Hook Cap Research Articles

Structure of the bacterial flagellar hook cap provides insights into a hook assembly mechanism

Assembly of bacterial flagellar hook requires FlgD, a protein known to form the hook cap. Symmetry mismatch between the hook and the hook cap is believed to drive efficient assembly of the hook in a way similar to the filament cap helping filament assembly. However, the hook cap dependent mechanism of hook assembly has remained poorly understood. Here, we report the crystal structure of the hook cap composed of five subunits of FlgD from Salmonella enterica at 3.3 Å resolution. The pentameric structure of the hook cap is divided into two parts: a stalk region composed of five N-terminal domains; and a petal region containing five C-terminal domains. Biochemical and genetic analyses show that the N-terminal domains of the hook cap is essential for the hook-capping function, and the structure now clearly reveals why. A plausible hook assembly mechanism promoted by the hook cap is proposed based on the structure.

A triangular loop of domain D1 of FlgE is essential for hook assembly but not for the mechanical function

The bacterial flagellar hook is a short, curved tubular structure made of FlgE. The hook connects the basal body as a rotary motor and the filament as a helical propeller and functions as a universal joint to smoothly transmit torque produced by the motor to the filament. Salmonella FlgE consists of D0, Dc, D1 and D2 domains. Axial interactions between a triangular loop of domain D1 (D1-loop) and domain D2 are postulated to be responsible for hook supercoiling. In contrast, Bacillus FlgE lacks the D1-loop and domain D2. Here, to clarify the roles of the D1-loop and domain D2 in the mechanical function, we carried out deletion analysis of Salmonella FlgE. A deletion of the D1-loop conferred a loss-of-function phenotype whereas that of domain D2 did not. The D1-loop deletion inhibited hook polymerization. Suppressor mutations of the D1-loop deletion was located within FlgD, which acts as the hook cap to promote hook assembly. This suggests a possible interaction between the D1-loop of FlgE and FlgD. Suppressor mutant cells produced straight hooks, but retained the ability to form a flagellar bundle behind a cell body, suggesting that the loop deletion does not affect the bending flexibility of the Salmonella hook.

Structure and Stability of FlgD from the Pathogenic 26695 Strain of Helicobacter pylori

Flagellin component D (FlgD) is a hook capping protein involved in the proper structural assembly of bacterial flagella but is not present in the mature flagella. We report here the crystal structure of the truncated form of FlgD from the strain 26695 of Helicobacter pylori (HpFlgD_26695) in the P2 space group at 2.8 A resolution, along with data on protein stability. The structure includes only the central globular part of the protein composed of conserved Fn-III and tudor domains, while the N- and C-terminal regions are absent. This structure, together with the tetragonal crystal structure of HpFlgD_26695 and the monoclinic crystal structure of HpFlgD_G27 reported previously, revealed a different mutual orientation of the two domains in the HpFlgD structures in respect to FlgD from Xanthomonas campestris and Pseudomonas aeruginosa. In addition, investigation of protein stability in solution showed higher protein stability then in other bacterial orthologues reported so far.

Global transcriptome and mutagenic analyses of the acid tolerance response of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the leading causative agents of food-borne bacterial gastroenteritis. Swift invasion through the intestinal tract and successful establishment in systemic organs are associated with the adaptability of S. Typhimurium to different stress environments. Low-pH stress serves as one of the first lines of defense in mammalian hosts, which S. Typhimurium must efficiently overcome to establish an infection. Therefore, a better understanding of the molecular mechanisms underlying the adaptability of S. Typhimurium to acid stress is highly relevant. In this study, we have performed a transcriptome analysis of S. Typhimurium under the acid tolerance response (ATR) and found a large number of genes (∼47%) to be differentially expressed (more than 1.5-fold or less than -1.5-fold; P < 0.01). Functional annotation revealed differentially expressed genes to be associated with regulation, metabolism, transport and binding, pathogenesis, and motility. Additionally, our knockout analysis of a subset of differentially regulated genes facilitated the identification of proteins that contribute to S. Typhimurium ATR and virulence. Mutants lacking genes encoding the K(+) binding and transport protein KdpA, hypothetical protein YciG, the flagellar hook cap protein FlgD, and the nitrate reductase subunit NarZ were significantly deficient in their ATRs and displayed varied in vitro virulence characteristics. This study offers greater insight into the transcriptome changes of S. Typhimurium under the ATR and provides a framework for further research on the subject.

