Regulation of Streptococcus pneumoniae clp genes and their role in competence development and stress survival

In vitro mariner transposon mutagenesis of Streptococcus pneumoniae chromos omal DNA was used to isolate regulatory mutants affecting expression of the comCDE operon, encoding the peptide quorum-sensing two-component signal tr ansduction system controlling competence development. A transposon insertio n leading to increased comC expression was found to lie directly upstream f rom the S. pneumoniae clpP gene, encoding the proteolytic subunit of the Cl p ATP-dependent protease, whose expression in Bacillus subtilis is controll ed by the CtsR repressor. In order to examine clp gene regulation in S. pne umoniae, a detailed analysis of the complete genome sequence was performed, indicating that there are five likely CtsR-binding sites located upstream from the clpE, clpP, and clpL genes and the ctsR-clpC and groESL operons. T he S. pneumoniae ctsR gene was cloned under the control of an inducible pro moter and used to demonstrate regulation of the S. pneumoniae clpP and clpE genes and the clpC and groESL operons by using B. subtilis as a heterologo us host. The CtsR protein of S. pneumoniae was purified and shown to bind s pecifically to the clpP, clpC, clpE, and groESL regulatory regions. S. pneu moniae Delta ctsR, Delta clpP, Delta clpC, and Delta clpE mutants were cons tructed by gene deletion/replacement. ClpP was shown to act as a negative r egulator, preventing competence gene expression under inappropriate conditi ons. Phenotypic analyses also indicated that ClpP and ClpE are both require d for thermotolerance. Contrary to a previous report, we found that ClpC do es not play a major role in competence development, autolysis, pneumolysin production, or growth at high temperature of S. pneumoniae.