Vaughn S. Cooper

The population genetics of ecological specialization in evolving Escherichia coli populations

When organisms adapt genetically to one environment, they may lose fitness in other environments. Two distinct population genetic processes can produce ecological specialization—mutation accumulation and antagonistic pleiotropy. In mutation accumulation, mutations become fixed by genetic drift in genes that are not maintained by selection; adaptation to one environment and loss of adaptation to another are caused by different mutations. Antagonistic pleiotropy arises from trade-offs, such that the same mutations that are beneficial in one environment are detrimental in another. In general, it is difficult to distinguish between these processes. We analysed the decay of unused catabolic functions in 12 lines of Escherichia coli propagated on glucose for 20,000 generations. During that time, several lines evolved high mutation rates. If mutation accumulation is important, their unused functions should decay more than the other lines, but no significant difference was observed. Moreover, most catabolic losses occurred early in the experiment when beneficial mutations were being rapidly fixed, a pattern predicted by antagonistic pleiotropy. Thus, antagonistic pleiotropy appears more important than mutation accumulation for the decay of unused catabolic functions in these populations.

Punctuated Evolution Caused by Selection of Rare Beneficial Mutations

For more than two decades there has been intense debate over the hypothesis that most morphological evolution occurs during relatively brief episodes of rapid change that punctuate much longer periods of stasis. A clear and unambiguous case of punctuated evolution is presented for cell size in a population of Escherichia coli evolving for 3000 generations in a constant environment. The punctuation is caused by natural selection as rare, beneficial mutations sweep successively through the population. This experiment shows that the most elementary processes in population genetics can give rise to punctuated evolutionary dynamics.

Mechanisms Causing Rapid and Parallel Losses of Ribose Catabolism in Evolving Populations of Escherichia coli B

Twelve populations of Escherichia coli B all lost D-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs(-) mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs(+) progenitor. These Rbs(-) mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 x 10(-5) per cell generation, of mutation from Rbs(+) to Rbs(-), which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbs genes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150 element located immediately upstream of the rbs operon. The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS150 copy and the one upstream of the rbs operon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functional rbs operon to two Rbs(-) mutants, and we constructed another Rbs(-) strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs(-) mutants have a competitive advantage relative to their Rbs(+) counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs(-) mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.

TRADEOFF BETWEEN HORIZONTAL AND VERTICAL MODES OF TRANSMISSION IN BACTERIAL PLASMIDS

It has been hypothesized that there is a fundamental conflict between horizontal (infectious) and vertical (intergenerational) modes of parasite transmission. Activities of a parasite that increase its rate of infectious transmission are presumed to reduce its hosts fitness. This reduction in host fitness impedes vertical transmission of the parasite and causes a tradeoff between horizontal and vertical transmission. Given this tradeoff, and assuming no multiple infections (no within‐host competition among parasites), a simple model predicts that the density of uninfected hosts in the environment should determine the optimum balance between modes of parasite transmission. When susceptible hosts are abundant, selection should favor increased rates of horizontal transfer, even at the expense of reduced vertical transmission. Conversely, when hosts are rare, selection should favor increased vertical transmission even at the expense of lower horizontal transfer. We tested the tradeoff hypothesis and these evolutionary predictions using conjugative plasmids and the bacteria that they infect. Plasmids were allowed to evolve for 500 generations in environments with different densities of susceptible hosts. The plasmids rate of horizontal transfer by conjugation increased at the expense of host fitness, indicating a tradeoff between horizontal and vertical transmission. Also, reductions in conjugation rate repeatedly coincided with the loss of a particular plasmid‐encoded antibiotic resistance gene. However, susceptible host density had no significant effect on the evolution of horizontal versus vertical modes of plasmid transmission. We consider several possible explanations for the failure to observe such an effect.

Tangled bank of experimentally evolved Burkholderia biofilms reflects selection during chronic infections

How diversity evolves and persists in biofilms is essential for understanding much of microbial life, including the uncertain dynamics of chronic infections. We developed a biofilm model enabling long-term selection for daily adherence to and dispersal from a plastic bead in a test tube. Focusing on a pathogen of the cystic fibrosis lung, Burkholderia cenocepacia, we sequenced clones and metagenomes to unravel the mutations and evolutionary forces responsible for adaptation and diversification of a single biofilm community during 1,050 generations of selection. The mutational patterns revealed recurrent evolution of biofilm specialists from generalist types and multiple adaptive alleles at relatively few loci. Fitness assays also demonstrated strong interference competition among contending mutants that preserved genetic diversity. Metagenomes from five other independently evolved biofilm lineages revealed extraordinary mutational parallelism that outlined common routes of adaptation, a subset of which was found, surprisingly, in a planktonic population. These mutations in turn were surprisingly well represented among mutations that evolved in cystic fibrosis isolates of both Burkholderia and Pseudomonas. These convergent pathways included altered metabolism of cyclic diguanosine monophosphate, polysaccharide production, tricarboxylic acid cycle enzymes, global transcription, and iron scavenging. Evolution in chronic infections therefore may be driven by mutations in relatively few pathways also favored during laboratory selection, creating hope that experimental evolution may illuminate the ecology and selective dynamics of chronic infections and improve treatment strategies.

