Correction: Plasmids manipulate bacterial behaviour through translational regulatory crosstalk.

[This corrects the article DOI: 10.1371/journal.pbio.3001988.].

Compensatory mutations reducing the fitness cost of plasmid carriage occur in plant rhizosphere communities.

Plasmids drive bacterial evolutionary innovation by transferring ecologically important functions between lineages, but acquiring a plasmid often comes at a fitness cost to the host cell. Compensatory mutations, which ameliorate the cost of plasmid carriage, promote plasmid maintenance in simplified laboratory media across diverse plasmid-host associations. Whether such compensatory evolution can occur in more complex communities inhabiting natural environmental niches where evolutionary paths may be more constrained is, however, unclear. Here, we show a substantial fitness cost of carrying the large conjugative plasmid pQBR103 in Pseudomonas fluorescens SBW25 in the plant rhizosphere. This plasmid fitness cost could be ameliorated by compensatory mutations affecting the chromosomal global regulatory system gacA/gacS, which arose rapidly in plant rhizosphere communities and were exclusive to plasmid carriers. These findings expand our understanding of the importance of compensatory evolution in plasmid dynamics beyond simplified lab media. Compensatory mutations contribute to plasmid survival in bacterial populations living within complex microbial communities in their environmental niche.

Plasmids manipulate bacterial behaviour through translational regulatory crosstalk.

Beyond their role in horizontal gene transfer, conjugative plasmids commonly encode homologues of bacterial regulators. Known plasmid regulator homologues have highly targeted effects upon the transcription of specific bacterial traits. Here, we characterise a plasmid translational regulator, RsmQ, capable of taking global regulatory control in Pseudomonas fluorescens and causing a behavioural switch from motile to sessile lifestyle. RsmQ acts as a global regulator, controlling the host proteome through direct interaction with host mRNAs and interference with the host's translational regulatory network. This mRNA interference leads to large-scale proteomic changes in metabolic genes, key regulators, and genes involved in chemotaxis, thus controlling bacterial metabolism and motility. Moreover, comparative analyses found RsmQ to be encoded on a large number of divergent plasmids isolated from multiple bacterial host taxa, suggesting the widespread importance of RsmQ for manipulating bacterial behaviour across clinical, environmental, and agricultural niches. RsmQ is a widespread plasmid global translational regulator primarily evolved for host chromosomal control to manipulate bacterial behaviour and lifestyle.

Horizontal gene transfer and ecological interactions jointly control microbiome stability.

Genes encoding resistance to stressors, such as antibiotics or environmental pollutants, are widespread across microbiomes, often encoded on mobile genetic elements. Yet, despite their prevalence, the impact of resistance genes and their mobility upon the dynamics of microbial communities remains largely unknown. Here we develop eco-evolutionary theory to explore how resistance genes alter the stability of diverse microbiomes in response to stressors. We show that adding resistance genes to a microbiome typically increases its overall stability, particularly for genes on mobile genetic elements with high transfer rates that efficiently spread resistance throughout the community. However, the impact of resistance genes upon the stability of individual taxa varies dramatically depending upon the identity of individual taxa, the mobility of the resistance gene, and the network of ecological interactions within the community. Nonmobile resistance genes can benefit susceptible taxa in cooperative communities yet damage those in competitive communities. Moreover, while the transfer of mobile resistance genes generally increases the stability of previously susceptible recipient taxa to perturbation, it can decrease the stability of the originally resistant donor taxon. We confirmed key theoretical predictions experimentally using competitive soil microcosm communities. Here the stability of a susceptible microbial community to perturbation was increased by adding mobile resistance genes encoded on conjugative plasmids but was decreased when these same genes were encoded on the chromosome. Together, these findings highlight the importance of the interplay between ecological interactions and horizontal gene transfer in driving the eco-evolutionary dynamics of diverse microbiomes.

Ecological and evolutionary solutions to the plasmid paradox.

