Detection of mutator subpopulations in Salmonella typhimurium LT2 by reversion of his alleles

Defects in the methyl-directed mismatch repair lead to both the hypermutability phenotype and removal of a barrier to genetic exchange between species. Mutator bacteria carrying such defects occur frequently among bacterial pathogens, suggesting that subpopulations of mutators are contained within pathogen clones and give rise to the genetic variants that are acted upon by selective forces to allow survival or successful infection. We report here on the detection of the mutator subpopulation in Salmonella typhimurium and determination of its frequency in laboratory cultures. The analysis involved screening for mutators among revertants of S. typhimurium histidine auxotrophs selected for the His⁺ phenotype, since the frequency of mutators is expected to be increased in the selected mutant population they helped to spawn. The increases in spontaneous reversion of histidine mutations were first measured in isogenic strains carrying mismatch repair-defective mutH, mutL, mutS, or uvrD alleles, relative to their mismatch repair-proficient counterparts. Screening for the mutator phenotype in nearly 12,000 revertants of repair-proficient strains carrying his mutations highly stimulated for reversion in mutator backgrounds, the base substitution in hisG428 and frameshift in hisC3076, yielded five mutator strains (0.04%). the his⁺ reversion mutations contained within the newly-arisen mutator strains were characteristic of the predominant nucleotide changes expected in such mutators, as assessed by comparison with the spectra for reversion events in wild-type and mismatch correction-defective backgrounds. The results show that subpopulations of mutators, residing in normal populations at a finite frequency, can be culled from the culture by strong selection for a required phenotype. We calculate that the frequency of mutators in the unselected population of S. typhimurium is 1–4×10⁻⁶, an incidence of 10-fold lower than that expected based on studies of laboratory cultures of Escherichia coli.     

FIXATION OF MUTATORS IN ASEXUAL POPULATIONS: THE ROLE OF GENETIC DRIFT AND EPISTASIS

We study the evolutionary dynamics of an asexual population of nonmutators and mutators on a class of epistatic fitness landscapes. We consider the situation in which all mutations are deleterious and mutators are produced from nonmutators continually at a constant rate. We find that in an infinitely large population, a minimum nonmutator‐to‐mutator conversion rate is required to fix the mutators but an arbitrarily small conversion rate results in the fixation of mutators in a finite population. We calculate analytical expressions for the mutator fraction at mutation‐selection balance and fixation time for mutators in a finite population when the difference between the mutation rate for mutator and nonmutator is smaller (regime I) and larger (regime II) than the selection coefficient. Our main result is that in regime I, the mutator fraction and the fixation time are independent of epistasis but in regime II, mutators are rarer and take longer to fix when the decrease in fitness with the number of deleterious mutations occurs at an accelerating rate (synergistic epistasis) than at a diminishing rate (antagonistic epistasis). Our analytical results are compared with numerics and their implications are discussed.     

Mutators, population size, adaptive landscape and the adaptation of asexual populations of bacteria.

Selection of mutator alleles, increasing the mutation rate up to 10, 000-fold, has been observed during in vitro experimental evolution. This spread is ascribed to the hitchhiking of mutator alleles with favorable mutations, as demonstrated by a theoretical model using selective parameters corresponding to such experiments. Observations of unexpectedly high frequencies of mutators in natural isolates suggest that the same phenomenon could occur in the wild. But it remains questionable whether realistic in natura parameter values could also result in selection of mutators. In particular, the main parameters of adaptation, the size of the adapting population and the height and steepness of the adaptive peak characterizing adaptation, are very variable in nature. By simulation approach, we studied the effect of these parameters on the selection of mutators in asexual populations, assuming additive fitness. We show that the larger the population size, the more likely the fixation of mutator alleles. At a large population size, at least four adaptive mutations are needed for mutator fixation; moreover, under stronger selection stronger mutators are selected. We propose a model based on multiple mutations to illustrate how second-order selection can optimize population fitness when few favorable mutations are required for adaptation.     

