FliH and FliI help FlhA bring strict order to flagellar protein export in Salmonella

Abstract

The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The FlhA ring is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly, but the mechanism remains a mystery. Here, we report that the highly conserved GYXLI motif of FlhA is important for ordered protein export by the fT3SS. The fT3SS with the flhA(Y369A/R370A/L371A/I372A) (AAAA) or flhA(Y369G/R370G/L371G/I372G) (GGGG) mutation did not switch the substrate specificity at an appropriate timing of hook assembly. The A372V/T and G372V substitutions recovered the export switching function of the AAAA and GGGG mutants, respectively, in the presence but not in the absence of FliH and FliI, components of the flagellar ATPase complex. Interestingly, a filament-type substrate, FlgL, was secreted via the fT3SS with the AAAA or GGGG mutation during hook assembly in the absence of FliH and FliI but not in their presence. These observations suggest that FlhA requires the flagellar ATPase complex not only to efficiently remodel its ring structure responsible for the substrate specificity switching of the fT3SS but also to correct substrate recognition errors that occur during flagellar assembly.