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Michio Homma - One of the best experts on this subject based on the ideXlab platform.
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The Helix Rearrangement in the Periplasmic Domain of the Flagellar Stator B Subunit Activates Peptidoglycan Binding and Ion Influx
Structure (London England : 1993), 2018Co-Authors: Seiji Kojima, Michio Homma, Masato Takao, Gaby Almira, Ikumi Kawahara, Mayuko Sakuma, Chojiro Kojima, Katsumi ImadaAbstract:Summary The stator of the bacterial flagellar motor couples ion flow with torque generation. The ion-conducting stator channel opens only when incorporated into and anchored around the rotor via the peptidoglycan (PG) binding domain of the B subunit (MotB C ). However, no direct evidence of PG binding coupled with channel activation has been presented. Here, we report the structural rearrangements of MotB C responsible for this coupling process. A MotB C fragment with the L119P replacement, which is known to cause channel activation, was able to bind PG. Nuclear magnetic resonance analysis of MotB C and the crystal structure of the MotB C -L119P dimer revealed major structural changes in helix α1. In vivo crosslinking results confirm that a major rearrangement occurs. Our results suggest that, upon stator incorporation into the motor, helix α1 of MotB C changes into an extended non-helical structure. We propose that this change allows the stator both to bind PG and to open its proton channel.
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Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium.
Scientific reports, 2015Co-Authors: Norihiro Takekawa, Seiji Kojima, Masayoshi Nishiyama, Tsuyoshi Kaneseki, Tamotsu Kanai, Haruyuki Atomi, Michio HommaAbstract:Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s−1 at 85 °C. We expressed the A. aeolicus mot genes (motA and MotB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na+. As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na+ for energy coupling of the flagellar motor. The Na+-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.
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Functional chimeras of flagellar stator proteins between E. coli MotB and Vibrio PomB at the periplasmic region in Vibrio or E. coli
MicrobiologyOpen, 2015Co-Authors: Yuuki Nishino, Yasuhiro Onoue, Seiji Kojima, Michio HommaAbstract:The bacterial flagellar motor has a stator and a rotor. The stator is composed of two membrane proteins, MotA and MotB in Escherichia coli and PomA and PomB in Vibrio alginolyticus. The Vibrio motor has a unique structure, the T ring, which is composed of MotX and MotY. Based on the structural information of PomB and MotB, we constructed three chimeric proteins between PomB and MotB, named PotB91, PotB129, and PotB138, with various chimeric junctions. When those chimeric proteins were produced with PomA in a ΔmotAB strain of E. coli or in ΔpomAB and ΔpomAB ΔmotX strains of Vibrio, all chimeras were functional in E. coli or Vibrio, either with or without the T ring, although the motilities were very weak in E. coli. Furthermore, we could isolate some suppressors in E. coli and identified the mutation sites on PomA or the chimeric B subunit. The weak function of chimeric PotBs in E. coli is derived mainly from the defect in the rotational switching of the flagellar motor. In addition, comparing the motilities of chimera strains in ΔpomAB, PotB138 had the highest motility. The difference between the origin of the α1 and α2 helices, E. coli MotB or Vibro PomB, seems to be important for motility in E. coli and especially in Vibrio.
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Ion-coupling Determinants of Na1-driven and H1-driven Flagellar Motors
2014Co-Authors: Yukako Asai, Ikuro Kawagishi, Toshiharu Yakushi, Michio HommaAbstract:The bacterial flagellar motor is a tiny molecular machine that uses a trans-membrane flux of Hþ or Naþ ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectiv-ity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DpomAB Vibrio alginolyticus. Most hyper-motile segregants isolated from this weakly motile strain contain mutations in MotB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7E) func-tioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7E) supported sodium-driven motility in motA or MotB E. coli cells, and the swimming speed was even higher than with the original stator of E. coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7E) supported sodium-driven motility in a MotX/Y-depen-dent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor
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Disulphide cross-linking between the stator and the bearing components in the bacterial flagellar motor
Journal of Biochemistry, 2010Co-Authors: Yohei Hizukuri, Seiji Kojima, Michio HommaAbstract:The flagellar motor is composed of the stator and the rotor, and the interaction between the stator and the rotor at the cytoplasmic region is believed to produce mechanical force for the rotation of flagella. The periplasmic region of the stator has been proposed to play an important role in assembly around and incorporation into the motor. In this study, we provide evidence suggesting that the periplasmic region of the stator component MotB interacts with the P-ring component FlgI, which functions as a bearing for the rotor along with the L-ring protein FlgH, from a site-directed disulphide cross-linking approach. First, we prepared four FlgI and three MotB cysteine-substituted mutant proteins and co-expressed them in various combinations in Escherichia coli. We detected cross-linked combinations of FlgI G11C and MotB S248C when treated with the oxidant Cu-phenanthroline or bismaleimide cross-linkers. Furthermore, we performed Cys-scanning mutagenesis around these two residues and found additional combinations of cross-linked residues. Treatment with a protonophore CCCP significantly reduced the cross-linking efficiency between FlgI and MotB in flagellated cells, but not in non-flagellated cells. These results suggest a direct contact between MotB and FlgI upon assembly of the stator into a motor.
