Natural transformation is a mechanism for genetic exchange in many bacterial genera. It proceeds through the uptake of exogenous DNA and subsequent homology-dependent integration into the genome. In Streptococcus pneumoniae, this integration requires the ubiquitous recombinase, RecA, and DprA, a protein of unknown function widely conserved in bacteria. To unravel the role of DprA, we have studied the properties of the purified S. pneumoniae protein and its Bacillus subtilis ortholog (Smf). We report that DprA and Smf bind cooperatively to single-stranded DNA (ssDNA) and that these proteins both self-interact and interact with RecA. We demonstrate that DprA-RecA-ssDNA filaments are produced and that these filaments catalyze the homology-dependent formation of joint molecules. Finally, we show that while the Escherichia coli ssDNA-binding protein SSB limits access of RecA to ssDNA, DprA lowers this barrier. We propose that DprA is a new member of the recombination-mediator protein family, dedicated to natural bacterial transformation.

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.