Coresident Plasmids Research Articles

Evolution, persistence, and host adaption of a gonococcal AMR plasmid that emerged in the pre-antibiotic era.

Plasmids are diverse extrachromosomal elements significantly that contribute to interspecies dissemination of antimicrobial resistance (AMR) genes. However, within clinically important bacteria, plasmids can exhibit unexpected narrow host ranges, a phenomenon that has scarcely been examined. Here we show that pConj is largely restricted to the human-specific pathogen, Neisseria gonorrhoeae. pConj can confer tetracycline resistance and is central to the dissemination of other AMR plasmids. We tracked pConj evolution from the pre-antibiotic era 80 years ago to the modern day and demonstrate that, aside from limited gene acquisition and loss events, pConj is remarkably conserved. Notably, pConj has remained prevalent in gonococcal populations despite cessation of tetracycline use, thereby demonstrating pConj adaptation to its host. Equally, pConj imposes no measurable fitness costs and is stably inherited by the gonococcus. Its maintenance depends on the co-operative activity of plasmid-encoded Toxin:Antitoxin (TA) and partitioning systems rather than host factors. An orphan VapD toxin encoded on pConj forms a split TA with antitoxins expressed from an ancestral co-resident plasmid or a horizontally-acquired chromosomal island, potentially explaining pConj's limited distribution. Finally, ciprofloxacin can induce loss of this highly stable plasmid, reflecting epidemiological evidence of transient reduction in pConj prevalence when fluoroquinolones were introduced to treat gonorrhoea.

Conjugation across Bacillus cereus and kin: A review.

Horizontal gene transfer (HGT) is a major driving force in shaping bacterial communities. Key elements responsible for HGT are conjugation-like events and transmissible plasmids. Conjugative plasmids can promote their own transfer as well as that of co-resident plasmids. Bacillus cereus and relatives harbor a plethora of plasmids, including conjugative plasmids, which are at the heart of the group species differentiation and specification. Since the first report of a conjugation-like event between strains of B. cereus sensu lato (s.l.) 40 years ago, many have studied the potential of plasmid transfer across the group, especially for plasmids encoding major toxins. Over the years, more than 20 plasmids from B. cereus isolates have been reported as conjugative. However, with the increasing number of genomic data available, in silico analyses indicate that more plasmids from B. cereus s.l. genomes present self-transfer potential. B. cereus s.l. bacteria occupy diverse environmental niches, which were mimicked in laboratory conditions to study conjugation-related mechanisms. Laboratory mating conditions remain nonetheless simplistic compared to the complex interactions occurring in natural environments. Given the health, economic and ecological importance of strains of B. cereus s.l., it is of prime importance to consider the impact of conjugation within this bacterial group.

Plasmid pND6-1 enhances the stability and conjugative transfer of co-resident companion plasmid pND6-2 in the naphthalene-degradative Pseudomonas putida strain ND6

BackgroundBacterial cells often harbor multiple plasmids, but the interaction between these plasmids is poorly understood. Pseudomonas putida ND6, one of the typical naphthalene-degrading bacteria, contains two annotated plasmids, pND6-1 and pND6-2. The conjugative plasmid pND6-2 can be used as a helper to mobilize the naphthalene-degrading plasmid pND6-1. The role of pND6-1 in functions other than naphthalene degradation has never been explored. Therefore, it is of great significance to study the possible effect of pND6-1 on the transfer rate or maintenance of pND6-2. ResultsTo investigate the interaction between pND6-1 and pND6-2, we successfully cured plasmid pND6-1 using the incompatibility method, generated the pND6-1-null strain ND6dp1, and first classified it into the IncP-7 group by curing experimental evidence. Subsequently, the pND6-1-cured mutant showed an insignificant increase in cell growth compared with the wild ND6, indicating that a lower cost was imposed on the host by pND6-1. The influences of pND6-1 on the stability and conjugation of plasmid pND6-2 were subsequently explored. The results showed that the presence of pND6-1 enhanced the stability of pND6-2 in natural host ND6 and heterologous host P. putida KT2440. In addition, the presence of pND6-1 in wild-type hosts promoted intra-specific and inter-specific conjugative transfer efficiency of pND6-2 by 3.9–6.3-fold with P. putida, P. resinovans, and P. fluorescens as the recipients, respectively. ConclusionsCollectively, these results suggest that the plasmid pND6-1 improves the pND6-2 plasmid's stability and transfer rate in the ND6 strain with the selected recipient strains.How to cite: Wang S, Liu M, Wang D, et al. Plasmid pND6-1 enhances the stability and conjugative transfer of co-resident companion plasmid pND6-2 in the naphthalene-degradative Pseudomonas putida strain ND6. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.08.002.

