The replication origin of a repABC plasmid

• Main Authors: Cevallos Miguel A, Pérez-Segura Gabriela, Pedraza-López Francisco, Cervantes-Rivera Ramón
• Format: Article
• Language: English
• Published: BMC 2011-06-01
• Series: BMC Microbiology
• Online Access: http://www.biomedcentral.com/1471-2180/11/158

<p>Abstract</p> <p>Background</p> <p><it>repABC </it>operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: <it>repA</it>, <it>repB</it>, and <it>repC</it>. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the <it>repB-repC </it>genes modulates <it>repC </it>expression.</p> <p>Results</p> <p>To identify the minimal region of the <it>Rhizobium etli </it>p42d plasmid that is capable of autonomous replication, we amplified different regions of the <it>repABC </it>operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into <it>R. etli </it>strains that did or did not contain p42d. The minimal replicon consisted of a <it>repC </it>open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of <it>repC </it>revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into <it>R. etli </it>strain containing p42d, but similar constructs that carried <it>repC </it>from <it>Sinorhizobium meliloti </it>pSymA or the linear chromosome of <it>Agrobacterium tumefaciens </it>replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d.</p> <p>Conclusions</p> <p>RepC is the only element encoded in the <it>repABC </it>operon of the <it>R. etli </it>p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The <it>oriV </it>of this plasmid resides within the <it>repC </it>gene and is located close to or inside of a large A+T region. RepC can act as an incompatibility factor, and the last 39 amino acid residues of the carboxy-terminal region of this protein are involved in promoting this phenotype.</p>