Rod-to-hook transition for extracellular flagellum assembly is catalyzed by the L-ring-dependent rod scaffold removal.

In Salmonella, the rod substructure of the flagellum is a periplasmic driveshaft that couples the torque generated by the basal body motor to the extracellular hook and filament. The rod subunits self-assemble, spanning the periplasmic space and stopping at the outer membrane when a mature length of ~22 nm is reached. Assembly of the extracellular hook and filament follow rod completion. Hook initiation requires that a pore forms in the outer membrane and that the rod-capping protein, FlgJ, dislodges from the tip of the distal rod and is replaced with the hook-capping protein, FlgD. Approximately 26 FlgH subunits form the L-ring around the distal rod that creates the pore through which the growing flagellum will elongate from the cell body. The function of the L-ring in the mature flagellum is also thought to act as a bushing for the rotating rod. Work presented here demonstrates that, in addition to outer membrane pore formation, L-ring formation catalyzes the removal of the FlgJ rod cap. Rod cap removal allows the hook cap to assemble at the rod tip and results in the transition from rod completion in the periplasm to extracellular hook polymerization. By coupling the rod-to-hook switch to outer membrane penetration, FlgH ensures that hook and filament polymerization is initiated at the appropriate spatial and temporal point in flagellar biosynthesis.

Cryoelectron tomography reveals the sequential assembly of bacterial flagella in Borrelia burgdorferi

Periplasmic flagella are essential for the distinctive morphology, motility, and infectious life cycle of the Lyme disease spirochete Borrelia burgdorferi. In this study, we genetically trapped intermediates in flagellar assembly and determined the 3D structures of the intermediates to 4-nm resolution by cryoelectron tomography. We provide structural evidence that secretion of rod substrates triggers remodeling of the central channel in the flagellar secretion apparatus from a closed to an open conformation. This open channel then serves as both a gateway and a template for flagellar rod assembly. The individual proteins assemble sequentially to form a modular rod. The hook cap initiates hook assembly on completion of the rod, and the filament cap facilitates filament assembly after formation of the mature hook. Cryoelectron tomography and mutational analysis thus combine synergistically to provide a unique structural blueprint of the assembly process of this intricate molecular machine in intact cells.

Role of the Dc domain of the bacterial hook protein FlgE in hook assembly and function.

The bacterial flagellar hook acts as a universal joint to smoothly transmit torque produced by the motor to the filament. The hook protein FlgE assembles into a 55 nm tubular structure with the help of the hook cap (FlgD). FlgE consists of four domains, D0, Dc, D1 and D2, arranged from the inner to the outer part of the tubular structure of the hook. The Dc domain contributes to the structural stability of the hook, but it is unclear how this Dc domain is responsible for the universal joint mechanism. Here, we carried out a deletion analysis of the FlgE Dc domain. FlgEΔ4/5 with deletion of residues 30 to 49 was not secreted into the culture media. FlgEΔ5 and FlgEΔ6 with deletions of residues 40 to 49 and 50 to 59, respectively, still formed hooks, allowing the export apparatus to export the hook-filament junction proteins FlgK and FlgL and flagellin FliC. However, these deletions inhibited the replacement of the FlgD hook cap by FlgK at the hook tip, thereby abolishing filament formation. Deletion of residues 50 to 59 significantly affected hook morphology. These results suggest that the Dc domain is responsible not only for hook assembly but also for FlgE export, the interaction with FlgK, and the polymorphic supercoiling mechanism of the hook.

Molecular Characterization of the Flagellar Hook in Bacillus subtilis

The structure of the Gram-positive flagellum is poorly understood, and Bacillus subtilis encodes three proteins homologous to the flagellar hook protein from Salmonella enterica. Here we generated a modified B. subtilis hook protein that could be fluorescently stained using a cysteine-reactive dye. We used the fluorescently labeled hook to demonstrate that FlgE is the hook structural protein and that FliK regulated hook length. We further demonstrate that two proteins of unknown function, FlhO and FlhP, and the putative hook cap, FlgD, were required for hook assembly, such that when flhO, flhP, or flgD was mutated, hook protein was secreted into the supernatant. All mutants defective in hook completion resulted in homogeneously reduced σ(D)-dependent gene expression due to the action of the anti-sigma factor FlgM.