Spreading Advantages of Coresident Plasmids blaCTX-M-Bearing IncFII and mcr-1-Bearing IncI2 in Escherichia coli.

ABSTRACTTwo diverse conjugative plasmids can interact within bacterial cells. However, to the best of our knowledge, the interaction between blaCTX-M-bearing IncFII plasmid and mcr-1-carrying IncI2 plasmid colocated on the same bacterial host has not been reported. This study was initiated to explore the interaction and to analyze the reasons that these two plasmids are often coresident in multidrug-resistant Escherichia coli. To assess the interactions on plasmid stabilities, fitness costs, and transfer rates, we constructed two groups of isogenic derivatives, C600FII, C600I2, and C600FII+I2 of E. coli C600 and J53FII, J53I2, and J53FII+I2 of E. coli J53, respectively. We found that carriage of FII and I2 plasmids, independently and together, had not impaired the growth of the bacterial host. It was difficult for the single plasmid FII or I2 in E. coli C600 to reach stable persistence for a long time in an antibiotic-free environment, while the stability would be striking improved when they coresided. Meanwhile, plasmids FII and I2, whether together or apart, could notably enhance the fitness advantage of the host; moreover, E. coli coharboring plasmids FII and I2 presented more obvious fitness advantage than that carrying single plasmid FII. Coresident plasmids FII and I2 could accelerate horizontal cotransfer by conjugation. The transfer rates from a strain carrying coresident FII and I2 plasmids increased significantly when it mated with a recipient cell carrying one of them. Our findings highlight the advantages of coinhabitant FII and I2 plasmids in E. coli to drive the persistence and spread of plasmid-carried blaCTX-M and mcr-1 genes, although the molecular mechanisms of their coresidence warrant further study.IMPORTANCE More and more Enterobacteriaceae carry both blaCTX-M and mcr-1, which are usually located on IncFII-type and IncI2-type plasmids in the same bacterial host, respectively. However, the study on advantages of coresident plasmids in bacterial host is still sparse. Here, we investigated the stability, fitness cost, and cotransfer traits associated with coresident IncFII-type and IncI2-type plasmids in E. coli. Our results show that coinhabitant plasmids in E. coli are more stable, confer more fitness advantages, and are easier to transfer and cotransfer than a single plasmid IncFII or IncI2. Our findings confirm the advantages of coresident plasmids of blaCTX-M-bearing IncFII and mcr-1-bearing IncI2 in clinical E. coli, which will pose a serious threat to clinical therapy and public health.

Plasmid co-infection: linking biological mechanisms to ecological and evolutionary dynamics.

As infectious agents of bacteria and vehicles of horizontal gene transfer, plasmids play a key role in bacterial ecology and evolution. Plasmid dynamics are shaped not only by plasmid–host interactions but also by ecological interactions between plasmid variants. These interactions are complex: plasmids can co-infect the same cell and the consequences for the co-resident plasmid can be either beneficial or detrimental. Many of the biological processes that govern plasmid co-infection—from systems that exclude infection by other plasmids to interactions in the regulation of plasmid copy number—are well characterized at a mechanistic level. Modelling plays a central role in translating such mechanistic insights into predictions about plasmid dynamics and the impact of these dynamics on bacterial evolution. Theoretical work in evolutionary epidemiology has shown that formulating models of co-infection is not trivial, as some modelling choices can introduce unintended ecological assumptions. Here, we review how the biological processes that govern co-infection can be represented in a mathematical model, discuss potential modelling pitfalls, and analyse this model to provide general insights into how co-infection impacts ecological and evolutionary outcomes. In particular, we demonstrate how beneficial and detrimental effects of co-infection give rise to frequency-dependent selection on plasmid variants.This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Replication of theSalmonella Genomic Island 1 (SGI1) triggered by helper IncC conjugative plasmids promotes incompatibility and plasmid loss.

