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Keiichi Namba - One of the best experts on this subject based on the ideXlab platform.
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Structure of the native supercoiled flagellar Hook as a universal joint
Nature communications, 2019Co-Authors: Takayuki Kato, Fumiaki Makino, Tomoko Miyata, Péter Horváth, Keiichi NambaAbstract:The Bacterial flagellar Hook is a short supercoiled tubular structure made from a helical assembly of the Hook Protein FlgE. The Hook acts as a universal joint that connects the flagellar basal body and filament, and smoothly transmits torque generated by the rotary motor to the helical filament propeller. In peritrichously flagellated bacteria, the Hook allows the filaments to form a bundle behind the cell for swimming, and for the bundle to fall apart for tumbling. Here we report a native supercoiled Hook structure at 3.6 A resolution by cryoEM single particle image analysis of the polyHook. The atomic model built into the three-dimensional (3D) density map reveals the changes in subunit conformation and intersubunit interactions that occur upon compression and extension of the 11 protofilaments during their smoke ring-like rotation. These observations reveal how the Hook functions as a dynamic molecular universal joint with high bending flexibility and twisting rigidity. The bacterial flagellar Hook is a molecular universal joint that connects the rotary motor and long helical propeller of the bacterial flagellum. Here the authors present the 3.6 A resolution cryo-EM structure of the native supercoiled Salmonella Hook that provides insights into the dynamic changes of subunit conformations and intermolecular interactions of the Hook Protein FlgE.
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Structure of Salmonella Flagellar Hook Reveals Intermolecular Domain Interactions for the Universal Joint Function.
Biomolecules, 2019Co-Authors: Péter Horváth, Tomoko Miyata, Takayuki Kato, Keiichi NambaAbstract:The bacterial flagellum is a motility organelle consisting of a rotary motor and a long helical filament as a propeller. The flagellar Hook is a flexible universal joint that transmits motor torque to the filament in its various orientations that change dynamically between swimming and tumbling of the cell upon switching the motor rotation for chemotaxis. Although the structures of the Hook and Hook Protein FlgE from different bacterial species have been studied, the structure of Salmonella Hook, which has been studied most over the years, has not been solved at a high enough resolution to allow building an atomic model of entire FlgE for understanding the mechanisms of self-assembly, stability and the universal joint function. Here we report the structure of Salmonella polyHook at 4.1 A resolution by electron cryomicroscopy and helical image analysis. The density map clearly revealed folding of the entire FlgE chain forming the three domains D0, D1 and D2 and allowed us to build an atomic model. The model includes domain Dc with a long β-hairpin structure that connects domains D0 and D1 and contributes to the structural stability of the Hook while allowing the flexible bending of the Hook as a molecular universal joint.
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Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III Protein export
Science advances, 2018Co-Authors: Naoya Terahara, Yusuke V. Morimoto, Keiichi Namba, Katsumi Imada, Yumi Inoue, Noriyuki Kodera, Takayuki Uchihashi, Toshio Ando, Tohru MinaminoAbstract:The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the Hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III Protein export apparatus and coordinates flagellar Protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the Protein export apparatus from the Hook Protein to the filament Protein upon completion of the Hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the Hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC.
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straight and rigid flagellar Hook made by insertion of the flgg specific sequence into flge
Scientific Reports, 2017Co-Authors: Koichi D Hiraoka, Yusuke V. Morimoto, Keiichi Namba, Tohru Minamino, Fumiaki Makino, Tomoko Miyata, Yumi Inoue, Takashi FujiiAbstract:: The bacterial flagellar Hook connects the helical flagellar filament to the rotary motor at its base. Bending flexibility of the Hook allows the helical filaments to form a bundle behind the cell body to produce thrust for bacterial motility. The Hook Protein FlgE shows considerable sequence and structural similarities to the distal rod Protein FlgG; however, the Hook is supercoiled and flexible as a universal joint whereas the rod is straight and rigid as a drive shaft. A short FlgG specific sequence (GSS) has been postulated to confer the rigidity on the FlgG rod, and insertion of GSS at the position between Phe-42 and Ala-43 of FlgE actually made the Hook straight. However, it remains unclear whether inserted GSS confers the rigidity as well. Here, we provide evidence that insertion of GSS makes the Hook much more rigid. The GSS insertion inhibited flagellar bundle formation behind the cell body, thereby reducing motility. This indicates that the GSS insertion markedly reduced the bending flexibility of the Hook. Therefore, we propose that the inserted GSS makes axial packing interactions of FlgE subunits much tighter in the Hook to suppress axial compression and extension of the protofilaments required for bending flexibility.
