FIG. 5.

Accessibility of TolC to proteinase K reflects its recruitment to T1SS. The proteinase K (PK) accessibility of TolC was assessed on permeabilized cells expressing combinations of T1SS components and substrates as indicated for each panel. Cultures (5 ml) were permeabilized and treated with protease at 10 μg/ml for 10 min at 30°C. Protease treatment was terminated with 0.1 mM PMSF. Proteins were resolved by 11% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, and detected with an anti-TolC polyclonal antiserum. TolC and TolC*, which corresponds to the 46-kDa degradation product generated by treatment with proteinase K, are indicated. The same strains were streak onto SDS-supplemented plates and incubated overnight at 37°C. Growth was recorded as follows: R, SDS resistant; S, SDS sensitive.