The mobilizable resistance island Salmonella genomic island 1 (SGI1) is specifically mobilized by IncA and IncC conjugative plasmids. SGI1, its variants and IncC plasmids propagate multidrug resistance in pathogenic enterobacteria such as Salmonella enterica serovars and Proteus mirabilis. SGI1 modifies and uses the conjugation apparatus encoded by the helper IncC plasmid, thus enhancing its own propagation. Remarkably, although SGI1 needs a coresident IncC plasmid to excise from the chromosome and transfer to a new host, these elements have been reported to be incompatible. Here, the stability of SGI1 and its helper IncC plasmid, each expressing a different fluorescent reporter protein, was monitored using fluorescence-activated cell sorting (FACS). Without selective pressure, 95% of the cells segregated into two subpopulations containing either SGI1 or the helper plasmid. Furthermore, FACS analysis revealed a high level of SGI1-specific fluorescence in IncC+ cells, suggesting that SGI1 undergoes active replication in the presence of the helper plasmid. SGI1 replication was confirmed by quantitative PCR assays, and extraction and restriction of its plasmid form. Deletion of genes involved in SGI1 excision from the chromosome allowed a stable coexistence of SGI1 with its helper plasmid without selective pressure. In addition, deletion of S003 (rep) or of a downstream putative iteron-based origin of replication, while allowing SGI1 excision, abolished its replication, alleviated the incompatibility with the helper plasmid and enabled its cotransfer to a new host. Like SGI1 excision functions, rep expression was found to be controlled by AcaCD, the master activator of IncC plasmid transfer. Transient SGI1 replication seems to be a key feature of the life cycle of this family of genomic islands. Sequence database analysis revealed that SGI1 variants encode either a replication initiator protein with a RepA_C domain, or an alternative replication protein with N-terminal replicase and primase C terminal 1 domains.

Molecular genetic analysis of an XDR Pseudomonas aeruginosa ST664 clone carrying multiple conjugal plasmids

A group of ST664 XDR Pseudomonas aeruginosa strains have been isolated from a burn clinic. Here we decipher their resistomes and likely mechanisms of resistance acquisition. The complete nucleotide sequences of representative isolates were determined, by PacBio and Illumina MiSeq sequencing, and analysed for antimicrobial resistance (AMR) genes as well as sequence variations. S1-PFGE was used to determine the sizes and numbers of plasmids harboured by the isolates. Purified plasmid DNA was further sequenced by PacBio technology, closed manually and annotated by RAST. The mobility of plasmids was determined by conjugation assays. The XDR P. aeruginosa ST664 clone carries 11 AMR genes, including a blaKPC-2 gene that confers resistance to carbapenems. Most of the ST664 isolates carry three coexisting plasmids. blaKPC-2 and a cluster of three AMR genes (aadB-cmlA1-sul1) are encoded on a 475 kb megaplasmid pNK546a, which codes for an IncP-3-like replication and partitioning mechanism, but has lost the conjugative transfer system. Interestingly, however, pNK546a is mobilizable and can be transferred to P. aeruginosa PAO1 with the help of a co-residing IncP-7 conjugative plasmid. The blaKPC-2 gene is carried by an IS6100-ISKpn27-blaKPC-2-ΔISKpn6-Tn1403 mobile element, which might be brought into the ST664 clone by another co-resident IncP-1α plasmid, which is inclined to be lost. Moreover, pNK546a harbours multiple heavy metal (mercury, tellurite and silver) resistance modules. To the best of our knowledge, pNK546a is the first fully sequenced blaKPC-2-carrying megaplasmid from P. aeruginosa. These results give new insights into bacterial adaptation and evolution during nosocomial infections.