Seiji Kojima - One of the best experts on this subject based on the ideXlab platform.
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The Helix Rearrangement in the Periplasmic Domain of the Flagellar Stator B Subunit Activates Peptidoglycan Binding and Ion Influx
Structure (London England : 1993), 2018Co-Authors: Seiji Kojima, Michio Homma, Masato Takao, Gaby Almira, Ikumi Kawahara, Mayuko Sakuma, Chojiro Kojima, Katsumi ImadaAbstract:Summary The stator of the bacterial flagellar motor couples ion flow with torque generation. The ion-conducting stator channel opens only when incorporated into and anchored around the rotor via the peptidoglycan (PG) binding domain of the B subunit (MotB C ). However, no direct evidence of PG binding coupled with channel activation has been presented. Here, we report the structural rearrangements of MotB C responsible for this coupling process. A MotB C fragment with the L119P replacement, which is known to cause channel activation, was able to bind PG. Nuclear magnetic resonance analysis of MotB C and the crystal structure of the MotB C -L119P dimer revealed major structural changes in helix α1. In vivo crosslinking results confirm that a major rearrangement occurs. Our results suggest that, upon stator incorporation into the motor, helix α1 of MotB C changes into an extended non-helical structure. We propose that this change allows the stator both to bind PG and to open its proton channel.
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The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium.
Scientific reports, 2016Co-Authors: Norihiro Takekawa, Seiji Kojima, Naoya Terahara, Takayuki Kato, Mizuki Gohara, Kouta Mayanagi, Atsushi Hijikata, Yasuhiro Onoue, Tsuyoshi Shirai, Keiichi NambaAbstract:Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx, and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB, and for the first time, demonstrate the cytoplasmic structure of the stator.
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Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium.
Scientific reports, 2015Co-Authors: Norihiro Takekawa, Seiji Kojima, Masayoshi Nishiyama, Tsuyoshi Kaneseki, Tamotsu Kanai, Haruyuki Atomi, Michio HommaAbstract:Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s−1 at 85 °C. We expressed the A. aeolicus mot genes (motA and MotB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na+. As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na+ for energy coupling of the flagellar motor. The Na+-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.
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Functional chimeras of flagellar stator proteins between E. coli MotB and Vibrio PomB at the periplasmic region in Vibrio or E. coli
MicrobiologyOpen, 2015Co-Authors: Yuuki Nishino, Yasuhiro Onoue, Seiji Kojima, Michio HommaAbstract:The bacterial flagellar motor has a stator and a rotor. The stator is composed of two membrane proteins, MotA and MotB in Escherichia coli and PomA and PomB in Vibrio alginolyticus. The Vibrio motor has a unique structure, the T ring, which is composed of MotX and MotY. Based on the structural information of PomB and MotB, we constructed three chimeric proteins between PomB and MotB, named PotB91, PotB129, and PotB138, with various chimeric junctions. When those chimeric proteins were produced with PomA in a ΔmotAB strain of E. coli or in ΔpomAB and ΔpomAB ΔmotX strains of Vibrio, all chimeras were functional in E. coli or Vibrio, either with or without the T ring, although the motilities were very weak in E. coli. Furthermore, we could isolate some suppressors in E. coli and identified the mutation sites on PomA or the chimeric B subunit. The weak function of chimeric PotBs in E. coli is derived mainly from the defect in the rotational switching of the flagellar motor. In addition, comparing the motilities of chimera strains in ΔpomAB, PotB138 had the highest motility. The difference between the origin of the α1 and α2 helices, E. coli MotB or Vibro PomB, seems to be important for motility in E. coli and especially in Vibrio.
