Testing optimality with experimental evolution: lysis time in a bacteriophage

Optimality models collapse the vagaries of genetics into simple trade-offs to calculate phenotypes expected to evolve by natural selection. Optimality approaches are commonly criticized for this neglect of genetic details, but resolution of this disagreement has been difficult. The importance of genetic details may be tested by experimental evolution of a trait for which an optimality model exists and in which genetic details can be studied. Here we evolved lysis time in bacteriophage T7, a virus of Escherichia coli. Lysis time is equivalent to the age of reproduction in an organism that reproduces once and then dies. Delaying lysis increases the number of offspring but slows generation time, and this trade-off renders the optimum sensitive to environmental conditions: earlier lysis is favored when bacterial hosts are dense, later lysis is favored when hosts are sparse. In experimental adaptations, T7 evolved close to the optimum in conditions favoring early lysis but not in conditions favoring late lysis. One of the late lysis adaptations exhibited no detectable phenotypic evolution despite genetic evolution; the other evolved only partly toward the expected optimum. Overall, the lysis time of the adapted phages remained closer to their starting values than predicted by the model. From the perspective of the optimality model, the experimental conditions were expected to select changes only along the postulated trade-off, but a trait outside the trade-off evolved as well. Evidence suggests that the model's failure ultimately stems from a violation of the trade-off, rather than a paucity of mutations.     

The population genetics of adaptation: the adaptation of DNA sequences

I describe several patterns characterizing the genetics of adaptation at the DNA level. Following Gillespie (1983, 1984, 1991), I consider a population presently fixed for the ith best allele at a locus and study the sequential substitution of favorable mutations that results in fixation of the fittest DNA sequence locally available. Given a wild type sequence that is less than optimal, I derive the fitness rank of the next allele typically fixed by natural selection as well as the mean and variance of the jump in fitness that results when natural selection drives a substitution. Looking over the whole series of substitutions required to reach the best allele, I show that the mean fitness jumps occurring throughout an adaptive walk are constrained to a twofold window of values, assuming only that adaptation begins from a reasonably fit allele. I also show that the first substitution and the substitution of largest effect account for a large share of the total fitness increase during adaptation. I further show that the distribution of selection coefficients fixed throughout such an adaptive walk is exponential (ignoring mutations of small effect), a finding reminiscent of that seen in Fisher's geometric model of adaptation. Last, I show that adaptation by natural selection behaves in several respects as the average of two idealized forms of adaptation, perfect and random.     

Natural transformation increases the rate of adaptation in the human pathogen Helicobacter pylori

Gene exchange between individuals can lead to profound evolutionary effects at both the genomic and population levels. These effects have sparked widespread interest in examining the specific adaptive benefits of recombination. Although this work has primarily focused on the benefits of sex in eukaryotes, it is assumed that similar benefits of genetic exchange apply across eukaryotes and prokaryotes. Here we report a direct test of this assumption using the naturally transformable human gastric pathogen Helicobacter pylori as a model organism. We show that genetic exchange accelerates adaptation to a novel laboratory environment within bacterial populations and that a general adaptive advantage exists for naturally transformable strains when transfer occurs among conspecific backgrounds. This finding demonstrates that there are generalized benefits to adaptation in both eukaryotes and prokaryotes even though the underlying processes are mechanistically different.     

Evolutionary perspectives in a mutualism of sepiolid squid and bioluminescent bacteria: combined usage of microbial experimental evolution and temporal population genetics

The symbiosis between marine bioluminescent Vibrio bacteria and the sepiolid squid Euprymna is a model for studying animal-bacterial Interactions. Vibrio symbionts native to particular Euprymna species are competitively dominant, capable of outcompeting foreign Vibrio strains from other Euprymna host species. Despite competitive dominance, secondary colonization events by invading nonnative Vibrio fischeri have occurred. Competitive dominance can be offset through superior nonnative numbers and advantage of early start host colonization by nonnatives, granting nonnative vibrios an opportunity to establish beachheads in foreign Euprymna hosts. Here, we show that nonnative V. fischeri are capable of rapid adaptation to novel sepiolid squid hosts by serially passaging V. fischeri JRM200 (native to Hawaiian Euprymna scolopes) lines through the novel Australian squid host E. tasmanica for 500 generations. These experiments were complemented by a temporal population genetics survey of V. fischeri, collected from E. tasmanica over a decade, which provided a perspective from the natural history of V. fischeri evolution over 15,000-20,000 generations in E. tasmanica. No symbiont anagenic evolution within squids was observed, as competitive dominance does not purge V. fischeri genetic diversity through time. Instead, abiotic factors affecting abundance of V. fischeri variants in the planktonic phase sustain temporal symbiont diversity, a property itself of ecological constraints imposed by V. fischeri host adaptation.     

