SummaryOf the 13 two-component signal transduction systems (TCS) identified in Streptococcus pneumoniae , two, ComDE and CiaRH, are known to affect competence for natural genetic transformation. ComD and ComE act together with the comC-encoded competence-stimulating peptide (CSP) and with ComAB, the CSP-dedicated exporter, to co-ordinate activation of genes required for differentiation to competence. Several lines of evidence suggest that the CiaRH TCS and competence regulation are interconnected, including the observation that inactivation of the CiaR response regulator derepresses competence. However, the nature of the interconnection remains poorly understood. Interpretation of previous transcriptome analyses of ciaR mutants was complicated by competence derepression in the mutants. To circumvent this problem, we have used microarray analysis to investigate the transition from noncompetence to competence in a comC -null wild-type strain and its ciaR derivative after the addition of CSP. This study increased the number of known CSPinduced genes from ª ª ª ª 47 to 105 and revealed ª ª ª ª 42 genes with reduced expression in competent cells. Induction of the CiaR regulon, as well as the entire HrcA and part of the CtsR stress response regulons, was observed in wild-type competent cells. Enhanced induction of stress response genes was detected in ciaR competent cells. In line with these observations, CSP was demonstrated to trigger growth arrest and stationary phase autolysis in ciaR cells. Taken together, these data strongly suggest that differentiation to competence imposes a temporary stress on cells, and that the CiaRH TCS is required for the cells to exit normally from the competent state.

SummaryA secreted competence-stimulating peptide (CSP), encoded by comC, constitutes, together with the twocomponent system ComD-ComE, the master switch for competence induction in Streptococcus pneumoniae. Interaction between CSP and its membrane-bound histidine-kinase receptor, ComD, is believed to lead to autophosphorylation of ComD, which then transphosphorylates the ComE response regulator to activate transcription of a limited set of genes, including the comCDE operon. This generates a positive feedback loop, amplifying the signal and co-ordinating competence throughout the population. On the other hand, the promoter(s) and proteins important for basal comCDE expression have not been defined. We now report that CSP-induced and basal comCDE transcription both initiate from the same promoter, P E; that basal expression necessitates the presence of both ComD and a phosphateaccepting form of ComE, but not CSP; and that overexpression of ComE R120S triggers ComDdependent transformation in the absence of CSP. These observations suggest that self-activation of ComD is required for basal comCDE expression. We also establish that transcriptional readthrough occurs across the tRNA Arg5 terminator and contributes significantly to comCDE expression. Finally, we demonstrate by various means, including single-cell competence analysis with GFP, that readthrough is crucial to avoid the stochastic production of CSP non-responsive cells lacking ComD or ComE.