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When bacteria are exposed to adverse conditions, they adapt complex strategies and tactical methods to cause an adaptive response. One of these processes often referred to as stress-Induced Mutagenesis (SIM). These periodic stress induced mutagenesis maybe increased due to product of various stress responses. Any disturbance of the normal biological processes or absence of essential substances to carry out these processes is defined as stress. These stresses include nutritional starvation, oxidation, UV radiation, DNA damage, osmolality shifts, temperature changes and antibiotic treatment (Tanaillon, et al, 2004,) these environmental changes; induces these stresses are by-products of aerobic respiration of the bacterial population or from other microorganisms. Said mutations can arise in base substitutions, small base pair additions and deletions and as much as whole chromosome rearrangements. Different organisms present different molecular mechanisms to combat, stress-induced mutagenesis (Foster P.L, 2007) Environmental factors can affect DNA therefore causing inhibition of anti-mutator DNA repair enzymes therefore increasing mutation rates, such as nitric oxide produced by macrophage damage and inhibits DNA glycosylase O6-methylguanine (McKenzie G.t, et al., 2001)
When bacteria occupy a new nutritional rich niche they ravenously consume all available nutrients, when deprived from nutrients they enter a stationary state whereby those bacteria have to revert to scavenge for nutrients in the dead biomass. Under starved conditions comK regulatory gene is essential for the control of the stress response that enables Bacillus subtilis to transform. (Sung H-M, 2002) The majority of studies have been done on E.coli strains for the study for stress induced mutagenesis. It was found that during nutritional starvation RpoS is required for starvation-induced excisions oh phage Mu, as well as base substitution mutagenesis is older colonies causing starvation-induced point mutations and transposons for starvation-induced gene amplification. (Lombardo M, et al, 2004). Transposons could be selected for as mutator genes through products of the mutations they produce. (Bjedov, I et al., 2003)
These stress responses forces periodic control or restriction of mutagenesis. This negative or positive control gives allowances for genetic stability when cells and organisms are not stressed; and thus the rate of mutagenesis is increased, therefore during periods of induced stress the potential of evolution is accelerated. This increase in the mutation rate maybe permitted due to a variety of reasons including a decreased in consistency of DNA replication and error repair. (Ross C, et al., 2006) An increase in mutation frequency during times of nutritional deprivation in the stationary phase has been thought of as being deleterious, suggesting that an increase in mutation frequency could potentially generate new alleles, this increases in alleles the side effects of deleterious mutations are taken over by other mutated genes. It has been highly theorised that new alleles may be sourced from dead cells in the culture. This gene transfer from the surrounding detritus, could be beneficial to bacteria taking it up. Over time as selective advantageous alleles are taken up, this will lead to the evolution of microbial diversity. (Giraud A, et al., 2001)
This starvation transfer has been stimulated by using a +1 frameshift allele of a lacIZ fusion genelocated in the F9128 plasmid in cells with a deletion of the chromosomal lac genes (Cairns, J., and Foster P. L., 1991) When cultured on lactose minimal medium, a few Lac1 mutant colonies were observed due to spontaneous generation-dependent mutations that may have occurred during growth. When these cells were re-cultured over along period of time it was noted that there was an increase in Lac1 mutants (Galhardo, S. R, et al., 2007) these cells were shown to undergo hypermutation. These hybrid cells are important to the formation of Lac+ stress induced mutants (Gonzales C. P., et al., 2008,) In adaptive amplifications , the leakly lac mutant gene is amplified, so sufficient β-galactosidase acitivity is produced for growth without gaining Lac+ point mutation. These point mutations differentiate adaptive point mutations and amplifications in growing organisms. The mutation occurs due to homologous protein recombination of RecBCD double stranded break system. the mutation is amplified during a period of mismatched protein deficiency starvation. Newly formed hybrids arise from a transient gemome-wide hypermutability as they present highly variable; unrelated genomes. (Godoy, V. G., et al., 2000)
E.coli may either increase point mutation rates or undergo extensive genomic re-arrangement in response to growth limiting conditions. These require an induction of the general starvation stress response controlled by RpoS. Point mutagenesis, but not amplification, also requires induction of the SOS DNA damage stress response. Single stranded DNA errors or damage induces the SOS response. SOS genes are controlled by the activated Co-protese as RecA binds to the single stranded DNA, as shown in the work Galhardo and his team, further proved that the DinB gene as the centromere of SOS regulated protein in Stress induced mutagenesis and combining cellular stress responses, gives allowances for temporary regulation of mutagenesis, limiting periods of stress. (Courcelle, J, et al., 2001)This confinement may lead to accelerated genetic changes, particularly when organisms have been maladapted to their environments. (Galhardo R S, et al, 2009)
DinB is the founding member of the most widespread subfamily of Y-family specialized DNA polymerases, DinB/ Pol IV can perform high-fidelity translesion DNA synthesis TLS) across a number of different DNA lesion substrates (Yuan B., et al., 2008), mutations in DinB can abolish its TLS activity, suggesting that mutagenesis and TLS are independent activities of Pol IV (Godoy, V. G., et al., 2000) caused by overexpression of DinB, Eighty-five percent of the stress-induced Lac1 point mutations generated in the nongrowing cells arise in a DinBdependent manner DinB does not affect adaptive amplification, growth dependant mutation, or bacterial survival after UV damage. (McKenzie, et al 2001)
