Purification and functional reconstitution of the Na^+-driven polar flagellar motor component of Vibrio alginolyticus

Sato, Kiyotaka; Homma, M.

doi:10.2142/biophys.39.s147_4

Cited by 66 publications

(105 citation statements)

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“…Reconstitution experiments with liposomes provided strong proof that purified V. alginolyticus MotA-MotB complexes catalyzed Na ϩ flux (157). Furthermore, these purification studies, as well as immunoprecipitation experiments, indicated that MotA and MotB physically interact and can be purified as a complex (157,203). Interestingly, the molar ratio of MotA to MotB was calculated to be 2:1.…”

Section: The Mota-motb Complex Translocates Sodium Ionsmentioning

confidence: 87%

“…Some mutations, such as a substitution at residue Asp31 in motA, caused a slow-motility phenotype, suggesting that the negative charge of this residue contributes to optimal speed or efficiency of the motor (94). Reconstitution experiments with liposomes provided strong proof that purified V. alginolyticus MotA-MotB complexes catalyzed Na ϩ flux (157). Furthermore, these purification studies, as well as immunoprecipitation experiments, indicated that MotA and MotB physically interact and can be purified as a complex (157,203).…”

Section: The Mota-motb Complex Translocates Sodium Ionsmentioning

confidence: 91%

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Polar Flagellar Motility of the Vibrionaceae

McCarter

2001

Microbiol Mol Biol Rev

313

356

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Polar flagella of Vibrio species can rotate at speeds as high as 100,000 rpm and effectively propel the bacteria in liquid as fast as 60 μm/s. The sodium motive force powers rotation of the filament, which acts as a propeller. The filament is complex, composed of multiple subunits, and sheathed by an extension of the cell outer membrane. The regulatory circuitry controlling expression of the polar flagellar genes of members of the Vibrionaceae is different from the peritrichous system of enteric bacteria or the polar system of Caulobacter crescentus. The scheme of gene control is also pertinent to other members of the gamma purple bacteria, in particular to Pseudomonas species. This review uses the framework of the polar flagellar system of Vibrio parahaemolyticus to provide a synthesis of what is known about polar motility systems of the Vibrionaceae. In addition to its propulsive role, the single polar flagellum of V. parahaemolyticus is believed to act as a tactile sensor controlling surface-induced gene expression. Under conditions that impede rotation of the polar flagellum, an alternate, lateral flagellar motility system is induced that enables movement through viscous environments and over surfaces. Although the dual flagellar systems possess no shared structural components and although distinct type III secretion systems direct the simultaneous placement and assembly of polar and lateral organelles, movement is coordinated by shared chemotaxis machinery

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Section: The Mota-motb Complex Translocates Sodium Ionsmentioning

confidence: 87%

Section: The Mota-motb Complex Translocates Sodium Ionsmentioning

confidence: 91%

Polar Flagellar Motility of the Vibrionaceae

McCarter

2001

Microbiol Mol Biol Rev

313

356

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“…The stator is a hetero-hexameric complex of two membrane proteins, A and B, with a stoichiometry of A 4 B 2 (8,9). The stator consists of MotA and MotB for the H + -driven motor of Escherichia coli and Salmonella enterica, and PomA and PomB for the Na + -driven motor of Vibrio and Shewanella spp.…”

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confidence: 99%

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor

Zhu

Takao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

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The torque of the bacterial flagellum is generated by the rotorstator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomB C ) at 2.0-Å resolution, and the structure suggests a conformational change in the N-terminal region of PomB C for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that the N-terminal disordered region (121-153) and following the N-terminal two-thirds of α1(154-164) in PomB C changes its conformation to form a functional stator around the rotor. The cross-linking did not affect the localization of the stator nor the ion conductivity, suggesting that the conformational change occurs in the final step of the stator assembly around the rotor.ion-driven motor | peptidoglycan binding | Vibrio alginolyticus | X-ray crystallography

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“…Each stator unit is a complex of two distinct membrane proteins, A and B, with an A 4 B 2 stoichiometry. The stator is composed of PomA and PomB in the sodium driven motor of Vibrio sp., and MotA and MotB in the proton motors of E. coli and Salmonella, and they generate torque by conducting the coupling ion into the cytoplasm (6)(7)(8)(9)(10)(11). Multiple stator complexes assemble around the rotor, although a single stator complex can generate torque (12)(13)(14)(15).…”

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confidence: 99%

Insight into the assembly mechanism in the supramolecular rings of the sodium-driven Vibrio flagellar motor from the structure of FlgT

Terashima

Sakuma

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Flagellar motility is a key factor for bacterial survival and growth in fluctuating environments. The polar flagellum of a marine bacterium, Vibrio alginolyticus, is driven by sodium ion influx and rotates approximately six times faster than the proton-driven motor of Escherichia coli. The basal body of the sodium motor has two unique ring structures, the T ring and the H ring. These structures are essential for proper assembly of the stator unit into the basal body and to stabilize the motor. FlgT, which is a flagellar protein specific for Vibrio sp., is required to form and stabilize both ring structures. Here, we report the crystal structure of FlgT at 2.0-Å resolution. FlgT is composed of three domains, the N-terminal domain (FlgT-N), the middle domain (FlgT-M), and the C-terminal domain (FlgT-C). FlgT-M is similar to the N-terminal domain of TolB, and FlgT-C resembles the N-terminal domain of FliI and the α/β subunits of F 1 -ATPase. To elucidate the role of each domain, we prepared domain deletion mutants of FlgT and analyzed their effects on the basal-body ring formation. The results suggest that FlgT-N contributes to the construction of the H-ring structure, and FlgT-M mediates the T-ring association on the LP ring. FlgT-C is not essential but stabilizes the H-ring structure. On the basis of these results, we propose an assembly mechanism for the basal-body rings and the stator units of the sodium-driven flagellar motor.X-ray crystallography | molecular motor T o find favorable conditions for survival and growth, bacteria swim by rotating their flagellum, a filamentous organelle for motility. The flagellum is a large macromolecular assembly composed of three major parts: the basal body, the hook, and the filament. The flagellar filament is driven by a rotary motor embedded in the cell membrane (1-4). The motor is powered by the electrochemical gradient of the coupling ion (H + or Na + ), which results in rotation of the filament like a screw. Whereas the proton motors of Escherichia coli and Salmonella enterica serovar Typhimurium spin up to 300 Hz, the sodium motor of Vibrio alginolyticus can rotate remarkably faster, up to 1,700 Hz (5).The flagellar motor is made of the basal body, which includes the rotor, and a dozen stator units that surround the rotor. Each stator unit is a complex of two distinct membrane proteins, A and B, with an A 4 B 2 stoichiometry. The stator is composed of PomA and PomB in the sodium driven motor of Vibrio sp., and MotA and MotB in the proton motors of E. coli and Salmonella, and they generate torque by conducting the coupling ion into the cytoplasm (6-11). Multiple stator complexes assemble around the rotor, although a single stator complex can generate torque (12-15).The basal body is a complex macromolecular assembly including several ring structures, such as the L, P, MS, and C rings, and a drive shaft, which is also called the rod (16). The L and P rings form a bushing for the rod, and the MS and C rings are a reversible rotor. The L and P rings are composed of FlgH an...

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Purification and functional reconstitution of the Na^+-driven polar flagellar motor component of Vibrio alginolyticus

Cited by 66 publications

References 0 publications

Polar Flagellar Motility of the Vibrionaceae

Polar Flagellar Motility of the Vibrionaceae

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor

Insight into the assembly mechanism in the supramolecular rings of the sodium-driven Vibrio flagellar motor from the structure of FlgT

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