Roles of the extreme N‐terminal region of FliH for efficient localization of the FliH–FliI complex to the bacterial flagellar type III export apparatus

Abstract

Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self-assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH-FliI complex also binds to the C ring of the basal body through a FliH-FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI-YFP by fluorescence microscopy. FliI-YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI-YFP formed a complex with FliHDelta1 (missing residues 2-10) but the complex did not show any localization. FliHDelta1 did not interact with FliN, and alanine-scanning mutagenesis revealed that only Trp-7 and Trp-10 of FliH are essential for the interaction with FliN. Overproduction of the FliH-FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)-FliI nor FliH(W10A)-FliI complexes did, suggesting that Trp-7 and Trp-10 of FliH are also required for efficient localization of the FliH-FliI complex to the export gate.

Quantitative Proteomic Analysis Reveals Formation of an EscL-EscQ-EscN Type III Complex in Enteropathogenic Escherichia coli

“…This suggests that, although EscL is not essential for EscN-EscQ interaction, it may stabilize the EscNEscQ complex. Similar results were reported for the flagellar system in Salmonella, where FliI can interact with the export apparatus in the absence of FliH (20), but are contradictory to recent reports that FliH is responsible for efficient localization of the FliI to the C-ring complex (9,19,22).…”

Insight into the flagella type III export revealed by the complex structure of the type III ATPase and its regulator

“…The extreme N-terminal region of FliH N is critical for binding to the C ring and is essential for export function (21). The first 10 residues in FliH are crucial to the interaction with FliN, and Trp-7 and Trp-10 directly interact with FliN (11). A recent structural study revealed that the FliH N-terminal 18 residues bind to FliM and FliN in an extended conformation (37).…”

“…The N-terminal region composed of residues 1-100 (FliH N ) is elongated, and the extreme N-terminal region is responsible for the interaction with FliN and FlhA to allow the FliI 6 ring to associate with the export gate (10)(11)(12)27). The middle region, residues 101-140, is essential for homodimer formation.…”

“…The FliI 6 ring has been identified to be located at the base of the flagellum by electron cryotomography (9). The FliI 6 ring associates with the basal body through the interactions of FliH with FlhA and FliN, a C-ring component of the basal body (10)(11)(12) (Fig. S1).…”

Type III secretion systems: the bacterial flagellum and the injectisome

“…In addition, the interaction network of the cytosolic components might well change between different functional states of the T3SS. A candidate for such a switch is FliH/SctL, the negative regulator of the ATPase, which can additionally interact with both the major export apparatus component and the C-ring [27,28,203].…”