FIG. 4.

Analysis of DDM-extracted membrane protein complexes by blue native electrophoresis. Strains C58, CB1002 (ΔvirB2), CB1005 (ΔvirB5), and CB1008 (ΔvirB8) and complemented variants were cultivated on AB minimal medium in the absence (−AS, lane 1) or in the presence of AS (+AS, lanes 2 to 7) for virulence gene induction, followed by cell lysis, sedimentation of the membranes and extraction with 2% DDM. The samples were separated by blue native PAGE on a 15% gel, followed by Western blotting with VirB2-, VirB5-, and Tzs-specific antiserum. Lanes: 1, C58 without AS; 2, C58 with AS; 3, CB1002; 4, CB1005; 5, CB1002 pTrcB2; 6, CB1005 pTrcB5; 7, CB1008. Arrowheads point to Tzs in a 100-kDa complex in CB1005 and CB1008, and molecular masses of reference proteins are shown on the right (in kilodaltons). This experiment was conducted twice with qualitatively similar results.