Interaction of the Extreme N-Terminal Region of FliH with FlhA Is Required for Efficient Bacterial Flagellar Protein Export

Abstract

The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH EN ) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH EN with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.

Type III secretion systems: the bacterial flagellum and the injectisome

“…Both the identified minimal export signal of the flagellin and fulllength anti-s 28 factor FlgM have been successfully used as export signals for substrates which could subsequently cleaved by TEV protease [190,191]. As in the injectisome, both the peptide sequence and an mRNA signal seem to play a role in substrate recognition [189].…”

“…In E. coli, a class 1 master operon, flhDC, regulated by metabolic state and cell cycle, controls the expression of class 2 operons, which encode for the proximal structural components of the flagellum including its T3SS core. After completion of the T3SS, the anti-s 28 factor FlgM, a T3SS substrate, is exported, which allows the expression of class 3 genes, including the filament subunit, by the alternative sigma factor s 28 [64].…”

“…Environmental inputs control not only whether a bacterium needs a flagellum, but also how [28,29]. Flagellar model based on the study of Minamino & Imada [30]; injectisome model modified from [31]; based on cryo-electromicroscopic data [32]; F 0 F 1 -ATP synthase and structural homologies based on Ibuki et al [33].…”

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

“…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.…”

Recognition and targeting mechanisms by chaperones in flagellum assembly and operation

“…The first 20 N-terminal residues of FliI mediate self-oligomerization (18) and are involved in binding FliH (19,20). FliH is homologous to the β and δ subunits of the F o F 1 ATP synthase (21) and is thought to provide the support point for the association of the FliI ring to the export gate (22). FliJ, which is essential for the export of flagellum building blocks, binds to the center of the hexametric FliI ring in a manner similar to the way the γ subunit binds to the β subunit of F o F 1 ATP synthase (23).…”