Isolation and characterization of replication elements froma small cryptic plasmid of the alfalfa symbiont Sinorhizobium meliloti

PISTORIO, M.; GIUSTI, M.A.; LOZANO, M.J.; TORRES TEJERIZO, G.A.; DRAGHI, W.O.; SAN JUAN, J.; LAGARES, A.

California, USA

Congreso; Plasmid Biology; 2006

International Symposium of Plasmid Biology

Gram negative bacteria that belong to the genera Rhizobium, Sinorhizobium, Azorhizobium, and Mesorhizobium grow in the soil in free-living conditions, and in symbiosis associated with theroot of legumes as nitrogen-fixing organisms. Current evidence shows that rhizobia carry in most cases variable number of plasmids related to their symbiosis and free-living, being a commonfeature the presence of several plasmid replicons within a same strain. Thus, several rhizobial plasmids have been characterized in their replication machinery. In S. meliloti, the symbiontof alfalfa, plasmid replication strategies and incompatibility mechanisms have been recently analyzed (Izquierdo et al., 2005; MacLellan et al., 2005; Watson and Heys, 2006). In our laboratory we have previously described the transmissibility properties of two cryptic plasmids from the strain S. meliloti LPU88, a local isolate from Argentina. One of them, pSmeLPU88b (approx. 40 kb), resulted to be mobilizable if helper functions were supplied in trans by an accompanying plasmid pSmeLPU88a (binary conjugal system) (Pistorio et al., 2003). To characterize the replication elements of the transmissible plasmid pSmeLPU88b, a plasmid library was constructed and sequenced using a shotgun strategy. From the collected sequence data two plasmid replication modules were identified in silico: one belonging to the repABC type, and the other related to the type A family of plasmid replication regions. Both regions resulted nearly identical to the homologous elements from plasmid pMBA9a present in an isolate from Canada. The contiguous repC and repABC modules present in pMBA9a are, however, separated by an IS-element in the transmissible plasmid pSmeLPU88b. It seems to be a common feature the presence of more than one type of replication system in the S. meliloti cryptic plasmids as it has also been observed in pSmeLPU88b, pMBA9a, and in the recently sequenced pSmeSM11a. By cloning each replication system in suicide plasmids we demonstrated that any of them can support plasmid replication in the S. meliloti genetic background. We do not know yet which replication system determines the plasmid copy number of pSmeLPU88b in the host rhizobia. While within the intergenic region repB-repC a consensus sequence to promoters of regulatoryctRNAs was identified, no consensus promoter sequence could be identified upstream the type A-related repC replicase as previously reported for the related plasmid plasmid pRmeGR4a.