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.     

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.     

Clonal interference and the periodic selection of new beneficial mutations in Escherichia coli

The conventional model of adaptation in asexual populations implies sequential fixation of new beneficial mutations via rare selective sweeps that purge all variation and preserve the clonal genotype. However, in large populations multiple beneficial mutations may co-occur, causing competition among them, a phenomenon called "clonal interference." Clonal interference is thus expected to lead to longer fixation times and larger fitness effects of mutations that ultimately become fixed, as well as to a genetically more diverse population. Here, we study the significance of clonal interference in populations consisting of mixtures of differently marked wild-type and mutator strains of Escherichia coli that adapt to a minimal-glucose environment for 400 generations. We monitored marker frequencies during evolution and measured the competitive fitness of random clones from each marker state after evolution. The results demonstrate the presence of multiple beneficial mutations in these populations and slower and more erratic invasion of mutants than expected by the conventional model, showing the signature of clonal interference. We found that a consequence of clonal interference is that fitness estimates derived from invasion trajectories were less than half the magnitude of direct estimates from competition experiments, thus revealing fundamental problems with this fitness measure. These results force a reevaluation of the conventional model of periodic selection for asexual microbes.     

Rates of DNA sequence evolution in experimental populations of Escherichia coli during 20,000 generations

We examined rates of DNA sequence evolution in 12 populations of Escherichia coli propagated in a glucose minimal medium for 20,000 generations. Previous work saw mutations mediated by mobile elements in these populations, but the extent of other genomic changes was not investigated. Four of the populations evolved defects in DNA repair and became mutators. Some 500 bp was sequenced in each of 36 genes for 50 clones, including 2 ancestral variants, 2 clones from each population at generation 10,000, and 2 from each at generation 20,000. Ten mutations were found in total, all point mutations including mostly synonymous substitutions and nonsynonymous polymorphisms; all 10 were found in mutator populations. We compared the observed sequence evolution to predictions based on different scenarios. The number of synonymous substitutions is lower than predicted from measured mutation rates in E. coli, but the number is higher than rates based on comparing E. coli and Salmonella genomes. Extrapolating to the entire genome, these data predict about 250 synonymous substitutions on average per mutator population, but only about 3 synonymous substitutions per nonmutator population, during 20,000 generations. These data illustrate the challenge of finding sequence variation among bacterial isolates that share such a recent ancestor. However, this limited variation also provides a useful baseline for research aimed at finding the beneficial substitutions in these populations.     

A method to infer positive selection from marker dynamics in an asexual population

MOTIVATION: The observation of positive selection acting on a mutant indicates that the corresponding mutation has some form of functional relevance. Determining the fitness effects of mutations thus has relevance to many interesting biological questions. One means of identifying beneficial mutations in an asexual population is to observe changes in the frequency of marked subsets of the population. We here describe a method to estimate the establishment times and fitnesses of beneficial mutations from neutral marker frequency data. RESULTS: The method accurately reproduces complex marker frequency trajectories. In simulations for which positive selection is close to 5% per generation, we obtain correlations upwards of 0.91 between correct and inferred haplotype establishment times. Where mutation selection coefficients are exponentially distributed, the inferred distribution of haplotype fitnesses is close to being correct. Applied to data from a bacterial evolution experiment, our method reproduces an observed correlation between evolvability and initial fitness defect.     

Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection

The genetic bases of adaptation are being investigated in 12 populations of Escherichia coli, founded from a common ancestor and serially propagated for 20,000 generations, during which time they achieved substantial fitness gains. Each day, populations alternated between active growth and nutrient exhaustion. DNA supercoiling in bacteria is influenced by nutritional state, and DNA topology helps coordinate the overall pattern of gene expression in response to environmental changes. We therefore examined whether the genetic controls over supercoiling might have changed during the evolution experiment. Parallel changes in topology occurred in most populations, with the level of DNA supercoiling increasing, usually in the first 2000 generations. Two mutations in the topA and fis genes that control supercoiling were discovered in a population that served as the focus for further investigation. Moving the mutations, alone and in combination, into the ancestral background had an additive effect on supercoiling, and together they reproduced the net change in DNA topology observed in this population. Moreover, both mutations were beneficial in competition experiments. Clonal interference involving other beneficial DNA topology mutations was also detected. These findings define a new class of fitness-enhancing mutations and indicate that the control of DNA supercoiling can be a key target of selection in evolving bacterial populations.     

Genotype‐by‐environment interactions due to antibiotic resistance and adaptation in Escherichia coli

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype‐by‐environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic‐free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic‐free conditions (LB medium), and did the same for the antibiotic‐sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant‐evolved genotypes were more phenotypically different from the ancestor and from each other than resistant‐nonevolved and sensitive‐evolved genotypes. This suggests genotype‐by‐environment interactions generated by antibiotic resistance mutations, observed previously in short‐term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long‐term selection on resistance mutations from fitness effects in a single environment.     