Genetic analysis of the bacterial hook-capping protein FlgD responsible for hook assembly

FlgD of Salmonella enterica is a 232 aa protein that acts as the hook cap to promote assembly of FlgE into the hook structure. The N-terminal 86 residues (FlgD(N)) complement flgD mutants, albeit to a small degree. However, little is known about the role of the C-terminal region of FlgD (FlgD(C)). Here we isolated pseudorevertants from Salmonella flgE mutants. About half of the extragenic mutations lay within FlgD(C) and only one in FlgD(N). These suppressor mutations prevented mutant FlgE subunits from leaking out to some degree. Two weakly motile flgD mutants encoding C-terminally truncated variants, FlgD₁₋₁₉₅ and FlgD((1-138f-s+4aa)), secreted larger amounts of FlgE into the culture medium than wild-type cells. Their hooks were shorter, and their length distributions were broader, with significant tailing towards smaller values. These results suggest that FlgD(C) contributes to efficient hook polymerization. Therefore, we propose that FlgD(N) attaches to the distal end of the hook to promote hook polymerization and that FlgD(C) blocks the exit of newly exported FlgE monomers into the culture medium, allowing FlgE to have more time to assemble into the hook.

Cloning, purification, crystallization and preliminary X-ray studies of flagellar hook scaffolding protein FlgD fromPseudomonas aeruginosaPAO1

FlgD regulates the assembly of the hook cap structure to prevent leakage of hook monomers into the medium and hook monomer polymerization and also plays a role in determination of the correct hook length, with the help of the FliK protein. In order to better elucidate the functions of FlgD in flagellar hook biosynthesis, the three-dimensional structure of FlgD is being determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of FlgD from P. aeruginosa are reported. The crystal belonged to space group I222 and diffracted to a resolution of 2.5 A, with unit-cell parameters a = 116.47, b = 118.71, c = 118.85 A. The crystals are most likely to contain three molecules in the asymmetric unit, with a V(M) value of 2.73 A(3) Da(-1).

Mutations in Flk, FlgG, FlhA, and FlhE That Affect the Flagellar Type III Secretion Specificity Switch in Salmonella enterica

Upon completion of the flagellar hook-basal body (HBB) structure, the flagellar type III secretion system switches from secreting rod/hook-type to filament-type substrates. The secretion specificity switch has been reported to occur prematurely (prior to HBB completion) in flk-null mutants (P. Aldridge, J. E. Karlinsey, E. Becker, F. F. Chevance, and K. T. Hughes, Mol. Microbiol. 60:630-643, 2006) and in distal rod gene gain-of-function mutants (flgG* mutants) that produce filamentous rod structures (F. F. Chevance, N. Takahashi, J. E. Karlinsey, J. Gnerer, T. Hirano, R. Samudrala, S. Aizawa, and K. T. Hughes, Genes Dev. 21:2326-2335, 2007). A fusion of beta-lactamase (Bla) to the C terminus of the filament-type secretion substrate FlgM was used to select for mutants that would secrete FlgM-Bla into the periplasmic space and show ampicillin resistance (Ap(r)). Ap(r) resulted from null mutations in the flhE gene, C-terminal truncation mutations in the flhA gene, null and dominant mutations in the flk gene, and flgG* mutations. All mutant classes required the hook length control protein (FliK) and the rod cap protein (FlgJ) for the secretion specificity switch to occur. However, neither the hook (FlgE) nor the hook cap (FlgD) protein was required for premature FlgM-Bla secretion in the flgG* and flk mutant strains, but it was in the flhE mutants. Unexpectedly, when deletions of either flgE or flgD were introduced into flgG* mutant strains, filaments were able to grow directly on the filamentous rod structures.

