pXO16, an unique conjugative plasmid from Bacillus thuringiensis serovar israelensis

Bacillus thuringiensis serovar israelensis is an entomopathogenic bacterium used as biopesticide to control dipteran insects, vectors of human and animal diseases. The strains of this serovar are quite homogenous and display several extrachromosomal molecules whose sizes span from 5 to more than 300 kb. The largest of these molecules is pXO16, a 350-kb conjugative plasmid. This unusual plasmid encodes an aggregation-mediated conjugation system and is able of transferring itself and other non-conjugative and non-mobilizable plasmids in a fast and very efficient manner. Although the conjugation kinetics of this element has been extensively studied, its genetic organization has only recently been uncovered. In this study, the pXO16 CDSs were predicted from its sequence and their putative functions were examined, with a particular attention to possible conjugative and aggregation functions. pXO16 displayed unexpected characteristics for a conjugative plasmid: it harbours 467 putative CDS, all oriented in the same direction, and more than two third showed no homology with proteins in the databases. Concerning the putative conjugative functions, no homologues of the type IV secretion system (T4SS) could be found, strongly suggesting that pXO16 encodes an unforeseen conjugative system. Using double-recombination mutagenesis, several knockouts were made, including a 25-kb region containing five cell-surface-associated CDS. The transfer frequency and aggregation phenotype of the mutants were analyzed, together with the modification of their host cell surface. The 25-kb cell-surface-associated knockout mutant was unable to produce macroscopic aggregation but the transfer of pXO16 was still detected, albeit at lower frequencies (1,000-fold less for conjugative transfer and 100-fold less for mobilization), indicating that aggregation is important but not essential in the transfer process