Hook Assembly Research Articles

Edwardsiella tarda flagellar protein FlgD: A protective immunogen against edwardsiellosis

Edwardsiella tarda is a gram-negative bacterium and a causative agent of edwardsiellosis, resulting to severe loss of the aquaculture industry. In this study, based on the reverse vaccinology, sixteen flagellar proteins were selected from highly pathogenic E. tarda EIB202 genome information and in silico analyzed as potential vaccine candidates. Among them, ten recombinant proteins were highly expressed in Escherichia coli and successfully purified. The immunoprotective potentials of these purified recombinant proteins were evaluated in zebrafish model. And recombinant FlgD and FliD were found to lead to a high relative percent survival (RPS, about 70%) against E. tarda EIB202. Furthermore, FlgD required in flagellum hook assembly brought about the similar immune protection in turbot. The immune responses of zebrafish and turbot to recombinant FlgD were also investigated, and the results indicated that its high protection was mainly involved in cellular mediated immune response, corresponding to the intracellular pathogenicity of E. tarda.

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.

Fatigue life simulation of a rear tow hook assembly of a passenger car

A comparison between laboratory test data on fatigue crack nucleation in a rear tow hook pin assembly of passenger vehicle and a computational methodology using commercial package software is presented. Fatigue damage is determined using local material response, measured during experimental tests. Experiments were performed simulating the actual conditions in the customer environment. Stress and strain were experimentally measured by using strain gages, bonded on the hook assembly. These experimental lives are compared with those obtained through numerical analysis using a commercial fatigue software. Fatigue analysis methods ( S– N curves, rainflow counting and Miner rule) were used to determine the fatigue damage imposed on the component. Interpretation and evaluation of the measured strain and stresses, simulation tests and fatigue life assessments, on the basis of S– N curve, are described in this paper.

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)

Two Parts of the T3S4 Domain of the Hook-length Control Protein FliK Are Essential for the Substrate Specificity Switching of the Flagellar Type III Export Apparatus

The switch in export specificity of the type III flagellar protein export apparatus from rod/hook type to filament type is believed to occur upon completion of hook assembly by way of an interaction of the type III secretion substrate specificity switch (T3S4) domain of the hook-length control protein FliK, with the integral membrane export apparatus component FlhB. The T3S4 domain of FliK (FliK T3S4) consisting of amino acid residues 265–405 has an unstable and flexible conformation in its last 35 residues (FliK CT). To investigate the role of FliK T3S4 in substrate specificity switching, we studied the effect of deletions and point mutations within this domain and characterized suppressor mutations. Deletions of ten amino acid residues within the region of residues 301–350 and five amino acids of residues 401–405 abolished switching of export specificity. Site directed mutagenesis showed that highly conserved residues, Val302, Ile304, Leu335, Val401 and Ala405, are essential, and that the five C terminal residues (401–405) are restricted in conformation for the switching process. Suppressor mutant analysis of the fliK(S319Y) mutant, which produces extended hooks with filaments attached due to delayed switching, suggested that FliK T3S4 interacts with the C terminal half of the cytoplasmic domain of FlhB (FlhB C). We propose a two step binding model of FliK T3S4 and FlhB C, in which residues 301–350 of FliK bind to FlhB C upon hook assembly completion at about 55 nm, and then unfolded FliK CT binds to FlhB C to trigger the switch in substrate specificity.

The Type III Flagellar Export Specificity Switch is Dependent on FliK Ruler and a Molecular Clock

Salmonella flagellar hook length is controlled at the level of export substrate specificity of the FlhB component of the type III flagellar export apparatus. FliK is believed to be the hook length sensor and interacts with FlhB to change its export specificity upon hook completion. To find properties of FliK expected of such a molecular ruler, we assayed binding of FliK to the hook and found that the N-terminal domain of FliK (FliK(N)) bound to the hook-capping protein FlgD with high affinity and to the hook protein FlgE with low affinity. To investigate a possible role of FlgE in hook length control, flgE mutants with partially impaired motility were isolated and analyzed. Eight flgE mutants obtained all formed flagellar filaments. The mutants produced significantly shorter hooks while the hook-type substrates such as FlgE, FliK and FlgD were secreted in large amounts, suggesting defective hook assembly with the mutant FlgE proteins. Upon overexpression, mutant FlgEs produced hooks of normal length and wild-type FlgE produced longer hooks. These results suggest that hook length is dependent on the hook polymerization rate and that the start of hook polymerization initiates a "time countdown" for the specificity switch to occur or for significant slow down of rod/hook-type export after hook length reaches around 55 nm for later infrequent FliK(C)-FlhB(C) interaction. We propose that FliK(N) acts as a flexible tape measure, but that hook length is also dependent on the hook elongation rate and a switch timing mechanism.

1P187 べん毛フック構築におけるFlgD-C末領域の役割(接着・運動・骨格・伝達・膜)))

1P187 べん毛フック構築におけるFlgD-C末領域の役割(接着・運動・骨格・伝達・膜)))

Domain Organization and Function of Salmonella FliK, a Flagellar Hook-length Control Protein

Salmonella hook-length control protein FliK, which consists of 405 amino acid residues, switches substrate specificity of the type III flagellar protein export apparatus from rod/ hook-type to filament-type by causing a conformational change in the cytoplasmic domain of FlhB (FlhBC) upon completion of the hook assembly. An N-terminal region of FliK contains an export signal, and a highly conserved C-terminal region consisting of amino acid residues 265–405 (FliK(265–405)) is directly involved in the switching of FlhBC. Here, we have investigated the structural properties of FliK. Gel filtration chromatography, multi-angle light scattering and analytical ultracentrifugation showed that FliK is monomeric in solution and has an elongated shape. Limited proteolysis showed that FliK consists of two domains, the N-terminal (FliKN) and C-terminal domains (FliKC), and that the first 203 and the last 35 amino acid residues are partially unfolded and subjected to proteolysis. Both FliKN and FliKC are more globular than full-length FliK, suggesting that these domains are connected in tandem. Overproduced His-FliK(199–405) failed to switch export specificity of the export apparatus. Affinity blotting revealed that FlhBC binds to FliK and FliK(1–147), but not to FliK(265–405). Based on these results, we propose that FliKN within the central channel of the hook-basal body during the export of FliK is the sensor and transmitter of hook completion information and that the binding interaction of FliKC to FlhBC is structurally regulated by FliKN so as to occur only when the hook has reached a preset length. The conformational flexibility of FliKC may play a role in interfering with switching at an inappropriate point of flagellar assembly.

Organization, expression, and function of Caulobacter crescentus genes needed for assembly and function of the flagellar hook.

This paper reports on the organization, expression, and function of the divergently transcribed flbG and flaN operons in the hook gene cluster of Caulobacter crescentus. The transcription initiation site of flbG was determined previously, and in this work the transcription map was completed by locating the 3' end of the mRNA using nuclease S1 protection assays. A previous genetic study had suggested that the flbG operon is comprised of four genes; however, the nucleotide sequence revealed three tandemly arranged ORFs that correspond to 5'-flbG, flbH, and flgE. FlbG is similar to FliK proteins which are required for termination of hook synthesis, FlbH is similar to FlgD proteins which are essential scaffolding proteins that cap the hook during its assembly, and FlgE corresponds to the hook structural protein. The divergently transcribed flaN gene codes for a hook associated protein I homolog based on its inferred amino acid sequence similarity to FlgK proteins. Based on the amino acid sequence similarities and phenotypes of mutants, flbG, flbH, and flaN have been renamed fliK, flgD, and flgK, FlgD, FlgE, and FlgK proteins, with apparent molecular masses of 23, 68, and 41 kDa, respectively, were expressed from plasmids in a cell-free coupled transcription-translation system, and a protein corresponding to FliK was identified as part of a 190-kDa FliK-LacZ fusion protein. We present evidence showing that, in addition to its role in termination of hook synthesis, FliK is also required for initiation of hook assembly.

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.

Effect of hook subunit concentration on assembly and control of length of the flagellar hook of Salmonella.

The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, a fliC (flagellin) mutant, or a fliD (hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.

Components of the Salmonella flagellar export apparatus and classification of export substrates.

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.

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.

Lufenuron, a chitin-synthesis inhibitor, interrupts development of Drosophila melanogaster.

The chitin-synthesis inhibitor lufenuron was administered to Drosophila melanogaster to better understand the effects of chitin-synthesis interruption during the development and reproduction of this insect. When larvae were fed a diet containing a low concentration (< 1 ppm) of lufenuron, mortality was observed during either larval or pupal development, depending on the dose. Survivor adults were unable to fly, probably due to abnormal cuticular development in the wing hinge regions of the thorax. Larvae fed a higher concentration (10 ppm) completed development within that instar, but died during ecdysis to the next instar, presumably due to inadequate cuticle synthesis. Third instar larvae pupariated, but the puparium was abnormal, and pupation did not occur. Adults fed 10 ppm showed normal viability but slightly depressed oogenesis; additionally, although their eggs were fertilized, they failed to hatch. Examination of the embryos showed that they completed development but were unable to perforate the surrounding vitelline membrane, probably due to a weakened chitinous mouth hook assembly that was insufficiently rigid to effect hatching. These results identify stages during D. melanogaster development when chitin synthesis and deposition are critical. This information will be useful for identifying chitin-synthesis mutants of this insect.

Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis.

During flagellar morphogenesis in Salmonella typhimurium, the genes involved in filament assembly are expressed fully only after completion of hook-basal body assembly. This coupling of gene expression to morphogenesis is achieved by exporting the flagellum-specific anti-sigma factor, FlgM, out of the cell through the mature hook-basal body structure. Therefore, the flagellum-specific export apparatus must be able to sense the assembly state of the flagellar structure and to turn on FlgM export at a specific stage of hook assembly. It has been suggested that FlhB may act as the molecular switch which mediates this ordered export. Here, I report genetic evidence that in addition to FlhB, the product of a newly identified gene, rflH, is involved in the negative regulation of FlgM export. FlgM is released through the basal body structure lacking the hook and the filament only when the flhB and rflH genes are both defective. Therefore, the export gate for FlgM should be double locked by FlhB and RflH. The rflH gene is located at around 52 min, where no flagellum-related gene has been found. I propose a revised model of the export-switching machinery which consists of two systems, the hook-length signal transduction pathway and the double-locked gate for FlgM export.

Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium

Mutations in the fliK gene of Salmonella typhimurium commonly cause failure to terminate hook assembly and initiate filament assembly (polyhook phenotype). Polyhook mutants give rise to pseudorevertants which are still defective in hook termination but have recovered the ability to assemble filament (polyhook-filament phenotype). The polyhook mutations have been found to be either frameshift or nonsense, resulting in truncation of the C terminus of FliK. Intragenic suppressors of frameshift mutations were found to be ones that restored the original frame (and therefore the C-terminal sequence), but in most cases with substantial loss of natural sequence and sometimes the introduction of artificial sequence; in no cases did intragenic suppression occur when significant disruption remained within the C-terminal region. By use of a novel PCR protocol, in-frame deletions affecting the N-terminal and central regions of FliK were constructed and the resulting phenotypes were examined. Small deletions resulted in almost normal hook length control and almost wild-type swarming. Larger deletions resulted in loss of control of hook length and poor swarming. The largest deletions severely affected filament assembly as well as hook length control. Extragenic suppressors map to an unlinked gene, flhB, which encodes an integral membrane protein (T. Hirano, S. Yamaguchi, K. Oosawa, and S.-I. Aizawa, J. Bacteriol. 176:5439-5449, 1994; K. Kutsukake, T. Minamino, and T. Yokoseki, J. Bacteriol. 176:7625-7629, 1994). They were either point mutations in the C-terminal cytoplasmic region of FlhB or frameshift or nonsense mutations close to the C terminus. The processes of hook and filament assembly and the roles of FliK and FlhB in these processes are discussed in light of these and other available data. We suggest that FliK measures hook length and, at the appropriate point, sends a signal to FlhB to switch the substrate specificity of export from hook protein to late proteins such as flagellin.

Structural Organization and Assembly of Flagellar Hook Protein from Salmonella typhimurium

The terminal regions of monomeric hook protein from Salmonella typhimuriumare known to be highly mobile and exposed to the solvent. Although hook protein exhibits an unusual far UV circular dichroism spectrum, resembling that of random coil structures, our calorimetric experiments clearly demonstrate that the molecule has a compact ordered core. The compact part probably consists of three domains as suggested by deconvolution analysis of the calorimetric melting profiles. Secondary structure prediction, together with the analysis of fat-UV circular dichroism spectra, has shown that the domains of monomeric hook protein contain β-sheeted structures without significant α-helical content. The polymerization of hook protein is accompanied by the stabilization of its disordered terminal regions into a predominantly α-helical domain. Evaluation of circular dichroism data suggests that about 45 terminal residues are involved in helical segments. Coiled-coil prediction indicates that whereas the whole carboxy-terminal helical region of hook protein has a strong bundle-forming potential, there is only a single short amino-terminal segment exhibiting weak coiled-coil forming tendencies. The formation of α-helical bundles in commonly believed to be a key event during the polymerization of the axial structure of bacterial flagella. To clarify the role of helical bundle formation in hook assembly, proteolytic fragments of hook protein with truncations of various lengths in their carboxy-terminal disordered regions were generated, and their polymerizational behavior was investigated. We found that even fragments completely lacking the main helix-forming carboxy-terminal regions can polymerize into filaments in vitrounder appropriately high salt concentrations. Our results suggest that, although helical bundle formation may occur during self-assembly, governing precise subunit packing and playing an important role in the stabilization of hook filaments, it is not the principal interaction mainly responsible for the development of their filamentous structure.

