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

Abstract

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.

Analysis of ParB-centromere interactions by multiplex SPR imaging reveals specific patterns for binding ParB in six centromeres of Burkholderiales chromosomes and plasmids

“…Thus, the binding of parS to ParB, crucial for the segregation process, looks also essential for discriminating between replicons in the multi-chromosomal genomes of the Burkholderiales. The same conclusion was drawn recently for the multipartite Rhizobium leguminosarum genome [29]. …”

RepB C-terminus mutation of an ori pRi vector affects plasmid copy number inAgrobacteriumand transgene copy number in plants

“…The increased copy number of the oriRi with the RepB Y299H mutation in Agrobacterium may result from decreased RepB Y299H binding activity to the inverted repeat (IR) in RepD [26]. The C-terminus of RepB from Rhizobium leguminosarum is responsible for RepB dimerization/oligomerization [19], and is conserved among the repABC family (Figure 2). However, whether the RepB Y299H mutation in our oriRi binary vector affects dimerization has not been experimentally determined.…”

“…However, whether the RepB Y299H mutation in our oriRi binary vector affects dimerization has not been experimentally determined. It is less likely that the RepB Y299H mutation reduces RepB interaction with RepA because the N-terminus of RepB is required for RepA interaction [19]. The repABC promoter 4 (P4) in Agrobacterium is repressed by RepA, whose activity is strongly enhanced by RepB [27].…”

“…RepB can form dimers and oligomers in solution. The middle part of RepB is responsible for parS binding and the C-terminus determines RepB dimerization/oligomerization [19]. The parS binding site identified in the plasmid pRiA4b is 28 bp downstream from the end of the repC ORF [20].…”

The specificity of ParR binding determines the compatibility of conjugative plasmids inClostridium perfringens

“…Likewise, Rhizobium leguminosarum bv. trifolii RepB (ParB homologue) proteins discriminate between similar parS centromeres to independently segregate and maintain a chromosome in addition to four plasmids (50). Unlike B. cenopacia and R. leguminosarum , where the selection pressure to maintain multiple chromosomes and plasmids seems to have driven the coevolution of separate partition specificities, the selective pressure that has resulted in the generation of so many parMRC alleles in these conjugative C. perfringens plasmids remains unclear.…”