FIGURE 3.

Expression of SecA1(K106R) and SecA2(K106R) causes a severe S-layer translocation defect. A, mid-log cultures of C. difficile strain 630 harboring pMTL960 (Vector), pRPF186 (WT SecA2), pRPF187 (SecA2(K106R)), pRPF193 (WT SecA1), or pRPF194 (SecA1(K106R)) were induced with ATc (500 ng/ml) for 4 h, and surface proteins were isolated using low pH glycine (39). Surface proteins were separated on 12% SDS-polyacrylamide gels and Coomassie Blue-stained. B, densitometry analysis of the gel shown in A. Densitometry was performed independently on the LMW and HMW SLPs; the average densitometry values with S.D. are shown. C and D, Western immunoblot analysis of the SlpA translocation defect in cultures expressing wild-type and dominant-negative SecA1 and SecA2. Cultures of C. difficile strain 630 harboring pRPF186 (WT SecA2), pRPF187 (SecA2(K106R)), pRPF193 (WT SecA1), or pRPF194 (SecA1(K106R)) were induced for 3 h with the indicated concentrations of ATc. Whole cell lysates were separated on 10% SDS-polyacrylamide gels and probed with anti-Strep-tag II or anti-LMW SLP antibody. E, [³⁵S]methionine labeling of de novo synthesized C. difficile surface proteins. Cultures of C. difficile strain 630 carrying pMTL960, pRPF187 (SecA2(K106R)), pRPF186 (WT SecA2), pRPF194 (SecA1(K106R)), and pRPF193 (WT SecA1) were ³⁵S-labeled as described under “Experimental Procedures.” Surface proteins were isolated using low pH glycine and visualized by SDS-PAGE, followed by autoradiography.