The 'plasmid paradox' arises because, although plasmids are common features of bacterial genomes, theoretically they should not exist: rates of conjugation were believed insufficient to allow plasmids to persist by infectious transmission, whereas the costs of plasmid maintenance meant that plasmids should be purged by negative selection regardless of whether they encoded beneficial accessory traits because these traits should eventually be captured by the chromosome, enabling the loss of the redundant plasmid. In the decade since the plasmid paradox was described, new data and theory show that a range of ecological and evolutionary mechanisms operate in bacterial populations and communities to explain the widespread distribution and stable maintenance of plasmids. We conclude, therefore, that multiple solutions to the plasmid paradox are now well understood. The current challenge for the field, however, is to better understand how these solutions operate in natural bacterial communities to explain and predict the distribution of plasmids and the dynamics of the horizontal gene transfer that they mediate in bacterial (pan)genomes.

Genetic variation is associated with differences in facilitative and competitive interactions in the Rhizobium leguminosarum species complex.

Competitive and facilitative interactions influence bacterial community composition, diversity and functioning. However, the role of genetic diversity for determining interactions between coexisting strains of the same, or closely related, species remains poorly understood. Here, we investigated the type (facilitative/inhibitory) and potential underlying mechanisms of pairwise interactions between 24 genetically diverse bacterial strains belonging to three genospecies (gsA,C,E) of the Rhizobium leguminosarum species complex. Interactions were determined indirectly, based on secreted compounds in cell-free supernatants, and directly, as growth inhibition in cocultures. We found supernatants mediated both facilitative and inhibitory interactions that varied greatly between strains and genospecies. Overall, gsE strains indirectly suppressed growth of gsA strains, while their own growth was facilitated by other genospecies' supernatants. Similar genospecies-level patterns were observed in direct competition, where gsA showed the highest susceptibility and gsE the highest inhibition capacity. At the genetic level, increased gsA susceptibility was associated with a non-random distribution of quorum sensing and secondary metabolite genes across genospecies. Together, our results suggest that genetic variation is associated with facilitative and competitive interactions, which could be important ecological mechanisms explaining R. leguminosarum diversity.

Why are rhizobial symbiosis genes mobile?

Rhizobia are one of the most important and best studied groups of bacterial symbionts. They are defined by their ability to establish nitrogen-fixing intracellular infections within plant hosts. One surprising feature of this symbiosis is that the bacterial genes required for this complex trait are not fixed within the chromosome, but are encoded on mobile genetic elements (MGEs), namely plasmids or integrative and conjugative elements. Evidence suggests that many of these elements are actively mobilizing within rhizobial populations, suggesting that regular symbiosis gene transfer is part of the ecology of rhizobial symbionts. At first glance, this is counterintuitive. The symbiosis trait is highly complex, multipartite and tightly coevolved with the legume hosts, while transfer of genes can be costly and disrupt coadaptation between the chromosome and the symbiosis genes. However, horizontal gene transfer is a process driven not only by the interests of the host bacterium, but also, and perhaps predominantly, by the interests of the MGEs that facilitate it. Thus understanding the role of horizontal gene transfer in the rhizobium-legume symbiosis requires a 'mobile genetic element's-eye view' on the ecology and evolution of this important symbiosis. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Why do plasmids manipulate the expression of bacterial phenotypes?

Conjugative plasmids play an important role in bacterial evolution by transferring niche-adaptive traits between lineages, thus driving adaptation and genome diversification. It is increasingly clear, however, that in addition to this evolutionary role, plasmids also manipulate the expression of a broad range of bacterial phenotypes. In this review, we argue that the effects that plasmids have on the expression of bacterial phenotypes may often represent plasmid adaptations, rather than mere deleterious side effects. We begin by summarizing findings from untargeted omics analyses, which give a picture of the global effects of plasmid acquisition on host cells. Thereafter, because many plasmids are capable of both vertical and horizontal transmission, we distinguish plasmid-mediated phenotypic effects into two main classes based upon their potential fitness benefit to plasmids: (i) those that promote the competitiveness of the host cell in a given niche and thereby increase plasmid vertical transmission, and (ii) those that promote plasmid conjugation and thereby increase plasmid horizontal transmission. Far from being mere vehicles for gene exchange, we propose that plasmids often act as sophisticated genetic parasites capable of manipulating their bacterial hosts for their own benefit. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Introducing a Novel, Broad Host Range Temperate Phage Family Infecting Rhizobium leguminosarum and Beyond.

