ComA can process and secrete BlpC independently of BlpA.

(A-C) BIRP1-lacZ transcriptional response in wildtype (blue circles), blpAFS (purple octagon) and blpAFS/comAB (red squares) mutant reporters during growth in broth. Closed symbols denote CSP addition, open symbols denote no peptide added. Cellular growth is shown as grey lines. (A) Response to CSP added at time 0 in THY. (B-C) Natural BIRP1-lacZ induction in competence permissive (B) and competence non-permissive C+Y media (C). (D) Western blot of cell lysate of BlpCFLAG expressing strains in ΔhtrA background: PSD127 (Wt), PSD131 (ΔblpA), PSD146 (ΔcomAB) and PSD147 (ΔblpA/comAB) after treatment with CSP or BlpCR6 or both BlpCR6/CSP. Preprocessed BlpCFLAG is denoted as pre-BlpC, the processed form is BlpC. Anti-pneumolysin (Ply) antibody was used as a loading control. Densitometry analysis was performed to determine % processed BlpCFLAG of the total BlpCFLAG detected in each strain.