Antagonistic coevolution limits population persistence of a virus in a thermally deteriorating environment

Understanding the conditions under which rapid evolutionary adaptation can prevent population extinction in deteriorating environments (i.e. evolutionary rescue) is a crucial aim in the face of global climate change. Despite a rapidly growing body of work in this area, little attention has been paid to the importance of interspecific coevolutionary interactions. Antagonistic coevolution commonly observed between hosts and parasites is likely to retard evolutionary rescue because it often reduces population sizes, and results in the evolution of costly host defence and parasite counter-defence. We used experimental populations of a bacterium Pseudomonas fluorescens SBW25 and a bacteriophage virus (SBW25Φ2), to study how host-parasite coevolution impacts viral population persistence in the face of gradually increasing temperature, an environmental stress for the virus but not the bacterium. The virus persisted much longer when it evolved in the presence of an evolutionarily constant host genotype (i.e. in the absence of coevolution) than when the bacterium and virus coevolved. Further experiments suggest that both a reduction in population size and costly infectivity strategies contributed to viral extinction as a result of coevolution. The results highlight the importance of interspecific evolutionary interactions for the evolutionary responses of populations to global climate change.     

Population subdivision and adaptation in asexual populations of Saccharomyces cerevisiae

Population subdivision limits competition between individuals, which can have a profound effect on adaptation. Subdivided populations maintain more genetic diversity at any given time compared to well-mixed populations, and thus "explore" larger parts of the genotype space. At the same time, beneficial mutations take longer to spread in such populations, and thus subdivided populations do not "exploit" discovered mutations as efficiently as well-mixed populations. Whether subdivision inhibits or promotes adaptation in a given environment depends on the relative importance of exploration versus exploitation, which in turn depends on the structure of epistasis among beneficial mutations. Here we investigate the relative importance of exploration versus exploitation for adaptation by evolving 976 independent asexual populations of budding yeast with several degrees of geographic subdivision. We find that subdivision systematically inhibits adaptation: even the luckiest demes in subdivided populations on average fail to discover genotypes that are fitter than those discovered by well-mixed populations. Thus, exploitation of discovered mutations is more important for adaptation in our system than a thorough exploration of the mutational neighborhood, and increasing subdivision slows adaptation.     

Restrictions to RNA virus adaptation: an experimental approach

Some basic properties of RNA viruses are their high mutation rate, their enormous population sizes and their short generation time. These properties allow RNA virus populations to quickly explore fitness landscapes. A great adaptability has been amply demonstrated in experimental, as well as in natural, populations of RNA viruses. However, at least from a theoretical point of view, a limit to the extent of viral adaptation may exist as a consequence of adaptive trade-offs arising during evolution in changing environmental conditions. Here, I review previously published results searching for such fitness trade-offs. The following scenario has been explored: the cost of host-range expansion, the cost of resistance to antiviral drugs, and the adaptation to different population densities. Despite the environmental conditions tested, results show a common pattern: whenever a virus adapt to a simple environmental situation it pays a cost in terms of adaptation to alternative situations. However, in those cases where the virus has been simultaneously adapted to different environmental conditions, this cost disappears or, at least, is greatly reduced. Finally, and as another factor imposing a limit to their speed of adaptation, I review results showing that clonal interference also plays an important role during viral evolution. This revised version was published online in August 2006 with corrections to the Cover Date.     

Compensatory adaptation and diversification subsequent to evolutionary rescue in a model adaptive radiation

Biological populations may survive lethal environmental stress through evolutionary rescue. The rescued populations typically suffer a reduction in growth performance and harbor very low genetic diversity compared with their parental populations. The present study addresses how population size and within‐population diversity may recover through compensatory evolution, using the experimental adaptive radiation of bacterium Pseudomonas fluorescens. We exposed bacterial populations to an antibiotic treatment and then imposed a one‐individual‐size population bottleneck on those surviving the antibiotic stress. During the subsequent compensatory evolution, population size increased and leveled off very rapidly. The increase of diversity was of slower paces and persisted longer. In the very early stage of compensatory evolution, populations of large sizes had a greater chance to diversify; however, this productivity–diversification relationship was not observed in later stages. Population size and diversity from the end of the compensatory evolution was not contingent on initial population growth performance. We discussed the possibility that our results be explained by the emergence of a “holey” fitness landscape under the antibiotic stress.     

