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

PISTORIO M., GIUSTI M.Á., LOZANO M., TORRES TEJERIZO, G., DRAGHI W., Y LAGARES A.

Fallen Leaf Lake, South Lake Tahoe, USA

Simposio; Plasmid Biology 2006; 2006

International Society for Plasmid Biology and other Mobile Genetic Elements

Gram negative bacteria that belong to the genera Rhizobium,Sinorhizobium, Azorhizobium, and Mesorhizobium grow in the soilin free-living conditions, and in symbiosis associated with theroot of legumes as nitrogen-fixing organisms. Current evidenceshows that rhizobia carry in most cases variable number ofplasmids related to their symbiosis and free-living, being a commonfeature the presence of several plasmid replicons within asame strain. Thus, several rhizobial plasmids have been characterizedin their replication machinery. In S. meliloti, the symbiontof alfalfa, plasmid replication strategies and incompatibilitymechanisms have been recently analyzed (Izquierdo et al., 2005;MacLellan et al., 2005; Watson and Heys, 2006). In our laboratorywe have previously described the transmissibility propertiesof two cryptic plasmids from the strain S. meliloti LPU88, a localisolate from Argentina. One of them, pSmeLPU88b (approx. 40kb), resulted to be mobilizable if helper functions were supplied intrans by an accompanying plasmid pSmeLPU88a (binary conjugalsystem) (Pistorio et al., 2003). To characterize the replicationelements of the transmissible plasmid pSmeLPU88b, aplasmid library was constructed and sequenced using a shotgunstrategy. From the collected sequence data two plasmid replicationmodules were identified in silico: one belonging to therepABC type, and the other related to the type A family ofplasmid replication regions. Both regions resulted nearly identicalto the homologous elements from plasmid pMBA9a present in anisolate from Canada. The contiguous repC and repABC modulespresent in pMBA9a are, however, separated by an IS-element inthe transmissible plasmid pSmeLPU88b. It seems to be a commonfeature the presence of more than one type of replicationsystem in the S. meliloti cryptic plasmids as it has also been observedin pSmeLPU88b, pMBA9a, and in the recently sequencedpSmeSM11a. By cloning each replication system in suicide plasmidswe demonstrated that any of them can support plasmidreplication in the S. meliloti genetic background. We do not knowyet which replication system determines the plasmid copy numberof pSmeLPU88b in the host rhizobia. While within the intergenicregion repB-repC a consensus sequence to promoters of regulatoryctRNAs was identified, no consensus promoter sequencecould be identified upstream the type A-related repC replicase aspreviously reported for the related plasmid plasmid pRmeGR4a