Substrate-specific binding of hook-associated proteins by FlgN and FliT, putative chaperones for flagellum assembly

During flagellum assembly by motile enterobacteria, flagellar axial protein s destined for polymerization into the cell surface structure are thought t o be exported through the 25-30 Angstrom flagellum central channel as parti ally unfolded monomers. How are premature folding and oligomerization in th e cytosol prevented? We have shown previously using hyperflagellated Proteu s mirabilis and a motile but nonswarming flgN transposon mutant that the ap parently cytosolic 16.5 kDa flagellar protein FlgN facilitates efficient fl agellum filament assembly. Here, we investigate further whether FlgN, predi cted to contain a C-terminal amphipathic helix typical of type III export c haperones, acts as a chaperone for axial proteins. Incubation of soluble ra diolabelled FlgN from Salmonella typhimurium with nitrocellulose-immobilize d cell lysates of wild-type S. typhimurium and a non-flagellate class 1 flh DC mutant indicated that FlgN binds to flagellar proteins. Identical affini ty blot analysis of culture supernatants from the wild-type and flhDC, flgI , flgK, flgL, fliC or fliD flagellar mutants showed that FlgN binds to the flagellar hook-associated proteins (HAPs) FlgK and FlgL. This was confirmed by blotting artificially expressed individual HAPs in Escherichia coli. An alysis of axial proteins secreted into the culture medium by the original P . mirabilis flgN mutant demonstrated that export of FlgK and FlgL was speci fically reduced, with concomitant increased release of unpolymerized flagel lin (FliC), the immediately distal component of the flagellum. These data s uggest that FlgN functions as an export chaperone for FlgK and FlgL. Parall el experiments showed that FliT, a similarly small (14 kDa), potentially he lical flagellar protein, binds specifically to the flagellar filament cap p rotein, FliD (HAPP), indicating that it too might be an export chaperone. F lagellar axial proteins all contain amphipathic helices at their termini. R emoval of the HAP C-terminal helical domains abolished binding by FlgN and FliT in each case, and polypeptides comprising each of the HAP C-termini we re specifically bound by FlgN and FliT. We suggest that FlgN and FliT are s ubstrate-specific flagellar chaperones that prevent oligomerization of the HAPs by binding to their helical domains before export.