ComE/ComE similar to P interplay dictates activation or extinction status of pneumococcal X-state (competence)

Since 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 similar to P of the early competence (com) genes, including comX which encodes the competence-specific sigma(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 Delta comD cells, an that in vitro ComE(D58E) exhibits significantly improved binding affinity for P-comCDE. We also provide evidence for a differential transcriptional activation and repression of P-comCDE and P-comX. Altogether, these data support the model of ComE similar to 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 P-comCDE observed even in cells lacking sigma(X) is due to the progressive accumulation of ComE, which outcompetes ComE similar to P.