Interplay among IncA and blaKPC-Carrying Plasmids in Citrobacter freundii.

We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, blaKPC-2 was harbored on a novel variant of a Tn4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, blaKPC-3 was on a Tn4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying blaKPC and the coresident IncA plasmids offers new information on plasmids coresident within clinically relevant enterobacteria.

Mechanistic Features of the Enterococcal pCF10 Sex Pheromone Response and the Biology of Enterococcus faecalis in Its Natural Habitat.

Conjugative transfer of plasmids in enterococci is promoted by intercellular communication using peptide pheromones. The regulatory mechanisms that control transfer have been extensively studied in vitro However, the complicated systems that regulate the spread of these plasmids did not evolve in the laboratory test tube, and remarkably little is known about this form of signaling in the intestinal tract, the primary niche of these organisms. Because the evolution of Enterococcus faecalis strains and their coresident pheromone-inducible plasmids, such as pCF10, have occurred in the gastrointestinal (GI) tract, it is important to consider the functions controlled by pheromones in light of this ecology. This review summarizes our current understanding of the pCF10-encoded pheromone response. We consider how selective pressures in the natural environment may have selected for the complex and very tightly regulated systems controlling conjugation, and we pay special attention to the ecology of enterococci and the pCF10 plasmid as a gut commensal. We summarize the results of recent studies of the pheromone response at the single-cell level, as well as those of the first experiments demonstrating a role for pheromone signaling in plasmid transfer and in GI tract competitive fitness. These results will serve as a foundation for further in vivo studies that could lead to novel interventions to reduce opportunistic infections and the spread of antibiotic resistance.

Rapid conjugative mobilization of a 100\xa0kb segment of Bacillus subtilis chromosomal DNA is mediated by a helper plasmid with no ability for self-transfer

BackgroundThe conjugative plasmid, pLS20, isolated from Bacillus subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the mobilization of an independently replicating co-resident plasmid.ResultsIn this study, the oriT sequence of pLS20cat (oriTLS20) was eliminated to obtain the plasmid, pLS20catΔoriT. This resulted in the complete loss of the conjugative transfer of the plasmid but still allowed it to mobilize a co-resident mobilizable plasmid. Moreover, pLS20catΔoriT was able to mobilize longer DNA segments, up to 113 kb of chromosomal DNA containing oriTLS20, after mixing the liquid cultures of the donor and recipient for only 15 min.ConclusionsThe chromosomal DNA mobilization mediated by pLS20catΔoriT will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.

PifC and Osa, Plasmid Weapons against Rival Conjugative Coupling Proteins.

Bacteria display a variety of mechanisms to control plasmid conjugation. Among them, fertility inhibition (FI) systems prevent conjugation of co-resident plasmids within donor cells. Analysis of the mechanisms of inhibition between conjugative plasmids could provide new alternatives to fight antibiotic resistance dissemination. In this work, inhibition of conjugation of broad host range IncW plasmids was analyzed in the presence of a set of co-resident plasmids. Strong FI systems against plasmid R388 conjugation were found in IncF/MOBF12 as well as in IncI/MOBP12 plasmids, represented by plasmids F and R64, respectively. In both cases, the responsible gene was pifC, known also to be involved in FI of IncP plasmids and Agrobacterium T-DNA transfer to plant cells. It was also discovered that the R388 gene osa, which affects T-DNA transfer, also prevented conjugation of IncP-1/MOBP11 plasmids represented by plasmids RP4 and R751. Conjugation experiments of different mobilizable plasmids, helped by either FI-susceptible or FI-resistant transfer systems, demonstrated that the conjugative component affected by both PifC and Osa was the type IV conjugative coupling protein. In addition, in silico analysis of FI proteins suggests that they represent recent acquisitions of conjugative plasmids, i.e., are not shared by members of the same plasmid species. This implies that FI are rapidly-moving accessory genes, possibly acting on evolutionary fights between plasmids for the colonization of specific hosts.

