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1.
We report, based on proteolytic experiments and high resolution 1H nuclear magnetic resonance studies that the terminal regions of the monomeric hook protein are highly mobile and exposed to the solvent. The disordered parts of the hook protein span approximately the first 70 and the last 30 amino acid residues. Although the amino acid sequences of flagellin and hook protein do not resemble each other at all, both proteins have now been shown to contain large disordered terminal regions. Sequential similarities of flagellin and hook protein, especially near the NH2 and COOH termini, to other axial components of bacterial flagellum suggest that terminal disorder may be a common structural feature of the axial proteins of the bacterial flagellum. 相似文献
2.
Flagellar hook and hook-associated proteins of Salmonella typhimurium and their relationship to other axial components of the flagellum 总被引:27,自引:0,他引:27
Within the bacterial flagellum the basal-body rod, the hook, the hook-associated proteins (HAPs), and the helical filament constitute an axial substructure whose elements share structural features and a common export pathway. We present here the amino acid sequences of the hook protein and the three HAPs of Salmonella typhimurium, as deduced from the DNA sequences of their structural genes (flgE, flgK, flgL and fliD, respectively). We compared these sequences with each other and with those for the filament protein (flagellin) and four rod proteins, which have been described previously (Joys, 1985; Homma et al., 1990; Smith & Selander, 1990). Hook protein most strongly resembled the distal rod protein (FlgG) and the proximal HAP (HAP1), which are thought to be attached to the proximal and distal ends of the hook, respectively; the similarities were most pronounced near the N and C termini. Hook protein and flagellin, which occupy virtually identical helical lattices, did not resemble each other strongly but showed some limited similarities near their termini. HAP3 and HAP2, which form the proximal and distal boundaries of the filament, showed few similarities to flagellin, each other, or the other axial proteins. With the exceptions of the N-terminal region of HAP2, and the C-terminal region of flagellin, proline residues were absent from the terminal regions of the axial proteins. Moreover, with the exception of the N-terminal region of HAP2, the terminal regions contained hydrophobic residues at intervals of seven residues. Together, these observations suggest that the axial proteins may have amphipathic alpha-helical structure at their N and C termini. In the case of the filament and the hook, the terminal regions are believed to be responsible for the quaternary interactions between subunits. We suggest that this is likely to be true of the other axial structures as well, and specifically that interaction between N-terminal and C-terminal alpha-helices may be important in the formation of the axial structures of the flagellum. Although consensus sequences were noted among some of the proteins, such as the rod, hook and HAP1, no consensus extended to the entire set of axial proteins. Thus the basis for recognition of a protein for export by the flagellum-specific pathway remains to be identified. 相似文献
3.
Localization and stoichiometry of hook-associated proteins within Salmonella typhimurium flagella. 总被引:8,自引:6,他引:2
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The localization of hook-associated proteins (HAP1, HAP2, and HAP3) in Salmonella typhimurium flagella was studied by using specific antibodies together with a second antibody conjugated with colloidal gold. HAP1 and HAP3 were localized at the hook-filament junction, as has been suggested previously. HAP2, however, was localized at the filament tip. This finding supports the idea that HAP2 acts to induce polymerization of endogenous flagellin at the filament tip, and HAP1 and HAP3 are junction proteins to connect hook with filament. Analysis of the protein composition of short flagella from a mutant indicated that a single flagellum contains about 10 to 20 HAP1, 10 to 20 HAP2, and 10 to 40 HAP3 molecules. 相似文献
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The flagellar hook is a short, curved, extracellular structure located between the basal body and the filament. The hook is composed of the FlgE protein. In this study, we analyzed flagellum assembly in a temperature-sensitive flgE mutant of Salmonella enterica serovar Typhimurium. When the mutant cells were grown at 30°C, they produced flagella of a normal length (71% of the population) and short hooks without filaments (26%). At 37°C, 70% of the basal bodies lacked hooks, and intact flagella made up only 6% of the population. Mutant cells secreted monomeric FlgE in abundance at 37°C, suggesting that the mutant FlgE protein might be defective in polymerization at higher temperatures. The average length of the hooks in intact filaments was 55 nm, whereas after acid treatment, it was 45 nm. SDS-PAGE analysis of the hook-basal body showed that HAP1 was missing in the mutant but not in the wild type. We concluded that hook length in the mutant is controlled in the same way as in the wild type, but the hook appeared short after acid treatment due to the lack of HAP1. We also learned that the true length of the hook is possibly 45 nm, not 55 nm, as has been believed. 相似文献
6.