Rapid increase in viability due to new beneficial mutations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

It is usually assumed that new beneficial mutations are extremely rare. Yet, few experiments have been performed in multicellular organisms that measure the effect of new beneficial mutations on viability and other measures of fitness. In most experiments, it is difficult to clearly distinguish whether adaptations have occurred due to selection on new beneficial mutations or on preexisting genetic variation. Using a modification of a Dobzhansky and Spassky (Evolution 1:191–216, 1947) assay to study change in viability over generations, we have observed an increase in viability in lines homozygous for the second and third chromosomes of Drosophila melanogaster in 6–26 generations due to the occurrence of new beneficial mutations in population sizes of 20, 100 and 1,000. The lines with the lowest initial viability responded the fastest to new beneficial mutations. These results show that new beneficial mutations, along with selection, can quickly increase viability and fitness even in small populations. Hence, new advantageous mutations may play an important role in adaptive evolution in higher organisms.     

Genetic Divergence and Fitness Convergence under Uniform Selection in Experimental Populations of Bacteria

Replicate populations of bacteria were propagated for 1000 generations in the laboratory. The growth substrate was periodically renewed, so that during most generations (cell doublings) it was not limiting. The final clones demonstrated about a 40% fitness increase when competed against their common ancestor. This increase was uniform both among and within populations despite extensive differentiation in correlated traits: cell size, resistance to starvation and dry mass of culture. It is suggested that genetic diversity developed because selection promoted any changes directing cell activity toward a higher maximum growth rate. Evolution of this trait halted at a similar level when some basic constraints on bacterial metabolism were met. The selective values of emerging mutations must have depended on the genetic background. They would be beneficial early in evolution but ineffective near the limit of adaptation. This hypothesis was tested for one mutation that affected both fitness and colony morphology. In some clones it was the first adaptive mutation and provided a third of the total fitness increase, but it was not assimilated by the clones that reached the adaptive ceiling in some other way. Near the limit of adaptation, epistasis levels off the fitnesses of genetically variable clones.     

Linking temperature dependence of fitness effects of mutations to thermal niche adaptation

Fitness effects of mutations may generally depend on temperature that influences all rate-limiting biophysical and biochemical processes. Earlier studies suggested that high temperatures may increase the availability of beneficial mutations (‘more beneficial mutations’), or allow beneficial mutations to show stronger fitness effects (‘stronger beneficial mutation effects’). The ‘more beneficial mutations’ scenario would inevitably be associated with increased proportion of conditionally beneficial mutations at higher temperatures. This in turn predicts that populations in warm environments show faster evolutionary adaptation but suffer fitness loss when faced with cold conditions, and those evolving in cold environments become thermal-niche generalists (‘hotter is narrower’). Under the ‘stronger beneficial mutation effects’ scenario, populations evolving in warm environments would show faster adaptation without fitness costs in cold environments, leading to a ‘hotter is (universally) better’ pattern in thermal niche adaptation. We tested predictions of the two competing hypotheses using an experimental evolution study in which populations of two model bacterial species, Escherichia coli and Pseudomonas fluorescens, evolved for 2400 generations at three experimental temperatures. Results of reciprocal transplant experiments with our P. fluorescens populations were largely consistent with the ‘hotter is narrower’ prediction. Results from the E. coli populations clearly suggested stronger beneficial mutation effects at higher assay temperatures, but failed to detect faster adaptation in populations evolving in warmer experimental environments (presumably because of limitation in the supply of genetic variation). Our results suggest that the influence of temperature on mutational effects may provide insight into the patterns of thermal niche adaptation and population diversification across thermal conditions.     

LONG-TERM EXPERIMENTAL EVOLUTION IN ESCHERICHIA COLI. VII. MECHANISMS MAINTAINING GENETIC VARIABILITY WITHIN POPULATIONS

Six replicate populations of the bacterium Escherichia coli were propagated for more than 10,000 generations in a defined environment. We sought to quantify the variation among clones within these populations with respect to their relative fitness, and to evaluate the roles of three distinct population genetic processes in maintaining this variation. On average, a pair of clones from the same population differed from one another in their relative fitness by approximately 4%. This within-population variation was small compared with the average fitness gain relative to the common ancestor, but it was statistically significant. According to one hypothesis, the variation in fitness is transient and reflects the ongoing substitution of beneficial alleles. We used Fisher's fundamental theorem to compare the observed rate of each population's change in mean fitness with the extent of variation for fitness within that population, but we failed to discern any correspondence between these quantities. A second hypothesis supposes that the variation in fitness is maintained by recurrent deleterious mutations that give rise to a mutation-selection balance. To test this hypothesis, we made use of the fact that two of the six replicate populations had evolved mutator phenotypes, which gave them a genomic mutation rate approximately 100-fold higher than that of the other populations. There was a marginally significant correlation between a population's mutation rate and the extent of its within-population variance for fitness, but this correlation was driven by only one population (whereas two of the populations had elevated mutation rates). Under a third hypothesis, this variation is maintained by frequency-dependent selection, whereby genotypes have an advantage when they are rare relative to when they are common. In all six populations, clones were more fit, on average, when they were rare than when they were common, although the magnitude of the advantage when rare was usually small (~1% in five populations and ~5% in the other). These three hypotheses are not mutually exclusive, but frequency-dependent selection appears to be the primary force maintaining the fitness variation within these experimental populations.     

