Functional insights into pG12, a cryptic rolling-circle replicating plasmid from Bacillus thuringiensis

Detailed functional analysis revealed the modular organization of pGl2, a 9 672 bp plasmid from Bacillus thuringiensis H1.1 that harbours the 4149 bp t ransposon Tn4430. whereas the pGl2 leading-strand replicon was identified t hrough deletion experiments, sequence comparisons indicated the presence of an sso(t)-like single-strand origin commonly found among Bacillus plasmids . Southern hybridization confirmed the existence of ssDNA intermediates, bu t only in the case of plasmid derivatives lacking the sso(t) site. Moreover the pGl2. replication protein Rep displayed significant similarity with th at of pTX 14-3. a 7.6 kb plasmid from B. thuringiensis serovar israelensis, suggesting that both elements are representatives of a new family of rolli ng-circle replicating (RCR) plasmids. In addition, both plasmids share a co nserved 320 bp region downstream of their rep genes which, in the case of p Gl2, appeared indispensable for replication. This region is therefore likel y to correspond to, or to be part of, the actual double-strand origin of bo th plasmids. Another interesting feature of pC12 is the presence of a mobil ization (Mob) protein, as demonstrated by its ability to be mobilized by th e conjugative plasmid pAW63 from B. thuringiensis serovar kurstaki HD73. Th e same transfer system was also used to unambiguously demonstrate similar p roperties of the related Mob-like protein from pTX14-3. A closer analysis o f this family of related Mob proteins suggested a subdomanial organization among its members. Finally, the 270 residue pGl2 ORF2 was shown to be relat ed 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 IS94 6 element. pGl2 appears thus far as an assemblage of functional modules wit h no obvious metabolic function, presumably acting as a reservoir of carrie r (rep and sso), rearrangement (Tn4430) or recruiting (Mob) entities for it s bacterial host.