Experimental adaptation of Salmonella typhimurium to mice

Experimental evolution is a powerful approach to study the dynamics and mechanisms of bacterial niche specialization. By serial passage in mice, we evolved 18 independent lineages of Salmonella typhimurium LT2 and examined the rate and extent of adaptation to a mainly reticuloendothelial host environment. Bacterial mutation rates and population sizes were varied by using wild-type and DNA repair-defective mutator (mutS) strains with normal and high mutation rates, respectively, and by varying the number of bacteria intraperitoneally injected into mice. After <200 generations of adaptation all lineages showed an increased fitness as measured by a faster growth rate in mice (selection coefficients 0.11-0.58). Using a generally applicable mathematical model we calculated the adaptive mutation rate for the wild-type bacterium to be >10(-6)/cell/generation, suggesting that the majority of adaptive mutations are not simple point mutations. For the mutator lineages, adaptation to mice was associated with a loss of fitness in secondary environments as seen by a reduced metabolic capability. During adaptation there was no indication that a high mutation rate was counterselected. These data show that S. typhimurium can rapidly and extensively increase its fitness in mice but this niche specialization is, at least in mutators, associated with a cost.     

Evolutionary genetics: The economics of mutation.

The presence of mutator genotypes in populations of bacteria may be favoured by selection because they produce rare beneficial mutations and thereby increase the rate of adaptive evolution. Recent work, however, shows that the relationship between mutation rates and adaptive evolution is more complicated.     

Mutational Spectrum Drives the Rise of Mutator Bacteria

Understanding how mutator strains emerge in bacterial populations is relevant both to evolutionary theory and to reduce the threat they pose in clinical settings. The rise of mutator alleles is understood as a result of their hitchhiking with linked beneficial mutations, although the factors that govern this process remain unclear. A prominent but underappreciated fact is that each mutator allele increases only a specific spectrum of mutational changes. This spectrum has been speculated to alter the distribution of fitness effects of beneficial mutations, potentially affecting hitchhiking. To study this possibility, we analyzed the fitness distribution of beneficial mutations generated from different mutator and wild-type Escherichia coli strains. Using antibiotic resistance as a model system, we show that mutational spectra can alter these distributions substantially, ultimately determining the competitive ability of each strain across environments. Computer simulation showed that the effect of mutational spectrum on hitchhiking dynamics follows a non-linear function, implying that even slight spectrum-dependent fitness differences are sufficient to alter mutator success frequency by several orders of magnitude. These results indicate an unanticipated central role for the mutational spectrum in the evolution of bacterial mutation rates. At a practical level, this study indicates that knowledge of the molecular details of resistance determinants is crucial for minimizing mutator evolution during antibiotic therapy.     

A trade-off between oxidative stress resistance and DNA repair plays a role in the evolution of elevated mutation rates in bacteria

The dominant paradigm for the evolution of mutator alleles in bacterial populations is that they spread by indirect selection for linked beneficial mutations when bacteria are poorly adapted. In this paper, we challenge the ubiquity of this paradigm by demonstrating that a clinically important stressor, hydrogen peroxide, generates direct selection for an elevated mutation rate in the pathogenic bacterium Pseudomonas aeruginosa as a consequence of a trade-off between the fidelity of DNA repair and hydrogen peroxide resistance. We demonstrate that the biochemical mechanism underlying this trade-off in the case of mutS is the elevated secretion of catalase by the mutator strain. Our results provide, to our knowledge, the first experimental evidence that direct selection can favour mutator alleles in bacterial populations, and pave the way for future studies to understand how mutation and DNA repair are linked to stress responses and how this affects the evolution of bacterial mutation rates.     

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.     

Evolution of mutation rates in bacteria

Evolutionary success of bacteria relies on the constant fine-tuning of their mutation rates, which optimizes their adaptability to constantly changing environmental conditions. When adaptation is limited by the mutation supply rate, under some conditions, natural selection favours increased mutation rates by acting on allelic variation of the genetic systems that control fidelity of DNA replication and repair. Mutator alleles are carried to high frequency through hitchhiking with the adaptive mutations they generate. However, when fitness gain no longer counterbalances the fitness loss due to continuous generation of deleterious mutations, natural selection favours reduction of mutation rates. Selection and counter-selection of high mutation rates depends on many factors: the number of mutations required for adaptation, the strength of mutator alleles, bacterial population size, competition with other strains, migration, and spatial and temporal environmental heterogeneity. Such modulations of mutation rates may also play a role in the evolution of antibiotic resistance.     

