Wu F, Levchenko I, Filutowicz M
Department of Bacteriology, University of Wisconsin, Madison 53706, USA.
J Bacteriol. 1995 Nov;177(22):6338-45. doi: 10.1128/jb.177.22.6338-6345.1995.
The plasmid R6K gamma origin consists of two adjacent modules, the enhancer and the core, and requires R6K initiator protein pi for replication. While the core alone can replicate at a low level of wild-type pi protein, we show here that host cells do not stably maintain core plasmids. The presence of the enhancer segment confers stable inheritance on core plasmids without a significant change in average plasmid copy number. Deletions and site-directed mutagenesis indicated that the stability of core plasmids is not mediated by binding sites or consensus sequences in the enhancer for DnaA, pi protein, gyrase, Fis, or Dcm methylase. Proper segregation of core plasmids requires only the R6K stb or stability-related region, which includes the 20-bp segment of the 100-bp enhancer adjacent to the core. The use of the pi 116 mutant protein, which increases plasmid copy number fourfold, does not stabilize core plasmids lacking the enhancer. We also show that at an elevated level of wild-type pi, the gamma-origin plasmid is unstable, even in the presence of the enhancer. We discuss the differences and similarities between the R6K stability system and those found in other plasmids.
质粒R6Kγ 复制起点由两个相邻模块组成，即增强子和核心区域，其复制需要R6K起始蛋白π。虽然仅核心区域在野生型π蛋白水平较低时就能进行低水平复制，但我们在此表明宿主细胞不能稳定维持核心质粒。增强子片段的存在赋予核心质粒稳定的遗传特性，而平均质粒拷贝数没有显著变化。缺失和定点诱变表明，核心质粒的稳定性不是由增强子中DnaA、π蛋白、促旋酶、Fis或Dcm甲基化酶的结合位点或共有序列介导的。核心质粒的正确分离仅需要R6K stb或稳定性相关区域，该区域包括与核心相邻的100 bp增强子的20 bp片段。使用使质粒拷贝数增加四倍的π 116突变蛋白，并不能使缺乏增强子的核心质粒稳定。我们还表明，在野生型π水平升高时，即使存在增强子，γ复制起点质粒也是不稳定的。我们讨论了R6K稳定性系统与其他质粒中发现的稳定性系统之间的异同。
