Identification and distribution of plasmid-type A replicator region in rhizobia

Sinorhizobium meliloti strain GR4 harbors two cryptic plasmids, named pRmeGR4a and pRmeGR4b in addition to the symbiotic megaplasmids. The replicator region of plasmid pRmeGR4a has been recently cloned and sequenced. By DNA hybridization, homology to former replicator region was found on plasmid pRmeGR4b as well as on other plasmids harbored by S. meliloti and S. fredii strains. In these former bacteria, a PCR product of 362 bp was generated using pRmeGR4a-repC derived primers (C1 and C2). DNA sequence analysis showed that the amplified repC fragments were closely related being classified into two subgroups designated A(I) and A(II). Similarly, a PCR product of 482 bp was obtained when primers (C3 and C5) derived from pRmeGR4a repC-upstream non-coding DNA region (IR) were used. DNA sequence analysis of the corresponding amplified products showed that, as occurs, with repC the IRs were also conserved. In addition, we designed a set of primers (P2/P4), derived from the S. meliloti and S. fredii consensus sequence encompassing the IRs and repC loci, that are able to recognize homologous plasmid-type A replicator regions in Rhizobium. By using these primers, we determined that the former replicator region is widespread in S. meliloti indigenous populations and that its frequency within the infective isolates depends on the host plant. Furthermore, it is shown that the replicator region type A is linked to cryptic plasmids in S. meliloti and S. fredii, whereas it is located in the pSym of R. tropici strains.