Temperate phages play important roles in bacterial communities but have been largely overlooked, particularly in non-pathogenic bacteria. In rhizobia the presence of temperate phages has the potential to have significant ecological impacts but few examples have been described. Here we characterize a novel group of 5 Rhizobium leguminosarum prophages, capable of sustaining infections across a broad host range within their host genus. Genome comparisons identified further putative prophages infecting multiple Rhizobium species isolated globally, revealing a wider family of 10 temperate phages including one previously described lytic phage, RHEph01, which appears to have lost the ability to form lysogens. Phylogenetic discordance between prophage and host phylogenies suggests a history of active mobilization between Rhizobium lineages. Genome comparisons revealed conservation of gene content and order, with the notable exception of an approximately 5 kb region of hypervariability, containing almost exclusively hypothetical genes. Additionally, several horizontally acquired genes are present across the group, including a putative antirepressor present only in the RHEph01 genome, which may explain its apparent inability to form lysogens. In summary, both phenotypic and genomic comparisons between members of this group of phages reveals a clade of viruses with a long history of mobilization within and between Rhizobium species.

Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation.

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid carriage are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expression level. By combining the results of experimental evolution with genetics and transcriptomics, we show here that fitness costs of 2 divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid carriage. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by up-regulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained up-regulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer (HGT) due to their propensity for amelioration by single compensatory mutations, helping to explain why plasmids are so common in bacterial genomes.

The dilution effect limits plasmid horizontal transmission in multispecies bacterial communities.

By transferring ecologically important traits between species, plasmids drive genomic divergence and evolutionary innovation in their bacterial hosts. Bacterial communities are often diverse and contain multiple coexisting plasmids, but the dynamics of plasmids in multi-species communities are poorly understood. Here, we show, using experimental multi-species communities containing two plasmids, that bacterial diversity limits the horizontal transmission of plasmids due to the 'dilution effect'; this is an epidemiological phenomenon whereby living alongside less proficient host species reduces the expected infection risk for a focal host species. In addition, plasmid horizontal transmission was also affected by plasmid diversity, such that the rate of plasmid conjugation was reduced from co-infected host cells carrying both plasmids. In diverse microbial communities, plasmid spread may be limited by the dilution effect and plasmid-plasmid interactions, reducing the rate of horizontal transmission.

Positive Selection Inhibits Plasmid Coexistence in Bacterial Genomes.

Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversification. Bacterial genomes commonly contain multiple, coexisting plasmid replicons, which could fuel adaptation by increasing the range of gene functions available to selection and allowing their recombination. However, plasmid coexistence is difficult to explain because the acquisition of plasmids typically incurs high fitness costs for the host cell. Here, we show that plasmid coexistence was stably maintained without positive selection for plasmid-borne gene functions and was associated with compensatory evolution to reduce fitness costs. In contrast, with positive selection, plasmid coexistence was unstable despite compensatory evolution. Positive selection discriminated between differential fitness benefits of functionally redundant plasmid replicons, retaining only the more beneficial plasmid. These data suggest that while the efficiency of negative selection against plasmid fitness costs declines over time due to compensatory evolution, positive selection to maximize plasmid-derived fitness benefits remains efficient. Our findings help to explain the forces structuring bacterial genomes: coexistence of multiple plasmids in a genome is likely to require either rare positive selection in nature or nonredundancy of accessory gene functions among the coexisting plasmids.IMPORTANCE Bacterial genomes often contain multiple coexisting plasmids that provide important functions like antibiotic resistance. Using lab experiments, we show that such plasmid coexistence within a genome is stable only in environments where the function they encode is useless but is unstable if the function is useful and beneficial for bacterial fitness. Where competing plasmids perform the same useful function, only the most beneficial plasmid is kept by the cell, a process that is similar to competitive exclusion in ecological communities. This process helps explain how bacterial genomes are structured: bacterial genomes expand in size by acquiring multiple plasmids when selection is relaxed but subsequently contract during periods of strong selection for the useful plasmid-encoded function.