Alternatively s (transcription) factor sS (RpoS) which plays a role in the SOS response in controlling mutagenesis in the Lac assay. This is a complex issue because several SOS-controlled genes are required for the process. dinB, recA, ruvA, and ruvB are all required for mutagenesis (He et al. 2006) and are all upregulated by SOS (Additionally, for effective recombination, adaptive mutation requires a functional SOS response for protein regulation. (McKenzie, G. J., 2000) The molecular mechanism of point mutagenesis in the Lac system is now considerably well understood. The Lac system produces adaptive point mutations presenting a Lac+ phenotype. (Hastings, P. J., 2007)
During periods of stress it switches from the normally high-fidelity DNA synthesis associated with recombination-dependent double-strand-break repair to an error-prone synthesis. As well ad the genes for induction of the SOS DNA-damage response there have been several other genetic requirements have been identified for stress-induced point mutagenesis, including DNA-recombination functions (He, A. S., et al., 2006) and the sS (RpoS) general starvation stress-response regulons, and the dinB gene encoding DNA polymerase (Pol) IV Pol IV is believed to participate in the mutagenesis of undamaged phage λ DNA during and infection of irradiated E.coli. Pol IV may also serve a function on DNA replication by DSBR recombination, the source of replication in adaptive mutation. (Lombardo M et al., 2004) Pol IV is required for most adaptive point mutations at Lac, but not for mutations in actively growing organisms, damage caused by UV or hydrogen peroxide or adaptive amplifications. In E.colipol IV involves genetic changes due to environmental stresses. (McKenzie, 2009)
Bacteria tend to alter their morphology, when they are exposed to stressful factors. These are regulated by Sigma (σ) factors. Sigma factors are a class of proteins which carry essential dissociable subunits of prokarayotic RNA polymerase. These promoters are polymerase specific and contribute to DNA strand separation. These sigma factors act independently to regulate large number of genes. (Kazmierczak M J, et al., 2005) In response to stress the dominant sigma factors σ70 is replaced by a sigma subuit of RNA polymerase σs (RopS) in the E.colimodel. This replacement causes a number of independent controlled genes are activated. The RpoS gene has been linked to be involved in the near UV resistance (nUV) and as a stationary phase inducible gene, RpoS was once thought to be only induced in the Stationary phase, recent studies have shown that it plays a critical role in different stress responses. (Cirz, R T and Floyd E. Romesberg, F E., 2007) Induction of RpoS is often accompanied by a reduction in or cessation of growth and provides the cells with the ability survive the actual stress, as well as additional stresses that they are yet to encounter, this is refered to as Cross-protection (Rosche T M, et al., 2006)
σs cause's changes in the cellular envelope as well as and alteration metabolic rate, where organisms shift from optimum growth and replication, to optimum metabolic preservation. σs also enhances genes that control apoptosis in the stationary phase which would be advantageous from survival for bacterial populations by sacrificing a small fraction of the population in order to be able to sustain the nutritional requirements of the remaining surviving organisms. σs levels are amplified in response to nitrogen, phosphate and amino acid starvation into the stationary phase. In the exponential phase σs is also induced with out causing a change in bacterial growth (Finkel, S E., 2006) during periods of high temperatures, σs is still induced even when bacterial growth is exponential. RpoS transcription is stimulated by downshifts in the growth rate. The stress response induces σs with minutes, by a complex network whose dismissal, additions and the general feedback loop are essential for its signal-integration. It has been concluded that the regulation of σs as soon as it has been recognised in the stationary phase was done by post-transitional mechanisms The levels of σs appear low bacterial populations expand, conversely, raised levels of RpoS mRNA are present and do not seem to change in response of any stimulated stress conditions this leads to a high σs protein levels. (Bashyam, M. D, and Hasnain S. E., 2004)
When most organic systems are in the stationary phase or under stressful conditions, inorganic polyphosphate is accumulated in E.colipopulations, resulting in a balance between synthesis catalysed by polyphosphate kinase, encoded by ppk and degeneration; catalysed by exopolyhosphate, encoded by ppx adnd gppA. Polyphosphate plays a major role in the mediation of ribosomal proteins, which may be essential for the uptake of intercellular amino acid reserves for abrupt carbon or amino acid starvation. Polyphosphate stimulates RpoS transcription this indicting that it contributes to stationary-phase induction of RpoS (Patten C. L., et al., 2004)
Bacteria are capable of resisting the action of antibiotics as a result of genetic alterations, including the physical exchange of genetic material with another organism (via plasmid conjugation, phage-based transduction, or horizontal transformation), the activation of latent mobile genetic elements (transposons or cryptic genes), and the mutagenesis of its own DNA ( Wright G D, 2007) chromosomal mutagenesis, may arise directly from either interactions between the chromosome and the antibacterial agent or antibiotic-induced oxidative stress, or indirectly from the bacterium's error-prone DNA polymerases during the repair of a broad spectrum of DNA lesions.
