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 pneumoniaeis known to be controlled by the ComD/ComEtwo-component regulatory system. The mechanism of induction is generally described as involving ComDautophosphorylation, transphosphorylation of ComEand transcriptional activation by ComE similar to Pof the early competence (com) genes, including comXwhich encodes the competence-specific sigma X. However, none of these features has been experimentally established. Here we document the autokinase activity of ComDproteins in vitro, and provide an estimate of the stoichiometry of ComDand ComEin vivo. We report that a phosphorylmimetic mutant, ComED58E, constructed because of the failure to detect transphosphorylation of purified ComEin vitro, displays full spontaneous competence in Delta comD cells, an that in vitroComED58E 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 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 PcomCDE observed even in cells lacking sigma X is due to the progressive accumulation of ComE, which outcompetes ComE similar to P.