Relative number of generations of hosts and parasites does not influence parasite local adaptation in coevolving populations of bacteria and phages

A potential consequence of host-parasite coevolution in spatially structured populations is parasite local adaptation: local parasites perform better than foreign parasites on their local host populations. It has been suggested that the generally shorter generation times of parasites compared with their hosts contributes to parasites, rather than hosts, being locally adapted. We tested the hypothesis that relative generation times of hosts and parasites affect local adaptation of hosts and parasites, using the bacterium Pseudomonas fluorescens and a lytic phage as host and parasite, respectively. Generation times were not directly manipulated, but instead one of the coevolving partners was regularly removed and replaced with a population from an earlier time point. Thus, one partner underwent more generations than the other. Manipulations were carried out at both early and later periods of coevolutionary interactions. At early stages of coevolution, host and parasites that underwent relatively more generations displayed higher levels of resistance and infectivity, respectively. However, the relative number of generations that bacteria and phages underwent did not change the level of local adaptation relative to control populations. This is likely because generalist hosts and parasites are favoured during early stages of coevolution, preventing local adaptation. By contrast, at later stages manipulations had no effect on either average levels of resistance or infectivity, or alter the level of local adaptation relative to the controls, possibly because traits other than resistance and infectivity were under strong selection. Taken together, these data suggest that the relative generation times of hosts and parasites may not be an important determinant of local adaptation in this system.     

Adaptive landscapes in evolving populations of Pseudomonas fluorescens

The repeatability of adaptive evolution depends on the ruggedness of the underlying adaptive landscape. We contrasted the relative ruggedness of two adaptive landscapes by measuring the variance in fitness and metabolic phenotype within and among genetically distinct strains of Pseudomonas fluorescens in two environments differing only in the carbon source provided (glucose vs. xylose). Fitness increased in all lines, plateauing in one environment but not the other. The pattern of variance in fitness among replicate lines was unique to the selection environment; it increased over the course of the experiment in xylose but not in glucose. Metabolic phenotypes displayed two results: (1) populations adapted via changes that were distinctive to their selection environment, and (2) endpoint phenotypes were less variable in glucose than in xylose. These results indicate that although the response to selection is highly repeatable at the level of fitness, the underlying genetic routes taken were different for each environment and more variable in xylose. We suggest that this reflects a more rugged adaptive landscape in xylose compared to glucose. Our study demonstrates the utility of using replicate selection lines with different evolutionary starting points to try and quantify the relative ruggedness of adaptive landscapes.     

Isolation and characterization of bacteriophages specific for Campylobacter jejuni

Human infection by Campylobacter jejuni is mainly through the consumption of contaminated poultry products, which results in gastroenteritis and, rarely, bacteremia and polyneuropathies. In this study, six C. jejuni -specific bacteriophages (CPS1–6) were isolated by the spot-on-the-lawn technique from chicken samples in Korea and characterized for potential use as biocontrol agents. All isolated bacteriophages exhibited a high specificity, being able to lyse only C. jejuni , but not other Gram–negative bacteria, including C. coli , Escherichia coli , Salmonella spp., and Gram–positive bacteria. Bacteriophages contain an icosahedral head and a contractile tail sheath in transmission electron microscopy, and possess ds-DNA with an average genome size of approximately 145 kb; therefore, all bacteriophages are categorized into the Myoviridae family. Bacterial lysis studies in liquid media revealed that CPS2 could be used to control the growth of C. jejuni .     

Adaptation of Escherichia coli to glucose promotes evolvability in lactose

The selective history of a population can influence its subsequent evolution, an effect known as historical contingency. We previously observed that five of six replicate populations that were evolved in a glucose‐limited environment for 2000 generations, then switched to lactose for 1000 generations, had higher fitness increases in lactose than populations started directly from the ancestor. To test if selection in glucose systematically increased lactose evolvability, we started 12 replay populations—six from a population subsample and six from a single randomly selected clone—from each of the six glucose‐evolved founder populations. These replay populations and 18 ancestral populations were evolved for 1000 generations in a lactose‐limited environment. We found that replay populations were initially slightly less fit in lactose than the ancestor, but were more evolvable, in that they increased in fitness at a faster rate and to higher levels. This result indicates that evolution in the glucose environment resulted in genetic changes that increased the potential of genotypes to adapt to lactose. Genome sequencing identified four genes—iclR, nadR, spoT, and rbs—that were mutated in most glucose‐evolved clones and are candidates for mediating increased evolvability. Our results demonstrate that short‐term selective costs during selection in one environment can lead to changes in evolvability that confer longer term benefits.     

Ploidy tug‐of‐war: Evolutionary and genetic environments influence the rate of ploidy drive in a human fungal pathogen

Variation in baseline ploidy is seen throughout the tree of life, yet the factors that determine why one ploidy level is maintained over another remain poorly understood. Experimental evolution studies using asexual fungal microbes with manipulated ploidy levels intriguingly reveals a propensity to return to the historical baseline ploidy, a phenomenon that we term “ploidy drive.” We evolved haploid, diploid, and polyploid strains of the human fungal pathogen Candida albicans under three different nutrient limitation environments to test whether these conditions, hypothesized to select for low ploidy levels, could counteract ploidy drive. Strains generally maintained or acquired smaller genome sizes (measured as total nuclear DNA through flow cytometry) in minimal medium and under phosphorus depletion compared to in a complete medium, while mostly maintained or acquired increased genome sizes under nitrogen depletion. Improvements in fitness often ran counter to changes in genome size; in a number of scenarios lines that maintained their original genome size often increased in fitness more than lines that converged toward diploidy (the baseline ploidy of C. albicans). Combined, this work demonstrates a role for both the environment and genotype in determination of the rate of ploidy drive, and highlights questions that remain about the force(s) that cause genome size variation.     

Multiple genetic pathways to similar fitness limits during viral adaptation to a new host

The gain in fitness during adaptation depends on the supply of beneficial mutations. Despite a good theoretical understanding of how evolution proceeds for a defined set of mutations, there is little understanding of constraints on net fitness-whether fitness will reach a limit despite ongoing selection and mutation, and if there is a limit, what determines it. Here, the dsDNA bacteriophage SP6, a virus of Salmonella, was adapted to Escherichia coli K-12. From an isolate capable of modest growth on E. coli, four lines were adapted for rapid growth by protocols differing in use of mutagen, propagation method, and duration, but using the same media, temperature, and a continual excess of the novel host. Nucleotide changes underlying those adaptations differed greatly in number and identity, but the four lines achieved similar absolute fitness at the end, an increase of more than 4000-fold phage descendants per hour. Thus, the fitness landscape allows multiple genetic paths to the same approximate fitness limit. The existence and causes of fitness limits have ramifications to genome engineering, vaccine design, and "lethal mutagenesis" treatments to cure viral infections.     

Experimental Evolution Yields Hundreds of Mutations in a Functional Viral Genome

Two lines of the bacteriophage T7 were grown to fix mutations indiscriminately, using a combination of population bottlenecks and mutagenesis. Complete genome sequences revealed 404 and 299 base substitutions in the two lines, the largest number characterized in functional microbial genomes so far. Missense substitutions outnumbered silent substitutions. Silent substitutions occurred at similar rates between essential and nonessential genes, but missense substitutions occurred at a higher rate in nonessential genes than in essential genes, as expected if they were less deleterious in the nonessential genes. Viral fitness declined during this protocol, and subsequent passaging of each mutated line in large population sizes restored some of the lost fitness. Substitution levels during these recoveries were less than 6% of those during the bottleneck phase, and only two changes during recovery were reversions of the original mutations. Exchanges of genomic fragments between the two recovered lines revealed that fitness effects of some substitutions were not additive—that interactions were accumulating which could lead to incompatibility between the diverged genomes. Based on these results, unprecedented high rates of nucleotide and functional divergence in viral genomes should be attainable experimentally by using repeated population bottlenecks at a high mutation rate interspersed with recovery. Present address (M. R. Badgett): Ambion, Inc., 2130 Woodward, Austin, TX 78744, USA Present address (D. Rokyta): Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA     

Experimental evolution of viruses: Microviridae as a model system

φX174 was developed as a model system for experimental studies of evolution because of its small genome size and ease of cultivation. It has been used extensively to address statistical questions about the dynamics of adaptive evolution. Molecular changes seen during experimental evolution of φX174 under a variety of conditions were compiled from 10 experiments comprising 58 lineages, where whole genomes were sequenced. A total of 667 substitutions was seen. Parallel evolution was rampant, with over 50 per cent of substitutions occurring at sites with three or more events. Comparisons of experimentally evolved sites to variation seen among wild phage suggest that at least some of the adaptive mechanisms seen in the laboratory are relevant to adaptation in nature. Elucidation of these mechanisms is aided by the availability of capsid and pro-capsid structures for φX174 and builds on years of genetic studies of the phage life history.     

