SEX PHEROMONE INDUCED PLASMID TRANSFER

粪肠球菌是一种重要的机会致病菌， 是引起菌血症的第二大原因， 心内膜炎编码交配反应的接合质粒 肽类性信息素在E.粪便，可能 对抗生素耐药性的传播起着重要作用 和该物种中的细胞溶解素（例如溶血素/细菌素）产生。 编码溶细胞素的质粒pAD 1是这种质粒的一个实例， 一直是PI实验室严格审查的对象， 好几年了 最近发现的关键调节基因和 测定它们的核苷酸序列，沿着与 生理学研究，使制定一个工作， 假设来解释可能被使用的电路， 诱导缀合反应。 该模型的一个关键方面 涉及通过负调节子控制traE 1的表达 由traA编码，影响转录通读， 终止位点TTS 1/TTS 2。 拟议研究的大部分内容旨在 以测试该模型并进一步表征相关过程。 更 具体而言，拟议的研究将：1）确定 iad启动子和TTS 1之间发生的转录事件， 强调检查记录之间的关系， 被指定为m3，m4和m5; 2）检查TraA在以下方面的作用： 调节转录超过TTS 1/TTS 2并进入traE 1决定簇 包括确定TraA是否直接结合cAD 1 肽; 3）检查其他因素（即TraA以外）， 在TTS 1/TTS 2处并影响转录通读; 4）确定是否 TraE 1在其自身的调节中起作用，通过控制 转录指定为m3 '; 5）检查关闭的动力学， 信息素反应; 6）表征Dry+/Dryc阶段的基础 变化机制，有助于绕过生理 7）确定TraA、TraE 1、RepA、RepB或其他 蛋白质相互作用; 8）继续我们的努力，克隆 并表征cad，cAD 1的染色体决定簇。

Enterococcus faecalis is an important opportunistic pathogen that is now the second leading cause of bacteremia and third leading cause of endocarditis in humans. Conjugative plasmids encoding a mating response to peptide sex pheromones are ubiquitous in E. faecalis and probably contribute significantly to the dissemination of antibiotic resistance and cytolysin (e.g. hemolysin/bacteriocin) production in this species. The cytolysin-encoding plasmid pAD1 is an example of such a plasmid and has been a subject of intense scrutiny in the laboratory of the PI for a number of years. Recent identification of key regulatory genes and determination of their nucleotide sequence, along with related physiological studies, has enabled the formulation of a working hypothesis to explain the circuitry which may be utilized during induction of the conjugation response. A key aspect of the model concerns the control of expression of traE1 by a negative regulator encoded by traA that influences transcriptional readthrough of the termination site TTS1/TTS2. The bulk of the proposed study is designed to test this model and further characterize related processes. More specifically the proposed studies will: 1) determine the nature of transcription events that occur between the iad promoter and TTS1 with an emphasis on examining the relationship between transcripts that have been designated m3, m4, and m5; 2) examine the role TraA plays in regulating transcription beyond TTS1/TTS2 and into the traE1 determinant including a determination of whether TraA directly binds to the cAD1 peptide; 3) examine additional factors (i.e. other than TraA) operating at TTS1/TTS2 and affecting transcriptional readthrough; 4) determine if TraE1 plays a role in its own regulation by controlling initiation of the transcript designated m3'; 5) examine the kinetics of shutdown of the pheromone response; 6) characterize the basis of the Dry+/Dryc phase variation mechanism that facilitates a bypass of the physiological response to cAD1; 7) determine if TraA, TraE1, RepA, RepB or other proteins interact with each other; and 8) continue our efforts to clone and characterize cad, the chromosomal determinant for cAD1.