The impact of intra-specific diversity in the rhizobia-legume symbiosis.

Rhizobia - nitrogen-fixing, root-nodulating bacteria - play a critical role in both plant ecosystems and sustainable agriculture. Rhizobia form intracellular infections within legumes roots where they produce plant accessible nitrogen from atmospheric nitrogen and thus reduce the reliance on industrial inputs. The rhizobia-legume symbiosis is often treated as a pairwise relationship between single genotypes, both in research and in the production of rhizobial inoculants. However in nature individual plants are infected by a high diversity of rhizobia symbionts. How this diversity affects productivity within the symbiosis is unclear. Here, we use a powerful statistical approach to assess the impact of diversity within the Rhizobium leguminosarum - clover symbiosis using a biodiversity-ecosystem function framework. Statistically, we found no significant impact of rhizobium diversity. However this relationship was weakly positive - rather than negative - indicating that there is no significant cost to increasing inoculant diversity. Productivity was influenced by the identity of the strains within an inoculant; strains with the highest individual performance showed a significant positive contribution within mixed inoculants. Overall, inoculant effectiveness was best predicted by the individual performance of the best inoculant member, and only weakly predicted by the worst performing member. Collectively, our data suggest that the Rhizobium leguminosarum - clover symbiosis displays a weak diversity-function relationship, but that inoculant performance can be improved through the inclusion of high performing strains. Given the wide environmental dependence of rhizobial inoculant quality, multi-strain inoculants could be highly successful as they increase the likelihood of including a strain well adapted to local conditions across different environments.

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 fluorescens SBW25, 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 introduced mer resistance operon. We found that P. fluorescens SBW25 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 by P. fluorescens SBW25. Nevertheless, the introduced merA gene 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.

Sexual and natural selection on pollen morphology in Taraxacum.

PREMISE: Spiny pollen has evolved independently in multiple entomophilous lineages. Sexual selection may act on exine traits that facilitate male mating success by influencing the transfer of pollen from the anther to the body of the pollinator, while natural selection acts to increase pollen survival. We postulated that relative to sexual congeners, apomictic dandelions undergo relaxed selection on traits associated with male mating success. METHODS: We explored sexual selection on exine traits by measuring the propensity for Taraxacum spp. pollen to attach to hairs of flower-visiting bumblebees (Bombus spp.) or flies (Diptera: Syrphidae and Muscoidea) and assessed natural selection by testing whether pollen traits defend against consumption. RESULTS: Pollen picked up by bumblebees exhibited a narrower subset of spine-spacing phenotypes, consistent with stabilizing selection. Flies picked up larger pollen from flowers than expected at random. Surveys of corbiculae (pollen basket) contents from foraging bumblebees and feces of flies showed that pollen grains consumed by both kinds of visitors are similar in spine characteristics and size to those produced by the donor. When bees visit inflorescences of apomictic T. officinale, they pick up pollen with spine-spacing phenotypes above the mean and shifted toward those of sexual T. ceratophorum. CONCLUSIONS: We demonstrate that traits under sexual selection during pollen pickup vary among pollinators, while natural selection for pollen defense is nil in T. ceratophorum. In hybrid zones between apomictic and sexual dandelions, pollen traits place apomictic donors at a dispersal disadvantage, potentially reinforcing reproductive isolation.

Extremely fast amelioration of plasmid fitness costs by multiple functionally diverse pathways.

