Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS
(A) The indicated strains of EPEC, complemented with the plasmid encoding GspD-C4 were cultured in medium to an OD600∼0.6 and arabinose was then added to the culture (0.1%, final concentration). At the indicated time-point cell extracts were prepared from the culture, resuspended in sample buffer containing Lumio reagent and analysed by SDS-PAGE. The polyacrylamide gels were then imaged by fluorimetry (Argon Blue 488 nm laser and 520 nm BP40 filter). Positions of molecular weight markers and the 21 kDa protein SlyD are indicated. (B) Wild-type EPEC (WT), ΔgspD mutant EPEC, and the ΔgspD mutant EPEC complemented with the plasmid encoding GspD-C4 were grown in culture and post-cell supernatants containing secreted proteins (200 µg of protein) were analyzed by SDS-PAGE and Coomassie blue staining. (C) Strains of EPEC: ΔgspD, ΔgspDΔyacC or ΔgspDΔaspS, were complemented with the plasmid encoding GspD-C4 under control of the tet promoter and were cultured to an OD600∼1.0, extracted and then fractionated by sucrose density centrifugation. Identical samples were analysed by SDS-PAGE for detection of GspD multimers with Lumio reagent and immunoblotting for the outer membrane protein BamA and the inner membrane β-subunit of the F1Fo-ATP synthase (F1β).