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中的双组分调节系统CiaRH涉及遗传能力、β-内酰胺抗性、自溶、细菌素的产生和毒力。这些表型主要受五种小的非编码RNA（称为csRNA）的影响，这些小的非编码RNA从CiaR调节子中最强的启动子转录。csRNA通过靶向comC（编码感受态刺激肽CSP的前体的基因）干扰感受态发育。虽然已经鉴定了另外五种csRNA靶基因，但仍然缺少涉及β-内酰胺抗性的靶标。此外，对CiaRH本身活性的调节知之甚少。反应调节剂CiaR在磷酸化时有活性，可通过同源激酶CiaH或乙酰磷酸获得其磷酸。但这些过程是如何调节的，在很大程度上是未知的。该提案解决了CiaRH的这些基本方面，csRNA的调节和系统活性的控制，因此分为两个部分。第一部分涉及csRNA介导的调节。定义新的csRNA调控基因，特别是那些参与β-内酰胺抗性的基因，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。第二部分研究了CiaRH活性的调控。CiaRH受到CiaR调节子的几个成员的广泛反馈调节，主要是通过csRNA和丝氨酸蛋白酶HtrA。此外，参与丙酮酸代谢的蛋白质乙酸激酶在不存在CiaH的情况下在控制CiaR活性中起特殊作用，并且添加乙酸能够将CiaH从激酶转换为磷酸酶。这些调节是如何实现的，以及它们是如何相互联系的，将结合遗传和生物化学方法进行分析。拟议项目的目标是尽可能完整地描述CiaRH监管的特征，以将各种监管途径联合收割机结合到这个复杂系统的监管的共同概念中。

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.