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

Abstract

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 ΔCC (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.