CHARACTERIZATION OF THE oriT REGION FROM A SMALL CRYPTIC PLASMID FROM Sinorhizobium meliloti LPU88.

GIUSTI, M. A.; PISTORIO, M.; LOZANO, M. J.; TORRES TEJERIZO, G. A.; SALAS, M, E.; MARTINI, C; DEL PAPA, M.F.; PEREZ MENDOZA, D.; SANJUAN, J.; LAGARES, A.

Bariloche

Congreso; International Plasmid Biology Conference 2010; 2010

International Society for Plasmid Biology and other Mobile Genetic Elements

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) (1).In a previous work we have identified elements of the Dtr region of plasmid pSmeLPU88b, including a putative relaxase which does not belong to any of the previously described groups of similar proteins. Immediately upstream of the putative relaxase there was a mobC homolog as reported for some members of the ColE1 superfamily. Upstream mobC a putative oriT was predicted by pairwise comparison against the homologue sequence from plasmid pRmeGR4, followed by a divergent ORF homolog to parA of plasmid pRmeGR4. Analysis of the parA-mobC region of strains LPU88 showed ca. 88% DNA sequence identity to the homolog region of pRmeGR4 (2, 3).We are currently working in the analysis of the parA-mobC intergenic sequence, and in the functional characterization of the relaxase. To evaluate if between parA and mobC there is a functional oriT sequence we cloned the intergenic (INT) region and evaluated its ability to convert a non-transmissible vector into a mobilizable plasmid. As a result, we found that the INT region could promote plasmid mobilization in strain LPU88 at a similar frequency than the observed for the complete pSmeLPU88b. This observation contrasts with the behavior of other rhizobial oriT which displayed a remarkable preference for a cis-location of the relaxase (4). In order to identify the minimal sequence able to promote mobilization we deleted the INT fragment from both sides and identified a 406 bp fragment still active as a functional oriT. Recently we have successfully expressed the complete putative relaxase (MobZ) and its N-terminal amino portion (241 amino acids) that will be used now to identify the nick site within the characterized oriT.