Diversity of repC plasmid-replication sequences in Rhizobium leguminosarum

Homologues of the plasmid replicator gene repC were detected and characterized in a sample of Rhizobium leguminosarum strains. Conserved PCR primers were designed from published sequences of repC; they amplified a fragment of about 750 bp from 39 out of 41 strains tested, and also from several Sinorhizobium strains, including S. meliloti. Restriction endonuclease digestion showed that the PCR product from individual strains, though uniform in size, was often heterogeneous in sequence. PCR products from 24 field isolates of R. leguminosarum from France, Germany and the UK were cloned and partially sequenced from both ends. Phylogenies constructed from the 5' and 3' ends (200 bp each) were largely congruent and demonstrated four clearly defined groups plus several unique strains. Published Agrobacterium repC sequences fall within the phylogeny of R. leguminosarum sequences, though not within any of the four groups. Specific pairs of PCR primers were designed for each of the four groups; 29 out of 41 R. leguminosarum strains gave a PCR product of the expected size with more than one group-specific primer pair. We hypothesize that the sequence groups correspond to incompatibility groups of Rhizobium plasmids.