FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium

Ohnishi, Kouhei; Ohto, Y.; Aizawa, Shin‐Ichi; Macnab, Robert M.; Iino, Tetsuo

doi:10.1128/jb.176.8.2272-2281.1994

Cited by 194 publications

(157 citation statements)

References 30 publications

(25 reference statements)

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“…The diameter of the channel is too small for a fully folded hook subunit to pass through, so the subunit must be unfolded, at least partially. These unfolded hook subunits would attain their final, folded conformation upon deposition at the growing tip, capped by FlgD (33), in the same way that flagellin is exported and refolded at the growing end of the filament, capped by FliD (34).…”

Section: Discussionmentioning

confidence: 99%

A partial atomic structure for the flagellar hook of Salmonella typhimurium

Shaikh

Thomas

Chen

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The axial proteins of the bacterial flagellum function as a drive shaft, universal joint, and propeller driven by the flagellar rotary motor; they also form the putative protein export channel. The Nand C-terminal sequences of the eight axial proteins were predicted to form interlocking ␣-domains generating an axial tube. We report on an Ϸ1-nm resolution map of the hook from Salmonella typhimurium, which reveals such a tube made from interdigitated, 1-nm rod-like densities similar to those seen in maps of the filament. Atomic models for the two outer domains of the hook subunit were docked into the corresponding outermost features of the map. The N and C termini of the hook subunit fragment are positioned next to each other and face toward the axis of the hook. The placement of these termini would permit the residues missing in the fragment to form the rod-like features that form the core domain of the hook. We also fit the hook atomic model to an Ϸ2-nm resolution map of the hook from Caulobacter crescentus. The hook protein sequence from C. crescentus is largely homologous to that of S. typhimurium except for a large insertion (20 kDa). According to difference maps and our fitting, this insertion is found on the outer surface of the hook, consistent with our modeling of the hook.bacterial chemotaxis ͉ bacterial motility ͉ electron cryomicroscopy T he bacterial flagellum is the organ of motility for many species of bacteria. About 40 genes are needed to assemble the structure; Ϸ22 of the genes contribute structural proteins found in the completed flagellum. Of these 22 proteins, six appear to be key components of the rotary motor. An additional protein is likely to function as an adaptor connecting the motor to the axial component (1). Nine more proteins make up the axial component consisting of a rod (drive shaft), hook (universal joint), junction, filament (propeller), and cap. Two of the remaining proteins make up rings, which serve as a bushing that allows passage of the drive shaft through the cell wall and outer membrane. The rest of the proteins are associated with the flagellar-specific protein export.The rotary motor, powered by the proton-motive gradient across the cell membrane, turns the filament, which converts torque into thrust. The helical hook of the bacterial flagellum acts as a universal joint, allowing the motor to drive the filament off-axis. The hook connects the rod to the hook-filament junction, which in turn is connected to the filament. The hook, which is assembled before the more distal segments of the axial component, plays a role in the assembly of the filament. The flagellar filament elongates by subunit addition at its distal tip (2, 3). Subunits exported by the cell are thought to diffuse along a channel in the hook (and also the rod within the basal body and partially assembled filament). Three-dimensional reconstructions reveal a 3-nm channel running along the axis of the rod (D.R.T., D. G. Morgan, and D.J.D., unpublished data), hook (4), and filament (5), although higher-resolu...

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Section: Discussionmentioning

confidence: 99%

A partial atomic structure for the flagellar hook of Salmonella typhimurium

Shaikh

Thomas

Chen

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The wild-type S. typhimurium SJW1103 is motile (15). Derived mutants carry lesions in flagellar genes flgN (7); flhDC [SJW1368 (16)]; fliOPQR (SJW192), flhA (SJW1616), flhB (SJW1684), fliJ (SJW135), and fliI (SJW2702) (17); and fliH [MKM11 (18)]. Recombinant proteins were expressed in E. coli BL21 (DE3) (19) or its derivative C41 (20) from isopropyl ␤-D-thiogalactoside-inducible plasmids pET15b (21) or pACT7 (22) or in S. typhimurium from Larabinose-inducible pBAD18 (23).…”

Section: Methodsmentioning

confidence: 99%

Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export

Thomas

Stafford

Hughes

2004

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial type III protein export underlies flagellum assembly and delivery of virulence factors into eukaryotic cells. The sequence of protein interactions underlying the export pathway are poorly characterized; in particular, it is not known how chaperoned substrates in the cytosol are engaged by the membrane-localized export apparatus. We have identified a stalled intermediate export complex in the flagellar type III export pathway of Salmonella typhimurium by generating dominant-negative chaperone variants that are export-defective and arrest flagellar assembly in the wild-type bacterium. These chaperone variants bound their specific export substrates strongly and severely reduced their export. They also attenuated export of other flagellar proteins, indicating that inhibition occurs at a common step in the pathway. Unlike the cytosolic wild-type chaperone, the variants localized to the inner membrane, but not in the absence of the flagellar type III export apparatus. Membrane localization persisted in fliOPQR, flhB, flhA, fliJ, and fliH null mutants lacking specific flagellar export components but depended on the presence of the membrane-associated ATPase FliI. After expression of the variant chaperones in Salmonella, a stalled intermediate export complex, which contained chaperone, substrate, and the FliI ATPase with its regulator FliH, was isolated. Neither chaperone nor substrate alone was able to interact with liposome-associated FliI, but the chaperone-substrate-FliI(FliH) complex was assembled when chaperone was prebound to its substrate. Our data establish a key event in the type III protein export mechanism, docking of the cytosolic chaperone-substrate complex at the ATPase of the membrane-export apparatus.

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“…Elongation of the hook is enabled by a scaffolding protein, FlgD, which associates with the tip of elongating hooks (30), much as FliD does for filament elongation (9,13,16). When the hook length reaches the mature, or defined, size, the replacement of FlgD by HAP1 (FlgK) is followed by the attachment of HAP3 (FlgL) and progressive filament elongation (9,14).…”

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confidence: 99%