The large stable plasmids of rhizobia play an important role in survival, environmental competition, and plant symbiosis. The six plasmids of Rhizobium leguminosarum bv. viciae VF39SM comprise nearly 35% of the bacterium’s genome and are all repABC replicons, which are defined by the presence of a highly unusual operon containing both replication (repC) and partitioning functions (repA and repB) under the control of a single promoter.
The six repABC operons were isolated and were found to confer the autonomous replication, incompatibility characteristics, and strong stability of the large repABC plasmid from which they were derived. The repABC operons of the three largest plasmids were found to have strong incompatibility determinants in the non-protein coding regions, and the location and number of these determinants were unique to each operon. The varying incompatibility determinants were predicted to be the centromere-like site of the plasmid, based on the presence of a 16 nucleotide consensus palindromic sequence. However, in all three repABC operons, the intergenic region between repB and repC was the strongest incompatibility factor, and is predicted to encode a counter-transcribed RNA (ctRNA). Mutations in the promoter region of the ctRNAs result in loss of incompatibility.
Deletion of the repA and repB genes of the repABC operons of the three largest plasmids abolished replicon stability, whereas deletion of the intergenic region increased the stability significantly, such that the intergenic deletion replicons were more stable than the entire repABC operon. Depending on the rhizobial host, the intergenic deletion replicon had increased stability due either to an increase in copy number (Agrobacterium
tumefaciens host), or a cointegration event with the chromosome or a resident plasmid (A. tumefaciens or R. leguminosarum host).
Genomic rearrangements are common in the rhizobial species in this study, but appear to be especially prevalent in R. leguminosarum VF39SM. Cointegration events to mitigate plasmid incompatibility or loss of plasmid stability are common and result in consistent and reproducible patterns, even in a RecA-deficient background.