Genetic basis of infectivity evolution in a bacteriophage

Antagonistic coevolution between hosts and parasites is probably ubiquitous. However, very little is known of the genetic changes associated with parasite infectivity evolution during adaptation to a coevolving host. We followed the phenotypic and genetic changes in a lytic virus population (bacteriophage; phage Φ2) that coevolved with its bacterial host, Pseudomonas fluorescens SBW25. First, we show the rapid evolution of numerous unique phage infectivity phenotypes, and that both phage host range and bacterial resistance to individual phage increased over coevolutionary time. Second, each of the distinct phage phenotypes in our study had a unique genotype, and molecular evolution did not act uniformly across the phage genome during coevolution. In particular, we detected numerous substitutions on the tail fibre gene, which is involved in the first step of the host-parasite interaction: host adsorption. None of the observed mutations could be directly linked with infection against a particular host, suggesting that the phenotypic effects of infectivity mutations are probably epistatic. However, phage genotypes with the broadest host ranges had the largest number of nonsynonymous amino acid changes on genes implicated in infectivity evolution. An understanding of the molecular genetics of phage infectivity has helped to explain the complex phenotypic coevolutionary dynamics in this system.     

Antagonistic coevolution between a bacterium and a bacteriophage

Antagonistic coevolution between hosts and parasites is believed to play a pivotal role in host and parasite population dynamics, the evolutionary maintenance of sex and the evolution of parasite virulence. Furthermore, antagonistic coevolution is believed to be responsible for rapid differentiation of both hosts and parasites between geographically structured populations. Yet empirical evidence for host-parasite antagonistic coevolution, and its impact on between-population genetic divergence, is limited. Here we demonstrate a long-term arms race between the infectivity of a viral parasite (bacteriophage; phage) and the resistance of its bacterial host. Coevolution was largely driven by directional selection, with hosts becoming resistant to a wider range of parasite genotypes and parasites infective to a wider range of host genotypes. Coevolution followed divergent trajectories between replicate communities despite establishment with isogenic bacteria and phage, and resulted in bacteria adapted to their own, compared with other, phage populations.     

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.     

Host-parasite coevolutionary arms races give way to fluctuating selection

Host-parasite coevolution is a key driver of biological diversity and parasite virulence, but its effects depend on the nature of coevolutionary dynamics over time. We used phenotypic data from coevolving populations of the bacterium Pseudomonas fluorescens SBW25 and parasitic phage SBW25Φ2, and genetic data from the phage tail fibre gene (implicated in infectivity evolution) to show that arms race dynamics, typical of short-term studies, decelerate over time. We attribute this effect to increasing costs of generalism for phages and bacteria with increasing infectivity and resistance. By contrast, fluctuating selection on individual host and parasite genotypes was maintained over time, becoming increasingly important for the phenotypic properties of parasite and host populations. Given that costs of generalism are reported for many other systems, arms races may generally give way to fluctuating selection in antagonistically coevolving populations.     

The Impact of Resource Availability on Bacterial Resistance to Phages in Soil

Resource availability can affect the coevolutionary dynamics between host and parasites, shaping communities and hence ecosystem function. A key finding from theoretical and in vitro studies is that host resistance evolves to greater levels with increased resources, but the relevance to natural communities is less clear. We took two complementary approaches to investigate the effect of resource availability on the evolution of bacterial resistance to phages in soil. First, we measured the resistance and infectivity of natural communities of soil bacteria and phage in the presence and absence of nutrient-providing plants. Second, we followed the real-time coevolution between defined bacteria and phage populations with resource availability manipulated by the addition or not of an artificial plant root exudate. Increased resource availability resulted in increases in bacterial resistance to phages, but without a concomitant increase in phage infectivity. These results suggest that phages may have a reduced impact on the control of bacterial densities and community composition in stable, high resource environments.     

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.     

Multiplicity of infection does not accelerate infectivity evolution of viral parasites in laboratory microcosms

Coinfection with multiple parasite genotypes [multiplicity of infection (MOI)] creates within-host competition and opportunities for parasite recombination and is therefore predicted to be important for both parasite and host evolution. We tested for a difference in the infectivity of viral parasites (lytic phage Φ2) and resistance of their bacterial hosts (Pseudomonas fluorescens SBW25) under both high and low MOI during coevolution in laboratory microcosms. Results show that MOI has no effect on infectivity and resistance evolution during coevolution over ～80 generations of host growth, and this is true when the experiment is initiated with wild-type viruses and hosts, or with viruses and hosts that have already been coevolving for ～330 generations. This suggests that MOI does not have a net effect of accelerating parasite adaptation to hosts through recombination, or slowing adaptation to hosts through between-parasite conflict in this system.     

