The daily life of a microscopic organism consists of adapting to a changing environment and altering its gene expression to regulate these changes in its cells. Bacteria and archaea are prokaryotic cells that sense and respond to changes in their surroundings. Gene regulation helps microorganisms compete in unfavourable, competitive environments by regulating the function of macromolecules such as protein to maximize their resources. Gene expression in bacteria is the response to the available nutrient sources, controlling the amount and type of protein or enzyme. The process of gene expression regulates protein synthesis through transcription by controlling the amount of mRNA available, or through translation which decides whether or not to translate mRNA. Enzyme synthesis and enzyme activity allow a cell to control its metabolism through regulatory processes. Chemotaxis is a process that allows motile bacteria to move towards attractants such as nutrients and away from repellents such as toxins (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, pp. 209 - 210). This paper explores methods of gene regulation in prokaryotic cells such as the lac operon, global control mechanisms of catabolite repression, omp system, alternative sigma factors, Chemotaxis systems, and quorum sensing. It also discusses the interaction of protein-nucleic acid, endospore formation, flagellar rotation and adaptation, and virulence factors in relation to these systems.
Protein-Nucleic Acid Interaction
Regulatory proteins are binding proteins responsible for regulating gene expressions and allow transcription to be turned on and off. A specific promoter on DNA must be recognized to allow a gene to be transcribed. Transcription is a microbial regulation mechanism used by prokaryotic cells. The location protein-nucleic acid interaction occurs depends if the binding protein has a specific binding site or not on the nucleic acid. The protein-nucleic acid interactions are essential to the gene regulation processes of replication, transcription, and translation. A DNA-binding protein; helix-turn-helix is formed of a polypeptide chain with two segments. A short sequence is connected to an α-helix secondary structure, which creates the turn in the structure. This helix-turn-helix structure used by DNA-binding proteins in Bacteria such as Escherichia coli (E. coli ) have lac and trp repressor proteins (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, pp. 210 - 212).
Negative control prevents transcription by repressing mRNA synthesis, which is the first step in the flow of biological information. A gene that is transcribed more often will have a larger amount of protein available in a cell, resulting in more RNA of that gene available for translation. If products such as amino acid arginine, used by E. coli are present in sufficient amounts in a medium, enzymes that catalyze the synthesis are not produced, they are repressed. Enzyme repression affects biosynthetic (anabolic) enzymes, the opposite of repression is induction which affects degradative (catabolic) enzymes. Induction occurs when a substrate is present and produces enzymes. The sugar lactose is used by E. coli as a source of carbon and energy. β-galactosidase is an enzyme that cleaves lactose into glucose and galactose and is necessary for E. coli to form on lactose. Synthesis of β-galactosidase is not induced until lactose is added and is encoded by the lac operon to allow bacteria to use lactose as a source of energy. The lactose operon, known as the lac operon, is used to ferment lactose. This is a control mechanism used to synthesize enzymes only when necessary (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 212).
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An inducer and corepressor are substances that induce and repress enzyme synthesis, respectively. These small molecules are known as effectors and are not substrates or end products of the synthesis. These substances affect transcription by indirectly binding to DNA-binding proteins. Arginine genes induce transcription from the arginine operon when cells need arginine and repress when the enzyme is abundant. Arginine binds to the corepressor in a cell and activates the protein, where the repressor protein can bind to the operator; a DNA region close to the promoter of the gene. This is a region of consecutive genes called the operon, controlled by one operator. The promoter initiates synthesis of mRNA, upstream from the operator. Transcription can be blocked if the repressor binds to the operator and the encoded polypeptides are repressed and not synthesized. When an inducer is absent, a repressor is active and can control induction, blocking transcription. The repressor protein becomes inactivated when an inducer is added and the effectors combine, overcoming inhibition and allowing transcription to continue (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 213).
Positive control activates transcription. The activator is a regulatory protein that binds RNA polymerase to DNA. In E. coli, maltose is synthesised by enzymes after maltose is added to the medium. The sequence of these enzymes is the same as β-galactosidase as mentioned above, using maltose instead of lactose to induce gene expression. Except, the synthesis of maltose-degrading enzymes is under positive control, instead of negative control with the lac operon, requiring an activator protein to bind to DNA. The maltose activator protein must first bind the maltose inducer before it can bind to DNA. RNA polymerase can then start transcription. Operons of Bacteria and Archaea have many control features; therefore, a network of interactions is vital for regulation (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, pp. 213-214).