Inefficient mismatch repair: genetic defects and down regulation

The mismatch repair system is involved in the maintenance of genomic integrity by editing DNA replication and recombination. However, although most mutations are neutral or deleterious, a mutator phenotype due to an inefficient mismatch repair may generate advantageous variants and may therefore be selected for. We review the evidence for inefficient mismatch repair due either to genetic defects in mismatch repair genes or to physiological conditions. Among natural isolates ofEscherichia coli andSalmonella enterica, about 1% are mutator bacteria, mostly deficient in mismatch repair (most of them defective in themutS gene). Characterization of mutators derived from laboratory strains led also to the isolation of mismatch repair mutants in which the most frequently found defects are inmutL andmutS. The correlation of the size of the antimutator genes with the frequency of their defective alleles amongE. coli andSalmonella strains reveals thatmutU mutants are underrepresented. Analysis of the progeny of a defined M13 phage heteroduplex DNA transfected intoE. coli cells shows that mismatch repair efficiency progressively decreases from the end of the exponential growth in K-12 and is variable among natural isolates. Implications of this defective mismatch repair activity for evolution and tumorigenesis will be discussed.     

Interplay between Pleiotropy and Secondary Selection Determines Rise and Fall of Mutators in Stress Response

Mutators are clones whose mutation rate is about two to three orders of magnitude higher than the rate of wild-type clones and their roles in adaptive evolution of asexual populations have been controversial. Here we address this problem by using an ab initio microscopic model of living cells, which combines population genetics with a physically realistic presentation of protein stability and protein-protein interactions. The genome of model organisms encodes replication controlling genes (RCGs) and genes modeling the mismatch repair (MMR) complexes. The genotype-phenotype relationship posits that the replication rate of an organism is proportional to protein copy numbers of RCGs in their functional form and there is a production cost penalty for protein overexpression. The mutation rate depends linearly on the concentration of homodimers of MMR proteins. By simulating multiple runs of evolution of populations under various environmental stresses—stationary phase, starvation or temperature-jump—we find that adaptation most often occurs through transient fixation of a mutator phenotype, regardless of the nature of stress. By contrast, the fixation mechanism does depend on the nature of stress. In temperature jump stress, mutators take over the population due to loss of stability of MMR complexes. In contrast, in starvation and stationary phase stresses, a small number of mutators are supplied to the population via epigenetic stochastic noise in production of MMR proteins (a pleiotropic effect), and their net supply is higher due to reduced genetic drift in slowly growing populations under stressful environments. Subsequently, mutators in stationary phase or starvation hitchhike to fixation with a beneficial mutation in the RCGs, (second order selection) and finally a mutation stabilizing the MMR complex arrives, returning the population to a non-mutator phenotype. Our results provide microscopic insights into the rise and fall of mutators in adapting finite asexual populations.     

Mutagenesis and repair in Bacillus anthracis: the effect of mutators

We have generated mutator strains of Bacillus anthracis Sterne by using directed gene knockouts to investigate the effect of deleting genes involved in mismatch repair, oxidative repair, and maintaining triphosphate pools. The single-knockout strains are deleted for mutS, mutY, mutM, or ndk. We also made double-knockout strains that are mutS ndk or mutY mutM. We have measured the levels of mutations in the rpoB gene that lead to the Rif(r) phenotype and have examined the mutational specificity. In addition, we examined the mutational specificity of two mutagens, 5-azacytidine and N-methyl-N'-nitro-N-nitroso-guanidine. The mutY and mutM single knockouts are weak mutators by themselves, but the combination of mutY mutM results in very high mutation rates, all due to G:C --> T:A transversions. The situation parallels that seen in Escherichia coli. Also, mutS knockouts are strong mutators and even stronger in the presence of a deletion of ndk. The number of sites in rpoB that can result in the Rif(r) phenotype by single-base substitution is more limited than in certain other bacteria, such as E. coli and Deinococcus radiodurans, although the average mutation rate per mutational site is roughly comparable. Hotspots at sites with virtually identical surrounding sequences are organism specific.     

