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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 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 natural environmental niche.

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 multispecies communities are poorly understood. Here, we show, using experimental multispecies communities containing two plasmids, that bacterial diversity limits the horizontal transmission of plasmids due to ‘the dilution effect’; 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 coinfected 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.

Plasmids are common in natural bacterial communities, facilitating bacterial evolution via horizontal gene transfer. Bacterial species vary in their proficiency to host plasmids: whereas plasmids are stably maintained in some species regardless of selection for plasmid-encoded genes, in other species, even beneficial plasmids are rapidly lost. It is, however, unclear how this variation in host proficiency affects plasmid persistence in communities. Here, we test this using multispecies bacterial soil communities comprising species varying in their proficiency to host a large conjugative mercury resistance plasmid, pQBR103. The plasmid reached higher community-level abundance where beneficial and when introduced to the community in a more proficient host species. Proficient plasmid host species were also better able to disseminate the plasmid to a wider diversity of host species. These findings suggest that the dynamics of plasmids in natural bacterial communities depend not only upon the plasmid's attributes and the selective environment but also upon the proficiency of their host species.

Mobile genetic elements (MGE) such as plasmids and transposons mobilise genes within and between species, playing a crucial role in bacterial evolution via horizontal gene transfer (HGT). Currently, we lack data on variation in MGE dynamics across bacterial host species. We tracked the dynamics of a large conjugative plasmid, pQBR103, and its Tn5042 mercury resistance transposon, in five diverse Pseudomonas species in environments with and without mercury selection. Plasmid fitness effects and stability varied extensively between host species and environments, as did the propensity for chromosomal capture of the Tn5042 mercury resistance transposon associated with loss of the plasmid. Whereas Pseudomonas fluorescens and Pseudomonas savastanoi stably maintained the plasmid in both environments, the plasmid was highly unstable in Pseudomonas aeruginosa and Pseudomonas putida, where plasmid-free genotypes with Tn5042 captured to the chromosome invaded to higher frequency under mercury selection. These data confirm that plasmid stability is dependent upon the specific genetic interaction of the plasmid and host chromosome rather than being a property of plasmids alone, and moreover imply that MGE dynamics in diverse natural communities are likely to be complex and driven by a subset of species capable of stably maintaining plasmids that would then act as hubs of HGT., Bacteria share genes via mobile genetic elements but the dynamics of these vary extensively between species.

Plasmids are well known for spreading antibiotic-resistance genes between bacterial strains. Recent experiments show that they can also act as catalysts for evolutionary innovation, promoting rapid evolution of novel antibiotic resistance.

Horizontal gene transfer is a fundamental process in bacterial evolution that can accelerate adaptation via the sharing of genes between lineages. Conjugative plasmids are the principal genetic elements mediating the horizontal transfer of genes, both within and between bacterial species. In some species, plasmids are unstable and likely to be lost through purifying selection, but when alternative hosts are available, interspecific plasmid transfer could counteract this and maintain access to plasmid-borne genes. To investigate the evolutionary importance of alternative hosts to plasmid population dynamics in an ecologically relevant environment, we established simple soil microcosm communities comprising two species of common soil bacteria, Pseudomonas fluorescens and Pseudomonas putida, and a mercury resistance (HgR) plasmid, pQBR57, both with and without positive selection [i.e., addition of Hg(II)]. In single-species populations, plasmid stability varied between species: although pQBR57 survived both with and without positive selection in P. fluorescens, it was lost or replaced by nontransferable HgR captured to the chromosome in P. putida. A simple mathematical model suggests these differences were likely due to pQBR57's lower intraspecific conjugation rate in P. putida. By contrast, in two-species communities, both models and experiments show that interspecific conjugation from P. fluorescens allowed pQBR57 to persist in P. putida via source-sink transfer dynamics. Moreover, the replacement of pQBR57 by nontransferable chromosomal HgR in P. putida was slowed in coculture. Interspecific transfer allows plasmid survival in host species unable to sustain the plasmid in monoculture, promoting community-wide access to the plasmid-borne accessory gene pool and thus potentiating future evolvability.

Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997–2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens., Author Summary Influenza A viruses are characterized by rapid antigenic drift: structural changes in B-cell epitopes that facilitate escape from pre-existing immunity. Consequently, seasonal influenza continues to impose a major burden on human health. Accurate quantification of the antigenic impact of specific amino acid substitutions is a pre-requisite for predicting the fitness and evolutionary outcome of variant viruses. Using assays to attribute antigenic variation to amino acid sequence changes we identify substitutions that contribute to antigenic drift and quantify their impact. We show that substitutions identified as low-impact are a critical component of virus antigenic evolution and by including these, as well as the high-impact substitutions often focused on, the accuracy of predicting antigenic phenotypes of emerging viruses from genotype is doubled.

A main determinant of prolonged Trypanosoma brucei infection and transmission and success of the parasite is the interplay between host acquired immunity and antigenic variation of the parasite variant surface glycoprotein (VSG) coat. About 0.1% of trypanosome divisions produce a switch to a different VSG through differential expression of an archive of hundreds of silent VSG genes and pseudogenes, but the patterns and extent of the trypanosome diversity phenotype, particularly in chronic infection, are unclear. We applied longitudinal VSG cDNA sequencing to estimate variant richness and test whether pseudogenes contribute to antigenic variation. We show that individual growth peaks can contain at least 15 distinct variants, are estimated computationally to comprise many more, and that antigenically distinct ‘mosaic’ VSGs arise from segmental gene conversion between donor VSG genes or pseudogenes. The potential for trypanosome antigenic variation is probably much greater than VSG archive size; mosaic VSGs are core to antigenic variation and chronic infection., Author Summary Trypanosoma brucei—a deadly parasite of humans and animals—owes its success to its ability to cope with host immunity, and the mechanism it uses to do so is a remarkable example of biological variation. Immune responses that develop against the parasite surface coat are only partially effective against the parasite population; some individual parasites will have already switched to a different variant of the coat antigen, and thus survive to prolong infection. Little is known about how the pattern of antigen variation unfolds, particularly after the early stage of infection. Here, we examined different antigen variants that appeared over the course of infection, to estimate their diversity and to see whether the parasites are able to generate new antigen variants by combination. We found antigen diversity was much greater than expected, and that ‘mosaic’ variants—produced by combining bits of more than one antigen gene—played a central role in the later stages of infection. These results provide important evidence for the robustness of this key survival strategy, provide clues about its evolution, and allow us to identify patterns in common with other antigenically variable pathogens.