Study of stability genes of E. faecalis plasmid pAD1

粪肠球菌是一种革兰氏阳性菌，是肠道正常菌群中的一员。 肠道。它还能够引起机会性感染，并已获得 最近因为有快速积累抗生素耐药性基因的倾向而臭名昭著。 这些基因通常位于可移动的遗传元件上，有助于它们的传播 对其他细菌物种的影响。这项提议的目标是确定分子 稳定遗传所需的遗传位点PAR的作用机制 信息素反应结合质粒pad1。PAR通过后种族隔离在杀戮中运作 机制，对任何失去质粒的细胞进行死亡编程。PAR由唯一的 涉及通过其消息与An相互作用来抑制毒素翻译的机制 反义RNA在多个分散的位置形成稳定的复合体。的目标是 这项建议的目标1是确定激活该复合体的机制 在无质粒分离物中的翻译。这将涉及对PAR的转录分析 RNAs，识别参与复杂降解的序列元件和RNase，以及 对毒素基因稳定性所需的序列元件进行评估。PAR的表达 毒素Fst破坏宿主细胞膜的完整性。这个目标2的目标是 建议确定FST的作用机制。FST函数的两个模型，一个 涉及细胞蛋白酶的加工，以及涉及内部选择性作用的加工 细胞质膜的表面，将被检测。此外，将确定Fst抗性突变体的特性，以努力确定Fst的特定靶点。 最后，将对Fst进行饱和突变，以努力确定哪种氨基 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.