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

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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.