The toxic guardians - multiple toxin-antitoxin systems provide stability, avoid deletions and maintain virulence genes of virulence plasmids.

BACKGROUND

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, 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.

RESULTS

The three virulence plasmids of 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 MITE caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10. Likewise, one-ended transposition of IS generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 × 10, of which 80% had lost virulence gene . These deletion derivatives were stably maintained in the population by replication mediated by , which is adjacent to IS. IS 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 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 and other bacteria.