Coevolving parasites enhance the diversity-decreasing effect of dispersal

High dispersal rates between patches in spatially structured populations can impede diversification and homogenize diversity. These homogenizing effects of dispersal are likely to be enhanced by coevolving parasites that impose strong selection on hosts for resistance. However, the interactive effects of dispersal and parasites on host diversification have never been tested. We used spatially structured, experimental populations of the bacterium Pseudomonas fluorescens, cultured with or without the phage SBW25Ф2 under three levels of dispersal (none, localized or global), and quantified diversity in terms of evolved bacterial colony morphologies after approximately 100 bacterial generations. We demonstrate that higher levels of colony morphology richness evolved in the presence of phage, and that dispersal reduced diversity most strongly in the presence of phage. Thus, our results suggest that, while parasites can drive host diversification, host populations coevolving with parasites are more prone to homogenization through dispersal.     

Hill–Robertson interference maintained by Red Queen dynamics favours the evolution of sex

Although it is well established theoretically that selective interference among mutations (Hill–Robertson interference) favours meiotic recombination, genomewide mean rates of mutation and strengths of selection appear too low to support this as the mechanism favouring recombination in nature. A possible solution to this discrepancy between theory and observation is that selection is at least intermittently very strong due to the antagonistic coevolution between a host and its parasites. The Red Queen theory posits that such coevolution generates fitness epistasis among loci, which generates negative linkage disequilibrium among beneficial mutations, which in turn favours recombination. This theory has received only limited support. However, Red Queen dynamics without epistasis may provide the ecological conditions that maintain strong and frequent selective interference in finite populations that indirectly selects for recombination. This hypothesis is developed here through the simulation of Red Queen dynamics. This approach required the development of a method to calculate the exact frequencies of multilocus haplotypes after recombination. Simulations show that recombination is favoured by the moderately weak selection of many loci involved in the interaction between a host and its parasites, which results in substitution rates that are compatible with empirical estimates. The model also reproduces the previously reported rapid increase in the rate of outcrossing in Caenorhabditis elegans coevolving with a bacterial pathogen.     

Plasmid carriage can limit bacteria–phage coevolution

Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria–phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria–phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo.     

Host population bottlenecks drive parasite extinction during antagonistic coevolution

Host–parasite interactions are often characterized by large fluctuations in host population size, and we investigated how such host bottlenecks affected coevolution between a bacterium and a virus. Previous theory suggests that host bottlenecks should provide parasites with an evolutionary advantage, but instead we found that phages were rapidly driven to extinction when coevolving with hosts exposed to large genetic bottlenecks. This was caused by the stochastic loss of sensitive bacteria, which are required for phage persistence and infectivity evolution. Our findings emphasize the importance of feedbacks between ecological and coevolutionary dynamics, and how this feedback can qualitatively alter coevolutionary dynamics.     

The impact of parasite dispersal on antagonistic host-parasite coevolution

Coevolving populations of hosts and parasites are often subdivided into a set of patches connected by dispersal. Higher relative rates of parasite compared with host dispersal are expected to lead to parasite local adaptation. However, we know of no studies that have considered the implications of higher relative rates of parasite dispersal for other aspects of the coevolutionary process, such as the rate of coevolution and extent of evolutionary escalation of resistance and infectivity traits. We investigated the effect of phage dispersal on coevolution in experimental metapopulations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage SBW25Phi2. Both the rate of coevolution and the breadth of evolved infectivity and resistance ranges peaked at intermediate rates of parasite dispersal. These results suggest that parasite dispersal can enhance the evolutionary potential of parasites through provision of novel genetic variation, but that high rates of parasite dispersal can impede the evolution of parasites by homogenizing genetic variation between patches, thereby constraining coevolution.     

