ComE / ComE ∼ P interplay dictates activation or extinction status of pneumococcal X ‐state (competence)

Summary Since 1996, induction of competence for genetic transformation of S treptococcus 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 σ 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 Δ 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 ∼ 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 σ X is due to the progressive accumulation of ComE , which outcompetes ComE ∼ P .