Mutator dynamics in fluctuating environments

Populations with high mutation rates (mutator clones) are being found in increasing numbers of species, and a clear link is being established between the presence of mutator clones and drug resistance. Mutator clones exist despite the fact that in a constant environment most mutations are deleterious, with the spontaneous mutation rate generally held at a low value. This implies that mutator clones have an important role in the adaptation of organisms to changing environments. Our study examines how mutator dynamics vary according to the frequency of environmental fluctuations. Although recent studies have considered a single environmental switch, here we investigate mutator dynamics in a regularly varying environment, seeking to mimic conditions present, for example, under certain drug or pesticide regimes. Our model provides four significant new insights. First, the results demonstrate that mutators are most prevalent under intermediate rates of environmental change. When the environment oscillates more rapidly, mutators are unable to provide sufficient adaptability to keep pace with the frequent changes in selection pressure and, instead, a population of 'environmental generalists' dominates. Second, our findings reveal that mutator dynamics may be complex, exhibiting limit cycles and chaos. Third, we demonstrate that when each beneficial mutation provides a greater gain in fitness, mutators achieve higher densities in more rapidly fluctuating environments. Fourth, we find that mutators of intermediate strength reach higher densities than very weak or strong mutators.     

High relatedness selects against hypermutability in bacterial metapopulations

Mutation rate and cooperation have important ecological and evolutionary consequences and, moreover, can affect pathogen virulence. While hypermutability accelerates adaptation to novel environments, hypermutable lineages ('mutators') are selected against in well-adapted populations. Using the model organism Pseudomonas aeruginosa, we previously demonstrated a further potential disadvantage to hypermutability, namely, that it can accelerate the breakdown of cooperation. We now investigate how this property of mutators can affect their persistence in metapopulations. Mutator and wild-type bacteria were competed for 250 generations in globally competing metapopulations, imposing conditions of high or low intra-deme relatedness. High relatedness favours cooperating groups, so we predicted that mutators should achieve lower equilibrium frequencies under high relatedness than under low relatedness. This was observed in our study. Consistent with our hypothesis, there was a positive correlation between mean mutator and cheat frequencies. We conclude that when dense population growth requires cooperation, and when cooperation is favoured (high relatedness), demes containing high frequencies of mutators are likely to be selected against because they also contain high frequencies of non-cooperating cheats. We have also identified conditions where mutator lineages are likely to dominate metapopulations; namely, when low relatedness reduces kin selection for cooperation. These results may help to explain clinical distributions of mutator bacteria.     

Direct selection for mutators in Escherichia coli

We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10(-5) in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.     

The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants

We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.     

Inactivation of mismatch repair increases the diversity of Vibrio parahaemolyticus

Inactivation of mismatch repair (MMR) has been shown to increase the accumulation of spontaneous mutations and frequency of recombination for diverse pathogenic bacteria. Currently, little is known regarding the role of mutator phenotypes for the diversification of natural populations of opportunistic human pathogens in marine environments. In this study, a higher frequency of mutators was detected among V. parahaemolyticus strains obtained from environmental sources compared with clinical sources. Inactivation of the MMR gene mutS caused increased antibiotic resistance and phase variation resulting in translucent colony morphologies. Increased nucleotide diversity in mutS and rpoB alleles from mutator compared with wild-type strains indicated a significant contribution of the mutator phenotype to the evolution of select genes. The results of this study indicate that the inactivation of MMR in V. parahaemolyticus leads to increased genetic and phenotypic diversity. This study is the first to report a higher frequency of natural mutators among Vibrio environmental strains and to provide evidence that inactivation of MMR increases the diversity of V. parahaemolyticus .     

