Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria.
Plasmid. 2011 Jan;65(1):70-6. doi: 10.1016/j.plasmid.2010.09.002. Epub 2010 Sep 18.
pHW126, pIGRK, pIGMS31 and pRAO1 are the only known members of a novel and as yet uncharacterised family of rolling circle plasmids. pHW126 contains only two open reading frames, of which one shows homology to pMV158-family mobilisation proteins. Here we provide evidence that the second open reading frame encodes a replication protein (Rep). Mutation or deletion of this gene resulted in replication deficient constructs, but providing functional Rep from a compatible vector rescued these constructs, indicating that Rep acts in trans. An approximately 300 bp cis-acting region representing the origin of replication was identified upstream of the rep gene. The origin was identified to be composed of three parts: an accessory region, a conserved stretch and four perfect tandem repeats. The two latter elements were essential for replication. Constructs with a deletion of the accessory region could still replicate, but their loss rate was high, indicating that the accessory region is necessary for plasmid maintenance under non-selective conditions. Interestingly, pHW126 could replicate in all Enterobacteriaceae tested while Agrobacterium tumefaciens and Pseudomonas syringae were inappropriate hosts. Thus, pHW126 seems to have a rather limited host range.
pHW126、pIGRK、pIGMS31 和 pRAO1 是一种新型且尚未被描述的滚环质粒家族中唯一已知的成员。pHW126 仅包含两个开放阅读框，其中一个与 pMV158 家族的移动蛋白具有同源性。在这里，我们提供了证据表明第二个开放阅读框编码复制蛋白 (Rep)。该基因的突变或缺失导致复制缺陷的构建体，但提供来自相容载体的功能 Rep 挽救了这些构建体，表明 Rep 以反式作用。在 rep 基因上游鉴定出约 300 bp 的顺式作用区，代表复制起点。该起点由三个部分组成：辅助区、保守区和四个完美串联重复。后两个元件对于复制是必需的。缺失辅助区的构建体仍能复制，但它们的丢失率很高，表明辅助区对于非选择性条件下质粒的维持是必要的。有趣的是，pHW126 可以在所有测试的肠杆菌科中复制，而根癌农杆菌和丁香假单胞菌则不是合适的宿主。因此，pHW126 的宿主范围似乎相当有限。
