Functional insights into pGI2, a cryptic rolling-circle replicating plasmid from Bacillus thuringiensis

Detailed functional analysis revealed the modular organization of pGI2, a 9672 bp plasmid from Bacillus thuringiensis H1.1 that harbours the 4149 bp transposon Tn4430. Whereas the pGI2 leading-strand replicon was identified through deletion experiments, sequence comparisons indicated the presence of an ssot-like single-strand origin commonly found among Bacillus plasmids. Southern hybridization confirmed the existence of ssDNA intermediates, but only in the case of plasmid derivatives lacking the ssot site. Moreover, the pGI2 replication protein Rep displayed significant similarity with that of pTX14-3, a 7-6 kb plasmid from B. thuringiensis serovar israelensis, suggesting that both elements are representatives of a new family of rolling-circle replicating (RCR) plasmids. In addition, both plasmids share a conserved 320 bp region downstream of their rep genes which, in the case of pGI2, appeared indispensable for replication. This region is therefore likely to correspond to, or to be part of, the actual double-strand origin of both plasmids. Another interesting feature of pGI2 is the presence of a mobilization (Mob) protein, as demonstrated by its ability to be mobilized by the conjugative plasmid pAW63 from B. thuringiensis serovar kurstaki HD73. The same transfer system was also used to unambiguously demonstrate similar properties of the related Mob-like protein from pTX14-3. A closer analysis of this family of related Mob proteins suggested a subdomanial organization among its members. Finally, the 270 residue pGI2 ORF2 was shown to be related to ORF43 of pMRC01, a 60 kb conjugative plasmid from Lactococcus lactis subsp. lactis. Although no function has been assigned to the putative ORF43 protein, it is located downstream of a bacteriocin operon, next to an IS946 element. pGI2 appears thus far as an assemblage of functional modules with no obvious metabolic function, presumably acting as a reservoir of carrier (rep and sso), rearrangement (Tn4430) or recruiting (Mob) entities for its bacterial host.