Enzymatic Characterization of FliI

Abstract

FliI is a protein needed for flagellar assembly in Salmonella typhimurium. It shows sequence similarity to the catalytic beta subunit of the F0F1-ATPase and is even more closely related to putative ATPases in Type III bacterial secretory pathways. A His-tagged version of FliI, which was fully functional in complementation tests, was purified to homogeneity. It had an ATPase activity of 0.16 s-1 at 25 degrees C and pH 7, and a Km for ATP of 0.3 mM; Mg2+ was required. The activity was not affected by inhibitors of the F-, V- or P-type ATPases, or inhibitors of the Type I or Type II bacterial secretory pathways. Mutations K188I and Y363S decreased the ATPase activity about 100-fold, increased the Km about 10-fold, blocked flagellar assembly, and were dominant. Other FliI mutations that disrupted flagellar protein export were found near the N terminus; they permitted essentially wild-type ATPase activity, were not dominant, and showed a dosage-dependent phenotype. We propose that FliI has a C-terminal ATPase domain and an N-terminal domain that interacts with other components in the flagellum-specific export apparatus.