Functional Reconstitution of the Na+-driven Polar Flagellar Motor Component of Vibrio alginolyticus

Sato, Ken; Homma, Michio

doi:10.1074/jbc.275.8.5718

Cited by 142 publications

(63 citation statements)

References 44 publications

(53 reference statements)

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“…A conserved aspartic acid residue in the transmembrane segment of MotB is predicted to be the proton-binding site and plays a crucial role for torque generation and bacterial motility (59). In V. alginolyticus and Shewanella spp., the MotA/MotB homologs PomA and PomB form the stator complex, which is driven by sodium motive force (60). The stoichiometry of the stator varies significantly among different species.…”

Section: Discussionmentioning

confidence: 99%

Imaging the Motility and Chemotaxis Machineries in Helicobacter pylori by Cryo-Electron Tomography

et al. 2017

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Helicobacter pylori is a bacterial pathogen that can cause many gastrointestinal diseases, including ulcers and gastric cancer. A unique chemotaxis-mediated motility is critical for H. pylori to colonize in the human stomach and to establish chronic infection, but the underlying molecular mechanisms are not well understood. Here, we employ cryo-electron tomography (cryo-ET) to reveal detailed structures of the H. pylori cell envelope, including the sheathed flagella and chemotaxis arrays. Notably, H. pylori possesses a distinctive periplasmic cage-like structure with 18-fold symmetry. We propose that this structure forms a robust platform for recruiting 18 torque generators, which likely provide the higher torque needed for swimming in high-viscosity environments. We also reveal a series of key flagellar assembly intermediates, providing structural evidence that flagellar assembly is tightly coupled with the biogenesis of the membrane sheath. Finally, we determine the structure of putative chemotaxis arrays at the flagellar pole, which have implications for how the direction of flagellar rotation is regulated. Together, our pilot cryo-ET studies provide novel structural insights into the unipolar flagella of H. pylori and lay a foundation for a better understanding of the unique motility of this organism.IMPORTANCE Helicobacter pylori is a highly motile bacterial pathogen that colonizes approximately 50% of the world's population. H. pylori can move readily within the viscous mucosal layer of the stomach. It has become increasingly clear that its unique flagella-driven motility is essential for successful gastric colonization and pathogenesis. Here, we use advanced imaging techniques to visualize novel in situ structures with unprecedented detail in intact H. pylori cells. Remarkably, H. pylori possesses multiple unipolar flagella, which are driven by one of the largest flagellar motors found in bacteria. These large motors presumably provide the higher torque needed by the bacterial pathogens to navigate in the viscous environment of the human stomach.

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Section: Discussionmentioning

confidence: 99%

Imaging the Motility and Chemotaxis Machineries in Helicobacter pylori by Cryo-Electron Tomography

et al. 2017

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“…Torque is generated by interactions between the rotor protein FliG, located at the intersection between the MS and C rings, and the stator units attached to the cell wall (5). Each unit is believed to contain two copies of MotB and four copies of MotA in proton-driven motors of E. coli, two copies of PomB and four copies of PomA in sodium-driven motors of Vibrio alginolyticus (6,7), and function as an ion channel (8,9). Ion flux through these channels powers the motor (10,11).…”

