cells are distributed on the plate properly, it can generally be assumed that each cell will give rise to a single colony. The colonies can then be counted and, based on the known volume of the culture that was spread on the plate, the cell concentration can be calculated. Bacterial colony counts made from plating dilutions of bacteria are useful to estimate the strength of bacterial infections; for example, a urinary tract bacterial infection.
As with hemocytometers or counting chambers, cultures need to be heavily diluted prior to plating. Otherwise, instead of obtaining single colonies that can be counted, a so-called “lawn” of thousands of colonies will form, all lying atop each other. Additionally, plating is the slowest method because most microorganisms need at least 12 hours to form visible colonies. These methods of direct counting do not require sophisticated instrumentation, so they can easily be performed in most laboratories.
Key Points
• Directly counting blood cells or tissue cells by using a hemocytometer can determine the concentration of a known volume.
• Counting the number of colonies that arise on a pour plate can calculate the concentration by multiplying the count by the volume spread on the pour plate.
• Direct counting methods are easy to perform and do not require highly specialized equipment, but are often slower than other methods.
Key Terms
• hemocytometer: A device that counts microscopic particles. The hemocytometer works by creating a volumetric grid divided into differently sized cubes for accurately counting the number of particles in a cube and calculating the concentration of the entire sample.
• streak plate: A petri dish with a growth medium.
6.8B: Viable Cell Counting
Plate counting is used to estimate the number of viable cells that are present in a sample.
LEARNING OBJECTIVES
Explain viable cell counting
Key Points
• The spread plate relies on bacteria growing a colony on a nutrient medium so that the colony becomes visible to the naked eye and the number of colonies on a plate can be counted.
• Selective media can be used to restrict the growth of non-target bacteria.
• The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air, for example water samples.
Key Terms
• plate count: A means to identify the number of actively growing cells in a sample.
Viable Cell Counting
There are a variety of ways to enumerate the number of bacteria in a sample. A viable cell count allows one to identify the number of actively growing/dividing cells in a sample. The plate count method or spread plate relies on bacteria growing a colony on a nutrient medium. The colony becomes visible to the naked eye and the number of colonies on a plate can be counted. To be effective, the dilution of the original sample must be arranged so that on average between 30 and 300 colonies of the target bacterium are grown. Fewer than 30 colonies makes the interpretation statistically unsound and greater than 300 colonies often results in overlapping colonies and imprecision in the count. To ensure that an appropriate number of colonies will be generated several dilutions are normally cultured. The laboratory procedure involves making serial dilutions of the sample (1:10, 1:100, 1:1000 etc. ) in sterile water and cultivating these on nutrient agar in a dish that is sealed and incubated. Typical media include Plate count agar for a general count or MacConkey agar to count gram-negative bacteria such as E. coli. Typically one set of plates is incubated at 22°C and for 24 hours and a second set at 37°C for 24 hours. The composition of the nutrient usually includes reagents that resist the growth of non-target organisms and make the target organism easily identified, often by a color change in the medium. Some recent methods include a fluorescent agent so that counting of the colonies can be automated. At the end of the incubation period the colonies are counted by eye, a procedure that takes a few moments and does not require a microscope as the colonies are typically a few millimeters across.
The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air. The initial analysis is done by mixing serial dilutions of the sample in liquid nutrient agar which is then poured into bottles. The bottles are then sealed and laid on their sides to produce a sloping agar surface. Colonies that develop in the body of the medium can be counted by eye after incubation. The total number of colonies is referred to as the Total Viable Count (TVC). The unit of measurement is cfu/ml (or colony forming units per milliliter) and relates to the original sample. Calculation of this is a multiple of the counted number of colonies multiplied by the dilution used. Examples of a viable cell count are spread plates from a serial dilution of a liquid culture and pour plates. With a spread plate one makes serial dilutions in liquid media and then spreads a known volume from the last tube in the dilution series. The colonies on the plate can then be counted and the concentration of bacteria in the original culture can be calculated. In the pour plate method a diluted bacterial sample is mixed with melted agar and then that mixture is poured into a petri dish. Again the colonies would be counted and the viable cell count calculated. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.08%3A_Counting_Bacteria/6.8A%3A_Direct_Counting.txt |
Learning Objectives
• Recall ways of measuring microbial mass
Bacterial growth follows three phases: the lag phase, the log phase, and the stationary phase. The measurement of an exponential bacterial growth curve in a batch culture was traditionally a part of the training of all microbiologists; the basic means requires bacterial enumeration (cell counting) by direct and individual (microscopic, flow cytometry), direct and bulk (biomass), indirect and individual (colony counting), or indirect and bulk (most probable number, turbidity, nutrient uptake) methods. Models reconcile theory with the measurements.
METHODS OF MEASUREMENT
There are several methods for measuring cell mass, including the gravimetermethod which uses ordinary balances to weigh a sample (dry weight/ml) after the water has been removed.
An indirect method for calculating cell mass is turbidimetry. Cell cultures are turbid: they absorb some of the light and let the rest of it pass through. The higher the cell concentration is, the higher the turbidity. Spectrophotometers are electrical appliances that can measure turbidity very accurately. The culture is placed in a translucent cuvette; the cuvette is placed in the machine and the turbidity measured immediately. Simple mathematical formulae help convert the detected turbidity to cell concentration. Using spectrophotometry for measuring the turbidity of cultures is known as turbidometry. Note the difference in spelling: turbidimetry and turbidometry are not the same word.
In spectrophotometry, cultures usually do not need to be diluted, although above a certain cell density the results lose reliability. Of all the electrical appliances used for counting cells, a spectrophotometer is the cheapest and its operation the fastest and most straightforward. This has made spectrophotometry the methods of choice for quick measurements of bacterial growth and related applications. There are spectrophotometers in which several cuvettes can be inserted at one time, reducing work time even more. Additionally, there are spectrophotometers that require extremely small volumes of culture, as little as 1 microliter. This, combined with the stochastic nature of liquid cultures, enables only an estimation of cell numbers.
An additional method for the measurement of microbial mass is the quantification of cells in a culture by plating the cells on a petri dish. If the cells are efficiently distributed on the plate, it can be generally assumed that each cell will give rise to a single colony. The colonies can then be counted, and based on the known volume of culture that was spread on the plate the cell concentration can be calculated.
As is with counting chambers, cultures usually need to be heavily diluted prior to plating; otherwise, instead of obtaining single colonies that can be counted, a so-called “lawn” will form, resulting in thousands of colonies lying over each other. Additionally, plating is the slowest method of all: most microorganisms need at least 12 hours to form visible colonies.
Key Points
• Calculating the dry weight of a sample enables one to calculate the cell count, but the sensitivity is limited to samples containing more than 10E8 bacteria per milliliter.
• Spectrophotometry is an indirect method for calculating cell concentrations by measuring the changes in turbidity.
• Bacteria can also be counted by using the plating method, which is based on the number of colonies formed in Petri dishes containing specific growth media.
Key Terms
• spectrophotometry: A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm – 2500nm using different controls and calibrations. [2] Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination. [3]
• flow cytometry: A technique used to sort and classify cells by using fluorescent markers on their surface.
• gravimeter: An instrument used to measure local variations in the gravitational field | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.08%3A_Counting_Bacteria/6.8C%3A_Measurements_of_Microbial_Mass.txt |
Culture media can be used to differentiate between different kinds of bacteria by detecting acid or gas production.
LEARNING OBJECTIVES
Show how microbial acid and gas production are detected
Key Points
• Differential media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators.
• To measure acid production one can use a pH indicator in the media.
• The Durham tube method is used to detect production of gas by microorganisms.
Key Terms
• differential media: Differential media or indicator media distinguish one microorganism type from another growing on the same media.
Cultures and Differential Media
A microbiological culture, or microbial culture, is created using a method for multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine an organism’s type, its abundance in the sample being tested, or both. It is one of the primary diagnostic techniques of microbiology, where it is used as a tool to determine the cause of infectious diseases by letting the agent multiply in a predetermined medium. A throat culture, for example, is taken by scraping the lining of tissue in the back of the throat and blotting the sample into a growing medium; this will allow analysis to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative agent of strep throat. The term “culture” can be used to refer to the process of culturing organisms, to the medium they’re grown in, and is more generally used informally to refer to “selectively growing” a specific kind of microorganism in the lab.
Differential media, also known as indicator media, distinguish one microorganism type from another growing on the same media. These types of media use the biochemical characteristics of a microorganism grown in the presence of specific nutrients or indicators that have been added to the medium to visibly indicate the defining characteristics of a microorganism. These indicators or nutrients include but are not limited to neutral red, phenol red, eosin y, and methylene blue. Differential media are used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.
Durham Cultures
The Durham tube method is used to detect production of gas by microorganisms. They are simply smaller test tubes inserted upside down in another test tube. This small tube is initially filled with the solution in which the microorganism is to be grown. If gas is produced after inoculation and incubation, a visible gas bubble will be trapped inside the small tube. The initial air gap produced when the tube is inserted upside down is lost during sterilization, usually performed at 121°C for 15 or so minutes
Escherichia coli
Escherichia coli (E. coli), a rod-shaped member of the coliform group, can be distinguished from most other coliforms by its ability to ferment lactose at 44°C in the fecal coliform test, and by its growth and color reaction on certain types of culture media. When cultured on an EMB (eosin methylene blue) plate, a positive result for E. coli is metallic green colonies on a dark purple media. Unlike the general coliform group, E. coli are almost exclusively of fecal origin and their presence is thus an effective confirmation of fecal contamination. Some strains of E. coli can cause serious illness in humans.
Sorbitol MacConkey Agar
Sorbitol MacConkey agar is a variant of the traditional MacConkey commonly used in the detection of E. coli O157:H7. Traditionally, MacConkey agar has been used to distinguish those bacteria that ferment lactose from those that do not.
This is an important distinction. Gut bacteria, such as Escherichia coli, can typically ferment lactose; important gut pathogens including Salmonella enterica and most shigellas are unable to ferment lactose. Shigella sonnei can ferment lactose, but only after prolonged incubation; it is referred to as a late-lactose fermenter.
During fermentation of sugar, acid is formed and the pH of the medium drops, changing the color of the pH indicator. Different formulations use different indicators; neutral red is often used when culturing gut bacteria because lactose fermenters turn a deep red when this pH indicator is used. Those bacteria unable to ferment lactose, often referred to as nonlactose fermenters (NLFs) metabolize the peptone in the medium. This releases ammonia, which raises the pH of the medium. Although some authors refer to NLFs as being colorless, in reality they turn neutral red a buffish color.
E. coli O157:H7 differs from most other strains of E. coli in being unable to ferment sorbitol. In sorbitol MacConkey agar, lactose is replaced by sorbitol. Most strains of E. coli ferment sorbitol to produce acid: E. coli O157:H7 can not ferment sorbitol, so this strain uses peptone to grow. This raises the pH of the medium, allowing the O157:H7 strain to be differentiated from other E. coli strains through the action of the pH indicator in the medium.
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Bacteria may grow across a wide range of temperatures, from very cold to very hot.
LEARNING OBJECTIVES
Describe how the growth of bacteria is affected by temperature and how bacterial growth can be measured
Key Points
• The basic means of measuring growth requires bacterial enumeration (cell counting).
• Methods for bacterial cell counting include: 1. direct and individual (microscopic, flow cytometry), 2. direct and bulk (biomass), 3. indirect and individual (colony counting), or 4. indirect and bulk (most probable number, turbidity, nutrient uptake).
• A mesophile is an organism that grows best in moderate temperature, neither too hot nor too cold. All human pathogens are mesophiles.
• Cold shock proteins help the cell to survive in temperatures lower than optimum growth temperature.
• Heat shock proteins help the cell to survive in temperatures greater than the optimum, possibly by condensation of the chromosome and organization of the prokaryotic nucleoid.
Key Terms
• mesophile: An organism, especially a microorganism, that lives and thrives at moderate temperatures.
• psychrophile: An organism that can live and thrive at temperatures much lower than normal; a form of extremophile.
• thermophile: An organism that lives and thrives at relatively high temperatures; a form of extremophile; many are members of the Archaea.
Growth Rate and Temperature
Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. Providing no mutational event occurs the resulting daughter cells are genetically identical to the original cell. Hence, local doubling of the bacterial population occurs. Both daughter cells from the division do not necessarily survive. However, if the number surviving exceeds unity on average, the bacterial population undergoes exponential growth. The measurement of an exponential bacterial growth curve in batch culture was traditionally a part of the training of all microbiologists. The basic means requires bacterial enumeration (cell counting) by direct and individual (microscopic, flow cytometry), direct and bulk (biomass), indirect and individual (colony counting), or indirect and bulk (most probable number, turbidity, nutrient uptake) methods. Models reconcile theory with the measurements.
Bacteria may grow across a wide range of temperatures, from very cold to very hot. A mesophile is an organism that grows best in moderate temperature, neither too hot nor too cold. All human pathogens are mesophiles. Organisms that prefer extreme environments are known as extremophiles: those that prefer cold environments are termed psychrophilic, those preferring warmer temperatures are termed thermophilic or thermotrophs and those thriving in extremely hot environments are hyperthermophilic.
For example, in molecular biology, the cold-shock domain (CSD) is a protein domain of about 70 amino acids which has been found in prokaryotic and eukaryotic DNA-binding proteins. Part of this domain is highly similar to the RNP-1 RNA-binding motif. When Escherichia coli is exposed to a temperature drop from 37 to 10 degrees Celsius, a four to five hour lag phase occurs and then growth is resumed at a reduced rate. During the lag phase, the expression of around 13 proteins, which contain cold shock domains is increased two- to ten-fold. These so-called cold shock proteins are thought to help the cell survive in temperatures lower than optimum growth temperature, by contrast with heat shock proteins, which help the cell survive in temperatures greater than the optimum, possibly by condensation of the chromosome and organization of the prokaryotic nucleoid. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.09%3A_Temperature_and_Microbial_Growth/6.9A%3A_Growth_Rate_and_Temperature.txt |
Bacteria can be classified on the basis of cell structure, metabolism or on differences in cell components.
LEARNING OBJECTIVES
Describe how bacteria can be classified on the basis of cell structure, cellular metabolism or differences in cell components such as DNA
Key Points
• A mesophile is an organism that grows best in moderate temperature, neither too hot nor too cold, typically between 20 and 45 °C (68 and 113 °F).The term is mainly applied to microorganisms.
• All bacteria have their own optimum environmental surroundings and temperatures in which they thrive the most.
• Thermophiles contain enzymes that can function at high temperatures. Some of these enzymes are used in molecular biology (for example, heat-stable DNA polymerases for PCR), and in washing agents.
Key Terms
• mesophile: An organism, especially a microorganism, that lives and thrives at moderate temperatures.
• thermophile: An organism that lives and thrives at relatively high temperatures; a form of extremophile; many are members of the Archaea.
Classification seeks to describe the diversity of bacterial species by naming and grouping organisms based on similarities. Bacteria can be classified on the basis of cell structure, cellular metabolism, or on differences in cell components such as DNA, fatty acids, pigments, antigens and quinones.
Bacteria can be classified by their optimal growth temperature. The following are the five classifications:
• Hyperthermophile (60 degrees C and upwards)
• Thermophile (optimal growth between 45 and 122 degrees)
• Mesophile (20 and 45 degrees C)
• Psychrotrophs (will survive at 0 degrees C, but prefer mesophilic temperature
• Psychrophiles (-15 and 10 degrees C or lower)
Methanopyrus kandleri
Methanopyrus kandleri can survive and reproduce at 122 °C.
A mesophile is an organism that grows best in moderate temperature, neither too hot nor too cold, typically between 20 and 45 °C (68 and 113 °F). The term is mainly applied to microorganisms.The habitats of these organisms include especially cheese, yogurt, and mesophile organisms are often included in the process of beer and wine making. Organisms that prefer cold environments are termed psychrophilic, those preferring warmer temperatures are termed thermophilic and those thriving in extremely hot environments are hyperthermophilic. All bacteria have their own optimum environmental surroundings and temperatures in which they thrive the most. A thermophile is an organism — a type of extremophile — that thrives at relatively high temperatures, between 45 and 122 °C (113 and 252 °F). Thermophilic eubacteria are suggested to have been among the earliest bacteria. Thermophiles are found in various geothermally heated regions of the Earth, such as hot springs like those in Yellowstone National Park. and deep sea hydrothermal vents, as well as decaying plant matter, such as peat bogs and compost.As a prerequisite for their survival, thermophiles contain enzymes that can function at high temperatures. Some of these enzymes are used in molecular biology (for example, heat-stable DNA polymerases for PCR), and in washing agents.
6.9C: The Heat-Shock Response
Heat shock response is a cell’s response to intense heat, including up-regulation of heat shock proteins.
LEARNING OBJECTIVES
Describe how the bacterial stress response enables bacteria to survive adverse and fluctuating conditions in their immediate surroundings such as increases in temperature
Key Points
• The bacterial stress response enables bacteria to survive adverse and fluctuating conditions in their immediate surroundings.
• A bacterial cell can react simultaneously to a wide variety of stresses and the various stress response systems interact with each other by a complex of global regulatory networks.
• The up-regulation of HSPs during heat shock is generally controlled by a single transcription factor; in eukaryotes this regulation is performed by heat shock factor (HSF), while σ32 is the heat shock sigma factor in Escherichia coli.
Key Terms
• heat shock response: The cellular response to heat shock.
The bacterial stress response enables bacteria to survive adverse and fluctuating conditions in their immediate surroundings. Various bacterial mechanisms recognize different environmental changes and mount an appropriate response. A bacterial cell can react simultaneously to a wide variety of stresses, and the various stress response systems interact with each other by a complex of global regulatory networks.
In biochemistry, heat shock is the “effect of subjecting a cell to a higher temperature than that of the ideal body temperature of the organism from which the cell line was derived. ”
Heat shock response is the cellular response to heat shock includes the transcriptional up-regulation of genes encoding heat shock proteins (HSPs) as part of the cell’s internal repair mechanism. HSPs are also called ‘stress-proteins’ and respond to heat, cold and oxygen deprivation by activating several cascade pathways. HSPs are also present in cells under perfectly normal conditions. Some HSPs, called ‘chaperones’, ensure that the cell’s proteins are in the right shape and in the right place at the right time. For example, HSPs help new or misfolded proteins to fold into their correct three-dimensional conformations, which is essential for their function. They also shuttle proteins from one compartment to another inside the cell and target old or terminally misfolded proteins to proteases for degradation. Additionally, heat shock proteins are believed to play a role in the presentation of pieces of proteins (or peptides) on the cell surface to help the immune system recognize diseased cells. The up-regulation of HSPs during heat shock is generally controlled by a single transcription factor; in eukaryotes this regulation is performed by heat shock factor (HSF), while σ32 is the heat shock sigma factor in Escherichia coli.
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Cells are grown and maintained at an appropriate temperature and gas mixture of oxygen, carbon dioxide, and nitrogen in a cell incubator.
LEARNING OBJECTIVES
Compare different gas requirements of various microbes
Key Points
• Culture conditions vary greatly for each cell type. The variation of conditions for a particular cell type can result in different phenotypes.
• Capnophiles are microorganisms that thrive in the presence of high concentrations of carbon dioxide.
• Diazotrophs are microorganisms that fix atmospheric nitrogen gas into a more usable form such as ammonia.
Key Terms
• capnophile: A microorganism that requires or grows best in presence of high concentrations of carbon dioxide.
• diazotroph: A microorganism that can fix nitrogen.
Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C and a mixture of oxygen, carbon dioxide, and nitrogen) in a cell incubator. Culture conditions vary greatly for each cell type. The variation of conditions for a particular cell type can result in different phenotypes.
Capnophiles are microorganisms that thrive in the presence of high concentrations of carbon dioxide. Typically, in a cell culture the CO2 concentration is around 5%. Some capnophiles may have a metabolic requirement for carbon dioxide, while others merely compete more successfully for resources under these conditions.
Diazotrophs are microorganisms that fix atmospheric nitrogen gas into a more usable form such as ammonia. A diazotroph is an organism that is able to grow without external sources of fixed nitrogen. Some example free-living diazotrophs include:
1) obligate anaerobes that cannot tolerate oxygen even if they are not fixing nitrogen. They live in habitats low in oxygen, such as soils and decaying vegetable matter.
2) Facultative anaerobes that can grow either with or without oxygen, but they only fix nitrogen anaerobically. Often, they respire oxygen as rapidly as it is supplied, keeping the amount of free oxygen low.
3) Aerobes that require oxygen to grow, yet their nitrogenase is still debilitated if exposed to oxygen.
4) Oxygenic photosynthetic bacteria generate oxygen as a by-product of photosynthesis, yet some are able to fix nitrogen as well.
5) And finally, Anoxygenic photosynthetic bacteria that do not generate oxygen during photosynthesis as they have only a single photosystem which cannot split water. In addition, nitrogenase is expressed under nitrogen limitation.
Some higher plants, and some animals (termites), have formed associations (symbioses) with diazotrophs. Examples of those diazotrophs include: rhizobia that associate with legumes, plants of the Fabaceae family, frankias, and cyanobacteria that associate with fungi as lichens, with liverworts, with a fern, and with a cycad.
6.10B: Osmotic Pressure
The correct osmotic pressure in the culture medium is essential for the survival of the cells.
LEARNING OBJECTIVES
Describe osmotic effects
Key Points
• Osmosis is the net movement of solvent molecules through a partially permeable membrane into a region of higher solute concentration in order to equalize the solute concentrations on the two sides.
• Osmosis provides the primary means by which water is transported into and out of cells.
• Osmoregulation is the homeostasis mechanism of an organism to reach balance in osmotic pressure.
• If the medium is hypotonic, the cells will gain water through osmosis.
• If the medium is hypertonic, the cells will lose water through osmosis.
Key Terms
• osmosis: the net movement of solvent molecules from a region of high solvent potential to a region of lower solvent potential through a partially permeable membrane
• hypotonic: Having a lower osmotic pressure than another.
• isotonic: Having the same osmotic pressure.
• hypertonic: Having a greater osmotic pressure than another.
• halophile: Organisms that thrive in high salt concentrations.
Osmotic pressure is an important factor that affects cells. Osmosis is the net movement of solvent molecules through a partially permeable membrane into a region of higher solute concentration. The intent of osmosis is to equalize the solute concentrations on the two sides. Osmosis is essential in biological systems because biological membranes are semi permeable. In general, these membranes are impermeable to large and polar molecules such as ions, proteins, and polysaccharides. However, they are permeable to non-polar and/or hydrophobic molecules like lipids as well as to small molecules like oxygen, carbon dioxide, nitrogen, nitric oxide, etc. Osmosis provides the primary means by which water is transported into and out of cells. Osmoregulation is the homeostasis mechanism of an organism to reach balance in osmotic pressure.
Having the correct osmotic pressure in the culture medium is essential. A cell can be influenced by a solution in three ways. Suppose a cell is placed in a solution of sugar or salt water. If the medium is hypotonic — a diluted solution with a higher water concentration than the cell — the cell will gain water through osmosis. If the medium is isotonic — a solution with exactly the same water concentration as the cell — there will be no net movement of water across the cell membrane. If the medium is hypertonic — a concentrated solution with a lower water concentration than the cell — the cell will lose water by osmosis.
Essentially, this means that if a cell is put in a solution that has a solute concentration higher than its own, then it will shrivel up. If it is put in a solution with a lower solute concentration than its own, the cell will expand and burst.
Obligate and Facultative Halophiles
A halophile is a microorganism that can survive and replicate in a high salt concentration environment (high osmotic pressure).
Obligate halophiles are microorganisms that can only survive in high salt concentration environments. Facultative halophiles are able to survive in bothhigh and normal salt concentration environments.
6.10C: Microbial Growth at Low or High pH
Microorganisms live and thrive within specific pH levels.
LEARNING OBJECTIVES
Differentiate microbial growth at high or low pHs
Key Points
• Neutrophiles are organisms that thrive in neutral (pH 7) environments.
• Alkaliphiles are microbes that thrive in alkaline (pH 9-11) environments.
• Acidophilic organisms are those that thrive under highly acidic conditions (usually at pH 2.0 or below).
Key Terms
• neutrophile: any organism that thrives in a relatively neutral pH
• alkaliphile: any organism that lives and thrives in an alkaline environment, such as a soda lake; a form of extremophile
• acidophile: an organism that lives and thrives under acidic conditions; a form of extremophile
In chemistry, pH is a measure of the activity of the (solvated) hydrogen ion. In other words, it is a measure of hydrogen ion concentration. Pure water has a pH very close to 7 at 25°C. Solutions with a pH less than 7 are said to be acidic, and solutions with a pH greater than 7 are said to be basic or alkaline. The pH scale is traceable to a set of standard solutions whose pH is established by international agreement. The pH of different cellular compartments, body fluids, and organs is usually tightly regulated in a process called acid-base homeostasis. Microorganisms live and thrive within specific pH levels.
Neutrophiles are organisms that thrive in neutral (pH 7) environments; extromophiles are organisms that thrive in extreme pH environments.
Alkaliphiles are microbes that thrive in alkaline environments with a pH of 9 to 11, such as playa lakes and carbonate-rich soils. To survive, alkaliphiles maintain a relatively low alkaline level of about 8 pH inside their cells by constantly pumping hydrogen ions in the form of hydronium ions (H3O+) across their cell membranes and into their cytoplasm.
Acidophilic organisms are those that thrive under highly acidic conditions (usually at pH 2.0 or below). Most acidophile organisms have evolved extremely efficient mechanisms to pump protons out of the intracellular space in order to keep the cytoplasm at or near neutral pH. Therefore, intracellular proteins do not need to develop acid stability through evolution. However, other acidophiles, such as Acetobacter aceti, have an acidified cytoplasm which forces nearly all proteins in the genome to evolve acid stability. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.10%3A_Other_Environmental_Growth_Factors/6.10A%3A_Gas_Requirements.txt |
Oxygen requirements vary among microorganisms.
LEARNING OBJECTIVES
Identify the role of oxygen in microbial growth
Key Points
• An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment.
• An anaerobic organism or anaerobe is any organism that does not require oxygen for growth.
• Normal microbial culturing occurs in an aerobic environment which poses a problem when culturing anaerobes; requiring one of a number of techniques to be used to keep oxygen out of the culturing setup.
Key Terms
• anaerobic: Without oxygen; especially of an environment or organism.
• aerobic respiration: metabolic reactions and processes that take place in the cells of organisms and require oxygen to convert biochemical energy from nutrients into adenosine triphosphate (ATP)
• aerobic: Living or occurring only in the presence of oxygen.
• aerotolerant anaerobe: an organism that does not require oxygen to sustain its metabolic processes, but is able to survive in the presence of oxygen
An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment. Several varietis of aerobes exist. Obligate aerobes require oxygen for aerobic cellular respiration. In a process known as cellular respiration, these organisms use oxygen to oxidize substrates (for example sugars and fats) in order to obtain energy. Facultative anaerobes can use oxygen, but also have anaerobic (i.e. not requiring oxygen) methods of energy production. Microaerophiles are organisms that may use oxygen, but only at low concentrations. Aerotolerant organisms can survive in the presence of oxygen, but they are anaerobic because they do not use it as a terminal electron acceptor.
An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. It could possibly react negatively and may even die if oxygen is present. For practical purposes there are three categories: obligate anaerobes, which cannot use oxygen for growth and are even harmed by it. Aerotolerant organisms, which cannot use oxygen for growth, but tolerate the presence of it. And finally, facultative anaerobes, which can grow without oxygen but can utilize oxygen if it is present.
Since normal microbial culturing occurs in atmospheric air, which is an aerobic environment, the culturing of anaerobes poses a problem. Therefore, a number of techniques are employed by microbiologists when culturing anaerobic organisms, for example, handling the bacteria in a glovebox filled with nitrogen or the use of other specially-sealed containers.
The GasPak System is an isolated container that achieves an anaerobic environment by the reaction of water with sodium borohydride and sodium bicarbonate tablets to produce hydrogen gas and carbon dioxide. Hydrogen then reacts with oxygen gas on a palladium catalyst to produce more water, thereby removing oxygen gas.
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Despite an apparent simplicity, bacteria can form complex associations with other organisms.
LEARNING OBJECTIVES
Contrast the ecological associations among microorganisms
Key Points
• Due to their small size, commensal bacteria are ubiquitous and grow on animals and plants exactly as they would grow on any other surface. Their growth can be increased by warmth and sweat; in humans, large populations of these organisms are the cause of body odor.
• Pathogenic bacteria are a major cause of human death and disease, and cause infections such as tetanus, typhoid fever, diphtheria, syphilis, cholera, foodborne illness, leprosy and tuberculosis.
• Certain bacteria form mutualistic associations, such as close spatial associations that are essential for their survival; for example, interspecies hydrogen transfer.
Key Terms
• mutualism: Any interaction between two species that benefits both; typically involves the exchange of substances or services.
• parasitism: Interaction between two organisms, in which one organism (the parasite) benefits and the other (the host) is harmed.
• commensalism: Describes a relationship between two living organisms where one benefits and the other is not significantly harmed or helped.
Ecological Associations Among Microorganisms
Despite an apparent simplicity, bacteria can form complex associations with other organisms. This process is known as symbiosis. These symbiotic associations can be divided into parasitism, mutualism and commensalism. Due to their small size, commensal bacteria are ubiquitous and grow on animals and plants exactly as they would grow on any other surface. However, their growth can be increased by warmth and sweat; in humans, large populations of these organisms are the cause of body odor.
PREDATORS
Some species of bacteria kill and then consume other microorganisms; these species are called predatory bacteria. These include organisms such asMyxococcus xanthus, which forms swarms of cells that kill and digest any bacteria they encounter. Other bacterial predators either attach to their prey in order to digest them and absorb nutrients, such as Vampirococcus, or invade another cell and multiply inside the cytosol, such as Daptobacter. These predatory bacteria are thought to have evolved from saprophages that consumed dead microorganisms through adaptations that allowed them to entrap and kill other organisms.
MUTUALISTS
Certain bacteria form close spatial associations that are essential for their survival. One such mutualistic association, called interspecies hydrogen transfer, occurs between clusters of anaerobic bacteria that consume organic acids, such as butyric acid or propionic acid, and produce hydrogen; and methanogenic Archaea, which consume hydrogen. The bacteria in this association are unable to consume the organic acids since this reaction produces hydrogen, which accumulates in the bacteria’s surroundings. Only the intimate association with the hydrogen-consuming Archaea keeps the hydrogen concentration low enough to allow the bacteria to grow.
In soil, microorganisms that reside in the rhizospehere (a zone that includes the root surface and the soil that adheres to the root after gentle shaking) carry out nitrogen fixation, converting nitrogen gas to nitrogenous compound. This serves to provide an easily absorbable form of nitrogen for many plants that cannot fix nitrogen themselves. Many other bacteria are found as symbionts in humans and other organisms. For example, the presence of over one thousand bacterial species in the normal human gut flora of the intestines can contribute to gut immunity. Synthesis vitamins such as folic acid, vitamin K, and biotin convert sugars to lactic acid (see Lactobacillus), as well as fermenting complex undigestible carbohydrates. The presence of this gut flora also inhibits the growth of potentially pathogenic bacteria (usually through competitive exclusion), and these beneficial bacteria are consequently sold as probioticdietary supplements.
PATHOGENS
If bacteria form a parasitic association with other organisms, they are classed as pathogens. Pathogenic bacteria are a major cause of human death and disease and cause infections such as tetanus, typhoid fever, diphtheria, syphilis, cholera, foodborne illness, leprosy, and tuberculosis. A pathogenic cause for a known medical disease may only be discovered many years after, as was the case with Helicobacter pylori and peptic ulcer disease. Bacterial diseases are also important in agriculture, with bacteria causing leaf spot, fire blight, and wilts in plants; as well as Johne’s disease, mastitis, salmonella, and anthrax in farm animals.
Each species of pathogen has a characteristic spectrum of interactions with its human hosts. Some organisms, such as Staphylococcus or Streptococcus, can cause skin infections, pneumonia, meningitis and even overwhelming sepsis, a systemic inflammatory response producing shock, massive vasodilation, and death. Yet these organisms are also part of the normal human flora and usually exist on the skin or in the nose without causing any disease at all. Other organisms invariably cause disease in humans, such as the Rickettsia, which are obligate intracellular parasites able to grow and reproduce only within the cells of other organisms. One species of Rickettsia causes typhus, while another causes Rocky Mountain spotted fever. Chlamydia, another phylum of obligate intracellular parasites, contains species that can cause pneumonia, or urinary tract infection and may be involved in coronary heart disease. Finally, some species such as Pseudomonas aeruginosa, Burkholderia cenocepacia, and Mycobacterium avium are opportunistic pathogens and cause disease mainly in people suffering from immunosuppression or cystic fibrosis. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.11%3A_Microbial_Growth_in_Communities/6.11A%3A_Ecological_Associations_Among_Microorganisms.txt |
Learning Objectives
• Describe biofilms
Biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface. These cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS, also referred to as slime, is a polymeric conglomeration composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial, and hospital settings.
The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single cells that may float or swim in liquid. Microbes form a biofilm in response to many factors, including cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or exposure of planktonic cells to sub-inhibitory concentrations of antibiotics. When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior in which large suites of genes are differentially regulated.
Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists initially form a weak, reversible adhesion to the surface via van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili. Some species are not able to attach to a surface on their own but are able to anchor themselves to the matrix or directly to earlier colonists. It is during this colonization that the cells are able to communicate via quorum sensing using such products as AHL. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The final stage of biofilm formation is known as development; this is the stage in which the biofilm is established and may change only in shape and size. The development of a biofilm may allow for an aggregate cell colony (or colonies) to be antibiotic-resistant.
In sum, the five stages of biofilm development are as follows:
1. Initial attachment
2. Irreversible attachment
3. Maturation I
4. Maturation II
5. Dispersion
Dispersal of cells from the biofilm colony is an essential stage of the biofilm life cycle. Dispersal enables biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm extracellular matrix, such as dispersin B and deoxyribonuclease, may play a role in biofilm dispersal. Biofilm matrix-degrading enzymes may be useful as anti-biofilm agents. Recent evidence has shown that one fatty acid messenger, cis-2-decenoic acid, is capable of inducing dispersion and inhibiting growth of biofilm colonies. Secreted by Pseudomonas aeruginosa, this compound induces cyclo heteromorphic cells in several species of bacteria and the yeast Candida albicans. Nitric oxide has also been shown to trigger the dispersal of biofilms of several bacteria species at sub-toxic concentrations, so it shows potential for use in the treatment of patients that suffer from chronic infections caused by biofilms.
Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased a thousandfold. Lateral gene transfer is also greatly facilitated in biofilms and leads to a more stable structure.
However, biofilms are not always less susceptible to antibiotics. For instance, the biofilm form of Pseudomonas aeruginosa has no greater resistance to antimicrobials than do stationary-phase planktonic cells, although when the biofilm is compared to logarithmic-phase planktonic cells, the biofilm does show greater resistance to antimicrobials. This resistance to antibiotics in both stationary phase cells and biofilms may be due to the presence of persister cells.
Key Points
• Microbes form a biofilm in response to many factors, including cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or exposure of planktonic cells to sub-inhibitory concentrations of antibiotics.
• Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists initially adhere to the surface through weak, reversible adhesion via van der Waals forces.
• If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili.
Key Terms
• biofilm: an aggregate of microorganisms in which cells adhere to each other on a surface
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Controlling microbial growth is important in many fields but the degree of acceptable microbial levels can be quite different.
LEARNING OBJECTIVES
Discover considerations in microbial control
Key Points
• Controlling microbial growth is important in the medical field, pharmaceutical and biotechnology industries, academic research, and food industry.
• The degree of acceptable microbial presence can differ based on the circumstances. Sterilization as a definition means that all life was terminated, whereas sanitization and disinfection terminates selectively and partially.
• Chemical agents that can eliminate or suppress microbial life are separated in different groups based on their use. The major groups are disinfectants, antiseptics, and antibiotics.
• Antibacterials are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth.
Key Terms
• sterilization: Any process that eliminates or kills all forms of microbial life present on a surface, solution, or solid compound.
• microbicides: Compounds or substances whose purpose is to reduce the infectivity of microbes, such as viruses or bacteria.
• parenteral: Administered by some means other than oral intake, particularly intravenously or by injection.
Considerations in Microbial Control
Ever since microbes were shown to cause diseases, people have invented different techniques to control their spread. Controlling microbial growth is important in the medical field, pharmaceutical and biotechnology industries, academic research, and food industry. Each antimicrobial substance or agent achieves a different level of microbial elimination by a certain mechanism.
TYPES OF MICROBIAL CONTROL
Sterilization (or sterilisation ) is a term referring to any process that eliminates (removes) or kills all forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, and spore forms) present on a surface, contained in a fluid, in medication, or in a compound. Sterilization can be achieved by applying the proper combinations of heat, chemicals, irradiation, high pressure, and filtration.
Chemical agents that can eliminate or suppress microbial life are separated in different groups based on their use.
Disinfectants are substances that are applied to non-living objects to destroy microorganisms that are living on them. Disinfection does not necessarily kill all microorganisms, especially resistant bacterial spores, so it is less effective than sterilisation. Disinfectants are different from other antimicrobial agents such as antibiotics, which destroy microorganisms within the body. Disinfectants are also different from biocides, as these are intended to destroy all forms of life, not just microorganisms. Disinfectants work by destroying the cell wall of microbes or interfering with their metabolism.
Antiseptics are antimicrobial substances that are applied to living tissue or skin to reduce the possibility of infection, sepsis, or putrefaction. Antiseptics are generally distinguished from antibiotics by the latter’s ability to be transported through the lymphatic system to destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects.
The term antibiotic was first used in 1942 by Selman Waksman and his collaborators in journal articles to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. This definition excluded substances that kill bacteria, but are not produced by microorganisms (such as gastric juices and hydrogen peroxide). It also excluded synthetic antibacterial compounds such as the sulfonamides. With advances in medicinal chemistry, most of today’s antibacterials chemically are semisynthetic modifications of various natural compounds.
Many antibacterial compounds are classified on the basis of chemical or biosynthetic origin into natural, semisynthetic, and synthetic. Another classification system is based on biological activity. In this classification, antibacterials are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, andbacteriostatic agents slow down or stall bacterial growth.
Microbicides which destroy virus particles are called viricides or antivirals.
LEVEL OF MICROBIAL PRESENCE
The degree of acceptable microbial presence can differ based on the circumstances. Sterilization as a definition means that all life was terminated, whereas sanitization and disinfection terminates selectively and partially. Both sanitization and disinfection reduce the number of targeted pathogenic organisms to what are considered “acceptable” levels – levels that a reasonably healthy, intact body can deal with.
In general, surgical instruments and medications that enter an already aseptic part of the body (such as the bloodstream, or penetrate the skin) must be sterilized to a high sterility assurance level (SAL). Examples of such instruments include scalpels, hypodermic needles, and artificial pacemakers. For example, medical device manufacturers design their sterilization processes for an extremely low SAL. Their “one in a million” devices should be nonsterile.
This is also essential in the manufacture of parenteral pharmaceuticals. Preparation of injectable medications and intravenous solutions for fluid replacement therapy requires not only a high sterility assurance level, but also well-designed containers to prevent entry of adventitious agents after the initial product sterilization.
Food preservation is another field where the presence of microorganisms is taken under consideration. The process usually involves preventing the growth of bacteria, fungi (such as yeasts), and other microorganisms (although some methods work by introducing benign bacteria or fungi to the food). | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.12%3A_Control_in_Microbial_Death/6.12A%3A_Considerations_in_Microbial_Control.txt |
Learning Objectives
• Describe microbial death rates
The rate of microbial death can be determined. It is important in order to develop standard protocols for disinfection which will facilitate the sterilization routine in many industries. The goal is to find out what is the minimum time needed to achieve acceptable level of sterilization for a specific purpose. The killing agent can be different (e.g., heat, chemical with certain concentration) depending on the specific application.
When the killing factor is heat, the phrase thermal death can be used. Thermal death time is a concept used to determine how long it takes to kill a specific bacteria at a specific temperature. It was originally developed for food canning and has found applications in cosmetics, and in producing salmonella-free feeds for animals (e.g. poultry, and pharmaceuticals).
In the food industry, it is important to reduce the amount of microbes in products to ensure proper food safety. This is usually done by thermal processing and finding ways to reduce the number of bacteria in the product. Time-temperature measurements of bacterial reduction is determined by a D-value, meaning how long it would take to reduce the bacterial population by 90% or one log10 at a given temperature. This D-value reference (DR) point is 121°C.