Fitness evolution and the rise of mutator alleles in experimental Escherichia coli populations

We studied the evolution of high mutation rates and the evolution of fitness in three experimental populations of Escherichia coli adapting to a glucose-limited environment. We identified the mutations responsible for the high mutation rates and show that their rate of substitution in all three populations was too rapid to be accounted for simply by genetic drift. In two of the populations, large gains in fitness relative to the ancestor occurred as the mutator alleles rose to fixation, strongly supporting the conclusion that mutator alleles fixed by hitchhiking with beneficial mutations at other loci. In one population, no significant gain in fitness relative to the ancestor occurred in the population as a whole while the mutator allele rose to fixation, but a substantial and significant gain in fitness occurred in the mutator subpopulation as the mutator neared fixation. The spread of the mutator allele from rarity to fixation took >1000 generations in each population. We show that simultaneous adaptive gains in both the mutator and wild-type subpopulations (clonal interference) retarded the mutator fixation in at least one of the populations. We found little evidence that the evolution of high mutation rates accelerated adaptation in these populations.     

Hypermutability impedes cooperation in pathogenic bacteria

When the supply of beneficial mutations limits adaptation, bacterial mutator alleles can reach high frequencies by hitchhiking with advantageous mutations. However, when populations are well adapted to their environments, the increased rate of deleterious mutations makes hypermutability selectively disadvantageous. Here, we consider a further cost of hypermutability: its potential to break down cooperation (group-beneficial behavior that is costly to the individual). This probably occurs for three reasons. First, an increased rate at which 'cheating' genotypes are generated; second, an increased probability of producing efficient cheats; and third, a decrease in relatedness (not addressed in the present study). We used Pseudomonas aeruginosa's production of extracellular iron-scavenging molecules, siderophores, to determine if cheating evolved more readily in mutator populations. Siderophore production is costly to individual bacteria but benefits all nearby cells. Siderophore-deficient cheats therefore have a selective advantage within populations. We observed the de novo evolution and subsequent increase in frequency of siderophore cheats within both wild-type and mutator populations for 200 generations. Cheats appeared and increased in frequency more rapidly in mutator populations. The presence of cheats was costly to the group, as shown by a negative correlation between cheat frequency and population density.     

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.     

The evolution of bacterial mutation rates under simultaneous selection by interspecific and social parasitism

Many bacterial populations harbour substantial numbers of hypermutable bacteria, in spite of hypermutation being associated with deleterious mutations. One reason for the persistence of hypermutators is the provision of novel mutations, enabling rapid adaptation to continually changing environments, for example coevolving virulent parasites. However, hypermutation also increases the rate at which intraspecific parasites (social cheats) are generated. Interspecific and intraspecific parasitism are therefore likely to impose conflicting selection pressure on mutation rate. Here, we combine theory and experiments to investigate how simultaneous selection from inter- and intraspecific parasitism affects the evolution of bacterial mutation rates in the plant-colonizing bacterium Pseudomonas fluorescens. Both our theoretical and experimental results suggest that phage presence increases and selection for public goods cooperation (the production of iron-scavenging siderophores) decreases selection for mutator bacteria. Moreover, phages imposed a much greater growth cost than social cheating, and when both selection pressures were imposed simultaneously, selection for cooperation did not affect mutation rate evolution. Given the ubiquity of infectious phages in the natural environment and clinical infections, our results suggest that phages are likely to be more important than social interactions in determining mutation rate evolution.     

Competition between Transposable Elements and Mutator Genes in Bacteria

Although both genotypes with elevated mutation rate (mutators) and mobilization of insertion sequence (IS) elements have substantial impact on genome diversification, their potential interactions are unknown. Moreover, the evolutionary forces driving gradual accumulation of these elements are unclear: Do these elements spread in an initially transposon-free bacterial genome as they enable rapid adaptive evolution? To address these issues, we inserted an active IS1 element into a reduced Escherichia coli genome devoid of all other mobile DNA. Evolutionary laboratory experiments revealed that IS elements increase mutational supply and occasionally generate variants with especially large phenotypic effects. However, their impact on adaptive evolution is small compared with mismatch repair mutator alleles, and hence, the latter impede the spread of IS-carrying strains. Given their ubiquity in natural populations, such mutator alleles could limit early phase of IS element evolution in a new bacterial host. More generally, our work demonstrates the existence of an evolutionary conflict between mutation-promoting mechanisms.     