Global Control Mechanisms of Catabolite Repression
Global control systems are regulatory mechanisms that regulate gene transcription as a response to a signal in an environment. The lac operon responds to global controls and their own negative and positive controls. Multiple usable sources of carbon may be present for bacteria, E. coli can use various sugars. Catabolite repression is a global control mechanism that chooses the best source of carbon to be used if more than one is present. When glucose is available, it is used first because E.coli forms quicker on glucose compared to other sources of carbon. Catabolite repression uses the best source of carbon and energy first for an organism and is known as the glucose effect. The synthesis of enzymes used in breaking down carbon sources other than glucose are repressed when growing E. coli in glucose-containing medium. Induction is therefore repressed by the presence of the best source of carbon. Global control of the catabolite repression indicates the use of the best source of energy by preventing other catabolic operons, such as lactose and maltose from being expressed while glucose is available. Genes of flagella synthesis are also controlled by catabolite repression due to bacteria having a sufficient source of carbon and not having to swim to find other nutrients. Diauxic growth is a potential consequence of catabolite repression. When two usable sources of energy are present, the better source; glucose will be consumed first, the cell will stop growing when the resource is depleted. After a lag period, the cells use the second energy source; lactose, cells grow faster with glucose than lactose. The synthesis of proteins used by the lac operon rely on catabolite repression and is not expressed in the presence of glucose, lactose is not used. β-galactosidase is necessary for including cells to use lactose. Abolishment of catabolite repression occurs when the glucose is depleted and lac operon is expressed, cells can then use lactose to grow (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 215).
Catabolite repression depends on a regulatory protein that acts as a positive control, called cyclic AMP receptor protein (CRP). RNA polymerases bind to a promoter if CRP binds to DNA and gene encoding catabolite repression enzymes are expressed. DNA must first bind to cyclic adenosine monophosphate (cyclic AMP, also known as cAMP)before it can bind to CRP. Cyclic AMP is a regulatory nucleotide and is synthesized by adenylate cyclase from ATP while its synthesis can be disrupted by glucose, potentially stimulating the transport of cyclic AMP outside the cell wall. The level of cyclic AMP is lowered when glucose is added to the cell. Polymerase can not bind to promoters of catabolite repression when levels of cyclic AMP are low due to CRP not being able to bind DNA. A low concentration of cyclic AMP influences catabolite repression, which is impacted due to glucose being a better source of energy. Transcription of the lac operon relies on the absence of glucose and a high concentration of cyclic AMP. This allows CRP to bind to the CRP-binding site, and an inducer such as lactose must prevent the lactose repressor binding to the operator, blocking transcription (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, pp. 216 - 217).
Two-component regulatory systems contain a sensor kinase protein and a response regulator protein located in the cytoplasmic membrane and cytoplasm, respectfully. Autophosphorylation is a process used by sensor kinases; histidine kinases enzymes, to phosphorylate themselves when an environmental signal is detected. Kinase uses phosphate from ATP to phosphorylate compounds and transfers the protein from a sensor into the cell to the response regulator; another protein. Transcription is regulated by a DNA-binding protein as the phosphorylated response regulation functions either act as an inducer by allowing transcription or acts as a repressor, binding DNA and blocking transcription. If repressed, transcription can occur when the response regulator is released and dephosphorylated. A feedback loop is required for a regulatory system to operate correctly. This system uses a phosphatase enzyme to remove phosphate from the response regulator and terminate the response, resetting the cycle of the system (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 219).
There are close to 50 two-component systems in E. coli. The levels of OmpC and OmpF in the outer membrane of E. coli are controlled by the osmolarity of the environment. OmpC and OmpF are porins, proteins that are part of the Omp system, allowing metabolites to pass through the outer membrane of Gram-negative bacteria. Synthesis of these proteins depends on osmotic pressure. OmpC is a porin that has a small pore; synthesis is larger in quantities if pressure is high, and OmpF is a porin that has a large pore; synthesis increases when pressure is low. Located in the cytoplasmic membrane is a sensor histidine kinase called EnvZ, it detects osmotic pressure changes and moves phosphate to OmpR, the system’s response regulator. OmpR-P; phosphorylated OmpR, activates transcription of ompF gene in low osmotic pressure conditions. Omp-R activates transcription of OmpC in high-pressure osmotic conditions and represses transcription of the ompF gene. Regulatory RNA is a control mechanism that regulates the expression of ompF (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 219).
