Interaction of the C-Terminal Tail of FliF with FliG from the Na+-Driven Flagellar Motor of Vibrio alginolyticus
Rotation of the polar flagellum ofVibrio alginolyticusis driven by a Na+-type flagellar motor. FliG, one of the essential rotor proteins located at the upper rim of the C ring, binds to the membrane-embedded MS ring. The MS ring is composed of a single membrane protein, FliF, and serves as a foundation for flagellar assembly. Unexpectedly, about half of theVibrioFliF protein produced at high levels inEscherichia coliwas found in the soluble fraction. Soluble FliF purifies as an oligomer of ∼700 kDa, as judged by analytical size exclusion chromatography. By using fluorescence correlation spectroscopy, an interaction between a soluble FliF multimer and FliG was detected. This binding was weakened by a series of deletions at the C-terminal end of FliF and was nearly eliminated by a 24-residue deletion or a point mutation at a highly conserved tryptophan residue (W575). Mutations in FliF that caused a defect in FliF-FliG binding abolish flagellation and therefore confer a nonmotile phenotype. As data fromin vitrobinding assays using the soluble FliF multimer correlate with data fromin vivofunctional analyses, we conclude that the C-terminal region of the soluble form of FliF retains the ability to bind FliG. Our study confirms that the C-terminal tail of FliF provides the binding site for FliG and is thus required for flagellation inVibrio, as reported for other species. This is the first report of detection of the FliF-FliG interaction in the Na+-driven flagellar motor, bothin vivoandin vitro.