Locations of hook-associated proteins in flagellar structures of Salmonella typhimurium. 总被引:13,自引:10,他引:3
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Hooks of the flagella of Salmonella typhimurium were purified from an flaL mutant. Hook-associated proteins, namely HAP1, HAP2, and HAP3, were separated from them, and the antibody against each HAP was prepared. By immunoelectron microscopic observation, these three kinds of antiHAP antibodies were found to bind on the distal ends of hooks of filamentless mutants consistently with their composition of HAPs. The antiHAP2 antibody bound to the very tops of the claw-shaped ends of the hooks which contain all three HAPS. The antibodies against HAP1 and HAP3 bound to the basal areas and the middle areas, respectively, of the claw-shaped ends. The order of disassembly of the component proteins by heat treatment of the hook structure from the filamentless mutants was (HAP2, HAP3) greater than HAP1 greater than hook protein. These observations were consistent with our layered structure model: HAP1, HAP3, and HAP2 are assembled at the distal end of the hook in this sequence. All three HAPs were detected in the hook-filament complexes prepared from a flagellate strain. When the hook-filament structure was treated with antibody against HAP1 and with the anti-rabbit immunoglobulin G antibody, the antibody aggregate was observed in the region corresponding to the boundary between filament and hook. This observation strongly suggests that HAP1 is the protein connecting filament with hook. The locations of HAP2 and HAP3 in the hook-filament structure were not clarified with the same procedure. 相似文献
7.
Hinderlich S Salama I Eisenberg I Potikha T Mantey LR Yarema KJ Horstkorte R Argov Z Sadeh M Reutter W Mitrani-Rosenbaum S 《FEBS letters》2004,568(1-3):105-109
In vivo growth of bacterial flagellar filaments by self-assembly of flagellin is promoted by a capping structure composed of a pentameric assembly of hook associated protein 2 (HAP2). Isolated native filaments with intact HAP2 cap exhibited higher melting temperature (deltaTm = 4 degrees C) and significantly increased resistance against heat-induced depolymerization than non-capped ones. Reconstituted filaments were also stabilized by HAP2 binding, but the obtained filament-HAP2 complexes were less stable than native assemblies. Their fast depolymerization at elevated temperatures and sensitivity to proteolysis indicated that native-like filament-HAP2 complexes are rarely obtained by in vitro reconstitution. A procedure was developed to isolate perfectly capped native filaments to facilitate high-resolution structural analysis. 相似文献
8.
During flagellum assembly by motile enterobacteria, flagellar axial proteins destined for polymerization into the cell surface structure are thought to be exported through the 25–30 Å flagellum central channel as partially unfolded monomers. How are premature folding and oligomerization in the cytosol prevented? We have shown previously using hyperflagellated Proteus mirabilis and a motile but non-swarming flgN transposon mutant that the apparently cytosolic 16.5 kDa flagellar protein FlgN facilitates efficient flagellum filament assembly. Here, we investigate further whether FlgN, predicted to contain a C-terminal amphipathic helix typical of type III export chaperones, acts as a chaperone for axial proteins. Incubation of soluble radiolabelled FlgN from Salmonella typhimurium with nitrocellulose-immobilized cell lysates of wild-type S. typhimurium and a non-flagellate class 1 flhDC mutant indicated that FlgN binds to flagellar proteins. Identical affinity blot analysis of culture supernatants from the wild-type and flhDC, flgI, flgK, flgL, fliC or fliD flagellar mutants showed that FlgN binds to the flagellar hook-associated proteins (HAPs) FlgK and FlgL. This was confirmed by blotting artificially expressed individual HAPs in Escherichia coli. Analysis of axial proteins secreted into the culture medium by the original P. mirabilis flgN mutant demonstrated that export of FlgK and FlgL was specifically reduced, with concomitant increased release of unpolymerized flagellin (FliC), the immediately distal component of the flagellum. These data suggest that FlgN functions as an export chaperone for FlgK and FlgL. Parallel experiments showed that FliT, a similarly small (14 kDa), potentially helical flagellar protein, binds specifically to the flagellar filament cap protein, FliD (HAP2), indicating that it too might be an export chaperone. Flagellar axial proteins all contain amphipathic helices at their termini. Removal of the HAP C-terminal helical domains abolished binding by FlgN and FliT in each case, and polypeptides comprising each of the HAP C-termini were specifically bound by FlgN and FliT. We suggest that FlgN and FliT are substrate-specific flagellar chaperones that prevent oligomerization of the HAPs by binding to their helical domains before export. 相似文献
9.