Alternate Sigma Factors
Complex transduction systems exist that contain multiple regulatory elements. Nitrogen assimilation in bacteria is regulated by the Ntr regulatory system. Nitrogen regulator I (NRI) is the response regulator that activates transcription from promoters using the alternative sigma factor s54 (RpoN) to recognize RNA polymerase. In the Ntr system, a sensor kinase; nitrogen regulator II (NRII), is a protein regulated by a second protein called PII, who in turn, is regulated by the contribution of uridine monophosphate (UMP) groups. NRII acts as a kinase and a phosphatase, and the activity of kinase is promoted by the PII-UMP complex during periods of nitrogen starvation, where UMP connects to PII, resulting in phosphorylation of NRII. Phosphatase activity of NRII is promoted by the removal of UMP from PII (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 219 - 220).
Another two-component regulatory system with more than one regulator is the Nar regulatory system. During anaerobic respiration, the use of nitrate and/or nitrite as alternative electron acceptors are controlled by the Nar system. This system contains two different response regulators and sensor kinases, while fumarate nitrite regulator (FNR), controls all genes regulated by the Nar System. Complex chains of regulating systems such as two-component systems for bacteria to phosphorylate histidine residues are common for systems related to cellular metabolism (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 220).
Stringent response is a regulatory mechanism bacterium use to survive changes in their environment such as stresses, antibiotics, and nutrient deprivation. When stringent responses are triggered, stress survival pathways are activated (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 224). Alternative sigma factor RpoS (σs or σ38) is a general stress response that controls these pathways of Gram-negative bacteria cells, who have stress responses and the stringent responses. Gram-positive cells face environmental stresses by undergoing sporulation. These processes allow cells to adapt to environmental stressors and harsh conditions. RpoS regulates more than 400 genes and is the master controller of gene regulation in bacteria. RpoS regulon sense changes in the environment such as nutrients, biofilm formation and stresses and transfer signals to other regulators such as the heat shock response. The dinB gene of E. coli encodes DNA polymerase IV and is recognized by RpoS (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 226 & 228).
RpoH (σ32) is the alternative sigma factor that controls the expression of proteins in the heat shock response of E. coli. Degradation of RpoH synthesis often occurs within a couple of minutes. When cells are affected by heat shock, degradation is inhibited, and transcription of operons increases as more promoters are recognized. Free DnaK inactivates RpoH, influencing degradation. When proteins unfold due to heat stress, DnaK cannot degrade RpoH due to binding with unfolded proteins. The level of free DnaK is affected by the number of denatured proteins. With less free DnaK, the level of RpoH is higher which causes expression of the heat shock genes. DnaK inactivates RpoH when environmental stressors such as temperature disappear and the reduction of heat shock protein synthesis occurs (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 227).
Endospore formation is used by microorganisms as a response to harsh environmental conditions, forming spores from vegetative cells. Spores germinate when the conditions improve, and the organism’s cells return to a state of vegetation. Bacillus spp. are Gram-positive bacteria that form endospores within the mother cell and cells divide asymmetrically before endospore formation. Endospores are released once formed and the mother cells burst. Unfavourable environmental conditions trigger endospore formation of Bacillus subtilis (B. subtilis) which use five sensor kinases to monitor their environment. Sporulation factors are the successive phosphorylation of multiple proteins resulting from responding to cumulative adverse conditions. Spo0A is a sporulation factor that controls the expression of genes specific for sporulation. Sporulation occurs when Spo0A is highly phosphorylated. The σF sigma factor is an important part of the sporulation process. This product requires SpoIIE to remove phosphate from SpoIIAA, triggering the removal of SpoIIAB, the anti-sigma factor (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 248).
