Protein transport across the cytoplasmic membrane is coupled to energy derived from adenosine triphosphate hydrolysis or the protein motive force (pmf). A sophisticated, multi-component type III secretion system exports substrate proteins of both the bacterial flagellum and virulence-associated injectisome system of many Gram-negative pathogens. The type-III secretion system is primarily a pmf-driven protein exporter. Here, I describe methods to investigate the export of substrate proteins into the culture supernatant under conditions that manipulate the pmf.
Keywords: Bacterial flagellum; Carbonyl cyanide m-chlorophenylhydrazone (CCCP); Ionophore; Protein export; Proton motive force; Type III secretion system; Valinomycin; ∆pH gradient; ∆Ψ gradient.