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Role of the Dc domain of the bacterial Hook Protein FlgE in Hook assembly and function.
Biophysics, 2013Co-Authors: Nao Moriya, Hedda U. Ferris, Yusuke V. Morimoto, Masamichi Ashihara, Tohru Minamino, Takayuki Kato, Keiichi NambaAbstract: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.
Robert M Macnab - One of the best experts on this subject based on the ideXlab platform.
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Substrate Specificity Classes and the Recognition Signal for Salmonella Type III Flagellar Export
Journal of Bacteriology, 2003Co-Authors: Takanori Hirano, Tohru Minamino, Keiichi Namba, Robert M MacnabAbstract:Most flagellar Proteins of Salmonella are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar Proteins, most notably the Hook Protein (FlgE), the Hook-capping Protein (FlgD), and the filament Protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/Hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod Proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction Protein (FliE), or the muramidase and putative rod-capping Protein (FlgJ). In this study, we have measured the amounts of these Proteins exported before and after Hook completion. Their amounts in the culture supernatant from a flgE mutant (which is still at the Hook-type specificity stage) were much higher than those from a flgK mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the Hook-type Proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the Hook-capping Protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after Protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these Proteins suffices to mediate their recognition and export.
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Interactions among components of the Salmonella flagellar export apparatus and its substrates.
Molecular microbiology, 2000Co-Authors: Tohru Minamino, Robert M MacnabAbstract: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.
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FLIK, THE Protein RESPONSIBLE FOR FLAGELLAR Hook LENGTH CONTROL IN SALMONELLA, IS EXPORTED DURING Hook ASSEMBLY
Molecular Microbiology, 1999Co-Authors: Tohru Minamino, Shin-ichi Aizawa, Bertha González-pedrajo, Kenta Yamaguchi, Robert M MacnabAbstract: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 ≈ 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.
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Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook of Salmonella
Journal of Bacteriology, 1999Co-Authors: Kazumasa Muramoto, Shigeru Makishima, Shin-ichi Aizawa, Robert M MacnabAbstract: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.
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FliK, the Protein responsible for flagellar Hook length control in Salmonella, is exported during Hook assembly
Molecular microbiology, 1999Co-Authors: Tohru Minamino, Shin-ichi Aizawa, Bertha González-pedrajo, Kenta Yamaguchi, Robert M MacnabAbstract: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 ≈ 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.
Shin-ichi Aizawa - One of the best experts on this subject based on the ideXlab platform.
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The NMR Structure of FliK, the Trigger for the Switch of Substrate Specificity in the Flagellar Type III Secretion Apparatus
Journal of molecular biology, 2011Co-Authors: Shino Mizuno, Shin-ichi Aizawa, Hirokazu Amida, Naohiro Kobayashi, Shin-ichi TateAbstract:Abstract The flagellar cytoplasmic Protein FliK controls Hook elongation by two successive events: by determining Hook length and by stopping the supply of Hook Protein. These two distinct roles are assigned to different parts of FliK: the N-terminal half (FliKN) determines length and the C-terminal half (FliKC) switches secretion from the Hook Protein to the filament Protein. The interaction of FliKC with FlhB, the switchable secretion gate, triggers the switch. By NMR spectroscopy, we demonstrated that FliK is largely unstructured and determined the structure of a compact domain in FliKC. The compact domain, denoted the FliKC core domain, consists of two α-helices, a β-sheet with two parallel and two antiparallel strands, and several exposed loops. Based on the functional data obtained by a series of deletion mutants of the FliKC core domain, we constructed a model of the complex between the FliKC core domain and FlhBC. The model suggested that one of the FliKC loops has a high probability of interacting with the C-terminal domain of FlhB (FlhBC) as the FliK molecule enters the secretion gate. We suggest that the autocleaved NPTH sequence in FlhB contacts loop 2 of FliKC to trigger the switching event. This contact is sterically prevented when NPTH is not cleaved. Thus, the structure of FliK provides insight into the mechanism by which this bifunctional Protein triggers a switch in the export of substrates.
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completion of the Hook basal body complex of the salmonella typhimurium flagellum is coupled to flgm secretion and flic transcription
Molecular Microbiology, 2000Co-Authors: Joyce E Karlinsey, Shin-ichi Aizawa, Shigeru Yamaguchi, Shugo Tanaka, Vera Bettenworth, Winfried Boos, Kelly T. HughesAbstract:The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline-inducible promoter PtetA using the T-POP transposon. Cells containing this construct are motile in the presence of tetracycline and non-motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA Protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti-sigma factor in the growth medium. This also coincides with the completion of Hook-basal body structures. Rolling cells first appeared 35 min after induction, and excess Hook Protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the Hook-basal body structure.
