Synchronous Waves of Failed Soft Sweeps in the Laboratory: Remarkably Rampant Clonal Interference of Alleles at a Single Locus

It has increasingly been recognized that adapting populations of microbes contain not one, but many lineages continually arising and competing at once. This process, termed “clonal interference,” alters the rate and dynamics of adaptation and biases winning mutations toward those with the largest selective effect. Here we uncovered a dramatic example of clonal interference between multiple similar mutations occurring at the same locus within replicate populations of Methylobacterium extorquens AM1. Because these mutational events involved the transposition of an insertion sequence into a narrow window of a single gene, they were both readily detectable at low frequencies and could be distinguished due to differences in insertion sites. This allowed us to detect up to 17 beneficial alleles of this type coexisting in a single population. Despite conferring a large selective benefit, the majority of these alleles rose and then fell in frequency due to other lineages emerging that were more fit. By comparing allele-frequency dynamics to the trajectories of fitness gains by these populations, we estimated the fitness values of the genotypes that contained these mutations. Collectively across all populations, these alleles arose upon backgrounds with a wide range of fitness values. Within any single population, however, multiple alleles tended to rise and fall synchronously during a single wave of multiple genotypes with nearly identical fitness values. These results suggest that alleles of large benefit arose repeatedly in failed “soft sweeps” during narrow windows of adaptation due to the combined effects of epistasis and clonal interference.     

The genetic basis of parallel and divergent phenotypic responses in evolving populations of Escherichia coli

Pleiotropy plays a central role in theories of adaptation, but little is known about the distribution of pleiotropic effects associated with different adaptive mutations. Previously, we described the phenotypic effects of a collection of independently arising beneficial mutations in Escherichia coli. We quantified their fitness effects in the glucose environment in which they evolved and their pleiotropic effects in five novel resource environments. Here we use a candidate gene approach to associate the phenotypic effects of the mutations with the underlying genetic changes. Among our collection of 27 adaptive mutants, we identified a total of 21 mutations (18 of which were unique) encompassing five different loci or gene regions. There was limited resolution to distinguish among loci based on their fitness effects in the glucose environment, demonstrating widespread parallelism in the direct response to selection. However, substantial heterogeneity in mutant effects was revealed when we examined their pleiotropic effects on fitness in the five novel environments. Substitutions in the same locus clustered together phenotypically, indicating concordance between molecular and phenotypic measures of divergence.     

Model and test in a fungus of the probability that beneficial mutations survive drift

Determining the probability of fixation of beneficial mutations is critically important for building predictive models of adaptive evolution. Despite considerable theoretical work, models of fixation probability have stood untested for nearly a century. However, recent advances in experimental and theoretical techniques permit the development of models with testable predictions. We developed a new model for the probability of surviving genetic drift, a major component of fixation probability, for novel beneficial mutations in the fungus Aspergillus nidulans, based on the life-history characteristics of its colony growth on a solid surface. We tested the model by measuring the probability of surviving drift in 11 adapted strains introduced into wild-type populations of different densities. We found that the probability of surviving drift increased with mutant invasion fitness, and decreased with wild-type density, as expected. The model accurately predicted the survival probability for the majority of mutants, yielding one of the first direct tests of the extinction probability of beneficial mutations.     

Long-Term Experimental Evolution in Escherichia coli. XIII. Phylogenetic History of a Balanced Polymorphism

We investigated the phylogenetic history of a balanced polymorphism that evolved in an experimental population of Escherichia coli. Previous work showed that two ecologically and morphologically distinct types, designated L (large) and S (small), arose by generation 6000 and coexisted for more than 12,000 generations thereafter. Here, we performed RFLP analyses using Insertion Sequence elements to resolve the phylogenetic history of L and S. Specifically, we sought to determine whether the derived S morph was monophyletic, indicating a long history of coexistence with L or, alternatively, S was repeatedly regenerated from L, indicating a series of periods with only transiently stable coexistence. Phylogenetic analysis of some 200 clones collected throughout the history of this population demonstrates that S is monophyletic. We then performed competition assays using clones of both morphs from different generations to determine whether either or both lineages continued to undergo genetic adaptation. Indeed, both lineages continued to adapt, and their continued evolution contributed to fluctuations in their relative abundance over evolutionary time. Based on their phylogenetic history and independent evolutionary trajectories, S and L fulfill Cohan’s criteria for being different asexual species.[Reviewing Editor: Niles Lehman]     

