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.

Integrases are a family of tyrosine recombinases that are highly abundant in bacterial genomes, actively disseminating adaptive characters such as pathogenicity determinants and antibiotics resistance. Using comparative genomics and functional assays, we identified a novel type of mobile genetic element, the GInt, in many diverse bacterial groups but not in archaea. Integrated as genomic islands, GInts show a tripartite structure consisting of the ginABCD operon, a cargo DNA region from 2.5 to at least 70 kb, and a short AT-rich 3′ end. The gin operon is characteristic of GInts and codes for three putative integrases and a small putative helix-loop-helix protein, all of which are essential for integration and excision of the element. Genes in the cargo DNA are acquired mostly from phylogenetically related bacteria and often code for traits that might increase fitness, such as resistance to antimicrobials or virulence. GInts also tend to capture clusters of genes involved in complex processes, such as the biosynthesis of phaseolotoxin by Pseudomonas syringae. GInts integrate site-specifically, generating two flanking direct imperfect repeats, and excise forming circular molecules. The excision process generates sequence variants at the element attachment site, which can increase frequency of integration and drive target specificity.

Background: Virulence plasmids are critically exposed to genetic decay and loss, particularly in Pseudomonas syringae strains because of their high content of mobile genetic elements and their exploitation of environmental niches outside of the plant host. The demonstrated high plasticity and adaptability of P. syringae plasmids, involving the acquisition and loss of large DNA regions, contrasts with their usual high stability and the maintenance of key virulence genes in free living conditions. The identification of plasmid stability determinants and mechanisms will help to understand their evolution and adaptability to agroecosystems as well as to develop more efficient control measures. Results:We show that the three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain diverse functional stability determinants, including three toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C, whereas one of the two replicons of pPsv48C can confer stable inheritance by itself. Loss of pPsv48A increased by two orders of magnitude upon functional inactivation of its TA systems. However, inactivation of the TA systems from pPsv48C did not result in its curing but led to the recovery of diverse deletion derivatives. One type consisted in the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10 -3 , by recombination between two copies of MITEPsy2. Likewise, IS801 promoted the occurrence of deletions of variable size by one-ended transposition with a frequency of 5.5 ± 2.1 × 10 -4 , 80 % of which resulted in the loss of 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. Conclusions:Virulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Additionally, multiple TA systems favour the long-term survival and integrity of virulence plasmids, as well as the maintenance of pathogenicity genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.(MINECO), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER).