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（改编自研究者的摘要）：细菌具有 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 例如通过监管的毒素、抗生素和降解酶 网络。它们还诱导复杂的细胞分化过程，从而赋予 产生耐药细胞类型或能够获取外源 DNA 的感受态细胞。 在枯草芽孢杆菌中，srf 操纵子位于一个调控网络中， 控制由营养耗尽和高细胞密度引起的过程。 srf 编码表面活性素合成酶（一种抗生素生物合成操纵子）和 ComS（一种 控制能力发展的调节肽。该计划的主要目标是 该项目旨在了解 srf 和 comS 是如何监管的以及 ComS 如何刺激 能力发展。 srf 受到两个融合监管机构的控制 途径。一种介导群体感应控制并涉及两部分 调节器 ComP 和 ComA；磷酸化的 ComA 激活 srf 转录。这 其他途径，涉及 Phr 胞外肽和 SpoOK 寡肽通透酶，由饥饿和高细胞密度激活；这 通过 SpoOK 导入的 Phr 肽可抑制 Rap 磷酸酶，从而将 ComA 处于非活性形式，允许两条通路之间相互作用。 phr 表达的激活需要 SigmaH 形式的 RNA 聚合酶，即 其活性是由饥饿诱导的并且需要 ClpX，一种 ATP 依赖性 伴侣。 ClpX 在 E-SigmaH 激活中的作用将由以下因素确定 用于转录的体外 RNA 聚合酶的纯化和重构 含有纯化 ClpX 蛋白的反应。抑制的突变 clpX 突变体的表型将被表征以确定因素 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确定 clpX compP 和 clpX spoOK 双突变体对 srf 表达的影响。需要 ComS 释放 ComK，能力基因表达的转录激活剂， 来自能力抑制蛋白 MecA 和 ClpC。依赖 ComS 的 释放被认为可以将 ComK 免于受调节的蛋白水解作用。的集合 将分析 ComS 点突变以确定 ComS 在 能力基因表达的激活。这些研究将进一步 了解应激诱导的蛋白质和 原核细胞分化的调节。