Co-resident plasmids travel together

Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids.

Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids

Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free – a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such interactions may determine how antibiotic resistance disseminates in bacterial populations.

The tet39 Determinant and the msrE-mphE Genes in Acinetobacter Plasmids Are Each Part of Discrete Modules Flanked by Inversely Oriented pdif (XerC-XerD) Sites.

The tet39 tetracycline resistance determinant and the macrolide resistance genes msrE and mphE were found in an 18.2-kb plasmid, pS30-1, recovered from a global clone 2 (GC2) Acinetobacter baumannii isolate from Singapore, that conferred resistance to tetracycline and erythromycin. pS30-1 also contains mobA and mobC genes encoding MOBQ family proteins, but attempts to mobilize pS30-1 utilizing a coresident conjugative repAci6 plasmid were unsuccessful. Eight pdif sites, consisting of inversely oriented binding sites for the XerC and XerD recombinases separated by 6 bp, were detected in pS30-1. The tet39 determinant and the msrE-mphE gene pair are each surrounded by two pdif sites in inverse orientation. Identical regions in different contexts and many previously unnoticed pdif sites were found in a number of different plasmids in GenBank, showing that the tet39 and msrE-mphE dif modules are mobile. A putative toxin/antitoxin system, a gene encoding a serine recombinase, and open reading frames of unknown function were also part of dif modules in pS30-1. In general, modules with internal XerC or XerD sites alternate. Two copies of ISAjo2-1 (94% identical to ISAjo2) in pS30-1 were inserted 5 bp from a XerC site, and this appears to be the preferred insertion site for this insertion sequence (IS) group. Apparently, Acinetobacter plasmids exploit the Acinetobacter XerC-XerD recombinases to mobilize DNA units containing resistance and other genes, via an uncharacterized mechanism. The tet39 and msrE-mphE dif modules add to the oxa24 module and the oxa58 module redefined here, bringing the total of resistance gene-containing dif modules in Acinetobacter plasmids to four.

A Functional oriT in the Ptw Plasmid of Burkholderia cenocepacia Can Be Recognized by the R388 Relaxase TrwC.

Burkholderia cenocepacia is both a plant pathogen and the cause of serious opportunistic infections, particularly in cystic fibrosis patients. B. cenocepacia K56-2 harbors a native plasmid named Ptw for its involvement in the Plant Tissue Watersoaking phenotype. Ptw has also been reported to be important for survival in human cells. Interestingly, the presence of PtwC, a homolog of the conjugative relaxase TrwC of plasmid R388, suggests a possible function for Ptw in conjugative DNA transfer. The ptw region includes Type IV Secretion System genes related to those of the F plasmid. However, genes in the adjacent region shared stronger homology with the R388 genes involved in conjugative DNA metabolism. This region included the putative relaxase ptwC, a putative coupling protein and accessory nicking protein, and a DNA segment with high number of inverted repeats and elevated AT content, suggesting a possible oriT. Although we were unable to detect conjugative transfer of the Ptw resident plasmid, we detected conjugal mobilization of a co-resident plasmid containing the ptw region homologous to R388, demonstrating the cloned ptw region contains an oriT. A similar plasmid lacking ptwC could not be mobilized, suggesting that the putative relaxase PtwC must act in cis on its oriT. Remarkably, we also detected mobilization of a plasmid containing the Ptw oriT by the R388 relaxase TrwC, yet we could not detect PtwC-mediated mobilization of an R388 oriT-containing plasmid. Our data unambiguously show that the Ptw plasmid harbors DNA transfer functions, and suggests the Ptw plasmid may play a dual role in horizontal DNA transfer and eukaryotic infection.

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci.

Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-like oriT. These data indicate that the conjugative relaxase in trans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids. Understanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus multiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variant oriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase in trans mechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.

A Transmissible Plasmid-Borne Pathogenicity Island Confers Piscibactin Biosynthesis in the Fish Pathogen Photobacterium damselae subsp. piscicida.

