The adcCBA putative operon of Streptococcus pneumoniae, an important human pathogen, was identified in a search for transformation-deficient mutants. It was found to exhibit homology to ATP-binding cassette (ABC) transport operons encoding streptococcal adhesins such as FimA of Streptococcus parasanguis and PsaA of S. pneumoniae. The latter was recently shown to be essential for virulence as judged by intranasal or intraperitoneal challenge of mice. We suggested previously that AdcA, together with a set of 14 proteins, including PsaA and homologous adhesins, defines a new family of external solute-binding proteins specific for metals. In this work, Northern analysis revealed the existence of two adcB-adcA specific transcripts originating within adcC or further upstream, consistent with the hypothesis that adc is an operon. Investigation of growth of adc and psaA mutants in synthetic medium revealed that the addition of Zn improved the growth rate of the former, whereas the latter exhibited an absolute requirement for added Mn. A psaA-adc double mutant turned out to be essentially non-viable unless both metals were added in the appropriate ratio. Taken together, these results suggest a previously undocumented requirement of S. pneumoniae for Zn and Mn. The addition of Zn also restored near-normal spontaneous transformation of adc mutant cells in standard transformation medium. Zn was found to be specifically required soon after contact of cells with the competence-stimulating peptide, revealing an unsuspected need for Zn in transformation of S. pneumoniae. The removal of Mn from standard transformation medium also resulted in transformation deficiency of psaA mutant cells. Taken together, these results lead us to propose that Adc is an ABC-type Zn permease, the first such protein complex identified in any organism, and that Psa is an ABC-type Mn permease complex.

SummaryTwo operons, comAB and comCDE, play a key role in the co-ordination of spontaneous competence development in cultures of Streptococcus pneumoniae. ComAB is required for export of the comC-encoded competence-stimulating peptide (CSP). Upon CSP binding, the histidine kinase ComD activates ComE, its cognate response regulator, required for autoinduction of comCDE and for induction of the late competence genes. To understand better the early control of competence development, mutants upregulating comCDE (ComCDE UP ) were isolated 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 ComCDE UP mutants exhibited similar phenotypes.They differed from wild type in displaying trypsinresistant transformation, competence under acid growth conditions and expression of comCDE under microaerobiosis; increased production of CSP in the mutants could account for the various phenotypes. The ComCDE UP transposon mutations included four independent insertions in the ciaR gene, which encodes the response regulator of a two-component system previously found to affect competence, and two immediately upstream of the comAB operon. The latter two resulted in comAB overexpression, indicating that CSP export is rate limiting. Among comDE point mutations, a single amino acid change in ComD (T233I) conferred constitutive, CSP-independent competence and resulted in comAB overexpression, providing support for the hypothesis that ComE regulates 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. pneumoniae. They argue for a metabolic control of competence, mediated directly or indirectly by CiaR, and they suggest that both comAB and comCDE are potential targets for regulation.

Natural bacterial transformation is a genetically programmed process allowing genotype alterations that involves the internalization of DNA and its chromosomal integration catalyzed by the universal recombinase RecA, assisted by its transformation-dedicated loader, DNA processing protein A (DprA). In Streptococcus pneumoniae, the ability to internalize DNA, known as competence, is transient, developing suddenly and stopping as quickly. Competence is induced by the comC-encoded peptide, competence stimulating peptide (CSP), via a classic two-component regulatory system ComDE. Upon CSP binding, ComD phosphorylates the ComE response-regulator, which then activates transcription of comCDE and the competencespecific σ X , leading to a sudden rise in CSP levels and rendering all cells in a culture competent. However, how competence stops has remained unknown. We report that DprA, under σ X control, interacts with ComE∼P to block ComE-driven transcription, chiefly impacting σ X production. Mutations of dprA specifically disrupting interaction with ComE were isolated and shown to map mainly to the N-terminal domain of DprA. Wild-type DprA but not ComE interaction mutants affected in vitro binding of ComE to its promoter targets. Once introduced at the dprA chromosomal locus, mutations disrupting DprA interaction with ComE altered competence shut-off. The absence of DprA was found to negatively impact growth following competence induction, highlighting the importance of DprA for pneumococcal physiology. DprA has thus two key roles: ensuring production of transformants via interaction with RecA and competence shut-off via interaction with ComE, avoiding physiologically detrimental consequences of prolonged competence. Finally, phylogenetic analyses revealed that the acquisition of a new function by DprA impacted its evolution in streptococci relying on ComE to regulate comX expression.