Crystal structure of FlgD fromXanthomonas: insights into the hook capping for flagellar assembly

Crystal structure of FlgD from<i>Xanthomonas</i>: insights into the hook capping for flagellar assembly

Crystal Structure of the C-Terminal Domain of a Flagellar Hook-Capping Protein from Xanthomonas campestris

The crystal structure of the C-terminal domain of a hook-capping protein FlgD from the plant pathogen Xanthomonas campestris (Xc) has been determined to a resolution of ca 2.5 Å using X-ray crystallography. The monomer of whole FlgD comprises 221 amino acids with a molecular mass of 22.7 kDa, but the flexible N-terminus is cleaved for up to 75 residues during crystallization. The final structure of the C-terminal domain reveals a novel hybrid comprising a tudor-like domain interdigitated with a fibronectin type III domain. The C-terminal domain of XcFlgD forms three types of dimers in the crystal. In agreement with this, analytical ultracentrifugation and gel filtration experiments reveal that they form a stable dimer in solution. From these results, we propose that the Xc flagellar hook cap protein FlgD comprises two individual domains, a flexible N-terminal domain that cannot be detected in the current study and a stable C-terminal domain that forms a stable dimer.

2P-067 細菌べん毛フックキャップの結晶構造(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

2P-067 細菌べん毛フックキャップの結晶構造(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

Crystallization and X-ray analysis of a flagellar hook capping protein

Crystallization and X-ray analysis of a flagellar hook capping protein

2P133 べん毛フックキャップタンパク質FlgDの変異解析(細胞生物的課題 : 接着・運動・骨格・伝達・膜)

2P133 べん毛フックキャップタンパク質FlgDの変異解析(細胞生物的課題 : 接着・運動・骨格・伝達・膜)

Role of FliJ in flagellar protein export in Salmonella.

We isolated and characterized spontaneous mutants with defects in the 147-amino-acid Salmonella protein FliJ, which is a cytoplasmic component of the type III flagellar export apparatus. These mutants, including ones with null mutations, have the ability to form swarms on motility agar plates after prolonged incubation at 30 degrees C; i.e., they display a leaky motile phenotype. One mutant, SJW277, which formed significantly bigger swarms than the others, encoded only the N-terminal 73 amino acids of FliJ, one-half of the protein. At 30 degrees C, overproduction of this mutant protein improved, to wild-type levels, both motility and the ability to export both rod/hook-type (FlgD; hook capping protein) and filament-type (FliC; flagellin) substrates. At 42 degrees C, however, export was inhibited, indicating that the mutant FliJ protein was temperature sensitive. Taking advantage of this, we performed temperature upshift experiments, which demonstrated that FliJ is directly required for the export of FliC. Co-overproduction of FliJ and either of two export substrates, FliE or FlgG, hindered their aggregation in the cytoplasm. We conclude that FliJ is a general component of the flagellar export apparatus and has a chaperone-like activity for both rod/hook-type and filament-type substrates.

Interactions among components of the Salmonella flagellar export apparatus and its substrates.

We have examined the cytoplasmic components (FliH, FliI and FliJ) of the type III flagellar protein export apparatus, plus the cytoplasmic domains (FlhAC and FlhBC) of two of its six membrane components. FliH, FlhAC and FliJ, when overproduced, caused inhibition of motility of wild-type cells and inhibition of the export of substrates such as the hook protein FlgE. Co-overproduction of FliH and FliI substantially relieved the inhibition caused by FliH, suggesting that it is excess free FliH that is inhibitory and that FliH and FliI form a complex. We purified His-FLAG-tagged versions of: (i) export components FliH, FliI, FliJ, FlhAC and FlhBC; (ii) rod/hook-type export substrates FlgB (rod protein), FlgE (hook protein), FlgD (hook capping protein) and FliE (basal body protein); and (iii) filament-type export substrates FlgK and FlgL (hook-filament junction proteins) and FliC (flagellin). We tested for protein-protein interactions by affinity blotting. In many cases, a given protein interacted with more than one other component, indicating that there are likely to be multiple dynamic interactions or interactions that involve more than two components. Interactions of FlhBC with rod/hook-type substrates were strong, whereas those with filament-type substrates were very weak; this may reflect the role of FlhB in substrate specificity switching. We propose a model for the flagellar export apparatus in which FlhA and FlhB and the other four integral membrane proteins of the apparatus form a complex at the base of the flagellar motor. A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP-dependent process mediated by FliI.

FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly.

In wild-type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at approximately 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild-type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook-filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N-terminal region of FliK, but not by C-terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook-length control and the switching of export substrate specificity.

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.