Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone

Abstract

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti-σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far-UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.

Bacterial flagella grow through an injection-diffusion mechanism

“…A similar filament length-dependent effect on flagellin transport was also observed in a mutant of the flagellin-specific cytoplasmic chaperone FliS (Figure 1b). FliS promotes docking and subsequent unfolding of flagellin at the export apparatus (Kinoshita et al, 2013; Furukawa et al, 2016), suggesting that the flagellin injection rate at the export apparatus substantially contributes to the flagellum growth dynamics.…”

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

“…Such interaction would also rationalize the low‐affinity binding component of CesAB/EspA for EscV (i.e., Δ sepL , Fig J). In the flagellum system, the C‐terminal region of substrate loaded‐chaperones targets the chaperone/secretory protein complex to the C‐domain of the EscV homologue, FlhA with higher affinity than the chaperone alone (Bange et al , ; Minamino et al , ; Kinoshita et al , , ; Furukawa et al , ). Defining the structural basis of such interactions with EscV is a major challenge due to its multimeric and membrane‐embedded nature.…”

“…This effect could be mediated Fig 3J). In the flagellum system, the C-terminal region of substrate loaded-chaperones targets the chaperone/secretory protein complex to the C-domain of the EscV homologue, FlhA with higher affinity than the chaperone alone (Bange et al, 2010;Minamino et al, 2012;Kinoshita et al, 2013Kinoshita et al, , 2016Furukawa et al, 2016).…”

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

“…It has been reported that deletion of domain D4 of FlhA C allows filament-type substrates to be exported into the periplasm prior to hook completion . The FlgN/FlgK, FlgN/ FlgL, FliT/FliD and FliS/FliC chaperone/filament-type substrate complexes all bind to well-conserved Asp-456, Phe-459 and Thr-490 residues of FlhA C , promoting unfolding and translocation of the filament-type substrates into the lumen of the growing structure Kinoshita et al, 2013Kinoshita et al, , 2016Furukawa et al, 2016). This raises the possibility that domain D4 of FlhA C suppresses the interactions of these conserved residues with the flagellar chaperone/ filament-type substrate complexes during hook assembly.…”