Z or z-value is used to determine the time values with different D-values at different temperatures with its equation shown below:
\[z=T_2−T_1 \log D_1−\log D_2 z=T_2−T_1 \log D_1−\log D_2\]
where T is temperature in °C. Such death curves can be empirically established for all bactericidal agents. This D-value is affected by pH of the product where low pH has faster D values on various foods. The D-value at an unknown temperature can be calculated knowing the D-value at a given temperature provided the Z-value is known. The target of reduction in canning is the 12-D reduction of Clostridium botulinum, which means that processing time will reduce the amount of this bacteria by 1012 bacteria per gram or milliliter. The DR for C. botulinum is 12.6 seconds. A 12-D reduction will take 151 seconds.
Key Points
• When researching microbial death rate, the goal is usually to find out the minimum time needed to achieve acceptable level of sterilization for a specific purpose.
• Bacterial reduction is determined by a D-value, meaning how long it would take to reduce the bacterial population by 90% or one log10 at a given state of the killing agent.
• Microbial death curves have been developed for many agents and are used in numerous industries.
Key Terms
• D-value: The time needed to reduce the bacterial population by 90% or one log10 at a given temperature.
• 12-D reduction: The time needed to reduce the amount of bacteria by 1012 bacteria per gram or milliliter.
6.12C: Relative Resistance of Microbes
Different microbial structures and types of microbial cells have different level of resistance to antimicrobial agents.
LEARNING OBJECTIVES
Contrast the relative resistance of microbes
Key Points
• Endospores are considered the most resistant structure of microbes. They are resistant to most agents that would normally kill the vegetative cells they formed from.
• Mycobacterial infections are notoriously difficult to treat. Protozoa cysts are quite hard to eliminate too. Gram negative species have high levels of natural antibiotic resistance. Staphylococcus aureus is one of the major resistant human pathogens.
• Fungal cells as well as spores are more susceptible to treatments. Vegetative bacterial and yeasts cells are some of the easiest to eliminate with different treatment methods. Viruses, especially enveloped ones, are relatively easy to treat successfully with chemicals due to the presence of lipids.
Key Terms
• horizontal gene transfer: The transfer of genetic material from one organism to another one that is not its offspring; especially common among bacteria.
• endospores: An endospore is a dormant, tough, and non-reproductive structure produced by certain bacteria from the Firmicute phylum.
Different microbial structures and types of microbial cells have different level of resistance to antimicrobial agents used to eliminate them.
Endospores are considered the most resistant structure of microbes. They are resistant to most agents that would normally kill the vegetative cells from which they formed. Nearly all household cleaning products, alcohols, quaternary ammonium compounds and detergents have little effect. However, alkylating agents (e.g. ethylene oxide), and 10% bleach are effective against endospores. Endospores are able to survive boiling at 100°C for hours. Prolonged exposure to ionizing radiation, such as x-rays and gamma rays, will also kill most endospores.
Certain bacterial species are more resistant to treatment than others. Mycobacterial infections are notoriously difficult to treat. The organisms are hardy due to their cell wall, which is neither truly Gram negative nor positive. In addition, they are naturally resistant to a number of antibiotics that disrupt cell-wall biosynthesis, such as penicillin. Due to their unique cell wall, they can survive long exposure to acids, alkalis, detergents, oxidative bursts, lysis by complement, and many antibiotics. Most mycobacteria are susceptible to the antibiotics clarithromycin and rifamycin, but antibiotic-resistant strains have emerged.
Protozoa cysts are quite hard to eliminate too. As cysts, protozoa can survive harsh conditions, such as exposure to extreme temperatures or harmful chemicals, or long periods without access to nutrients, water, or oxygen for a period of time. Being a cyst enables parasitic species to survive outside of a host, and allows their transmission from one host to another. Protozoa cells are also hardy to eliminate.
Gram-negative bacteria have high natural resistance to some antibiotics. Examples include Pseudomonas spp. which are naturally resistant to penicillin and the majority of related beta-lactam antibiotics. This ability to thrive in harsh conditions is a result of their hardy cell wall that contains porins. Their resistance to most antibiotics is attributed to efflux pumps, which pump out some antibiotics before the antibiotics are able to act.
Staphylococcus aureus is one of the major resistant pathogens. Found on the mucous membranes and the human skin of around a third of the population, it is extremely adaptable to antibiotic pressure. It was one of the earlier bacteria in which penicillin resistance was found—in 1947, just four years after the drug started being mass-produced. Methicillin-resistant Staphylococcus aureus (MRSA) was first detected in Britain in 1961, and is now “quite common” in hospitals. A recent study demonstrated that the extent of horizontal gene transfer among Staphylococcus is much greater than previously expected—and encompasses genes with functions beyond antibiotic resistance and virulence, and beyond genes residing within the mobile genetic elements.
Fungal cells as well as spores are more susceptible to treatments. Vegetative bacterial and yeasts cells are some of the easiest to eliminate with numerous agents and methods. Viruses, especially enveloped ones, are relatively easy to treat successfully with chemicals due to the presence of lipids.
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Learning Objectives
• Illustrate the alteration of membrane permeability on microbial growth
As a phospholipid bilayer, the lipid portion of the outer membrane is impermeable to charged molecules. However, channels called porins are present in the outer membrane that allow for passive transport, across the outer membrane, of many ions, sugars, and amino acids. These molecules are present in the periplasm, the region between the cytoplasmic and outer membranes. The periplasm contains the peptidoglycan layer and also many proteins responsible for substrate binding or hydrolysis and the reception of extracellular signals. The periplasm is thought to exist as a gel-like state rather than a liquid due to the high concentration of proteins and peptidoglycan found within it. Because of its location between the cytoplasmic and outer membranes, signals received and substrates bound are available to be transported across the cytoplasmic membrane using transport and signalling proteins that are embedded there.
Antimicrobial Drugs
Examples of antimicrobial drugs that can target the microbial cell membrane to alter its functionality include polymyxin and gramicidin.
After binding to lipopolysaccharide (LPS) in the outer membrane of gram-negative bacteria, polymyxins disrupt both the outer and inner membranes. The hydrophobic tail is important in causing membrane damage, suggesting a detergent-like mode of action. Removal of the hydrophobic tail of polymyxin B yields polymyxin nonapeptide, which still binds to LPS but no longer kills the bacterial cell. However, it still increases the permeability of the bacterial cell wall to other antibiotics, indicating that it causes some degree of membrane disorganization. Gram-negative bacteria can develop resistance to polymyxins through various modifications of the LPS structure that inhibit the binding of polymyxins to LPS.
Gramicidin is a heterogeneous mixture of six antibiotic compounds, gramicidins A, B, and C, making up 80%, 6%, and 14% respectively, all of which are obtained from the soil bacterial species Bacillus brevis and called collectively gramicidin D. Gramicidin D is made of linear pentadecapeptides, chains made of 15 amino acids. This is in contrast to gramicidin S, which is a cyclic peptide chain.
Gramicidin is active against gram-positive bacteria, except for gram-positive bacilli, and against selective gram-negative organisms, such as Neisseria bacteria. The therapeutic use of gramicidin is limited to topical application, as it induces hemolysis in lower concentrations and then bacteria cell death, so cannot be administered internally. Since the exterior epidermis is composed of dead cells, applying it to the skin’s surface does not cause harm.
Gramicidin is used primarily as a topical antibiotic and is one of the three constituents of the consumer antibiotic polysporin ophthalmic solution. Gramicidin’s bactericidal activity is a result of increasing the permeability of the bacterial cell membrane, allowing inorganic monovalent cations (e.g. Na+) to travel through unrestricted and thereby destroy the ion gradient between the cytoplasm and the extracellular environment.
Gramicidin D functioning as a channel was demonstrated by Hladky and Haydon, who investigated the unit conductance channel. In general, gramicidin channels are ideally selective for monovalent cations and the single-channel conductances for the alkali cations are ranked in the same order as the aqueous mobility of these ions. Divalent cations like Ca2+ block the channel by binding near its mouth so that it is essentially impermeable to divalent cations, and also excludes anions. Cl− in particular is excluded from the channel because its hydration shell is thermodynamically stronger than the shells of most monovalent cations. The channel is permeable to most monovalent cations, which move through the channel in single file. The channel is filled with about six water molecules, almost all of which must be displaced when an ion is transported. Thus, ions moving through the gramicidin pore carry a single file of water molecules. The flow of ion molecules and water molecules is known as flux coupling. In the presence of a second type of permeable ion, the two ions couple their flux as well. Like valinomycin and nonactin, the gramicidin channel is selective for potassium over sodium but only slightly so. It has a permeability ratio of 2.9. Though it is impermeable to anions, there are conditions under which some anion permeation may be observed. Its ability to bind and transport cations is due to the presence of cation-binding sites, one strong and the other weak, in the channel.
Key Points
• Antimicrobial drugs can target the microbial cell membrane to alter its functionality. Examples include: polymyxin and gramicidin.
• After binding to lipopolysaccharide in the outer membrane of gram-negative bacteria, polymyxins disrupt both the outer and inner membranes.
• Gramicidin’s bactericidal activity is a result of increasing the permeability of the bacterial cell membrane, allowing inorganic monovalent cations (e.g. Na+) to travel through unrestricted and thereby destroy the ion gradient between the cytoplasm and the extracellular environment.
Key Terms
• membrane: A flexible enclosing or separating tissue forming a plane or film and separating two environments (usually in a plant or animal).
• antimicrobial: An antimicrobial substance kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes (microbiocidal) or prevent the growth of microbes (microbiostatic).
• permeable: Of or relating to substance, substrate, membrane or material that absorbs or allows the passage of fluids.
6.13B: Damage to Proteins and Nucleic Acids
Learning Objectives
• Explain how microbes are controlled through DNA and protein damage
A bacteriostatic agent or bacteriostat, abbreviated Bstatic, is a biological or chemical agent that stops bacteria from reproducing, while not necessarily harming them. Depending on their application, bacteriostatic antibiotics, disinfectants, antiseptics, and preservatives can be distinguished. Upon removal of the bacteriostat, the bacteria usually start to grow again. This is in contrast to bactericides, which kill bacteria.
Bacteriostats are often used in plastics to prevent growth of bacteria on surfaces. Bacteriostats commonly used in laboratory work include sodium azide (which is acutely toxic) and thiomersal (which is a mutagen in mammalian cells).
Bacteriostatic antibiotics limit the growth of bacteria by interfering with bacterial protein production, DNA replication, or other aspects of bacterial cellular metabolism. They must work together with the immune system to remove the microorganisms from the body. However, there is not always a precise distinction between them and bactericidal antibiotics. High concentrations of some bacteriostatic agents are also bactericidal, whereas low concentrations of some bacteriocidal agents are bacteriostatic.
This group of drugs include: Tetracyclines, sulfonamides, Spectinomycin, Trimethoprim, Chloramphenicol, Macrolides, and Lincosamides.
Tetracycline is a broad-spectrum polyketide antibiotic produced by the Streptomyces genus of Actinobacteria, indicated for use against many bacterial infections. It is a protein synthesis inhibitor. It is commonly used to treat acne today, and, more recently, rosacea, and is historically important in reducing the number of deaths from cholera. Tetracycline is marketed under the brand names Sumycin, Tetracyn, and Panmycin, among others. Actisite is a thread-like fiber formulation used in dental applications. It is also used to produce several semisynthetic derivatives, which together are known as the tetracycline antibiotics. The term “tetracycline” is also used to denote the four-ring system of this compound. “Tetracyclines” are related substances that contain the same four-ring system.
Key Points
• Bacteriostatic antibiotics limit the growth of bacteria by interfering with bacterial protein production, DNA replication, or other aspects of bacterial cellular metabolism.
• Upon removal of the bacteriostat, the bacteria usually start to grow again.
• Bacteriostatic antibiotics must work together with the immune system to remove the microorganisms from the body.
Key Terms
• bacteriostatic: A drug that prevents bacterial growth and reproduction but does not necessarily kill them. When it is removed from the environment the bacteria start growing again.
• bacteria: A type, species, or strain of bacterium.
• replication: Process by which an object, person, place or idea may be copied mimicked or reproduced.
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Learning Objectives
• Evaluate heat as an agent of microbrial control
Applying heat to bacterial media and utensils in research and the medical field as well as to sterilize food is one of the most common methods for control of bacterial growth. To achieve sterilization, different techniques and tools are used.
Moist Heat Sterilization
Moist heat causes destruction of micro- organisms by denaturation of macromolecules, primarily proteins. Autoclaving (pressure cooking) is a very common method for moist sterilization. It is effective in killing fungi, bacteria, spores, and viruses but does not necessarily eliminate prions. When sterilizing in this way, samples are placed into a steam chamber. The chamber is closed and heated so that steam forces air out of the vents or exhausts. Pressure is then applied so that the interior temperature reaches 121°C. This temperature is maintained for between 15 and 30 minutes. This elevated temperature and pressure is sufficient to sterilize samples of any commonly encountered microbes or spores. The chamber is then allowed to cool slowly or by passive heat dissipation.
Pressure sterilization is the prevailing method used for medical sterilization of heat-resistant tools. It is also used for sterilization of materials for microbiology and other fields calling for aseptic technique. To facilitate efficient sterilization by steam and pressure, there are several methods of verification and indication used; these include color-changing indicator tapes and biological indicators. For any method of moist heat sterilization, it is common to use biological indicators as a means of validation and confirmation. When using biological indicators, samples containing spores of heat-resistant microbes such as Geobacillus stearothermophilis are sterilized alongside a standard load, and are then incubated in sterile media (often contained within the sample in a glass ampoule to be broken after sterilization). A color change in the media (indicating acid production by bacteria; requires the medium to be formulated for this purpose) or the appearance of turbidity (cloudiness indicating light scattering by bacterial cells) indicates that sterilization was not achieved and the sterilization cycle may need revision or improvement. Other moist methods are boiling samples for certain period of time and Tyndallisation. Boiling is not efficient in eliminating spores. Tyndallisation inactivates spores as well, but is a more lengthy process.
Dry Heat Sterilization
Dry heat destroys microorganisms by causing coagulation of proteins. The dry heat sterilization process is accomplished by conduction; that is where heat is absorbed by the exterior surface of an item and then passed inward to the next layer. Eventually, the entire item reaches the proper temperature needed to achieve sterilization. The time and temperature for dry heat sterilization is 160°C for 2 hours or 170°C for 1 hour. Instruments should be dry before sterilization since water will interfere with the process. Other heat sterilization methods include flaming and incineration. Flaming is commonly used to sterilize small equipment used to manipulate bacteria aseptically. Leaving transfer loops in the flame of a Bunsen burner or alcohol lamp until it glows red ensures that any infectious agent gets inactivated. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below). However, during the initial heating infectious material may be “sprayed” from the wire surface before it is killed, contaminating nearby surfaces and objects. Therefore, special heaters have been developed that surround the inoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area. Another problem is that gas flames may leave residues on the object, e.g. carbon, if the object is not heated enough. A variation on flaming is to dip the object in 70% ethanol (or a higher concentration) and merely touch the object briefly to the Bunsen burner flame, but not hold it in the gas flame. The ethanol will ignite and burn off in a few seconds. 70% ethanol kills many, but not all, bacteria and viruses. It has the advantage that it leaves less residue than a gas flame. This method works well for the glass “hockey stick”-shaped bacteria spreaders. Incineration will also burn any organism to ash. It is used to sanitize medical and other bio hazardous waste before it is discarded with non-hazardous waste.
Key Points
• Different methods are used to achieve sterilization. One of the most common is applying moist heat which includes autoclaving (pressure cooking), boiling, and Tyndallisation.
• Dry heat sterilization is accomplished by conduction and is used widely for instruments.
• Other heat methods include flaming and incineration. Flaming is commonly used to sterilize small equipment used to manipulate bacteria aseptically.
Key Terms
• sterilization: Any process that eliminates or kills all forms of microbial life present on a surface, solution, or solid compound.
• Tyndallisation: Tyndallisation is the process of three successive steam treatments to achieve sterilization over the course of three days. This works by killing vegetative cells, allowing germination of surviving spores, and killing the resulting vegetative cells before they have time to form further spores. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.14%3A_Physical_Antimicrobial_Control/6.14A%3A_Heat.txt |
Learning Objectives
• Compare non-ionizing and ionizing radiation in terms of microbe inhibition
Both non-ionizing and ionizing radiation methods are applied for sterilization.
Non-ionizing Radiation Sterilization
Ultraviolet light irradiation (UV, from a germicidal lamp) is useful only for sterilization of surfaces and some transparent objects. Many objects that are transparent to visible light such as glass, absorb UV. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses, but is ineffective in shaded areas. The drawback of UV radiation is that it damages some plastics, such as polystyrene foam, if they are exposed for prolonged periods of time.
Ionizing Radiation Sterilization
Ionizing radiation could be a lethal health hazard if used inappropriately. The proper use of these methods is regulated and monitored by world and national safety organizations. Any incidents that have occurred in the past are documented and thoroughly analyzed to determine root cause and improvement potential.
• Gamma rays: Gamma rays are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets, and food. The gamma radiation is emitted from a radioisotope (usually cobalt-60 or cesium-137). Cesium-137 is used in small hospital units to treat blood before transfusion to prevent Graft-versus-host disease. Use of a radioisotope requires shielding to ensure the safety of the operators while in use and in storage as these radioisotopes continuously emits gamma rays (cannot be turned off). An incident in Decatur, Georgia where water soluble cesium-137 leaked into the source storage pool requiring NRC intervention has led to near elimination of this radioisotope; it has been replaced by the more costly, non-water soluble cobalt-60. Sterilization by irradiation with gamma rays may, in some cases affect material properties.
• Electron beams: Electron beam processing is also commonly used for sterilization. Electron beams use an on-off technology and provide a much higher dosing rate than gamma or x-rays. Due to the higher dose rate, less exposure time is needed and thereby any potential degradation to polymers is reduced. A limitation is that electron beams are less penetrating than either gamma or x-rays. Facilities rely on substantial concrete shields to protect workers and the environment from radiation exposure.
• X-rays: High-energy X-rays are a form of ionizing energy allowing to irradiate large packages and pallet loads of medical devices. X-ray sterilization is an electricity based process that does not require chemical or radioactive material. High energy and high power X-rays are generated by an X-ray machine that can be turned off when not in use, and therefore does not require any shielding when in storage. Irradiation with X-rays or gamma rays does not make materials radioactive.
• Subatomic particles: Subatomic particles may be more or less penetrating, and may be generated by a radioisotope or a device, depending on the type of particle. Irradiation with particles may make materials radioactive, depending on the type of particles, their energy, and the type of target material: neutrons and very high-energy particles can make materials radioactive but have good penetration, whereas lower energy particles (other than neutrons) cannot make materials radioactive, but have poorer penetration.
Irradiation is used by the United States Postal Service to sterilize mail in the Washington, DC area. Some foods (e.g. spices, ground meats) are irradiated for sterilization.
Key Points
• Ultraviolet light irradiation is a non-ionizing method useful only for sterilization of surfaces and some transparent objects.
• Common methods of ionizing radiation are gamma rays, electron beams, X-rays, and subatomic particles.
• However, ionizing radiation could be a lethal health hazard if used inappropriately. The proper use of these methods is regulated and monitored by world and national safety organizations.
Key Terms
• Graft-versus-host disease: A complication after tissue or organ transplant or blood transfusion if the blood was not irradiated. White blood cells of the transplanted tissue or organ (the graft) attack cells in the recipients body (the host).
• NRC: Nuclear Regulatory Commission | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.14%3A_Physical_Antimicrobial_Control/6.14B%3A_Radiation.txt |
Learning Objectives
• Identify how low temperatures are used for microbial control
Temperature is an important factor for microbial growth. Each species has its own optimal growth temperature at which it flourishes. Human microbial pathogens usually thrive at body temperature, 37ºC. Low temperatures usually inhibit or stop microbial growth and proliferation but often do not kill bacteria. Refrigeration (4ºC) and freezing (-20ºC or less) are commonly used in the food, pharmaceuticals and biotechnology industry.
Refrigeration preserves food by slowing down the growth and reproduction of microorganisms and the action of enzymes which cause food to rot. The introduction of commercial and domestic refrigerators drastically improved the diets of many in the 1930s by allowing foods such as fresh fruit, salads and dairy products to be stored safely for longer periods, particularly during warm weather. It also facilitated transportation of fresh food on long distances.
Refrigeration is also used to facilitate the preservation of liquid medicines or other substances used for research where microbial growth is undesirable, often combined with added preservatives. Fridge temperatures inhibit the proliferation of bacteria better than molds and fungi.
For longer periods of preservation, freezing temperatures are preferred to refrigeration. Since early times, farmers, fishermen, and trappers have preserved their game and produce in unheated buildings during the winter season. Freezing food slows down decomposition by turning residual moisture into ice, inhibiting the growth of most bacterial species.
Freezing temperatures curb the spoiling effect of microorganisms in food, but can also preserve some pathogens unharmed for long periods of time. While it kills some microorganisms by physical trauma, others are sublethally injured by freezing, and may recover to become infectious.
Frozen products do not require any added preservatives because microorganisms do not grow when the temperature of the food is below -9.5°C, which is sufficient in itself to prevent food spoilage. Long-term preservation of food may call for food storage at even lower temperatures.
Key Points
• Refrigeration (4ºC) and freezing (-20ºC or less) are commonly used in food, pharmaceutical and biotechnology industries.
• Refrigeration preserves food by slowing down the growth and reproduction of microorganisms as well as the action of enzymes which cause food to rot.
• Freezing food slows down decomposition by turning residual moisture into ice, inhibiting the growth of most bacterial species. Freezing kills some microorganisms by physical trauma, while sublethally injuring others which may recover to become infectious.
Key Terms
• proliferation: The process by which an organism produces others of its kind; breeding, propagation, procreation, reproduction.
6.14D: High Pressure
Under very high hydrostatic pressure(HHP) of up to 700 MPa, water inactivates pathogens such as E. coli and Salmonella.
LEARNING OBJECTIVES
Explain high pressure as a antimicrobial control
Key Points
• High pressure processing (HPP), pascalization or bridgmanization, is a method of preserving and sterilizing food, in which a product is processed under very high pressure, leading to the inactivation of certain microorganisms and enzymes in the food.
• The frist reports showed that bacterial spores were not always inactivated by pressure, while vegetative bacteria were usually killed. Later it was discovered that using moderate pressures was more effective in eliminating spores than using higher pressures.
• Experiments were also performed with anthrax, typhoid, and tuberculosis, which was a potential health risk for the researchers.
Key Terms
• bridgmanization: Pascalization is also known as bridgmanization, named for physicist Percy Williams Bridgman.
Under very high hydrostatic pressure of up to 700 MPa (100,000 psi), water inactivates pathogens such as Listeria, E. coli and Salmonella.
High pressure processing (HPP), pascalization or bridgmanization, is a method of preserving and sterilizing food, in which a product is processed under very high pressure, leading to the inactivation of certain microorganisms and enzymes in the food. The technique was named after Blaise Pascal, whose work included detailing the effects of pressure on fluids. Pascalization is preferred over heat treatment in the food industry as it eliminates changes in the quality of foods due to thermal degradation, resulting in fresher taste, texture, appearance and nutrition. Processing conveniently takes place at ambient or refrigeration temperatures.
Experiments into the effects of pressure on microorganisms were first recorded in the late nineteenth century. The frist reports showed that bacterial spores were not always inactivated by pressure, while vegetative bacteria were usually killed. Around 1970, researchers renewed their efforts in studying bacterial spores after it was discovered that using moderate pressures was more effective than using higher pressures. These spores, which caused a lack of preservation in the earlier experiments, were inactivated faster by moderate pressure, but in a manner different from what occurred with vegetative microbes. When subjected to moderate pressures, bacterial spores germinate, and the resulting spores are easily killed using pressure, heat, or ionizing radiation.
Research into the effects of high pressures on microorganisms was largely focused on deep-sea organisms until the 1980s, when advancements in ceramic processing were made. This resulted in the production of machinery that allowed for processing foods at high pressures at a large scale, and generated some interest in the technique, especially in Japan. Although commercial products preserved by pascalization first emerged in 1990, the technology behind pascalization is still being perfected for widespread use.
In pascalization, food products are sealed and placed into a steel compartment containing a liquid, often water, and pumps are used to create pressure. The pumps may apply pressure constantly or intermittently. During pascalization, more than 50,000 pounds per square inch (340 MPa) may be applied for around fifteen minutes, leading to the inactivation of yeast, mold, and bacteria. In the process, the food’s proteins are denatured, hydrogen bonds are fortified, and noncovalent bonds in the food are disrupted, while the product’s main structure remains intact. Because pascalization is not heat-based, covalent bonds are not affected, causing no change in the food’s taste.
Experiments were also performed with anthrax, typhoid, and tuberculosis, which was a potential health risk for the researchers. Indeed, before the process was improved, one employee of the Experimental Station became ill with typhoid fever. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.14%3A_Physical_Antimicrobial_Control/6.14C%3A_Low_Temperatures.txt |
Desiccation is the state of extreme dryness, or the process of extreme drying and can be used to control microbial growth.
LEARNING OBJECTIVES
Show how desiccation inhibits microbes
Key Points
• Microorganisms cannot grow and divide when desiccated, but can survive for certain periods of time, depending on their features. After the addition of water, the bacteria will start growing again, so desiccation does not provide complete sterilization.
• Pharmaceutical companies often use freeze-drying as a desiccation tool to increase the shelf life of products, such as vaccines and other injectables. Drying is also a method for food preservation.
• Freeze-drying is performed using special equipment.
Key Terms
• desiccation: the state of drying
• freeze-drying: Freeze-drying, also known as lyophilisation, lyophilization, or cryodesiccation, is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport.
Desiccation is the state of extreme dryness, or the process of extreme drying. In biology and ecology, desiccation refers to the drying out of a living organism. Microorganisms cannot grow and divide when desiccated, but can survive for certain periods of time, depending on their features. After the addition of water, the bacteria will start growing again, so desiccation does not provide complete sterilization.
Some bacteria, such as Deinococcus radiodurans and Mycobacterium , are extremely resistant to damage from prolonged desiccation while others, such as Neisseria gonorrhoeae, can survive only short periods of desiccation.
Pharmaceutical companies often use freeze-drying as a desiccation tool to increase the shelf life of products, such as vaccines and other injectables. By removing the water from the material and sealing the material in a vial, the material can be easily stored, shipped, and later reconstituted to its original form. Preservation is possible because the greatly reduced water content inhibits the action of microorganisms and enzymes that would normally spoil or degrade the substance. Another example from the pharmaceutical industry is the use of freeze-drying to produce tablets or wafers.
Drying is also a method for food preservation that works by removing water from the food, which inhibits the growth of microorganisms. Open air drying using sun and wind has been practiced since ancient times to preserve food. A solar or electric food dehydrator can greatly speed the drying process and ensure more consistent results. Water is usually removed by evaporation (air drying, sun drying, smoking, or wind drying) but, in the case of freeze-drying, food is first frozen and then the water is removed by sublimation. Bacteria, yeasts, and molds need the water in the food to grow, and drying effectively prevents them from surviving in food.
Freeze-drying is performed using special equipment. Two components are common to all types of freeze-dryers: a vacuum pump to reduce the ambient gas pressure in a vessel containing the substance to be dried, and a condenser to remove the moisture by condensation on a surface cooled to −40º to −80ºC.
6.14F: Osmotic Pressure
Osmotic pressure is the pressure which must be applied to a solution to prevent the inward flow of water across a semipermeable membrane.
LEARNING OBJECTIVES
Interpret osmotic pressure as a means of microbial control
Key Points
• Osmotic pressure is of vital importance in biology as the cell’s membrane is selective toward many of the solutes found in living organisms.
• When a cell is placed in a hypertonic solution, water actually flows out of the cell into the surrounding solution thereby causing the cells to shrink and lose its turgidity. Hypertonic solutions are used for antimicrobial control.
• Salt and sugar are used to create hypertonic environment for microorganisms and are commonly used as food preservatives.
Key Terms
• turgidity: Turgidity (turgor pressure) pushes the plasma membrane against the cell wall of plant, bacteria, and fungi cells as well as those protiat cells which have cell walls.
Osmotic pressure is the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane. It is also defined as the minimum pressure needed to nullify osmosis.The phenomenon of osmotic pressure arises from the tendency of a pure solvent to move through a semi-permeable membrane and into a solution containing a solute to which the membrane is impermeable. This process is of vital importance in biology as the cell’s membrane is selective toward many of the solutes found in living organisms.
Osmosis causes water to flow from an area of low solute concentration to an area of high solute concentration until the two areas have an equal ratio of solute to water. Normally, the solute diffuses toward equilibrium as well; however, all cells are surrounded by a lipid bilayer cell membrane which permits the flow of water in and out of the cell but restricts the flow of solute under many circumstances. As a result, when a cell is placed in a hypotonic solution, water rushes into the membrane, increasing its volume. Eventually, the cell’s membrane is enlarged such that it pushes against the cell’s rigid wall. In an isotonic solution, water flows into the cell at the same rate it flows out. When a cell is placed in a hypertonic solution, water actually flows out of the cell into the surrounding solution causing the cells to shrink and lose its turgidity. Two of the most common substances used to create hypertonic environment for microorganisms and prevent them from growing are salt and sugar. They are widely applied in food preservation.
Table salt (sodium chloride) is the primary ingredient used in meat curing. Removal of water and addition of salt to meat creates a solute-rich environment where osmotic pressure draws water out of microorganisms, thereby retarding their growth. Doing this requires a concentration of salt of nearly 20%.
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. The purpose of sugaring is to create an environment hostile to microbial life and prevent food spoilage. From time to time, sugaring has also been used for non-food preservation. For example, honey was used as part of the mummification process in some ancient Egyptian rites. However, the growth of molds and fungi is not suppressed as efficiently as the growth of bacteria. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.14%3A_Physical_Antimicrobial_Control/6.14E%3A_Desiccation.txt |
Fluids that would be damaged by heat, irradiation, or chemical sterilization can be sterilized by microfiltration using membrane filters.
LEARNING OBJECTIVES
Demonstrate microbial control using filtration
Key Points
• A typical microfiltration membrane pore size range is 0.1-10 µm, with the most commonly used being 0.2 µm and 0.45 µm, which is sufficient to eliminate bacteria and fungi.
• Quite often, when biological samples are processed, viruses must be removed or inactivated. Nanofilters with smaller pore sizes of 20-50 nm (nanofiltration) are used.
• Filtration is commonly used for heat labile pharmaceuticals and protein solutions in processing medicines. It is also increasingly used in the treatment of drinking water.
Key Terms
• ester: An ester is a chemical compound consisting of a carbonyl group adjacent to an ether linkage.
• polyethersulfone: Thermoplastic polymers that have low protein retention. They contain the subunit aryl-SO2-aryl, the defining feature of which is the sulfone group.
Fluids that would be damaged by heat (such as fluids containing proteins like large molecule drug products, but also wine and beer), irradiation, or chemical sterilization can only be sterilized by microfiltration using membrane filters. This method is commonly used for heat labile pharmaceuticals and for protein solutions in processing medicines.
The typical microfiltration membrane pore size range is 0.1-10 µm, with the most commonly used being 0.2 µm; and 0.45 µm is sufficient to eliminate bacteria and fungi.
Microfiltration is increasingly used in drinking water treatment. It effectively removes major pathogens and contaminants such as Giardia lamblia cysts, Cryptosporidium oocysts, and large bacteria. For this application, the filter has to be rated for 0.2 µm or smaller pore size.
Quite often when biological samples are processed, viruses must be removed or inactivated. Nanofilters with smaller pore sizes of 20-50 nm (nanofiltration) are used. The smaller the pore size, the lower the flow rate. To achieve higher total throughput or avoid premature blockage, pre-filters might be used to protect small pore membrane filters. Some studies have shown that prions can be removed or reduced by filtration.
Membrane filters used in production processes are commonly made from materials such as mixed cellulose ester or polyethersulfone. The filtration equipment and the filters may be purchased as pre-sterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure proper functioning of the filter, the membrane filters are integrity tested post-use or sometimes pre-use. A non-destructive integrity test assures the filter is undamaged, and is also a regulatory requirement enforced by agencies like the Food and Drug Administration, the European Medicines Agency, and others.
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A perfect disinfectant would offer full microbiological sterilisation, without harming humans and would also be non-corrosive.
LEARNING OBJECTIVES
Describe the rationale for selecting a disinfectant and how their effectiveness is rated
Key Points
• Most modern household disinfectants contain Bitrex, an exceptionally bitter substance added to discourage ingestion, as a safety measure.
• The choice of disinfectant to be used depends on the particular situation.
• One way to compare disinfectants is to compare how well they do against a known disinfectant, such as phenol, and rate them accordingly.
Key Terms
• non-corrosive: That does not cause corrosion.
• disinfectant: A substance which kills germs and/or viruses.
• sterilisation: Sterilization (or sterilisation) is a term referring to any process that eliminates (removes) or kills all forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media.
A perfect disinfectant would also offer complete and full microbiological sterilisation, without harming humans and useful forms of life. It would also be inexpensive and non-corrosive. Most disinfectants, however, are by nature, potentially harmful (even toxic) to humans or animals. Most modern household disinfectants contain Bitrex, an exceptionally bitter substance added to discourage ingestion, as a safety measure. Those that are used indoors should never be mixed with other cleaning products, or else chemical reactions can occur.
The choice of disinfectant depends on the particular situation. Some disinfectants have a wide spectrum and kill many different types of microorganisms, while others kill a smaller range of disease-causing organisms but are preferred for other instances (they may be non-corrosive, non-toxic, or inexpensive).
There are arguments for creating or maintaining conditions that are not conducive to bacterial survival and multiplication, rather than attempting to kill them with chemicals. Bacteria can increase in number very quickly, which enables them to evolve rapidly. Should some bacteria survive a chemical attack, they give rise to new generations composed completely of bacteria that are resistant to the particular chemical used. Under a sustained chemical attack, the surviving bacteria in successive generations are increasingly resistant to the chemical used, and ultimately the chemical is rendered ineffective. For this reason, some question the wisdom of impregnating cloths, cutting boards, and worktops in the home with bactericidal chemicals.
One way to compare disinfectants is to compare how well they do against a known disinfectant and rate them accordingly. Phenol is the standard disinfectant, and the corresponding rating system is called the “phenol coefficient. ” The disinfectant to be tested is compared with phenol on a standard microbe (usually Salmonella typhi or Staphylococcus aureus). Disinfectants that are more effective than phenol have a coefficient > 1. Those that are less effective have a coefficient < 1.
A less specific measurement of effectiveness is the United States Environmental Protection Agency’s (EPA) classification into either high, intermediate, or low level of disinfection. High-level disinfection kills all organisms, except high levels of bacterial spores, and is effected with a chemical germicide cleared for marketing as a sterilant by the U.S. Food and Drug Administration (FDA). Intermediate-level disinfection kills mycobacteria, most viruses, and bacteria with a chemical germicide registered as a “tuberculocide” by the EPA. Low-level disinfection kills some viruses and bacteria with a chemical germicide registered as a hospital disinfectant by the EPA. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.15%3A_Chemical_Antimicrobial_Control/6.15A%3A_Effective_Disinfection.txt |
Some antiseptics are germicides, capable of destroying microbes (bacteriocidal), while others are bacteriostatic and prevent their growth.
LEARNING OBJECTIVES
Describe how antiseptics can be bacteriocidal or bacteriostatic
Key Points
• Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction.
• Antibacterials are antiseptics that have the proven ability to act against bacteria.
• Antiseptics are generally distinguished from antibiotics by the latter’s ability to be transported through the lymphatic system to destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects.
Key Terms
• germicides: Some antiseptics are true germicides, capable of destroying microbes (bacteriocidal), while others are bacteriostatic and only prevent or inhibit their growth.
• microbes: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.
• antimicrobial: An agent that destroys microbes, inhibits their growth, or prevents or counteracts their pathogenic action.
Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction. Antiseptics are generally distinguished from antibiotics by the latter’s ability to be transported through the lymphatic system to destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects.
Some antiseptics are true germicides, capable of destroying microbes (bacteriocidal), while others are bacteriostatic and only prevent or inhibit their growth.
Antibacterials are antiseptics that have the proven ability to act against bacteria. Microbicides that destroy virus particles are called viricides or antivirals.
Most commonly used are ethanol (60–90%), 1-propanol (60–70%), and 2-propanol/isopropanol (70–80%) or mixtures of these alcohols. They are commonly referred to as “surgical alcohol. ” They are used to disinfect the skin before injections are given, often along with iodine (tincture of iodine) or some cationic surfactants (benzalkonium chloride 0.05–0.5%, chlorhexidine 0.2–4.0%, or octenidine dihydrochloride 0.1–2.0%).
Quaternary ammonium compounds include the chemicals benzalkonium chloride (BAC), cetyl trimethylammonium bromide (CTMB), cetylpyridinium chloride (Cetrim, CPC), and benzethonium chloride (BZT). Benzalkonium chloride is used in some pre-operative skin disinfectants (conc. 0.05–0.5%) and antiseptic towels. The antimicrobial activity of Quats is inactivated by anionic surfactants, such as soaps. Related disinfectants include chlorhexidine and octenidine.
Boric acid: Used in suppositories to treat yeast infections of the vagina, in eyewashes, and as an antiviral to shorten the duration of cold sore attacks. Put into creams for burns. Also common in trace amounts in eye contact solution. A triarylmethane dye still widely used as 1% ethanol solution in Eastern Europe and ex-USSR countries for treatment of small wounds and abscesses. Efficient against gram-positive bacteria.
Chlorhexidine Gluconate: A biguanidine derivative, used in concentrations of 0.5–4.0% alone or in lower concentrations in combination with other compounds, such as alcohols. Used as a skin antiseptic and to treat inflammation of the gums (gingivitis). The microbicidal action is somewhat slow, but remanent. It is a cationic surfactant, similar to Quats.
Hydrogen peroxide: Used as a 6% (20 Vols) solution to clean and deodorize wounds and ulcers. More common 3% solutions of hydrogen peroxide have been used in household first aid for scrapes, etc. However, even this less potent form is no longer recommended for typical wound care because the strong oxidization causes scar formation and increases healing time. Gentle washing with mild soap and water or rinsing a scrape with sterile saline is a better practice.
Novel iodine antiseptics containing povidone-iodine (an iodophor, complex of povidone, a water-soluble polymer, with triiodide anions I3-, containing about 10% of active iodine) are far better tolerated, don’t negatively affect wound healing, and leave a deposit of active iodine, thereby creating the so-called “remnant,” or persistent, effect. The great advantage of iodine antiseptics is their wide scope of antimicrobial activity, killing all principal pathogens and, given enough time, even spores, which are considered to be the most difficult form of microorganisms to be inactivated by disinfectants and antiseptics.
Mercurochrome: Not recognized as safe and effective by the U.S. Food and Drug Administration (FDA) due to concerns about its mercury content. Other obsolete organomercury antiseptics include bis-(phenylmercuric) monohydrogenborate (Famosept).
Manuka Honey: Recognized by the U.S. Food and Drug Administration (FDA) as a medical device for use in wounds and burns. Active +15 is equal to a 15% solution of phenol.
Octenidine dihydrochloride: A cationic surfactant and bis-(dihydropyridinyl)-decane derivative, used in concentrations of 0.1–2.0%. It is similar in its action to the Quats, but is of somewhat broader spectrum of activity. Octenidine is currently increasingly used in continental Europe as a QAC’s and chlorhexidine (with respect to its slow action and concerns about the carcinogenic impurity 4-chloroaniline) substitute in water- or alcohol-based skin, mucosa, and wound antiseptic. In aqueous formulations, it is often potentiated with addition of 2-phenoxyethanol.
Phenol is germicidal in strong solution, inhibitory in weaker ones. Used as a “scrub” for pre-operative hand cleansing. Used in the form of a powder as an antiseptic baby powder, where it is dusted onto the navel as it heals. Also used in mouthwashes and throat lozenges, where it has a painkilling effect as well as an antiseptic one. Example: TCP. Other phenolic antiseptics include historically important, but today rarely used (sometimes in dental surgery) thymol, today obsolete hexachlorophene, still used triclosan and sodium 3,5-dibromo-4-hydroxybenzenesulfonate (Dibromol).