Ecological succession in long-term experimentally evolved biofilms produces synergistic communities

Many biofilm populations are known for their exceptional biodiversity, but the relative contributions of the forces that could produce this diversity are poorly understood. This uncertainty grows in the old, well-established communities found on many natural surfaces and in long-term, chronic infections. If the prevailing interactions among species within biofilms are positive, productivity should increase with diversity, but if they tend towards competition or antagonism, productivity should decrease. Here, we describe the parallel evolution of synergistic communities derived from a clone of Burkholderia cenocepacia during ∼1500 generations of biofilm selection. This long-term evolution was enabled by a new experimental method that selects for daily cycles of colonization, biofilm assembly and dispersal. Each of the six replicate biofilm populations underwent a common pattern of adaptive morphological diversification, in which three ecologically distinct morphotypes arose in the same order of succession and persisted. In two focal populations, mixed communities were more productive than any monoculture and each variant benefited from the mixture. These gains in output resulted from asymmetrical cross-feeding between ecotypes and the expansion and partitioning of biofilm space that constructed new niches. Therefore, even in the absence of starting genetic variation, prolonged selection for surface colonization generates a dynamic of ecological succession that enhances productivity.

Why Genes Evolve Faster on Secondary Chromosomes in Bacteria

In bacterial genomes composed of more than one chromosome, one replicon is typically larger, harbors more essential genes than the others, and is considered primary. The greater variability of secondary chromosomes among related taxa has led to the theory that they serve as an accessory genome for specific niches or conditions. By this rationale, purifying selection should be weaker on genes on secondary chromosomes because of their reduced necessity or usage. To test this hypothesis we selected bacterial genomes composed of multiple chromosomes from two genera, Burkholderia and Vibrio, and quantified the evolutionary rates (dN and dS) of all orthologs within each genus. Both evolutionary rate parameters were faster among orthologs found on secondary chromosomes than those on the primary chromosome. Further, in every bacterial genome with multiple chromosomes that we studied, genes on secondary chromosomes exhibited significantly weaker codon usage bias than those on primary chromosomes. Faster evolution and reduced codon bias could in turn result from global effects of chromosome position, as genes on secondary chromosomes experience reduced dosage and expression due to their delayed replication, or selection on specific gene attributes. These alternatives were evaluated using orthologs common to genomes with multiple chromosomes and genomes with single chromosomes. Analysis of these ortholog sets suggested that inherently fast-evolving genes tend to be sorted to secondary chromosomes when they arise; however, prolonged evolution on a secondary chromosome further accelerated substitution rates. In summary, secondary chromosomes in bacteria are evolutionary test beds where genes are weakly preserved and evolve more rapidly, likely because they are used less frequently.

Rapid phenotypic change and diversification of a soil bacterium during 1000 generations of experimental evolution

Evolutionary pathways open to even relatively simple organisms, such as bacteria, may lead to complex and unpredictable phenotypic changes, both adaptive and non-adaptive. The evolutionary pathways taken by 18 populations of Ralstonia strain TFD41 while they evolved in defined environments for 1000 generations were examined. Twelve populations evolved in liquid media, while six others evolved on agar surfaces. Phenotypic analyses of these derived populations identified some changes that were consistent across all populations and others that differed among them. The evolved populations all exhibited morphological changes in their cell envelopes, including reductions of the capsule in each population and reduced prostheca-like surface structures in most populations. Mean cell length increased in most populations (in one case by more than fourfold), although a few populations evolved shorter cells. Carbon utilization profiles were variable among the evolved populations, but two distinct patterns were correlated with genetic markers introduced at the outset of the experiment. Fatty acid methyl ester composition was less variable across populations, but distinct patterns were correlated with the two physical environments. All 18 populations evolved greatly increased sensitivity to bile salts, and all but one had increased adhesion to sand; both patterns consistent with changes in the outer envelope. This phenotypic diversity contrasts with the fairly uniform increases in competitive fitness observed in all populations. This diversity may represent a set of equally probable adaptive solutions to the selective environment; it may also arise from the chance fixation of non-adaptive mutations that hitchhiked with a more limited set of beneficial mutations.

Timing of transmission and the evolution of virulence of an insect virus

We used the nuclear polyhedrosis virus of the gypsy moth, Lymantria dispar, to investigate whether the timing of transmission influences the evolution of virulence. In theory, early transmission should favour rapid replication and increase virulence, while late transmission should favour slower replication and reduce virulence. We tested this prediction by subjecting one set of 10 virus lineages to early transmission (Early viruses) and another set to late transmission (Late viruses). Each lineage of virus underwent nine cycles of transmission. Virulence assays on these lineages indicated that viruses transmitted early were significantly more lethal than those transmitted late. Increased exploitation of the host appears to come at a cost, however. While Early viruses initially produced more progeny, Late viruses were ultimately more productive over the entire duration of the infection. These results illustrate fitness trade-offs associated with the evolution of virulence and indicate that milder viruses can obtain a numerical advantage when mild and harmful strains tend to infect separate hosts.

The Environment Affects Epistatic Interactions to Alter the Topology of an Empirical Fitness Landscape

The fitness effect of mutations can be influenced by their interactions with the environment, other mutations, or both. Previously, we constructed 32 ( = 25) genotypes that comprise all possible combinations of the first five beneficial mutations to fix in a laboratory-evolved population of Escherichia coli. We found that (i) all five mutations were beneficial for the background on which they occurred; (ii) interactions between mutations drove a diminishing returns type epistasis, whereby epistasis became increasingly antagonistic as the expected fitness of a genotype increased; and (iii) the adaptive landscape revealed by the mutation combinations was smooth, having a single global fitness peak. Here we examine how the environment influences epistasis by determining the interactions between the same mutations in two alternative environments, selected from among 1,920 screened environments, that produced the largest increase or decrease in fitness of the most derived genotype. Some general features of the interactions were consistent: mutations tended to remain beneficial and the overall pattern of epistasis was of diminishing returns. Other features depended on the environment; in particular, several mutations were deleterious when added to specific genotypes, indicating the presence of antagonistic interactions that were absent in the original selection environment. Antagonism was not caused by consistent pleiotropic effects of individual mutations but rather by changing interactions between mutations. Our results demonstrate that understanding adaptation in changing environments will require consideration of the combined effect of epistasis and pleiotropy across environments.