阪崎肠杆菌噬菌体的分离及其生物学特性

[目的]以阪崎肠杆菌模式菌株及分离菌株为指示菌,从污水中分离出该菌噬菌体,并对其基本生物学特性进行研究.[方法]以双层琼脂法从污水中分离噬菌体,通过同属和同科参考菌株测定噬菌体的特异性和宿主谱;电镜观察噬菌体颗粒形态;随机扩增多态性DNA(RAPD)实验分析噬菌体的分子生物学特性.[结果]从污水中分离得到5株噬菌体,表现出较窄的宿主范围,仅裂解阪崎肠杆菌,以ATCC 51329分离的噬菌体SK2可裂解27株阪崎肠杆菌中的24株(89%),负染经电镜观察,5株噬菌体都是由多面体头部和尾部组成;随机引物(5′-GAAACGGGTG-3′)扩增DNA分析,5株噬菌体DNA明显不同.[结论]分离出的5株噬菌体仅对阪崎肠杆菌敏感,在阪崎肠杆菌的分型、预防、治疗、以及生态环境的净化等方面具有潜在用途.     

PARALLEL CHANGES IN HOST RESISTANCE TO VIRAL INFECTION DURING 45,000 GENERATIONS OF RELAXED SELECTION

The dynamics of host susceptibility to parasites are often influenced by trade‐offs between the costs and benefits of resistance. We assayed changes in the resistance to three viruses in six lines of Escherichia coli that had been evolving for almost 45,000 generations in their absence. The common ancestor of these lines was completely resistant to T6, partially resistant to T6* (a mutant of T6 with altered host range), and sensitive to λ. None of the populations changed with respect to resistance to T6, whereas all six evolved increased susceptibility to T6*, probably ameliorating a cost of resistance. More surprisingly, however, the majority of lines evolved complete resistance to λ, despite not encountering that virus during this period. By coupling our results with previous work, we infer that resistance to λ evolved as a pleiotropic effect of a beneficial mutation that downregulated an unused metabolic pathway. The strong parallelism between the lines implies that selection had almost deterministic effects on the evolution of these patterns of host resistance. The opposite outcomes for resistance to T6* and λ demonstrate that the evolution of host resistance under relaxed selection cannot be fully predicted by simple trade‐off models.     

Compensatory evolution in response to a novel RNA polymerase: orthologous replacement of a central network gene

A bacteriophage genome was forced to evolve a new system of regulation by replacing its RNA polymerase (RNAP) gene, a central component of the phage developmental pathway, with that of a relative. The experiment used the obligate lytic phage T7 and the RNAP gene of phage T3. T7 RNAP uses 17 phage promoters, which are responsible for all middle and late gene expression, DNA replication, and progeny maturation, but the enzyme has known physical contacts with only 2 other phage proteins. T3 RNAP was supplied in trans by the bacterial host to a T7 genome lacking its own RNAP gene and the phage population was continually propagated on naive bacteria throughout the adaptation. Evolution of the T3 RNAP gene was thereby prevented, and selection was for the evolution of regulatory signals throughout the phage genome. T3 RNAP transcribes from T7 promoters only at low levels, but a single mutation in the promoter confers high expression, providing a ready mechanism for reevolution of gene expression in this system. When selected for rapid growth, fitness of the engineered phage evolved from a low of 5 doublings/h to 33 doublings/h, close to the expected maximum of 37 doublings/h. However, the experiment was terminated before it could be determined accurately that fitness had reached an obvious plateau, and it is not known whether further adaptation could have resulted in complete recovery of fitness. More than 30 mutations were observed in the evolved genome, but changes were found in only 9 of the 16 promoters, and several coding changes occurred in genes with no known contacts with the RNAP. Surprisingly, the T7 genome adapted to T3 RNAP also maintained high fitness when using T7 RNAP, suggesting that the extreme incompatibility of T7 elements with T3 RNAP is not an invariant property of divergence in these expression systems.