Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities.

BackgroundPseudomonas syringae is a γ-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems.ResultsThe three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10− 3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 × 10− 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo.ConclusionsVirulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.Electronic supplementary materialThe online version of this article (10.1186/s13100-019-0149-4) contains supplementary material, which is available to authorized users.

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6×10−5 and 1.1×10−6, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

regulating the expression of other virulence genes. Therefore, gene ptz, but not gene idi, is essential for the biosynthesis of high amounts of cytokinins in culture; however, both ptz and idi are individually essential for the adequate development of tumors on olive plants by Psv NCPPB 3335.

Pseudomonas syringae pv. savastanoi NCPPB 3335 is the causal agent of olive knot disease and contains three virulence plasmids: pPsv48A (pA), 80 kb; pPsv48B (pB), 45 kb, and pPsv48C (pC), 42 kb. Here we show that pB contains a complete MPFT (previously type IVA secretion system) and a functional origin of conjugational transfer adjacent to a relaxase of the MOBP family; pC also contains a functional oriT-MOBP array, whereas pA contains an incomplete MPFI (previously type IVB secretion system), but not a recognizable oriT. Plasmid transfer occurred on solid and in liquid media, and on leaf surfaces of a non-host plant (Phaseolus vulgaris) with high (pB) or moderate frequency (pC); pA was transferred only occasionally after cointegration with pB. We found three plasmid-borne and three chromosomal relaxase genes, although the chromosomal relaxases did not contribute to plasmid dissemination. The MOBP relaxase genes of pB and pC were functionally interchangeable, although with differing efficiencies. We also identified a functional MOBQ mobilization region in pC, which could only mobilize this plasmid. Plasmid pB could be efficiently transferred to strains of six phylogroups of P. syringae sensu lato, whereas pC could only be mobilized to two strains of phylogroup 3 (genomospecies 2). In two of the recipient strains, pB was stably maintained after 21 subcultures in liquid medium. The carriage of several relaxases by the native plasmids of P. syringae impacts their transfer frequency and, by providing functional diversity and redundancy, adds robustness to the conjugation system.