The fish pathogen Photobacterium damselae subsp. piscicida produces the siderophore piscibactin. A gene cluster that resembles the Yersinia high-pathogenicity island (HPI) encodes piscibactin biosynthesis. Here, we report that this HPI-like cluster is part of a hitherto-uncharacterized 68-kb plasmid dubbed pPHDP70. This plasmid lacks homologs of genes that mediate conjugation, but we found that it could be transferred at low frequencies from P. damselae subsp. piscicida to a mollusk pathogenic Vibrio alginolyticus strain and to other Gram-negative bacteria, likely dependent on the conjugative functions of the coresident plasmid pPHDP60. Following its conjugative transfer, pPHDP70 restored the capacity of a vibrioferrin mutant of V. alginolyticus to grow under low-iron conditions, and piscibactin became detectable in its supernatant. Thus, pPHDP70 appears to harbor all the genes required for piscibactin biosynthesis and transport. P. damselae subsp. piscicida strains cured of pPHDP70 no longer produced piscibactin, had impaired growth under iron-limited conditions, and exhibited markedly decreased virulence in fish. Collectively, our findings highlight the importance of pPHDP70, with its capacity for piscibactin-mediated iron acquisition, in the virulence of P. damselae subsp. piscicida. Horizontal transmission of this plasmid-borne piscibactin synthesis gene cluster in the marine environment may facilitate the emergence of new pathogens.

Ecological dynamics and complex interactions of Agrobacterium megaplasmids.

As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it’s Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together.

Non-additive costs and interactions alter the competitive dynamics of co-occurring ecologically distinct plasmids.

Plasmids play an important role in shaping bacterial evolution and adaptation to heterogeneous environments. As modular genetic elements that are often conjugative, the selective pressures that act on plasmid-borne genes are distinct from those that act on the chromosome. Many bacteria are co-infected by multiple plasmids that impart niche-specific phenotypes. Thus, in addition to host-plasmid dynamics, interactions between co-infecting plasmids are likely to be important drivers of plasmid population dynamics, evolution and ecology. Agrobacterium tumefaciens is a facultative plant pathogen that commonly harbours two distinct megaplasmids. Virulence depends on the presence of the tumour-inducing (Ti) plasmid, with benefits that are primarily restricted to the disease environment. Here, we demonstrate that a second megaplasmid, the At plasmid, confers a competitive advantage in the rhizosphere. To assess the individual and interactive costs of these plasmids, we generated four isogenic derivatives: plasmidless, pAt only, pTi only and pAtpTi, and performed pairwise competitions under carbon-limiting conditions. These studies reveal a low cost to the virulence plasmid when outside of the disease environment, and a strikingly high cost to the At plasmid. In addition, the costs of pAt and pTi in the same host were significantly lower than predicted based on single plasmid costs, signifying the first demonstration of non-additivity between naturally occurring co-resident plasmids. Based on these empirically demonstrated costs and benefits, we developed a resource-consumer model to generate predictions about the frequencies of these genotypes in relevant environments, showing that non-additivity between co-residing plasmids allows for their stable coexistence across environments.

The pYV virulence plasmids of Yersinia pseudotuberculosis and Y. pestis contain a conserved DNA region responsible for the mobilization by the self‐transmissible plasmid pYE854

Pathogenic Yersinia strains possess a 70 kb virulence plasmid which can be transmitted to other strains by conjugation systems expressed by co-resident plasmids. We isolated a 720 bp fragment of the Yersinia pseudotuberculosis virulence plasmid p1340 that mediated mobilization of the vector pIV2 by the self-transmissible plasmid pYE854. The p1340 mobilization region contains the resolvase gene tnpR and its proposed resolution site res. Both elements are required for mobilization. Plasmid transfer was associated with the formation of co-integrates in which res was fused to pYE854 fragments by short nucleotide stretches similarly present within res. blast searches and PCR experiments revealed the presence of the mobilization region in the virulence plasmids of other Y. pseudotuberculosis and Y. pestis strains but not in pYV of Yersinia enterocolitica.