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FLIK, THE Protein RESPONSIBLE FOR FLAGELLAR Hook LENGTH CONTROL IN SALMONELLA, IS EXPORTED DURING Hook ASSEMBLY
Molecular Microbiology, 1999Co-Authors: Tohru Minamino, Shin-ichi Aizawa, Bertha González-pedrajo, Kenta Yamaguchi, Robert M MacnabAbstract: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 ≈ 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.
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Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook of Salmonella
Journal of Bacteriology, 1999Co-Authors: Kazumasa Muramoto, Shigeru Makishima, Shin-ichi Aizawa, Robert M MacnabAbstract: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.
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FliK, the Protein responsible for flagellar Hook length control in Salmonella, is exported during Hook assembly
Molecular microbiology, 1999Co-Authors: Tohru Minamino, Shin-ichi Aizawa, Bertha González-pedrajo, Kenta Yamaguchi, Robert M MacnabAbstract: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 ≈ 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.
Tohru Minamino - One of the best experts on this subject based on the ideXlab platform.
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Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III Protein export
Science advances, 2018Co-Authors: Naoya Terahara, Yusuke V. Morimoto, Keiichi Namba, Katsumi Imada, Yumi Inoue, Noriyuki Kodera, Takayuki Uchihashi, Toshio Ando, Tohru MinaminoAbstract:The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the Hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III Protein export apparatus and coordinates flagellar Protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the Protein export apparatus from the Hook Protein to the filament Protein upon completion of the Hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the Hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC.
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straight and rigid flagellar Hook made by insertion of the flgg specific sequence into flge
Scientific Reports, 2017Co-Authors: Koichi D Hiraoka, Yusuke V. Morimoto, Keiichi Namba, Tohru Minamino, Fumiaki Makino, Tomoko Miyata, Yumi Inoue, Takashi FujiiAbstract:: The bacterial flagellar Hook connects the helical flagellar filament to the rotary motor at its base. Bending flexibility of the Hook allows the helical filaments to form a bundle behind the cell body to produce thrust for bacterial motility. The Hook Protein FlgE shows considerable sequence and structural similarities to the distal rod Protein FlgG; however, the Hook is supercoiled and flexible as a universal joint whereas the rod is straight and rigid as a drive shaft. A short FlgG specific sequence (GSS) has been postulated to confer the rigidity on the FlgG rod, and insertion of GSS at the position between Phe-42 and Ala-43 of FlgE actually made the Hook straight. However, it remains unclear whether inserted GSS confers the rigidity as well. Here, we provide evidence that insertion of GSS makes the Hook much more rigid. The GSS insertion inhibited flagellar bundle formation behind the cell body, thereby reducing motility. This indicates that the GSS insertion markedly reduced the bending flexibility of the Hook. Therefore, we propose that the inserted GSS makes axial packing interactions of FlgE subunits much tighter in the Hook to suppress axial compression and extension of the protofilaments required for bending flexibility.
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Role of the Dc domain of the bacterial Hook Protein FlgE in Hook assembly and function.
Biophysics, 2013Co-Authors: Nao Moriya, Hedda U. Ferris, Yusuke V. Morimoto, Masamichi Ashihara, Tohru Minamino, Takayuki Kato, Keiichi NambaAbstract: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.
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Interaction of FliK with the bacterial flagellar Hook is required for efficient export specificity switching.
Molecular microbiology, 2009Co-Authors: Tohru Minamino, Nao Moriya, Kelly T. Hughes, Takanori Hirano, Keiichi NambaAbstract:Summary FliK–FlhB interaction switches export specificity of the bacterial flagellar Protein export apparatus to stop Hook Protein export at an appropriate timing for Hook length control. The Hook structure is required for the productive FliK–FlhB interaction to flip the switch but it remains unknown how it works. Here, we characterize the role of FliK in the switching probability in the absence of the Hook. When RflH/Flk was missing in the Hook mutants, the switching occurred at a low probability. Overproduction of FliK significantly increased the switching probability although not at the wild-type level. An in-frame deletion of residues 129 through 159 of FliK weakened the interaction with the Hook Protein but not with the Hook-capping Protein, producing polyHooks with filaments attached. We suggest that temporary association of FliK with the inner surface of the Hook during FliK secretion results in a pause in the secretion process to allow the C-terminal switch domain of FliK to be positioned and appropriately oriented near FlhB for catalysing the switch and that RflH/Flk interferes with premature switch by preventing access of cytoplasmic FliK to FlhB and even that of FliK during its secretion until Hook length reaches 55 nm; only then FliKC passes the RflH/Flk block.
