Domain Structure of Salmonella FlhB, a Flagellar Export Component Responsible for Substrate Specificity Switching

Abstract

We have investigated the properties of the cytoplasmic domain (FlhB C ) of the 383-amino-acid Salmonella membrane protein FlhB, a component of the type III flagellar export apparatus. FlhB, along with the hook-length control protein FliK, mediates the switching of export specificity from rod-and hook-type substrates to filament-type substrates during flagellar morphogenesis. Wild-type FlhB C was unstable (half-life, ca. 5 min), being specifically cleaved at Pro-270 into two polypeptides, FlhB CN and FlhB CC , which retained the ability to interact with each other after cleavage. Full-length wild-type FlhB was also subject to cleavage. Coproduction of the cleavage products, FlhB ⌬CC (i.e., the N-terminal transmembrane domain FlhB TM plus FlhB CN ) and FlhB CC , resulted in restoration of both motility and flagellar protein export to an flhB mutant host, indicating that the two polypeptides were capable of productive association. Mutant FlhB proteins that can undergo switching of substrate specificity even in the absence of FliK were much more resistant to cleavage (half-lives, 20 to 60 min). The cleavage products of wild-type FlhB C , existing as a FlhB CN -FlhB CC complex on an affinity blot membrane, bound the rod-and hook-type substrate FlgD more strongly than the filament-type substrate FliC. In contrast, the intact form of FlhB C (mutant or wild type) or the FlhB CC polypeptide alone bound FlgD and FliC to about the same extent. FlhB CN by itself did not bind substrates appreciably. We propose that FlhB C has two substrate specificity states and that a conformational change, mediated by the interaction between FlhB CN and FlhB CC , is responsible for the specificity switching process. FliK itself is an export substrate; its binding properties for FlhB C resemble those of FlgD and do not provide any evidence for a physical interaction beyond that of the export process.

Deletion of a previously uncharacterized flagellar-hook-length control gene fliK modulates the σ 54-dependent regulon in Campylobacter jejuni

“…Gene name Gene number that the switch in export specificity from hook to filament components in S. enterica serovar Typhimurium is irreversible, resulting from cleavage of FlhB upon activation of the export switch (Hirano et al, 2005;Minamino & Macnab, 2000); this implies that export-specificity reversal to produce alternating hook-like and filament-like structures is not possible. We conclude that there are alternate modes of packing of the FlgE protein into either hooks/ (Goon et al, 2003).…”

Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

“…The C-terminal subdomain of FlhB C (FlhB CC ) remains tightly associated with the rest of FlhB following processing (Minamino & Macnab, 2000). FlhB homologues in the virulence factor T3SS used by pathogenic bacteria also undergo autocleavage, and like those of FlhB, the C-terminal subdomains of these proteins remain associated with the rest of the protein after autocleavage (Deane et al, 2008;Minamino & Macnab, 2000;Zarivach et al, 2008). FlhB processing has been proposed to serve as a molecular clock to regulate temporal gene expression with the spatial assembly of the flagellum (Ferris et al, 2005).…”

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

“…Several studies have demonstrated the involvement of the YscU/FlhB family of protein in the substrate specificity switch (11,13,14,34). YscU is anchored in the inner membrane via four transmembrane segments.…”

“…After activation of the T3SS, the secretion of early substrates is modulated in favor of the secretion of translocator and effector proteins, the so called middle and late substrates, respectively (11,12). This phenomenon is described as the substrate specificity switch that was first identified by Macnab and coworkers in the flagellum (13,14).…”