Interaction of FliI, a component of the flagellar export apparatus, with flagellin and hook protein.
FliI is a key component of the flagellar export apparatus in Salmonella typhimurium. It catalyzes the hydrolysis of ATP which is necessary for flagellar assembly. Affinity blotting experiments showed that purified flagellin and hook protein, two flagellar axial proteins, interact specifically with FliI. The interaction of either of the two proteins with FliI, increases the intrinsic ATPase activity. The presence of either flagellin or hook protein stimulates ATPase activity in a specific and reversible manner. A Vmax of 0.12 nmol Pi min-1 microgram-1 and a Km for MgATP of 0.35 mM was determined for the unstimulated FliI; the presence of flagellin increased the Vmax to 0.35 nmol Pi min-1 microgram-1 and the Km for MgATP to 1.1 mM. The stimulation induced by the axial proteins was fully reversible suggesting a direct link between the catalytic activity of FliI and the export process. 相似文献
10.
Hook-associated proteins essential for flagellar filament formation in Salmonella typhimurium. 总被引:20,自引:14,他引:6
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The hooks of the flagella of Salmonella typhimurium were purified by a newly developed method, using a flaL mutant without a filament, and the hook components were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, we detected three protein species in addition to hook protein. We call these three proteins hook-associated proteins (HAPs). Their molecular weights were 59,000 for HAP1, 53,000 for HAP2, and 31,000 for HAP3. The HAP1/hook protein/HAP3/HAP2 molar ratio, calculated from their relative amounts and their molecular weights, was 1:10:1.1:0.53. The compositions of HAPs were analyzed in the hooks from the other filamentless mutants which were defective in H1 H2, flaV, flaU, or flaW. Hooks from the H1 H2 mutant had the same HAP composition as hooks from the flaL mutant. Hooks from the flaV mutants contained HAP1 and HAP3. Hooks from the flaU mutants contained HAP1. Hooks from the flaW mutants contained a very small amount of HAP3. From these results, the process of hook morphogenesis and the genes responsible for each step were postulated. Electron micrographs of hooks from the filamentless mutants showed that hooks which contained all three HAPs had a sharp clawlike tip, whereas hooks lacking any HAP had a flat tip. Electron micrographs of hooks treated with antibody against the hook protein showed that each claw-shaped end was not covered with antibody. These results strongly suggest that all three HAPs or at least some of them are located at the claw-shaped end and play an essential role in filament formation. 相似文献
11.
Termini of Salmonella flagellin are disordered and become organized upon polymerization into flagellar filament 总被引:5,自引:0,他引:5
The terminal regions of Salmonella flagellin are essential for polymerization to form the flagellar filament. It has recently been suggested, on the basis of results from circular dichroism spectroscopy and scanning calorimetry, that these regions are disordered in solution. We report here direct evidence for disorder and mobility in the terminal regions of flagellin using 400 MHz proton nuclear magnetic resonance (n.m.r.) spectroscopy. Comparison of the n.m.r. spectra of monomeric and polymeric flagellin shows that the terminal regions become organized when polymerized to form the filament. 相似文献
12.
Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook of Salmonella 总被引:3,自引:0,他引:3
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Kazumasa Muramoto Shigeru Makishima Shin-Ichi Aizawa Robert M. Macnab 《Journal of bacteriology》1999,181(18):5808-5813
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. 相似文献
13.