The acquisition of plasmids is often accompanied by fitness costs such that compensatory evolution is required to allow plasmid survival, but it is unclear whether compensatory evolution can be extensive or rapid enough to maintain plasmids when they are very costly. The mercury-resistance plasmid pQBR55 drastically reduced the growth of its host, Pseudomonas fluorescens SBW25, immediately after acquisition, causing a small colony phenotype. However, within 48 h of growth on agar plates we observed restoration of the ancestral large colony morphology, suggesting that compensatory mutations had occurred. Relative fitness of these evolved strains, in lab media and in soil microcosms, varied between replicates, indicating different mutational mechanisms. Using genome sequencing we identified that restoration was associated with chromosomal mutations in either a hypothetical DNA-binding protein PFLU4242, RNA polymerase or the GacA/S two-component system. Targeted deletions in PFLU4242, gacA or gacS recapitulated the ameliorated phenotype upon plasmid acquisition, indicating three distinct mutational pathways to compensation. Our data shows that plasmid compensatory evolution is fast enough to allow survival of a plasmid despite it imposing very high fitness costs upon its host, and indeed may regularly occur during the process of isolating and selecting individual plasmid-containing clones.

The Ecology and Evolution of Pangenomes.

Since the first genome-scale comparisons, it has been evident that the genomes of many species are unbound by strict vertical descent: Large differences in gene content can occur among genomes belonging to the same prokaryotic species, with only a fraction of genes being universal to all genomes. These insights gave rise to the pangenome concept. The pangenome is defined as the set of all the genes present in a given species and can be subdivided into the accessory genome, present in only some of the genomes, and the core genome, present in all the genomes. Pangenomes arise due to gene gain by genomes from other species through horizontal gene transfer and differential gene loss among genomes, and have been described in both prokaryotes and eukaryotes. Our current view of pangenome variation is phenomenological and incomplete. In this review, we outline the mechanistic, ecological and evolutionary drivers of and barriers to horizontal gene transfer that are likely to structure pangenomes. We highlight the key role of conflict between the host chromosome(s) and the mobile genetic elements that mediate gene exchange. We identify shortcomings in our current models of pangenome evolution and suggest directions for future research to allow a more complete understanding of how and why pangenomes evolve.

Mobile Compensatory Mutations Promote Plasmid Survival.

The global dissemination of plasmids encoding antibiotic resistance represents an urgent issue for human health and society. While the fitness costs for host cells associated with plasmid acquisition are expected to limit plasmid dissemination in the absence of positive selection of plasmid traits, compensatory evolution can reduce this burden. Experimental data suggest that compensatory mutations can be located on either the chromosome or the plasmid, and these are likely to have contrasting effects on plasmid dynamics. Whereas chromosomal mutations are inherited vertically through bacterial fission, plasmid mutations can be inherited both vertically and horizontally and potentially reduce the initial cost of the plasmid in new host cells. Here we show using mathematical models and simulations that the dynamics of plasmids depends critically on the genomic location of the compensatory mutation. We demonstrate that plasmid-located compensatory evolution is better at enhancing plasmid persistence, even when its effects are smaller than those provided by chromosomal compensation. Moreover, either type of compensatory evolution facilitates the survival of resistance plasmids at low drug concentrations. These insights contribute to an improved understanding of the conditions and mechanisms driving the spread and the evolution of antibiotic resistance plasmids. IMPORTANCE Understanding the evolutionary forces that maintain antibiotic resistance genes in a population, especially when antibiotics are not used, is an important problem for human health and society. The most common platform for the dissemination of antibiotic resistance genes is conjugative plasmids. Experimental studies showed that mutations located on the plasmid or the bacterial chromosome can reduce the costs plasmids impose on their hosts, resulting in antibiotic resistance plasmids being maintained even in the absence of antibiotics. While chromosomal mutations are only vertically inherited by the daughter cells, plasmid mutations are also provided to bacteria that acquire the plasmid through conjugation. Here we demonstrate how the mode of inheritance of a compensatory mutation crucially influences the ability of plasmids to spread and persist in a bacterial population.