SRF OPERON--REGULATION AND ROLE IN GENETIC COMPETENCE

SRF 操纵子——遗传能力的调节和作用

• 批准号: 6386178
• 负责人: PETER ZUBER
• 金额: $ 28.69万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386178
• 关键词: Bacillus; Escherichia coli; bacterial genetics; fusion gene; gene mutation; genetic regulation; genetic transcription; genetic translation; immunoelectron microscopy; molecular cloning; nucleic acid sequence; operon

DESCRIPTION (adapted from the investigator's abstract): Bacteria possess regulatory networks that sense environmental and metabolic conditions, and respond by generating and transducing signals that affect gene expression. Starvation and high cell density influence the production of virulence factors such as toxins, antibiotics, and degradative enzymes through regulatory networks. They also induce complex cell differentiation processes that give rise to resistant cell types or competent cells that can acquire exogenous DNA. In Bacillus subtilis, the srf operon resides within a regulatory network that governs processes induced by nutrient depletion and high cell density. srf encodes surfactin synthetase, an antibiotic biosynthesis operon, and ComS, a regulatory peptide that controls competence development. The major goal of the project is to understand how srf and comS are regulated and how ComS stimulates competence development. srf is under the control of two converging regulatory pathways. One mediates quorum-sensing control and involves the two-component regulators ComP and ComA; phorphorylated ComA activates srf transcription. The other pathway, involving the Phr extracellular peptide and the SpoOK oligopeptide permease, is activated by starvation and high cell density; the Phr peptide, imported via SpoOK, inhibits the Rap phosphatase that converts ComA to an inactive form, allowing interplay between the two pathways. Activation of phr expression requires the SigmaH form of RNA polymerase, the activity of which is induced by starvation and requires ClpX, an ATP-dependent chaperone. The role of ClpX in the activation of E-SigmaH will be determined by purification and reconstitution of RNA polymerase in vitro for transcription reactions containing purified ClpX proteins. Mutations which suppress the phenotype of a clpX mutant will be characterized to identify factors influencing ClpX-dependent activation of E-SigmaH. Other functions of ClpX in the activation of srf transcription will be identified by testing the effects of clpX comP and clpX spoOK double mutants on srf expression. ComS is required to release ComK, the transcriptional activator of competence gene expression, from the competence inhibitory proteins MecA and ClpC. The ComS-dependent release is thought to rescue ComK from regulated proteolysis. A collection of ComS point mutations will be analyzed to determine the function of ComS in the activation of competence gene expression. These studies will further understanding of the functional links between stress-induced proteins and the regulation of prokaryotic cellular differentiation.

描述（改编自调查员的摘要）：细菌拥有 感知环境和代谢条件的监管网络，以及 通过产生和转导影响基因表达的信号来做出反应。 饥饿和高细胞密度影响毒力因子的产生 例如毒素，抗生素和降解酶通过调节 网络。它们还引起复杂的细胞分化过程 抗抗性细胞类型或可以获取外源性DNA的胜任细胞。 在枯草芽孢杆菌中，SRF操纵子位于一个监管网络中 控制营养耗竭和高细胞密度引起的过程。 SRF 编码抗生素生物合成操纵子和coms的表面激素合成酶 控制能力发展的监管肽。主要目标 项目将了解如何调节SRF和COMS以及COMS如何刺激 能力发展。 SRF在两个收敛调节的控制之下 途径。一个介导群体感应控制，涉及两组 监管机构comp and Coma； Phorphoryed Coma激活SRF转录。这 其他途径，涉及PHR细胞外肽和怪异的途径 通过饥饿和高细胞密度激活寡肽渗透酶。这 通过Spook进口的PHR肽抑制转化的RAP磷酸酶 昏迷至不活跃的形式，允许在这两个途径之间相互作用。 PHR表达的激活需要RNA聚合酶的SIGMAH形式，即 其活性是由饥饿诱导的，需要CLPX，ATP依赖性 伴侣。 CLPX在E-Sigmah激活中的作用将由 RNA聚合酶在体外的纯化和重构进行转录 含有纯化的CLPX蛋白的反应。抑制的突变 CLPX突变体的表型将被表征为识别因素 影响E-Sigmah的CLPX依赖性激活。 Clpx的其他功能 SRF转录的激活将通过测试效果来识别 SRF表达上的Clpx Comp和Clpx Spook双突变体的of。需要com 要释放能力基因表达的转录激活因子， 来自能力抑制蛋白MECA和CLPC。 coms依赖性 释放被认为可以从受管制的蛋白水解中挽救昏迷。集合 将分析COMS点突变以确定COM在 能力基因表达的激活。这些研究将进一步 理解压力诱导的蛋白质与 核细胞分化的调节。