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

FlgD is known to be absolutely required for hook assembly, yet it has not been detected in the mature flagellum. We have overproduced and purified FlgD and raised an antibody against it. By using this antibody, we have detected FlgD in substantial amounts in isolated basal bodies from flgA, flgE, flgH, flgI, flgK, and fliK mutants, in much smaller amounts in those from the wild type and flgL, fliA, fliC, fliD, and fliE mutants, and not at all in those from flgB, flgD, flgG, and flgJ mutants. In terms of the morphological assembly pathway, these results indicate that FlgD is first added to the structure when the rod is completed and is discarded when the hook, having reached its mature length, has the first of the hook-filament junction proteins, FlgK, added to its tip. Immunoelectron microscopy established that FlgD initially is located at the distal end of the rod and eventually is located at the distal end of the hook. Thus, it appears to act as a hook-capping protein to enable assembly of hook protein subunits, much as another flagellar protein, FliD, does for the flagellin subunits of the filament. However, whereas FliD is associated with the filament tip indefinitely, FlgD is only transiently associated with the hook tip; i.e., it acts as a scaffolding protein. When FlgD was added to the culture medium of a flgD mutant, cells gained motility; thus, although the hook cap is normally added endogenously, it can be added exogenously. When culture media were analyzed for the presence of hook protein, it was found only with the flgD mutant and, in smaller amounts, the fliK (polyhook) mutant. Thus, although FlgD is needed for assembly of hook protein, it is not needed for its export.

Image reconstruction of the flagellar basal body of Caulobacter crescentus

The bacterium Caulobacter crescentus has a single polar flagellum, which is present for only a portion of its cell cycle. The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. The flagellum is composed of a transmembrane basal body, a hook and a filament. Single-particle averaging and image reconstruction methods were applied to the electron micrographs of negatively stained basal bodies from C. crescentus. These basal bodies have five rings threaded on a rod. The L and P rings are connected by a bridge of material at their outer radii. The E ring is a thin, flat disk. The S ring has a triangular cross section, the sides of the triangle abutting the E ring, the rod and the M ring. The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. With one method, the M ring makes a snug contact with the S ring and is often capped by an axial button, a new component apparently distinct from the M ring. With the other method, the M ring is similar to that of S. typhimurium; that is, it contacts the S ring only at an outer radius and lacks the button. Averages of the rod-hook-filament subassembly ejected by swarmer cells reveal that the rod consists of two parts with the E ring marking the approximate position of the break. The structures of basal bodies from two mutants defective in the hook assembly were found to be indistinguishable from wild-type basal bodies, suggesting that the assembly of the basal body is independent of the hook or filament assembly.

Regulation of periodic protein synthesis in the cell cycle: Control of initiation and termination of flagellar gene expression

Conditional mutations and drugs that specifically block DNA synthesis and cell division in Caulobacter crescentus have been used to analyze the role of the cell cycle in differentiation. The results suggest that the DNA synthetic pathway, and not an intact cell division pathway, acts to time the periodic synthesis of flagellin A, flagellin B and the hook protein. The expression of these flagellar genes is apparently controlled at the level of transcription, with initiation of the synthetic periods dependent on a specific stage of chromosome replication. The required stage of replication, or DNA execution point, occurs near mid-S phase at approximately 0.65 of the swarmer cell cycle. Examination of a C. crescentus polyhook mutant PCM103 indicates, however, that the DNA synthetic pathway is only one level of regulation over flagellum formation. In the mutant, hook protein synthesis is coupled to DNA synthesis and is initiated at the expected time in the cell cycle, but hook protein synthesis is not repressed at the normal time, hook assembly fails to terminate and only a low level of flagellin is detected. Thus the defect in this strain, which grows and divides normally, appears to affect a sequence of developmental events at a point after the induction of the hook protein. These findings are consistent with a hierarchy of controls that regulates the periodic synthesis of flagellar proteins and their assembly in the flagellum. Initiation of hook protein and flagellin synthesis requires a specific stage of chromosome replication, while later stages of flagellum formation, including the termination of hook protein synthesis, also require a second level of regulation that is defective in strain PCM 103.