FIG 2.
Mapping the location of the TcdC C terminus with cysteine accessibility analysis. (A) Schematic representation of the C-terminally 3× Myc-tagged TcdC construct used for the cysteine accessibility analysis. The different domains of TcdC are represented: the transmembrane domain (TM), the dimerization domain (DM), and the OB-fold. The 3× Myc tag and the cysteines residues present on TcdC are represented. (B) Western blot analysis of anti-TcdC antibody specificity in C. difficile 630Δerm lysates harboring pLDJ1 (Ptet-tcdC-3×myc) before (lane T0) and after (lane T2 [time point 2]) induction with 200-ng/ml anhydrotetracycline for 2 h. Full-length TcdC is indicated with an arrowhead. (C) Cysteine labeling analysis of the TcdC-3×Myc construct. The strain harboring the C-terminally 3× Myc-tagged TcdC construct (38 kDa) was induced for 2 h. Samples were collected and either not treated with MPB (lane −) or treated with 1 mM MPB (lane +). Samples were immunoprecipitated and immunoblotted with anti-Myc for TcdC-3×Myc protein detection (top) and antibiotin for biotinylated protein detection (bottom). Cysteine biotinylation of TcdC-3×Myc was observed. MW, molecular weight.