FIG. 3.

The PAL peptide competes with PAL for binding to TolB. (A) Purified PAL and TolBHis proteins can be cross-linked in vitro. Purified PAL and TolBHis proteins were mixed (PAL + TolB) or treated independently with 1% formaldehyde (see Materials and Methods). An in vivo cross-linking was performed as a control to localize the Tol-PAL complex on whole cells overproducing the TolB and PAL proteins (JC7752 pBP). The samples were analyzed by SDS–12% PAGE and by immunoblotting with antibodies directed against TolB and PAL (Ab anti-TolB and Ab anti-PAL). (B) Purified PAL, purified TolBHis, and increasing amounts of the PAL peptide (0 to 100 μg) or 250 μg of the TolR peptide were treated with 1% formaldehyde as described in Materials and Methods. The samples were analyzed by SDS–12% PAGE and by immunoblotting with an antiserum directed against TolB (Ab anti-TolB). The positions of the TolB and PAL proteins, of the TolB-PAL complex, and of the molecular mass markers are indicated.