Parallel compensatory evolution stabilizes plasmids across the parasitism-mutualism continuum

Plasmids drive genomic diversity in bacteria via horizontal gene transfer [1 and 2]; nevertheless, explaining their survival in bacterial populations is challenging [3]. Theory predicts that irrespective of their net fitness effects, plasmids should be lost: when parasitic (costs outweigh benefits), plasmids should decline due to purifying selection [4, 5 and 6], yet under mutualism (benefits outweigh costs), selection favors the capture of beneficial accessory genes by the chromosome and loss of the costly plasmid backbone [4]. While compensatory evolution can enhance plasmid stability within populations [7, 8, 9, 10, 11, 12, 13, 14 and 15], the propensity for this to occur across the parasitism-mutualism continuum is unknown. We experimentally evolved Pseudomonas fluorescens and its mercury resistance mega-plasmid, pQBR103 [ 16], across an environment-mediated parasitism-mutualism continuum. Compensatory evolution stabilized plasmids by rapidly ameliorating the cost of plasmid carriage in all environments. Genomic analysis revealed that, in both parasitic and mutualistic treatments, evolution repeatedly targeted the gacA/gacS bacterial two-component global regulatory system while leaving the plasmid sequence intact. Deletion of either gacA or gacS was sufficient to completely ameliorate the cost of plasmid carriage. Mutation of gacA/gacS downregulated the expression of ∼17% of chromosomal and plasmid genes and appears to have relieved the translational demand imposed by the plasmid. Chromosomal capture of mercury resistance accompanied by plasmid loss occurred throughout the experiment but very rarely invaded to high frequency, suggesting that rapid compensatory evolution can limit this process. Compensatory evolution can explain the widespread occurrence of plasmids and allows bacteria to retain horizontally acquired plasmids even in environments where their accessory genes are not immediately useful

Rapid compensatory evolution promotes the survival of conjugative plasmids

Conjugative plasmids play a vital role in bacterial adaptation through horizontal gene transfer. Explaining how plasmids persist in host populations however is difficult, given the high costs often associated with plasmid carriage. Compensatory evolution to ameliorate this cost can rescue plasmids from extinction. In a recently published study we showed that compensatory evolution repeatedly targeted the same bacterial regulatory system, GacA/GacS, in populations of plasmid-carrying bacteria evolving across a range of selective environments. Mutations in these genes arose rapidly and completely eliminated the cost of plasmid carriage. Here we extend our analysis using an individual based model to explore the dynamics of compensatory evolution in this system. We show that mutations which ameliorate the cost of plasmid carriage can prevent both the loss of plasmids from the population and the fixation of accessory traits on the bacterial chromosome. We discuss how dependent the outcome of compensatory evolution is on the strength and availability of such mutations and the rate at which beneficial accessory traits integrate on the host chromosome

Bacteriophages limit the existence conditions for conjugative plasmids

Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome

Conflicting selection alters the trajectory of molecular evolution in a tripartite bacteria-plasmid-phage interaction

Bacteria engage in a complex network of ecological interactions, which includes mobile genetic elements (MGEs) such as phages and plasmids. These elements play a key role in microbial communities as vectors of horizontal gene transfer but can also be important sources of selection for their bacterial hosts. In natural communities bacteria are likely to encounter multiple MGEs simultaneously and conflicting selection among MGEs could alter the bacterial evolutionary response to each MGE. Here we test the effect of interactions with multiple MGEs on bacterial molecular evolution in the tripartite interaction between the bacterium, Pseudomonas fluorescens, the lytic bacteriophage SBW25φ2 and conjugative plasmid, pQBR103, using genome sequencing of experimentally evolved bacteria. We show that, individually, both plasmids and phages impose selection leading to bacterial evolutionary responses that are distinct from bacterial populations evolving without MGEs, but that together, plasmids and phages impose conflicting selection on bacteria, constraining the evolutionary responses observed in pairwise interactions. Our findings highlight the likely difficulties of predicting evolutionary responses to multiple selective pressures from the observed evolutionary responses to each selective pressure alone. Understanding evolution in complex microbial communities comprising many species and MGEs will require that we go beyond studies of pairwise interactions

Multidrug resistance plasmids commonly reprogram the expression of metabolic genes in <i>Escherichia coli</i>

Multidrug-resistant Escherichia coli is a leading cause of global mortality. Transfer of plasmids carrying genes encoding beta-lactamases, carbapenamases, and colistin resistance between lineages is driving the rising rates of hard-to-treat nosocomial and community infections. Multidrug resistance (MDR) plasmid acquisition commonly causes transcriptional disruption, and while a number of studies have shown strain-specific fitness and transcriptional effects of an MDR plasmid across diverse bacterial lineages, fewer studies have compared the impacts of different MDR plasmids in a common bacterial host. As such, our ability to predict which MDR plasmids are the most likely to be maintained and spread in bacterial populations is limited. Here, we introduced eight diverse MDR plasmids encoding resistances against a range of clinically important antibiotics into E. coli K-12 MG1655 and measured their fitness costs and transcriptional impacts. The scale of the transcriptional responses varied substantially between plasmids, ranging from &gt;650 to &lt;20 chromosomal genes being differentially expressed. However, the scale of regulatory disruption did not correlate significantly with the magnitude of the plasmid fitness cost, which also varied between plasmids. The identities of differentially expressed genes differed between transconjugants, although the expression of certain metabolic genes and functions were convergently affected by multiple plasmids, including the downregulation of genes involved in L-methionine transport and metabolism. Our data show the complexity of the interaction between host genetic background and plasmid genetic background in determining the impact of MDR plasmid acquisition on E. coli.IMPORTANCE: The increase in infections that are resistant to multiple classes of antibiotics, including those isolates that carry carbapenamases, beta-lactamases, and colistin resistance genes, is of global concern. Many of these resistances are spread by conjugative plasmids. Understanding more about how an isolate responds to an incoming plasmid that encodes antibiotic resistance will provide information that could be used to predict the emergence of MDR lineages. Here, the identification of metabolic networks as being particularly sensitive to incoming plasmids suggests the possible targets for reducing plasmid transfer. </p

