SEX PHEROMONE INDUCED PLASMID TRANSFER

粪肠球菌是一种重要的条件致病菌。 细菌血症的第二大原因和第三大原因 人类的心内膜炎。编码交配反应的接合质粒 多肽性信息素在粪肠球菌中普遍存在，可能 极大地促进了抗生素耐药性的传播 和细胞溶素(例如，溶血素/细菌素)在该物种中的产生。 细胞溶素编码质粒PAD1是这种质粒的例子，并且 一直是国际刑警组织实验室严密审查的对象 好几年了。最近发现的关键调控基因和 它们的核苷酸序列的测定，以及相关的 生理学研究，使制定一项工作成为可能 一种假说，用来解释在 诱导接合反应。该模型的一个关键方面 涉及负调控因子对trae1表达的调控 由TraA编码，影响转录通读 终端站点TTS1/TTS2。拟议研究的大部分内容都是经过设计的 以检验这一模型，并进一步表征相关过程。更多 具体而言，拟议的研究将：1)确定 IAD启动子和TTS1之间发生的转录事件 重点考察具有以下特征的成绩单之间的关系 被指定为M3、M4和M5；2)检查TraA在 调节转录超出TTS1/TTS2并进入TraE1决定簇 包括确定TraA是否直接与cAD1结合 多肽；3)检查其他因素(即，除TRAA以外)的作用 在TTS1/TTS2并影响转录通读；4)确定是否 TraE1在其自身的调节中发挥作用，它通过控制 标记为M3‘的文本；5)检查关闭的动力学 信息素反应；6)表征了干+/干湿相的基础 有助于生理学旁路的变异机制 对cAD1的响应；7)确定TraA、TraE1、REPA、RepB或其他 蛋白质相互作用；8)继续努力克隆 并对cad1的染色体决定因素cad进行了表征。

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.