RepB proteins of the multipartite Rhizobium leguminosarum bv. trifolii genome discriminate between centromere-like parS sequences for plasmid segregational stability.

The plasmids of the Rhizobiaceae family members and other Alphaproteobacteria are usually large, low copy-number and contain all elements necessary for active segregation and replication located in one operon comprising repABC genes. The genome of Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) consists of a chromosome and four plasmids (pRleTA1a-d) with repABC operons. In this work, centromere-binding RepB proteins of four RtTA1 plasmids were studied. Stability assays of the truncated derivatives of repABC cassettes demonstrated that RepA, RepB proteins and parS-like elements constituted plasmid partitioning systems, while RepC were sufficient for their replication. Individual RepB proteins bound specifically to centromere-like parS elements of the parental plasmids, which was crucial step toward the proper segregation of plasmids into daughter cells. RtTA1 RepB proteins formed dimers and oligomers in the solution. The C-terminal part of RepB was responsible for dimerization, while the domain engaged in parS binding was located in the middle of the protein. It was concluded that the specific interaction between individual RepB proteins and their target sequences together with the substantial diversity of the Rep proteins and parS originating from different plasmids strongly contributed to the coexistence of several plasmids equipped with similar repABC cassettes in the multipartite bacterial genome.