Long-term experimental evolution in Escherichia coli. V. Effects of recombination with immigrant genotypes on the rate of bacterial evolution

This study builds upon an earlier experiment that examined the dynamics of mean fitness in evolving populations of Escherichia coli in which mutations were the sole source of genetic variation. During thousands of generations in a constant environment, the rate of improvement in mean fitness of these asexual populations slowed considerably from an initially rapid pace. In this study, we sought to determine whether sexual recombination with novel genotypes would reaccelerate the rate of adaption in these populations. To that end, treatment populations were propagated for an additional 1000 generations in the same environment as their ancestors, but they were periodically allowed to mate with an immigrant pool of genetically distinct Hfr (high frequency recombination) donors. These donors could transfer genes to the resident populations by conjugation, but the donors themselves could not grow in the experimental environment. Control populations were propagated under identical conditions, but in the absence of sexual recombination with the donors. All twelve control populations retained the ancestral alleles at every locus that was scored. In contrast, the sexual recombination treatment yielded dramatic increases in genetic variation. Thus, there was a profound effect of recombination on the rate of genetic change. However, the increased genetic variation in the treatment populations had no significant effect on the rate of adaptive evolution, as measured by changes in mean fitness relative to a common competitor. We then considered three hypotheses that might reconcile these two outcomes: recombination pressure, hitchhiking of recombinant genotypes in association with beneficial mutations, and complex selection dynamics whereby certain genotypes may have a selective advantage only within a particular milieu of competitors. The estimated recombination rate was too low to explain the observed rate of genetic change, either alone or in combination with hitchhiking effects. However, we documented comple x ecological interactions among some recombinant genotypes, suggesting that our method for estimating fitness relative to a common competitor might have underestimated the rate of adaptive evolution in the treatment populations.     

Adaptation of proteins to different environments: a comparison of proteome structural properties in Bacillus subtilis and Escherichia coli

We compared amino acid solvent accessibilities and helix propensities in data sets of Escherichia coli and Bacillus subtilis proteins. These species reside in very different environments and hold very different physiological properties. From the observations, it was proposed that the cytoplasm of B. subtilis is more ion-rich compared to the cytoplasm of E. coli, which might be more hydrophobic; therefore, during evolution these differences have resulted in different protein folding tracks. Such inherent differences imply that the results of bioinformatic analyses of protein structures might depend on the species from which the proteins are picked. It is also suggested that different cytoplasmic environments cause E. coli and B. subtilis to be appropriate for expression of distinct types of proteins.     

Interactions between motile Escherichia coli and glass in media with various ionic strengths, as observed with a three-dimensional-tracking microscope.

Escherichia coli bacteria have been observed to swim along a glass surface for several minutes at a time. Settling velocities of nonmotile cells and a computer simulation of motile cells confirmed that an attractive force kept the bacteria near the surface. The goal of this study was to evaluate whether this attractive force could be explained by reversible adhesion of E. coli to the surface in the secondary energy minimum, according to the theory of Derjaguin, Landan, Verwey, and Overbeek (DLVO theory). This theory describes interactions between colloidal particles by combining attractive van der Waals forces with repulsive electrostatic forces. A three-dimensional-tracking microscope was used to follow both wild-type and smooth-swimming E. coli bacteria as they interacted with a glass coverslip in media of increasing ionic strengths, which corresponded to increasing depths of the secondary energy minimum. We found no quantifiable changes with ionic strength for either the tendencies of individual bacteria to approach the surface or the overall times bacteria spent near the surface. One change in bacterial behavior which was observed with the change in ionic strength was that the diameters of the circles which the smooth-swimming bacteria traced out on the glass increased in low-ionic-strength solution.     

Changes in intracellular metabolite pools, and acetate formation in Escherichia coli are associated with a cell-density-dependent metabolic switch

A cell density-dependent metabolic switch in amino acid metabolism occurs in E. coli W3110 batch cultures at 1.15 g dry wt l^–1 (Han L, Doverskog M, Enfors S-O, Häggström L, 2002, J. Biotechnol.92: 237–249). A two- to three-fold decrease of the concentration of most glycolytic and citric acid cycle metabolites, and an increase in acetyl-CoA concentration after the switch, indicates that the central metabolism also is affected. The specific acetate production rate decreases throughout the culture, except for a temporary increase at the switch point. The intracellular acetate concentration remains relatively constant during the culture.     