Antimicrobial compound suitable for clinical use in critically colonized or infected acute and chronic wounds. The physicochemical action on the bacterial envelope prevents or impedes the development of resistant bacterial strains. | textbooks/bio/Microbiology/Microbiology_(Boundless)/06%3A_Culturing_Microorganisms/6.15%3A_Chemical_Antimicrobial_Control/6.15B%3A_Factors_that_Affect_Germicidal_Activity_of_Chemicals.txt |
There are multiple types of disinfectants, including but not limited to air disinfectants, alcohols, and oxidizing agents.
LEARNING OBJECTIVES
List the types of disinfectants available
Key Points
• Air disinfectants are typically chemical substances capable of disinfecting microorganisms suspended in the air.
• Alcohols, usually ethanol or isopropanol, are sometimes used as a disinfectant, but more often as an antiseptic.
• Oxidizing agents act by oxidizing the cell membrane of microorganisms, which results in a loss of structure and leads to cell lysis and death.
Key Terms
• disinfectants: Disinfectants are substances that are applied to non-living objects to destroy microorganisms that are living on the objects. Disinfectants are substances that are applied to non-living objects to destroy microorganisms that are living on the objects.
• microorganisms: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.
• antiseptic: Any substance that inhibits the growth and reproduction of microorganisms. Generally includes only those that are used on living objects (as opposed to disinfectants) and aren’t transported by the lymphatic system to destroy bacteria in the body (as opposed to antibiotics).
Types of disinfectants include: Air disinfectants, Alcohols, Aldehydes, Oxidizing agents, Phenolics, Quaternary ammonium compounds, Silver, and Copper alloy surfaces.
Air Disinfectants
Air disinfectants are typically chemical substances capable of disinfecting microorganisms suspended in the air. Disinfectants are often assumed to be limited to use on surfaces, but that is not the case. In 1928, a study found that airborne microorganisms could be killed using mists of dilute bleach. An air disinfectant must be dispersed either as an aerosol or vapor at a sufficient concentration in the air to cause the number of viable infectious microorganisms to be significantly reduced.
In the 1940s and early 1950s, further studies showed inactivation of diverse bacteria, influenza virus, and Penicillium chrysogenum (previously P. notatum) mold fungus using various glycols, principally propylene glycol and triethylene glycol. In principle, these chemical substances are ideal air disinfectants because they have both high lethality to microorganisms and low mammalian toxicity.
Although glycols are effective air disinfectants in controlled laboratory environments, it is more difficult to use them effectively in real-world environments because the disinfection of air is sensitive to continuous action. Continuous action in real-world environments with outside air exchanges at door, HVAC, and window interfaces, and in the presence of materials that adsorb and remove glycols from the air, poses engineering challenges that are not critical for surface disinfection. The engineering challenges associated with creating a sufficient concentration of the glycol vapors in the air have not to date been sufficiently addressed.
Alcohol Disinfectants
Alcohols, usually ethanol or isopropanol, are sometimes used as a disinfectant, but more often as an antiseptic, the distinction being that alcohol tends to be used on living tissue rather than nonliving surfaces. These alcohols are non-corrosive but can be a fire hazard. They also have limited residual activity due to evaporation, which results in brief contact times unless the surface is submerged. They also have a limited activity in the presence of organic material.
Alcohols are most effective when combined with purified water to facilitate diffusion through the cell membrane; 100% alcohol typically denatures only external membrane proteins. A mixture of 70% ethanol or isopropanol diluted in water is effective against a wide spectrum of bacteria, though higher concentrations are often needed to disinfect wet surfaces. Additionally, high-concentration mixtures (such as 80% ethanol + 5% isopropanol) are required to effectively inactivate lipid-enveloped viruses (such as HIV, hepatitis B, and hepatitis C). Alcohol is only partly effective against most non-enveloped viruses (such as hepatitis A), and is not at all effective against fungal and bacterial spores.
The efficacy of alcohol is enhanced when in solution with the wetting agent dodecanoic acid (coconut soap). The synergistic effect of 29.4% ethanol with dodecanoic acid is effective against a broad spectrum of bacteria, fungi, and viruses. Further testing is being performed against Clostridium difficile (C. Diff) spores using higher concentrations of ethanol and dodecanoic acid, which has been indicated to be effective with a contact time of ten minutes.
Aldehydes, such as formaldehyde and glutaraldehyde, have a wide microbiocidal activity and are sporocidal and fungicidal. They are partly inactivated by organic matter and have slight residual activity. Some bacteria have developed resistance to glutaraldehyde; it has also been found that glutaraldehyde can cause asthma and other health hazards, hence ortho-phthalaldehyde is replacing glutaraldehyde.
Oxidizing Disinfectants
Oxidizing agents act by oxidizing the cell membrane of microorganisms, which results in a loss of structure and leads to cell lysis and death. A large number of disinfectants operate in this way. Chlorine and oxygen are strong oxidizers, so their compounds figure heavily here.
Phenolics are active ingredients in some household disinfectants. They are also found in some mouthwashes and in disinfectant soap and handwashes.
Quaternary ammonium compounds (quats), such as benzalkonium chloride, are a large group of related compounds. Some concentrated formulations have been shown to be effective low-level disinfectants. Typically, quats do not exhibit effectiveness against difficult to kill non-enveloped viruses such as norovirus, rotavirus, or polio virus.
6.15D: Biological Control of Microbes
Learning Objectives
• Describe the types of antimicrobial agents available for controlling the growth of microbes
A wide variety of chemicals called antimicrobial agents are available for controlling the growth of microbes. For example:
1. Chemotherapeutic agents, including antibiotics, are administered into the infected body.
2. Disinfectants are chemical agents used on inanimate objects to lower the level of microbes present on the object. These are not capable of sterilizing, typically because they fail to kill endospores, some viruses, and organisms such as Mycobacteriumtuberculosis.
3. Antiseptics are chemicals used on living tissue to decrease the number of microbes present in that tissue.
Disinfectants and antiseptics affect bacteria in many ways. Those that result in bacterial death are called bactericidal agents. Those causing temporary inhibition of growth are bacteriostatic agents. No single antimicrobial agent is most effective for use in all situations – different situations may call for different agents. A number of factors affect selection of the best agent for any given situation – Antimicrobial agents must be selected with specific organisms and environmental conditions in mind. Additional variables to consider in the selection of an antimicrobial agent include pH, solubility, toxicity, organic material present, and cost.
Once an agent has been selected, it is important to evaluate it’s effectiveness. In evaluating the effectiveness of antimicrobial agents, the concentration, length of contact, and whether it is lethal (-cidal) or inhibiting (-static) at that concentration of exposure are the important criteria.
Prior to the advent of antibiotics, live organisms were used directly in attempts to control microbial infections. Examples of such biological control included bacteriotherapy, bacteriophage therapy, malaria therapy, probiotics, and the use of living maggots. In all cases the organisms themselves rather than a product of their metabolism were used as the potentially curative agent. The biological control of human infections was largely restricted to the treatment of surface infections of the skin and mucous membrane. Additionally, attempts were made to alter the microflora of the human intestinal tract to favor the growth of benign or beneficial bacteria or yeasts. Modern studies suggest that the use of biological control in the treatment of human infections should be re-evaluated in the light of the increasing world-wide occurrence of antibiotic-resistant bacteria, and the opportunities provided by recent developments in gene technology.
Key Points
• Most of the examples of biologic control of microbes predate the sulphonamides and penicillin.
• Maggot therapy, although repellent by modern standards, proved to be surprisingly effective.
• Today, a wide variety of chemicals called antimicrobial agents are available for controlling the growth of microbes. These include chemotherapeutic agents, disinfectants, and antiseptics.
Key Terms
• probiotics: live microorganisms that may confer a health benefit on the host.
• antibiotics: agents that inhibit bacterial growth or kill bacteria.
LICENSES AND ATTRIBUTIONS
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Learning Objectives
• Explain the basic features of bacterial genomes
Bacterial genomes are generally smaller and less variant in size between species when compared with genomes of animals and single cell eukaryotes. Bacterial genomes can range in size anywhere from 139 kbp to 13,000 kbp. Recent advances in sequencing technology led to the discovery of a high correlation between the number of genes and the genome size of bacteria, suggesting that bacteria have relatively small amounts of junk DNA.
Studies have since shown that a large number of bacterial species have undergone genomic degradation resulting in a decrease in genome size from their ancestral state. Over the years, researchers have proposed several theories to explain the general trend of bacterial genome decay and the relatively small size of bacterial genomes. Compelling evidence indicates that the apparent degradation of bacterial genomes is owed to a deletional bias.
In prokaryotes, most of the genome (85-90%) is non-repetitive DNA, which means coding DNA mainly forms it, while non-coding regions only take a small part. Most biological entities that are more complex than a virus sometimes or always carry additional genetic material besides that which resides in their chromosomes. In some contexts, such as sequencing the genome of a pathogenic microbe, “genome” is meant to include information stored on this auxiliary material, which is carried in plasmids. In such circumstances then, “genome” describes all of the genes and information on non-coding DNA that have the potential to be present.
Amongst species of bacteria, there is relatively little variation in genome size when compared with the genome sizes of other major groups of life. Genome size is of little relevance when considering the number of functional genes in eukaryotic species. In bacteria however, the strong correlation between the number of genes and the genome size makes the size of bacterial genomes an interesting topic for research and discussion. The general trends of bacterial evolution indicate that bacteria started as free-living organisms. Evolutionary paths led some bacteria to become pathogens and symbionts.
Figure 7.1A: Graph of variation in estimated genome sizes in base pairs.: Unlike eukaryotes, bacteria show a strong correlation between genome size and number of functional genes in a genome. Genome size ranges (in base pairs) of various life forms. (CC BY-SA 4.0; Abizar).
The lifestyles of bacteria play an integral role in their respective genome sizes. Free-living bacteria have the largest genomes out of the three types of bacteria; however, they have fewer pseudogenes than bacteria that have recently acquired pathogenicity. Facultative and recently evolved pathogenic bacteria exhibit a smaller genome size than free-living bacteria, yet they have more pseudogenes than any other form of bacteria. Obligate bacterial symbionts or pathogens have the smallest genomes and the fewest number of pseudogenes of the three groups. The relationship between life-styles of bacteria and genome size raises questions as to the mechanisms of bacterial genome evolution.
Researchers have developed several theories to explain the patterns of genome size evolution amongst bacteria. One theory predicts that bacteria have smaller genomes due to a selective pressure on genome size to ensure faster replication. The theory is based upon the logical premise that smaller bacterial genomes will take less time to replicate. Subsequently, smaller genomes will be selected preferentially due to enhanced fitness.
Deletional bias selection is but one process involved in evolution. Two other major processes (mutation and genetic drift) can be used to explain the genome sizes of various types of bacteria.
Evidence of a deletional bias is present in the respective genome sizes of free-living bacteria, facultative and recently derived parasites and obligate parasites and symbionts. Free-living bacteria tend to have large population sizes and are subject to more opportunity for gene transfer. As such, selection can effectively operate on free-living bacteria to remove deleterious sequences resulting in a relatively small number of pseudogenes. Continually, further selective pressure is evident as free-living bacteria must produce all gene-products independent of a host. Given that there is sufficient opportunity for gene transfer to occur and there are selective pressures against even slightly deleterious deletions, it is intuitive that free-living bacteria should have the largest bacterial genomes of all bacteria types. Recently formed parasites undergo severe bottlenecks and can rely on host environments to provide gene products. As such, in recently formed and facultative parasites, there is an accumulation of pseudogenes and transposable elements due to a lack of selective pressure against deletions. The population bottlenecks reduce gene transfer and as such, deletional bias ensures the reduction of genome size in parasitic bacteria.
Key Points
• In prokaryotes, most of the genome (85-90%) is non-repetitive, coding DNA, while the remaining DNA is non-coding.
• The genome of a pathogenic microbe, “genome” is meant to include information stored on this auxiliary material, which is carried in plasmids.
• The lifestyles of bacteria play an integral role in their respective genome sizes. Free-living bacteria have the largest genomes out of the three types of bacteria; however, they have fewer pseudogenes than bacteria that have recently acquired pathogenicity.
Key Terms
• genome: The complete genetic information (either DNA or, in some viruses, RNA) of an organism, typically expressed in the number of basepairs. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.01%3A_Genes/7.1A%3A_Bacterial_Genomes.txt |
Prokaryotic DNA is replicated by DNA polymerase III in the 5′ to 3′ direction at a rate of 1000 nucleotides per second.
LEARNING OBJECTIVES
Explain the functions of the enzymes involved in prokaryotic DNA replication
Key Points
• Helicase separates the DNA to form a replication fork at the origin of replication where DNA replication begins.
• Replication forks extend bi-directionally as replication continues.
• Okazaki fragments are formed on the lagging strand, while the leading strand is replicated continuously.
• DNA ligase seals the gaps between the Okazaki fragments.
• Primase synthesizes an RNA primer with a free 3′-OH, which DNA polymerase III uses to synthesize the daughter strands.
Key Terms
• DNA replication: a biological process occuring in all living organisms that is the basis for biological inheritance
• helicase: an enzyme that unwinds the DNA helix ahead of the replication machinery
• origin of replication: a particular sequence in a genome at which replication is initiated
DNA Replication in Prokaryotes
DNA replication employs a large number of proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them, similar to ATP which has three phosphate groups attached. When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.
There are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks at the origin of replication are extended bi-directionally as replication proceeds. Single-strand binding proteins coat the strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. DNA polymerase is able to add nucleotides only in the 5′ to 3′ direction (a new DNA strand can be extended only in this direction). It also requires a free 3′-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next nucleotide. This means that it cannot add nucleotides if a free 3′-OH group is not available. Another enzyme, RNA primase, synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA, priming DNA synthesis. A primer provides the free 3′-OH end to start replication. DNA polymerase then extends this RNA primer, adding nucleotides one by one that are complementary to the template strand.
The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase can only extend in the 5′ to 3′ direction, which poses a slight problem at the replication fork. As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5′ to 3′ direction and the other is oriented in the 3′ to 5′ direction. One strand (the leading strand), complementary to the 3′ to 5′ parental DNA strand, is synthesized continuously towards the replication fork because the polymerase can add nucleotides in this direction. The other strand (the lagging strand), complementary to the 5′ to 3′ parental DNA, is extended away from the replication fork in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them.
The leading strand can be extended by one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3′ to 5′, while that of the leading strand will be 5′ to 3′. The sliding clamp (a ring-shaped protein that binds to the DNA) holds the DNA polymerase in place as it continues to add nucleotides. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, while the gaps are filled in by deoxyribonucleotides. The nicks that remain between the newly-synthesized DNA (that replaced the RNA primer) and the previously-synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of phosphodiester linkage between the 3′-OH end of one nucleotide and the 5′ phosphate end of the other fragment.
The table summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.
Prokaryotic DNA Replication: Enzymes and Their Function
Enzyme/protein Specific Function
DNA pol I Exonuclease activity removes RNA primer and replaces with newly synthesized DNA
DNA pol II Repair function
DNA pol III Main enzyme that adds nucletides in the 5′ – 3′ direction
Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Ligase Seals the gaps between the Okazaki fragments to create one continuous DNA strand
Primase Synthesizes RNA primers needed to start replication
Sliding Clamp Helps to hold the DNA polymerase in place when nucleotides are being added
Topoisomerase Helps relieve the stress on DNA when unwinding by causing breaks and then resealing the DNA
Single-strand binding proteins (SSB) Binds to single-stranded DNA to avoid DNA rewinding back. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.01%3A_Genes/7.1B%3A_DNA_Replication_in_Prokaryotes.txt |
Learning Objectives
• Explain how gene sequence inversions can have a regulatory effect
Recombining sequences in site-specific reactions are usually short and occur at a single target site within the recombining sequence. For this to occur, there is typically one or more cofactors (to name a few: DNA-binding proteins and the presence or absence of DNA binding sites) and a site specific recombinase. There is also a change in orientation of the DNA that will affect gene expression or the structure of the gene product. This is done by changing the spatial arrangement of the promoter or the regulatory elements.
Through the utilization of specific recombinases, a particular DNA sequence is inverted, resulting in an ON to OFF switch, and vice versa, of the gene located within or next to this switch. Many bacterial species can utilize inversion to change the expression of certain genes for the benefit of the bacterium during infection. The inversion event can be simple by involving the toggle in expression of one gene, like E. coli pilin expression; or more complicated by involving multiple genes in the expression of multiple types of flagellin by S. typhimurium. Fimbrial adhesion by the type I fimbriae in E. coli undergoes site specific inversion to regulate the expression of fimA, the major subunit of the pili, depending on the stage of infection. The invertible element has a promoter within it that depending on the orientation will turn on or off the transcription of fimA. The inversion is mediated by two recombinases, FimB and FimE, and regulatory proteins H-NS, Integration Host Factor (IHF) and Leucine responsive protein (LRP). The FimE recombinase has the capability to only invert the element and turn expression from on to off, while FimB can mediate the inversion in both directions.
Key Points
• Recombining sequences in site-specific reactions are usually short and occur at a single target site. For this to occur, there is typically one or more cofactors (to name a few: DNA -binding proteins and the presence or absence of DNA binding sites) and a site specific recombinase.
• Many bacterial species can utilize inversion to change the expression of certain genes for the benefit of the bacterium during infection.
• The inversion event can be simple by involving the toggle in expression of one gene, like E. coli pilin expression; or more complicated by involving multiple genes in the expression of multiple types of flagellin by S. typhimurium.
Key Terms
• recombinase: Any of several enzymes that mediate recombination of DNA fragments between maternal and paternal chromosomes in prokaryotes.
7.1D: Slipped-Strand Mispairing
Slipped strand mispairing (SSM) is a process that produces mispairing of short repeat sequences during DNA synthesis.
LEARNING OBJECTIVES
Explain how slipped-strand mispairing can be used as a mechanism to regulate gene expression
Key Points
• Altered gene expression is a result of SSM and depending where the increase or decrease of the short repeat sequences occurs in relation to the promoter will either regulate at the level of transcription or translation. The outcome is an ON or OFF phase of a gene or genes.
• SSM can result in an increase or decrease in the number of short repeat sequences. The short repeat sequences are 1 to 7 nucleotides and can be homogeneous or heterogeneous repetitive DNA sequences.
• Transcriptional regulation can occur if the repeats are located in the promoter region at the RNA polymerase binding site, -10 and -35 upstream of the gene(s).
• SSM induces transcriptional regulation is by changing the short repeat sequences located outside the promoter. If there is a change in the short repeat sequence it can affect the binding of a regulatory protein, such as an activator or repressor.
Key Terms
• Slipped strand mispairing: a process that produces mispairing off short repeat sequences between the mother and daughter strand during DNA synthesis.
Slipped strand mispairing (SSM) is a process that produces mispairing of short repeat sequences between the mother and daughter strand during DNA synthesis. This RecA-independent mechanism can transpire during either DNA replication or DNA repair and can be on the leading or lagging strand and can result in an increase or decrease in the number of short repeat sequences. The short repeat sequences are 1 to 7 nucleotides and can be homogeneous or heterogeneous repetitive DNA sequences.
Altered gene expression is a result of SSM and depending where the increase or decrease of the short repeat sequences occurs in relation to the promoter will either regulate at the level of transcription or translation. The outcome is an ON or OFF phase of a gene or genes.
Transcriptional regulation occurs in several ways. One possibility is if the repeats are located in the promoter region at the RNA polymerase binding site, -10 and -35, upstream of the gene(s). The opportunistic pathogen H. influenzae has two divergently oriented promoters in fimbriae geneshifA and hifB. The overlapping promoter regions have repeats of the dinucleotide TA in the -10 and -35 sequences. Through SSM the TA repeat region can undergo addition or subtraction of TA dinucleotides which results in the reversible ON phase or OFF phase of transcription of the hifA and hifB. The second way that SSM induces transcriptional regulation is by changing the short repeat sequences located outside the promoter. If there is a change in the short repeat sequence, it can affect the binding of a regulatory protein, such as an activator or repressor. It can also lead to differences in post-transcriptional stability of mRNA.
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Learning Objectives
• Evaluate the nucleoid in prokaryotes
The Nucleoid
The nucleoid (meaning nucleus-like) is an irregularly-shaped region within the cell of a prokaryote that contains all or most of the genetic material. In contrast to the nucleus of a eukaryotic cell, it is not surrounded by a nuclear membrane. The genome of prokaryotic organisms generally is a circular, double-stranded piece of DNA, of which multiple copies may exist at any time. The length of a genome varies widely, but is generally at least a few million base pairs.
The nucleoid can be clearly visualized on an electron micrograph at high magnification, where it is clearly visible against the cytosol. Sometimes even strands of what is thought to be DNA are visible. The nucleoid can also be seen under a light microscope.by staining it with the Feulgen stain, which specifically stains DNA. The DNA-intercalating stains DAPI and ethidium bromide are widely used for fluorescence microscopy of nucleoids.
Experimental evidence suggests that the nucleoid is largely composed of about 60% DNA, plus a small amount of RNA and protein. The latter two constituents are likely to be mainly messenger RNA and the transcription factor proteins found regulating the bacterial genome. Proteins helping to maintain the supercoiled structure of the nucleic acid are known as nucleoid proteins or nucleoid-associated proteins, and are distinct from histones of eukaryotic nuclei. In contrast to histones, the DNA-binding proteins of the nucleoid do not form nucleosomes, in which DNA is wrapped around a protein core. Instead, these proteins often use other mechanisms, such as DNA looping, to promote compaction.
The Genophore
A genophore is the DNA of a prokaryote. It is commonly referred to as a prokaryotic chromosome. The term “chromosome” is misleading, because the genophore lacks chromatin. The genophore is compacted through a mechanism known as supercoiling, but a chromosome is additionally compacted through the use of chromatin. The genophore is circular in most prokaryotes, and linear in very few. The circular nature of the genophore allows replication to occur without telomeres. Genophores are generally of a much smaller size than Eukaryotic chromosomes. A genophore can be as small as 580,073 base pairs (Mycoplasma genitalium). Many eukaryotes (such as plants and animals) carry genophores in organelles such as mitochondria and chloroplasts. These organelles are very similar to true prokaryotes.
Key Points
• The genome of prokaryotic organisms generally is a circular, double-stranded piece of DNA, multiple copies of which may exist at any time.
• The length of a genome varies widely, but is generally at least a few million base pairs.
• A genophore is the DNA of a prokaryote. It is commonly referred to as a prokaryotic chromosome.
Key Terms
• nucleoid: The irregularly-shaped region within a prokaryote cell where the genetic material is localized.
• prokaryote: An organism characterized by the absence of a nucleus or any other membrane-bound organelles.
• genome: The complete genetic information (either DNA or, in some viruses, RNA) of an organism, typically expressed in the number of basepairs.
7.2B: Supercoiling
Learning Objectives
• Assess the role of supercoiling in prokaryotic genomes
DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on that strand. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to facilitate functions such as DNA replication or transcription. Mathematical expressions are used to describe supercoiling by comparing different coiled states to relaxed B-form DNA.
As a general rule, the DNA of most organisms is negatively supercoiled. In a “relaxed” double-helical segment of B-DNA, the two strands twist around the helical axis once every 10.4 to 10.5 base pairs of sequence. Adding or subtracting twists, as some enzymes can do, imposes strain. If a DNA segment under twist strain were closed into a circle by joining its two ends and then allowed to move freely, the circular DNA would contort into a new shape, such as a simple figure-eight. Such a contortion is a supercoil.
The simple figure eight is the simplest supercoil, and is the shape a circular DNA assumes to accommodate one too many or one too few helical twists. The two lobes of the figure eight will appear rotated either clockwise or counterclockwise with respect to one another, depending on whether the helix is over or underwound. For each additional helical twist being accommodated, the lobes will show one more rotation about their axis.
The noun form “supercoil” is rarely used in the context of DNA topology. Instead, global contortions of a circular DNA, such as the rotation of the figure-eight lobes above, are referred to as writhe. The above example illustrates that twist and writhe are interconvertible. “Supercoiling” is an abstract mathematical property representing the sum of twist and writhe. The twist is the number of helical turns in the DNA and the writhe is the number of times the double helix crosses over on itself (these are the supercoils).
Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. Many topoisomerase enzymes sense supercoiling and either generate or dissipate it as they change DNA topology. In part because chromosomes may be very large, segments in the middle may act as if their ends are anchored. As a result, they may be unable to distribute excess twist to the rest of the chromosome or to absorb twist to recover from underwinding—the segments may become supercoiled, in other words. In response to supercoiling, they will assume an amount of writhe, just as if their ends were joined.
Supercoiled DNA forms two structures; a plectoneme or a toroid, or a combination of both. A negatively supercoiled DNA molecule will produce either a one-start left-handed helix, the toroid, or a two-start right-handed helix with terminal loops, the plectoneme. Plectonemes are typically more common in nature, and this is the shape most bacterial plasmids will take. For larger molecules, it is common for hybrid structures to form – a loop on a toroid can extend into a plectoneme. If all the loops on a toroid extend, it becomes a branch point in the plectonemic structure.
The Importance of DNA Supercoiling
DNA supercoiling is important for DNA packaging within all cells. Because the length of DNA can be thousands of times that of a cell, packaging this genetic material into the cell or nucleus (in eukaryotes ) is a difficult feat. Supercoiling of DNA reduces the space and allows for much more DNA to be packaged. In prokaryotes, plectonemic supercoils are predominant, because of the circular chromosome and relatively small amount of genetic material. In eukaryotes, DNA supercoiling exists on many levels of both plectonemic and solenoidal supercoils, with the solenoidal supercoiling proving the most effective in compacting the DNA. Solenoidal supercoiling is achieved with histones to form a 10 nm fiber. This fiber is further coiled into a 30 nm fiber, and further coiled upon itself numerous times more.
DNA packaging is greatly increased during nuclear division events such as mitosis or meiosis, where DNA must be compacted and segregated to daughter cells. Condensins and cohesins are structural maintenance of chromosome (SMC) proteins that aid in the condensation of sister chromatids and the linkage of the centromere in sister chromatids. These SMC proteins induce positive supercoils.
Supercoiling is also required for DNA and RNA synthesis. Because DNA must be unwound for DNA and RNA polymerase action, supercoils will result. The region ahead of the polymerase complex will be unwound; this stress is compensated with positive supercoils ahead of the complex. Behind the complex, DNA is rewound and there will be compensatory negative supercoils. It is important to note that topoisomerases such as DNA gyrase (Type II Topoisomerase) play a role in relieving some of the stress during DNA and RNA synthesis.
Key Points
• As a general rule, the DNA of most organisms is negatively supercoiled.
• The simple figure eight is the simplest supercoil, and is the shape a circular DNA assumes to accommodate one too many or one too few helical twists.
• DNA supercoiling is important for DNA packaging within all cells.
Key Terms
• supercoiling: The coiling of the DNA helix upon itself; can cause disruption to transcription and lead to cell death.
• DNA: A biopolymer of deoxyribonucleic acids (a type of nucleic acid) that has four different chemical groups, called bases: adenine, guanine, cytosine, and thymine.
• chromosome: A structure in the cell nucleus that contains DNA, histone protein, and other structural proteins. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.02%3A_Prokaryotic_Genomes/7.2A%3A_Bacterial_Chromosomes_in_the_Nucleoid.txt |
Learning Objectives
• Explain prokaryotic genome size variation and ORFs
In molecular genetics, an open reading frame (ORF) is the part of a reading frame that contains no stop codons. The transcription termination pause site is located after the ORF, beyond the translation stop codon, because if transcription were to cease before the stop codon, an incomplete protein would be made during translation.
Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop codons.
Open reading frames are used as one piece of evidence to assist in gene prediction. Long ORFs are often used, along with other evidence, to initially identify candidate protein coding regions in a DNA sequence. The presence of an ORF does not necessarily mean that the region is ever translated. For example, in a randomly generated DNA sequence with an equal percentage of each nucleotide, a stop-codon would be expected once every 21 codons. A simple gene prediction algorithm for prokaryotes might look for a start codon followed by an open reading frame that is long enough to encode a typical protein, where the codon usage of that region matches the frequency characteristic for the given organism ‘s coding regions. Even a long open reading frame by itself is not conclusive evidence for the presence of a gene.
If a portion of a genome has been sequenced (e.g. 5′-ATCTAAAATGGGTGCC-3′), ORFs can be located by examining each of the three possible reading frames on each strand. In this sequence two out of three possible reading frames are entirely open, meaning that they do not contain a stop codon:
…A TCT AAA ATG GGT GCC…
…AT CTA AAA TGG GTG CC…
…ATC TAA AAT GGG TGC C…
Possible stop codons in DNA are “TGA”, “TAA”, and “TAG”. Thus, the last reading frame in this example contains a stop codon (TAA), unlike the first two.
Bacterial genomes display variation in size, even among strains of the same species. These microorganisms have very little noncoding or repetitive DNA, as the variation in their genome size usually reflects differences in gene repertoire. Some species, particularly bacterial parasites and symbionts, have undergone massive genome reduction and simply contain a subset of the genes present in their ancestors.
However, in free-living bacteria, such gene loss cannot explain the observed disparities in genome size because ancestral genomes would have had to contain improbably large numbers of genes. Surprisingly, a substantial fraction of the difference in gene contents in free-living bacteria is due to the presence of ORFans, that is, open reading frames (ORFs) that have no known homologs and are consequently of no known function.
The high numbers of ORFans in bacterial genomes indicate that, with the exception of those species with highly reduced genomes, much of the observed diversity in gene inventories does not result from either the loss of ancestral genes or the transfer from well-characterized organisms (processes that result in a patchy distribution of orthologs but not in unique genes) or from recent duplications (which would likely yield homologs within the same or closely related genome).
Key Points
• Open reading frames are used as one piece of evidence to assist in gene prediction.
• If a portion of a genome has been sequenced, ORFs can be located by examining each of the three possible reading frames on each strand.
• Bacterial genomes display variation in size, even among strains of the same species.
Key Terms
• gene: A unit of heredity; a segment of DNA or RNA that is transmitted from one generation to the next. It carries genetic information such as the sequence of amino acids for a protein.
• codons: The genetic code is the set of rules by which information encoded within genetic material (DNA or mRNA sequences) is translated into proteins (amino acid sequences) by living cells. Biological decoding is accomplished by the ribosome, which links amino acids in an order specified by mRNA, using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a time. The genetic code is highly similar among all organisms, and can be expressed in a simple table with 64 entries.
• open reading frame: A sequence of DNA triplets, between the initiator and terminator codons, that can be transcribed into mRNA and later translated into protein. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.02%3A_Prokaryotic_Genomes/7.2C%3A_Size_Variation_and_ORF_Contents_in_Genomes.txt |
Bioinformatics is the study of methods for storing, retrieving and analyzing biological data.
LEARNING OBJECTIVES
Describe the purposes and applications of bioinformatics
Key Points
• The primary goal of bioinformatics is to increase the understanding of biological processes.
• Bioinformatics entails the creation and advancement of databases, algorithms, computational and statistical techniques and theory to solve problems arising from the management and analysis of biological data.
• Gene Ontology, or GO, is a major bioinformatics initiative to unify the representation of gene and gene product attributes across all species.
Key Terms
• bioinformatics: Bioinformatics is a branch of biological science which deals with the study of methods for storing, retrieving and analyzing biological data like nucleic acid (DNA/RNA) and protein sequence, structure, function, pathways and genetic interactions.
• algorithms: In mathematics and computer science, an algorithm is a step-by-step procedure for calculations. Algorithms are used for calculation, data processing, and automated reasoning.
• gene ontology: The Gene Ontology is a major bioinformatics initiative to unify the representation of gene and gene product attributes across all species.
Bioinformatics is a branch of biological science dealing with the study of storing, retrieving and analyzing biological data like nucleic acid (DNA/RNA) and protein sequence, structure, function, pathways and genetic interactions. It generates new knowledge that is useful in such fields as drug design and development of new software tools. Bioinformatics also deals with algorithms, databases and information systems, web technologies, artificial intelligence and soft computing, information and computation theory, structural biology, software engineering, data mining, image processing, modeling and simulation, discrete mathematics, control and system theory, circuit theory, and statistics.
At the beginning of the “genomic revolution,” the term bioinformatics refered to the creation and maintenance of a database to store biological information like nucleotide and amino acid sequences. Development of this type of database involved not only design issues but the development of complex interfaces whereby researchers could access existing data as well as submit new or revised data.
In order to study how normal cellular activities are altered in different disease states, the biological data must be combined to form a comprehensive picture of these activities. Therefore, the field of bioinformatics has evolved such that the most pressing task now involves the analysis and interpretation of various types of data. This includes nucleotide and amino acid sequences, protein domains and protein structures. The actual process of analyzing and interpreting data is referred to as computational biology. Important sub-disciplines within bioinformatics and computational biology include:
• the development of tools that enable efficient use of various types of information
• the development of new algorithms (mathematical formulas) and statistics with which to assess relationships among members of large data sets. For example, methods to locate a gene within a sequence (gene distributions), predict protein structure and/or function, and cluster protein sequences into families of related sequences.
The primary goal of bioinformatics is to increase the understanding of biological processes. What sets it apart from other approaches, however, is its focus on developing and applying computationally intensive techniques to achieve this goal. Examples include pattern recognition, data mining, machine learning algorithms, and visualization. Major research efforts in the field include sequence alignment, gene finding, genome assembly, drug design, drug discovery, protein structure alignment, and the modeling of evolution.
Gene Ontology, or GO, is a major bioinformatics initiative to unify the representation of gene and gene product attributes across all species. More specifically, the project aims to:
• maintain and develop its controlled vocabulary of gene and gene product attributes
• annotate genes and gene products and assimilate and disseminate annotation data
• offer tools for easy access to all aspects of the data provided by the project
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Thumbnail: DNA Double Helix. (Public Domain; Apers0n).
07: Microbial Genetics
• 7.3A: Basics of DNA Replication
DNA replication uses a semi-conservative method that results in a double-stranded DNA with one parental strand and a new daughter strand.
• 7.3B: DNA Replication in Eukaryotes
Because eukaryotic genomes are quite complex, DNA replication is a very complicated process that involves several enzymes and other proteins. It occurs in three main stages: initiation, elongation, and termination. DNA replication in eukaryotes occurs in three stages: initiation, elongation, and termination, which are aided by several enzymes.
7.03: DNA Replication
DNA replication uses a semi-conservative method that results in a double-stranded DNA with one parental strand and a new daughter strand.
LEARNING OBJECTIVES
Explain how the Meselson and Stahl experiment conclusively established that DNA replication is semi-conservative.
Key Points
• There were three models suggested for DNA replication: conservative, semi-conservative, and dispersive.
• The conservative method of replication suggests that parental DNA remains together and newly-formed daughter strands are also together.
• The semi-conservative method of replication suggests that the two parental DNA strands serve as a template for new DNA and after replication, each double-stranded DNA contains one strand from the parental DNA and one new (daughter) strand.
• The dispersive method of replication suggests that, after replication, the two daughter DNAs have alternating segments of both parental and newly-synthesized DNA interspersed on both strands.
• Meselson and Stahl, using E. coli DNA made with two nitrogen istopes (14N and 15N) and density gradient centrifugation, determined that DNA replicated via the semi-conservative method of replication.
Key Terms
• DNA replication: a biological process occuring in all living organisms that is the basis for biological inheritance
• isotope: any of two or more forms of an element where the atoms have the same number of protons, but a different number of neutrons within their nuclei
Basics of DNA Replication
Watson and Crick’s discovery that DNA was a two-stranded double helix provided a hint as to how DNA is replicated. During cell division, each DNA molecule has to be perfectly copied to ensure identical DNA molecules to move to each of the two daughter cells. The double-stranded structure of DNA suggested that the two strands might separate during replication with each strand serving as a template from which the new complementary strand for each is copied, generating two double-stranded molecules from one.
Models of Replication
There were three models of replication possible from such a scheme: conservative, semi-conservative, and dispersive. In conservative replication, the two original DNA strands, known as the parental strands, would re-basepair with each other after being used as templates to synthesize new strands; and the two newly-synthesized strands, known as the daughter strands, would also basepair with each other; one of the two DNA molecules after replication would be “all-old” and the other would be “all-new”. In semi-conservative replication, each of the two parental DNA strands would act as a template for new DNA strands to be synthesized, but after replication, each parental DNA strand would basepair with the complementary newly-synthesized strand just synthesized, and both double-stranded DNAs would include one parental or “old” strand and one daughter or “new” strand. In dispersive replication, after replication both copies of the new DNAs would somehow have alternating segments of parental DNA and newly-synthesized DNA on each of their two strands.
To determine which model of replication was accurate, a seminal experiment was performed in 1958 by two researchers: Matthew Meselson and Franklin Stahl.
Meselson and Stahl
Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N) that is incorporated into nitrogenous bases and, eventually, into the DNA. The E. coliculture was then shifted into medium containing the common “light” isotope of nitrogen (14N) and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge in a tube in which a cesium chloride density gradient had been established. Some cells were allowed to grow for one more life cycle in 14N and spun again.
During the density gradient ultracentrifugation, the DNA was loaded into a gradient (Meselson and Stahl used a gradient of cesium chloride salt, although other materials such as sucrose can also be used to create a gradient) and spun at high speeds of 50,000 to 60,000 rpm. In the ultracentrifuge tube, the cesium chloride salt created a density gradient, with the cesium chloride solution being more dense the farther down the tube you went. Under these circumstances, during the spin the DNA was pulled down the ultracentrifuge tube by centrifugal force until it arrived at the spot in the salt gradient where the DNA molecules’ density matched that of the surrounding salt solution. At the point, the molecules stopped sedimenting and formed a stable band. By looking at the relative positions of bands of molecules run in the same gradients, you can determine the relative densities of different molecules. The molecules that form the lowest bands have the highest densities.
DNA from cells grown exclusively in 15N produced a lower band than DNA from cells grown exclusively in 14N. So DNA grown in 15N had a higher density, as would be expected of a molecule with a heavier isotope of nitrogen incorporated into its nitrogenous bases. Meselson and Stahl noted that after one generation of growth in 14N (after cells had been shifted from 15N), the DNA molecules produced only single band intermediate in position in between DNA of cells grown exclusively in 15N and DNA of cells grown exclusively in 14N. This suggested either a semi-conservative or dispersive mode of replication. Conservative replication would have resulted in two bands; one representing the parental DNA still with exclusively 15N in its nitrogenous bases and the other representing the daughter DNA with exclusively 14N in its nitrogenous bases. The single band actually seen indicated that all the DNA molecules contained equal amounts of both 15N and 14N.
The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N and the other corresponded to the band of exclusively 14N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Dispersive replication would have resulted in exclusively a single band in each new generation, with the band slowly moving up closer to the height of the 14N DNA band. Therefore, dispersive replication could also be ruled out.
Meselson and Stahl’s results established that during DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are synthesized. The new strand will be complementary to the parental or “old” strand and the new strand will remain basepaired to the old strand. So each “daughter” DNA actually consists of one “old” DNA strand and one newly-synthesized strand. When two daughter DNA copies are formed, they have the identical sequences to one another and identical sequences to the original parental DNA, and the two daughter DNAs are divided equally into the two daughter cells, producing daughter cells that are genetically identical to one another and genetically identical to the parent cell. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.03%3A_DNA_Replication/7.3A%3A_Basics_of_DNA_Replication.txt |
Learning Objectives
• Describe how DNA is replicated in eukaryotes
Because eukaryotic genomes are quite complex, DNA replication is a very complicated process that involves several enzymes and other proteins. It occurs in three main stages: initiation, elongation, and termination. DNA replication in eukaryotes occurs in three stages: initiation, elongation, and termination, which are aided by several enzymes.
Initiation
Eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes. During initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. There are specific chromosomal locations called origins of replication where replication begins. In some eukaryotes, like yeast, these locations are defined by having a specific sequence of basepairs to which the replication initiation proteins bind. In other eukaryotes, like humans, there does not appear to be a consensus sequence for their origins of replication. Instead, the replication initiation proteins might identify and bind to specific modifications to the nucleosomes in the origin region.
Certain proteins recognize and bind to the origin of replication and then allow the other proteins necessary for DNA replication to bind the same region. The first proteins to bind the DNA are said to “recruit” the other proteins. Two copies of an enzyme called helicase are among the proteins recruited to the origin. Each helicase unwinds and separates the DNA helix into single-stranded DNA. As the DNA opens up, Y-shaped structures called replication forks are formed. Because two helicases bind, two replication forks are formed at the origin of replication; these are extended in both directions as replication proceeds creating a replication bubble. There are multiple origins of replication on the eukaryotic chromosome which allow replication to occur simultaneously in hundreds to thousands of locations along each chromosome.
