Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate

Abstract

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate.

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

“…This suggests that FliS by itself does not induce unfolding of FliC subunit. Since FliS binds to FlhA C (Kinoshita et al ., 2013) but neither to FliH, FliI nor FliJ (Evans et al ., ; Sajó et al ., ), we propose that a physical interaction between FliS and FlhA C not only efficiently transfers FliC to the export gate complex but also contributes to efficient unfolding of FliC for its rapid export.…”

“…Loss of FliS results in a reduction in the export rate of FliC, thereby conferring a short flagellar filament phenotype (Yokoseki et al ., ). The FliS–FliC complex binds to FlhA through an interaction between FliS and the C‐terminal cytoplasmic domain of FlhA (FlhA C ) (Bange et al ., ; Kinoshita et al ., ) but neither to FliH, FliI nor FliJ (Evans et al ., ; Sajó et al ., ). Tyr‐10 of FliS, a highly conserved residue in the extreme N‐terminal segment is directly involved in the interaction with FlhA C (Kinoshita et al ., ).…”

Beyond a ribosomal RNA methyltransferase, the wider role of MraW in DNA methylation, motility and colonization inEscherichia coliO157:H7

“…FliR is one of the six integral membrane proteins of the export apparatus. FliJ participates in forming an ATPase ring complex at the export gate that also plays an important role in substrate recognition with FliI (34, 35). FliK takes part in switching flagellar secretion mode from hook construction to filament elongation and controlling the length of the hook (36).…”

Type III secretion systems: the bacterial flagellum and the injectisome

“…Beside the low specificity of export signals, this notion is supported by the fact that loss of some conserved cytosolic components of the T3SS can be compensated for. As an example, the flagellar ATPase FliI is not absolutely required for the formation and rotation of flagella, especially in the absence of its negative regulator FliH and at increased proton-motive force (PMF) [204,205], an effect that was to some extent also observed for a catalytically inactive injectisome ATPase [204]. Similarly, absence of the flagellar C-ring component FliMN could be compensated by overexpression of the flagellar main transcription factor FlhDC [206] or even the ATPase FliI alone [207], although similar experiments did not lead to the same outcome for the injectisome (A Diepold 2009, unpublished data).…”