Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apartado Postal 565-A, Cuernavaca, Morelos, México.
BMC Microbiol. 2011 Jun 30;11:158. doi: 10.1186/1471-2180-11-158.
repABC 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: repA, repB, and repC. 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 repB-repC genes modulates repC expression.
To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC 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 R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens 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.
RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The oriV of this plasmid resides within the repC 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.
repABC 操纵子存在于至少 19 种α变形菌属的大型低拷贝数质粒和一些次级染色体上，负责这些复制子的复制和分离特性。这些操纵子由三个基因组成：repA、repB 和 repC。RepA 和 RepB 参与质粒分配和自身转录的负调控，RepC 是复制的限制因素。编码在 repB-repC 基因之间的反义 RNA 调节 repC 的表达。
为了鉴定 Rhizobium etli p42d 质粒能够自主复制的最小区域，我们使用 PCR 扩增了 repABC 操纵子的不同区域，并将这些区域克隆到自杀载体中。然后将这些载体引入含有或不含有 p42d 的 R.etli 菌株中。最小复制子由一个受组成型启动子控制的 repC 开放阅读框组成，启动子带有我们设计的 Shine-Dalgarno 序列。RepC 的序列分析表明，存在一个富含 A+T 的区域，但没有回文序列或 DnaA 盒。修饰该区域 A+T 含量的沉默突变消除了质粒的复制能力。最小复制子不能引入含有 p42d 的 R.etli 菌株，但携带 Sinorhizobium meliloti pSymA 或 Agrobacterium tumefaciens 线性染色体 repC 的类似构建体在含有或不含有 p42d 的情况下都能复制，表明 RepC 是一种不相容性因子。表达来自 p42d RepC 的前 362 个氨基酸残基和来自 SymA 的 RepC 的最后 39 个氨基酸残基的 RepC 杂种基因构建体能够在 p42d 的存在下复制。
RepC 是 R.etli p42d 质粒 repABC 操纵子中唯一编码的、对质粒复制必不可少且充分的元件，可能是起始蛋白。该质粒的 oriV 位于 repC 基因内，位于富含 A+T 的大片段附近或内部。RepC 可以作为一种不相容性因子，并且该蛋白羧基末端区域的最后 39 个氨基酸残基参与促进这种表型。