Coevolutionary dynamics shape the structure of bacteria‐phage infection networks

Coevolution—reciprocal evolutionary change among interacting species driven by natural selection—is thought to be an important force in shaping biodiversity. This ongoing process takes place within tangled networks of species interactions. In microbial communities, evolutionary change between hosts and parasites occurs at the same time scale as ecological change. Yet, we still lack experimental evidence of the role of coevolution in driving changes in the structure of such species interaction networks. Filling this gap is important because network structure influences community persistence through indirect effects. Here, we quantified experimentally to what extent coevolutionary dynamics lead to contrasting patterns in the architecture of bacteria–phage infection networks. Specifically, we look at the tendency of these networks to be organized in a nested pattern by which the more specialist phages tend to infect only a proper subset of those bacteria infected by the most generalist phages. We found that interactions between coevolving bacteria and phages become less nested over time under fluctuating dynamics, and more nested under arms race dynamics. Moreover, when coevolution results in high average infectivity, phages and bacteria differ more from each other over time under arms race dynamics than under fluctuating dynamics. The tradeoff between the fitness benefits of evolving resistance/infectivity traits and the costs of maintaining them might explain these differences in network structure. Our study shows that the interaction pattern between bacteria and phages at the community level depends on the way coevolution unfolds.     

Ongoing phenotypic and genomic changes in experimental coevolution of RNA bacteriophage Qβ and Escherichia coli

According to the Red Queen hypothesis or arms race dynamics, coevolution drives continuous adaptation and counter-adaptation. Experimental models under simplified environments consisting of bacteria and bacteriophages have been used to analyze the ongoing process of coevolution, but the analysis of both parasites and their hosts in ongoing adaptation and counter-adaptation remained to be performed at the levels of population dynamics and molecular evolution to understand how the phenotypes and genotypes of coevolving parasite-host pairs change through the arms race. Copropagation experiments with Escherichia coli and the lytic RNA bacteriophage Qβ in a spatially unstructured environment revealed coexistence for 54 days (equivalent to 163-165 replication generations of Qβ) and fitness analysis indicated that they were in an arms race. E. coli first adapted by developing partial resistance to infection and later increasing specific growth rate. The phage counter-adapted by improving release efficiency with a change in host specificity and decrease in virulence. Whole-genome analysis indicated that the phage accumulated 7.5 mutations, mainly in the A2 gene, 3.4-fold faster than in Qβ propagated alone. E. coli showed fixation of two mutations (in traQ and csdA) faster than in sole E. coli experimental evolution. These observations suggest that the virus and its host can coexist in an evolutionary arms race, despite a difference in genome mutability (i.e., mutations per genome per replication) of approximately one to three orders of magnitude.     

Population mixing promotes arms race host–parasite coevolution

The consequences of host–parasite coevolution are highly contingent on the qualitative coevolutionary dynamics: whether selection fluctuates (fluctuating selection dynamic; FSD), or is directional towards increasing infectivity/resistance (arms race dynamic; ARD). Both genetics and ecology can play an important role in determining whether coevolution follows FSD or ARD, but the ecological conditions under which FSD shifts to ARD, and vice versa, are not well understood. The degree of population mixing is thought to increase host exposure to parasites, hence selecting for greater resistance and infectivity ranges, and we hypothesize this promotes ARD. We tested this by coevolving bacteria and viruses in soil microcosms and found that population mixing shifted bacteria–virus coevolution from FSD to ARD. A simple theoretical model produced qualitatively similar results, showing that mechanisms that increase host exposure to parasites tend to push dynamics towards ARD. The shift from FSD to ARD with increased population mixing may help to explain variation in coevolutionary dynamics between different host–parasite systems, and more specifically the observed discrepancies between laboratory and field bacteria–virus coevolutionary studies.     

THE MAINTENANCE OF SEX BY PARASITISM AND MUTATION ACCUMULATION UNDER EPISTATIC FITNESS FUNCTIONS

The mutation accumulation hypothesis predicts that sex functions to reduce the population mutational load, while the Red Queen hypothesis holds that sex is adaptive as a defense against coevolving pathogens. We used computer simulations to examine the combined and separate effects of selection against deleterious mutations and host-parasite coevolution on the spread of a clone into an outcrossing sexual population. The results suggest that the two processes operating simultaneously may select for sex independent of the exact shape of the function that maps mutation number onto host fitness.     