Adaptive mgl-regulatory mutations and genetic diversity evolving in glucose-limited Escherichia coli populations

The mutational adaptation of E. coli to low glucose concentrations was studied in chemostats over 280 generations of growth. All members of six independent populations acquired increased fitness through the acquisition of mutations at the mgl locus, increasing the binding protein-dependent transport of glucose. These mutations provided a strong fitness advantage (up to 10-fold increase in glucose affinity) and were present in most isolates after 140 generations. mgl constitutivity in some isolates was caused by base substitution, short duplication, small deletion and IS1 insertion in the 1041 bp gene encoding the repressor of the mgl system, mglD (galS). But an unexpectedly large proportion of mutations were located in the short mgl operator sequence (mglO), and the majority of mutations were in mglO after 280 generations of selection. The adaptive mglO substitutions in several independent populations were at exactly the positions conserved in the two 8 bp half-sites of the mgl operator, with the nature of the base changes also completely symmetrical. Either mutations were directed to the operator or the particular operator mutations had a selective advantage under glucose limitation. Indeed, isolates carrying mglO mutations showed greater rates of transport for glucose and galactose at low concentrations than those carrying mglD null mutations. mglO mutations avoid cross-talk by members of the GalR-Lacl repressor family, reducing transporter expression and providing a competitive advantage in a glucose-limited environment. Another interesting aspect of these results was that each adapted population acquired multiple mgl alleles, with several populations containing at least six different mgl-regulatory mutations co-existing after 200 generations. The diversity of mutations in the mglO/mglD region, generally in combination with mutations at other loci regulating glucose uptake (malT, mlc, ptsG), provided evidence for multiple clones in each population. Increased fitness was accompanied by the generation of genetic diversity and not the evolution of a single winner clone, as predicted by the periodic selection model of bacterial populations.     

Combining mathematical models and statistical methods to understand and predict the dynamics of antibiotic-sensitive mutants in a population of resistant bacteria during experimental evolution

Temporarily discontinuing the use of antibiotics has been proposed as a means to eliminate resistant bacteria by allowing sensitive clones to sweep through the population. In this study, we monitored a tetracycline-sensitive subpopulation that emerged during experimental evolution of E. coli K12 MG1655 carrying the multiresistance plasmid pB10 in the absence of antibiotics. The fraction of tetracycline-sensitive mutants increased slowly over 500 generations from 0.1 to 7%, and loss of resistance could be attributed to a recombination event that caused deletion of the tet operon. To help understand the population dynamics of these mutants, three mathematical models were developed that took into consideration recurrent mutations, increased host fitness (selection), or a combination of both mechanisms (full model). The data were best explained by the full model, which estimated a high mutation frequency (lambda = 3.11 x 10(-5)) and a significant but small selection coefficient (sigma = 0.007). This study emphasized the combined use of experimental data, mathematical models, and statistical methods to better understand and predict the dynamics of evolving bacterial populations, more specifically the possible consequences of discontinuing the use of antibiotics.     

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.     

The Minimal Complexity of Adapting Agents Increases with Fitness

What is the relationship between the complexity and the fitness of evolved organisms, whether natural or artificial? It has been asserted, primarily based on empirical data, that the complexity of plants and animals increases as their fitness within a particular environment increases via evolution by natural selection. We simulate the evolution of the brains of simple organisms living in a planar maze that they have to traverse as rapidly as possible. Their connectome evolves over 10,000s of generations. We evaluate their circuit complexity, using four information-theoretical measures, including one that emphasizes the extent to which any network is an irreducible entity. We find that their minimal complexity increases with their fitness.     

The Properties of Adaptive Walks in Evolving Populations of Fungus

The rarity of beneficial mutations has frustrated efforts to develop a quantitative theory of adaptation. Recent models of adaptive walks, the sequential substitution of beneficial mutations by selection, make two compelling predictions: adaptive walks should be short, and fitness increases should become exponentially smaller as successive mutations fix. We estimated the number and fitness effects of beneficial mutations in each of 118 replicate lineages of Aspergillus nidulans evolving for approximately 800 generations at two population sizes using a novel maximum likelihood framework, the results of which were confirmed experimentally using sexual crosses. We find that adaptive walks do indeed tend to be short, and fitness increases become smaller as successive mutations fix. Moreover, we show that these patterns are associated with a decreasing supply of beneficial mutations as the population adapts. We also provide empirical distributions of fitness effects among mutations fixed at each step. Our results provide a first glimpse into the properties of multiple steps in an adaptive walk in asexual populations and lend empirical support to models of adaptation involving selection towards a single optimum phenotype. In practical terms, our results suggest that the bulk of adaptation is likely to be accomplished within the first few steps.     