mentioning

confidence: 99%

The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11

Reid

Leake

Chandler

et al. 2006

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

253

268

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Torque is generated in the rotary motor at the base of the bacterial flagellum by ion translocating stator units anchored to the peptidoglycan cell wall. Stator units are composed of the proteins MotA and MotB in proton-driven motors, and they are composed of PomA and PomB in sodium-driven motors. Strains of Escherichia coli lacking functional stator proteins produce flagella that do not rotate, and induced expression of the missing proteins leads to restoration of motor rotation in discrete speed increments, a process known as ''resurrection.'' Early work suggested a maximum of eight units. More recent indications that WT motors may contain more than eight units, based on recovery of disrupted motors, are inconclusive. Here we demonstrate conclusively that the maximum number of units in a motor is at least 11. Using back-focal-plane interferometry of 1-m polystyrene beads attached to flagella, we observed at least 11 distinct speed increments during resurrection with three different combinations of stator proteins in E. coli. The average torques generated by a single unit and a fully induced motor were lower than previous estimates. Speed increments at high numbers of units are smaller than those at low numbers, indicating that not all units in a fully induced motor are equivalent.T he flagellar motor is the mechanism of propulsion for most swimming bacteria (1-5). In Escherichia coli Ϸ40 gene products are required for motor assembly, with Ϸ20 of them being present in the final structure. Each motor drives a helical filament up to Ϸ10 m long. The flagellar motor spans the inner and outer bacterial membranes (Fig. 1). Torque is generated by interactions between the rotor protein FliG, located at the intersection between the MS and C rings, and the stator units attached to the cell wall (5). Each unit is believed to contain two copies of MotB and four copies of MotA in proton-driven motors of E. coli, two copies of PomB and four copies of PomA in sodium-driven motors of Vibrio alginolyticus (6, 7), and function as an ion channel (8, 9). Ion flux through these channels powers the motor (10, 11). Functional chimeras have been engineered containing components of proton-and sodium-driven motors (12), indicating that the structure and mechanisms of both types of motor are very similar. The torque-speed relationship of the motor has been measured by using electrorotation of tethered cells (13) and attaching varying viscous loads (14-16). A notable feature is a regime between stall and a speed of Ϸ175 Hz in WT E. coli at room temperature, over which torque falls linearly with increasing speed to Ϸ90% of the value at stall. At higher speeds torque falls more steeply, eventually to zero at a speed of Ϸ350 Hz.Successive incorporation of torque-generating units to restore rotation in paralyzed motors is known as resurrection (17). The maximum number of speed increments previously seen during resurrection of a single motor, using inducible plasmids to express missing stator proteins, was eight (18). However, in experiments w...

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“…The functions of MotX and MotY are not clear, although it is thought that they may be involved in ion recognition. MotX and MotY do not co-purify with the PomA/PomB complex (19). This observation may mean that MotX and MotY are not associated with the PomA/PomB complex, that the association is weak, or that MotX and MotY are unstable during purification.…”

Section: Discussionmentioning

confidence: 89%

“…The D148Y and P16S mutations combined to produce a synergistic increase in resistance to phenamil and impaired motor function much more severely than the individual mutations in the absence of inhibitor (17). We have also shown that PomA and PomB physically interact with each other (18), and that Na ϩ influx can be detected in reconstituted proteoliposomes containing purified PomA/PomB complex (19). The molar ratio of the isolated complex is calculated to be 2PomA/1PomB, and PomA seems to form a stable dimer.…”

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confidence: 70%

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Intermolecular Cross-linking between the Periplasmic Loop3–4 Regions of PomA, a Component of the Na+-driven Flagellar Motor of Vibrio alginolyticus

Yorimitsu

Asai

Sato

et al. 2000

Journal of Biological Chemistry

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PomA and PomB form a complex that conducts sodium ions and generates the torque for the Na ؉ -driven polar flagellar motor of Vibrio alginolyticus. PomA has four transmembrane segments. One periplasmic loop (loop 1-2 ) connects segments 1 and 2, and another (loop 3-4), in which cysteine-scanning mutagenesis had been carried out, connects segments 3 and 4. When PomA with an introduced Cys residue (Cys-PomA) in the Cterminal periplasmic loop (loop 3-4 ) was examined without exposure to a reducing reagent, a 43-kDa band was observed, whereas only a 25-kDa band, which corresponds to monomeric PomA, was observed under reducing conditions. The intensity of the 43-kDa band was enhanced in most mutants by the oxidizing reagent CuCl 2 . The 43-kDa band was strongest in the P172C mutant. The motility of the P172C mutant was severely reduced, and P172C showed a dominant-negative effect, whereas substitution of Pro with Ala, Ile, or Ser at this position did not affect motility. In the presence of DTT, the ability to swim was partially restored, and the amount of 43-kDa protein was reduced. These results suggest that the disulfide cross-link disturbs the function of PomA. When the mutated Cys residue was modified with N-ethylmaleimide, only the 25-kDa PomA band was labeled, demonstrating that the 43-kDa form is a cross-linked homodimer and suggesting that the loops 3-4 of adjacent subunits of PomA are close to each other in the assembled motor. We propose that this loop region is important for dimer formation and motor function.

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Functional Reconstitution of the Na+-driven Polar Flagellar Motor Component of Vibrio alginolyticus

Cited by 142 publications

References 44 publications

Imaging the Motility and Chemotaxis Machineries in Helicobacter pylori by Cryo-Electron Tomography

Imaging the Motility and Chemotaxis Machineries in Helicobacter pylori by Cryo-Electron Tomography

The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11

Intermolecular Cross-linking between the Periplasmic Loop3–4 Regions of PomA, a Component of the Na+-driven Flagellar Motor of Vibrio alginolyticus

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