The efficacy of inhibiting essential bacterial processes by antibiotics, and thus their capacity to prevent infection, is diminished following any of the aforementioned resistance-conferring events. This is due to the microbe's new-found ability to modify or destroy the structure of a given drug, reduce access to the drug target by an alteration in permeability/active transport, or abolish stable interactions between the drug molecule and its target (Alekshun MN, Levy SB., 2007) Major responses that are causative factors in inducing mutagenesis include: the SOS-DNA stress response, the heat-shock protein stress response, and the oxidative stress response (ROS). ROS is a major contributor to the mutational burden experienced by microbes during periods of oxidative stress. This notion is supported by the existence of several overlapping enzymatic mechanisms employed by bacteria to combat ROS toxicity. (Imlay JA., 2008)
For the most part antibiotic drugs predominantly inhibit ribosome function, targeting both the 30S (tetracycline family and aminocyclitol family) and 50S (macrolide family and chloramphenicol) ribosome subunits (Chopra I et al., 2003) Mutagenic stress induced by ROS include physical damage to the DNA base and the sugar- phosphate backbone of integrated or unincorporated nucleotides, as well as single-stranded and double-stranded breaks within the double helix. DNA damage can be come about as by-products of lipid peroxidation. A wide variety of base additions have been described following exposure to ROS, with the most prevalent of these being 7,8-dihydro-8-oxoguanine (8- oxoG or GO), 2,6-diamino-4-hydroxy-5-formamido-pyrimidine (FapyG), and thymine glycol. (Beckman K B, and Ames B N, 1997)
The majority of core SOS genes play a part in the physical repair of damaged DNA. Repair may occur via nucleotide excision, base excision, or recombination pathways, depending on the type and number of lesions. The repair process also involves the activity of specialized DNA polymerases, DNA pol II (product of polB/dinA), IV (product of dinB), and V (product of umuC and umuD), which catalyze error-prone DNA synthesis across lesions (translesion synthesis, or TLS) that are physically essential to the normal replicative polymerase, DNA pol III [ and the expression of pol V is SOS-dependent and its activity is RecA-dependent, while expression of pol II and pol IV is SOS-independent yet increased approximately 10-fold upon SOS induction (Godoy, V. G, et al.,2007)
Prolonged exposure to antibiotics resurrects dormant cells called persisters. Persisters are a set of cells that lay dormant. These dormant cells survive the various stressed including lethal exposure to antibiotics that organism are exposed. After the impact of the stress these dormant cells are woken up and revitalize a population. Bacterial antibiotics promote the generation of toxic hydroxal radicals as the end product of a common oxidative damage of cellular death pathway, involved in metabolic-related NADH depletion, destabilization of iron -sulphur clusters. The free radicals produced and the SOS response induces the mutagenic survival properties of bacteria. Single cells analyses have shown that the SOS network produces modulated impulses of activity in response to DNA damage. (Kohanski M A., 2007)
Mutagenic miscoding DNA structures which could end up in replication errors due to a number of chemical and physical agents such ionizing radiation. Environmental agents have a profound implication on DNA, causing an increase rate in mutagenesis, by inhibiting antimutator DNA repair enzymes, the nitric oxide produced by macrophages damages DNA and inhibits Fpg DNA glycosylase, O6-methyl-guanine-DNA methyltransferase and DNA ligase Pyrimidine dimers produced by UV irradiation block replicative DNA polymerases induce the SOS system, causing DNA lesions. (Weiss B., 2006)
Induced stresses, such as starvation, high osmolarity, shits in temperature and pH, induce the RpoS regulon resulting in various morphological and physiological alterations that increase the capability of cells to resist those stresses and survive. (Foster P.L., 2007) These stresses can lead to induce the mobility of transposons and insertion sequences, which can lead to gene inactivation or activation. The mechanisms control genetic control of the mutagenesis. Transposons can increase their opportunity for transmission to other bacteria by increasing their copy number in the chromosome, conjugative plasmid and prophage genomes of their bacterial host. (Tenaillon O, et al., 2004) Bacteria will probably die under stress, but their transposon-infested DNA can be transmitted before or after cellular death. Gene amplification leads to drug resistance, a common mechanism adopted by eukaryotic organisms, including bacteria. The amplification leads to an overexpression of multi grip transporters and drug targets.