title: Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.
abstract: For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hara
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
authors.forename: Tohru
authors.initials: T
authors.lastname: Minamino
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics*; D005407:Flagella / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis / Q000235:genetics; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D021381:Protein Transport; D019779:Salmonella enterica / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; C080688:FlhA protein, Bacteria; D008565:Membrane Proteins; D050505:Mutant Proteins; D000409:Alanine