TEMPORAL DYNAMICS OF OUTCROSSING AND HOST MORTALITY RATES IN HOST–PATHOGEN EXPERIMENTAL COEVOLUTION

Cross‐fertilization is predicted to facilitate the short‐term response and the long‐term persistence of host populations engaged in antagonistic coevolutionary interactions. Consistent with this idea, our previous work has shown that coevolving bacterial pathogens (Serratia marcescens) can drive obligately selfing hosts (Caenorhabditis elegans) to extinction, whereas the obligately outcrossing and partially outcrossing populations persisted. We focused the present study on the partially outcrossing (mixed mating) and obligately outcrossing hosts, and analyzed the changes in the host resistance/avoidance (and pathogen infectivity) over time. We found that host mortality rates increased in the mixed mating populations over the first 10 generations of coevolution when outcrossing rates were initially low. However, mortality rates decreased after elevated outcrossing rates evolved during the experiment. In contrast, host mortality rates decreased in the obligately outcrossing populations during the first 10 generations of coevolution, and remained low throughout the experiment. Therefore, predominant selfing reduced the ability of the hosts to respond to coevolving pathogens compared to outcrossing hosts. Thus, we found that host–pathogen coevolution can generate rapid evolutionary change, and that host mating system can influence the outcome of coevolution at a fine temporal scale.     

Parasite diversity drives rapid host dynamics and evolution of resistance in a bacteria‐phage system

Host–parasite evolutionary interactions are typically considered in a pairwise species framework. However, natural infections frequently involve multiple parasites. Altering parasite diversity alters ecological and evolutionary dynamics as parasites compete and hosts resist multiple infection. We investigated the effects of parasite diversity on host–parasite population dynamics and evolution using the pathogen Pseudomonas aeruginosa and five lytic bacteriophage parasites. To manipulate parasite diversity, bacterial populations were exposed for 24 hours to either phage monocultures or diverse communities containing up to five phages. Phage communities suppressed host populations more rapidly but also showed reduced phage density, likely due to interphage competition. The evolution of resistance allowed rapid bacterial recovery that was greater in magnitude with increases in phage diversity. We observed no difference in the extent of resistance with increased parasite diversity, but there was a profound impact on the specificity of resistance; specialized resistance evolved to monocultures through mutations in a diverse set of genes. In summary, we demonstrate that parasite diversity has rapid effects on host–parasite population dynamics and evolution by selecting for different resistance mutations and affecting the magnitude of bacterial suppression and recovery. Finally, we discuss the implications of phage diversity for their use as biological control agents.     

The evolution of specificity in evolving and coevolving antagonistic interactions between a bacteria and its phage

The evolution of exploitative specificity can be influenced by environmental variability in space and time and the intensity of trade-offs. Coevolution, the process of reciprocal adaptation in two or more species, can produce variability in host exploitation and as such potentially drive patterns in host and parasite specificity. We employed the bacterium Pseudomonas fluorescens SBW25 and its DNA phage Phi2 to investigate the role of coevolution in the evolution of phage infectivity range and its relation with phage growth rate. At the phage population level, coevolution led to the evolution of broader infectivity range, but without an associated decrease in phage growth rate relative to the ancestor, whereas phage evolution in the absence of bacterial evolution led to an increased growth rate but no increase in infectivity range. In contrast, both selection regimes led to phage adaptation (in terms of growth rates) to their respective bacterial hosts. At the level of individual phage genotypes, coevolution resulted in within-population diversification in generalist and specialist infectivity range types. This pattern was consistent with a multilocus gene-for-gene interaction, further confirmed by an observed cost of broad infectivity range for individual phage. Moreover, coevolution led to the emergence of bacterial genotype by phage genotype interactions in the reduction of bacterial growth rate by phage. Our study demonstrates that the strong reciprocal selective pressures underlying the process of coevolution lead to the emergence and coexistence of different strategies within populations and to specialization between selective environments.     

Effects of antagonistic coevolution on parasite‐mediated host coexistence

Parasites can promote diversity by mediating coexistence between a poorer and superior competitor, if the superior competitor is more susceptible to parasitism. However, hosts and parasites frequently undergo antagonistic coevolution. This process may result in the accumulation of pleiotropic fitness costs associated with host resistance, and could breakdown coexistence. We experimentally investigated parasite‐mediated coexistence of two genotypes of the bacterium Pseudomonas fluorescens, where one genotype underwent coevolution with a parasite (a virulent bacteriophage), whereas the other genotype was resistant to the evolving phages at all time points, but a poorer competitor. In the absence of phages, the resistant genotype was rapidly driven extinct in all populations. In the presence of the phages, the resistant genotype persisted in four of six populations and eventually reached higher frequencies than the sensitive genotype. The coevolving genotype showed a reduction in the growth rate, consistent with a cost of resistance, which may be responsible for a decline in its relative fitness. These results demonstrate that the stability of parasite‐mediated coexistence of resistant and susceptible species or genotypes is likely to be affected if parasites and susceptible hosts coevolve.