Quantifying Substrate Protein Secretion via the Type III Secretion System of the Bacterial Flagellum

Protein transport across the cytoplasmic membrane is coupled to energy derived from ATP hydrolysis or the proton motive force. A sophisticated, multi-component type III secretion system (T3SS) exports substrate proteins of both the bacterial flagellum and virulence-associated injectisome system of many Gram-negative pathogens. The T3SS is primarily a proton motive force-driven protein exporter. Here, we describe a method to investigate the export of substrate proteins of the flagellar T3SS into the culture supernatant under conditions that manipulate the proton motive force. Further, we describe methods to precisely quantify flagellar protein export into the culture supernatant using a split NanoLuc luciferase, and how fluorescence labeling of the extracellular flagellar filament can bring insights into the protein export rate of individual flagellar T3SS.