ABSTRACTThe type III secretion system (T3SS) forms the functional core of injectisomes, protein transporters that allow bacteria to deliver virulence factors into their hosts for infection, and flagella, which are critical for many pathogens to reach the site of infection. In spite of intensive genetic and biochemical studies, the T3SS protein export mechanism remains unclear due to the difficulty of accurate measurement of protein exportin vivo. Here, we developed anin vitroflagellar T3S protein transport assay system using an inverted cytoplasmic membrane vesicle (IMV) for accurate and controlled measurements of flagellar protein export. We show that the flagellar T3SS in the IMV fully retains export activity. The flagellar hook was constructed inside the lumen of the IMV by adding purified component proteins externally to the IMV solution. We reproduced the hook length control and export specificity switch in the IMV consistent with that seen in the native cell. Previousin vivoanalyses showed that flagellar protein export is driven by proton motive force (PMF) and facilitated by ATP hydrolysis by FliI, a T3SS-specific ATPase. Ourin vitroassay recapitulated these previousin vivoobservations but furthermore clearly demonstrated that even ATP hydrolysis by FliI alone can drive flagellar protein export. Moreover, this assay showed that addition of the FliH2/FliI complex to the assay solution at a concentration similar to that in the cell dramatically enhanced protein export, confirming that the FliH2/FliI complex in the cytoplasm is important for effective protein transport.IMPORTANCEThe type III secretion system (T3SS) is the functional core of the injectisome, a bacterial protein transporter used to deliver virulence proteins into host cells, and bacterial flagella, critical for many pathogens. The molecular mechanism of protein transport is still unclear due to difficulties in accurate measurements of protein transport under well-controlled conditionsin vivo. We succeeded in developing anin vitrotransport assay system of the flagellar T3SS using inverted membrane vesicles (IMVs). Flagellar hook formation was reproduced in the IMV, suggesting that the export apparatus in the IMV retains a protein transport activity similar to that in the cell. Using this system, we revealed that ATP hydrolysis by the T3SS ATPase can drive protein export without PMF.
The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export