Elongation
During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3′ end of the newly synthesized polynucleotide strand. The template strand specifies which of the four DNA nucleotides (A, T, C, or G) is added at each position along the new chain. Only the nucleotide complementary to the template nucleotide at that position is added to the new strand.
DNA polymerase contains a groove that allows it to bind to a single-stranded template DNA and travel one nucleotide at at time. For example, when DNA polymerase meets an adenosine nucleotide on the template strand, it adds a thymidine to the 3′ end of the newly synthesized strand, and then moves to the next nucleotide on the template strand. This process will continue until the DNA polymerase reaches the end of the template strand.
DNA polymerase cannot initiate new strand synthesis; it only adds new nucleotides at the 3′ end of an existing strand. All newly synthesized polynucleotide strands must be initiated by a specialized RNA polymerase called primase. Primase initiates polynucleotide synthesis and by creating a short RNA polynucleotide strand complementary to template DNA strand. This short stretch of RNA nucleotides is called the primer. Once RNA primer has been synthesized at the template DNA, primase exits, and DNA polymerase extends the new strand with nucleotides complementary to the template DNA.
Eventually, the RNA nucleotides in the primer are removed and replaced with DNA nucleotides. Once DNA replication is finished, the daughter molecules are made entirely of continuous DNA nucleotides, with no RNA portions.
The Leading and Lagging Strands
DNA polymerase can only synthesize new strands in the 5′ to 3′ direction. Therefore, the two newly-synthesized strands grow in opposite directions because the template strands at each replication fork are antiparallel. The “leading strand” is synthesized continuously toward the replication fork as helicase unwinds the template double-stranded DNA.
The “lagging strand” is synthesized in the direction away from the replication fork and away from the DNA helicase unwinds. This lagging strand is synthesized in pieces because the DNA polymerase can only synthesize in the 5′ to 3′ direction, and so it constantly encounters the previously-synthesized new strand. The pieces are called Okazaki fragments, and each fragment begins with its own RNA primer.
Termination
Eukaryotic chromosomes have multiple origins of replication, which initiate replication almost simultaneously. Each origin of replication forms a bubble of duplicated DNA on either side of the origin of replication. Eventually, the leading strand of one replication bubble reaches the lagging strand of another bubble, and the lagging strand will reach the 5′ end of the previous Okazaki fragment in the same bubble.
DNA polymerase halts when it reaches a section of DNA template that has already been replicated. However, DNA polymerase cannot catalyze the formation of a phosphodiester bond between the two segments of the new DNA strand, and it drops off. These unattached sections of the sugar-phosphate backbone in an otherwise full-replicated DNA strand are called nicks.
Once all the template nucleotides have been replicated, the replication process is not yet over. RNA primers need to be replaced with DNA, and nicks in the sugar-phosphate backbone need to be connected.
The group of cellular enzymes that remove RNA primers include the proteins FEN1 (flap endonulcease 1) and RNase H. The enzymes FEN1 and RNase H remove RNA primers at the start of each leading strand and at the start of each Okazaki fragment, leaving gaps of unreplicated template DNA. Once the primers are removed, a free-floating DNA polymerase lands at the 3′ end of the preceding DNA fragment and extends the DNA over the gap. However, this creates new nicks (unconnected sugar-phosphate backbone).
In the final stage of DNA replication, the enyzme ligase joins the sugar-phosphate backbones at each nick site. After ligase has connected all nicks, the new strand is one long continuous DNA strand, and the daughter DNA molecule is complete.
DNA Replication: This is a clip from a PBS production called “DNA: The Secret of Life.” It details the latest research (as of 2005) concerning the process of DNA replication.
Key Points
• During initiation, proteins bind to the origin of replication while helicase unwinds the DNA helix and two replication forks are formed at the origin of replication.
• During elongation, a primer sequence is added with complementary RNA nucleotides, which are then replaced by DNA nucleotides.
• During elongation the leading strand is made continuously, while the lagging strand is made in pieces called Okazaki fragments.
• During termination, primers are removed and replaced with new DNA nucleotides and the backbone is sealed by DNA ligase.
Key Terms
• origin of replication: a particular sequence in a genome at which replication is initiated
• leading strand: the template strand of the DNA double helix that is oriented so that the replication fork moves along it in the 3′ to 5′ direction
• lagging strand: the strand of the template DNA double helix that is oriented so that the replication fork moves along it in a 5′ to 3′ manner
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Learning Objectives
• Outline the utility of plasmids
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952. A plasmid is a DNA molecule that is separate from, and can replicate independently of the chromosomal DNA. They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in archaea, and even in eukaryotic organisms (e.g., the 2-micrometre ring in Saccharomyces cerevisiae). Plasmid sizes vary from 1 to over 1,000 kbp. The number of identical plasmids in a single cell can range anywhere from one to thousands under some circumstances. Plasmids can be considered part of the mobilome because they are often associated with conjugation, a mechanism of horizontal gene transfer.
Plasmids are considered replicons. They can be found in all three major domains: Archaea, Bacteria, and Eukarya. Similar to viruses, plasmids are not considered by some to be a form of life. Unlike viruses, they are naked DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer. Plasmid host-to-host transfer requires direct mechanical transfer by conjugation, or changes in incipient host gene expression allowing the intentional uptake of the genetic element by transformation. Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, because each implies the presence of an independent species living in a commensal or detrimental state with the host organism. Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may act as toxins under similar circumstances. Plasmids can also provide bacteria with the ability to fix elemental nitrogen or to degrade recalcitrant organic compounds that provide an advantage when nutrients are scarce.
Key Points
• Plasmids can be found in all three major domains: Archaea, Bacteria, and Eukarya. Similar to viruses, plasmids are not considered by some to be a form of life.
• Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state.
• Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may act as toxins under similar circumstances.
Key Terms
• plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.
• mobilome: The entirety of the mobile (transposable) elements of a genome.
• replicons: a region of DNA or RNA, that replicates from a single origin of replication.
7.4B: Types of Plasmids and Their Biological Significance
Plasmids are commonly used to multiply (make many copies of) or express particular genes.
LEARNING OBJECTIVES
Recognize the characteristics of, and thus the functions, of plasmids
Key Points
• The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics, and into a multiple cloning site (MCS, or polylinker), allowing the easy insertion of DNA fragments.
• A major use of plasmids is to make large amounts of proteins. Bacterium can be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for; for example, insulin or even antibiotics.
• It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. However, related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions.
Key Terms
• Col plasmids: These plasmids contain genes that code for bacteriocins, proteins that can kill other bacteria.
• F-plasmid: Fertility F-plasmids contain tra genes and are capable of conjugation resulting in the expression of sex pilli.
• Resistance plasmids: These plasmids contain genes that provide resistance against antibiotics or poisons.
Types of Plasmids
Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics. The gene is also inserted into a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments.
Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria that take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way, the antibiotics act as a filter, selecting only the modified bacteria. Finally, these bacteria can be grown in large amounts, harvested, and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for; for example, insulin or even antibiotics.
One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium. Non-conjugative plasmids are incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can parasitize a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA, and may possibly be a tool for curing many diseases.
It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. However, related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Thus, plasmids can be assigned into incompatibility groups.
Another way to classify plasmids is by function. There are five main classes:
• Fertility F-plasmids, which contain tra genes. They are capable of conjugation and result in the expression of sex pilli.
• Resistance plasmids, which contain genes that provide resistance against antibiotics or poisons. They were historically known as R-factors, before the nature of plasmids was understood.
• Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen.
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Elongation synthesizes pre-mRNA in a 5′ to 3′ direction, and termination occurs in response to termination sequences and signals.
LEARNING OBJECTIVES
Describe what is happening during transcription elongation and termination
Key Points
• RNA polymerase II (RNAPII) transcribes the major share of eukaryotic genes.
• During elongation, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome.
• Transcription elongation occurs in a bubble of unwound DNA, where the RNA Polymerase uses one strand of DNA as a template to catalyze the synthesis of a new RNA strand in the 5′ to 3′ direction.
• RNA Polymerase I and RNA Polymerase III terminate transcription in response to specific termination sequences in either the DNA being transcribed (RNA Polymerase I) or in the newly-synthesized RNA (RNA Polymerase III).
• RNA Polymerase II terminates transcription at random locations past the end of the gene being transcribed. The newly-synthesized RNA is cleaved at a sequence-specified location and released before transcription terminates.
Key Terms
• nucleosome: any of the subunits that repeat in chromatin; a coil of DNA surrounding a histone core
• histone: any of various simple water-soluble proteins that are rich in the basic amino acids lysine and arginine and are complexed with DNA in the nucleosomes of eukaryotic chromatin
• chromatin: a complex of DNA, RNA, and proteins within the cell nucleus out of which chromosomes condense during cell division
Transcription through Nucleosomes
Following the formation of the pre-initiation complex, the polymerase is released from the other transcription factors, and elongation is allowed to proceed with the polymerase synthesizing RNA in the 5′ to 3′ direction. RNA Polymerase II (RNAPII) transcribes the major share of eukaryotic genes, so this section will mainly focus on how this specific polymerase accomplishes elongation and termination.
Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the eukaryotic DNA template is more complex. When eukaryotic cells are not dividing, their genes exist as a diffuse, but still extensively packaged and compacted mass of DNA and proteins called chromatin. The DNA is tightly packaged around charged histone proteins at repeated intervals. These DNA–histone complexes, collectively called nucleosomes, are regularly spaced and include 146 nucleotides of DNA wound twice around the eight histones in a nucleosome like thread around a spool.
For polynucleotide synthesis to occur, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome. This is accomplished by a special protein dimer called FACT, which stands for “facilitates chromatin transcription.” FACT partially disassembles the nucleosome immediately ahead (upstream) of a transcribing RNA Polymerase II by removing two of the eight histones (a single dimer of H2A and H2B histones is removed.) This presumably sufficiently loosens the DNA wrapped around that nucleosome so that RNA Polymerase II can transcribe through it. FACT reassembles the nucleosome behind the RNA Polymerase II by returning the missing histones to it. RNA Polymerase II will continue to elongate the newly-synthesized RNA until transcription terminates.
The FACT protein dimer allows RNA Polymerase II to transcribe through packaged DNA: DNA in eukaryotes is packaged in nucleosomes, which consist of an octomer of 4 different histone proteins. When DNA is tightly wound twice around a nucleosome, RNA Polymerase II cannot access it for transcription. FACT removes two of the histones from the nucleosome immediately ahead of RNA Polymerase, loosening the packaging so that RNA Polymerase II can continue transcription. FACT also reassembles the nucleosome immediately behindd the RNA Polymerase by returning the missing histones.
Elongation
RNA Polymerase II is a complex of 12 protein subunits. Specific subunits within the protein allow RNA Polymerase II to act as its own helicase, sliding clamp, single-stranded DNA binding protein, as well as carry out other functions. Consequently, RNA Polymerase II does not need as many accessory proteins to catalyze the synthesis of new RNA strands during transcription elongation as DNA Polymerase does to catalyze the synthesis of new DNA strands during replication elongation.
However, RNA Polymerase II does need a large collection of accessory proteins to initiate transcription at gene promoters, but once the double-stranded DNA in the transcription start region has been unwound, the RNA Polymerase II has been positioned at the +1 initiation nucleotide, and has started catalyzing new RNA strand synthesis, RNA Polymerase II clears or “escapes” the promoter region and leaves most of the transcription initiation proteins behind.
All RNA Polymerases travel along the template DNA strand in the 3′ to 5′ direction and catalyze the synthesis of new RNA strands in the 5′ to 3′ direction, adding new nucleotides to the 3′ end of the growing RNA strand.
RNA Polymerases unwind the double stranded DNA ahead of them and allow the unwound DNA behind them to rewind. As a result, RNA strand synthesis occurs in a transcription bubble of about 25 unwound DNA basebairs. Only about 8 nucleotides of newly-synthesized RNA remain basepaired to the template DNA. The rest of the RNA molecules falls off the template to allow the DNA behind it to rewind.
RNA Polymerases use the DNA strand below them as a template to direct which nucleotide to add to the 3′ end of the growing RNA strand at each point in the sequence. The RNA Polymerase travels along the template DNA one nucleotide at at time. Whichever RNA nucleotide is capable of basepairing to the template nucleotide below the RNA Polymerase is the next nucleotide to be added. Once the addition of a new nucleotide to the 3′ end of the growing strand has been catalyzed, the RNA Polymerase moves to the next DNA nucleotide on the template below it. This process continues until transcription termination occurs.
Termination
Transcription termination by RNA Polymerase II on a protein-encoding gene.: RNA Polymerase II has no specific signals that terminate its transcription. In the case of protein-encoding genes, a protein complex will bind to two locations on the growing pre-mRNA once the RNA Polymerase has transcribed past the end of the gene. CPSF in the complex will bind a AAUAAA sequence, and CstF in the complex will bind a GU-rich sequence (top figure). CPSF in the complex will cleave the pre-mRNA at a site between the two bound sequences, releasing the pre-mRNA (middle figure). Poly(A) Polymerase is a part of the same complex and will begin to add a poly-A tail to the pre-mRNA. At the same time, Xrn2 protein, which is an exonuclease, attacks the 5′ end of the RNA strand still associated with the RNA Polymerase. Xrn2 will start digesting the non-released portion of the newly synthesized RNA until Xrn2 reaches the RNA Polymerase, where it aids in displacing the RNA Polymerase from the template DNA strand. This terminates transcription at some random location downstream from the true end of the gene (bottom figure).
The termination of transcription is different for the three different eukaryotic RNA polymerases.
The ribosomal rRNA genes transcribed by RNA Polymerase I contain a specific sequence of basepairs (11 bp long in humans; 18 bp in mice) that is recognized by a termination protein called TTF-1 (Transcription Termination Factor for RNA Polymerase I.) This protein binds the DNA at its recognition sequence and blocks further transcription, causing the RNA Polymerase I to disengage from the template DNA strand and to release its newly-synthesized RNA.
The protein-encoding, structural RNA, and regulatory RNA genes transcribed by RNA Polymerse II lack any specific signals or sequences that direct RNA Polymerase II to terminate at specific locations. RNA Polymerase II can continue to transcribe RNA anywhere from a few bp to thousands of bp past the actual end of the gene. However, the transcript is cleaved at an internal site before RNA Polymerase II finishes transcribing. This releases the upstream portion of the transcript, which will serve as the initial RNA prior to further processing (the pre-mRNA in the case of protein-encoding genes.) This cleavage site is considered the “end” of the gene. The remainder of the transcript is digested by a 5′-exonuclease (called Xrn2 in humans) while it is still being transcribed by the RNA Polymerase II. When the 5′-exonulease “catches up” to RNA Polymerase II by digesting away all the overhanging RNA, it helps disengage the polymerase from its DNA template strand, finally terminating that round of transcription.
In the case of protein-encoding genes, the cleavage site which determines the “end” of the emerging pre-mRNA occurs between an upstream AAUAAA sequence and a downstream GU-rich sequence separated by about 40-60 nucleotides in the emerging RNA. Once both of these sequences have been transcribed, a protein called CPSF in humans binds the AAUAAA sequence and a protein called CstF in humans binds the GU-rich sequence. These two proteins form the base of a complicated protein complex that forms in this region before CPSF cleaves the nascent pre-mRNA at a site 10-30 nucleotides downstream from the AAUAAA site. The Poly(A) Polymerase enzyme which catalyzes the addition of a 3′ poly-A tail on the pre-mRNA is part of the complex that forms with CPSF and CstF.
The tRNA, 5S rRNA, and structural RNAs genes transcribed by RNA Polymerase III have a not-entirely-understood termination signal. The RNAs transcribed by RNA Polymerase III have a short stretch of four to seven U’s at their 3′ end. This somehow triggers RNA Polymerase III to both release the nascent RNA and disengage from the template DNA strand. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.05%3A_RNA_Synthesis_-_Transcription/7.5A%3A_Elongation_and_Termination_in_Eukaryotes.txt |
Learning Objectives
• Describe the role of promoters in RNA transcription
Genes are organized to make the control of gene expression easier. The promoter region is immediately upstream of the coding sequence. This region can be short (only a few nucleotides in length) or quite long (hundreds of nucleotides long). The longer the promoter, the more available space for proteins to bind. This also adds more control to the transcription process. The length of the promoter is gene-specific and can differ dramatically between genes. Consequently, the level of control of gene expression can also differ quite dramatically between genes. The purpose of the promoter is to bind transcription factors that control the initiation of transcription.
Within the promoter region, just upstream of the transcriptional start site, resides the TATA box. This box is simply a repeat of thymine and adenine dinucleotides (literally, TATA repeats). RNA polymerase binds to the transcription initiation complex, allowing transcription to occur. To initiate transcription, a transcription factor (TFIID) is the first to bind to the TATA box. Binding of TFIID recruits other transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH to the TATA box. Once this transcription initiation complex is assembled, RNA polymerase can bind to its upstream sequence. When bound along with the transcription factors, RNA polymerase is phosphorylated. This releases part of the protein from the DNA to activate the transcription initiation complex and places RNA polymerase in the correct orientation to begin transcription; DNA-bending protein brings the enhancer, which can be quite a distance from the gene, in contact with transcription factors and mediator proteins.
In addition to the general transcription factors, other transcription factors can bind to the promoter to regulate gene transcription. These transcription factors bind to the promoters of a specific set of genes. They are not general transcription factors that bind to every promoter complex, but are recruited to a specific sequence on the promoter of a specific gene. There are hundreds of transcription factors in a cell that each bind specifically to a particular DNA sequence motif. When transcription factors bind to the promoter just upstream of the encoded gene, they are referred to as cis-acting elements because they are on the same chromosome, just next to the gene. The region that a particular transcription factor binds to is called the transcription factor binding site. Transcription factors respond to environmental stimuli that cause the proteins to find their binding sites and initiate transcription of the gene that is needed.
Key Points
• The purpose of the promoter is to bind transcription factors that control the initiation of transcription.
• The promoter region can be short or quite long; the longer the promoter is, the more available space for proteins to bind.
• To initiate transcription, a transcription factor (TFIID) binds to the TATA box, which causes other transcription factors to subsequently bind to the TATA box.
• Once the transcription initiation complex is assembled, RNA polymerase can bind to its upstream sequence and is then phosphorylated.
• Phosphorylation of RNA polymerase releases part of the protein from the DNA to activate the transcription initiation complex and places RNA polymerase in the correct orientation to begin transcription.
• Transcription factors respond to environmental stimuli that cause the proteins to find their binding sites and initiate transcription of the gene that is needed.
Key Terms
• TATA box: a DNA sequence (cis-regulatory element) found in the promoter region of genes in archaea and eukaryotes
• transcription factor: a protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to mRNA
• promoter: the section of DNA that controls the initiation of RNA transcription
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Learning Objectives
• Describe how pre-rRNAs and pre-tRNAs are processed into mature rRNAs and tRNAs.
The tRNAs and rRNAs are structural molecules that have roles in protein synthesis; however, these RNAs are not themselves translated. In eukaryotes, pre-rRNAs are transcribed, processed, and assembled into ribosomes in the nucleolus, while pre-tRNAs are transcribed and processed in the nucleus and then released into the cytoplasm where they are linked to free amino acids for protein synthesis.
Ribosomal RNA (rRNA)
The four rRNAs in eukaryotes are first transcribed as two long precursor molecules. One contains just the pre-rRNA that will be processed into the 5S rRNA; the other spans the 28S, 5.8S, and 18S rRNAs. Enzymes then cleave the precursors into subunits corresponding to each rRNA. In bacteria, there are only three rRNAs and all are transcribed in one long precursor molecule that is cleaved into the individual rRNAs. Some of the bases of pre-rRNAs are methylated for added stability. Mature rRNAs make up 50-60% of each ribosome. Some of a ribosome’s RNA molecules are purely structural, whereas others have catalytic or binding activities.
The eukaryotic ribosome is composed of two subunits: a large subunit (60S) and a small subunit (40S). The 60S subunit is composed of the 28S rRNA, 5.8S rRNA, 5S rRNA, and 50 proteins. The 40S subunit is composed of the 18S rRNA and 33 proteins. The bacterial ribosome is composed of two similar subunits, with slightly different components. The bacterial large subunit is called the 50S subunit and is composed of the 23S rRNA, 5S rRNA, and 31 proteins, while the bacterial small subunit is called the 30S subunit and is composed of the 16S rRNA and 21 proteins.
The two subunits join to constitute a functioning ribosome that is capable of creating proteins.
Transfer RNA (tRNA)
Each different tRNA binds to a specific amino acid and transfers it to the ribosome. Mature tRNAs take on a three-dimensional structure through intramolecular basepairing to position the amino acid binding site at one end and the anticodon in an unbasepaired loop of nucleotides at the other end. The anticodon is a three-nucleotide sequence, unique to each different tRNA, that interacts with a messenger RNA (mRNA) codon through complementary base pairing.
There are different tRNAs for the 21 different amino acids. Most amino acids can be carried by more than one tRNA.
.
In all organisms, tRNAs are transcribed in a pre-tRNA form that requires multiple processing steps before the mature tRNA is ready for use in translation. In bacteria, multiple tRNAs are often transcribed as a single RNA. The first step in their processing is the digestion of the RNA to release individual pre-tRNAs. In archaea and eukaryotes, each pre-tRNA is transcribed as a separate transcript.
The processing to convert the pre-tRNA to a mature tRNA involves five steps.
1. The 5′ end of the pre-tRNA, called the 5′ leader sequence, is cleaved off.
2. The 3′ end of the pre-tRNA is cleaved off.
3. In all eukaryote pre-tRNAs, but in only some bacterial and archaeal pre-tRNAs, a CCA sequence of nucleotides is added to the 3′ end of the pre-tRNA after the original 3′ end is trimmed off. Some bacteria and archaea pre-tRNAs already have the CCA encoded in their transcript immediately upstream of the 3′ cleavage site, so they don’t need to add one. The CCA at the 3′ end of the mature tRNA will be the site at which the tRNA’s amino acid will be added.
4. Multiple nucleotides in the pre-tRNA are chemically modified, altering their nitorgen bases. On average about 12 nucleotides are modified per tRNA. The most common modifications are the conversion of adenine (A) to pseudouridine (ψ), the conversion of adenine to inosine (I), and the conversion of uridine to dihydrouridine (D). But over 100 other modifications can occur.
5. A significant number of eukaryotic and archaeal pre-tRNAs have introns that have to be spliced out. Introns are rarer in bacterial pre-tRNAs, but do occur occasionally and are spliced out.
After processing, the mature pre-tRNA is ready to have its cognate amino acid attached. The cognate amino acid for a tRNA is the one specified by its anticodon. Attaching this amino acid is called charging the tRNA. In eukaryotes, the mature tRNA is generated in the nucleus, and then exported to the cytoplasm for charging.
Processing of a pre-tRNA.: A typical pre-tRNA undergoing processing steps to generate a mature tRNA ready to have its cognate amino acid attached. Nucleotides that are cleaved away are shown in green. Chemically-modified nucleotides are in yellow, as is the CAA trinucleotide that is added to the 3′ end of the pre-tRNA during processing. The anticodon nucleotides are shown in a lighter shade of red.
Key Points
• Ribosomal RNA (rRNA) is a structural molecule that makes up over half of the mass of a ribosome and aids in protein synthesis.
• Transfer RNA (tRNA) recognizes a codon on mRNA and brings the appropriate amino acid to that site.
• rRNAs are processed from larger pre-rRNAs by trimming the larger rRNAs down and methylating some of the nucleotides.
• tRNAs are processed from pre-tRNAs by trimming both ends of the pre-tRNA, adding a CCA trinucleotide to the 3′ end, if needed, removing any introns present, and chemically modified 12 nucleotides on average per tRNA.
Key Terms
• anticodon: a sequence of three nucleotides in transfer RNA that binds to the complementary triplet (codon) in messenger RNA, specifying an amino acid during protein synthesis | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.06%3A_Translation-_Protein_Synthesis/7.6A%3A_Processing_of_tRNAs_and_rRNAs.txt |
Protein synthesis, or translation of mRNA into protein, occurs with the help of ribosomes, tRNAs, and aminoacyl tRNA synthetases.
LEARNING OBJECTIVES
Explain the role played by ribosomes, tRNA, and aminoacyl tRNA synthetases in protein synthesis
Key Points
• Ribosomes, macromolecular structures composed of rRNA and polypeptide chains, are formed of two subunits (in bacteria and archaea, 30S and 50S; in eukaryotes, 40S and 60S), that bring together mRNA and tRNAs to catalyze protein synthesis.
• Fully assembled ribosomes have three tRNA binding sites: an A site for incoming aminoacyl-tRNAs, a P site for peptidyl-tRNAs, and an E site where empty tRNAs exit.
• tRNAs (transfer ribonucleic acids), which serve to deliver the appropriate amino acid to the growing peptide chain, consist of a modified RNA chain with the appropriate amino acid covalently attached.
• tRNAs have a loop of unbasepaired nucleotides at one end of the molecule that contains three nucleotides that act as the anticodon that basepairs to the mRNA codon.
• Aminoacyl tRNA synthetases are enzymes that load the individual amino acids onto the tRNAs.
Key Terms
• ribosome: protein/mRNA complexes found in all cells that are involved in the production of proteins by translating messenger RNA
The Protein Synthesis Machinery
In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component may vary across species. For instance, ribosomes may consist of different numbers of rRNAs and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to archaea to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.
Ribosomes
A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the synthesis and assembly of rRNAs occurs in the nucleolus.
Ribosomes exist in the cytoplasm in prokaryotes and in the cytoplasm and on rough endoplasmic reticulum membranes in eukaryotes. Mitochondria and chloroplasts also have their own ribosomes, and these look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the cytoplasmic ribosomes. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation.E. coli have a 30S small subunit and a 50S large subunit, for a total of 70S when assembled (recall that Svedberg units are not additive). Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs.
In bacteria, archaea, and eukaryotes, the intact ribosome has three binding sites that accomodate tRNAs: The A site, the P site, and the E site. Incoming aminoacy-tRNAs (a tRNA with an amino acid covalently attached is called an aminoacyl-tRNA) enter the ribosome at the A site. The peptidyl-tRNA carrying the growing polypeptide chain is held in the P site. The E site holds empty tRNAs just before they exit the ribosome.
Each mRNA molecule is simultaneously translated by many ribosomes, all reading the mRNA from 5′ to 3′ and synthesizing the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome.
tRNAs in eukaryotes
The tRNA molecules are transcribed by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Specific tRNAs bind to codons on the mRNA template and add the corresponding amino acid to the polypeptide chain. (More accurately, the growing polypeptide chain is added to each new amino acid bound in by a tRNA.)
The transfer RNAs (tRNAs) are structural RNA molecules. In eukaryotes, tRNA mole are transcribed from tRNA genes by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. (More accurately, the growing polypeptide chain is added to each new amino acid brought in by a tRNA.) Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins.
Of the 64 possible mRNA codons (triplet combinations of A, U, G, and C) three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain. Of the three termination codons, one (UGA) can also be used to encode the 21st amino acid, selenocysteine, but only if the mRNA contains a specific sequence of nucleotides known as a SECIS sequence. Of the 61 non-termination codons, one codon (AUG) also encodes the initiation of translation.
Each tRNA polynucleotide chain folds up so that some internal sections basepair with other internal sections. If just diagrammed in two dimensions, the regions where basepairing occurs are called stems, and the regions where no basepairs form are called loops, and the entire pattern of stems and loops that forms for a tRNA is called the “cloverleaf” structure. All tRNAs fold into very similar cloverleaf structures of four major stems and three major loops.
If viewed as a three-dimensional structure, all the basepaired regions of the tRNA are helical, and the tRNA folds into a L-shaped structure.
Each tRNA has a sequence of three nucleotides located in a loop at one end of the molecule that can basepair with an mRNA codon. This is called the tRNA’s anticodon. Each different tRNA has a different anticodon. When the tRNA anticodon basepairs with one of the mRNA codons, the tRNA will add an amino acid to a growing polypeptide chain or terminate translation, according to the genetic code. For instance, if the sequence CUA occurred on a mRNA template in the proper reading frame, it would bind a tRNA with an anticodon expressing the complementary sequence, GAU. The tRNA with this anticodon would be linked to the amino acid leucine.
Aminoacyl tRNA Synthetases
The process of pre-tRNA synthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule. The corresponding amino acid must be added later, once the tRNA is processed and exported to the cytoplasm. Through the process of tRNA “charging,” each tRNA molecule is linked to its correct amino acid by a group of enzymes called aminoacyl tRNA synthetases. When an amino acid is covalently linked to a tRNA, the resulting complex is known as an aminoacyl-tRNA. At least one type of aminoacyl tRNA synthetase exists for each of the 21 amino acids; the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze the formation of a covalent bond between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is expelled in this reaction. This is called “activating” the amino acid. The same enzyme then catalyzes the attachment of the activated amino acid to the tRNA and the simultaneous release of AMP. After the correct amino acid covalently attached to the tRNA, it is released by the enzyme. The tRNA is said to be charged with its cognate amino acid. (the amino acid specified by its anticodon is a tRNA’s cognate amino acid.) | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.06%3A_Translation-_Protein_Synthesis/7.6B%3A_The_Protein_Synthesis_Machinery.txt |
Learning Objectives
• Describe the events of prokaryotic transcription initiation
Overview of Prokaryotic Transcription
Prokaryotic transcription is the process in which messenger RNA transcripts of genetic material in prokaryotes are produced, to be translated for the production of proteins. Prokaryotic transcription occurs in the cytoplasm alongside translation. Prokaryotic transcription and translation can occur simultaneously. This is impossible in eukaryotes, where transcription occurs in a membrane-bound nucleus while translation occurs outside the nucleus in the cytoplasm. In prokaryotes genetic material is not enclosed in a membrane-enclosed nucleus and has access to ribosomes in the cytoplasm.
Transcription is controlled by a variety of regulators in prokaryotes. Many of these transcription factors are homodimers containing helix-turn-helix DNA-binding motifs.
Steps of Transcription Initiation
The following steps occur, in order, for transcription initiation:
• RNA polymerase (RNAP) binds to one of several specificity factors, σ, to form a holoenzyme. In this form, it can recognize and bind to specific promoter regions in the DNA. The -35 region and the -10 (“Pribnow box”) region comprise the basic prokaryotic promoter, and |T| stands for the terminator.
• The DNA on the template strand between the +1 site and the terminator is transcribed into RNA, which is then translated into protein. At this stage, the DNA is double-stranded (“closed”). This holoenzyme/wound-DNA structure is referred to as the closed complex.
• The DNA is unwound and becomes single-stranded (“open”) in the vicinity of the initiation site (defined as +1). This holoenzyme/unwound-DNA structure is called the open complex.
• The RNA polymerase transcribes the DNA (the beta subunit initiates the synthesis), but produces about 10 abortive (short, non-productive) transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the σ-factor.The σ-factor eventually dissociates from the holoenzyme, and elongation proceeds.
Additional Transcription Factors
Promoters can differ in “strength”; that is, how actively they promote transcription of their adjacent DNA sequence. Promoter strength is in many (but not all) cases, a matter of how tightly RNA polymerase and its associated accessory proteins bind to their respective DNA sequences. The more similar the sequences are to a consensus sequence, the stronger the binding is.
Additional transcription regulation comes from transcription factors that can affect the stability of the holoenzyme structure at initiation. Most transcripts originate using adenosine-5′-triphosphate (ATP) and, to a lesser extent, guanosine-5′-triphosphate (GTP) (purine nucleoside triphosphates) at the +1 site. Uridine-5′-triphosphate (UTP) and cytidine-5′-triphosphate (CTP) (pyrimidine nucleoside triphosphates) are disfavoured at the initiation site.
Two termination mechanisms are well known: Intrinsic termination (also called Rho-independent transcription termination) involves terminator sequences within the RNA that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic sequence that forms a stem-loop hairpin structure that leads to the dissociation of the RNAP from the DNA template. Rho-dependent termination uses a termination factor called ρ factor(rho factor) which is a protein to stop RNA synthesis at specific sites. This protein binds at a rho utilisation site on the nascent RNA strand and runs along the mRNA towards the RNAP. A stem loop structure upstream of the terminator region pauses the RNAP, when ρ-factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription.
Key Points
• In prokaryotes genetic material is not enclosed in a membrane-enclosed nucleus and has access to ribosomes in the cytoplasm.
• Transcription is known to be controlled by a variety of regulators in prokaryotes. Many of these transcription factors are homodimers containing helix-turn-helix DNA -binding motifs.
• Additional transcription regulation comes from transcription factors that can affect the stability of the holoenzyme structure at initiation.
Key Terms
• transcription: The synthesis of RNA under the direction of DNA. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.06%3A_Translation-_Protein_Synthesis/7.6C%3A_Prokaryotic_Transcription_and_Translation_Are_Coupled.txt |
Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard.
LEARNING OBJECTIVES
Describe the process and function of posttranslational modification
Key Points
• During protein synthesis, 20 different amino acids can be incorporated to become a protein.
• Posttranslational modification of amino acids change the chemical nature of an amino acid (e.g., citrullination), or make structural changes (e.g., formation of disulfide bridges).
• Non-standard amino acids either are not found in proteins (e.g., carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery.
Key Terms
• Posttranslational modification: the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins.
• translation: A process occurring in the ribosome, in which a strand of messenger RNA (mRNA) guides assembly of a sequence of amino acids to make a protein.
• amino acid: Any organic compound containing both an amino and a carboxylic acid functional group.
Posttranslational modification (PTM) is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins. A protein (also called a polypeptide) is a chain of amino acids. During protein synthesis, 20 different amino acids can be incorporated to become a protein. After translation, the posttranslational modification of amino acids extends the range of functions of the protein by attaching it to other biochemical functional groups (such as acetate, phosphate, various lipids, and carbohydrates), changing the chemical nature of an amino acid (e.g., citrullination), or making structural changes (e.g., formation of disulfide bridges).
Also, enzymes may remove amino acids from the amino end of the protein, or cut the peptide chain in the middle. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds. Also, most nascent polypeptides start with the amino acid methionine because the “start” codon on mRNA also codes for this amino acid. This amino acid is usually taken off during post-translational modification.
Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard. Those either are not found in proteins (e.g., carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery (e.g., hydroxyproline and selenomethionine).
Non-standard amino acids that are found in proteins are formed by post-translational modification, which is modification after translation during protein synthesis. These modifications are often essential for the function or regulation of a protein. For example, the carboxylation of glutamate allows for better binding of calcium cations, and the hydroxylation of proline is critical for maintaining connective tissues.
Another example is the formation of hypusine in the translation initiation factor EIF5A through modification of a lysine residue. Such modifications can also determine the localization of the protein. For instance, the addition of long hydrophobic groups can cause a protein to bind to a phospholipid membrane.
It is important to compare the structures of alanine and beta alanine. In alanine, the side-chain is a methyl group; in beta alanine, the side-chain contains a methylene group connected to an amino group, and the alpha carbon lacks an amino group. The two amino acids, therefore, have the same formulae but different structures.
Some nonstandard amino acids are not found in proteins. Examples include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in the metabolic pathways for standard amino acids. For example, ornithine and citrulline occur in the urea cycle, which is part of amino acid catabolism. A rare exception to the dominance of α-amino acids in biology is the β-amino acid beta alanine (3-aminopropanoic acid), which is used in plants and microorganisms in the synthesis of pantothenic acid (vitamin B5), a component of coenzyme A. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.06%3A_Translation-_Protein_Synthesis/7.6D%3A_The_Incorporation_of_Nonstandard_Amino_Acids.txt |
Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard.
LEARNING OBJECTIVES
Describe the process and function of posttranslational modification
Key Points
• During protein synthesis, 20 different amino acids can be incorporated to become a protein.
• Posttranslational modification of amino acids change the chemical nature of an amino acid (e.g., citrullination), or make structural changes (e.g., formation of disulfide bridges).
• Non-standard amino acids either are not found in proteins (e.g., carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery.
Key Terms
• Posttranslational modification: the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins.
• translation: A process occurring in the ribosome, in which a strand of messenger RNA (mRNA) guides assembly of a sequence of amino acids to make a protein.
• amino acid: Any organic compound containing both an amino and a carboxylic acid functional group.
Posttranslational modification (PTM) is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins. A protein (also called a polypeptide) is a chain of amino acids. During protein synthesis, 20 different amino acids can be incorporated to become a protein. After translation, the posttranslational modification of amino acids extends the range of functions of the protein by attaching it to other biochemical functional groups (such as acetate, phosphate, various lipids, and carbohydrates), changing the chemical nature of an amino acid (e.g., citrullination), or making structural changes (e.g., formation of disulfide bridges).
Also, enzymes may remove amino acids from the amino end of the protein, or cut the peptide chain in the middle. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds. Also, most nascent polypeptides start with the amino acid methionine because the “start” codon on mRNA also codes for this amino acid. This amino acid is usually taken off during post-translational modification.
Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard. Those either are not found in proteins (e.g., carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery (e.g., hydroxyproline and selenomethionine).
Non-standard amino acids that are found in proteins are formed by post-translational modification, which is modification after translation during protein synthesis. These modifications are often essential for the function or regulation of a protein. For example, the carboxylation of glutamate allows for better binding of calcium cations, and the hydroxylation of proline is critical for maintaining connective tissues.
Another example is the formation of hypusine in the translation initiation factor EIF5A through modification of a lysine residue. Such modifications can also determine the localization of the protein. For instance, the addition of long hydrophobic groups can cause a protein to bind to a phospholipid membrane.
It is important to compare the structures of alanine and beta alanine. In alanine, the side-chain is a methyl group; in beta alanine, the side-chain contains a methylene group connected to an amino group, and the alpha carbon lacks an amino group. The two amino acids, therefore, have the same formulae but different structures.
Some nonstandard amino acids are not found in proteins. Examples include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in the metabolic pathways for standard amino acids. For example, ornithine and citrulline occur in the urea cycle, which is part of amino acid catabolism. A rare exception to the dominance of α-amino acids in biology is the β-amino acid beta alanine (3-aminopropanoic acid), which is used in plants and microorganisms in the synthesis of pantothenic acid (vitamin B5), a component of coenzyme A.
LICENSES AND ATTRIBUTIONS
CC LICENSED CONTENT, SPECIFIC ATTRIBUTION | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.06%3A_Translation-_Protein_Synthesis/7.6E%3A_Unsticking_Stuck_Ribosomes.txt |
Eukaryotic pre-mRNA receives a 5′ cap and a 3′ poly (A) tail before introns are removed and the mRNA is considered ready for translation.
LEARNING OBJECTIVES
Outline the steps of pre-mRNA processing
Key Points
• A 7-methylguanosine cap is added to the 5′ end of the pre-mRNA while elongation is still in progress. The 5′ cap protects the nascent mRNA from degradation and assists in ribosome binding during translation.
• A poly (A) tail is added to the 3′ end of the pre-mRNA once elongation is complete. The poly (A) tail protects the mRNA from degradation, aids in the export of the mature mRNA to the cytoplasm, and is involved in binding proteins involved in initiating translation.
• Introns are removed from the pre-mRNA before the mRNA is exported to the cytoplasm.
Key Terms
• intron: a portion of a split gene that is included in pre-RNA transcripts but is removed during RNA processing and rapidly degraded
• moiety: a specific segment of a molecule
• spliceosome: a dynamic complex of RNA and protein subunits that removes introns from precursor mRNA
Pre-mRNA Processing
The eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated. The additional steps involved in eukaryotic mRNA maturation create a molecule with a much longer half-life than a prokaryotic mRNA. Eukaryotic mRNAs last for several hours, whereas the typical E. coli mRNA lasts no more than five seconds.
Pre-mRNAs are first coated in RNA-stabilizing proteins; these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus. The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5′ and 3′ ends of the molecule, and the removal of intervening sequences that do not specify the appropriate amino acids. In rare cases, the mRNA transcript can be “edited” after it is transcribed.
5′ Capping
While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is added to the 5′ end of the growing transcript by a 5′-to-5′ phosphate linkage. This moiety protects the nascent mRNA from degradation. In addition, initiation factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.
3′ Poly-A Tail
While RNA Polymerase II is still transcribing downstream of the proper end of a gene, the pre-mRNA is cleaved by an endonuclease-containing protein complex between an AAUAAA consensus sequence and a GU-rich sequence. This releases the functional pre-mRNA from the rest of the transcript, which is still attached to the RNA Polymerase. An enzyme called poly (A) polymerase (PAP) is part of the same protein complex that cleaves the pre-mRNA and it immediately adds a string of approximately 200 A nucleotides, called the poly (A) tail, to the 3′ end of the just-cleaved pre-mRNA. The poly (A) tail protects the mRNA from degradation, aids in the export of the mature mRNA to the cytoplasm, and is involved in binding proteins involved in initiating translation.
