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

GIUSTI M.Á., PISTORIO M., 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

Rhizobia are Gram-negative bacteria with ability to fix atmosphericN2 in symbiotic association with the root of legumes.Such associative life style of rhizobia alternates with free-livingperiods of the bacteria in the underground. Thus, rhizobiabecame an attractive model to investigate the biology and evolutionary genomics of symbiotic soil bacteria. A remarkablecharacteristic of rhizobia is that, in many cases, they carry theirrelevant symbiotic information in megaplasmids. Several othertraits not yet extensively characterized are frequently encoded inaccompanying cryptic replicons of variable size.In our laboratory we constructed in the last years a collectionof Sinorhizobium meliloti isolates (the symbiont of alfalfa) whichwere initially characterized in their plasmid content and diversity.A consistent search for transmissible replicons allowed us for theidentification of several plasmids that were either conjugative, ormobilizable. A model binary system conformed by two plasmids,one of them mobilizable (pSmeLPU88b) and the other carryingcompatible helper functions (pSmeLPU88a), was selected forfurther investigation at the functional and molecular level (Pistorioet al., 2003). The ability of representative strains from thecollection to mobilize pSmeLPU88b was evaluated, and the resultcompared with the ability of the same strains to transfer any oftheir own plasmids. A promiscuous conjugative activity wasevident with only two groups of mobilization. With the aim ofinvestigating the structure of the Dtr region present in the modelplasmid pSmeLPU88b we constructed a partial library and carryout a shot-gun sequencing approach. DNA elements associatedto the replication (see communication by Pistorio et al.) andmobilization could be identified. By plasmid walking we completedthe sequence of a putative nickase related to homologousconjugal proteins present in the strain Mesorhizobium lotiMAFF303099, and in the strain M. loti BNC1. The use of consensusprimers in a PCR assay resulted in the detection of similarsequences 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 accompanyinghelper plasmid.The putative nickase do not belong to any of the groups previouslyproposed by Francia et al. (2004). Nevertheless, thecentral region of the protein presented a motif III-like sequence,with partial sequence similarity to those present in the MOBP andMOBQ superfamily of nickases. Though immediately upstreamthe putative nickase there was a mobC homolog as reported forsome members of the ColE1 superfamily, the protein that weidentified did not show significant sequence similarity with suchfamily of relaxases. In addition, upstream the mobC a putativeoriT was identified followed by a divergent parA homolog as inthe parA-mobC region of plasmid pRmeGR4. Analysis of theparA-mobC region of strains LPU88, BNC1, and GR4 showed ahighly conserved stretch of 62 bp close to mobC (84% identity),with the maximal sequence divergence within 4 bp that possibleinclude the nick site as predicted by pairwise comparison againstthe homologue sequence from plasmid pTiC58. We are currentlyworking in the functional characterization of the nickase, and inthe analysis of parA-(oriT)-mobC intergenic sequences present indifferent S. meliloti plasmids from our strain collection and fromother geographic origins.