Deciphering transfer mechanisms of the conjugative plasmid pXO16

Bacillus thuringiensis serovar israelensis preparations are widely used as a bioinsecticide against Dipteran larvae. In this serovar, the entomopathogenic toxin genes are carried by the large pBtoxis plasmid and the plasmid transfer ability is associated with the large conjugative pXO16 plasmid (350 kb). pXO16 encodes an unusual conjugation mechanism with some unique features: its transfer is accompanied by an impressive macroscopic aggregation phenotype in liquid matings, and its conjugation is very fast and efficient, especially in B. thuringiensis sv. israelensis matings, where transfer lasts less than 4 min and reaches 100 % efficiency in just a few hours. The conjugative system of pXO16 is also able to mobilize and retro-mobilize any plasmid, including those usually classified as “non-mobilizable”, hence demanding in-depth study. In this work, identification and analysis of pXO16 sequence in available B. thuringiensis sv. israelensis genomes were first carried out. In order to identify the genes involved in the conjugative transfer, deletion mutants were obtained. A “transfer israelensis plasmid” (tip) locus containing an FtsK ATPase gene essential for transfer, was accordingly proposed and revealed potential Type IV Secretion System (T4SS) genes. Additionally, the analysis of natural restriction-modification (RM) systems in conjugative partners gave credit to a single-stranded DNA transfer mechanism. Moreover, identification of pXO16 genes necessary for aggregation was also performed by deletion of a candidate “agr” region, which resulted in impairing the aggregation phenomenon. The pXO16 aggregation phenotype and its corresponding genetic determinants proved to greatly benefit conjugative transfer while not being mandatory. Correspondingly, the transfer of pXO16 to recipient strains displaying no aggregation phenotype was assessed and revealed an unforeseen extended host range of this conjugative plasmid. Since no co-resident plasmids have yet appeared to be “safeguarded” against mobilization by pXO16, mobilization of chromosomal loci was also investigated using an original approach of “mating between close-relatives”. Interestingly, pXO16 conjugative system proved to transfer chromosomal regions (ranging from 2.5 to 791 kb) at frequencies of ca. 10-5 - 10-6 transconjugants per donor cell. This observation further emphasizes the biological role of pXO16 as key player in horizontal gene transfers and potential bacterial adaptation and evolution.