Poly (A) Polymerase adds a 3′ poly (A) tail to the pre-mRNA.: The pre-mRNA is cleaved off the rest of the growing transcript before RNA Polymerase II has stopped transcribing. This cleavage is done by an endonuclease-containing protein complex that binds to an AAUAAA sequence upstream of the cleavage site and to a GU-rich sequence downstream of the cut site. Immediately after the cleavage, Poly (A) Polymerase (PAP), which is also part of the protein complex, catalyzes the addition of up to 200 A nucleotides to the 3′ end of the just-cleaved pre-mRNA.
Pre-mRNA Splicing
Eukaryotic genes are composed of exons, which correspond to protein-coding sequences (ex-on signifies that they are expressed), and intervening sequences called introns (int-ron denotes their intervening role), which may be involved in gene regulation, but are removed from the pre-mRNA during processing. Intron sequences in mRNA do not encode functional proteins.
Discovery of Introns
The discovery of introns came as a surprise to researchers in the 1970s who expected that pre-mRNAs would specify protein sequences without further processing, as they had observed in prokaryotes. The genes of higher eukaryotes very often contain one or more introns. While these regions may correspond to regulatory sequences, the biological significance of having many introns or having very long introns in a gene is unclear. It is possible that introns slow down gene expression because it takes longer to transcribe pre-mRNAs with lots of introns. Alternatively, introns may be nonfunctional sequence remnants left over from the fusion of ancient genes throughout evolution. This is supported by the fact that separate exons often encode separate protein subunits or domains. For the most part, the sequences of introns can be mutated without ultimately affecting the protein product.
Intron Processing
All introns in a pre-mRNA must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional. The process of removing introns and reconnecting exons is called splicing. Introns are removed and degraded while the pre-mRNA is still in the nucleus. Splicing occurs by a sequence-specific mechanism that ensures introns will be removed and exons rejoined with the accuracy and precision of a single nucleotide. The splicing of pre-mRNAs is conducted by complexes of proteins and RNA molecules called spliceosomes.
Each spliceosome is composed of five subunits called snRNPs (for small nuclear ribonucleoparticles, and pronounced “snurps”.) Each snRNP is itself a complex of proteins and a special type of RNA found only in the nucleus called snRNAs (small nuclear RNAs). Spliceosomes recognize sequences at the 5′ end of the intron because introns always start with the nucleotides GU and they recognize sequences at the 3′ end of the intron because they always end with the nucleotides AG. The spliceosome cleaves the pre-mRNA’s sugar phosphate backbone at the G that starts the intron and then covalently attaches that G to an internal A nucleotide within the intron. Then the spliceosme connects the 3′ end of the first exon to the 5′ end of the following exon, cleaving the 3′ end of the intron in the process. This results in the splicing together of the two exons and the release of the intron in a lariat form. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.07%3A_Protein_Modification_Folding_Secretion_and_Degradation/7.7A%3A_mRNA_Processing.txt |
Denaturation is a process in which proteins lose their shape and, therefore, their function because of changes in pH or temperature.
LEARNING OBJECTIVES
Discuss the process of protein denaturation
Key Points
• Proteins change their shape when exposed to different pH or temperatures.
• The body strictly regulates pH and temperature to prevent proteins such as enzymes from denaturing.
• Some proteins can refold after denaturation while others cannot.
• Chaperone proteins help some proteins fold into the correct shape.
Key Terms
• chaperonin: proteins that provide favorable conditions for the correct folding of other proteins, thus preventing aggregation
• denaturation: the change of folding structure of a protein (and thus of physical properties) caused by heating, changes in pH, or exposure to certain chemicals
Each protein has its own unique sequence of amino acids and the interactions between these amino acids create a specify shape. This shape determines the protein’s function, from digesting protein in the stomach to carrying oxygen in the blood.
Changing the Shape of a Protein
If the protein is subject to changes in temperature, pH, or exposure to chemicals, the internal interactions between the protein’s amino acids can be altered, which in turn may alter the shape of the protein. Although the amino acid sequence (also known as the protein’s primary structure) does not change, the protein’s shape may change so much that it becomes dysfunctional, in which case the protein is considered denatured. Pepsin, the enzyme that breaks down protein in the stomach, only operates at a very low pH. At higher pHs pepsin’s conformation, the way its polypeptide chain is folded up in three dimensions, begins to change. The stomach maintains a very low pH to ensure that pepsin continues to digest protein and does not denature.
Enzymes
Because almost all biochemical reactions require enzymes, and because almost all enzymes only work optimally within relatively narrow temperature and pH ranges, many homeostatic mechanisms regulate appropriate temperatures and pH so that the enzymes can maintain the shape of their active site.
Reversing Denaturation
It is often possible to reverse denaturation because the primary structure of the polypeptide, the covalent bonds holding the amino acids in their correct sequence, is intact. Once the denaturing agent is removed, the original interactions between amino acids return the protein to its original conformation and it can resume its function.
However, denaturation can be irreversible in extreme situations, like frying an egg. The heat from a pan denatures the albumin protein in the liquid egg white and it becomes insoluble. The protein in meat also denatures and becomes firm when cooked.
Chaperone proteins (or chaperonins ) are helper proteins that provide favorable conditions for protein folding to take place. The chaperonins clump around the forming protein and prevent other polypeptide chains from aggregating. Once the target protein folds, the chaperonins disassociate.
7.7C: Protein Folding Modification and Targeting
In order to function, proteins must fold into the correct three-dimensional shape, and be targeted to the correct part of the cell.
LEARNING OBJECTIVES
Discuss how post-translational events affect the proper function of a protein
Key Points
• Protein folding is a process in which a linear chain of amino acids attains a defined three-dimensional structure, but there is a possibility of forming misfolded or denatured proteins, which are often inactive.
• Proteins must also be located in the correct part of the cell in order to function correctly; therefore, a signal sequence is often attached to direct the protein to its proper location, which is removed after it attains its location.
• Protein misfolding is the cause of numerous diseases, such as mad cow disease, Creutzfeldt-Jakob disease, and cystic fibrosis.
Key Terms
• prion: a self-propagating misfolded conformer of a protein that is responsible for a number of diseases that affect the brain and other neural tissue
• chaperone: a protein that assists the non-covalent folding/unfolding of other proteins
Protein Folding
After being translated from mRNA, all proteins start out on a ribosome as a linear sequence of amino acids. This linear sequence must “fold” during and after the synthesis so that the protein can acquire what is known as its native conformation. The native conformation of a protein is a stable three-dimensional structure that strongly determines a protein’s biological function. When a protein loses its biological function as a result of a loss of three-dimensional structure, we say that the protein has undergone denaturation. Proteins can be denatured not only by heat, but also by extremes of pH; these two conditions affect the weak interactions and the hydrogen bonds that are responsible for a protein’s three-dimensional structure. Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape if abnormal temperature or pH conditions prevent it from folding correctly. The denatured state of the protein does not equate with the unfolding of the protein and randomization of conformation. Actually, denatured proteins exist in a set of partially-folded states that are currently poorly understood. Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding.
Protein Modification and Targeting
During and after translation, individual amino acids may be chemically modified and signal sequences may be appended to the protein. A signal sequence is a short tail of amino acids that directs a protein to a specific cellular compartment. These sequences at the amino end or the carboxyl end of the protein can be thought of as the protein’s “train ticket” to its ultimate destination. Other cellular factors recognize each signal sequence and help transport the protein from the cytoplasm to its correct compartment. For instance, a specific sequence at the amino terminus will direct a protein to the mitochondria or chloroplasts (in plants). Once the protein reaches its cellular destination, the signal sequence is usually clipped off.
Misfolding
It is very important for proteins to achieve their native conformation since failure to do so may lead to serious problems in the accomplishment of its biological function. Defects in protein folding may be the molecular cause of a range of human genetic disorders. For example, cystic fibrosis is caused by defects in a membrane-bound protein called cystic fibrosis transmembrane conductance regulator (CFTR). This protein serves as a channel for chloride ions. The most common cystic fibrosis-causing mutation is the deletion of a Phe residue at position 508 in CFTR, which causes improper folding of the protein. Many of the disease-related mutations in collagen also cause defective folding.
A misfolded protein, known as prion, appears to be the agent of a number of rare degenerative brain diseases in mammals, like the mad cow disease. Related diseases include kuru and Creutzfeldt-Jakob. The diseases are sometimes referred to as spongiform encephalopathies, so named because the brain becomes riddled with holes. Prion, the misfolded protein, is a normal constituent of brain tissue in all mammals, but its function is not yet known. Prions cannot reproduce independently and not considered living microoganisms. A complete understanding of prion diseases awaits new information about how prion protein affects brain function, as well as more detailed structural information about the protein. Therefore, improved understanding of protein folding may lead to new therapies for cystic fibrosis, Creutzfeldt-Jakob, and many other diseases. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.07%3A_Protein_Modification_Folding_Secretion_and_Degradation/7.7B%3A_Denaturation_and_Protein_Folding.txt |
A cell can rapidly change the levels of proteins in response to the environment by adding specific chemical groups to alter gene regulation.
LEARNING OBJECTIVES
Explain how chemical modifications affect protein activity and longevity
Key Points
• Proteins can be chemically modified by adding methyl, phosphate, acetyl, and ubiquitin groups.
• Protein longevity can be affected by altering stages of gene regulation, including but not limited to altering: accessibility to chromosomal DNA for transcription, rate of translation, nuclear shuttling, RNA stability, and post-translational modifications.
• Ubiquitin is added to a protein to mark it for degradation by the proteasome.
Key Terms
• ubiquitin: a small polypeptide present in the cells of all eukaryotes; it plays a part in modifying and degrading proteins
• proteasome: a complex protein, found in bacterial, archaeal and eukaryotic cells, that breaks down other proteins via proteolysis
Chemical Modifications, Protein Activity, and Longevity
Proteins can be chemically modified with the addition of methyl, phosphate, acetyl, and ubiquitin groups. The addition or removal of these groups from proteins regulates their activity or the length of time they exist in the cell. Sometimes these modifications can regulate where a protein is found in the cell; for example, in the nucleus, the cytoplasm, or attached to the plasma membrane.
Chemical modifications occur in response to external stimuli such as stress, the lack of nutrients, heat, or ultraviolet light exposure. These changes can alter protein function, epigenetic accessibility, transcription, mRNA stability, or translation; all resulting in changes in expression of various genes. This is an efficient way for the cell to rapidly change the abundance levels of specific proteins in response to the environment. Because proteins are involved in every stage of gene regulation, the phosphorylation of a protein (depending on the protein that is modified) can alter accessibility to the chromosome, can alter translation (by altering transcription factor binding or function), can change nuclear shuttling (by influencing modifications to the nuclear pore complex), can alter RNA stability (by binding or not binding to the RNA to regulate its stability), can modify translation (increase or decrease), or can change post-translational modifications (add or remove phosphates or other chemical modifications). All of these protein activities are affected by the phosphorylation process. The enzymes which are responsible for phosphorylation are known as protein kinases. The addition of a phosphate group to a protein can result in either activation or deactivation; it is protein dependent.
Another example of chemical modifications affecting protein activity include the addition or removal of methyl groups. Methyl groups are added to proteins via the process of methylation; this is the most common form of post-translational modification. The addition of methyl groups to a protein can result in protein-protein interactions that allows for transcriptional regulation, response to stress, protein repair, nuclear transport, and even differentiation processes. Methylation on side chain nitrogens is considered largely irreversible while methylation of the carboxyl groups is potentially reversible. Methylation in the proteins negates the negative charge on it and increases the hydrophobicity of the protein. Methylation on carboxylate side chains covers up a negative charge and adds hydrophobicity. The addition of this chemical group changes the property of the protein and, thus, affects it activity.
The addition of an ubiquitin group to a protein marks that protein for degradation. Ubiquitin acts like a flag indicating that the protein lifespan is complete. These proteins are moved to the proteasome, an organelle that functions to remove proteins to be degraded. One way to control gene expression is to alter the longevity of the protein: ubiquitination shortens a protein’s lifespan.
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Archaea usually have a single circular chromosome.
LEARNING OBJECTIVES
Describe the characteristics of Archaeal chromosomes and their replication
Key Points
• Archaea are genetically distinct from bacteria and eukaryotes, with up to 15% of the proteins encoded by any one archaeal genome being unique to the domain, although most of these unique genes have no known function.
• DNA replication in archaea requires a specific primase that shares similarities to the RNA recognition motif (RRM) and to viral RNA dependent RNA polymerases.
• The circular chromosomes of archaea contain multiple origins of replication for initiation of DNA synthesis.
Key Terms
• chromosome: A structure in the cell nucleus that contains DNA, histone protein, and other structural proteins.
• eukaryote: Any of the single-celled or multicellular organisms, of the taxonomic domain Eukaryota, whose cells contain at least one distinct nucleus.
• bacteria: A type, species, or strain of bacterium.
Archaeal Genetics
Archaea usually have a single circular chromosome, the size of which may be as great as 5,751,492 base pairs in Methanosarcina acetivorans, which boasts the largest known archaean genome. One-tenth of this size is the tiny 490,885 base-pair genome of Nanoarchaeum equitans, which possesses the smallest archaean genome known; it is estimated to contain only 537 protein-encoding genes. Smaller independent pieces of DNA, called plasmids, are also found in archaea. Plasmids may be transferred between cells by physical contact, in a process that may be similar to bacterial conjugation.
Asexual Reproduction
The means of asexual reproduction that are used by Archaea include binary reproduction, multiple fission, fragmentation, or budding. The cell division process is controlled by the cell cycle; the chromosomes within the Archaea are replicated to produce two daughter chromosomes. Archaea typically have a single circular chromosome. The two daughter chromosomes are then separated and the cell divides. This process in Archaea appears to be similar to both bacterial and eukaryotic systems. The circular chromosomes contain multiple origins of replication, using DNA polymerases that resemble eukaryotic enzymes. However, the proteins involved that direct cell division are similar to those of bacterial systems.
DNA replication, similar in all systems, involves initiation, elongation, and termination. The replication of DNA, beginning at the origins of replication present on the circular chromosomes, requires initiator proteins. The recruitment of additional proteins by way of the initiator proteins allows the separation of the circular DNA and results in the formation of a bubble.
The DNA replication system in Archaea, similar to all systems, requires a free 3’OH group before synthesis is initiated. The primase used to synthesize a short RNA primer from the free 3’OH group varies in Archaea when compared to that of bacterial and eukaryotic systems. The primase used by archaea is a highly derived version of the RNA recognition motif (RRM). It is structurally similar to viral RNA dependent RNA polymerases, reverse transcriptases, cyclic nucleotide generating cyclases, and DNA polymerases involved in DNA replication and repair. Once the RNA primase has performed its job, DNA synthesis continues in a similar fashion by which the eukaryotic system and the DNA is replicated.
7.8B: Shared Features of Bacteria and Archaea
Most of the metabolic pathways, which comprise the majority of an organism’s genes, are common between Archaea and Bacteria.
LEARNING OBJECTIVES
Describe the evidence for the evolution of the Archaea from Bacteria
Key Points
• Within prokaryotes, archaeal cell structure is most similar to that of Gram-positive bacteria, largely because both have a single lipid bilayer and usually contain a thick sacculus of varying chemical composition.
• It has been proposed that the Archaea evolved from Gram-positive bacteria in response to antibiotic selection pressure.
• The evolution of Archaea in response to antibiotic selection, or any other competitive selective pressure, could also explain their adaptation to extreme environments (such as high temperature or acidity).
Key Terms
• prokaryotes: ( /proʊkæri.oʊts/, pro-kah-ree-otes or /proʊkæriəts/, pro-kah-ree-əts) a group of organisms whose cells lack a cell nucleus (karyon), or any other membrane-bound organelles. Most prokaryotes are unicellular organisms, although a few such as myxobacteria have multicellular stages in their life cycles.
• archaea: a taxonomic domain of single-celled organisms lacking nuclei that are fundamentally from bacteria.
• bacteria: Bacteria constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals. Bacteria were among the first life forms to appear on Earth, and are present in most habitats on the planet.
The relationship between the three domains is of central importance for understanding the origin of life. Most of the metabolic pathways, which comprise the majority of an organism ‘s genes, are common between Archaea and Bacteria, while most genes involved in genome expression are common between Archaea and Eukarya. Within prokaryotes, archaeal cell structure is most similar to that of Gram-positive bacteria, largely because both have a single lipid bilayer and usually contain a thick sacculus of varying chemical composition. In phylogenetic trees based upon different gene/ protein sequences of prokaryotic homologs, the archaeal homologs are more closely related to those of Gram-positive bacteria. Archaea and Gram-positive bacteria also share conserved indels in a number of important proteins, such as Hsp70 and glutamine synthetase I.
R.S. Gupta has proposed that the Archaea evolved from Gram-positive bacteria in response to antibiotic selection pressure. This is suggested by the observation that archaea are resistant to a wide variety of antibiotics that are primarily produced by Gram-positive bacteria, and that these antibiotics primarily act on the genes that distinguish Archaea from Bacteria. His proposal is that the selective pressure towards resistance generated by the Gram-positive antibiotics was eventually sufficient to cause extensive changes in many of the antibiotics’ target genes, and that these strains represented the common ancestors of present-day Archaea. The evolution of Archaea in response to antibiotic selection, or any other competitive selective pressure, could also explain their adaptation to extreme environments (such as high temperature or acidity) as the result of a search for unoccupied niches to escape from antibiotic-producing organisms; Cavalier-Smith has made a similar suggestion. Gupta’s proposal is also supported by other work investigating protein structural relationships and studies that suggest that Gram-positive bacteria may constitute the earliest branching lineages within the prokaryotes. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.08%3A_Archaeal_Genetics/7.8A%3A_Chromosomes_and_DNA_Replication_in_the_Archaea.txt |
Archaea possess genes and several metabolic pathways that are more closely related to those of eukaryotes than prokaryotes.
LEARNING OBJECTIVES
Describe the similarities between archaea, eukaryotes, and bacteria
Key Points
• Archaea exhibit a great variety of chemical reactions in their metabolism and use many sources of energy.
• The energy released generates adenosine triphosphate (ATP) through chemiosmosis, in the same basic process that happens in the mitochondrion of eukaryotic cells.
• The chromosomes replicate from multiple starting-points (origins of replication) using DNA polymerases that resemble the equivalent eukaryotic enzymes.
Key Terms
• eukaryote: Any of the single-celled or multicellular organisms, of the taxonomic domain Eukaryota, whose cells contain at least one distinct nucleus.
• mitochondrion: a spherical or ovoid organelle found in the cytoplasm of eukaryotic cells, contains genetic material separate from that of the host; it is responsible for the conversion of food to usable energy in the form of ATP
• archaea: a taxonomic domain of single-celled organisms lacking nuclei that are fundamentally from bacteria.
• metabolism: The complete set of chemical reactions that occur in living cells.
The evolutionary relationship between archaea and eukaryotes remains unclear. Aside from the similarities in cell structure and function that are discussed below, many genetic trees group the two.
Complicating factors include claims that the relationship between eukaryotes and the archaeal phylum Crenarchaeota is closer than the relationship between the Euryarchaeotaand the phylum Crenarchaeota, and the presence of archaean-like genes in certain bacteria, such as Thermotoga maritima, from horizontal gene transfer. The leading hypothesis is that the ancestor of the eukaryotes diverged early from the Archaea, and that eukaryotes arose through fusion of an archaean and eubacterium, which became the nucleus and cytoplasm. This explains various genetic similarities but runs into difficulties when it comes to explaining cell structure.
Despite this visual similarity to bacteria, archaea possess genes and several metabolic pathways that are more closely-related to those of eukaryotes, notably the enzymes involved in transcription and translation.
Archaea exhibit a great variety of chemical reactions in their metabolism and use many sources of energy. These reactions are classified into nutritional groups, depending on energy and carbon sources. Some archaea obtain energy from inorganic compounds such as sulfur or ammonia (they are lithotrophs). These include nitrifiers, methanogens and anaerobic methane oxidisers. In these reactions, one compound passes electrons to another (in a redox reaction), releasing energy to fuel the cell’s activities. One compound acts as an electron donor and another as an electron acceptor. The energy released generates adenosine triphosphate (ATP) through chemiosmosis, in the same basic process that happens in the mitochondrion of eukaryotic cells.
The chromosomes replicate from multiple starting-points (origins of replication) using DNA polymerases that resemble the equivalent eukaryotic enzymes. However, the proteins that direct cell division, such as the protein FtsZ, which forms a contracting ring around the cell, and the components of the septum that is constructed across the center of the cell, are similar to their bacterial equivalents.
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The cell cycle allows multiicellular organisms to grow and divide and single-celled organisms to reproduce.
LEARNING OBJECTIVES
Explain the role of the cell cycle in carrying out the cell’s essential functions
Key Points
• All multicellular organisms use cell division for growth and the maintenance and repair of cells and tissues.
• Single-celled organisms use cell division as their method of reproduction.
• Somatic cells divide regularly; all human cells (except for the cells that produce eggs and sperm) are somatic cells.
• Somatic cells contain two copies of each of their chromosomes (one copy from each parent).
• The cell cycle has two major phases: interphase and the mitotic phase.
• During interphase, the cell grows and DNA is replicated; during the mitotic phase, the replicated DNA and cytoplasmic contents are separated and the cell divides.
Key Terms
• somatic cell: any normal body cell of an organism that is not involved in reproduction; a cell that is not on the germline
• interphase: the stage in the life cycle of a cell where the cell grows and DNA is replicated
• mitotic phase: replicated DNA and the cytoplasmic material are divided into two identical cells
Introduction: Cell Division and Reproduction
A human, as well as every sexually-reproducing organism, begins life as a fertilized egg or zygote. Trillions of cell divisions subsequently occur in a controlled manner to produce a complex, multicellular human. In other words, that original single cell is the ancestor of every other cell in the body. Once a being is fully grown, cell reproduction is still necessary to repair or regenerate tissues. For example, new blood and skin cells are constantly being produced. All multicellular organisms use cell division for growth and the maintenance and repair of cells and tissues. Cell division is tightly regulated because the occasional failure of regulation can have life-threatening consequences. Single-celled organisms use cell division as their method of reproduction.
While there are a few cells in the body that do not undergo cell division, most somatic cells divide regularly. A somatic cell is a general term for a body cell: all human cells, except for the cells that produce eggs and sperm (which are referred to as germ cells), are somatic cells. Somatic cells contain two copies of each of their chromosomes (one copy received from each parent). Cells in the body replace themselves over the lifetime of a person. For example, the cells lining the gastrointestinal tract must be frequently replaced when constantly “worn off” by the movement of food through the gut. But what triggers a cell to divide and how does it prepare for and complete cell division?
The cell cycle is an ordered series of events involving cell growth and cell division that produces two new daughter cells. Cells on the path to cell division proceed through a series of precisely timed and carefully regulated stages of growth, DNA replication, and division that produces two identical (clone) cells. The cell cycle has two major phases: interphase and the mitotic phase. During interphase, the cell grows and DNA is replicated. During the mitotic phase, the replicated DNA and cytoplasmic contents are separated and the cell divides. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.09%3A_Eukaryotic_Genetics/7.9A%3A_The_Role_of_the_Cell_Cycle.txt |
Proteins, encoded by individual genes, orchestrate nearly every function of the cell.
LEARNING OBJECTIVES
Describe transcription and translation
Key Points
• Genes are composed of DNA arranged on chromosomes.
• Some genes encode structural or regulatory RNAs. Other genes encode proteins.
• Replication copies DNA; transcription uses DNA to make complementary RNAs; translation uses mRNAs to make proteins.
• In eukaryotic cells, replication and transcription take place within the nucleus while translation takes place in the cytoplasm.
• In prokaryotic cells, replication, transcription, and translation occur in the cytoplasm.
Key Terms
• DNA: a biopolymer of deoxyribonucleic acids (a type of nucleic acid) that has four different chemical groups, called bases: adenine, guanine, cytosine, and thymine
• messenger RNA: Messenger RNA (mRNA) is a molecule of RNA that encodes a chemical “blueprint” for a protein product.
• protein: any of numerous large, complex naturally-produced molecules composed of one or more long chains of amino acids, in which the amino acid groups are held together by peptide bonds
Genes and Proteins
Since the rediscovery of Mendel’s work in 1900, the definition of the gene has progressed from an abstract unit of heredity to a tangible molecular entity capable of replication, transcription, translation, and mutation. Genes are composed of DNA and are linearly arranged on chromosomes. Some genes encode structural and regulatory RNAs. There is increasing evidence from research that profiles the transcriptome of cells (the complete set all RNA transcripts present in a cell) that these may be the largest classes of RNAs produced by eukaryotic cells, far outnumbering the protein-encoding messenger RNAs (mRNAs), but the 20,000 protein-encoding genes typically found in animal cells, and the 30,o00 protein-encoding genes typically found in plant cells, nonetheless have huge impacts on cellular functioning.
Protein-encoding genes specify the sequences of amino acids, which are the building blocks of proteins. In turn, proteins are responsible for orchestrating nearly every function of the cell. Both protein-encoding genes and the proteins that are their gene products are absolutely essential to life as we know it.
Replication, Transcription, and Translation are the three main processes used by all cells to maintain their genetic information and to convert the genetic information encoded in DNA into gene products, which are either RNAs or proteins, depending on the gene. In eukaryotic cells, or those cells that have a nucleus, replication and transcription take place within the nucleus while translation takes place outside of the nucleus in cytoplasm. In prokaryotic cells, or those cells that do not have a nucleus, all three processes occur in the cytoplasm.
Replication is the basis for biological inheritance. It copies a cell’s DNA. The enzyme DNA polymerase copies a single parental double-stranded DNA molecule into two daughter double-stranded DNA molecules. Transcription makes RNA from DNA. The enzyme RNA polymerase creates an RNA molecule that is complementary to a gene-encoding stretch of DNA. Translation makes protein from mRNA. The ribosome generates a polypeptide chain of amino acids using mRNA as a template. The polypeptide chain folds up to become a protein.
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Most mistakes during replication are corrected by DNA polymerase during replication or by post-replication repair mechanisms.
LEARNING OBJECTIVES
Explain how errors during replication are repaired
Key Points
• Mismatch repair enzymes recognize mis-incorporated bases, remove them from DNA, and replace them with the correct bases.
• In nucleotide excision repair, enzymes remove incorrect bases with a few surrounding bases, which are replaced with the correct bases with the help of a DNA polymerase and the template DNA.
• When replication mistakes are not corrected, they may result in mutations, which sometimes can have serious consequences.
• Point mutations, one base substituted for another, can be silent (no effect) or may have effects ranging from mild to severe.
• Mutations may also involve insertions (addition of a base), deletion (loss of a base), or translocation (movement of a DNA section to a new location on the same or another chromosome ).
Key Terms
• mismatch repair: a system for recognizing and repairing some forms of DNA damage and erroneous insertion, deletion, or mis-incorporation of bases that can arise during DNA replication and recombination
• nucleotide excision repair: a DNA repair mechanism that corrects damage done by UV radiation, including thymine dimers and 6,4 photoproducts that cause bulky distortions in the DNA
Errors during Replication
DNA replication is a highly accurate process, but mistakes can occasionally occur as when a DNA polymerase inserts a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms can correct the mistakes, but in rare cases mistakes are not corrected, leading to mutations; in other cases, repair enzymes are themselves mutated or defective.
Mutations: In this interactive, you can “edit” a DNA strand and cause a mutation. Take a look at the effects!
Most of the mistakes during DNA replication are promptly corrected by DNA polymerase which proofreads the base that has just been added. In proofreading, the DNA pol reads the newly-added base before adding the next one so a correction can be made. The polymerase checks whether the newly-added base has paired correctly with the base in the template strand. If it is the correct base, the next nucleotide is added. If an incorrect base has been added, the enzyme makes a cut at the phosphodiester bond and releases the incorrect nucleotide. This is performed by the exonuclease action of DNA pol III. Once the incorrect nucleotide has been removed, a new one will be added again.
Some errors are not corrected during replication, but are instead corrected after replication is completed; this type of repair is known as mismatch repair. The enzymes recognize the incorrectly-added nucleotide and excise it; this is then replaced by the correct base. If this remains uncorrected, it may lead to more permanent damage. How do mismatch repair enzymes recognize which of the two bases is the incorrect one? In E. coli, after replication, the nitrogenous base adenine acquires a methyl group; the parental DNA strand will have methyl groups, whereas the newly-synthesized strand lacks them. Thus, DNA polymerase is able to remove the incorrectly-incorporated bases from the newly-synthesized, non-methylated strand. In eukaryotes, the mechanism is not very well understood, but it is believed to involve recognition of unsealed nicks in the new strand, as well as a short-term continuing association of some of the replication proteins with the new daughter strand after replication has been completed.
In another type of repair mechanism, nucleotide excision repair, enzymes replace incorrect bases by making a cut on both the 3′ and 5′ ends of the incorrect base. The segment of DNA is removed and replaced with the correctly-paired nucleotides by the action of DNA pol. Once the bases are filled in, the remaining gap is sealed with a phosphodiester linkage catalyzed by DNA ligase. This repair mechanism is often employed when UV exposure causes the formation of pyrimidine dimers.
DNA Damage and Mutations
Errors during DNA replication are not the only reason why mutations arise in DNA. Mutations, variations in the nucleotide sequence of a genome, can also occur because of damage to DNA. Such mutations may be of two types: induced or spontaneous. Induced mutations are those that result from an exposure to chemicals, UV rays, X-rays, or some other environmental agent. Spontaneous mutations occur without any exposure to any environmental agent; they are a result of natural reactions taking place within the body.
Mutations may have a wide range of effects. Some mutations are not expressed; these are known as silent mutations. Point mutations are those mutations that affect a single base pair. The most common nucleotide mutations are substitutions, in which one base is replaced by another. These can be of two types: transitions or transversions. Transition substitution refers to a purine or pyrimidine being replaced by a base of the same kind; for example, a purine such as adenine may be replaced by the purine guanine. Transversion substitution refers to a purine being replaced by a pyrimidine or vice versa; for example, cytosine, a pyrimidine, is replaced by adenine, a purine. Mutations can also be the result of the addition of a base, known as an insertion, or the removal of a base, known as a deletion. Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome.
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In homologous recombination, a type of genetic recombination, nucleotide sequences are exchanged between two similar molecules of DNA.
LEARNING OBJECTIVES
Explain the process of homologous recombination in bacteria
Key Points
• Homologous recombination can vary among different organisms and cell types, but most forms involve the same basic steps.
• Homologous recombination is a major DNA repair process in bacteria. It is also important for producing genetic diversity in bacterial populations.
• Homologous recombination has been most studied and is best understood for Escherichia coli.
Key Terms
• recombination: The formation of genetic combinations in offspring that are not present in the parents
• genetic: Relating to genetics or genes.
• homologous: Showing a degree of correspondence or similarity.
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.
Homologous recombination can vary among different organisms and cell types, but most forms involve the same basic steps. After a double-strand break occurs, sections of DNA around the 5′ ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3′ end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. After strand invasion, one or two cross-shaped structures called Holliday junctions connect the two DNA molecules. Depending on how the two junctions are cut by enzymes, the type of homologous recombination that occurs in meiosis results in either chromosomal crossover or non-crossover. Homologous recombination that occurs during DNA repair tends to result in non-crossover products, in effect restoring the damaged DNA molecule as it existed before the double-strand break.
Homologous recombination is conserved across all three domains of life as well as viruses. Homologous recombination is also used in gene targeting, a technique for introducing genetic changes into target organisms.
Homologous recombination is a major DNA repair process in bacteria. It is also important for producing genetic diversity in bacterial populations. Homologous recombination has been most studied and is best understood for Escherichia coli. Double-strand DNA breaks in bacteria are repaired by the RecBCD pathway of homologous recombination. Breaks that occur on one of the two DNA strands, known as single-strand gaps, are thought to be repaired by the RecF pathway. Both the RecBCD and RecF pathways include a series of reactions known as branch migration, in which single DNA strands are exchanged between two intercrossed molecules of duplex DNA, and resolution, in which those two intercrossed molecules of DNA are cut apart and restored to their normal double-stranded state.
The RecBCD pathway is the main recombination pathway used in bacteria to repair double-strand breaks in DNA. These double-strand breaks can be caused by UV light and other radiation, as well as chemical mutagens. Double-strand breaks may also arise by DNA replication through a single-strand nick or gap. Such a situation causes what is known as a collapsed replication fork and is fixed by several pathways of homologous recombination including the RecBCD pathway.
In this pathway, a three-subunit enzyme complex called RecBCD initiates recombination by binding to a blunt or nearly blunt end of a break in double-strand DNA. After RecBCD binds the DNA end, the RecB and RecD subunits begin unzipping the DNA duplex through helicase activity. The RecB subunit also has a nuclease domain, which cuts the single strand of DNA that emerges from the unzipping process. This unzipping continues until RecBCD encounters a specific nucleotide sequence (5′-GCTGGTGG-3′) known as a Chi site.
Upon encountering a Chi site, the activity of the RecBCD enzyme changes drastically. DNA unwinding pauses for a few seconds and then resumes at roughly half the initial speed. This is likely because the slower RecB helicase unwinds the DNA after Chi, rather than the faster RecD helicase, which unwinds the DNA before Chi. Recognition of the Chi site also changes the RecBCD enzyme so that it cuts the DNA strand with Chi and begins loading multiple RecA proteins onto the single-stranded DNA with the newly generated 3′ end. The resulting RecA-coated nucleoprotein filament then searches out similar sequences of DNA on a homologous chromosome. The search process induces stretching of the DNA duplex, which enhances homology recognition (a mechanism termed conformational proofreading). Upon finding such a sequence, the single-stranded nucleoprotein filament moves into the homologous recipient DNA duplex in a process called strand invasion. The invading 3′ overhang causes one of the strands of the recipient DNA duplex to be displaced, to form a D-loop. If the D-loop is cut, another swapping of strands forms a cross-shaped structure called a Holliday junction.Resolution of the Holliday junction by some combination of RuvABC or RecG can produce two recombinant DNA molecules with reciprocal genetic types, if the two interacting DNA molecules differ genetically. Alternatively, the invading 3′ end near Chi can prime DNA synthesis and form a replication fork. This type of resolution produces only one type of recombinant (non-reciprocal). | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.11%3A_Genetic_Transfer_in_Prokaryotes/7.11A%3A_Generalized_Recombination_and_RecA.txt |
Transformation is the direct uptake, incorporation and expression of exogenous genetic material from its surroundings.
LEARNING OBJECTIVES
Differentiate between natural and artificial transformation
Key Points
• Transformation results in the genetic alteration of the recipient cell.
• Exogenous DNA is taken up into the recipient cell from its surroundings through the cell membrane (s).
• Transformation occurs naturally in some species of bacteria, but it can also be affected by artificial means in other cells.
Key Terms
• eukaryotic: Having complex cells in which the genetic material is organized into membrane-bound nuclei.
• transformation: In molecular biology transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
• expression: Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product.
• exogenous: Produced or originating outside of an organism.
• translocase: An enzyme that assists in moving another molecule, usually across a membrane.
Genetic Alteration
In molecular biology, transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
Transformation: Illustration of bacterial transformation. DNA from dead cells gets cut into fragments and exits the cell. The free-floating DNA can then be picked up by competent cells. The exogenous DNA is incorporated into the host cell’s chromosome via recombination.
NATURAL TRANSFORMATION
Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells. For transformation to happen, bacteria must be in a state of competence, which might occur as a time-limited response to environmental conditions such as starvation and cell density. Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell; the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact), and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium).
Transformation” may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because “transformation” has a special meaning in relation to animal cells, indicating progression to a cancerous state, the term should be avoided for animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA into eukaryotic cells is often called “transfection“.
Bacterial transformation may be referred to as a stable genetic change, brought about by the uptake of naked DNA (DNA without associated cells or proteins ). Competence refers to the state of being able to take up exogenous DNA from the environment. There are two forms of competence: natural and artificial.
About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; more may be able to take it up in their natural environments. DNA material can be transferred between different strains of bacteria in a process that is called horizontal gene transfer.
Some species, upon cell death, release their DNA to be taken up by other cells; however, transformation works best with DNA from closely-related species. These naturally-competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogeneous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.
GRAM-POSITIVE AND GRAM-NEGATIVE DIFFERENCES
Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells. However, most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase. Only single-stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process.
In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence however, its role is uncertain. The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.
ARTIFICIAL TRANSFER
Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA, by exposing it to conditions that do not normally occur in nature. Typically, the cells are incubated in a solution containing divalent cations; most commonly, calcium chloride solution under cold condition, which is then exposed to a pulse of heat shock. However, the mechanism of the uptake of DNA via chemically-induced competence in this calcium chloride transformation method is unclear.
The surface of bacteria such as E. coli is negatively-charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively-charged. One function of the divalent cation therefore, would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface. It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.
Electroporation is another method of promoting competence. Using this method, the cells are briefly shocked with an electric field of 10-20 kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell’s membrane-repair mechanisms.
O. T. Avery, et al. were first to demonstrate that “rough” colonies of S. pneumoniae could be transformed to “smooth” (capsule producing) colonies by addition of DNA extracts of the former to the latter, thus “transforming” them. (See Lederberg below)
1. Lederberg, Joshua (1994). The Transformation of Genetics by DNA: An Anniversary Celebration of AVERY, MACLEOD and MCCARTY(1944) in Anecdotal, Historical and Critical Commentaries on Genetics. The Rockfeller University, New York, New York 10021-6399. PMID 8150273. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.11%3A_Genetic_Transfer_in_Prokaryotes/7.11B%3A_Bacterial_Transformation.txt |
Transduction is the process by which DNA is transferred from one bacterium to another by a virus.
LEARNING OBJECTIVES
Differentiate between generalized and specialized transduction
Key Points
• Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation), and it is DNAase resistant.
• Transduction happens through either the lytic cycle or the lysogenic cycle.
• Transduction is especially important because it explains one mechanism by which antibiotic drugs become ineffective due to the transfer of antibiotic-resistance genes between bacteria.
Key Terms
• lytic cycle: The normal process of viral reproduction involving penetration of the cell membrane, nucleic acid synthesis, and lysis of the host cell.
• lysogenic cycle: A form of viral reproduction involving the fusion of the nucleic acid of a bacteriophage with that of a host, followed by proliferation of the resulting prophage.
• transduction: Transduction is the process by which DNA is transferred from one bacterium to another by a virus.
Transduction
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation), and it is DNAase resistant (transformation is susceptible to DNAase). Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell’s genome.
Transduction: Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector.
When bacteriophages (viruses that infect bacteria) infect a bacterial cell, their normal mode of reproduction is to harness the replicational, transcriptional, and translation machinery of the host bacterial cell to make numerous virions, or complete viral particles, including the viral DNA or RNA and the protein coat.
Transduction is especially important because it explains one mechanism by which antibiotic drugs become ineffective due to the transfer of antibiotic-resistance genes between bacteria. In addition, hopes to create medical methods of genetic modification of diseases such as Duchenne/Becker Muscular Dystrophy are based on these methodologies.
The Lytic Cycle and the Lysogenic Cycle
Transduction happens through either the lytic cycle or the lysogenic cycle. If the lysogenic cycle is adopted, the phage chromosome is integrated (by covalent bonds) into the bacterial chromosome, where it can remain dormant for thousands of generations. If the lysogen is induced (by UV light for example), the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles. The lytic cycle leads to the production of new phage particles which are released by lysis of the host.
Transduction is a method for transferring genetic material. The packaging of bacteriophage DNA has low fidelity and small pieces of bacterial DNA, together with the bacteriophage genome, may become packaged into the bacteriophage genome. At the same time, some phage genes are left behind in the bacterial chromosome.
There are generally three types of recombination events that can lead to this incorporation of bacterial DNA into the viral DNA, leading to two modes of recombination.
Generalized transduction is the process by which any bacterial gene may be transferred to another bacterium via a bacteriophage, and typically carries only bacterial DNA and no viral DNA. In essence, this is the packaging of bacterial DNA into a viral envelope. This may occur in two main ways, recombination and headful packaging.
If bacteriophages undertake the lytic cycle of infection upon entering a bacterium, the virus will take control of the cell’s machinery for use in replicating its own viral DNA. If by chance bacterial chromosomal DNA is inserted into the viral capsid which is usually used to encapsulate the viral DNA, the mistake will lead to generalized transduction.
