Loftie-Eaton Wesley, Rawlings Douglas E
Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa.
J Bacteriol. 2009 Oct;191(20):6436-46. doi: 10.1128/JB.00864-09. Epub 2009 Aug 14.
Plasmids pRAS3.1 and pRAS3.2 are two closely related, natural variants of the IncQ-2 plasmid family that have identical plasmid backbones except for two differences. Plasmid pRAS3.1 has five 6-bp repeat sequences in the promoter region of the mobB gene and four 22-bp iterons in its oriV region, whereas pRAS3.2 has only four 6-bp repeats and three 22-bp iterons. Plasmid pRAS3.1 was found to have a higher copy number than pRAS3.2, and we show that the extra 6-bp repeat results in an increase in mobB and downstream mobA/repB expression. Placement of repB (primase) behind an arabinose-inducible promoter in trans resulted in an increase in repB expression and an approximately twofold increase in the copy number of plasmids with identical numbers of 22-bp iterons. The pRAS3 plasmids were shown to have a previously unrecognized toxin-antitoxin plasmid stability module within their replicons. The ability of the pRAS3 plasmids to mobilize the oriT regions of two other plasmids of the IncQ-2 family, pTF-FC2 and pTC-F14, suggested that the mobilization proteins pRAS3 are relaxed and can mobilize oriT regions with substantially different sequences. Plasmids pRAS3.1 and pRAS3.2 were highly incompatible with plasmids pTF-FC2 and pTC-F14, and this incompatibility was removed on inactivation of an open reading frame situated downstream of the mobCDE mobilization genes rather than being due to the 22-bp oriV-associated iterons. We propose that the pRAS3 plasmids represent a third, gamma incompatibility group within the IncQ-2 family plasmids.
质粒pRAS3.1和pRAS3.2是IncQ - 2质粒家族两个密切相关的天然变体，除了两个差异外，它们具有相同的质粒骨架。质粒pRAS3.1在mobB基因的启动子区域有五个6碱基对的重复序列，在其oriV区域有四个22碱基对的迭代子，而pRAS3.2只有四个6碱基对的重复序列和三个22碱基对的迭代子。发现质粒pRAS3.1的拷贝数高于pRAS3.2，并且我们表明额外的6碱基对重复导致mobB及其下游mobA/repB表达增加。将repB（引发酶）置于反式阿拉伯糖诱导型启动子之后导致repB表达增加，并且对于具有相同数量22碱基对迭代子的质粒，其拷贝数增加约两倍。pRAS3质粒在其复制子内显示具有一个先前未被识别的毒素 - 抗毒素质粒稳定模块。pRAS3质粒能够动员IncQ - 2家族的另外两个质粒pTF - FC2和pTC - F14的oriT区域，这表明pRAS3的动员蛋白较为宽松，能够动员具有显著不同序列的oriT区域。质粒pRAS3.1和pRAS3.2与质粒pTF - FC2和pTC - F14高度不相容，并且这种不相容性在位于mobCDE动员基因下游的一个开放阅读框失活后消除，而不是由于与22碱基对oriV相关的迭代子。我们提出pRAS3质粒代表IncQ - 2家族质粒中的第三个γ不相容组。
