Correction: Plasmids manipulate bacterial behaviour through translational regulatory crosstalk.

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

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.

IMG/PR: a database of plasmids from genomes and metagenomes with rich annotations and metadata.

Plasmids are mobile genetic elements found in many clades of Archaea and Bacteria. They drive horizontal gene transfer, impacting ecological and evolutionary processes within microbial communities, and hold substantial importance in human health and biotechnology. To support plasmid research and provide scientists with data of an unprecedented diversity of plasmid sequences, we introduce the IMG/PR database, a new resource encompassing 699 973 plasmid sequences derived from genomes, metagenomes and metatranscriptomes. IMG/PR is the first database to provide data of plasmid that were systematically identified from diverse microbiome samples. IMG/PR plasmids are associated with rich metadata that includes geographical and ecosystem information, host taxonomy, similarity to other plasmids, functional annotation, presence of genes involved in conjugation and antibiotic resistance. The database offers diverse methods for exploring its extensive plasmid collection, enabling users to navigate plasmids through metadata-centric queries, plasmid comparisons and BLAST searches. The web interface for IMG/PR is accessible at https://img.jgi.doe.gov/pr. Plasmid metadata and sequences can be downloaded from https://genome.jgi.doe.gov/portal/IMG_PR.

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.

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.

A tale of two plasmids: contributions of plasmid associated phenotypes to epidemiological success among Shigella.

Dissemination of antimicrobial resistance (AMR) genes by horizontal gene transfer (HGT) mediated through plasmids is a major global concern. Genomic epidemiology studies have shown varying success of different AMR plasmids during outbreaks, but the underlying reasons for these differences are unclear. Here, we investigated two Shigella plasmids (pKSR100 and pAPR100) that circulated in the same transmission network but had starkly contrasting epidemiological outcomes to identify plasmid features that may have contributed to the differences. We used plasmid comparative genomics to reveal divergence between the two plasmids in genes encoding AMR, SOS response alleviation and conjugation. Experimental analyses revealed that these genomic differences corresponded with reduced conjugation efficiencies for the epidemiologically successful pKSR100, but more extensive AMR, reduced fitness costs, and a reduced SOS response in the presence of antimicrobials, compared with the less successful pAPR100. The discrepant phenotypes between the two plasmids are consistent with the hypothesis that plasmid-associated phenotypes contribute to determining the epidemiological outcome of AMR HGT and suggest that phenotypes relevant in responding to antimicrobial pressure and fitness impact may be more important than those around conjugation in this setting. Plasmid phenotypes could thus be valuable tools in conjunction with genomic epidemiology for predicting AMR dissemination.

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.

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.

Source-sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities.

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 (Hg(R)) 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 Hg(R) 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 Hg(R) 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.

Plasmid stability is enhanced by higher-frequency pulses of positive selection.

Plasmids accelerate bacterial adaptation by sharing ecologically important traits between lineages. However, explaining plasmid stability in bacterial populations is challenging owing to their associated costs. Previous theoretical and experimental studies suggest that pulsed positive selection may explain plasmid stability by favouring gene mobility and promoting compensatory evolution to ameliorate plasmid cost. Here we test how the frequency of pulsed positive selection affected the dynamics of a mercury-resistance plasmid, pQBR103, in experimental populations of Pseudomonas fluorescens SBW25. Plasmid dynamics varied according to the frequency of Hg2+ positive selection: in the absence of Hg2+ plasmids declined to low frequency, whereas pulses of Hg2+ selection allowed plasmids to sweep to high prevalence. Compensatory evolution to ameliorate the cost of plasmid carriage was widespread across the entire range of Hg2+ selection regimes, including both constant and pulsed Hg2+ selection. Consistent with theoretical predictions, gene mobility via conjugation appeared to play a greater role in promoting plasmid stability under low-frequency pulses of Hg2+ selection. However, upon removal of Hg2+ selection, plasmids which had evolved under low-frequency pulse selective regimes declined over time. Our findings suggest that temporally variable selection environments, such as those created during antibiotic treatments, may help to explain the stability of mobile plasmid-encoded resistance.

Migration promotes plasmid stability under spatially heterogeneous positive selection.

Bacteria-plasmid associations can be mutualistic or antagonistic depending on the strength of positive selection for plasmid-encoded genes, with contrasting outcomes for plasmid stability. In mutualistic environments, plasmids are swept to high frequency by positive selection, increasing the likelihood of compensatory evolution to ameliorate the plasmid cost, which promotes long-term stability. In antagonistic environments, plasmids are purged by negative selection, reducing the probability of compensatory evolution and driving their extinction. Here we show, using experimental evolution of Pseudomonas fluorescens and the mercury-resistance plasmid, pQBR103, that migration promotes plasmid stability in spatially heterogeneous selection environments. Specifically, migration from mutualistic environments, by increasing both the frequency of the plasmid and the supply of compensatory mutations, stabilized plasmids in antagonistic environments where, without migration, they approached extinction. These data suggest that spatially heterogeneous positive selection, which is common in natural environments, coupled with migration helps to explain the stability of plasmids and the ecologically important genes that they encode.

Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses.

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.

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.

The evolution of plasmid stability: Are infectious transmission and compensatory evolution competing evolutionary trajectories?

Conjugative plasmids are widespread and play an important role in bacterial evolution by accelerating adaptation through horizontal gene transfer. However, explaining the long-term stability of plasmids remains challenging because segregational loss and the costs of plasmid carriage should drive the loss of plasmids though purifying selection. Theoretical and experimental studies suggest two key evolutionary routes to plasmid stability: First, the evolution of high conjugation rates would allow plasmids to survive through horizontal transmission as infectious agents, and second, compensatory evolution to ameliorate the cost of plasmid carriage can weaken purifying selection against plasmids. How these two evolutionary strategies for plasmid stability interact is unclear. Here, we summarise the literature on the evolution of plasmid stability and then use individual based modelling to investigate the evolutionary interplay between the evolution of plasmid conjugation rate and cost amelioration. We find that, individually, both strategies promote plasmid stability, and that they act together to increase the likelihood of plasmid survival. However, due to the inherent costs of increasing conjugation rate, particularly where conjugation is unlikely to be successful, our model predicts that amelioration is the more likely long-term solution to evolving stable bacteria-plasmid associations. Our model therefore suggests that bacteria-plasmid relationships should evolve towards lower plasmid costs that may forestall the evolution of highly conjugative, 'infectious' plasmids.

Multi-host environments select for host-generalist conjugative plasmids.

BACKGROUND: Conjugative plasmids play an important role in bacterial evolution by transferring ecologically important genes within and between species. A key limit on interspecific horizontal gene transfer is plasmid host range. Here, we experimentally test the effect of single and multi-host environments on the host-range evolution of a large conjugative mercury resistance plasmid, pQBR57. Specifically, pQBR57 was conjugated between strains of a single host species, either P. fluorescens or P. putida, or alternating between P. fluorescens and P. putida. Crucially, the bacterial hosts were not permitted to evolve allowing us to observe plasmid evolutionary responses in isolation. RESULTS: In all treatments plasmids evolved higher conjugation rates over time. Plasmids evolved in single-host environments adapted to their host bacterial species becoming less costly, but in the case of P. fluorescens-adapted plasmids, became costlier in P. putida, suggesting an evolutionary trade-off. When evolved in the multi-host environment plasmids adapted to P. fluorescens without a higher cost in P. putida. CONCLUSION: Whereas evolution in a single-host environment selected for host-specialist plasmids due to a fitness trade-off, this trade-off could be circumvented in the multi-host environment, leading to the evolution of host-generalist plasmids.

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 catabolize sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by 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 among co-occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity.

Mosaic VSGs and the scale of Trypanosoma brucei antigenic variation.

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.

Exploiting lung adaptation and phage steering to clear pan-resistant Pseudomonas aeruginosa infections in vivo.

Pseudomonas aeruginosa is a major nosocomial pathogen that causes severe disease including sepsis. Carbapenem-resistant P. aeruginosa is recognised by the World Health Organisation as a priority 1 pathogen, with urgent need for new therapeutics. As such, there is renewed interest in using bacteriophages as a therapeutic. However, the dynamics of treating pan-resistant P. aeruginosa with phage in vivo are poorly understood. Using a pan-resistant P. aeruginosa in vivo infection model, phage therapy displays strong therapeutic potential, clearing infection from the blood, kidneys, and spleen. Remaining bacteria in the lungs and liver displays phage resistance due to limiting phage adsorption. Yet, resistance to phage results in re-sensitisation to a wide range of antibiotics. In this work, we use phage steering in vivo, pre-exposing a pan resistant P. aeruginosa infection with a phage cocktail to re-sensitise bacteria to antibiotics, clearing the infection from all organs.

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.

What makes a megaplasmid?

Naturally occurring plasmids come in different sizes. The smallest are less than a kilobase of DNA, while the largest can be over three orders of magnitude larger. Historically, research has tended to focus on smaller plasmids that are usually easier to isolate, manipulate and sequence, but with improved genome assemblies made possible by long-read sequencing, there is increased appreciation that very large plasmids-known as megaplasmids-are widespread, diverse, complex, and often encode key traits in the biology of their host microorganisms. Why are megaplasmids so big? What other features come with large plasmid size that could affect bacterial ecology and evolution? Are megaplasmids 'just' big plasmids, or do they have distinct characteristics? In this perspective, we reflect on the distribution, diversity, biology, and gene content of megaplasmids, providing an overview to these large, yet often overlooked, mobile genetic elements. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.