FliH, a soluble component of the type III flagellar export apparatus of Salmonella, forms a complex with Flil and inhibits its ATPase activity

Both FliH and the ATPase Flil are cytoplasmic components of the Salmonella type III flagellar export apparatus. Dominance and inhibition data have suggested that the N-terminus of Flil interacts with FliH and that this interaction is important,for the ATPase function of the C-terminal domain of Flil. N-terminally histidine-tagged, wild-type Fill retarded untagged FliH in a Ni-NTA affinity chromatography assay, as did N-His-tagged versions of Flil carrying catalytic mutations. In contrast, N-His-tagged Flil carrying the double mutation R7C/L12P did not, further indicating that the N-terminus of Flil is responsible for interaction with FliH. Native agarose gel electrophoresis confirmed that FliH and Fill form a complex. Analytical gel filtration with in-line multiangle light scattering indicated that FliH alone forms a dimer, Flil alone remains as a monomer, and FliH and Flil together form a (FliH)(2)Flil complex. NI-NTA affinity chromatography using N-His-tagged Flil and a large excess of untagged FliH confirmed that FliH forms a homodimer. The ATPase activity of the FliH-Flil complex was about 10-fold lower than that of Fill alone; the presence or absence of ATP did not affect the formation of the complex. We propose that FliH functions as a negative regulator to prevent Flil from hydrolysing ATP until the flagellar export apparatus is competent to link this hydrolysis to the translocation of export substrates across the plane of the cytoplasmic membrane into the lumen of the nascent flagellar structure.