Enzymatic characterization of FliI. An ATPase involved in flagellar assembly in Salmonella typhimurium.
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.
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA. robert_macnab@qm.yale.edu
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000737:chemistry*; D006180:Proton-Translocating ATPases; D012394:Rosaniline Dyes; D012486:Salmonella typhimurium; D013696:Temperature
D001426:Bacterial Proteins; D011506:Proteins; D012394:Rosaniline Dyes; C005095:malachite green; C069251:fliI protein, bacteria; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases