The six plasmids of Rhizobium leguminosarum VF39SM comprise nearly 35% of the bacterium's genome and are all repABC replicons. The repABC operons of the three largest plasmids of VF39SM were found to have strong incompatibility determinants in the non-protein coding regions. However, in all three repABC operons, the intergenic region between repB and repC was the strongest incompatibility factor; this intergenic region has been shown, for most repABC plasmids, to encode a counter-transcribed RNA (ctRNA) that regulates RepC abundance and therefore also rate of initiation of replication. To understand the way in which the ctRNA regulates replication and incompatibility, we carried out mutagenesis on this region from all three plasmids, using error-prone PCR. Mutants with altered incompatibility were detected by screening for their ability to co-exist in the same cell as the parent plasmid. Mutations that abolished the strong incompatibility phenotype were nearly all localized to the predicted ctRNA promoter regions. RT-PCR analysis confirmed that ctRNA was still produced in these promoter mutants, but transcriptional fusions of these mutated promoters to a gusA reporter gene showed a 10- to 50-fold decrease in activity when compared with the wild type promoter. For the repABC operons in this study, the intergenic region is critical in establishing incompatibility, and this appears to require a high level of transcription of the ctRNA.
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