The role of pleiotropy in the maintenance of sex in yeast

In facultatively sexual species, lineages that reproduce asexually for a period of time can accumulate mutations that reduce their ability to undergo sexual reproduction when sex is favorable. We propagated Saccharomyces cerevisiae asexually for approximately 800 generations, after which we measured the change in sexual fitness, measured as the proportion of asci observed in sporulation medium. The sporulation rate in cultures propagated asexually at small population size declined by 8%, on average, over this time period, indicating that the majority of mutations that affect sporulation rate are deleterious. Interestingly, the sporulation rate in cultures propagated asexually at large population size improved by 11%, on average, indicating that selection on asexual function effectively eliminated most of the mutations deleterious to sporulation ability. These results suggest that pleiotropy between mutations' effects on asexual fitness and sexual fitness was predominantly positive, at least for the mutations accumulated in this experimental evolution study. A positive correlation between growth rate and sporulation rate among lines also provided evidence for positive pleiotropy. These results demonstrate that, at least under certain circumstances, selection acting on asexual fitness can help to maintain sexual function.     

The speed of evolution and maintenance of variation in asexual populations

BACKGROUND: The rate at which beneficial mutations accumulate determines how fast asexual populations evolve, but this is only partially understood. Some recent clonal-interference models suggest that evolution in large asexual populations is limited because smaller beneficial mutations are outcompeted by larger beneficial mutations that occur in different lineages within the same population. This analysis assumes that the important mutations fix one at a time; it ignores multiple beneficial mutations that occur in the lineage of an earlier beneficial mutation, before the first mutation in the series can fix. We focus on the effects of such multiple mutations. RESULTS: Our analysis predicts that the variation in fitness maintained by a continuously evolving population increases as the logarithm of the population size and logarithm of the mutation rate and thus yields a similar logarithmic increase in the speed of evolution. To test these predictions, we evolved asexual budding yeast in glucose-limited media at a range of population sizes and mutation rates. CONCLUSIONS: We find that their evolution is dominated by the accumulation of multiple mutations of moderate effect. Our results agree with our theoretical predictions and are inconsistent with the one-by-one fixation of mutants assumed by recent clonal-interference analysis.     

Genetic variation and the fate of beneficial mutations in asexual populations

The fate of a newly arising beneficial mutation depends on many factors, such as the population size and the availability and fitness effects of other mutations that accumulate in the population. It has proved difficult to understand how these factors influence the trajectories of particular mutations, since experiments have primarily focused on characterizing successful clones emerging from a small number of evolving populations. Here, we present the results of a massively parallel experiment designed to measure the full spectrum of possible fates of new beneficial mutations in hundreds of experimental yeast populations, whether these mutations are ultimately successful or not. Using strains in which a particular class of beneficial mutation is detectable by fluorescence, we followed the trajectories of these beneficial mutations across 592 independent populations for 1000 generations. We find that the fitness advantage provided by individual mutations plays a surprisingly small role. Rather, underlying "background" genetic variation is quickly generated in our initially clonal populations and plays a crucial role in determining the fate of each individual beneficial mutation in the evolving population.     

Frequent beneficial mutations during single-colony serial transfer of Streptococcus pneumoniae

The appearance of new mutations within a population provides the raw material for evolution. The consistent decline in fitness observed in classical mutation accumulation studies has provided support for the long-held view that deleterious mutations are more common than beneficial mutations. Here we present results of a study using a mutation accumulation design with the bacterium Streptococcus pneumoniae in which the fitness of the derived populations increased. This rise in fitness was associated specifically with adaptation to survival during brief stationary phase periods between single-colony population bottlenecks. To understand better the population dynamics behind this unanticipated adaptation, we developed a maximum likelihood model describing the processes of mutation and stationary-phase selection in the context of frequent population bottlenecks. Using this model, we estimate that the rate of beneficial mutations may be as high as 4.8×10(-4) events per genome for each time interval corresponding to the pneumococcal generation time. This rate is several orders of magnitude higher than earlier estimates of beneficial mutation rates in bacteria but supports recent results obtained through the propagation of small populations of Escherichia coli. Our findings indicate that beneficial mutations may be relatively frequent in bacteria and suggest that in S. pneumoniae, which develops natural competence for transformation, a steady supply of such mutations may be available for sampling by recombination.