Summary auto-generated
This study presents a detailed functional analysis of pGI2, a 9,672 base pair cryptic plasmid from Bacillus thuringiensis H1.1 that contains the transposon Tn4430. Through deletion experiments and sequence comparisons, researchers identified pGI2 as a rolling-circle replicating (RCR) plasmid with a modular organization. The minimal replicon comprises the rep gene (ORF1, encoding a 204 amino acid replication protein) and a putative double-strand origin located downstream of the rep gene. Southern hybridization confirmed the presence of single-strand DNA intermediates in plasmid derivatives lacking the single-strand origin (sso). The pGI2 replication protein shows significant similarity to that of pTX14-3 from B. thuringiensis serovar israelensis, suggesting both plasmids represent a new family of RCR elements. Notably, pGI2 possesses a mobilization (Mob) protein that enables conjugative transfer via the plasmid pAW63. The pGI2 ORF2 protein shares homology with a protein from a Lactococcus lactis plasmid. Overall, pGI2 functions as an assembly of distinct genetic modules involved in replication, strand conversion, and conjugative transfer, with no identified metabolic function, serving potentially as a genetic reservoir for its host.
Key findings
- pGI2 is a rolling-circle replicating plasmid with a modular architecture containing separate leading-strand and lagging-strand replication modules, plus a mobilization function
- The double-strand origin of replication is located 310 base pairs downstream of the rep gene in a highly conserved 320 bp region shared with pTX14-3, a structural organization not previously reported
- pGI2 possesses a functional mobilization protein enabling conjugative transfer via the helper plasmid pAW63, with similar Mob-like proteins found in related plasmids showing subdomanial organization
- The pGI2 Rep protein and pTX14-3 Rep protein define a new family of rolling-circle replicating plasmids distinct from previously characterized families
- ssDNA intermediates are only detected in plasmid derivatives lacking the single-strand origin, consistent with rolling-circle replication mechanism
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Abstract
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 sso(t)-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 sso(t) 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.