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Endospore development of B. subtilis are controlled by four σfactors. σF and σG activate genes required for developing forespores, while σE and σK activate genes required for the mother cell that surrounds the endospore. σF is bound by an anti-sigma factor, therefore, inactive in the forespore, and becomes activated by the Spo0A transmitting a spore signal. Transcription is promoted when σF binds to RNA polymerase and produces the gene encoding σG sigma factor. σE is important for transcribing genes for σK and requires to be activated by genes for proteins entering the mother cell, required for the sporulation process. This process also requires sigma factors σG and σK to later transcribe genes. After a long process, spore coats and structures are formed, and the endospore can be released. Sporulation in Bacillus is often triggered by the limitation of nutrients in the environment (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 248).
Chemotaxis regulation is the movement of Bacteria and Archaea cells away from repellants, towards attractants as they respond to changes in the environment such as toxin accumulation and limited nutrients. Prokaryotic cells sense and respond to temporal gradient changes in the concentration of a chemical over time, they are not large enough to sense the absolute concentration of a chemical. Bacteria have a unique, modified two-component system that regulates the direction of flagellar rotation by sensing temporal changes within repellants or attractants. Instead of encoding flagellar proteins by controlling transcription, this system regulates the activity of the flagellum protein complex. Sensory proteins called methyl-accepting chemotaxis proteins (MCPs) interact with cytoplasmic sensor kinases in the cytoplasmic membrane for monitoring concentrations of substances. MCPs sense attractants or repellants which drive the mechanism of chemotaxis through signals from proteins (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, pp. 220 – 221).
The Tar MCP transmembrane production of E. coli sense cobalt and nickel as repellents and maltose and aspartate as attractants. Flagellar rotation is influenced through interactions between MCPs binding attractants or repellents to periplasmic binding proteins which interact with cytoplasmic proteins. Chemoreceptors are made of thousands of clustered MCPs that contact CheA and CheW cytoplasmic proteins. In chemotaxis, the sensor kinase is CheA, and CheW helps with autophosphorylation of CheA to CheA-P after MCP binds a chemical. The concentration of an attractant affects the rate of autophosphorylation; as a concentration increases, autophosphorylation decreases, and with an increase in concentration of repellent or decrease in attractant, increases autophosphorylation. Flagellar rotation is controlled by a response regulator that forms when Phosphate is passed from CheA-P to CheY creating CheY-P. Phosphate can be transferred from CheA-P to CheB, which is important for adaptation (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 221).
Flagellar Rotation and Adaptation
CheY controls the direction of flagellar rotation and is a central protein of the regulatory system. After phosphorylation occurs, CheY induces clockwise rotation of the flagellum causing the cell to tumble. If CheY is phosphorylated (CheY-P), the motor of the flagellum can not bind and turns counter-clockwise, sending the cell to run; swimming smoothly. Runs occur when CheZ dephosphorylates CheY preventing cells from tumbling. Tumbling relates to an increase in the level of Chey-P, which depends on an increase in concentration of repellent or decrease of attractant. Runs are promoted by moving towards an attractant, tumbling is then suppressed due to the lower level of CheY-P. After responding to stimulus, an organism resets the sensory system through a feedback loop relying on CheB, this is known as adaptation of the chemotaxis system as the organisms must wait for another signal (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 221 - 222).
MCPs are sensitive towards repellents and when methylated, they do not respond to attractants. MCPs respond to attractants and not to repellents when unmethylated. Methylation and demethylation from CheR and CheB-P cause variation in levels of methylation, allowing adaptation to sensory signals. Autophosphorylation rate of CheA is low in relation to a high concentration of attractant. This causes cells to swim smoothly due to unphosphorylated CheY and CheB. At this time, methylation of MCPs increases as they can not be demethylated by CheB-P, as CheB-P is not around. When fully methylated, MCPs do not respond to an attractant causing the cell to tumble due to the high, constant level of attractant. When CheB is phosphorylated, CheB-P can demethylate the MCPs resetting the receptors, allowing them to respond to changes in levels of attractant. When fully methylated, MCPs signal for a cell to begin tumbling as they respond to an increase in gradient of a repellent. MCPs slowly demethylate as cells tumble in random directions. Chemotaxis successfully monitors small changes of concentrations over time in repellents and attractants through this mechanism for adaptation (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 221 - 222).
