Isolation and characterization of Dtr elements from a mobilizable cryptic plasmid of the alfalfa symbiont Sinorhizobium meliloti

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

California, USA

Congreso; International Symposium of Plasmid Biology; 2006

Rhizobia are Gram-negative bacteria with ability to fix atmospheric N2 in symbiotic association with the root of legumes. Such associative life style of rhizobia alternates with free-living periods of the bacteria in the underground. Thus, rhizobia became an attractive model to investigate the biology andevolutionary genomics of symbiotic soil bacteria. A remarkable characteristic of rhizobia is that, in many cases, they carry their relevant symbiotic information in megaplasmids. Several other traits not yet extensively characterized are frequently encoded in accompanying cryptic replicons of variable size. In our laboratory we constructed in the last years a collectionof Sinorhizobium meliloti isolates (the symbiont of alfalfa) which were initially characterized in their plasmid content and diversity. A consistent search for transmissible replicons allowed us for the identification of several plasmids that were either conjugative, or mobilizable. A model binary system conformed by two plasmids, one of them mobilizable (pSmeLPU88b) and the other carrying compatible helper functions (pSmeLPU88a), was selected for further investigation at the functional and molecular level (Pistorio et al., 2003). The ability of representative strains from thecollection to mobilize pSmeLPU88b was evaluated, and the result compared with the ability of the same strains to transfer any of their own plasmids. A promiscuous conjugative activity wasevident with only two groups of mobilization. With the aim of investigating the structure of the Dtr region present in the model plasmid pSmeLPU88b we constructed a partial library and carryout a shot-gun sequencing approach. DNA elements associated to the replication (see communication by Pistorio et al.) and mobilization could be identified. By plasmid walking we completed the sequence of a putative nickase related to homologous conjugal proteins present in the strain Mesorhizobium loti MAFF303099, and in the strain M. loti BNC1. The use of consensus primers in a PCR assay resulted in the detection of similar sequences in many members of our strain collection. As expected, site directed mutagenesis of the putative nickase in strain LPU88 abolished the mobilization of pSmeLPU88b by its accompanying helper plasmid.The putative nickase do not belong to any of the groups previously proposed by Francia et al. (2004). Nevertheless, the central region of the protein presented a motif III-like sequence,with partial sequence similarity to those present in the MOBP and MOBQ superfamily of nickases. Though immediately upstream the putative nickase there was a mobC homolog as reported for some members of the ColE1 superfamily, the protein that we identified did not show significant sequence similarity with such family of relaxases. In addition, upstream the mobC a putative oriT was identified followed by a divergent parA homolog as in the parA-mobC region of plasmid pRmeGR4. Analysis of the parA-mobC region of strains LPU88, BNC1, and GR4 showed a highly conserved stretch of 62 bp close to mobC (84% identity), with the maximal sequence divergence within 4 bp that possible include the nick site as predicted by pairwise comparison against the homologue sequence from plasmid pTiC58. We are currently working in the functional characterization of the nickase, and in the analysis of parA-(oriT)-mobC intergenic sequences present in different S. meliloti plasmids from our strain collection and from other geographic origins.