Kusano K, Nakayama K, Nakayama H
Department of Microbiology, School of Dentistry, Kyushu University, Fukuoka, Japan.
J Mol Biol. 1989 Oct 20;209(4):623-34. doi: 10.1016/0022-2836(89)90000-4.
Apparent plasmid instability, i.e. progressive plasmid loss in a bacterial culture growing in the absence of selection for the plasmid, in an Escherichia coli recBC sbcBC mutant was investigated with two different ColE1 derivatives (pMB9 and pBR322) and a mini-F plasmid. The instability was most striking for pMB9 and much less, but still significant, for pBR322 and the mini-F. It was also dependent upon a subset of the genes involved in the RecF recombination pathway: in addition to the previously reported recA, recF and recJ mutations, a recO and a recQ mutation showed a total and a partial suppression, respectively, of the instability. Other recF-family mutations, recN and ruv, were without such an effect. Population analyses of the recBC sbcBC strain carrying pMB9 or the mini-F, as carried out by plating and Coulter counting, revealed marked loss of viability in plasmid-carrying cells, strongly implicating plasmid-mediated cell death in the apparent defect in plasmid maintenance. Analysis of intracellular plasmid DNA by pulsed-field gel electrophoresis combined with the in-agarose cell lysis technique showed that the instability was associated with the formation of plasmid multimers, with a good correlation between the degree of the instability and the amount of the multimers. The multimer formation was also dependent on the same subset of the RecF pathway genes as in the instability phenomenon. These results strongly suggest that the lethality is somehow caused by the multimer formation. Various DNase treatments of cell lysates showed that such multimers of pMB9 DNA comprised molecules of exonuclease-sensitive and exonuclease-resistant types. It was inferred that the former class, which showed electrophoretic mobilities corresponding to plain linear duplexes of approximately 200 x 10(3) to 2200 x 10(3) base-pairs, represented linear multimers possibly carrying circular structures at one end. The latter class, which remained in the origin, was thought to consist of circular multimers and/or linear multimers protected by circular structures at both ends against exonucleolytic attack.
利用两种不同的ColE1衍生物（pMB9和pBR322）以及一个微型F质粒，对大肠杆菌recBC sbcBC突变体中明显的质粒不稳定性进行了研究，即在没有对质粒进行选择的情况下，细菌培养物中质粒的逐渐丢失。pMB9的不稳定性最为显著，pBR322和微型F质粒的不稳定性则小得多，但仍然很明显。它还取决于RecF重组途径中涉及的一部分基因：除了先前报道的recA、recF和recJ突变外，recO和recQ突变分别完全和部分抑制了不稳定性。其他recF家族突变，recN和ruv，没有这种作用。通过平板接种和库尔特计数对携带pMB9或微型F的recBC sbcBC菌株进行群体分析，结果显示携带质粒的细胞活力明显丧失，这强烈表明质粒介导的细胞死亡与质粒维持方面的明显缺陷有关。通过脉冲场凝胶电泳结合琼脂糖内细胞裂解技术对细胞内质粒DNA进行分析，结果表明不稳定性与质粒多聚体的形成有关，不稳定性程度与多聚体数量之间具有良好的相关性。多聚体的形成也取决于与不稳定性现象中相同的RecF途径基因子集。这些结果有力地表明，致死性在某种程度上是由多聚体的形成引起的。对细胞裂解物进行的各种DNA酶处理表明，pMB9 DNA的此类多聚体包括核酸外切酶敏感型和核酸外切酶抗性型分子。据推测，前者电泳迁移率对应于约200×10³至2200×10³碱基对的普通线性双链体，代表可能一端带有环状结构的线性多聚体。后者留在原点，被认为由环状多聚体和/或两端受环状结构保护免受核酸外切酶攻击的线性多聚体组成。
