(A) Inactivation of comX1 or comX2 does not impact transformation efficiency. Error bars represent triplicate repeats. (B) Inactivation of comX1 or comX2 delays the shut-off of competence. Data is plotted as Relative Light Units, corrected by optical density (RLU/OD). Error bars represent triplicate repeats. Differences in transformation efficiency of mutant strains determined as non-significant compared to wild-type using GraphPad Prism software (p=0.954 for comX1-, p=0.983 for comX2-). (C) Visual representation of experiment exploring the impact of pre-competence production of DprA and σX on induction. (D) BIP induction of cells lacking CEPIIR-comX does not alter competence induction. Cells possessing comC-luc and CEPlac-dprA (R4511) were treated as described in Panel C. Error bars represent triplicate repeats. (E) Production of σX and DprA prior to competence induction antagonizes competence. Cells possessing comC-luc and CEPlac-dprA and CEPIIR-comX (R4500) were treated as in panel C. Error bars represent triplicate repeats. (F) Inactivation of comW does not alter the antagonization of competence induction mediated by early DprA and σX production. Cells possessing comC-luc, CEPlac-dprA, CEPIIR-comX and ΔcomW::trim (R4509) were treated as in panel C. Error bars represent triplicate repeats. (G) Polar competence induction in Streptococcus pneumoniae. (1) Extracellular CSP interacts with ComD at the cell poles, prompting ComD autophosphorylation. (2) Patches of ComE navigate around the cell membrane. (3) Polar ComD phosphorylates ComE. (4) Active ComE~P dimers leave the cell poles to interact with genomic targets, inducting the early com regulon and (5) launching an autocatalytic feedback loop. (6) Among the early com genes, comX1, comX2, and comW produce σX and its activator ComW, which induce the late com regulon, including DprA. Orange arrows, early com promoters; purple arrows, late com promoters. C, comC; D, comD; E, comE; X1/2, comX1/comX2; W, comW; RNAP, RNA polymerase. (H) Polar shut-off of pneumococcal competence. (1) σX interacts directly with DprA, promoting accumulation of DprA at the cell pole, generating a polar DprA ‘cloud’ in competent cells, near ComD (2). (3) Polar DprA interacts directly with neosynthesized ComE~P, preventing the regulator from accessing its genomic targets. (4) Unphosphorylated ComE interacts with early com promoters, acting as a repressor to prevent induction (Martin et al., 2013), promoting extinction of the competence signal, resulting in competence shut-off (5).