FIGURE 4.

Expression of SecA1(K106R) and SecA2(K106R) impairs the translocation of several CWPs. A, C. difficile strain 630ΔrecV ON (14) harboring pMTL960 (Vector), pRPF186 (WT SecA2), pRPF187 (SecA2(K106R)), pRPF193 (WT SecA1), or pRPF194 (SecA1(K106R)) was induced with ATc (500 ng/ml) for 4 h. Surface proteins were isolated using low pH glycine (39) and analyzed by SDS-PAGE. B, whole cell lysates of the same cultures were also prepared and analyzed for the presence of full-length CwpV using an antibody raised against the 41.2-kDa N-terminal fragment of the protein. C, the surface protein samples described above were further analyzed using antibodies recognizing two additional CWPs (Cwp66 and Cwp84). The positions of the two CwpV subunits (C-term and N-term), Cwp2, HMW and LMW SLPs, and full-length CwpV, Cwp66, and Cwp84 are indicated.