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Substrate Specificity Classes and the Recognition Signal for Salmonella Type III Flagellar Export
Journal of Bacteriology, 2003Co-Authors: Takanori Hirano, Tohru Minamino, Keiichi Namba, Robert M MacnabAbstract:Most flagellar Proteins of Salmonella are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar Proteins, most notably the Hook Protein (FlgE), the Hook-capping Protein (FlgD), and the filament Protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/Hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod Proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction Protein (FliE), or the muramidase and putative rod-capping Protein (FlgJ). In this study, we have measured the amounts of these Proteins exported before and after Hook completion. Their amounts in the culture supernatant from a flgE mutant (which is still at the Hook-type specificity stage) were much higher than those from a flgK mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the Hook-type Proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the Hook-capping Protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after Protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these Proteins suffices to mediate their recognition and export.
Georges Dreyfus - One of the best experts on this subject based on the ideXlab platform.
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The Hook Gene (flgE) Is Expressed from the flgBCDEF Operon in Rhodobacter sphaeroides: Study of an flgE Mutant
Journal of bacteriology, 2001Co-Authors: Teresa Ballado, Eugenia Silva-herzog, Bertha González-pedrajo, Laura Camarena, Georges DreyfusAbstract:In this work we identified the flgE gene encoding the flagellar Hook Protein from Rhodobacter sphaeroides. Our results show that this gene is part of a flagellar cluster that includes the genes flgB, flgC, flgD, flgE, and flgF. Two different types of mutants in the flgE gene were isolated, and both showed a Fla(-) phenotype, indicating the functionality of this sequence. Complementation studies of these mutant strains suggest that flgE is included in a single transcriptional unit that starts in flgB and ends in flgF. In agreement with this possibility, a specific transcript of approximately 3.5 kb was identified by Northern blot. This mRNA is large enough to represent the complete flgBCDEF operon. FlgE showed a relatively high proline content; in particular, a region of 12 amino acids near the N terminus, in which four prolines were identified. Cells expressing a mutant FlgE Protein lacking this region showed abnormal swimming behavior, and their Hooks were curved. These results suggest that this region is involved in the characteristic quaternary structure of the Hook of R. sphaeroides and also imply that a straight Hook, or perhaps the rigidity associated with this feature, is important for an efficient swimming behavior in this bacterium.
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Interaction of FliI, a component of the flagellar export apparatus, with flagellin and Hook Protein.
Biochimica et biophysica acta, 1999Co-Authors: Eugenia Silva-herzog, Georges DreyfusAbstract: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.
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Structural and genetic analysis of a mutant of Rhodobacter sphaeroides WS8 deficient in Hook length control.
Journal of bacteriology, 1997Co-Authors: Bertha González-pedrajo, Teresa Ballado, Andrés Campos, R E Sockett, Laura Camarena, Georges DreyfusAbstract:Motility in the photosynthetic bacterium Rhodobacter sphaeroides is achieved by the unidirectional rotation of a single subpolar flagellum. In this study, transposon mutagenesis was used to obtain nonmotile flagellar mutants from this bacterium. We report here the isolation and characterization of a mutant that shows a polyHook phenotype. Morphological characterization of the mutant was done by electron microscopy. PolyHooks were obtained by shearing and were used to purify the Hook Protein monomer (FlgE). The apparent molecular mass of the Hook Protein was 50 kDa. N-terminal amino acid sequencing and comparisons with the Hook Proteins of other flagellated bacteria indicated that the Rhodobacter Hook Protein has consensus sequences common to axial flagellar components. A 25-kb fragment from an R. sphaeroides WS8 cosmid library restored wild-type flagellation and motility to the mutant. Using DNA adjacent to the inserted transposon as a probe, we identified a 4.6-kb SalI restriction fragment that contained the gene responsible for the polyHook phenotype. Nucleotide sequence analysis of this region revealed an open reading frame with a deduced amino acid sequence that was 23.4% identical to that of FliK of Salmonella typhimurium, the polypeptide responsible for Hook length control in that enteric bacterium. The relevance of a gene homologous to fliK in the uniflagellated bacterium R. sphaeroides is discussed.