Formation of recombinant lacZ+ DNA in conjugational crosses with a recB mutant of Escherichia coli K12 depends on recF, recJ, and recO

Summary Conjugational recombination in Escherichia coli was investigated by monitoring synthesis of the lacZ ⁺ product, -galactosidase, in crosses between lacZ mutants. We report here that mutation of recB and any combination of recF, recJ, or recO reduces enzyme production by a factor of between 10- and 25-fold whereas mutation of only one of these genes or any combination of recF, recJ, or recO has no more than a 2-fold effect. Mutation of recN has no effect either alone or in combination with the other mutations. We suggest that the products of recF, recJ, and recO may provide an efficient alternative to the RecBCD enzyme for the initiation of recombination in conjugational crosses but that RecBCD activity is needed in this case to produce a viable recombinant product.     

Frameshift events associated with the lysyl-tRNA and the rare arginine codon, AGA, in Escherichia coli: a case study involving the human Relaxin 2 protein

Human Relaxin 2 is an insulin-related peptide hormone with a mass of 19,084 Da. The mRNA contains a number of arginine codons that are rarely used by Escherichia coli to produce highly expressed proteins. As a result, expressing this recombinant protein in E. coli is problematic. When human Relaxin 2 was expressed in E. coli BL21 (DE3), several forms of the protein were made. One species had the expected molecular weight (19,084 Da). A second species observed had a molecular weight of 21,244 Da. A third minor species had a molecular weight of 17,118 Da. These aberrant molecular weights can be explained as follows. First, a sequence CGA-AAA-AAG-AGA, containing the rare arginine codons CGA and AGA was the site of the +1 frameshift that generated the 21,244 Da species. Since there was a limited supply of this arginyl-tRNA, the peptidyl-tRNA moved +1 nucleotide to occupy the codon and resumed protein synthesis. Second, a -1 frameshift associated with 'slippery A' sequence XXA-AAA-AAG accounted for 10% of the product with a mass of 17,118 Da. Presumably, the shift to -1 also occurred because there was a paucity of the arginyl-tRNAArgucu. Introduction of a plasmid coding for the cognate tRNA for AGA and site directed mutagenesis prevented the formation of both frameshift species.     

Evaluation of the pH-dependent,stationary-phase acid tolerance in Listeria monocytogenes and Salmonella Typhimurium DT104 induced by culturing in media with 1% glucose: a comparative study with Escherichia coli O157:H7

AIMS: To comparatively evaluate the adaptive stationary-phase acid tolerance response (ATR) in food-borne pathogens induced by culturing in glucose-containing media, as affected by strain variability and antibiotic resistance, growth temperature, challenge pH and type of acidulant. METHODS AND RESULTS: Antibiotic resistant or sensitive strains of Listeria monocytogenes, Salmonella including S. Typhimurium DT104, and Escherichia coli O157:H7 were cultured (30 degrees C for 24 h; 10 degrees C for up to 14 days) in trypticase soya broth with yeast extract (TSBYE) with 1% or without glucose to induce or prevent acid adaptation, respectively. Cultures were subsequently exposed to pH 3.5 or 3.7 with lactic or acetic acid at 25 degrees C for 120 min. Acid-adapted cultures were more acid tolerant than nonadapted cultures, particularly those of L. monocytogenes and Salmonella. No consistent, positive or negative, influence of antibiotic resistance on the pH-inducible ATR or acid resistance (AR) was observed. Compared with 30 degrees C cultures, growth and acid adaptation of L. monocytogenes and S. Typhimurium DT104 at 10 degrees C markedly reduced their ATR and AR in stationary phase. E. coli O157:H7 had the greatest AR, relying less on acid adaptation. A 0.2 unit difference in challenge pH (3.5-3.7) caused great variations in survival of acid-adapted and nonadapted cells. CONCLUSIONS: Culturing L. monocytogenes and Salmonella to stationary phase in media with 1% glucose induces a pH-dependent ATR and enhances their survival to organic acids; thus, this method is suitable for producing acid-adapted cultures for use in food challenge studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial pathogens may become acid-adapted in foods containing glucose or other fermentable carbohydrates. Low storage temperatures may substantially decrease the stationary-phase ATR of L. monocytogenes and S. Typhimurium DT104, but their effect on ATR of E. coli O157:H7 appears to be far less dramatic.