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M (2009) The peptidoglycan-binding (PGB) domain of the Escherichia coli Pal protein can also function as the PGB domain in E. coli flagellar motor protein MotB. J Biochem 146: 219–229. Accession codes Coordinates and structure factors have been depos
2015Co-Authors: Yohei Hizukuri, Seiji Kojima, Toshiharu Yakushi, John Frederick Morton, Michio HommayAbstract:The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought to be anchored to the peptidoglycan by the C-terminal conserved peptido-glycan-binding (PGB) motif of MotB. To clarify the role of the C-terminal region, we performed systematic cysteine mutagenesis and constructed a chimeric MotB protein which was replaced with the peptidoglycan-associated lipoprotein Pal. Although this chimera could not restore motility to a MotB strain, we were able to isolate two motile revertants. One was F172V in the Pal region and the other was P159L in the MotB region. Furthermore, we attempted to map the MotB Cys muta-tions in the crystal structure of Escherichia coli Pal. We found that the MotB muta-tions that affected motility nearly overlapped with the predicted PG-binding residues of Pal. Our results indicate that, although the functions of MotB and Pal are very different, the PGB region of Pal is interchangeable with the PGB region of MotB
Keiichi Namba - One of the best experts on this subject based on the ideXlab platform.
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GFP Fusion to the N-Terminus of MotB Affects the Proton Channel Activity of the Bacterial Flagellar Motor in Salmonella
Biomolecules, 2020Co-Authors: Yusuke V. Morimoto, Keiichi Namba, Tohru MinaminoAbstract:The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-MotBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-MotBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.
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The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium.
Scientific reports, 2016Co-Authors: Norihiro Takekawa, Seiji Kojima, Naoya Terahara, Takayuki Kato, Mizuki Gohara, Kouta Mayanagi, Atsushi Hijikata, Yasuhiro Onoue, Tsuyoshi Shirai, Keiichi NambaAbstract:Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx, and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB, and for the first time, demonstrate the cytoplasmic structure of the stator.
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Genetic analysis of revertants isolated from the rod-fragile fliF mutant of Salmonella
Biophysics and physicobiology, 2016Co-Authors: Hitomi Komatsu, Keiichi Namba, Fumio Hayashi, Masahiro Sasa, Koji Shikata, Shigeru Yamaguchi, Kenji OosawaAbstract:FliF is the protein comprising the MS-ring of the bacterial flagellar basal body, which is the base for the assembly of flagellar axial structures. From a fliF mutant that easily releases the rod-hook-filament in viscous environments, more than 400 revertants that recovered their swarming ability in viscous conditions, were isolated. The second-site mutations were determined for approximately 70% of them. There were three regions where the mutations were localized: two in Region I, 112 in Region II, and 71 in Region III including the true reversion. In Region I, second-site mutations were found in FlgC and FlgF of the proximal rod, suggesting that they affect the interaction between the MS-ring and the rod. In Region II, there were 69 and 42 mutations in MotA and MotB, respectively, suggesting that the second-site mutations in MotA and MotB may decrease the rotational speed of the flagellar motor to reduce the probability of releasing the rod under this condition. One exception is a mutation in FlhC that caused a down regulation of the flagellar proteins production but it may directly affect transcription or translation of motA and MotB. In Region III, there were 44, 24, and 3 mutations in FliG, FliM, and FliF, respectively. There were no second-site mutations identified in FliN although it is involved in torque generation as a component of the C-ring. Many of the mutations were involved in the motor rotation, and it is suggested that such reduced speeds result in stabilizing the filament attachment to the motor.
