The flagellar protein export apparatus switches its substrate specificity when hook length has reached approximately 55 nm in Salmonella. The C-terminal cytoplasmic domain of FlhB (FlhBC ) is involved in this switching process. FlhBC consists of FlhBCN and FlhBCC polypeptides. FlhBCC has a flexible C-terminal tail (FlhBCCT ). FlhBCC is involved in substrate recognition, and conformational rearrangements of FlhBCN -FlhBCC boundary are postulated to be required for the export switching. However, it remains unknown how it occurs. To clarify this question, we carried out mutational analysis of highly conserved residues in FlhBC . The flhB(E230A) mutation reduced the FlhB function. The flhB(E11S) mutation restored the protein transport activity of the flhB(E230A) mutant to the wild-type level, suggesting that the interaction of FlhBCN with the extreme N-terminal region of FlhB is required for flagellar protein export. The flhB(R320A) mutation affected hydrophobic interaction networks in FlhBCC , thereby increasing insolubility of FlhBC . The R320A mutation also affected the export switching, thereby producing longer hooks with the filament attached. C-terminal truncations of FlhBCCT induced a conformational change of FlhBCN -FlhBCC boundary, resulting in a loose hook length control. We propose that FlhBCCT may control conformational arrangements of FlhBCN -FlhBCC boundary through the hydrophobic interaction networks of FlhBCC .
Keywords: FlhB; bacterial flagella; hook; substrate specificity switching; type III protein export.
© 2019 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.