Deciphering the conjugation system of pXO16 from Bacillus thuringiensis serovar israelensis

The entomopathogenic Bacillus thuringiensis serovar israelensis displays peculiar transfer capabilities associated with pXO16, a large conjugative plasmid of 350 kb. The efficient and fast transfer is accompanied by a spectacular macroscopic aggregation of bacterial partners. Moreover, pXO16 is capable of efficient mobilization and retro-transfer of both mobilizable and “non-mobilizable” plasmids. Analysis of pXO16 sequence did not reveal any obvious type IV secretion system (T4SS) homologs, suggesting the presence of an unusual conjugative system. However, pXO16 harbors an FtsK/SpOIIIE homolog that could be reminiscent of either the Streptomyces spp. TraB-mediated dsDNA transfer system or the Clostridium perfringens tcp conjugation locus. In this work, we aimed to identify the genes responsible for pXO16 transfer mechanism and to gain better insights into pXO16 unique aggregation system. The construction of ftsK/spOIIIE and adjacent CDS knockout (KO) mutants was performed using homologous recombination. A similar strategy was used to delete a 25-kb region potentially involved in pXO16 aggregation phenotype. The presence of natural restriction-modification (RM) systems in both conjugative partners was also used to discriminate between ssDNA versus dsDNA transfer of pXO16. Several CDSs in the neighborhood of the ftsK/spOIIIE gene proved to be necessary for all pXO16 conjugative transfers. Analysis of the RM systems also strongly suggested the transfer of ssDNA between partners. Moreover, deletion of the “aggregation” region indicated that this phenomenon is important but not essential for pXO16 transfer, although additional experiments also demonstrated that the “disaggregation” of the mating pair requires the transfer of pXO16 into the recipient cells. Indirect proofs of pXO16 ssDNA transfer indicate more resemblance to the C. perfringens conjugative system than to that of Streptomyces spp. More specifically, pXO16 ftsK/spOIIIE could play the role of a coupling protein in this transfer system, which therefore appears to be distantly related to the other previously described T4SS