If the virus replicates using “headful packaging,” it attempts to fill the nucleocapsid with genetic material. If the viral genome results in spare capacity, viral packaging mechanisms may incorporate bacterial genetic material into the new virion.
The new virus capsule, now loaded with part bacterial DNA, continues to infect another bacterial cell. This bacterial material may become recombined into another bacterium upon infection.
Fates of DNA Inserted into the Recipient Cell
When the new DNA is inserted into this recipient cell it can fall to one of three fates: the DNA will be absorbed by the cell and be recycled for spare parts; if the DNA was originally a plasmid, it will recirculate inside the new cell and become a plasmid again; if the new DNA matches with a homologous region of the recipient cell’s chromosome, it will exchange DNA material similar to the actions in conjugation. This type of recombination is random and the amount recombined depends on the size of the virus being used.
Specialized transduction is the process by which a restricted set of bacterial genes are transferred to another bacterium. The genes that get transferred (donor genes) depend on where the phage genome is located on the chromosome. Specialized transduction occurs when the prophage excises imprecisely from the chromosome so that bacterial genes lying adjacent to the prophage are included in the excised DNA. The excised DNA is then packaged into a new virus particle, which can then deliver the DNA to a new bacterium, where the donor genes can be inserted into the recipient chromosome or remain in the cytoplasm, depending on the nature of the bacteriophage.
When the partially encapsulated phage material infects another cell and becomes a “prophage” (is covalently bonded into the infected cell’s chromosome), the partially coded prophage DNA is called a “heterogenote. ” Example of specialized transduction is λ phages in Escherichia coli, which was discovered by Esther Lederberg. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.11%3A_Genetic_Transfer_in_Prokaryotes/7.11C%3A_Bacterial_Transduction.txt |
Prokaryotes reproduce asexually by binary fission; they can also exchange genetic material by transformation, transduction, and conjugation.
LEARNING OBJECTIVES
Distinguish among the types of reproduction in prokaryotes
Key Points
• Binary fission is a type of reproduction in which the chromosome is replicated and the resultant prokaryote is an exact copy of the parental prokaryate, thus leaving no opportunity for genetic diversity.
• Transformation is a type of prokaryotic reproduction in which a prokaryote can take up DNA found within the environment that has originated from other prokaryotes.
• Transduction is a type of prokaryotic reproduction in which a prokaryote is infected by a virus which injects short pieces of chromosomal DNA from one bacterium to another.
• Conjugation is a type of prokaryotic reproduction in which DNA is transferred between prokaryotes by means of a pilus.
Key Terms
• transformation: the alteration of a bacterial cell caused by the transfer of DNA from another, especially if pathogenic
• transduction: horizontal gene transfer mechanism in prokaryotes where genes are transferred using a virus
• binary fission: the process whereby a cell divides asexually to produce two daughter cells
• conjugation: the temporary fusion of organisms, especially as part of sexual reproduction
• pilus: a hairlike appendage found on the cell surface of many bacteria
Reproduction
Reproduction in prokaryotes is asexual and usually takes place by binary fission. The DNA of a prokaryote exists as as a single, circular chromosome. Prokaryotes do not undergo mitosis; rather the chromosome is replicated and the two resulting copies separate from one another, due to the growth of the cell. The prokaryote, now enlarged, is pinched inward at its equator and the two resulting cells, which are clones, separate. Binary fission does not provide an opportunity for genetic recombination or genetic diversity, but prokaryotes can share genes by three other mechanisms.
In transformation, the prokaryote takes in DNA found in its environment that is shed by other prokaryotes. If a nonpathogenic bacterium takes up DNA for a toxin gene from a pathogen and incorporates the new DNA into its own chromosome, it, too, may become pathogenic. In transduction, bacteriophages, the viruses that infect bacteria, sometimes also move short pieces of chromosomal DNA from one bacterium to another. Transduction results in a recombinant organism. Archaea are not affected by bacteriophages, but instead have their own viruses that translocate genetic material from one individual to another. In conjugation, DNA is transferred from one prokaryote to another by means of a pilus, which brings the organisms into contact with one another. The DNA transferred can be in the form of a plasmid or as a hybrid, containing both plasmid and chromosomal DNA.
Reproduction can be very rapid: a few minutes for some species. This short generation time, coupled with mechanisms of genetic recombination and high rates of mutation, result in the rapid evolution of prokaryotes, allowing them to respond to environmental changes (such as the introduction of an antibiotic) very rapidly.
7.11E: Complementation
Learning Objectives
• Explain the mechanism of genetic complementation
Complementation refers to a relationship between two different strains of an organism which both have homozygous recessive mutations that produce the same phenotype (for example, a change in wing structure in flies) but which do not reside on the same (homologous) gene.
These strains are true breeding for their mutation. If, when these strains are crossed with each other, some offspring show recovery of the wild-type phenotype, they are said to show “genetic complementation”. When this occurs, each strain’s haploid supplies a wild-type allele to “complement” the mutated allele of the other strain’s haploid, causing the offspring to have heterozygous mutations in all related genes. Since the mutations are recessive, the offspring will display the wild-type phenotype.
A complementation test (sometimes called a “cis-trans” test) refers to this experiment, developed by American geneticist Edward B. Lewis. It answers the question: “Does a wild-type copy of gene X rescue the function of the mutant allele that is believed to define gene X?”. If there is an allele with an observable phenotype whose function can be provided by a wild type genotype (i.e., the allele is recessive), one can ask whether the function that was lost because of the recessive allele can be provided by another mutant genotype. If not, the two alleles must be defective in the same gene. The beauty of this test is that the trait can serve as a read-out of gene function even without knowledge of what the gene is doing at a molecular level.
Complementation arises because loss of function in genes responsible for different steps in the same metabolic pathway can give rise to the same phenotype. When strains are bred together, offspring inherit wildtype versions of each gene from either parent. Because the mutations are recessive, there is a recovery of function in that pathway, so offspring recover the wild-type phenotype. Thus, the test is used to decide if two independently derived recessive mutant phenotypes are caused by mutations in the same gene or in two different genes. If both parent strains have mutations in the same gene, no normal versions of the gene are inherited by the offspring; they express the same mutant phenotype and complementation has failed to occur.
In other words, if the combination of two haploid genomes containing different recessive mutations yields a mutant phenotype, then there are three possibilities: Mutations occur in the same gene; One mutation affects the expression of the other; One mutation may result in an inhibitory product. If the combination of two haploid genomes containing different recessive mutations yields the wild type phenotype, then the mutations must be in different genes.
Key Points
• A complementation test answers the question: “Does a wild-type copy of gene X rescue the function of the mutant allele that is believed to define gene X? “.
• Complementation arises because loss of function in genes responsible for different steps in the same metabolic pathway can give rise to the same phenotype.
• When strains are bred together, offspring inherit wildtype versions of each gene from either parent.
Key Terms
• Complementation: In genetics, complementation refers to a relationship between two different strains of an organism which both have homozygous recessive mutations that produce the same phenotype (for example, a change in wing structure in flies) but which do not reside on the same (homologous) gene.
• mutation: Any heritable change of the base-pair sequence of genetic material.
• homozygous: of an organism in which both copies of a given gene have the same allele | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.11%3A_Genetic_Transfer_in_Prokaryotes/7.11D%3A_Prokaryotic_Reproduction.txt |
Archaea are distinct from bacteria and eukaryotes, but genetic material can be transferred between them and between Archaea themselves.
LEARNING OBJECTIVES
Describe the mechanisms of gene transfer in Archaea
Key Takeaways
Key Points
• Archaea while being very different from eukaryotes and bacteria, there are many commonalities at the the genetic level between them.
• Horizontal gene transfer can explain the similarities between the genes found in the three domains of life and indeed there is evidence that horizontal gene transfer occurs with Archaea species.
• Archaea can be infected by double-stranded DNA viruses, which can account for gene transfers, as well like bacteria, Archaea may conjugate.
Key Terms
• archaea: a taxonomic domain of single-celled organisms lacking nuclei that are fundamentally from bacteria.
• translation: Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text.
• transcription: Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes.
Archaea are genetically distinct from bacteria and eukaryotes, but are poorly understood: many of the genes that Archaea encode are of unknown function. Transcription and translation in archaea resemble the same processes more closely in eukaryotes than in bacteria, with the archaean RNA polymerase and ribosomes being very close to their equivalents in eukaryotes.
Although Archaea only have one type of RNA polymerase, its structure and function in transcription is similar to that of the eukaryotic RNA polymerase II, with similar protein assemblies (the general transcription factors) directing the binding of the RNA polymerase to a gene’s promoter. However, other archaean transcription factors are closer to those found in bacteria. Post-transcriptional modification is simpler than in eukaryotes, since most archaean genes lack introns, although there are many introns in their transfer RNA and ribosomal RNA genes, and introns may occur in a few protein-encoding genes. This is all to say there are many similarities in the genes shared between Archaea and the other domains of life, suggesting there was a transfer of genetic material between the domains of life. This phenomenon is described as horizontal gene transfer.
Horizontal gene transfer (HGT) refers to the transfer of genes between organisms in a manner other than traditional reproduction. Also termed lateral gene transfer, it contrasts with vertical transfer, the transmission of genes from the parental generation to offspring via sexual or asexual reproduction. HGT has been shown to be an important factor in the evolution of many organisms, including bacteria, plants and humans.
Archaea show high levels of horizontal gene transfer between lineages. Some researchers suggest that individuals can be grouped into species-like populations given highly similar genomes and infrequent gene transfer to/from cells with less-related genomes, as in the Archaea genus Ferroplasma. On the other hand, studies in Halorubrum found significant genetic transfer to/from less-related populations. These gene transfers are identified by sequencing the DNA of various Archaea species; through the similarities and differences of the DNA of the different types of Archaea it is determined if the gene was perfectly transferred or from a common ancestor. The elucidation of this can be controversial.
How genetic material can move from one Archaea to another is poorly understood. In bacteria the natural ways in which this occurs is through either bacterial conjugation or viral transfer, also known as transduction. Conjugation is where two (sometimes distantly related) bacteria transfer genetic material by direct contact. Transduction occurs when a virus “picks up” some DNA from its host and when infecting a new host, moves that genetic material to the new host. It is thought that conjugation can occur in Archaea, though unlike bacteria the mechanism is not well understood. As well Archaea can be infected by viruses. In fact Archaea can be infected by double-stranded DNA viruses that are unrelated to any other form of virus and have a variety of unusual shapes, including bottles, hooked rods, or teardrops. Taken together it is clear that gene transfer happens in Archaea, and probably is similar to horizontal gene transfer seen in the other domains of life.
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Molecular cloning permits the replication of a specific DNA sequence in a living microorganism.
LEARNING OBJECTIVES
Show some of the methods and uses of recombinant DNA
Key Points
• Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector.
• E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.
• Modern bacterial cloning vectors (e.g. pUC19) use the blue-white screening system to distinguish colonies ( clones ) of transgenic cells from those that contain the parental vector.
Key Terms
• polymerase chain reaction: A technique in molecular biology for creating multiple copies of DNA from a sample; used in genetic fingerprinting etc.
• molecular cloning: a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.
• restriction enzyme: An endonuclease that catalyzes double-strand cleavage of DNA containing a specific sequence.
Recombinant DNA technology also referred to as molecular cloning is similar to polymerase chain reaction ( PCR ) in that it permits the replication of a specific DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.
In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps:
1. Choice of host organism and cloning vector
2. Preparation of vector DNA
3. Preparation of DNA to be cloned
4. Creation of recombinant DNA
5. Introduction of recombinant DNA into host organism
6. Selection of organisms containing recombinant DNA
7. Screening for clones with desired DNA inserts and biological properties
Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with that at the ends of the foreign DNA.
Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI. Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and is located within a gene (frequently beta-galactosidase) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme (alkaline phosphatase) that dephosphorylates the vector ends. Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site.
For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (RT-PCR) sequences prior to molecular cloning. The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA (linkers) containing desired restriction sites may be added to create end structures that are compatible with the vector. The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together. This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism. The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g. transformation, transduction, transfection, electroporation).
When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation, and cells that are in a physiological state such that they can take up DNA are said to be competent. When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the vector will survive when exposed to the antibiotic, while those that have failed to take up vector sequences will die. Modern bacterial cloning vectors (e.g. pUC19) use the blue-white screening system to distinguish colonies (clones) of transgenic cells from those that contain the parental vector.
In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of beta-galactosidase, an enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme, so that colonies containing recombinant plasmids remain colorless (white). Therefore, recombinant clones are easily identified. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.12%3A_Tools_of_Genetic_Engineering/7.12A%3A_Recombinant_DNA_Technology.txt |
A selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells.
LEARNING OBJECTIVES
Identify the purpose of selection in genetic engineering
Key Points
• Recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell’s DNA in the transgenic organism.
• Artificial genetic selection is the process in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.
• When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin.
Key Terms
• molecular cloning: a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.
• PCR: polymerase chain reaction
Scientists who do experimental genetics employ artificial selection experiments that permit the survival of organisms with user-defined phenotypes. Artificial selection is widely used in the field of microbial genetics, especially molecular cloning.
DNA recombination has been used to create gene replacements, deletions, insertions, inversions. Gene cloning and gene/protein tagging is also common. For gene replacements or deletions, usually a cassette encoding a drug-resistance gene is made by PCR.
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest. It is then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria ). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically-modified microorganisms (GMO). This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as “clones”. Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them.
Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell’s DNA in the transgenic organism.
Molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of a specific DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells. Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive. When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the vector will survive when exposed to the antibiotic, while those that fail to take up vector sequences die. When mammalian cells (e.g. human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the kanMX cassette) confers resistance to the antibiotic Geneticin. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.12%3A_Tools_of_Genetic_Engineering/7.12B%3A_Selection.txt |
Mutations are accidental changes in a genomic sequence of DNA; this includes the DNA sequence of a cell’s genome or the DNA or RNA sequence.
LEARNING OBJECTIVES
Explain genetic manipulation through mutations
Key Points
• Mutations are caused by radiation, viruses, transposons, and mutagenic chemicals. They are also caused by errors that occur during meiosis or DNA replication.
• Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule.
• The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation. It is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest.
Key Terms
• site-directed mutagenesis: Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule.
• mutation: Any heritable change of the base-pair sequence of genetic material.
In molecular biology and genetics, mutations are accidental changes in a genomic sequence of DNA: the DNA sequence of a cell’s genome or the DNA or RNA sequence in some viruses. These random sequences can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons, and mutagenic chemicals. They are also caused by errors that occur during meiosis or DNA replication. They can also be induced by the organism itself, through cellular processes such as hypermutation.
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known. It is commonly used in protein engineering.
The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site. It is then introduced into a host cell as a vector and cloned. Finally, mutants are selected.
The original method using single-primer extension was inefficient due to a lower yield of mutants. The resulting mixture may contain both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of a mismatch repair system which favors the methylated template DNA. Many approaches have since been developed to improve the efficiency of mutagenesis.
7.12D: Reproductive Cloning
Reproductive cloning, possible through artificially-induced asexual reproduction, is a method used to make a clone of an entire organism.
LEARNING OBJECTIVES
Differentiate reproductive cloning from cellular and molecular cloning
Key Points
• A form of asexual reproduction, parthenogenesis, occurs when an embryo grows and develops without the fertilization of the egg.
• In reproductive cloning, if the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of an individual of the same species, it will become a zygote that is genetically identical to the donor.
• Reproductive cloning has become successful, but still has limitations as cloned individuals often exhibit facial, limb, and cardiac abnormalities.
• Therapeutic cloning, the cloning of human embryos as a source of embryonic stem cells, has been attempted in order to produce cells that can be used to treat detrimental diseases or defects.
Key Terms
• clone: a living organism produced asexually from a single ancestor, to which it is genetically identical
• stem cell: a primal undifferentiated cell from which a variety of other cells can develop through the process of cellular differentiation
• parthenogenesis: a form of asexual reproduction where growth and development of embryos occur without fertilization
Reproductive Cloning
Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism. Most multicellular organisms undergo reproduction by sexual means, which involves genetic hybridization of two individuals (parents), making it impossible to generate an identical copy or clone of either parent. Recent advances in biotechnology have made it possible to artificially induce asexual reproduction of mammals in the laboratory.
Parthenogenesis, or “virgin birth,” occurs when an embryo grows and develops without the fertilization of the egg occurring; this is a form of asexual reproduction. An example of parthenogenesis occurs in species in which the female lays an egg. If the egg is fertilized, it is a diploid egg and the individual develops into a female; if the egg is not fertilized, it remains a haploid egg and develops into a male. The unfertilized egg is called a parthenogenic, or virgin, egg. Some insects and reptiles lay parthenogenic eggs that can develop into adults.
Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote results. The zygote nucleus contains the genetic information to produce a new individual. However, early embryonic development requires the cytoplasmic material contained in the egg cell. This idea forms the basis for reproductive cloning. If the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species (called a donor), it will become a zygote that is genetically identical to the donor. Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an enucleated egg. It can be used for either therapeutic cloning or reproductive cloning.
The first cloned animal was Dolly, a sheep who was born in 1996. The success rate of reproductive cloning at the time was very low. Dolly lived for seven years and died of respiratory complications. There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect the life expectancy of a cloned individual. Since Dolly, several animals (e.g. horses, bulls, and goats) have been successfully cloned, although these individuals often exhibit facial, limb, and cardiac abnormalities. There have been attempts at producing cloned human embryos as sources of embryonic stem cells. Sometimes referred to as cloning for therapeutic purposes, the technique produces stem cells that attempt to remedy detrimental diseases or defects (unlike reproductive cloning, which aims to reproduce an organism). Still, therapeutic cloning efforts have met with resistance because of bioethical considerations. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.12%3A_Tools_of_Genetic_Engineering/7.12C%3A_Mutation.txt |
Basic techniques used in genetic material manipulation include extraction, gel electrophoresis, PCR, and blotting methods.
LEARNING OBJECTIVES
Distinguish among the basic techniques used to manipulate DNA and RNA
Key Points
• The first step to study or work with nucleic acids includes the isolation or extraction of DNA or RNA from cells.
• Gel electrophoresis depends on the negatively-charged ions present on nucleic acids at neutral or basic pH to separate molecules on the basis of size.
• Specific regions of DNA can be amplified through the use of polymerase chain reaction for further analysis.
• Southern blotting involves the transfer of DNA to a nylon membrane, while northern blotting is the transfer of RNA to a nylon membrane; these techniques allow samples to be probed for the presence of certain sequences.
Key Terms
• denaturation: the change of folding structure of a protein (and thus of physical properties) caused by heating, changes in pH, or exposure to certain chemicals
• electrophoresis: a method for the separation and analysis of large molecules, such as proteins or nucleic acids, by migrating a colloidal solution of them through a gel under the influence of an electric field
• polymerase chain reaction: a technique in molecular biology for creating multiple copies of DNA from a sample
Basic Techniques to Manipulate Genetic Material (DNA and RNA)
To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base) linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. The two strands can be separated by exposure to high temperatures (DNA denaturation) and can be reannealed by cooling. The DNA can be replicated by the DNA polymerase enzyme. Unlike DNA, which is located in the nucleus of eukaryotic cells, RNA molecules leave the nucleus. The most common type of RNA that is analyzed is the messenger RNA (mRNA) because it represents the protein -coding genes that are actively expressed.
DNA and RNA Extraction
To study or manipulate nucleic acids, the DNA or RNA must first be isolated or extracted from the cells. This can be done through various techniques. Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as degradation of unwanted molecules and separation from the DNA sample). Cells are broken using a lysis buffer (a solution that is mostly a detergent); lysis means “to split.” These enzymes break apart lipid molecules in the membranes of the cell and the nucleus. Macromolecules are inactivated using enzymes such as proteases that break down proteins, and ribonucleases (RNAses) that break down RNA. The DNA is then precipitated using alcohol. Human genomic DNA is usually visible as a gelatinous, white mass. Samples can be stored at –80°C for years.
RNA analysis is performed to study gene expression patterns in cells. RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves the use of various buffers and enzymes to inactivate macromolecules and preserve the RNA.
Gel Electrophoresis
Because nucleic acids are negatively-charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field. Gel electrophoresis is a technique used to separate molecules on the basis of size using this charge and may be separated as whole chromosomes or fragments. The nucleic acids are loaded into a slot near the negative electrode of a porous gel matrix and pulled toward the positive electrode at the opposite end of the gel. Smaller molecules move through the pores in the gel faster than larger molecules; this difference in the rate of migration separates the fragments on the basis of size. There are molecular-weight standard samples that can be run alongside the molecules to provide a size comparison. Nucleic acids in a gel matrix can be observed using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the top of the gel (the negative electrode end) on the basis of their size.
Amplification of Nucleic Acid Fragments by Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis. PCR is used for many purposes in laboratories, such as the cloning of gene fragments to analyze genetic diseases, identification of contaminant foreign DNA in a sample, and the amplification of DNA for sequencing. More practical applications include the determination of paternity and detection of genetic diseases.
DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR). The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.
Hybridization, Southern Blotting, and Northern Blotting
Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences. Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting. The nucleic acid fragments that are bound to the surface of the membrane can then be probed with specific radioactively- or fluorescently-labeled probe sequences. When DNA is transferred to a nylon membrane, the technique is called Southern blotting; when RNA is transferred to a nylon membrane, it is called northern blotting. Southern blots are used to detect the presence of certain DNA sequences in a given genome, and northern blots are used to detect gene expression. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.12%3A_Tools_of_Genetic_Engineering/7.12E%3A_Basic_Techniques_to_Manipulate_Genetic_Material_%28DNA_and_RNA%29.txt |
Molecular cloning reproduces the desired regions or fragments of a genome, enabling the manipulation and study of genes.
LEARNING OBJECTIVES
Describe the process of molecular cloning
Key Points
• Cloning small fragments of a genome allows specific genes, their protein products, and non-coding regions to be studied in isolation.
• A plasmid, also known as a vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA; it can be used to provide a “folder” in which to insert a desired DNA fragment.
• Recombinant DNA molecules are plasmids with foreign DNA inserted into them; they are created artificially as they do not occur in nature.
• Bacteria and yeast naturally produce clones of themselves when they replicate asexually through cellular cloning.
Key Terms
• recombinant DNA: DNA that has been engineered by splicing together fragments of DNA from multiple species and introduced into the cells of a host
• molecular cloning: a biological method that creates many identical DNA molecules and directs their replication within a host organism
• plasmid: a circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa
Molecular Cloning
In general, the word “cloning” means the creation of a perfect replica; however, in biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning.
Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products) or noncoding regions in isolation. A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, the plasmid molecules can be used to provide a “folder” in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA (or a transgene) to differentiate it from the DNA of the bacterium, which is called the host DNA.
Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly-available restriction endonucleases. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease.
Recombinant DNA Molecules
Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. Proteins that are expressed from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors so that scientists can control the expression of the recombinant proteins.
Cellular Cloning
Unicellular organisms, such as bacteria and yeast, naturally produce clones of themselves when they replicate asexually by binary fission; this is known as cellular cloning. The nuclear DNA duplicates by the process of mitosis, which creates an exact replica of the genetic material. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.12%3A_Tools_of_Genetic_Engineering/7.12F%3A_Molecular_and_Cellular_Cloning.txt |
Plasmids can be used as cloning vectors, allowing the insertion of exogenous DNA into a bacterial target.
LEARNING OBJECTIVES
Illustrate how plasmids can be used as cloning vectors
Key Points
• All engineered vectors have an origin of replication, a multi- cloning site, and a selectable marker.
• Expression vectors (expression constructs) express the transgene in the target cell, and they have a promoter sequence that drives expression of the transgene.
• Transcription is needed for a plasmid to function, without the proper sequences to transcribe parts of a plasmid it will not be expressed or even maintained in host cells.
• Vectors can have many additional sequences that can be used for downstream applications—purification of proteins encoded by the plasmid and expressing proteins targeted to be exported or to a certain compartment of the cell.
Key Terms
• Kozak sequence: a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG. The Kozak consensus sequence plays a major role in the initiation of the translation process. The sequence was named after the person who brought it to prominence, Marilyn Kozak.
• transcription: The synthesis of RNA under the direction of DNA.
• polyadenylation: The formation of a polyadenylate, especially that of a nucleic acid
Vectors
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. All engineered vectors have an origin of replication, a multi-cloning site, and a selectable marker. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence, which serves as the “backbone” of the vector. The purpose of a vector that transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) express the transgene in the target cell, and they generally have a promoter sequence that drives expression of the transgene.
Plasmids
Plasmids are double-stranded, generally circular DNA sequences capable of automatically replicating in a host cell. Plasmid vectors minimally consist of the transgene insert and an origin of replication, which allows for semi-independent replication of the plasmid in the host. Modern plasmids generally have many more features, notably a “multiple cloning site”—with nucleotide overhangs for insertion of an insert—and multiple restriction enzyme consensus sites on either side of the insert. Plasmids may be conjugative/transmissible or non-conjugative. Conjugative plasmids mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population. Nonconjugative plasmids do not mediate DNA through conjugation.
Transcription
Transcription is a necessary component in all vectors. The purpose of a vector is to multiply the insert, although expression vectors also drive the translation of the multiplied insert. Even stable expression is determined by stable transcription, which depends on promoters in the vector. However, expression vectors have a two expression patterns: constitutive (consistent expression) or inducible (expression only under certain conditions or chemicals). Expression is based on different promoter activities, not post-transcriptional activities, meaning these two different types of expression vectors depend on different types of promoters. Expression vectors require translation of the vector’s insert, thus requiring more components than simpler transcription-only vectors.
Expression vectors require sequences that encode for:
• A polyadenylation tail at the end of the transcribed pre-mRNA: This protects the mRNA from exonucleases and ensures transcriptional and translational termination and stabilizes mRNA production.
• Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, so the shortest UTRs are encoded for in optimal expression vectors.
• Kozak sequence: a vector should encode for a Kozak sequence in the mRNA, which assembles the ribosome for translation of the mRNA.
The above conditions are necessary for expression vectors in eukaryotes, not prokaryotes.
Modern vectors may encompass additional features besides the transgene insert and a backbone:
• Promoter: a necessary component for all vectors, used to drive transcription of the vector’s transgene.
• Genetic markers: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA.
• Antibiotic resistance: Vectors with antibiotic-resistance allow for survival of cells that have taken up the vector in growth media containing antibiotics through antibiotic selection.
• Epitope: A vector containing a sequence for a specific epitope that is incorporated into the expressed protein. Allows for antibody identification of cells expressing the target protein.
• Reporter genes: Some vectors may contain a reporter gene that allow for identification of plasmid that contains inserted DNA sequence. An example is lacZ-α which codes for the N-terminus fragment of β-galactosidase, an enzyme that digests galactose.
• Targeting sequence: Expression vectors may include encoding for a targeting sequence in the finished protein that directs the expressed protein to a specific organelle in the cell or specific location such as the periplasmic space of bacteria.
• Protein purification tags: Some expression vectors include proteins or peptide sequences that allows for easier purification of the expressed protein. Examples include polyhistidine-tag, glutathione-S-transferase, and maltose binding protein. Some of these tags may also allow for increased solubility of the target protein. The target protein is fused to the protein tag, but a protease cleavage site positioned in the polypeptide linker region between the protein and the tag allows the tag to be removed later.
LICENSES AND ATTRIBUTIONS
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The strategies used for sequencing genomes include the Sanger method, shotgun sequencing, pairwise end, and next-generation sequencing.
LEARNING OBJECTIVES
Compare the different strategies used for whole-genome sequencing: Sanger method, shotgun sequencing, pairwise-end sequencing, and next-generation sequencing
Key Points
• The Sanger method is a basic sequencing technique that uses fluorescently-labeled dideoxynucleotides (ddNTPs) during DNA replication which results in multiple short strands of replicated DNA that terminate at different points, based on where the ddNTP was incorporated.
• Shotgun sequencing is a method that randomly cuts DNA fragments into smaller pieces and then, with the help of a computer, takes the DNA fragments, analyzes them for overlapping sequences, and reassembles the entire DNA sequence.
• Pairwise-end sequencing is a type of shotgun sequencing which is used for larger genomes and analyzes both ends of the DNA fragments for overlap.
• Next-generation sequencing is a type of sequencing which is automated and relies on sophisticated software for rapid DNA sequencing.
Key Terms
• fluorophore: a molecule or functional group which is capable of fluorescence
• contig: a set of overlapping DNA segments, derived from a single source of genetic material, from which the complete sequence may be deduced
• dideoxynucleotide: any nucleotide formed from a deoxynucleotide by loss of an a second hydroxyl group from the deoxyribose group
Strategies Used in Sequencing Projects
The basic sequencing technique used in all modern day sequencing projects is the chain termination method (also known as the dideoxy method), which was developed by Fred Sanger in the 1970s. The chain termination method involves DNA replication of a single-stranded template with the use of a primer and a regular deoxynucleotide (dNTP), which is a monomer, or a single unit, of DNA. The primer and dNTP are mixed with a small proportion of fluorescently-labeled dideoxynucleotides (ddNTPs). The ddNTPs are monomers that are missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a chain. Each ddNTP is labeled with a different color of fluorophore. Every time a ddNTP is incorporated in the growing complementary strand, it terminates the process of DNA replication, which results in multiple short strands of replicated DNA that are each terminated at a different point during replication. When the reaction mixture is processed by gel electrophoresis after being separated into single strands, the multiple, newly-replicated DNA strands form a ladder due to their differing sizes. Because the ddNTPs are fluorescently labeled, each band on the gel reflects the size of the DNA strand and the ddNTP that terminated the reaction. The different colors of the fluorophore-labeled ddNTPs help identify the ddNTP incorporated at that position. Reading the gel on the basis of the color of each band on the ladder produces the sequence of the template strand.
Early Strategies: Shotgun Sequencing and Pair-Wise End Sequencing
In the shotgun sequencing method, several copies of a DNA fragment are cut randomly into many smaller pieces (somewhat like what happens to a round shot cartridge when fired from a shotgun). All of the segments are then sequenced using the chain-sequencing method. Then, with the help of a computer, the fragments are analyzed to see where their sequences overlap. By matching overlapping sequences at the end of each fragment, the entire DNA sequence can be reformed. A larger sequence that is assembled from overlapping shorter sequences is called a contig. As an analogy, consider that someone has four copies of a landscape photograph that you have never seen before and know nothing about how it should appear. The person then rips up each photograph with their hands, so that different size pieces are present from each copy. The person then mixes all of the pieces together and asks you to reconstruct the photograph. In one of the smaller pieces you see a mountain. In a larger piece, you see that the same mountain is behind a lake. A third fragment shows only the lake, but it reveals that there is a cabin on the shore of the lake. Therefore, from looking at the overlapping information in these three fragments, you know that the picture contains a mountain behind a lake that has a cabin on its shore. This is the principle behind reconstructing entire DNA sequences using shotgun sequencing.
Originally, shotgun sequencing only analyzed one end of each fragment for overlaps. This was sufficient for sequencing small genomes. However, the desire to sequence larger genomes, such as that of a human, led to the development of double-barrel shotgun sequencing, more formally known as pairwise-end sequencing. In pairwise-end sequencing, both ends of each fragment are analyzed for overlap. Pairwise-end sequencing is, therefore, more cumbersome than shotgun sequencing, but it is easier to reconstruct the sequence because there is more available information.
Next-generation Sequencing
Since 2005, automated sequencing techniques used by laboratories are under the umbrella of next-generation sequencing, which is a group of automated techniques used for rapid DNA sequencing. These automated, low-cost sequencers can generate sequences of hundreds of thousands or millions of short fragments (25 to 500 base pairs) in the span of one day. Sophisticated software is used to manage the cumbersome process of putting all the fragments in order. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.13%3A_Bioinformatics/7.13A%3A_Strategies_Used_in_Sequencing_Projects.txt |
Genome annotation is the identification and understanding of the genetic elements of a sequenced genome.
LEARNING OBJECTIVES
Define genome annotation
Key Points
• Once a genome is sequenced, all of the sequencings must be analyzed to understand what they mean.
• Critical to annotation is the identification of the genes in a genome, the structure of the genes, and the proteins they encode.
• Once a genome is annotated, further work is done to understand how all the annotated regions interact with each other.
Key Terms
• BLAST: In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences.
• in silico: In computer simulation or in virtual reality
Genome projects are scientific endeavors that ultimately aim to determine the complete genome sequence of an organism (be it an animal, a plant, a fungus, a bacterium, an archaean, a protist, or a virus). They annotate protein-coding genes and other important genome-encoded features. The genome sequence of an organism includes the collective DNA sequences of each chromosome in the organism. For a bacterium containing a single chromosome, a genome project will aim to map the sequence of that chromosome.
Once a genome is sequenced, it needs to be annotated to make sense of it. An annotation (irrespective of the context) is a note added by way of explanation or commentary. Since the 1980’s, molecular biology and bioinformatics have created the need for DNA annotation. DNA annotation or genome annotation is the process of identifying the locations of genes and all of the coding regions in a genome and determining what those genes do.
Genome annotation is the process of attaching biological information to sequences. It consists of two main steps: identifying elements on the genome, a process called gene prediction, and attaching biological information to these elements. Automatic annotation tools try to perform all of this by computer analysis, as opposed to manual annotation (a.k.a. curation) which involves human expertise. Ideally, these approaches co-exist and complement each other in the same annotation pipeline (process). The basic level of annotation is using BLAST for finding similarities, and then annotating genomes based on that. However, nowadays more and more additional information is added to the annotation platform. The additional information allows manual annotators to deconvolute discrepancies between genes that are given the same annotation. Some databases use genome context information, similarity scores, experimental data, and integrations of other resources to provide genome annotations through their Subsystems approach. Other databases rely on both curated data sources as well as a range of different software tools in their automated genome annotation pipeline.
Structural annotation consists of the identification of genomic elements: ORFs and their localization, gene structure, coding regions, and the location of regulatory motifs. Functional annotation consists of attaching biological information to genomic elements: biochemical function, biological function, involved regulation and interactions, and expression.
These steps may involve both biological experiments and in silico analysis. Proteogenomics based approaches utilize information from expressed proteins, often derived from mass spectrometry, to improve genomics annotations. A variety of software tools have been developed to permit scientists to view and share genome annotations. Genome annotation is the next major challenge for the Human Genome Project, now that the genome sequences of human and several model organisms are largely complete. Identifying the locations of genes and other genetic control elements is often described as defining the biological “parts list” for the assembly and normal operation of an organism. Scientists are still at an early stage in the process of delineating this parts list and in understanding how all the parts “fit together. ” | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.13%3A_Bioinformatics/7.13B%3A_Annotating_Genomes.txt |
Learning Objectives
• Distinguish homologs, orthologs and paralogs
Homology forms the basis of organization for comparative biology. A homologous trait is often called a homolog (also spelled homologue). In genetics, the term “homolog” is used both to refer to a homologous protein and to the gene ( DNA sequence) encoding it. As with anatomical structures, homology between protein or DNA sequences is defined in terms of shared ancestry. Two segments of DNA can have shared ancestry because of either a speciation event (orthologs) or a duplication event (paralogs). Homology among proteins or DNA is often incorrectly concluded on the basis of sequence similarity. The terms “percent homology” and “sequence similarity” are often used interchangeably. As with anatomical structures, high sequence similarity might occur because of convergent evolution, or, as with shorter sequences, because of chance. Such sequences are similar, but not homologous. Sequence regions that are homologous are also called conserved. This is not to be confused with conservation in amino acid sequences in which the amino acid at a specific position has been substituted with a different one with functionally equivalent physicochemical properties. One can, however, refer to partial homology where a fraction of the sequences compared (are presumed to) share descent, while the rest does not. For example, partial homology may result from a gene fusion event.
Homologous sequences are orthologous if they were separated by a speciation event: when a species diverges into two separate species, the copies of a single gene in the two resulting species are said to be orthologous. Orthologs, or orthologous genes, are genes in different species that originated by vertical descent from a single gene of the last common ancestor. For instance, the plant Flu regulatory protein is present both in Arabidopsis (multicellular higher plant) and Chlamydomonas (single cell green algae). The Chlamydomonas version is more complex: it crosses the membrane twice rather than once, contains additional domains, and undergoes alternative splicing. However, it can fully substitute the much simpler Arabidopsis protein, if transferred from algae to plant genome by means of gene engineering. Significant sequence similarity and shared functional domains indicate that these two genes are orthologous genes, inherited from the shared ancestor. Orthologous sequences provide useful information in taxonomic classification and phylogenetic studies of organisms. The pattern of genetic divergence can be used to trace the relatedness of organisms. Two organisms that are very closely related are likely to display very similar DNA sequences between two orthologs. Conversely, an organism that is further removed evolutionarily from another organism is likely to display a greater divergence in the sequence of the orthologs being studied.
Homologous sequences are paralogous if they were separated by a gene duplication event: if a gene in an organism is duplicated to occupy two different positions in the same genome, then the two copies are paralogous. Paralogous genes often belong to the same species, but this is not necessary. For example, the hemoglobin gene of humans and the myoglobin gene of chimpanzees are paralogs. Paralogs can be split into in-paralogs (paralogous pairs that arose after a speciation event) and out-paralogs (paralogous pairs that arose before a speciation event). Between species out-paralogs are pairs of paralogs that exist between two organisms due to duplication before speciation. Within species out-paralogs are pairs of paralogs that exist in the same organism, but whose duplication event happened after speciation. Paralogs typically have the same or similar function, but sometimes do not. Due to lack of the original selective pressure upon one copy of the duplicated gene, this copy is free to mutate and acquire new functions. Paralogous sequences provide useful insight into the way genomes evolve. The genes encoding myoglobin and hemoglobin are considered to be ancient paralogs. Similarly, the four known classes of hemoglobins (hemoglobin A, hemoglobin A2, hemoglobin B, and hemoglobin F) are paralogs of each other. While each of these proteins serves the same basic function of oxygen transport, they have already diverged slightly in function: fetal hemoglobin (hemoglobin F) has a higher affinity for oxygen than adult hemoglobin. However, function is not always conserved. Human angiogenin diverged from ribonuclease, for example, and while the two paralogs remain similar in tertiary structure, their functions within the cell are now quite different.
Key Points
• A homologous gene (or homolog) is a gene inherited in two species from a common ancestor. While homologous genes can be similar in sequence, similar sequences are not necessarily homologous.
• Orthologous are homologous genes where a gene diverges after a speciation event, but the gene and its main function are conserved.
• If a gene is duplicated in a species, the resulting duplicated genes are paralogs of each other, even though over time they might become different in sequence composition and function.
Key Terms
• conserved: In biology, conserved sequences are similar or identical sequences that occur within nucleic acid sequences (such as RNA and DNA sequences), protein sequences, protein structures.
• selective pressure: Any cause that reduces reproductive success in a proportion of a population, potentially exerts evolutionary pressure or selection pressure. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.13%3A_Bioinformatics/7.13C%3A_Homologs_Orthologs_and_Paralogs.txt |
Learning Objectives
• Outline the methods and uses of DNA synthesis
To understand bacterial genetics, the underlying genetic material (i.e. DNA) must be understood. DNA must be synthesized to study genes, the sequence of genomes, and many other studies. This occurs in two fashions, by polymerase chain reaction (PCR) which is enzymatic and chemical synthesis. PCR is covered in another atom. Here we will focus on chemical synthesis of DNA, which is also known as oligonucleotide synthesis.
Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids, both DNA and RNA with a defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence. Whereas enzymes synthesize DNA and RNA in a 5′ to 3′ direction, chemical oligonucleotide synthesis is carried out in the opposite, 3′ to 5′ direction.
Currently, the process is implemented as solid -phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2′-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g. LNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. The process has been fully automated since the late 1970’s. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized. Products are often isolated by HPLC to obtain the desired oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-stranded DNA or RNA molecules around 15–25 bases in length. Oligonucleotides find a variety of applications in molecular biology and medicine. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.
A further application of oligosynthesis is to make artificial genes. Artificial gene synthesis is the process of synthesizing a gene in vitro without the need for initial template DNA samples. The main method is currently by oligonucleotide synthesis (also used for other applications) from digital genetic sequences and subsequent annealing of the resultant fragments. In contrast, natural DNA replication requires existing DNA templates for synthesizing new DNA.
Key Points
• DNA and RNA are at their essence chemical structures, and as such complex chemical reactions can be used to synthesize them.
• There are enzymatic ways to amplify DNA, notably PCR, while DNA sequences can be chemically synthesized by a process known as oligosynthesis.
• Oligosynthesis can be used to make artificial genes, which allows scientists to design and synthesis novel gene products, without relying a template of a gene found in nature.
Key Terms
• phosphoramidite: Any of a class of organic compounds formally derived from a phosphite by replacing a >P-O-R with a >P-N<R2 group; used in the synthesis of nucleic acids, etc.
• HPLC: High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.13%3A_Bioinformatics/7.13D%3A_Synthesizing_DNA.txt |
Learning Objectives
• Illustrate the applications, components and steps of PCR
The polymerase chain reaction (PCR) is a biochemical technology in molecular biology used to amplify a single, or a few copies, of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Applications
Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications including the following:
• DNA cloning for sequencing; DNA-based phylogeny, or functional analysis of genes
• The diagnosis of hereditary diseases
• The identification of genetic fingerprints (used in forensic sciences and paternity testing)
• The detection and diagnosis of infectious diseases
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region, along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.
Components
PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size. The reaction produces a limited amount of final amplified product that is governed by the available reagents in the reaction, and the feedback-inhibition of the reaction products. A basic PCR set up requires the following components and reagents:
• DNA template that contains the DNA region (target) to be amplified
• Two primers that are complementary to the 3′ (three prime) ends of each of the sense and anti-sense strand of the DNA target
• Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C
• Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand
• Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.Divalent cations, magnesium or manganese ions; generally Mg2+
• Monovalent cation potassium ions
Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of two to three discrete temperature steps, usually three. The temperatures used, and the length of time they are applied in each cycle, depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.
Steps
The following are the steps of PCR:
1. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
2. Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template.
3. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5′ to 3′ direction, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the nascent (extending) DNA strand.
After elongation, the cycle goes back to step one, usually for 20-40 cycles. Under optimum conditions (i.e., if there are no limitations due to limiting substrates or reagents) at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
Key Points
• PCR is used to amplify a specific region of DNA.
• PCR typically consists of three steps: denaturation, annealing, and elongation.
• The amplified DNA can be used for many purposes, such as identifying different genes and species of bacteria.
Key Terms
• annealing: Annealing, in genetics, means for complementary sequences of single-stranded DNA or RNA to pair by hydrogen bonds to form a double-stranded polynucleotide. The term is often used to describe the binding of a DNA probe, or the binding of a primer to a DNA strand during a polymerase chain reaction (PCR). The term is also often used to describe the reformation (renaturation) of complementary strands that were separated by heat (thermally denatured). Proteins such as RAD52 can help DNA anneal. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.13%3A_Bioinformatics/7.13E%3A_Amplifying_DNA_-_The_Polymerase_Chain_Reaction.txt |
Learning Objectives
• Recall dideoxynucleotide sequencing
Sanger sequencing, also known as chain-termination sequencing, refers to a method of DNA sequencing developed by Frederick Sanger in 1977. This method is based on amplification of the DNA fragment to be sequenced by DNA polymerase and incorporation of modified nucleotides – specifically, dideoxynucleotides (ddNTPs).
The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation. These chain-terminating nucleotides lack a 3′-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labelled for detection in automated sequencing machines.The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. This is frequently performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C). The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.
Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5′ end with a fluorescent dye. Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. The later development by Leroy Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing. Chain-termination methods have greatly simplified DNA sequencing. More recently, dye-terminator sequencing has been developed. Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths.
Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day. DNA sequencers carry out capillary electrophoresis for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Automation has lead to the sequencing of entire genomes.
Key Points
• The lack of the second deoxy group on an dNTP making it ddNTP, stops the incorporation of further nucleotides, this termination creates DNA lengths stopped at every nucleotide, this is central to further identifying each nucleotide.
• Different labels can be used, ddNTPS, dNTPs and primers can all be labelled with radioactivity and fluorescently.
• Using fluorescent labels, dideoxy sequencing can be automated allowing high-throughput methods which have been utilized to sequence entire genomes.
Key Terms
• chromatogram: The visual output from a chromatograph. Usually a graphical display or histogram.
• dideoxynucleotide: Any nucleotide formed from a deoxynucleotide by loss of an a second hydroxy group from the deoxyribose group | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.13%3A_Bioinformatics/7.13F%3A_DNA_Sequencing_Based_on_Sanger_Dideoxynucleotides.txt |
Metagenomics is the study of genetic material derived from environmental samples.
LEARNING OBJECTIVES
Summarize the utility of metagenomics
Key Points
• While previous work needed cultivation of single microbes before they could be sequenced and identified, metagenomics attempts to more completely identify many of the microbes that inhabit a given environmental location.
• The first attempts at metagenomics was to sequence one gene from a sample. The changes in that one gene helped determine the microbial diversity in a sample.
• High-throughput sequencing allows the complete sequencing and assembly of entire genomes of the microbes that inhabit a given environment, giving unprecedented depth into understanding the microbial diversity of the world around us.
Key Terms
• solid: SOLiD (Sequencing by Oligonucleotide Ligation and Detection) is a next-generation DNA sequencing technology developed by Life Technologies and has been commercially available since 2008. This next generation technology generates hundreds of millions to billions of small sequence reads at one time.
• gigabase: One billion bases (nucleotides) as a unit of length of a nucleic acid
• pyrosequencing: A technique used to sequence DNA using chemiluminescent enzymatic reactions
Metagenomics
Metagenomics is the study of metagenomes; genetic material recovered directly from environmental samples. The broad field may also be referred to as environmental genomics, ecogenomics or community genomics. While traditional microbiology and microbial genome sequencing and genomics rely upon cultivated clonal cultures, early environmental gene sequencing cloned specific genes (often the 16S rRNA gene) to produce a profile of diversity in a natural sample. Such work revealed that the vast majority of microbial biodiversity had been missed by cultivation-based methods. Recent studies use “shotgun” Sanger sequencing or massively parallel pyrosequencing to get largely unbiased samples of all genes from all the members of the sampled communities. Due to its ability to reveal the previously hidden diversity of microscopic life, metagenomics offers a powerful lens for viewing the microbial world that has the potential to revolutionize understanding of the entire living world.
Conventional Sequencing Studies
Conventional sequencing begins with a culture of identical cells as a source of DNA. However, early metagenomic studies revealed that there are probably large groups of microorganisms in many environments that cannot be cultured and thus cannot be sequenced. These early studies focused on 16S ribosomal RNA sequences which are relatively short, often conserved within a species, and generally different between species. Many 16S rRNA sequences have been found which do not belong to any known cultured species, indicating that there are numerous non-isolated organisms. These surveys of ribosomal RNA (rRNA) genes taken directly from the environment revealed that cultivation based methods find less than 1% of the bacterial and archaeal species in a sample.
Shotgun Metagenomics
Advances in bioinformatics, refinements of DNA amplification, and the proliferation of computational power have greatly aided the analysis of DNA sequences recovered from environmental samples, This allows the adaptation of shotgun sequencing to metagenomic samples. The approach, used to sequence many cultured microorganisms and the human genome, randomly shears DNA, sequences many short sequences, and reconstructs them into a consensus sequence. Shotgun sequencing and screens of clone libraries reveal genes present in environmental samples. This provides information both on which organisms are present and what metabolic processes are possible in the community. This can be helpful in understanding the ecology of a community, particularly if multiple samples are compared to each other.
Shotgun metagenomics is also capable of sequencing nearly complete microbial genomes directly from the environment. As the collection of DNA from an environment is largely uncontrolled, the most abundant organisms in an environmental sample are most highly represented in the resulting sequence data. To achieve the high coverage needed to fully resolve the genomes of under-represented community members, large samples are needed. On the other hand, the random nature of shotgun sequencing ensures that many of these organisms, which would otherwise go unnoticed using traditional culturing techniques, will be represented by at least some small sequence segments.
High-Throughput Sequencing
The first metagenomic studies conducted using high-throughput sequencing used massively parallel 454 pyrosequencing. Two other technologies commonly applied to environmental sampling are the Illumina Genome Analyzer II and the Applied Biosystems SOLiD system. These techniques for sequencing DNA generate shorter fragments than Sanger sequencing; 454 pyrosequencing typically produces ~400 bp reads, Illumina and SOLiD produce 25-75 bp reads. These read lengths are significantly shorter than the typical Sanger sequencing read length of ~750 bp.
However, this limitation is compensated for by the much larger number of sequence reads. Pyrosequenced metagenomes generate 200–500 megabases, while Illumina platforms generate around 20–50 gigabases. An additional advantage to short read sequencing is that this technique does not require cloning the DNA before sequencing, removing one of the main biases in environmental sampling. As most short-read assembly software was not designed for metagenomic applications, specialized methods have been developed to utilize mate-read data in metagenomic assembly. From these studies the microbial fauna that might reside in a sample of soil, even on the surface of a keyboard, can be more accurately and efficiently identified. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.13%3A_Bioinformatics/7.13G%3A_Metagenomics.txt |
A reporter fusion is the hybrid of a gene or portion of a gene with a tractable marker.
LEARNING OBJECTIVES
Explain reporter fusions
Key Points
• A reporter construct allows the study of gene ‘s function and localization of a gene product.
• The promoter reporter constructs allow a protein to be expressed under the control of a target gene.
• Reporter fusions can fuse a protein of interest to a protein with a property of interest, therefore allowing the tagged protein to be further studied.
Key Terms
• substrate analog: Substrate analogs (substrate state analogues), are chemical compounds with a chemical structure that resemble the substrate molecule in an enzyme-catalyzed chemical reaction.
• luminescent: Emitting light by luminescence.
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals, or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.
To introduce a reporter gene into an organism, scientists place the reporter gene and the gene of interest in the same DNA construct to be inserted into the cell or organism. For bacteria or prokaryotic cells in culture, this is usually in the form of a circular DNA molecule called a plasmid. It is important to use a reporter gene that is not natively expressed in the cell or organism under study, since the expression of the reporter is being used as a marker for successful uptake of the gene of interest. Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue light, the enzyme luciferase, which catalyzes a reaction with luciferin to produce light, and the red fluorescent protein from the gene dsRed. A common reporter in bacteria is the E. coli lacZ gene, which encodes the protein beta-galactosidase. This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal. An example of a selectable-marker which is also a reporter in bacteria is the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol.
Reporter genes can also be used to assay for the expression of the gene of interest, which may produce a protein that has little obvious or immediate effect on the cell culture or organism. In these cases the reporter is directly attached to the gene of interest to create a gene fusion. The two genes are under the same promoter elements and are transcribed into a single messenger RNA molecule. The mRNA is then translated into protein. In these cases it is important that both proteins be able to properly fold into their active conformations and interact with their substrates despite being fused. In building the DNA construct, a segment of DNA coding for a flexible polypeptide linker region is usually included so that the reporter and the gene product will only minimally interfere with one another. This is often done with GFP. The resulting protein-GFP hybrid transcribed from the reporter construct now has a protein attached to GFP. In the case of GFP which fluorescence one can deduce that the attached protein is wherever the fluorescence is. This allows a researched to determine where in a cell a protein may be localized in a cell.
LICENSES AND ATTRIBUTIONS
CC LICENSED CONTENT, SPECIFIC ATTRIBUTION | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.13%3A_Bioinformatics/7.13H%3A_Reporter_Fusions.txt |
The methods used to get DNA into cells are varied (e.g., transformation, transduction, transfection, and electroporation).
LEARNING OBJECTIVES
Describe the methods of introducing foreign DNA into cells
Key Points
• When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation.
• In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection.
• Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present).
Key Terms
• transformation: The alteration of a bacterial cell caused by the transfer of DNA from another, especially if pathogenic.
• microorganisms: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.
The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g., transformation, transduction, transfection, electroporation).
When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation, and cells that are in a physiological state such that they can take up DNA, are said to be competent. In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection. Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific species and cell types that are used.
Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). In contrast, transduction involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.
Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.
When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the vector will survive when exposed to the antibiotic, while those that have failed to take up vector sequences will die. When mammalian cells (e.g., human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the kanMX cassette) confers resistance to the antibiotic Geneticin. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.14%3A_Cloning_Techniques/7.14A%3A_Putting_Foreign_DNA_into_Cells.txt |
When cloning genomic DNA, the DNA to be cloned is extracted from the organism of interest.
LEARNING OBJECTIVES
Explain the methods of obtaining DNA for molecular cloning experiments and the process of creating a recombinant DNA molecule
Key Points
• The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted.
• DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (complementary DNA or cDNA cloning), or in the form of synthetic DNA (artificial gene synthesis).
• The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process.
Key Terms
• DNA: A biopolymer of deoxyribonucleic acids (a type of nucleic acid) that has four different chemical groups, called bases: adenine, guanine, cytosine, and thymine.
• cloning: The production of a cloned embryo by transplanting the nucleus of a somatic cell into an ovum.
• cloning vector: A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, into which a foreign DNA fragment can be inserted.
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The word cloning in this context refers to the fact that the method involves the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA.
For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Virtually any tissue source can be used (even tissues from extinct animals) as long as the DNA is not extensively degraded. The DNA is then purified using simple methods to remove contaminating proteins (extraction with phenol), RNA (ribonuclease) and smaller molecules (precipitation and/or chromatography). Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (by a process known as Reverse-Transcription or RT-PCR) sequences prior to molecular cloning using primers or short DNA sequences specific for the region of interest. DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (complementary DNA or cDNA cloning), or in the form of synthetic DNA (artificial gene synthesis). cDNA cloning is usually used to obtain clones representative of the mRNA population of the cells of interest, while synthetic DNA is used to obtain any precise sequence defined by the designer.
Although a very large number of host organisms and molecular cloning vectors are used, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available and offer rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.
The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with that at the ends of the foreign DNA. Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme. Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and are not located within the gene of interest to be cloned.
The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are treated with restriction enzymes to generate fragments with ends capable of being linked to those of the vector and they are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together. This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism for amplification (a process known as transformation ). In mammalian cell culture, the analogous process of introducing DNA into cells is commonly known as transfection. Both transformation and transfection usually require preparation of the cells through a special growth regimen and chemical treatment process that will vary with the specific species and cell types that are used. Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic, typically ampicillin, that would otherwise kill the cells. Cells harboring the cloning vector will survive when exposed to the antibiotic, while those that have failed to take up cloning vector will die. The former can therefore be amplified and screened for the presence of the gene of interest in the cloning vector by restriction digest analysis. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.14%3A_Cloning_Techniques/7.14B%3A_Obtaining_DNA.txt |
The majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) as the host.
LEARNING OBJECTIVES
Describe the features of a typical cloning vector
Key Points
• E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.
• If the DNA to be cloned is exceptionally large, then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.
• Specialized applications may call for specialized host -vector systems.
Key Terms
• Escherichia coli: Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms).
• plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.
• molecular cloning: a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.
A very large number of host organisms and molecular cloning vectors are in use, but the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.
Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an expression vector is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example transfer of DNA from bacteria to plants), then a multiple host range vector (also termed shuttle vector) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.
Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility–(1) an origin of DNA replication is necessary for the vector (and recombinant sequences linked to it) to replicate inside the host organism, (2) one or more unique restriction endonuclease recognition sites that serves as sites where foreign DNA may be introduced, (3) a selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences, and (4) an additional gene that can be used for screening which cells contain foreign DNA.
7.14D: Shuttle Vectors and Expression Vectors
An expression vector is generally a plasmid that is used to introduce a specific gene into a target cell.
LEARNING OBJECTIVES
Explain the structure and function of shuttle and expression vectors
Key Points
• The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.
• Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a portable translation initiation sequence.
• Expression vectors are used for molecular biology techniques such as site-directed mutagenesis.
Key Terms
• plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.
• expression vector: An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell.
• transcription: The synthesis of RNA under the direction of DNA.
An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA, and in extension, proteins. Expression vectors are basic tools for biotechnology and the production of proteins such as insulin, which is important for the treatment of diabetes.
After expression of the gene product, the purification of the protein is required; but since the vector is introduced to a host cell, the protein of interest should be purified from the proteins of the host cell. Therefore, to make the purification process easy, the cloned gene should have a tag. This tag could be histidine (His) tag or any other marker peptide.
Expression vectors are used for molecular biology techniques such as site-directed mutagenesis. Cloning vectors, which are very similar to expression vectors, involve the same process of introducing a new gene into a plasmid, but the plasmid is then added into bacteria for replication purposes. In general, DNA vectors that are used in many molecular-biology gene-cloning experiments need not result in the expression of a protein.
Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).
A shuttle vector is a vector that can propagate in two different host species, hence, inserted DNA can be tested or manipulated in two different cell types. The main advantage of these vectors is that they can be manipulated in E. coli and then used in a system which is more difficult or slower to use.
Shuttle vectors can be used in both eukaryotes and prokaryotes. Shuttle vectors are frequently used to quickly make multiple copies of the gene in E. coli (amplification). They can also be used for in vitro experiments and modifications such as mutagenesis and PCR. One of the most common types of shuttle vectors is the yeast shuttle vector that contains components allowing for the replication and selection in both E. coli cells and yeast cells. The E. coli component of a yeast shuttle vector includes an origin of replication and a selectable marker, such as an antibiotic resistance like beta lactamase. The yeast component of a yeast shuttle vector includes an autonomously replicating sequence (ARS), a yeast centromere (CEN), and a yeast selectable marker. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.14%3A_Cloning_Techniques/7.14C%3A_Hosts_for_Cloning_Vectors.txt |
Enterobacteria phage λ (lambda phage, coliphage λ) is a bacterial virus that infects the bacterial species Escherichia coli.
LEARNING OBJECTIVES
Describe the life cycle of lambda phage
Key Points
• Lambda phage consists of a virus particle including a head (also known as a capsid), tail and tail fibers.
• Specialized transduction is the process by which a restricted set of bacterial genes are transferred to another bacterium.
• The genes that get transferred (donor genes) depend on where the phage genome is located on the chromosome.
Key Terms
• transduction: Transduction is the process by which DNA is transferred from one bacterium to another by a virus.
• lysogeny: the process by which a bacteriophage incorporates its nucleic acids into a host bacterium
• Lambda phage: Enterobacteria phage λ (lambda phage, coliphage λ) is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. This virus is temperate and may reside within the genome of its host through lysogeny.
Enterobacteria phage λ (lambda phage, coliphage λ) is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. This virus is temperate and may reside within the genome of its host through lysogeny.
Lambda phage consists of a virus particle including a head (also known as a capsid), tail and tail fibers. The head contains the phage’s double-stranded circular DNA genome. The phage particle recognizes and binds to its host, E. coli, causing DNA in the head of the phage to be ejected through the tail into the cytoplasm of the bacterial cell. Usually, a “lytic cycle” ensues, where the lambda DNA is replicated many times and the genes for head, tail and lysis proteins are expressed. This leads to assembly of multiple new phage particles within the cell and subsequent cell lysis, releasing the cell contents, including virions that have been assembled, into the environment. However, under certain conditions, the phage DNA may integrate itself into the host cell chromosome in the lysogenic pathway. In this state, the λ DNA is called a prophage and stays resident within the host’s genome without apparent harm to the host. The host can be termed a lysogen when a prophage is present.
Lambda phage has been of major importance in the study of specialized transduction.
Specialized transduction is the process by which a restricted set of bacterial genes are transferred to another bacterium. The genes that get transferred (donor genes) depend on where the phage genome is located on the chromosome. Specialized transduction occurs when the prophage excises imprecisely from the chromosome so that bacterial genes lying adjacent to the prophage are included in the excised DNA. The excised DNA is then packaged into a new virus particle, which delivers the DNA to a new bacterium where the donor genes can be inserted into the recipient chromosome or remain in the cytoplasm, depending on the nature of the bacteriophage. When the partially encapsulated phage material infects another cell and becomes a “prophage” (is covalently bonded into the infected cell’s chromosome), the partially coded prophage DNA is called a “heterogenote. ” | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.14%3A_Cloning_Techniques/7.14E%3A_Bacteriophage_Lambda_as_a_Cloning_Vector.txt |
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell.
LEARNING OBJECTIVES
Differentiate between expression vectors and transcription vectors
Key Points
• The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the “backbone” of the vector.
• The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.
• Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.
Key Terms
• vector: A carrier of a disease-causing agent.
• plasmids: Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell.
• chromosomes: A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements, and other nucleotide sequences.
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.
The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) are specifically for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene. Simpler vectors called transcription vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify their insert.
Insertion of a vector into the target cell is usually called transformation for bacterial cells, and transfection for eukaryotic cells, although the insertion of a viral vector is often called transduction.
Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert. Modern plasmids generally have many more features, notably including a “multiple cloning site” which includes nucleotide overhangs for insertion of an insert, and multiple restriction enzyme consensus sites to either side of the insert.
In the case of plasmids utilized as transcription vectors, incubating bacteria with plasmids generates hundreds or thousands of copies of the vector within the bacteria in hours. The vectors can be extracted from the bacteria, and the multiple cloning sites can be cut by restriction enzymes to excise the hundredfold or thousandfold amplified insert. These plasmid transcription vectors characteristically lack crucial sequences that code for polyadenylation sequences and translation termination sequences in translated mRNAs, making protein expression from transcription vectors impossible.
Plasmids may be conjugative / transmissible and non-conjugative. Conjugative vectors mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population, such as the F plasmid, as well as many R and some col plasmids. Non-conjugative vectors do not mediate DNA through conjugation, such as many R and col plasmids.
Viral vectors are generally genetically-engineered viruses carrying modified viral DNA or RNA that has been rendered noninfectious, but still contain viral promoters and also the transgene. This allows for the translation of the transgene through a viral promoter. However, because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Viral vectors are often designed for permanent incorporation of the insert into the host genome, and thus leave distinct genetic markers in the host genome after incorporating the transgene. For example, retroviruses leave a characteristic retroviral integration pattern after insertion that is detectable and indicates that the viral vector has incorporated into the host genome.
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Gene families are groups of functionally related genes arising from a duplicated gene.
LEARNING OBJECTIVES
Distinguish gene families from gene complexes
Key Points
• Genes duplicate over evolutionary times. As they duplicate this can lead to families of related genes. Since they come from the same progenitor gene, they often have related biochemical functions.
• Gene families can expand and contract over evolutionary time scales.
• Gene complexes are groups of genes that work in a fashion similar to gene families; however, they need not arise from gene duplication.
Key Terms
• phylogenetic: Of or relating to the evolutionary development of organisms.
• secondary structure: The general three-dimensional structure of a biopolymer such as DNA or a protein.
A gene family is a set of several similar genes, formed by duplication of a single original gene, that generally have similar biochemical functions. One such family are the genes for human haemoglobin subunits. The 10 genes are in two clusters on different chromosomes, called the α-globin and β-globin loci. Genes are categorized into families based on shared nucleotide or protein sequences. Phylogenetic techniques can be used as a more rigorous test. The positions of exons within the coding sequence can be used to infer common ancestry. Knowing the sequence of the protein encoded by a gene can allow researchers to apply methods that find similarities among protein sequences that provide more information than similarities or differences among DNA sequences. Furthermore, knowledge of the protein’s secondary structure gives further information about ancestry, since the organization of secondary structural elements presumably would be conserved even if the amino acid sequence changes considerably.
These methods often rely upon predictions based upon the DNA sequence. If the genes of a gene family encode proteins, the term protein family is often used in an analogous manner to gene family. The expansion or contraction of gene families along a specific lineage can be due to chance or can be the result of natural selection. To distinguish between these two cases is often difficult in practice. Recent work uses a combination of statistical models and algorithmic techniques to detect gene families that are under the effect of natural selection.
In contrast, gene complexes are simply tightly linked groups of genes, often created via gene duplication (sometimes called segmental duplication if the duplicates remain side-by-side). Here, each gene has a similar though slightly diverged function.
7.15B: Genomics and Biofuels
Learning Objectives
• Explain the process of creating new biofuels by using microbial genomics
Knowledge of the genomics of microorganisms is being used to find better ways to harness biofuels from algae and cyanobacteria. The primary sources of fuel today are coal, oil, wood, and other plant products, such as ethanol. Although plants are renewable resources, there is still a need to find more alternative renewable sources of energy to meet our population ‘s energy demands. The microbial world is one of the largest resources for genes that encode new enzymes and produce new organic compounds, and it remains largely untapped.
For microbial biomass breakdown, many candidates have already been identified. These include Clostridia species for their ability to degrade cellulose, and fungi that express genes associated with the decomposition of the most recalcitrant features of the plant cell wall, lignin, the phenolic “glue” that imbues the plant with structural integrity and pest resistance. The white rot fungus Phanerochaete chrysosporium produces unique extracellular oxidative enzymes that effectively degrade lignin by gaining access through the protective matrix surrounding the cellulose microfibrils of plant cell walls.
Another fungus, the yeast Pichia stipitis, ferments the five-carbon “wood sugar” xylose abundant in hardwoods and agricultural harvest residue. Pichia‘s recently-sequenced genome has revealed insights into the metabolic pathways responsible for this process, guiding efforts to optimize this capability in commercial production strains. Pathway engineering promises to produce a wider variety of organisms able to ferment the full repertoire of sugars derived from cellulose and hemicellulose and tolerate higher ethanol concentrations to optimize fuel yields. For instance, the hindgut contents of nature’s own bioreactor, the termite, has yielded more than 500 genes related to the enzymatic deconstruction of cellulose and hemicellulose.
Key Points
• Microorganisms can encode new enzymes and produce new organic compounds that can be used as biofuels.
• Genomic analysis of the fungus Pichia will allow optimization of its use in fermenting ethanol fuels.
• Analysis of the microbes in the hindgut of termites have found 500 genes that may be useful in enzymatic destruction of cellulose.
• Genetic markers have been used in forensic analysis, like in 2001 when the FBI used microbial genomics to determine a specific strain of anthrax that was found in several pieces of mail.
• Genomics is used in agriculture to develop plants with more desirable traits, such as drought and disease resistance.
Key Terms
• renewable resource: a natural resource such that it is replenished by natural processes at a rate comparable to its rate of consumption by humans or other users
• biofuel: any fuel that is obtained from a renewable biological resource | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.15%3A_Genome_Evolution/7.15A%3A_Gene_Families.txt |
Learning Objectives
• Review genome reduction
Genome size is the total amount of DNA contained within one copy of a single genome. Over evolutionary times, genomes tend to increase in size due to the accumulation of duplication of the genome and an increase in genetic elements. The opposite or genome reduction also occurs. Genome reduction, also known as genome degradation, is the process by which a genome shrinks relative to its ancestor. Genomes fluctuate in size regularly; however, genome size reduction is most significant in bacteria.The most evolutionary significant cases of genome reduction may be the eukaryotic organelles that are derived from bacteria: the mitochondrion and plastid. These organelles are descended from endosymbionts, which can only survive within the host cell and which the host cell likewise needs for survival. Many mitochondria have less than 20 genes in their entire genome, whereas a free-living bacterium generally has at least 1000 genes. Many genes have been transferred to the host nucleus, while others have simply been lost and their function replaced by host processes.Other bacteria have become endosymbionts or obligate intracellular pathogens and have experienced extensive genome reduction as a result. This process seems to be dominated by genetic drift resulting from small population size, low recombination rates, and high mutation rates, as opposed to selection for smaller genomes. Some free-living marine bacterioplanktons also shows signs of genome reduction, which are hypothesized to be driven by natural selection.
Obligate endosymbiotic species are characterized by a complete inability to survive outside their host environment. These species have become a considerable threat to human health, as they are often highly capable of evading human immune systems and manipulating the host environment to acquire nutrients. A common explanation for these keen manipulative abilities is the compact and efficient genomic structure consistently found in obligate endosymbionts. This compact genome structure is the result of massive losses of extraneous DNA – an occurrence that is exclusively associated with the loss of a free-living stage. In fact, as much as 90% of the genetic material can be lost when a species makes the evolutionary transition from a free-living to obligate intracellular lifestyle. Common examples of species with reduced genomes include: Buchnera aphidicola, Rickettsia prowazekii and Mycobacterium leprae. One obligate endosymbiont of psyllid, Candidatus Carsonella ruddii, has the smallest genome currently known among cellular organisms at 160kb. It is important to note, however, that some obligate intracellular species have positive fitness effects on their hosts. The reductive evolution model has been proposed as an effort to define the genomic commonalities seen in all obligate endosymbionts. This model illustrates four general features of reduced genomes and obligate intracellular species: ‘genome streamlining’ resulting from relaxed selection on genes that are superfluous in the intracellular environment; a bias towards deletions (rather than insertions), which heavily affects genes that have been disrupted by accumulation of mutations (pseudogenes); very little or no capability for acquiring new DNA; and considerable reduction of effective population size in endosymbiotic populations, particularly in species that rely on vertical transmission. Based on this model, it is clear that endosymbionts face different adaptive challenges than free-living species.
Key Points
• Genomes tend to increase in size over time, however exceptions occur.
• Genome reduction is observed in species that depend on a host for survival, the most extreme examples are eukaryotic organelles that have bacterial origins such as mitochondria.
• As an endosymbiont becomes dependent on its host, it becomes an obligate endosymbiont; during this time the genome is reduced due to deletions of genes not needed to live in the host. Parts of the genome can be transferred to the host as well, leading to further genome reduction.
Key Terms
• genome: The complete genetic information (either DNA or, in some viruses, RNA) of an organism, typically expressed in the number of basepairs.
• endosymbiont: An organism that lives within the body or cells of another organism.
7.15D: Pathogenicity Islands
Learning Objectives
• Describe pathogenicity islands
Pathogenicity islands (PAIs) are a distinct class of genomic islands acquired by microorganisms through horizontal gene transfer. They are incorporated in the genome of pathogenic organisms, but are usually absent from those nonpathogenic organisms of the same or closely related species. These mobile genetic elements may range from 10-200 kb and encode genes which contribute to the virulence of the respective pathogen. Typical examples are adherence factors, toxins, iron uptake systems, invasion factors, and secretion systems. Pathogenicity islands are discrete genetic units flanked by direct repeats, insertion sequences or tRNA genes, which act as sites for recombination into the DNA. Cryptic mobility genes may also be present, indicating the provenance as transduction. One species of bacteria may have more than one PAI (i.e. Salmonella has at least 5). They are transferred through horizontal gene transfer events such as transfer by a plasmid, phage, or conjugative transposon.
Pathogenicity islands carry genes encoding one or more virulence factors, including, but not limited to, adhesins, toxins, or invasins. They may be located on a bacterial chromosome or may be transferred within a plasmid. The GC-content of pathogenicity islands often differs from that of the rest of the genome, potentially aiding in their detection within a given DNA sequence. PAIs are flanked by direct repeats; the sequence of bases at two ends of the inserted sequence is the same. They carry functional genes, such as integrases, transposases, or part of insertion sequences, to enable insertion into host DNA. PAIs are often associated with tRNA genes, which target sites for this integration event. They can be transferred as a single unit to new bacterial cells, thus conferring virulence to formerly benign strains.
Key Points
• A host may have more than one pathogenicity island.
• Pathogenicity islands are transferred horizontally, through plasmids or transposons.
• The addition of a pathogenicity island to a non-invasive species can make the non-invasive species pathogenic.
Key Terms
• horizontal gene transfer: The transfer of genetic material from one organism to another one that is not its offspring; especially common among bacteria.
• transposon: A segment of DNA that can move to a different position within a genome.
• integrase: Any enzyme that integrates viral DNA into that of an infected cell.
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Using metagenomics, the microbial constituents of the world can be identified by culturing each individual species.
LEARNING OBJECTIVES
Recognize the methods used to detect uncultured microorganisms
Key Points
• Many organisms that can cause diseases are not culturable, so a unicellular culture cannot be obtained.
• Initial studies of microbial fauna showed that they are more diverse than expected and contained many microbes that were not culturable.
• Advances in molecular biological techniques allow the sequencing of all or at least many of the genomes of microbes found in a sample.
Key Terms
• shotgun sequencing: A DNA sequencing technique in which a large number of small fragments of a long DNA strand are generated at random, sequenced, and reassembled to form a sequence of the original strand.
• culturable: Able to be cultured (grown in a suitable environment).
• high-throughput sequencing: Technologies that parallelize the sequencing process, producing thousands or millions of sequences at once.
Identification of bacteria in the laboratory is particularly relevant in medicine, where the correct treatment is determined by the bacterial species causing an infection. Consequently, the need to identify human pathogens was a major impetus for the development of techniques to identify bacteria.
Early studies have shown that the microbial life around us in the air, sea, and soil is very diverse and only a small fraction of the species are known. One limitation of identifying human pathogens or conventional sequencing begins with a culture of identical cells as a source of DNA. However, early metagenomic studies revealed that there are probably large groups of microorganisms in many environments that cannot be cultured and thus cannot be sequenced. These early studies focused on 16S ribosomal RNA sequences which are relatively short, often conserved within a species, and generally different between species. Many 16S rRNA sequences have been found which do not belong to any known cultured species, indicating that there are numerous non-isolated organisms out there. These surveys of ribosomal RNA (rRNA) genes taken directly from the environment revealed that cultivation based methods find less than 1% of the bacterial and archaeal species in a sample.
The discovery of such diversity led to the field of metagenomics, which is the study of metagenomes, genetic material recovered directly from environmental samples. Rather than culturing a microbe, this approach takes a sample and identifies the different species in it by sequencing all the species simultaneously. However, recovery of DNA sequences longer than a few thousand base pairs from environmental samples was very difficult until recent advances in molecular biological techniques. More specifically, the construction of libraries in bacterial artificial chromosomes (BACs) provided better vectors for molecular cloning.
Advances in bioinformatics, refinements of DNA amplification, and the proliferation of computational power have greatly aided the analysis of DNA sequences recovered from environmental samples. These advances have allowed the adaptation of shotgun sequencing to metagenomic samples. The approach, used to sequence many cultured microorganisms and the human genome, randomly shears DNA, sequences many short sequences, and reconstructs them into a consensus sequence.
Shotgun sequencing and screens of clone libraries reveal genes present in environmental samples. This can be helpful in understanding the ecology of a community, particularly if multiple samples are compared to each other. This was further followed by high-throughput sequencing which did the same process as the shotgun sequencing but at a much bigger scale in terms of the amount of DNA that could sequenced from one sample. This provides information both on which organisms are present and what metabolic processes are possible in the community. Using metagenomics, and the resultant sequencing of uncultured microbes, metagenomics has the potential to advance knowledge in a wide variety of fields. It can also be applied to solve practical challenges in medicine, engineering, agriculture, and sustainability. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.16%3A_Environmental_Genomics/7.16A%3A_Detecting_Uncultured_Microorganisms.txt |
Viruses are the most abundant biological entity on earth, they outnumber all other lifeforms on earth combined.
LEARNING OBJECTIVES
Recognize viral genomic diversity in nature
Key Points
• Viruses infect all types of cellular life including animals, plants, bacteria, and fungi.
• Metagenomic sequencing has identified that there are an enormous number of species of virus in nature, showing that a a teaspoon of water has a million species of viruses.
• Viruses kill about 20% of the microbes that constitute the oceans microbial fauna, each day. This viral-driven biological turn over is needed for for life on earth to continue.
• Viruses can transfer genetic material from one species to another, and as such viruses may be a main driving force of evolution.
Key Terms
• metagenomics: The study of genomes recovered from environmental samples; especially the differentiation of genomes from multiple organisms or individuals, either in a symbiotic relationship, or at a crime scene.
• stool: Feces; excrement.
Viruses are by far the most abundant biological entities on earth and they outnumber all the others put together. They infect all types of cellular life including animals, plants, bacteria, and fungi. However, different types of viruses can infect only a limited range of hosts and many are species-specific. Some, such as smallpox virus for example, can infect only one species – in this case humans, and are said to have a narrow host range. Other viruses, such as rabies virus, can infect different species of mammals and are said to have a broad range. The viruses that infect plants are harmless to animals, and most viruses that infect other animals are harmless to humans. The host range of some bacteriophages is limited to a single strain of bacteria and they can be used to trace the source of outbreaks of infections by a method called phage typing.
Metagenomic sequencing is particularly useful in the study of viral communities. As viruses lack a shared universal phylogenetic marker (as 16S RNA for bacteria and archaea, and 18S RNA for eukarya), the only way to access the genetic diversity of the viral community from an environmental sample is through metagenomics. Viral metagenomes (also called viromes) should therefore provide more and more information about viral diversity and evolution. In 2002, Mya Breitbart, Forest Rohwer, and colleagues used environmental shotgun sequencing to show that 200 liters of seawater contains over 5,000 different viruses. Subsequent studies showed that there are more than a thousand viral species in human stool and possibly a million different viruses per kilogram of marine sediment, including many bacteriophages. Essentially all of the viruses in these studies were new species.
To understand the complex diversity of viruses a further look at viruses in aquatic environments shows, a teaspoon of seawater contains about one million viruses. They are essential to the regulation of saltwater and freshwater ecosystems. Most of these viruses are bacteriophages, which are harmless to plants and animals. They infect and destroy the bacteria in aquatic microbial communities, comprising the most important mechanism of recycling carbon in the marine environment. The organic molecules released from the bacterial cells by the viruses stimulate fresh bacterial and algal growth. Microorganisms constitute more than 90% of the biomass in the sea. It is estimated that viruses kill approximately 20% of this biomass each day and that there are 15 times as many viruses in the oceans as there are bacteria and archaea. Viruses are the main agents responsible for the rapid destruction of harmful algal blooms, which often kill other marine life. The number of viruses in the oceans decreases further offshore and deeper into the water, where there are fewer host organisms. The effects of marine viruses are far-reaching. By increasing the amount of photosynthesis in the oceans, viruses are indirectly responsible for reducing the amount of carbon dioxide in the atmosphere by approximately three gigatonnes of carbon per year. Like any organism, marine mammals are susceptible to viral infections. In 1988 and 2002, thousands of harbor seals were killed in Europe by phocine distemper virus. Many other viruses, including caliciviruses, herpesviruses, adenoviruses, and parvoviruses circulate in marine mammal populations.
Viruses are an important natural means of transferring genes between different species, which increases genetic diversity and drives evolution. It is thought that viruses played a central role in the early evolution, before the diversification of bacteria, archaea, and eukaryotes and at the time of the last universal common ancestor of life on earth. Viruses are still one of the largest reservoirs of unexplored genetic diversity on earth.
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Learning Objectives
Explain how an activator works to increase transcription of a gene
Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the operator sequences that act as a positive regulator to turn genes on and activate them. For example, when glucose is scarce, E. colibacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate genes must be transcribed. This type of process can be seen in the lac operon which is turned on in the presence of lactose and absence of glucose.
When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. When glucose levels decline in the cell, accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that control the processing of alternative sugars, such as the lac operon. The CAP assists in production in the absence of glucose. CAP is a transcriptional activator that exists as a homodimer in solution, with each subunit comprising a ligand-binding domain at the N-terminus, which is also responsible for the dimerization of the protein and a DNA-binding domain at the C-terminus. Two cAMP molecules bind dimeric CAP with negative cooperativity and function as allosteric effectors by increasing the protein’s affinity for DNA. CAP has a characteristic helix-turn-helix structure that allows it to bind to successive major grooves on DNA. This opens up the DNA molecule, allowing RNA polymerase to bind and transcribe the genes involved in lactose catabolism. When cAMP binds to CAP, the complex binds to the promoter region of the genes that are needed to use the alternate sugar sources. In these operons, a CAP-binding site is located upstream of the RNA-polymerase-binding site in the promoter. This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes. As cAMP-CAP is required for transcription of the lac operon, this requirement reflects the greater simplicity with which glucose may be metabolized in comparison to lactose.
Key Points
• Catabolite activator protein (CAP) must bind to cAMP to activate transcription of the lac operon by RNA polymerase.
• CAP is a transcriptional activator with a ligand-binding domain at the N-terminus and a DNA -binding domain at the C-terminus.
• cAMP molecules bind to CAP and function as allosteric effectors by increasing CAP’s affinity to DNA.
Key Terms
• RNA polymerase: a DNA-dependent RNA polymerase, an enzyme, that produces RNA
• operon: a unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together
• promoter: the section of DNA that controls the initiation of RNA transcription
7.17B: The Initiation Complex and Translation Rate
The first step of translation is ribosome assembly, which requires initiation factors.
LEARNING OBJECTIVES
Discuss how eukaryotes assemble ribosomes on the mRNA to begin translation
Key Points
• The components involved in ribosome assembly are brought together by the help of proteins called initiation factors which bind to the small ribosomal subunit.
• Initiator tRNA is used to locate the start codon AUG (the amino acid methionine) which establishes the reading frame for the mRNA strand.
