SRF OPERON--REGULATION AND ROLE IN GENETIC COMPETENCE

描述（改编自研究者摘要）：细菌具有 感知环境和代谢状况的调节网络，以及 通过产生和传递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， 网络.它们还诱导复杂的细胞分化过程， 产生抗性细胞类型或能够获得外源DNA的感受态细胞。 在枯草芽孢杆菌中，srf操纵子存在于一个调控网络中， 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，抗生素生物合成操纵子，和ComS， 控制感受态发育的调节肽。的主要目标 该项目旨在了解srf和comS是如何调节的，以及comS如何刺激 能力发展。SRF受两个趋同监管机构的控制， 途径。一种是介导群体感应控制， 调节子ComP和ComA;磷酸化ComA激活srf转录。的 其他途径，涉及Phr胞外肽和SpoOK 寡肽通透酶，被饥饿和高细胞密度激活; 通过SpoOK导入的Phr肽抑制Rap磷酸酶， 昏迷到一个不活跃的形式，允许两个途径之间的相互作用。 phr表达的激活需要RNA聚合酶的SigmaH形式， 其活性由饥饿诱导并需要ClpX，一种ATP依赖性的 监护人ClpX在E-SigmaH活化中的作用将通过以下测定： RNA聚合酶的体外纯化和重组 含有纯化的ClpX蛋白的反应。突变抑制了 clpX突变体的表型将被表征以鉴定因子 影响E-SigmaH的ClpX依赖性活化。ClpX的其他功能 SRF转录的激活将通过测试 clpX comP和clpX spoOK双突变体对srf表达的影响。ComS是必需的 为了释放ComK，感受态基因表达的转录激活因子， 来自感受态抑制蛋白MecA和ClpC。依赖Coms 释放被认为是将ComK从受调节的蛋白水解中拯救出来。的集合 将分析ComS点突变以确定ComS在细胞中的功能 感受态基因表达的激活。这些研究将进一步 了解应激诱导蛋白与应激蛋白之间的功能联系， 原核细胞分化的调节。

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.