Substrate complexes and domain organization of the Salmonella flagellar export chaperones FlgN and FliT

The flagellar proteins FlgN and FliT have been proposed to act as substrate -specific export chaperones, facilitating incorporation of the enterobacter ial hook-associated axial proteins (HAPs) FlgK/FlgL and FliD into the growi ng flagellum, In Salmonella typhimurium flgN and fliT mutants, the export o f target HAPs was reduced, concomitant with loss of unincorporated flagelli n into the surrounding medium. Gel filtration chromatography of wild-type S . typhimurium cell extracts identified stable pools of FlgN and FliT homodi mers in the cytosol, but no chaperone-substrate complexes were evident. Nev ertheless, stable unique complexes were assembled efficiently in vitro by c o-incubation of FlgN and FliT with target HAPs purified from recombinant Es cherichia coil, The sizes of the chaperone-substrate complexes indicated th at, in each case, a chaperone homodimer binds to a substrate monomer. FlgN prevented in vitro aggregation of FlgK monomers, generating a soluble form of the HAP, Recombinant polypeptides spanning the potentially amphipathic C -terminal regions of FlgN or FliT could not complement in trans the chapero ne deficiency of the respective flgN and fliT mutants, but efficient flagel lar assembly was restored by homodimeric translational fusions of these dom ains to glutathione S-transferase, which bound FlgK and FlgL like the wild- type FlgN, These data provide further evidence for the substrate-specific c haperone function of FlgN and FliT and indicate that these chaperones compr ise common N- and C-terminal domains mediating homodimerization and HAP sub strate binding respectively. In support of this view, the flgN mutation was specifically complemented by a hybrid chaperone comprising the N-terminal half of FliT and the C-terminal half of FlgN.