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Effect of the MotB(D33N) mutation on stator assembly and rotation of the proton-driven bacterial flagellar motor
Biophysics (Nagoya-shi Japan), 2014Co-Authors: Shuichi Nakamura, Tohru Minamino, Nobunori Kami-ike, Seishi Kudo, Keiichi NambaAbstract:The bacterial flagellar motor generates torque by converting the energy of proton translocation through the transmembrane proton channel of the stator complex formed by MotA and MotB. The MotA/B complex is thought to be anchored to the peptidoglycan (PG) layer through the PG-binding domain of MotB to act as the stator. The stator units dynamically associate with and dissociate from the motor during flagellar motor rotation, and an electrostatic interaction between MotA and a rotor protein FliG is required for efficient stator assembly. However, the association and dissociation mechanism of the stator units still remains unclear. In this study, we analyzed the speed fluctuation of the flagellar motor of Salmonella enterica wild-type cells carrying a plasmid encoding a nonfunctional stator complex, MotA/B(D33N), which lost the proton conductivity. The wild-type motor rotated stably but the motor speed fluctuated considerably when the expression level of MotA/B(D33N) was relatively high compared to MotA/B. Rapid accelerations and decelerations were frequently observed. A quantitative analysis of the speed fluctuation and a model simulation suggested that the MotA/B(D33N) stator retains the ability to associate with the motor at a low affinity but dissociates more rapidly than the MotA/B stator. We propose that the stator dissociation process depends on proton translocation through the proton channel.
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load sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor
Molecular Microbiology, 2014Co-Authors: Yusuke V. Morimoto, Keiichi Namba, Shuichi Nakamura, Yong-suk Che, Nobunori Kamiike, Tohru MinaminoAbstract:Summary The Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild-type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild-type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their ‘unplugged’ forms were only half of the conductivity of the wild-type channel. These results suggest that the D33E mutation induces a load-dependent inactivation of the MotA/B complex. We propose that the stator element is a load-sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co-ordinated proton translocation.
Yukako Asai - One of the best experts on this subject based on the ideXlab platform.
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Ion-coupling Determinants of Na1-driven and H1-driven Flagellar Motors
2014Co-Authors: Yukako Asai, Ikuro Kawagishi, Toshiharu Yakushi, Michio HommaAbstract:The bacterial flagellar motor is a tiny molecular machine that uses a trans-membrane flux of Hþ or Naþ ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectiv-ity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DpomAB Vibrio alginolyticus. Most hyper-motile segregants isolated from this weakly motile strain contain mutations in MotB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7E) func-tioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7E) supported sodium-driven motility in motA or MotB E. coli cells, and the swimming speed was even higher than with the original stator of E. coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7E) supported sodium-driven motility in a MotX/Y-depen-dent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor
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Ion-coupling determinants of Na+-driven and H+-driven flagellar motors.
Journal of molecular biology, 2003Co-Authors: Yukako Asai, Ikuro Kawagishi, Toshiharu Yakushi, Michio HommaAbstract:The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H þ or Na þ ions to drive flagellar rotation. In protondriven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DpomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in MotB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7 E ) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7 E ) supported sodium-driven motility in motA or MotB E. coli cells, and the swimming speed was even higher than with the original stator of E. coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7 E ) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor. q 2003 Elsevier Science Ltd. All rights reserved
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Coupling ion specificity of chimeras between H(+)- and Na(+)-driven motor proteins, MotB and PomB, in Vibrio polar flagella.
The EMBO journal, 2000Co-Authors: Yukako Asai, Ikuro Kawagishi, R. Elizabeth Sockett, Michio HommaAbstract:We have shown that a hybrid motor consisting of proton‐type Rhodobacter sphaeroides MotA and sodium‐type Vibrio alginolyticus PomB, MotX and MotY, can work as a sodium‐driven motor in Vibrio cells. In this study, we tried to substitute the B subunits, which contain a putative ion‐binding site in the transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N‐terminal MotB and C‐terminal PomB. MomB proteins, with the entire transmembrane region derived from the H + ‐type MotB, gave rise to an Na + motor with MotA. The other two MomB proteins, in which the junction sites were within the transmembrane region, also formed Na + motors with PomA, but were changed for Na + or Li + specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na + ‐ and H + ‐type subunits and this can affect the ion specificity. This is the first report to have changed the specificity of the coupling ions in a bacterial flagellar motor.
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coupling ion specificity of chimeras between h and na driven motor proteins MotB and pomb in vibrio polar flagella
The EMBO Journal, 2000Co-Authors: Yukako Asai, Ikuro Kawagishi, Elizabeth R Sockett, Michio HommaAbstract:We have shown that a hybrid motor consisting of proton‐type Rhodobacter sphaeroides MotA and sodium‐type Vibrio alginolyticus PomB, MotX and MotY, can work as a sodium‐driven motor in Vibrio cells. In this study, we tried to substitute the B subunits, which contain a putative ion‐binding site in the transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N‐terminal MotB and C‐terminal PomB. MomB proteins, with the entire transmembrane region derived from the H + ‐type MotB, gave rise to an Na + motor with MotA. The other two MomB proteins, in which the junction sites were within the transmembrane region, also formed Na + motors with PomA, but were changed for Na + or Li + specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na + ‐ and H + ‐type subunits and this can affect the ion specificity. This is the first report to have changed the specificity of the coupling ions in a bacterial flagellar motor.
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Hybrid Motor with H+- and Na+-Driven Components Can Rotate Vibrio Polar Flagella by Using Sodium Ions
Journal of bacteriology, 1999Co-Authors: Yukako Asai, Ikuro Kawagishi, R. Elizabeth Sockett, Michio HommaAbstract:The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium Rhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembrane region of PomB. To examine whether the R. sphaeroides motor genes can function in place of the pomA and pomB genes of V. alginolyticus, we constructed plasmids including both motA and MotB or motA alone and transformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand, pomB mutants were not restored to motility by any plasmid containing motA and/or MotB. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of R. sphaeroides can generate torque by coupling with the sodium ion flux in place of PomA of V. alginolyticus.
Tohru Minamino - One of the best experts on this subject based on the ideXlab platform.
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GFP Fusion to the N-Terminus of MotB Affects the Proton Channel Activity of the Bacterial Flagellar Motor in Salmonella
Biomolecules, 2020Co-Authors: Yusuke V. Morimoto, Keiichi Namba, Tohru MinaminoAbstract:The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-MotBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-MotBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.
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Effect of the MotB(D33N) mutation on stator assembly and rotation of the proton-driven bacterial flagellar motor
Biophysics (Nagoya-shi Japan), 2014Co-Authors: Shuichi Nakamura, Tohru Minamino, Nobunori Kami-ike, Seishi Kudo, Keiichi NambaAbstract:The bacterial flagellar motor generates torque by converting the energy of proton translocation through the transmembrane proton channel of the stator complex formed by MotA and MotB. The MotA/B complex is thought to be anchored to the peptidoglycan (PG) layer through the PG-binding domain of MotB to act as the stator. The stator units dynamically associate with and dissociate from the motor during flagellar motor rotation, and an electrostatic interaction between MotA and a rotor protein FliG is required for efficient stator assembly. However, the association and dissociation mechanism of the stator units still remains unclear. In this study, we analyzed the speed fluctuation of the flagellar motor of Salmonella enterica wild-type cells carrying a plasmid encoding a nonfunctional stator complex, MotA/B(D33N), which lost the proton conductivity. The wild-type motor rotated stably but the motor speed fluctuated considerably when the expression level of MotA/B(D33N) was relatively high compared to MotA/B. Rapid accelerations and decelerations were frequently observed. A quantitative analysis of the speed fluctuation and a model simulation suggested that the MotA/B(D33N) stator retains the ability to associate with the motor at a low affinity but dissociates more rapidly than the MotA/B stator. We propose that the stator dissociation process depends on proton translocation through the proton channel.
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Structure and Function of the Bi-Directional Bacterial Flagellar Motor
Biomolecules, 2014Co-Authors: Yusuke V. Morimoto, Tohru MinaminoAbstract:The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor-stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor.
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load sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor
Molecular Microbiology, 2014Co-Authors: Yusuke V. Morimoto, Keiichi Namba, Shuichi Nakamura, Yong-suk Che, Nobunori Kamiike, Tohru MinaminoAbstract:Summary The Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild-type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild-type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their ‘unplugged’ forms were only half of the conductivity of the wild-type channel. These results suggest that the D33E mutation induces a load-dependent inactivation of the MotA/B complex. We propose that the stator element is a load-sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co-ordinated proton translocation.
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Structure and Function of the Bi-Directional Bacterial Flagellar Motor
MDPI AG, 2014Co-Authors: Yusuke V. Morimoto, Tohru MinaminoAbstract:The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor–stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor