title: Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB.
abstract: FlhB, an integral membrane protein, gates the type III flagellar export pathway of Salmonella. It permits export of rod/hook-type proteins before hook completion, whereupon it switches specificity to recognize filament-type proteins. The cytoplasmic C-terminal domain of FlhB (FlhBC) is cleaved between Asn-269 and Pro-270, defining two subdomains: FlhBCN and FlhBCC. Here, we show that subdomain interactions and cleavage within FlhB are central to substrate-specificity switching. We found that deletions between residues 216 and 240 of FlhBCN permitted FlhB cleavage but abolished function, whereas a deletion spanning Asn-269 and Pro-270 abolished both. The mutation N269A prevented cleavage at the FlhBCN-FlhBCC boundary. Cells producing FlhB(N269A) exported the same amounts of hook-capping protein as cells producing wild-type FlhB. However, they exported no flagellin, even when the fliC gene was being expressed from a foreign promoter to circumvent regulation of expression by FlgM, which is itself a filament-type substrate. Electron microscopy revealed that these cells assembled polyhook structures lacking filaments. Thus, FlhB(N269A) is locked in a conformation specific for rod/hook-type substrates. With FlhB(P270A), cleavage was reduced but not abolished, and cells producing this protein were weakly motile, exported reduced amounts of flagellin and assembled polyhook filaments.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Gillian M
authors.initials: GM
authors.lastname: Fraser
authors.forename: Takanori
authors.initials: T
authors.lastname: Hirano
authors.forename: Hedda U
authors.initials: HU
authors.lastname: Ferris
authors.forename: Lara L
authors.initials: LL
authors.lastname: Devgan
authors.forename: May
authors.initials: M
authors.lastname: Kihara
authors.forename: Robert M
authors.initials: RM
authors.lastname: Macnab
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins; D005407:Flagella / Q000378:metabolism*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012475:Salmonella / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C085668:FlhB protein, Bacteria; D008565:Membrane Proteins; D015534:Trans-Activators; C090552:flhC protein, E coli