• GTP carried by eIF2 is the energy source used for loading the initiator tRNA carried by the small ribosomal subunit on the correct start codon in the mRNA.
• GTP carried by eIF5 is the energy source for assembling the large and small ribosomal subunits together.
Key Terms
• reading frame: either of three possible triplets of codons in which a DNA sequence could be transcribed
• phosphorylation: the addition of a phosphate group to a compound; often catalyzed by enzymes
Ribosome Assembly and Translation Rate
Like transcription, translation is controlled by proteins that bind and initiate the process. In translation, before protein synthesis can begin, ribosome assembly has to be completed. This is a multi-step process.
In ribosome assembly, the large and small ribosomal subunits and an initiator tRNA (tRNAi) containing the first amino acid of the final polypeptide chain all come together at the translation start codon on an mRNA to allow translation to begin. First, the small ribosomal subunit binds to the tRNAi which carries methionine in eukaryotes and archaea and carries N-formyl-methionine in bacteria. (Because the tRNAi is carrying an amino acid, it is said to be charged.) Next, the small ribosomal subunit with the charged tRNAi still bound scans along the mRNA strand until it reaches the start codon AUG, which indicates where translation will begin. The start codon also establishes the reading frame for the mRNA strand, which is crucial to synthesizing the correct sequence of amino acids. A shift in the reading frame results in mistranslation of the mRNA. The anticodon on the tRNAi then binds to the start codon via basepairing. The complex consisting of mRNA, charged tRNAi, and the small ribosomal subunit attaches to the large ribosomal subunit, which completes ribosome assembly. These components are brought together by the help of proteins called initiation factors which bind to the small ribosomal subunit during initiation and are found in all three domains of life. In addition, the cell spends GTP energy to help form the initiation complex. Once ribosome assembly is complete, the charged tRNAi is positioned in the P site of the ribosome and the empty A site is ready for the next aminoacyl-tRNA. The polypeptide synthesis begins and always proceeds from the N-terminus to the C-terminus, called the N-to-C direction.
In eukaryotes, several eukaryotic initiation factor proteins (eIFs) assist in ribosome assembly. The eukaryotic initiation factor-2 (eIF-2) is active when it binds to guanosine triphosphate (GTP). With GTP bound to it, eIF-2 protein binds to the small 40S ribosomal subunit. Next, the initiatior tRNA charged with methionine (Met-tRNAi) associates with the GTP-eIF-2/40S ribosome complex, and once all these components are bound to each other, they are collectively called the 43S complex.
Eukaryotic initiation factors eIF1, eIF3, eIF4, and eIF5 help bring the 43S complex to the 5′-m7G cap of an mRNA be translated. Once bound to the mRNA’s 5′ m7G cap, the 43S complex starts travelling down the mRNA until it reaches the initiation AUG codon at the start of the mRNA’s reading frame. Sequences around the AUG may help ensure the correct AUG is used as the initiation codon in the mRNA.
Once the 43S complex is at the initiation AUG, the tRNAi-Met is positioned over the AUG. The anticodon on tRNAi-Met basepairs with the AUG codon. At this point, the GTP bound to eIF2 in the 43S complexx is hydrolyzed to GDP + phosphate, and energy is released. This energy is used to release the eIF2 (with GDP bound to it) from the 43S complex, leaving the 40S ribosomal subunit and the tRNAi-Met at the translation start site of the mRNA.
Next, eIF5 with GTP bound binds to the 40S ribosomal subunit complexed to the mRNA and the tRNAi-Met. The eIF5-GTP allows the 60S large ribosomal subunit to bind. Once the 60S ribosomal subunit arrives, eIF5 hydrolyzes its bound GTP to GDP + phosphate, and energy is released. This energy powers assembly of the two ribosomal subunits into the intact 80S ribosome, with tRNAi-Met in its P site while also basepaired to the initiation AUG codon on the mRNA. Translation is ready to begin.
The binding of eIF-2 to the 40S ribosomal subunit is controlled by phosphorylation. If eIF-2 is phosphorylated, it undergoes a conformational change and cannot bind to GTP. Therefore, the 43S complex cannot form properly and translation is impeded. When eIF-2 remains unphosphorylated, it binds the 40S ribosomal subunit and actively translates the protein.
The ability to fully assemble the ribosome directly affects the rate at which translation occurs. But protein synthesis is regulated at various other levels as well, including mRNA synthesis, tRNA synthesis, rRNA synthesis, and eukaryotic initiation factor synthesis. Alteration in any of these components affects the rate at which translation can occur.
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The genetic code is a degenerate, non-overlapping set of 64 codons that encodes for 21 amino acids and 3 stop codons.
LEARNING OBJECTIVES
Describe the genetic code and how the nucleotide sequence prescribes the amino acid and the protein sequence
Key Points
• The relationship between DNA base sequences and the amino acid sequence in proteins is called the genetic code.
• There are 61 codons that encode amino acids and 3 codons that code for chain termination for a total of 64 codons.
• Unlike, eukayrotes, a bacterial chromosome is a covalently-closed circle.
• The DNA double helix must partially unwind for transcription to occur; this unwound region is called a transcription bubble.
Key Terms
• nucleotide: the monomer comprising DNA or RNA molecules; consists of a nitrogenous heterocyclic base that can be a purine or pyrimidine, a five-carbon pentose sugar, and a phosphate group
• amino acid: Any of 20 naturally occurring α-amino acids (having the amino, and carboxylic acid groups on the same carbon atom), and a variety of side chains, that combine, via peptide bonds, to form proteins.
• redundancy: duplication of components, such as amino acid codons, to provide survival of the total system in case of failure of single components
The Genetic Code: Nucleotide sequences prescribe the amino acids
The genetic code is the relationship between DNA base sequences and the amino acid sequence in proteins. Features of the genetic code include:
• Amino acids are encoded by three nucleotides.
• It is non-overlapping.
• It is degenerate.
There are 21 genetically-encoded amino acids universally found in the species from all three domains of life. ( There is a 22nd genetically-encooded amino acid, Pyl, but so far it has only been found in a handful of Archaea and Bacteria species.) Yet there are only four different nucleotides in DNA or RNA, so a minimum of three nucleotides are needed to code each of the 21 (or 22) amino acids. The set of three nucleotides that codes for a single amino acid is known as a codon. There are 64 codons in total, 61 that encode amino acids and 3 that code for chain termination. Two of the codons for chain termination can, under certain circumstances, instead code for amino acids.
Genetic Code Table.: A codon is made of three nucleotides. Consequently there are 43 (=64) different codons. The 64 codons encode 22 different amino acids and three termination codons, also called stop codons.
Degeneracy is the redundancy of the genetic code. The genetic code has redundancy, but no ambiguity. For example, although codons GAA and GAG both specify glutamic acid (redundancy), neither of them specifies any other amino acid (no ambiguity). The codons encoding one amino acid may differ in any of their three positions. For example, the amino acid glutamic acid is specified by GAA and GAG codons (difference in the third position); the amino acid leucine is specified by UUA, UUG, CUU, CUC, CUA, CUG codons (difference in the first or third position); while the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, AGC (difference in the first, second or third position). These properties of the genetic code make it more fault-tolerant for point mutations.
Origin of transcription on prokaryotic organisms
Prokaryotes are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. The central region of the cell in which prokaryotic DNA resides is called the nucleoid region. Bacterial and Archaeal chromosomes are covalently-closed circles that are not as extensively compacted as eukaryotic chromosomes, but are compacted nonetheless as the diameter of a typical prokaryotic chromosome is larger than the diameter of a typical prokaryotic cell. Additionally, prokaryotes often have abundant plasmids, which are shorter, circular DNA molecules that may only contain one or a few genes and often carry traits such as antibiotic resistance.
Transcription in prokaryotes (as in eukaryotes) requires the DNA double helix to partially unwind in the region of RNA synthesis. The region of unwinding is called a transcription bubble. Transcription always proceeds from the same DNA strand for each gene, which is called the template strand. The RNA product is complementary to the template strand and is almost identical to the other (non-template) DNA strand, called the sense or coding strand. The only difference is that in RNA all of the T nucleotides are replaced with U nucleotides.
The nucleotide on the DNA template strand that corresponds to the site from which the first 5′ RNA nucleotide is transcribed is called the +1 nucleotide, or the initiation site. Nucleotides preceding, or 5′ to, the template strand initiation site are given negative numbers and are designated upstream. Conversely, nucleotides following, or 3′ to, the template strand initiation site are denoted with “+” numbering and are called downstream nucleotides. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.18%3A_Global_Regulatory_Mechanisms/7.18A%3A_Transcription_in_Prokaryotes.txt |
Learning Objectives
• Explain the relationship between structure and function of an operon and the ways in which repressors regulate gene expression
Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon. If tryptophan is present in the environment, then E. coli does not need to synthesize it; the switch controlling the activation of the genes in the trp operon is turned off. However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized.
A DNA sequence that codes for proteins is referred to as the coding region. The five coding regions for the tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon. Just before the coding region is the transcriptional start site. This is the region of DNA to which RNA polymerase binds to initiate transcription. The promoter sequence is upstream of the transcriptional start site. Each operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and regulate transcription.
A DNA sequence called the operator sequence is encoded between the promoter region and the first trp-coding gene. This operator contains the DNA code to which the repressor protein can bind. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding and transcribing the downstream genes.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators.
Key Points
• The operator sequence is encoded between the promoter region and the first trp-coding gene.
• The trp operon is repressed when tryptophan levels are high by binding the repressor protein to the operator sequence via a corepressor which blocks RNA polymerase from transcribing the trp-related genes.
• The trp operon is activated when tryptophan levels are low by dissociation of the repressor protein to the operator sequence which allows RNA polymerase to transcribe the trp genes in the operon.
Key Terms
• repressor: any protein that binds to DNA and thus regulates the expression of genes by decreasing the rate of transcription
• operon: a unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together
7.18C: The Stringent Response
The stringent response is a stress response that occurs in bacteria in reaction to amino-acid starvation or other stress conditions.
LEARNING OBJECTIVES
Explain the function of the alarmone (p)ppGpp in the stringent response
Key Points
• The stringent response is signaled by the alarmone (p)ppGpp.
• In Escherichia coli, (p)ppGpp production is mediated by the ribosomal protein L11 and the ribosome-associated protein RelA.
• In other bacteria, stringent response is mediated by a variety of RelA/SpoT Homologue (RSH) proteins. Some only have synthetic, hydrolytic, or both (Rel) activities.
Key Terms
• stringent response: The stringent response, also called stringent control, is a stress response that occurs in bacteria and plant chloroplasts in reaction to amino-acid starvation, fatty acid limitation, iron limitation, heat shock, and other stress conditions.
• alarmone: Alarmone is an intracellular signal molecule that is produced due to harsh environmental factors.
• amino-acid starvation: The amino acid response pathway is triggered by a shortage of any essential amino acid.
The stringent response, also called stringent control, is a stress response that occurs in bacteria and plant chloroplasts in reaction to amino-acid starvation, fatty acid limitation, iron limitation, heat shock, and other stress conditions. The stringent response is signaled by the alarmone (p)ppGpp and modulating transcription of up to 1/3 of all genes in the cell. This in turn causes the cell to divert resources away from growth and division and toward amino acid synthesis in order to promote survival until nutrient conditions improve.
In Escherichia coli, (p)ppGpp production is mediated by the ribosomal protein L11. The ribosome-associated protein RelA with the A-site bound deacylated tRNA is the ultimate inducer. RelA converts GTP and ATP into pppGpp by adding the pyrophosphate from ATP onto the 3′ carbon of the ribose in GTP releasing AMP. pppGpp is converted to ppGpp by the gpp gene product, releasing Pi. ppGpp is converted to GDP by the spoT gene product, releasing pyrophosphate (PPi). GDP is converted to GTP by the ndk gene product. Nucleoside triphosphate (NTP) provides the Pi. It is converted to nucleoside diphosphate (NDP).
In other bacteria, stringent response is mediated by a variety of RelA/SpoT Homologue (RSH) proteins, with some having only synthetic, hydrolytic, or both (Rel) activities. The disable of stringent response by distruption of relA and spoT in Pseudomonas aeruginosa, produces infectious cells and biofilms that have nutrient limitations. They are more susceptible to antibiotics.
During the stringent response, (p)ppGpp accumulation affects the resource-consuming cell processes replication, transcription, and translation. (p)ppGpp is thought to bind RNA polymerase and alter the transcriptional profile, decreasing the synthesis of translational machinery (such as rRNA and tRNA), and increasing the transcription of biosynthetic genes. Additionally, the initiation of new rounds of replication is inhibited and the cell cycle arrests until nutrient conditions improve. Translational GTP involved in protein biosynthesis are also affected by ppGpp, with Initiation Factor 2 (IF2) being the main target.
Chemical reaction catalyzed by RelA: ATP+GTP→AMP+pppGppATP+GTP→AMP+pppGpp
Chemical reaction catalyzed by SpoT: ppGpp→GDP+PPi | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.18%3A_Global_Regulatory_Mechanisms/7.18B%3A_The_trp_Operon_-_A_Repressor_Operon.txt |
Repression of anabolic pathways is regulated by altering transcription rates.
LEARNING OBJECTIVES
Differentiate between inducible and repressible systems in gene regulation
Key Points
• Regulation of transcription controls when transcription occurs and how much RNA is created.
• Gene regulation is either controlled by an inducible system or a repressible system.
• In prokaryotes, regulation of transcription is needed for the cell to quickly adapt to the ever-changing outer environment.
Key Terms
• anabolic pathways: Anabolism describes the set of metabolic pathways that construct molecules from smaller units.
• transcription: The synthesis of RNA under the direction of DNA.
• gene: A unit of heredity; a segment of DNA or RNA that is transmitted from one generation to the next. It carries genetic information such as the sequence of amino acids for a protein.
Repression of anabolic pathways is regulated by altering transcription rates. Transcriptional regulation is the change in gene expression levels by altering transcription rates.
Regulation of transcription controls when transcription occurs and how much RNA is created. Transcription of a gene by RNA polymerase can be regulated by at least five mechanisms:
• Specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e. sigma factors used in prokaryotic transcription).
• Repressors bind to non-coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase’s progress along the strand, thus impeding the expression of the gene.
• General transcription factors position RNA polymerase at the start of a protein -coding sequence and then release the polymerase to transcribe the mRNA.
• Activators enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA.
• Enhancers are sites on the DNA helix that are bound to by activators in order to loop the DNA bringing a specific promoter to the initiation complex.
Regulatory protein is a term used in genetics to describe a protein involved in regulating gene expression. Such proteins are usually bound to a DNA binding site which is sometimes located near the promoter although this is not always the case. Sites of DNA sequences where regulatory proteins bind are called enhancer sequences. Regulatory proteins are often needed to be bound to a regulatory binding site to switch a gene on (activator) or to shut off a gene (repressor). Generally, as the organism grows more sophisticated, its cellular protein regulation becomes more complicated and, indeed, some human genes can be controlled by many activators and repressors working together.
In prokaryotes, regulation of transcription is needed for the cell to quickly adapt to the ever-changing outer environment. The presence or the quantity and type of nutrients determines which genes are expressed; in order to do that, genes must be regulated in some fashion. In prokaryotes, repressors bind to regions called operators that are generally located downstream from and near the promoter (normally part of the transcript). Activators bind to the upstream portion of the promoter, such as the CAP region (completely upstream from the transcript). A combination of activators, repressors and rarely enhancers (in prokaryotes) determines whether a gene is transcribed.
Gene regulation can be summarized as how genes respond: inducible systems or repressible systems. An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to “induce expression. ” The manner in which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells. A repressible system is on except in the presence of some molecule (called a corepressor) that suppresses gene expression. The molecule is said to “repress expression. ” The manner in which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
For example, when E. coli bacteria are subjected to heat stress, the σ32 subunit of its RNA polymerase changes in such a way that the enzyme binds to a specialized set of promoters that precede genes for heat-shock response proteins.
Another example is when a cell contains a surplus amount of the amino acid tryptophan, the acid binds to a specialized repressor protein (tryptophan repressor). The binding changes the structural conformity of the repressor such that it binds to the operator region for the operon that synthesizes tryptophan, preventing their expression and thus suspending production. This is a form of negative feedback.
In bacteria, the lac repressor protein blocks the synthesis of enzymes that digest lactose when there is no lactose to feed on. When lactose is present, it binds to the repressor, causing it to detach from the DNA strand.
7.18E: The AraC Regulator
The L-arabinose operon, also called ara operon, encodes enzymes needed for the catabolism of arabinose to xylulose 5-phosphate.
LEARNING OBJECTIVES
Describe the regulatory mechanism of the AraC protein in the presence and absence of arabinose
Key Takeaways
Key Points
• The structural gene, which encodes arabinose breakdown enzymes, is araBAD.
• The ara operon is regulated by the AraC protein.
• When arabinose is present, arabinose binds AraC and prevents it from interacting.
Key Terms
• operon: A unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together.
• L-arabinose: Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group.
• catabolism: Destructive metabolism, usually includes the release of energy and breakdown of materials.
The L-arabinose operon, also called ara operon, is a gene sequence encoding enzymes needed for the catabolism of arabinose to xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. It has both positive and negative regulation. The operon is found in Escherichia coli (E. coli).
It has been a focus for research in molecular biology since 1970, and has been investigated extensively at its genetic, biochemical, physiological, and biophysical levels.
The structural gene, which encodes arabinose breakdown enzymes, is araBAD. The regulator gene is araC. The genes, araBAD and araC, are transcribed in opposite directions.
The operators are araI and araO2. The operators lie between the AraC.
AraI lies between the structural genes and the operator. The araI1 and araI2 are DNA -binding sites that, when occupied by AraC, induce expression.
Sequence of the Operon: 5′—–araC—–araO—–araI—–araB—–araA—–araD—–3′
The ara operon is regulated by the AraC protein. If arabinose is absent, the dimer AraC protein represses the structural gene by binding to araI1 and araO2 and the DNA forms a loop, which prevents RNA polymerase from binding to the promoter of the ara operon, thereby blocking transcription.
When arabinose is present, arabinose binds AraC and prevents it from interacting. This breaks the DNA loop. The two AraC-arabinose complexes bind to the araI site which promotes transcription. When arabinose is present, AraC acts as an activator and it builds a complex: AraC + arabinose. This complex is needed for RNA polymerase to bind to the promoter and transcribe the araoperon.
Also for activation, the binding of another structure to araI is needed: CRP (formerly known as CAP) + cyclic AMP. Thus the activation depends on the presence of arabinose and cAMP.
LICENSES AND ATTRIBUTIONS
CC LICENSED CONTENT, SPECIFIC ATTRIBUTION | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.18%3A_Global_Regulatory_Mechanisms/7.18D%3A_Repression_of_Anabolic_Pathways.txt |
Antisense RNAs are single-stranded RNA molecules that can bind and inhibit specific mRNA translation to protein.
Learning Objectives
• Explain RNA regulation via antisense RNA
Key Points
• Antisense RNAs are specific to mRNAs based on the principle of complementary base pairs.
• Antisense RNAs bind to mRNAs and inhibit the ability of these mRNAs to be translated into functioning protein.
• Naturally occurring antisense RNAs have been identified in E. coli, which carry an R1 plasmid and control the hok/sok system that involves the production of toxic and antitoxic products.
Key Terms
• morphogenesis: The differentiation of tissues and growth in organisms.
Gene regulation, the ability to control whether a gene is expressed or not, is critical in controlling cellular and metabolic processes and contributes to diversity and variation in organisms. Furthermore, it is the key determinant in cellular differentiation and morphogenesis. There are specific types of RNA molecules that can be utilized to control gene regulation, including messenger RNAs (mRNAs), small RNAs such as microRNAs and lastly, antisense RNAs. The following is a brief overview of antisense RNAs and their role in RNA regulation. Antisense RNAs have been recently investigated as a new class of antiviral drugs.
Antisense RNAs are single-stranded RNA molecules that exhibit a complementary relationship to specific mRNAs. Antisense RNAs are utilized for gene regulation and specifically target mRNA molecules that are used for protein synthesis. The antisense RNA can physically pair and bind to the complementary mRNA, thus inhibiting the ability of the mRNA to be processed in the translation machinery. Pairing antisense RNA is a technique that can be utilized within the laboratory for gene regulation — however, it is not without limitations. Naturally occurring antisense RNAs have been isolated in a various microbes, including the E. coli RI plasmid, which uses a hok/sok system. A hok/sok system is a mechanism employed by E. coli that is used as a postsegregational killing mechanism. The hok gene is a toxic gene and the sok gene is an antitoxin. Hence, E. coli utilizing this system can regulate the expression of hok (toxin) and inhibits its translation by producing sok RNA (antitoxin). The outcome is the repression of hok mRNA translation.
7.19B: Attenuation
Learning Objectives
• Compare transcriptional and translational attenuation
Attenuation is a regulatory mechanism used in bacterial operons to ensure proper transcription and translation. In bacteria, transcription and translation are capable of proceeding simultaneously. The need to prevent unregulated and unnecessary gene expression can be prevented by attenuation, which is characterized as a regulatory mechanism.
The process of attenuation involves the presence of a stop signal that indicates premature termination. The stop signal, referred to as the attenuator, prevents the proper function of the ribosomal complex, stopping the process. The attenuator is transcribed from the appropriate DNA sequence and its effects are dependent on the metabolic environment. In times of need, the attenuator within the mRNA sequence will be bypassed by the ribosome and proper translation will occur. However, if there is not a need for a mRNA molecule to be translated but the process was simultaneously initiated, the attenuator will prevent further transcription and cause a premature termination. Hence, attenuators can function in either transcription-attenuation or translation-attenuation.
Transcription-attenuation is characterized by the presence of 5′-cis acting regulatory regions that fold into alternative RNA structures which can terminate transcription. These RNA structures dictate whether transcription will proceed successfully or be terminated early, specifically, by causing transcription-attenuation. The result is a misfolded RNA structure where the Rho-independent terminator disrupts transcription and produced a non-functional RNA product. This characterizes the mechanisms of transcription-attenuation. The other RNA structure produced will be an anti-terminator that allows transcription to proceed.
Translation-attenuation is characterized by the sequestration of the Shine-Dalgarno sequence, which is a bacterial specific sequence that indicates the site for ribosomal binding to allow for proper translation to occur. However, in translation-attenuation, the attenuation mechanism results in the Shine-Dalgarno sequence forming as a hairpin-loop structure. The formation of this hairpin-loop structure results in the inability of the ribosomal complexes to form and proceed with proper translation. Hence, this specific process is referred to as translation-attenuation.
Key Points
• Attenuators are characterized by the presence of stop signals within the DNA sequence that can result in either transcriptional- attenuation or translational-attenuation.
• Transcriptional-attenuation is characterized by the presence of an attenuator within the DNA sequence that results in formation of mRNA-stem loops that prevent further transcription from occurring. The non-functional RNA produced prevents proper transcription.
• Translational-attenuation is characterized by the misfolding of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence, responsible for ribosomal binding to allow proper translation, is inaccessible because it is folded into a hairpin-loop structure, thus, translation cannot occur.
Key Terms
• operons: A unit of genetic material that functions in a coordinated manner and is transcribed as one unit. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.19%3A_RNA-Based_Regulation/7.19A%3A_RNA_Regulation_and_Antisense_RNA.txt |
Learning Objectives
• Describe riboswitches
Riboswitches are specific components of an mRNA molecule that regulates gene expression. The riboswitch is a part of an mRNA molecule that can bind and target small target molecules. An mRNA molecule may contain a riboswitch that directly regulates its own expression. The riboswitch displays the ability to regulate RNA by responding to concentrations of its target molecule. The riboswitches are naturally occurring RNA molecules that allow for RNA regulation. Hence, the existence of RNA molecules provide evidence to the RNA world hypothesis that RNA molecules were the original molecules, and that proteins developed later in evolution.
Riboswitches are found in bacteria, plants, and certain types of fungi. The various mechanisms by which riboswitches function can be divided into two major parts including an aptamer and an expression platform. The aptamer is characterized by the ability of the riboswitch to directly bind to its target molecule. The binding of the aptamer to the target molecule results in a conformational change of the expression platform, thus affecting gene expression. The expression platforms, which control gene expression, can either be turned off or activated depending on the specific function of the small molecule. Various mechanisms by which riboswitches function include, but are not limited to the following:
• The ability to function as a ribozyme and cleave itself if a sufficient concentration of its metabolite is present
• The ability to fold the mRNA in such a way the ribosomal binding site is inaccessible and prevents translation from occurring
• The ability to affect the splicing of the pre-mRNA molecule
The riboswitch, dependent on its specific function, can either inhibit or activate gene expression.
Key Points
• The mechanisms by which riboswitches regulate RNA expression, can be divided into two major processes, including aptamer and expression platform.
• The aptamer is characterized by the direct binding of the small molecule to its target.
• The expression platform is characterized by the conformational change, which occurs in the target upon binding of an aptamer, resulting in either inhibition or activation of gene expression.
Key Terms
• aptamer: Any nucleic acid or protein that is used to bind to a specific target molecule.
7.19D: Regulation of Sigma Factor Activity
Learning Objectives
• Analyze the regulation of sigma factor activation
Sigma factors are proteins that function in transcription initiation. Specifically, in bacteria, sigma factors are necessary for recognition of RNA polymerase to the gene promoter site. The sigma factor allows the RNA polymerase to properly bind to the promoter site and initiate transcription which will result in the production of an mRNA molecule. The type of sigma factor that is used in this process varies and depends on the gene and on the cellular environment. The sigma factors identified to date are characterized based on molecular weight and have shown diversity between bacterial species as well. Once the role of the sigma factor is completed, the protein leaves the complex and RNA polymerase will continue with transcription.
The regulation of sigma factor activity is critical and necessary to ensure proper initiation of transcription. The activity of sigma factors within a cell is controlled in numerous ways. Sigma factor synthesis is controlled at the levels of both transcription and translation. Often times, sigma factor expression or activity is dependent on specific growth phase transitions of the organism. If transcription of genes involved in growth is necessary, the sigma factors will be translated to allow for transcription initiation to occur. However, if transcription of genes is not required, sigma factors will not be active.
In specific instances when transcriptional activity needs to be inhibited, there are anti-sigma factors which perform this function. The anti-sigma factors will bind to the RNA polymerase and prevent its binding to sigma factors present at the promoter site. The anti-sigma factors are responsible for regulating inhibition of transcriptional activity in organisms that require sigma factor for proper transcription initiation.
Key Points
• Sigma factor proteins promote binding of RNA polymerase to promoter sites within DNA sequences to allow for initiation of transcription.
• Sigma factors are specific for the gene and are affected by the cellular environment.
• Sigma factors can regulate at both a transcription and translational level.
• Anti-sigma factors are responsible for inhibiting sigma factor function thus, inhibiting transcription.
Key Terms
• growth phase transitions: The various phases required for bacterial growth include: lag, exponential, and stationary phases. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.19%3A_RNA-Based_Regulation/7.19C%3A__Riboswitches.txt |
Sigma factors are proteins that regulate gene expression that are controlled at various levels, including at the translational level.
Learning Objectives
• Explain the regulation of sigma factor translation
Key Points
• Sigma factor expression is often associated with environmental changes that cause changes in gene expression.
• The translational control of sigma factors is critical in its role in transcription regulation.
• Sigma factor translation is controlled by small noncoding RNAs that can either activate or inhibit translation.
Key Terms
• oxidative stress: Damage caused to cells or tissue by reactive oxygen species.
• sigma factor: A sigma factor (σ factor) is a protein needed only for initiation of RNA synthesis.
• RpoS protein: RpoS is a central regulator of the general stress response and operates in both a retroactive and a proactive manner: not only does it allow the cell to survive environmental challenges, but it also prepares the cell for subsequent stresses (cross-protection).
Sigma factors are groups of proteins that regulate transcription and therefore function in house-keeping, metabolic, and regulation of growth processes in bacteria. Sigma factor expression is often associated with environmental changes that cause changes in gene expression. The regulation of expression of sigma factors occurs at transcriptional, translational, and post-translational levels as dictated by the cellular environment and the presence or absence of numerous cofactors.
Sigma factors include numerous types of factors. The most commonly studied sigma factors are often referred to as a RpoS proteins as the rpoS genes encode for sigma proteins of various sizes. In E. coli, the RpoS is the regulator of growth phase genes, specifically in the stationary phase. The RpoS is critical in the general stress responses and can either function in promoting survival during environmental stresses, but can also prepare the cell for stresses. Specifically, the translational control of the sigma factor is a major level of control.
The translational control of sigma factors involves the presence and function of small noncoding RNAs. Using RpoS proteins as the focus, the RpoS expression and transcription is regulated at the translational level. Small noncoding RNAs are able to sense environmental changes and stresses resulting in increased expression of RpoS protein. The small noncoding RNAs are able to specifically increase the amount of rpoS mRNA that undergoes translation.
The resultant increase of RpoS protein is based on the cellular environment and its needs. There are numerous classes of small noncoding RNAs that function in RpoS regulation, including DsrA, RprA and OxyS. These small noncoding RNAs are capable of sensing changes in temperature (DsrA), cell surface stress (RprA) and oxidative stress (OxyS). These RNAs can induce activation of rpoS translation. However, there are small noncoding RNAs, such as LeuO, that are capable of inhibiting rpoS translation as well via repression mechanisms. The regulation of rpoS translation is complex and involves cross-signaling and networking of numerous proteins and the regulatory small noncoding RNAs.
7.19F: Proteolytic Degradation
Proteolytic degradation, or proteolysis, is a key factor that controls protein concentration and function.
LEARNING OBJECTIVES
Describe protein degradation
Key Points
• The major mechanism of proteolytic degradation utilized by the cell, is via the proteasomal pathway. Proteins that are degraded via the proteasomal complex are tagged via the addition of a ubiquitin signal.
• An additional mechanism utilized for proteolytic degradation is via the lysosomal pathway. The lysosome contains proteases which target proteins for degradation.
• Proteolysis is necessary to control protein concentration and prevent abnormal accumulation.
• Upon protein degradation, the amino acids are typically reused and recycled for the synthesis of new proteins.
Key Terms
• ubiquitin: A small regulatory protein sequence that directs proteins to specific compartments within the cell. Specifically, a ubiquitin tag directs the protein to a proteasome, which destroys and recycles the components.
• proteases: A class of enzymes that can cleave proteins.
Proteolytic degradation is necessary in the regulation of cellular processes and function. The breakdown of proteins into smaller polypeptides, or its respective amino acids, are necessary for metabolic and cellular homeostasis. Polypeptides are commonly broken down via hydrolysis of the peptide bonds by utilizing a class of enzymes called proteases. However, proteolytic degradation can also occur utilizing various mechanisms, including intramolecular digestion and non-enzymatic methods. The mechanisms of proteolytic degradation are necessary for obtaining amino acids via degradation of digested proteins, preventing accumulation or abnormal concentrations of proteins, and by regulating cellular processes by removing proteins no longer needed.
Proteasomes are protein complexes that function in the degradation of unneeded or damaged proteins via proteolysis. The proteasomes are a major component of a complex and highly regulated mechanism. The proteasome is able to degrade proteins based on the presence of a ubiquitinprotein. This ubiquitin sequence is a modification to proteins that are targeted for degradation. The recognition of this ubiquitin signal by the proteasome results in degradation of the protein into its amino acids, which are then recycled and reused for the synthesis of new proteins. The proteasomal degradation pathway is the major pathway utilized to ensure proteolytic degradation. It is necessary for homeostasis functioning in controlling cell cycle and gene expression, for example.
In addition to proteasomal complexes, the organelle, the lysosomes are also used to ensure protein degradation. The intracellular process that utilizes lysosomes involves autophagy. The lysosomal pathway, in comparison to the proteasomal pathway, is typically non-selective. The lysosome contains proteases that are able to target and degrade proteins. | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.19%3A_RNA-Based_Regulation/7.19E%3A__Regulation_of_Sigma_Factor_Translation.txt |
Proteolytic degradation, or proteolysis, is a key factor that controls protein concentration and function.
LEARNING OBJECTIVES
Describe protein degradation
Key Points
• The major mechanism of proteolytic degradation utilized by the cell, is via the proteasomal pathway. Proteins that are degraded via the proteasomal complex are tagged via the addition of a ubiquitin signal.
• An additional mechanism utilized for proteolytic degradation is via the lysosomal pathway. The lysosome contains proteases which target proteins for degradation.
• Proteolysis is necessary to control protein concentration and prevent abnormal accumulation.
• Upon protein degradation, the amino acids are typically reused and recycled for the synthesis of new proteins.
Key Terms
• ubiquitin: A small regulatory protein sequence that directs proteins to specific compartments within the cell. Specifically, a ubiquitin tag directs the protein to a proteasome, which destroys and recycles the components.
• proteases: A class of enzymes that can cleave proteins.
The Process of Protein Degradation in a Proteosome: Schematic of the proteolytic degradation pathway that utilizes proteasomal complexes. The protein is tagged with several ubiquitin signals that target the proteasome. Once the protein arrives at the proteasome, the protein is degraded into its amino acids which are then reused for synthesis of new proteins.
Proteolytic degradation is necessary in the regulation of cellular processes and function. The breakdown of proteins into smaller polypeptides, or its respective amino acids, are necessary for metabolic and cellular homeostasis. Polypeptides are commonly broken down via hydrolysis of the peptide bonds by utilizing a class of enzymes called proteases. However, proteolytic degradation can also occur utilizing various mechanisms, including intramolecular digestion and non-enzymatic methods. The mechanisms of proteolytic degradation are necessary for obtaining amino acids via degradation of digested proteins, preventing accumulation or abnormal concentrations of proteins, and by regulating cellular processes by removing proteins no longer needed.
Proteasomes are protein complexes that function in the degradation of unneeded or damaged proteins via proteolysis. The proteasomes are a major component of a complex and highly regulated mechanism. The proteasome is able to degrade proteins based on the presence of a ubiquitinprotein. This ubiquitin sequence is a modification to proteins that are targeted for degradation. The recognition of this ubiquitin signal by the proteasome results in degradation of the protein into its amino acids, which are then recycled and reused for the synthesis of new proteins. The proteasomal degradation pathway is the major pathway utilized to ensure proteolytic degradation. It is necessary for homeostasis functioning in controlling cell cycle and gene expression, for example.
In addition to proteasomal complexes, the organelle, the lysosomes are also used to ensure protein degradation. The intracellular process that utilizes lysosomes involves autophagy. The lysosomal pathway, in comparison to the proteasomal pathway, is typically non-selective. The lysosome contains proteases that are able to target and degrade proteins.
LICENSES AND ATTRIBUTIONS
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• Bacterial small RNA. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Bacterial_small_RNA. License: CC BY-SA: Attribution-ShareAlike
• Regulation of gene expression. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Regulat...ene_expression. License: CC BY-SA: Attribution-ShareAlike
• Antisense RNA. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Antisense_RNA. License: CC BY-SA: Attribution-ShareAlike
• Hok/sok system. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Hok/sok_system. License: CC BY-SA: Attribution-ShareAlike
• morphogenesis. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/morphogenesis. License: CC BY-SA: Attribution-ShareAlike
• Hok sok system R1 plasmid present. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:Ho...id_present.gif. License: Public Domain: No Known Copyright
• Attenuator (genetics). Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Attenuator_(genetics). License: CC BY-SA: Attribution-ShareAlike
• operons. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/operons. License: CC BY-SA: Attribution-ShareAlike
• Hok sok system R1 plasmid present. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:Ho...id_present.gif. License: Public Domain: No Known Copyright
• File:Trp operon attenuation.svg - Wikipedia, the free encyclopedia. Provided by: Wikipedia. Located at: en.Wikipedia.org/w/index.php?...ion.svg&page=1. License: CC BY-SA: Attribution-ShareAlike
• Riboswitch. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Riboswi...rld_hypothesis. License: CC BY-SA: Attribution-ShareAlike
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• aptamer. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/aptamer. License: CC BY-SA: Attribution-ShareAlike
• Hok sok system R1 plasmid present. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:Ho...id_present.gif. License: Public Domain: No Known Copyright
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• TPP riboswitch pdb-2hoj. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:TP...h_pdb-2hoj.png. License: CC BY-SA: Attribution-ShareAlike
• RpoS. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/RpoS. License: CC BY-SA: Attribution-ShareAlike
• Sigma factor. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Sigma_factor. License: CC BY-SA: Attribution-ShareAlike
• Anti-sigma factors. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Anti-sigma_factors. License: CC BY-SA: Attribution-ShareAlike
• Bacterial small RNA. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Bacterial_small_RNA. License: CC BY-SA: Attribution-ShareAlike
• growth phase transitions. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/growth%...%20transitions. License: CC BY-SA: Attribution-ShareAlike
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• TPP riboswitch pdb-2hoj. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:TP...h_pdb-2hoj.png. License: CC BY-SA: Attribution-ShareAlike
• PDB 1h3l EBI. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...B_1h3l_EBI.jpg. License: Public Domain: No Known Copyright
• RpoS. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/RpoS%23...ontrol_of_rpoS. License: CC BY-SA: Attribution-ShareAlike
• sigma factor. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/sigma%20factor. License: CC BY-SA: Attribution-ShareAlike
• RpoS protein. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/RpoS%20protein. License: CC BY-SA: Attribution-ShareAlike
• oxidative stress. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/oxidative_stress. License: CC BY-SA: Attribution-ShareAlike
• Hok sok system R1 plasmid present. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:Ho...id_present.gif. License: Public Domain: No Known Copyright
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• TPP riboswitch pdb-2hoj. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:TP...h_pdb-2hoj.png. License: CC BY-SA: Attribution-ShareAlike
• PDB 1h3l EBI. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...B_1h3l_EBI.jpg. License: Public Domain: No Known Copyright
• Image:Ta7.jpg - OpenWetWare. Provided by: Open Wetware. Located at: http://openwetware.org/wiki/Image:Ta7.jpg. License: CC BY-SA: Attribution-ShareAlike
• Proteolysis. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Proteol...in_degradation. License: CC BY-SA: Attribution-ShareAlike
• Proteasome. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Proteasome%23Proteolysis. License: CC BY-SA: Attribution-ShareAlike
• ubiquitin. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/ubiquitin. License: CC BY-SA: Attribution-ShareAlike
• proteases. Provided by: Wiktionary. Located at: en.wiktionary.org/wiki/proteases. License: CC BY-SA: Attribution-ShareAlike
• Hok sok system R1 plasmid present. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:Ho...id_present.gif. License: Public Domain: No Known Copyright
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• TPP riboswitch pdb-2hoj. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:TP...h_pdb-2hoj.png. License: CC BY-SA: Attribution-ShareAlike
• PDB 1h3l EBI. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...B_1h3l_EBI.jpg. License: Public Domain: No Known Copyright
• Image:Ta7.jpg - OpenWetWare. Provided by: Open Wetware. Located at: http://openwetware.org/wiki/Image:Ta7.jpg. License: CC BY-SA: Attribution-ShareAlike
• Ubiquitylation. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...uitylation.png. License: CC BY-SA: Attribution-ShareAlike
• Housekeeping gene. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Housekeeping_gene. License: CC BY-SA: Attribution-ShareAlike
• Antisense RNA. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Antisense_RNA. License: CC BY-SA: Attribution-ShareAlike
• Bacterial small RNA. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/Bacterial_small_RNA. License: CC BY-SA: Attribution-ShareAlike
• RNA polymerase. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/RNA%20polymerase. License: CC BY-SA: Attribution-ShareAlike
• Hok sok system R1 plasmid present. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:Ho...id_present.gif. License: Public Domain: No Known Copyright
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• TPP riboswitch pdb-2hoj. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:TP...h_pdb-2hoj.png. License: CC BY-SA: Attribution-ShareAlike
• PDB 1h3l EBI. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...B_1h3l_EBI.jpg. License: Public Domain: No Known Copyright
• Image:Ta7.jpg - OpenWetWare. Provided by: Open Wetware. Located at: http://openwetware.org/wiki/Image:Ta7.jpg. License: CC BY-SA: Attribution-ShareAlike
• Ubiquitylation. Provided by: Wikimedia. Located at: commons.wikimedia.org/wiki/Fi...uitylation.png. License: CC BY-SA: Attribution-ShareAlike
• Antisense DNA oligonucleotide. Provided by: Wikipedia. Located at: en.Wikipedia.org/wiki/File:An...nucleotide.png. License: CC BY: Attribution | textbooks/bio/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.19%3A_RNA-Based_Regulation/7.19G%3A__Small_Regulatory_RNAs.txt |
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