Spontaneously arising mutL mutators in evolving Escherichia coli populations are the result of changes in repeat length

Over the course of thousands of generations of growth in a glucose-limited environment, 3 of 12 experimental populations of Escherichia coli spontaneously and independently evolved greatly increased mutation rates. In two of the populations, the mutations responsible for this increased mutation rate lie in the same region of the mismatch repair gene mutL. In this region, a 6-bp repeat is present in three copies in the gene of the wild-type ancestor of the experimental populations but is present in four copies in one of the experimental populations and two copies in the other. These in-frame mutations either add or delete the amino acid sequence LA in the MutL protein. We determined that the replacement of the wild-type sequence with either of these mutations was sufficient to increase the mutation rate of the wild-type strain to a level comparable to that of the mutator strains. Complementation of strains bearing the mutator mutations with wild-type copies of either mutL or the mismatch repair gene uvrD rescued the wild-type mutation rate. The position of the mutator mutations-in the region of MutL known as the ATP lid-suggests a possible deficiency in MutL's ATPase activity as the cause of the mutator phenotype. The similarity of the two mutator mutations (despite the independent evolutionary histories of the populations that gave rise to them) leads to a discussion of the potential adaptive role of DNA repeats.     

Lethal mutagenesis of bacteria

Lethal mutagenesis, the killing of a microbial pathogen with a chemical mutagen, is a potential broad-spectrum antiviral treatment. It operates by raising the genomic mutation rate to the point that the deleterious load causes the population to decline. Its use has been limited to RNA viruses because of their high intrinsic mutation rates. Microbes with DNA genomes, which include many viruses and bacteria, have not been considered for this type of treatment because their low intrinsic mutation rates seem difficult to elevate enough to cause extinction. Surprisingly, models of lethal mutagenesis indicate that bacteria may be candidates for lethal mutagenesis. In contrast to viruses, bacteria reproduce by binary fission, and this property ensures their extinction if subjected to a mutation rate >0.69 deleterious mutations per generation. The extinction threshold is further lowered when bacteria die from environmental causes, such as washout or host clearance. In practice, mutagenesis can require many generations before extinction is achieved, allowing the bacterial population to grow to large absolute numbers before the load of deleterious mutations causes the decline. Therefore, if effective treatment requires rapid population decline, mutation rates 0.69 may be necessary to achieve treatment success. Implications for the treatment of bacteria with mutagens, for the evolution of mutator strains in bacterial populations, and also for the evolution of mutation rate in cancer are discussed.     

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.     

Volatility of Mutator Phenotypes at Single Cell Resolution

Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.     

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.     

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.     

The effect of population bottlenecks on mutation rate evolution in asexual populations

In the absence of recombination, a mutator allele can spread through a population by hitchhiking with beneficial mutations that appear in its genetic background. Theoretical studies over the past decade have shown that the survival and fixation probability of beneficial mutations can be severely reduced by population size bottlenecks. Here, we use computational modelling and evolution experiments with the yeast S. cerevisiae to examine whether population bottlenecks can affect mutator dynamics in adapting asexual populations. In simulation, we show that population bottlenecks can inhibit mutator hitchhiking with beneficial mutations and are most effective at lower beneficial mutation supply rates. We then subjected experimental populations of yeast propagated at the same effective population size to three different bottleneck regimes and observed that the speed of mutator hitchhiking was significantly slower at smaller bottlenecks, consistent with our theoretical expectations. Our results, thus, suggest that bottlenecks can be an important factor in mutation rate evolution and can in certain circumstances act to stabilize or, at least, delay the progressive elevation of mutation rates in asexual populations. Additionally, our findings provide the first experimental support for the theoretically postulated effect of population bottlenecks on beneficial mutations and demonstrate the usefulness of studying mutator frequency dynamics for understanding the underlying dynamics of fitness‐affecting mutations.     

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10⁻⁵. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.