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.

SummarySince 1996, induction of competence for genetic transformation of Streptococcus pneumoniae is known to be controlled by the ComD/ComE two-component regulatory system. The mechanism of induction is generally described as involving ComD autophosphorylation, transphosphorylation of ComE and transcriptional activation by ComE~P of the early competence (com) genes, including comX which encodes the competence-specific s X . However, none of these features has been experimentally established. Here we document the autokinase activity of ComD proteins in vitro, and provide an estimate of the stoichiometry of ComD and ComE in vivo. We report that a phosphorylmimetic mutant, ComE D58E , constructed because of the failure to detect transphosphorylation of purified ComE in vitro, displays full spontaneous competence in DcomD cells, an that in vitro ComE D58E exhibits significantly improved binding affinity for PcomCDE. We also provide evidence for a differential transcriptional activation and repression of PcomCDE and PcomX. Altogether, these data support the model of ComE~P-dependent activation of transcription. Finally, we establish that ComE antagonizes expression of the early com genes and propose that the rapid deceleration of transcription from PcomCDE observed even in cells lacking s X is due to the progressive accumulation of ComE, which outcompetes ComE~P.

Transformation promotes genome plasticity in bacteria via RecAdriven homologous recombination. In the Gram-positive human pathogen Streptococcus pneumoniae, the transformasome a multiprotein complex, internalizes, protects, and processes transforming DNA to generate chromosomal recombinants. Double-stranded DNA is internalized as single strands, onto which the transformation-dedicated DNA processing protein A (DprA) ensures the loading of RecA to form presynaptic filaments. We report that the structure of DprA consists of the association of a sterile alpha motif domain and a Rossmann fold and that DprA forms tail-totail dimers. The isolation of DprA self-interaction mutants revealed that dimerization is crucial for the formation of nucleocomplexes in vitro and for genetic transformation. Residues important for DprARecA interaction also were identified and mutated, establishing this interaction as equally important for transformation. Positioning of key interaction residues on the DprA structure revealed an overlap of DprA-DprA and DprA-RecA interaction surfaces. We propose a model in which RecA interaction promotes rearrangement or disruption of the DprA dimer, enabling the subsequent nucleation of RecA and its polymerization onto ssDNA.bacterial transformation | genetic exchange | recombinase loader | recombination mediator protein | horizontal gene transfer