Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2.
Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2.
复制标题
广泛宿主范围质粒 RK2 的最小质粒稳定系统的定义。
DOI：
10.1128/jb.174.24.8119-8132.1992
复制
发表时间：
1992
影响因子：
3.2
通讯作者：
Helinski,DR
中科院分区：
文献类型：
--
作者：
Roberts,RC;Helinski,DR
宽宿主范围质粒RK2的稳定遗传至少部分是由于位于坐标32.8至35.9 kb的区域内的功能，称为RK2位点。该位点在两个操作子中编码四个先前发现的基因（parCBA和parD； M. Gerlitz， O. Hrabak, and H. Schwab， J. Bacteriol. 172:6194-6203, 1990； R. C. Roberts， R. Burioni, and D. R. Helinski， J. Bacteriol. 172:6204-6216, 1990）。parba操纵子在将质粒多聚体分解为单体方面起作用。然而，对该区域缺失的质粒稳定能力的分析表明，该多分辨操纵子仅在某些大肠杆菌菌株和特定生长条件下才需要稳定。缺失分析进一步允许重新定义最小功能区域，长度为790 bp，由parD基因（243 bp）及其启动子以及parD下游序列组成。这个最小区域稳定了几种不同革兰氏阴性细菌中rk2衍生的微型复制子，至少在大肠杆菌中，以载体无关的方式稳定了rk2衍生的微型复制子。通过插入诱变，发现parD基因和下游（3’）区域都是质粒稳定所必需的。下游DNA序列包含一个开放阅读框，随后通过转录和翻译融合显示编码一个预测大小为11,698 Da的蛋白质，称为ParE。由于parDE操纵子需要parba操纵子的存在才能在某些生长条件下有效稳定，因此通过将ColE1位点替换为parba操纵子来测试多时间分辨率在质粒稳定中的潜在作用。虽然cer位点确实具有分解质粒多聚体的功能，但在需要parCBA操纵子来维持质粒稳定性的生长条件下，它不足以恢复parDE操纵子的稳定活性。这表明RK2 par位点的质粒稳定依赖于一个复杂的机制，代表了一个多方面的稳定系统，其中多时间分辨率是有条件地不可或缺的组成部分，并且parDE操纵子编码的功能是必不可少的。
The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, O. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vector-independent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColE1 cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.