The two-component regulatory system in Streptococcus pneumoniae: Regulation by the CiaR-dependent non-coding csRNAs and control of its activity.

人类病原体肺炎链球菌的双组分调控系统CiaRH与遗传能力、ß-内酰胺耐药性、自溶、细菌素的产生和毒力有关。这些表型主要受五种小的非编码rna的影响，称为csrna，它们是从CiaR调控中最强的启动子转录而来。csRNAs通过靶向comC基因来干扰能力发展，comC基因是编码能力刺激肽CSP前体的基因。虽然已经确定了另外5个csRNA靶基因，但与ß-内酰胺耐药有关的靶基因仍然缺失。此外，对CiaRH自身活性的调控知之甚少。反应调节因子CiaR在磷酸化时具有活性，可通过同源激酶CiaH或乙酰磷酸获得其磷酸。但是这些过程是如何被调控的在很大程度上是未知的。该提案涉及CiaRH的这些基本方面，即csRNAs的调控和对系统活动的控制，因此分为两个部分。第一部分介绍了csrna介导的调控。定义新的受csrna调控的基因，特别是那些与ß-内酰胺抗性有关的基因，是一个焦点。此外，将更详细地研究已知靶基因的调控，以确定调控的要求和机制。第二部分将研究CiaRH活性的控制。CiaRH受多个CiaR调控成员的广泛反馈调控，主要受csrna和丝氨酸蛋白酶HtrA的调控。此外，一种参与丙酮酸代谢的蛋白质醋酸激酶在缺乏CiaH的情况下对控制CiaR活性起特殊作用，并且醋酸的加入能够将CiaH从激酶转换为磷酸酶。这些调控是如何实现的，它们是如何相互联系的，将通过结合遗传和生物化学的方法来分析。拟议项目的目标是尽可能完整地描述CiaRH监管，将各种监管途径结合起来，形成对这一复杂系统监管的共同概念。

The two-component regulatory system CiaRH in the human pathogen Streptococcus pneumoniae is implicated in genetic competence, ß-lactam resistance, autolysis, production of bacteriocins, and virulence. These phenotypes are mainly influenced by five small non-coding RNAs, designated csRNAs, which are transcribed from the strongest promoters in the CiaR regulon. The csRNAs interfere with competence development by targeting comC, the gene encoding the precursor for the competence stimulating peptide CSP. While additional five csRNA target genes have been identified, targets involved in ß-lactam resistance are still missing. Furthermore, relatively little is known on the regulation of the activity of CiaRH itself. The response regulator CiaR, which is active upon phosphorylation, may obtain its phosphate by the cognate kinase CiaH or by acetyl phosphate. But how these processes are regulated is largely unknown. The proposal addresses these fundamental aspects of CiaRH, regulation by the csRNAs and control of the activity of the system, and is therefore divided into two parts. The first part deals with csRNA-mediated regulation. Defining new csRNA-regulated genes, especially those involved in ß-lactam resistance, is one focus. In addition, regulation of known target genes will be studied in more detail to define requirements and mechanisms of regulation. In the second part, the control of CiaRH activity will be studied. CiaRH is subject to extensive feed-back regulation by several members of the CiaR regulon, mainly by the csRNAs and the serine protease HtrA. Moreover, a protein involved in pyruvate metabolism, acetate kinase, plays a special role in controlling CiaR activity in the absence of CiaH and the addition of acetate is able to switch CiaH from kinase to phosphatase. How these regulations are achieved and how they are interconnected will be analyzed by combining genetic and biochemical methods. The goal of the proposed project is a characterization of CiaRH regulation as complete as possible to combine various regulatory pathways to a common concept of regulation of this complex system.