Cross-regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae

Two operons, comAB and comCDE, play a key role in the co-ordination of spon taneous competence development in cultures of Streptococcus pneumoniae, Com AB is required for export of the comC-encoded competence-stimulating peptid e (CSP), Upon CSP binding, the histidine kinase ComD activates ComE, its co gnate response regulator, required for autoinduction of comCDE and for indu ction of the late competence genes, To understand better the early control of competence development, mutants upregulating comCDE (ComCDE(UP)) were is olated using a comC-lacZ transcriptional fusion, Mutants were generated by polymerase chain reaction mutagenesis of the comCDE region and by in vitro transposon mutagenesis of the chromosome. Both types of ComCDEUP mutants ex hibited similar phenotypes, They differed from wild type in displaying tryp sin-resistant transformation, competence under acid growth conditions and e xpression of comCDE under microaerobiosis; increased production of CSP in t he mutants could account for the various phenotypes, The ComCDEUP transposo n mutations included four independent insertions in the ciaR gene, which en codes the response regulator of a two-component system previously found to affect competence, and two immediately upstream of the comAB operon, The la tter two resulted in comAB overexpression, indicating that CSP export is ra te limiting, Among comDE point mutations, a single amino acid change in Com D (T2331) conferred constitutive, CSP-independent competence and resulted i n comAB overexpression, providing support for the hypothesis that ComE regu lates comAB; a ComE mutant (R120S) exhibited altered kinetics of competence shut-off, Collectively, these data indicate that pheromone autoinduction, cross-regulation of the comAB and comCDE operons and, possibly, competence shut-off contribute to the early control of competence development in S, pn eumoniae, They argue for a metabolic control of competence, mediated direct ly or indirectly by CiaR, and they suggest that both comAB and comCDE are p otential targets for regulation.