Quorum means sufficient numbers. Quorum-sensing systems in bacteria respond to other cells of the same species around the cell, and the density of cells can control the regulatory pathway. This is known as quorum sensing. Some species of Archaea also contain quorum-sensing systems. Mechanisms of quorum sensing assess density of populations, bacteria use this to make sure the proper cell density is present before operating, correctly. Individual cells of toxin secreting pathogenic bacterium would waste resources as it would not have an effect on a host. Although, coordinated expression toxin-secreting bacteria cell may cause illness, thriving from collecting resources from the host, benefiting the pathogen. An autoinducer is a signal molecule synthesized through quorum sensing used by both Gram-positive and Gram-negative bacteria but is more common in the latter. The molecule diffuses in either direction across the cell envelope, causing the autoinducer to increase in concentration within the cell if multiple cells making the same autoinducer are around. The autoinducer triggers gene transcription binding to a transcriptional activator protein or sensor kinase of a two-component system. Gram-negative bacteria contain different lengths of acyl groups containing carbonyl and alkyl called Acyl homoserine lactones (AHLs). Autoinducer 2 (AI-2) containing cyclic furan derivative is made by some Gram-negative bacteria and is used between bacteria species as a common autoinducer. On the other hand, Gram-positive bacteria use short peptide autoinducers (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 222).
Bioluminescent light emissions in bacteria are regulated by a quorum sensing mechanism. Aliivibrio fischeri is a marine bacterial species that emit light generated by an enzyme called luciferase. Proteins needed for bioluminescence are encoded by lux operons controlled by activator protein LuxR. Bioluminescence is induced by an increase in concentration of N-3-oxohexanoyl homoserine lactone, a specific AHL of A. fischeri. The luxI gene synthesises AHL through the encoded enzyme (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 222).
Quorum sensing control genes of pathogenic E. coli 0157:H7, a Shiga toxin-producing foodborne pathogen induces virulence genes by making AI-3. While the population of E. coli cells in the intestine increases; producing AI-3, the stress hormones norepinephrine and epinephrine are made by host cells in the intestine. In the cytoplasmic membrane of E. coli, the signal molecules become bound to sensor kinases. This activates two transcriptional activator proteins which activate transcription of genes that secrete toxins, encode proteins causing lesions on intestinal mucosa of a host, encode the function of motility, and phosphorylation. This system regulates gene expression by sensing chemical signals of both bacterial and eukaryotic cells (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 223).
Autoinducing peptide (AIP) is a small peptide used by the quorum-sensing system to control virulence factors that encode genes and impact the immune system of a host organism. Staphylococcus aureus is a pathogenic bacterium that uses extracellular peptides to affect cells of an organism. The argD gene is the autoinducer that encodes AIP, and after its, synthesis the peptide is trimmed into the active form of AIP by the membrane-bound protein ArgB and the peptide used by S. aureus is excreted outside the cell. Concentration of AIP increases along with cell density of S. aureus. Autophosphorylation occurs from the binding of AIP to the membrane-bound sensor kinase ArgC, producing ArgC-P and transfers its phosphate to a transcriptional activator called ArgA, producing ArgA-P. Production of virulence proteins is controlled by an RNA molecule transcribed by argABCD genes. Transcription of these genes is influenced by an increase of ArgA-P, encoding the signal transduction systems. Eukaryotic organisms have been known to disrupt quorum sensing in bacteria by producing molecules that mimic AHLs or AI-2, interfering with the behaviour of bacteria. These quorum-sensing disruptors can potentially be used to impede virulence gene expression and disperse bacterial biofilms (Madigan, Bender, Buckley, Sattley, & Stahl, 2018, p. 223).
Regulatory mechanisms of gene expression are used by prokaryotic cells to sense signals in their environment and bind to DNA. Prokaryotic cells respond to environmental sensors and stressors by turning genes off and on. Endospore formation is used as in Gram-positive bacteria to enter a state of dormancy when unfavourable conditions arise. It is important to understand how these systems work to ensure the correct gene is present and it can be turned on in the available conditions. Understanding how these systems work and how they work with microorganisms in their natural environment is vital for bioremediation and designing applications for industrial use (Noble, 2021).
Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2018). Brock biology of microorganisms (15th ed.). New York: Pearson.
Noble, N. M. (2021). Lecture 1 – How do cells sense the environment.doc. Course Notes and Lecture. ENSC317. Victoria BC: Royal Roads University. https://moodle.royalroads.ca/moodle/mod/forum/discuss.php?
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