title: Counter-transcribed RNAs of Rhizobium leguminosarum repABC plasmids exert incompatibility effects only when highly expressed.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4.
authors.forename: Cynthia B
authors.initials: CB
authors.lastname: Yip
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4.
authors.forename: Hao
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4. Electronic address: hynes@ucalgary.ca.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Hynes
mesh_terms: D021901:DNA, Intergenic; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D009876:Operon; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense; D016961:Rhizobium leguminosarum / Q000235:genetics*
chemical_list: D021901:DNA, Intergenic; D016372:RNA, Antisense
keywords: Incompatibility; Plasmid; Replication; Rhizobium; ctRNA; repABC