Enrichment and elimination of mutY mutators in Escherichia coli populations

The kinetics of mutator sweeps was followed in two independent populations of Escherichia coli grown for up to 350 generations in glucose-limited continuous culture. A rapid elevation of mutation rates was observed in both populations within 120-150 generations, as was apparent from major increases in the proportion of the populations with unselected mutations in fhuA. The increase in mutation rates was due to sweeps by mutY mutators. In both cultures, the enrichment of mutators resulted from hitchhiking with identified beneficial mutations increasing fitness under glucose limitation; mutY hitchhiked with mgl mutations in one culture and ptsG in the other. In both cases, mutators were enriched to constitute close to 100% of the population before a periodic selection event reduced the frequency of unselected mutations and mutators in the cultures. The high proportion of mutators persisted for 150 generations in one population but began to be eliminated within 50 generations in the other. The persistence of mutator, as well as experimental data showing that mutY bacteria were as fit as near-isogenic mutY(+) bacteria in competition experiments, suggest that mutator load by deleterious mutations did not explain the rapidly diminishing proportion of mutators in the populations. The nonmutators sweeping out mutators were also unlikely to have arisen by reversion or antimutator mutations; the mutY mutations were major deletions in each case and the bacteria sweeping out mutators contained intact mutY. By following mgl allele frequencies in one population, we discovered that mutators were outcompeted by bacteria that had rare mgl mutations previously as well as additional beneficial mutation(s). The pattern of appearance of mutY, but not its elimination, conforms to current models of mutator sweeps in bacterial populations. A mutator with a narrow mutational spectrum like mutY may be lost if the requirement for beneficial mutations is for changes other than GC --> TA transversions. Alternatively, epistatic interactions between mutator mutation and beneficial mutations need to be postulated to explain mutator elimination.     

Mutator suppression and escape from replication error-induced extinction in yeast

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10⁻³ inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer.     

Contrasting dynamics of a mutator allele in asexual populations of differing size

Mutators have been shown to hitchhike in asexual populations when the anticipated beneficial mutation supply rate of the mutator subpopulation, NU(b) (for subpopulation of size N and beneficial mutation rate U(b)) exceeds that of the wild-type subpopulation. Here, we examine the effect of total population size on mutator dynamics in asexual experimental populations of Saccharomyces cerevisiae. Although mutators quickly hitchhike to fixation in smaller populations, mutator fixation requires more and more time as population size increases; this observed delay in mutator hitchhiking is consistent with the expected effect of clonal interference. Interestingly, despite their higher beneficial mutation supply rate, mutators are supplanted by the wild type in very large populations. We postulate that this striking reversal in mutator dynamics is caused by an interaction between clonal interference, the fitness cost of the mutator allele, and infrequent large-effect beneficial mutations in our experimental populations. Our work thus identifies a potential set of circumstances under which mutator hitchhiking can be inhibited in natural asexual populations, despite recent theoretical predictions that such populations should have a net tendency to evolve ever-higher genomic mutation rates.     

The evolution of mutator genes in bacterial populations: the roles of environmental change and timing

Recent studies have found high frequencies of bacteria with increased genomic rates of mutation in both clinical and laboratory populations. These observations may seem surprising in light of earlier experimental and theoretical studies. Mutator genes (genes that elevate the genomic mutation rate) are likely to induce deleterious mutations and thus suffer an indirect selective disadvantage; at the same time, bacteria carrying them can increase in frequency only by generating beneficial mutations at other loci. When clones carrying mutator genes are rare, however, these beneficial mutations are far more likely to arise in members of the much larger nonmutator population. How then can mutators become prevalent? To address this question, we develop a model of the population dynamics of bacteria confronted with ever-changing environments. Using analytical and simulation procedures, we explore the process by which initially rare mutator alleles can rise in frequency. We demonstrate that subsequent to a shift in environmental conditions, there will be relatively long periods of time during which the mutator subpopulation can produce a beneficial mutation before the ancestral subpopulations are eliminated. If the beneficial mutation arises early enough, the overall frequency of mutators will climb to a point higher than when the process began. The probability of producing a subsequent beneficial mutation will then also increase. In this manner, mutators can increase in frequency over successive selective sweeps. We discuss the implications and predictions of these theoretical results in relation to antibiotic resistance and the evolution of mutation rates.     