Synthesis and assembly of flagellar components by Caulobacter crescentus motility mutants 总被引:17,自引:9,他引:8
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Cultures of wild-type Caulobacter crescentus and strains with fla mutations representing 24 genes were pulse-labeled with 14C-amino acids and analyzed by immunoprecipitation to study the synthesis of flagellar components. Most fla mutants synthesize flagellin proteins at a reduced rate, suggesting the existence of some mechanism to prevent the accumulation of unpolymerized flagellin subunits. Two strains contain deletions that appear to remove a region necessary for this regulation. The hook protein does not seem to be subject to this type of regulation and, in addition, appears to be synthesized as a faster-sedimenting precursor. Mutations in a number of genes result in the appearance of degradation products of either the flagellin or the hook proteins. Mutations in flaA, -X, -Y, or -Z result in the production of filaments (stubs) that contain altered ratios of the flagellin proteins. In some flaA mutants, other flagellin-related proteins were assembled into the stub structures in addition to the flagellins normally present. Taken together, these analyses have begun to provide insight into the roles of individual fla genes in flagellum biogenesis in C. crescentus. 相似文献
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The organization of the Caulobacter crescentus flagellar filament 总被引:10,自引:0,他引:10
The structural organization of the flagellar filament of Caulobacter crescentus, as revealed by immunoelectron microscopy, shows five antigenically distinct regions within the hook-filament complex. The first region is the hook. The second region is adjacent to the hook and is approximately 10 nm in length. On the basis of its location in the hook-filament complex, this region may contain hook-associated proteins. Next to this is the third region, which is approximately 60 nm in length. Antibody decoration experiments using mutant strains with deletions of the structural gene for the 29 x 10(3) Mr flagellin (flgJ) showed that the presence of this region is correlated with the expression of the 29 x 10(3) Mr flagellin gene. The next region (region IV), of length approximately 1 to 2 microns, appears to contain the 27.5 x 10(3) Mr flagellin, but at its distal end includes, in gradually increasing amounts, the 25 x 10(3) Mr flagellin. The rest of the filament (region V) is made up predominantly, if not completely, of the 25 x 10(3) Mr flagellin. Except for the hook, there are no morphological features that would otherwise distinguish these regions. A functional flagellum, having the wild-type length and morphology, is assembled by mutant strains deficient in the 29 x 10(3) Mr flagellin and 27.5 x 10(3) Mr flagellin. 相似文献
17.
Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments. 相似文献
18.
The hypervariable D3 domain of Salmonella flagellin, composed of residues 190-283, is situated at the outer surface of flagellar filaments. A flagellin mutant deprived of the complete D3 domain (ΔD3_FliC) exhibited a significantly decreased thermal stability (Tm 41.9 °C) as compared to intact flagellin (Tm 47.3 °C). However, the stability of filaments formed from ΔD3_FliC subunits was virtually identical with that of native flagellar filaments. While D3 comprises the most stable part of monomeric flagellin playing an important role in the stabilization of the other two (D1 and D2) domains, the situation is reversed in the polymeric state. Upon filament formation, ordering of the disordered terminal regions of flagellin in the core part of the filament results in the stabilization of the radially arranged D1 and D2 domains, and there is a substantial increase of stability even in the distant outermost D3 domain, which is connected to D2 via a pair of short antiparallel β-strands. Our experiments revealed that crosslinking the ends of the isolated D3 domain through a disulfide bridge gives rise to a stabilization effect reminiscent of that observed upon polymerization. It appears that the short interdomain linker between domains D2 and D3 serves as a stabilization center that facilitates propagation of the conformational signal from the filament core to the outer part of filament. Because D3 is a largely independent part of flagellin, its replacement by heterologous proteins or domains might offer a promising approach for creation of various fusion proteins possessing polymerization ability. 相似文献
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Using spectrophotometrical titration, chemical modification, and ultraviolet difference spectral methods, the existence of at least two distinct tyrosine groups in the isolated flagellin of Proteus mirabilis flagella has been established. Three of the five flagellin tyrosines are buried in the protein matrix, whereas the other two seem to lie on the protein surface accessible to perturbants. Also about two tyrosine residues, presumably the latter ones exposed to the environment, can be nitrated with tetranitromethane in the monomeric flagellin with a concomitant loss of the polymerization ability after about one tyrosine per mol flagellin has been nitrated. Nitrated flagellin, homogeneous with respect to molecular weight, degree of nitration and isoelectric point, could be isolated and characterized. On the other hand, it could be shown that in the polymeric flagellum the phenolic groups of all five tyrosine residues are inaccessible to perturbing and modifying reagents. It seems, therefore, that the integrity of the phenolic groups is necessary for the proper folding and aggregation of the flagellin subunits to form the stable helical flagella. 相似文献