Study of stability genes of E. faecalis plasmid pAD1

粪肠球菌是一种革兰氏阳性细菌，是肠道正常植物群的成员。 肠道它也能引起机会性感染， 最近，由于抗生素抗性基因的快速积累倾向， 这些基因通常位于便于其传播的移动的遗传元件上 其他细菌物种。本建议的目的是确定分子 作用机制的遗传位点，PAR，所需的稳定遗传的 信息素应答接合质粒pAD 1。Par通过一种后隔离的杀戮方式运作 机制，编程死亡任何细胞失去质粒。Par由一个独特的 涉及通过其信息与一种蛋白质相互作用来抑制毒素翻译的机制 反义RNA在多个分散的位点上，并形成稳定的复合物。的目标 本提案的目的1是确定复合物被激活的机制， 在无质粒分离子体中的翻译。这将涉及对par的转录分析。 RNA，鉴定参与复杂降解的序列元件和RNA酶，以及 评价毒素mRNA稳定性所需的序列元件。面值的表示 毒素Fst破坏宿主细胞膜的完整性。目标2的目标 建议是确定FST的作用机制。Fst函数的两种模型，一种 一种涉及细胞蛋白酶的加工，另一种涉及内部的选择性作用， 细胞膜的表面，将被测试。此外，Fst抗性突变体的特征将被确定，以确定Fst的特异性靶标。 最后，将对Fst进行饱和诱变，以鉴定哪个氨基酸与Fst结合。 酸是其活性所必需的。

Enterococcus faecalis is a Gram-positive bacterium and a member of the normal flora of the intestinal tract. It is also capable of causing opportunistic infection and has gained notoriety recently because of a propensity to rapidly accumulate antibiotic resistance genes. These genes are commonly located on mobile genetic elements that facilitate their dissemination to other bacterial species. The objective of this proposal is to determine the molecular mechanism of action of a genetic locus, par, required for the stable inheritance of the pheromone-responsive conjugative plasmid pAD1. Par operates via a post-segregationat killing mechanism, programming for death any cell that loses the plasmid. Par is regulated by a unique mechanism involving inhibition of toxin translation by interaction of its message with an antisense RNA at multiple dispersed sites and the formation of a stable complex. The goal of Aim 1 of this proposal is to determine the mechanism by which the complex is activated for translation in plasmid-free segregants. This will involve transcriptional analysis of the par RNAs, identification of sequence elements and RNases involved in complex degradation, and evaluation of sequence elements required for stability of the toxin mRNA. Expression of the par toxin, Fst, disrupts the integrity of the host cell membrane. The goal of Aim 2 of this proposal is to determine the mechanism of action of Fst. Two models of Fst function, one involving processing by cellular proteases and one involving selective action at the internal surface of the cytoplasmic membrane, will be tesed. In addition, the characteristics of Fst-resistant mutants will be determined in an effort to identify the specific target of Fst. Finally, saturation mutagenesis will be performed on Fst in an effort to identify which amino acids are essential for its activity.