MSH2 missense mutations alter cisplatin cytotoxicity and promote cisplatin-induced genome instability

Defects in the mismatch repair protein MSH2 cause tolerance to DNA damage. We report how cancer-derived and polymorphic MSH2 missense mutations affect cisplatin cytotoxicity. The chemotolerance phenotype was compared with the mutator phenotype in a yeast model system. MSH2 missense mutations display a strikingly different effect on cell death and genome instability. A mutator phenotype does not predict chemotolerance or vice versa. MSH2 mutations that were identified in tumors (Y109C) or as genetic variations (L402F) promote tolerance to cisplatin, but leave the initial mutation rate of cells unaltered. A secondary increase in the mutation rate is observed after cisplatin exposure in these strains. The mutation spectrum of cisplatin-resistant mutators identifies persistent cisplatin adduction as the cause for this acquired genome instability. Our results demonstrate that MSH2 missense mutations that were identified in tumors or as polymorphic variations can cause increased cisplatin tolerance independent of an initial mutator phenotype. Cisplatin exposure promotes drug-induced genome instability. From a mechanistical standpoint, these data demonstrate functional separation between MSH2-dependent cisplatin cytotoxicity and repair. From a clinical standpoint, these data provide valuable information on the consequences of point mutations for the success of chemotherapy and the risk for secondary carcinogenesis.     

The evolution of low mutation rates in experimental mutator populations of Saccharomyces cerevisiae

Mutation is the source of both beneficial adaptive variation and deleterious genetic load, fueling the opposing selective forces than shape mutation rate evolution. This dichotomy is well illustrated by the evolution of the mutator phenotype, a genome-wide 10- to 100-fold increase in mutation rate. This phenotype has often been observed in clonally expanding populations exposed to novel or frequently changing conditions. Although studies of both experimental and natural populations have shed light on the evolutionary forces that lead to the spread of the mutator allele through a population, significant gaps in our understanding of mutator evolution remain. Here we use an experimental evolution approach to investigate the conditions required for the evolution of a reduction in mutation rate and the mechanisms by which populations tolerate the accumulation of deleterious mutations. We find that after ～6,700 generations, four out of eight experimental mutator lines had evolved a decreased mutation rate. We provide evidence that the accumulation of deleterious mutations leads to selection for reduced mutation rate clones in populations of mutators. Finally, we test the long-term consequences of the mutator phenotype, finding that mutator lines follow different evolutionary trajectories, some of which lead to drug resistance.     

Papillation in Bacillus anthracis colonies: a tool for finding new mutators

Colonies of Bacillus anthracis Sterne allow the growth of papillation after 6 days of incubation at 30°C on Luria–Bertani medium. The papillae are due to mutations that allow the cells to overcome the barriers to continued growth. Cells isolated from papillae display two distinct gross phenotypes (group A and group B). We determined that group A mutants have mutations in the nprR gene including frameshifts, deletions, duplications and base substitutions. We used papillation as a tool for finding new mutators as the mutators generate elevated levels of papillation. We discovered that disruption of yycJ or recJ leads to a spontaneous mutator phenotype. We defined the nprR/papillation system as a new mutational analysis system for B. anthracis. The mutational specificity of the new mutator yycJ is similar to that of mismatch repair‐deficient strains (MMR^‐) such as those with mutations in mutL or mutS. Deficiency in recJ results in a unique specificity, generating only tandem duplications.     

Ploidy controls the success of mutators and nature of mutations during budding yeast evolution

BACKGROUND: We used the budding yeast Saccharomyces cerevisiae to ask how elevated mutation rates affect the evolution of asexual eukaryotic populations. Mismatch repair defective and nonmutator strains were competed during adaptation to four laboratory environments (rich medium, low glucose, high salt, and a nonfermentable carbon source). RESULTS: In diploids, mutators have an advantage over nonmutators in all conditions, and mutators that win competitions are on average fitter than nonmutator winners. In contrast, haploid mutators have no advantage when competed against haploid nonmutators, and haploid mutator winners are less fit than nonmutator winners. The diploid mutator winners were all superior to their ancestors both in the condition they had adapted to, and in two of the other conditions. This phenotype was due to a mutation or class of mutations that confers a large growth advantage during the respiratory phase of yeast cultures that precedes stationary phase. This generalist mutation(s) was not selected in diploid nonmutator strains or in haploid strains, which adapt primarily by fixing specialist (condition-specific) mutations. In diploid mutators, such mutations also occur, and the majority accumulates after the fixation of the generalist mutation. CONCLUSIONS: We conclude that the advantage of mutators depends on ploidy and that diploid mutators can give rise to beneficial mutations that are inaccessible to nonmutators and haploid mutators.