Evolutionary responses to acquiring a multidrug resistance plasmid are dominated by metabolic functions across diverse <i>Escherichia coli</i> lineages

Multidrug resistance (MDR) plasmids drive the spread of antibiotic resistance between bacterial lineages. The immediate impact of MDR plasmid acquisition on fitness and cellular processes varies among bacterial lineages, but how the evolutionary processes enabling the genomic integration of MDR plasmids vary is less well understood, particularly in clinical pathogens. Using diverse Escherichia coli lineages experimentally evolved for ~700 generations, we show that the evolutionary response to gaining the MDR plasmid pLL35 was dominated by chromosomal mutations affecting metabolic and regulatory functions, with both strain-specific and shared mutational targets. The expression of several of these functions, such as anaerobic metabolism, is known to be altered upon acquisition of pLL35. Interactions with resident mobile genetic elements, notably several IS-elements, potentiated parallel mutations, including insertions upstream of hns that were associated with its upregulation and the downregulation of the plasmid-encoded extended-spectrum beta-lactamase gene. Plasmid parallel mutations targeted conjugation-related genes, whose expression was also commonly downregulated in evolved clones. Beyond their role in horizontal gene transfer, plasmids can be an important selective force shaping the evolution of bacterial chromosomes and core cellular functions. IMPORTANCE Plasmids drive the spread of antimicrobial resistance genes between bacterial genomes. However, the evolutionary processes allowing plasmids to be assimilated by diverse bacterial genomes are poorly understood, especially in clinical pathogens. Using experimental evolution with diverse E. coli lineages and a clinical multidrug resistance plasmid, we show that although plasmids drove unique evolutionary paths per lineage, there was a surprising degree of convergence in the functions targeted by mutations across lineages, dominated by metabolic functions. Remarkably, these same metabolic functions show higher evolutionary rates in MDR-lineages in nature and in some cases, like anaerobic metabolism, their expression is directly manipulated by the plasmid. Interactions with other mobile elements resident in the genomes accelerated adaptation by disrupting genes and regulatory sequences that they inserted into. Beyond their role in horizontal gene transfer, plasmids are an important selective force driving the evolution of bacterial genomes and core cellular functions. </p

The Impact of Mercury Selection and Conjugative Genetic Elements on Community Structure and Resistance Gene Transfer

Carriage of resistance genes can underpin bacterial survival, and by spreading these genes between species, mobile genetic elements (MGEs) can potentially protect diversity within microbial communities. The spread of MGEs could be affected by environmental factors such as selection for resistance, and biological factors such as plasmid host range, with consequences for individual species and for community structure. Here we cultured a focal bacterial strain,Pseudomonas fluorescensSBW25, embedded within a soil microbial community, with and without mercury selection, and with and without mercury resistance plasmids (pQBR57 or pQBR103), to investigate the effects of selection and resistance gene introduction on (1) the focal species; (2) the community as a whole; (3) the spread of the introducedmerresistance operon. We found thatP. fluorescensSBW25 only escaped competitive exclusion by other members of community under mercury selection, even when it did not begin with a mercury resistance plasmid, due to its propensity to acquire resistance from the community by horizontal gene transfer. Mercury pollution had a significant effect on community structure, decreasing alpha diversity within communities while increasing beta diversity between communities, a pattern that was not affected by the introduction of mercury resistance plasmids byP. fluorescensSBW25. Nevertheless, the introducedmerAgene spread to a phylogenetically diverse set of recipients over the 5 weeks of the experiment, as assessed by epicPCR. Our data demonstrates how the effects of MGEs can be experimentally assessed for individual lineages, the wider community, and for the spread of adaptive traits.Peer reviewe

Competitive species interactions constrain abiotic adaptation in a bacterial soil community

Studies of abiotic adaptation often consider single species in isolation, yet natural communities contain many coexisting species which could limit or promote abiotic adaptation. Here we show, using soil bacterial communities, that evolving in the presence of a competitor constrained abiotic adaptation. Specifically, Pseudomonas fluorescens evolved alone was fitter than P. fluorescens evolved alongside Pseudomonas putida, when P. putida was absent. Genome analyses indicated this was due to mutation of the acetate scavenger actP, which occurred exclusively, and almost universally, in single‐species‐evolved clones. actP disruption was associated with increased growth in soil compared with wild‐type actP, but this benefit was abolished when P. putida was present, suggesting a role for carbon scavenging transporters in species interactions, possibly through nutrient competition. Our results show that competitive species interactions can limit the evolutionary response to abiotic selection, because the fitness benefits of abiotic adaptive mutations were negated in more complex communities

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

Environmentally co-occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context-dependent fitness effects

Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co-occurring pQBR-family environmental mercury resistance plasmids and measured their effects on competitive fitness of a Pseudomonas fluorescens SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid-borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus-encoding cluster, and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolise sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by the different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity amongst co-occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity