Torque-speed relationship of the Na+-driven flagellar motor of Vibrio alginolyticus

The torque-speed relationship of the Na(+)-driven flagellar motor of Vibrio alginolyticus was investigated. The rotation rate of the motor was measured by following the position of a bead, attached to a flagellar filament, using optical nanometry. In the presence of 50mM NaCl, the generated torque was relatively constant ( approximately 3800pNnm) at lower speeds (speeds up to approximately 300Hz) and then decreased steeply, similar to the H(+)-driven flagellar motor of Escherichia coli. When the external NaCl concentration was varied, the generated torque of the flagellar motor was changed over a wide range of speeds. This result could be reproduced using a simple kinetic model, which takes into consideration the association and dissociation of Na(+) onto the motor. These results imply that for a complete understanding of the mechanism of flagellar rotation it is essential to consider both the electrochemical gradient and the absolute concentration of the coupling ion.     

Amiloride at pH 7.0 inhibits the Na(+)-driven flagellar motors of Vibrio alginolyticus but allows cell growth.

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus, is known to inhibit secondarily the growth of alkalophiles. The motility of a marine Vibrio, V. alginolyticus, was almost completely inhibited by 2 mM amiloride either at pH 7.0 or 8.5. We found that this concentration of amiloride inhibited the cell growth completely at pH 8.5 but only slightly at pH 7.0. Kinetic analysis of the inhibition of motility by amiloride at pH 7.0 showed that the inhibition was competitive with Na+ in the medium. Thus, amiloride at pH 7.0 is really a specific and useful tool for the analysis of the Na(+)-driven flagellar motors of Vibrio.     

Na+-driven bacterial flagellar motors

Bacterial flagellar motors are the reversible rotary engine which propels the cell by rotating a helical flagellar filament as a screw propeller. The motors are embedded in the cytoplasmic membrane, and the energy for rotation is supplied by the electrochemical potential of specific ions across the membrane. Thus, the analysis of motor rotation at the molecular level is linked to an understanding of how the living system converts chemical energy into mechanical work. Based on the coupling ions, the motors are divided into two types; one is the H⁺-driven type found in neutrophiles such asBacillus subtilis andEscherichia coli and the other is the Na⁺-driven type found in alkalophilicBacillus and marineVibrio. In this review, we summarize the current status of research on the rotation mechanism of the Na⁺-driven flagellar motors, which introduces several new aspects in the analysis.     

Na(+)-driven flagellar motor of Vibrio

Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. Bacteria move using rotating helical flagellar filaments. The flagellar motor is located at the base of the filament and is buried in the cytoplasmic membrane. Flagellar motors are classified into two types according to the coupling ion: namely the H(+)-driven motor and the Na(+)-driven motor. Analysis of the flagellar motor at the molecular level is far more advanced in the H(+)-driven motor than in the Na(+)-driven motor. Recently, the genes of the Na(+)-driven motor have been cloned from a marine bacterium of Vibrio sp. and some of the motor proteins have been purified and characterized. In this review, we summarize recent studies of the Na(+)-driven flagellar motor.     

Functional reconstitution of the Na(+)-driven polar flagellar motor component of Vibrio alginolyticus

The bacterial flagellar motor is a molecular machine that couples the influx of specific ions to the generation of the force necessary to drive rotation of the flagellar filament. Four integral membrane proteins, PomA, PomB, MotX, and MotY, have been suggested to be directly involved in torque generation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. In the present study, we report the isolation of the functional component of the torque-generating unit. The purified protein complex appears to consist of PomA and PomB and contains neither MotX nor MotY. The PomA/B protein, reconstituted into proteoliposomes, catalyzed (22)Na(+) influx in response to a potassium diffusion potential. Sodium uptake was abolished by the presence of Li(+) ions and phenamil, a sodium channel blocker. This is the first demonstration of a purification and functional reconstitution of the bacterial flagellar motor component involved in torque generation. In addition, this study demonstrates that the Na(+)-driven motor component, PomA and PomB, forms the Na(+)-conducting channel.     

Ion-coupling determinants of Na+-driven and H+-driven flagellar motors

The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H(+) or Na(+) 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 selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DeltapomAB 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.     

Characterization of PomA mutants defective in the functional assembly of the Na(+)-driven flagellar motor in Vibrio alginolyticus

The polar flagellar motor of Vibrio alginolyticus rotates using Na(+) influx through the stator, which is composed of 2 subunits, PomA and PomB. About a dozen stators dynamically assemble around the rotor, depending on the Na(+) concentration in the surrounding environment. The motor torque is generated by the interaction between the cytoplasmic domain of PomA and the C-terminal region of FliG, a component of the rotor. We had shown previously that mutations of FliG affected the stator assembly around the rotor, which suggested that the PomA-FliG interaction is required for the assembly. In this study, we examined the effects of various mutations mainly in the cytoplasmic domain of PomA on that assembly. All mutant stators examined, which resulted in the loss of motor function, assembled at a lower level than did the wild-type PomA. A His tag pulldown assay showed that some mutations in PomA reduced the PomA-PomB interaction, but other mutations did not. Next, we examined the ion conductivity of the mutants using a mutant stator that lacks the plug domain, PomA/PomB(ΔL)(Δ41-120), which impairs cell growth by overproduction, presumably because a large amount of Na(+) is conducted into the cells. Some PomA mutations suppressed this growth inhibition, suggesting that such mutations reduce Na(+) conductivity, so that the stators could not assemble around the rotor. Only the mutation H136Y did not impair the stator formation and ion conductivity through the stator. We speculate that this particular mutation may affect the PomA-FliG interaction and prevent activation of the stator assembly around the rotor.     

Characterization of the periplasmic region of PomB, a Na+-driven flagellar stator protein in Vibrio alginolyticus

The stator proteins PomA and PomB form a complex that couples Na⁺ influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB_C). To investigate the function of PomB_C, a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB_C fragment consisting of residues 135 to 315 (PomB_C5) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB_ΔL) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na⁺ influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB_ΔL stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal α-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a “plug” segment is important for the control of Na⁺ influx through the stator complex as well as for stator assembly.     

Multimeric structure of the PomA/PomB channel complex in the Na+-driven flagellar motor of Vibrio alginolyticus

It is known that PomA and PomB form a complex that functions as a Na(+) channel and generates the torque of the Na(+)-driven flagellar motor of Vibrio alginolyticus. It has been suggested that PomA works as a dimer and that the PomA/PomB complex is composed of four PomA and two PomB molecules. PomA does not have any Cys residues and PomB has three Cys residues. Therefore, a mutant PomB (PomB(cl)) whose three Cys residues were replaced by Ala was constructed and found to be motile as well. We carried out gel filtration analysis and examined the effect of cross-linking between the Cys residues of PomB on the formation of the PomA/PomB complex. In the presence of dithiothreitol (DTT), the elution profile of the PomA/PomB complex was shifted to a lower apparent molecular mass fraction similar to that of the complex of the wild-type PomA and PomB(cl) mutant. Next, to analyze the arrangement of PomA molecules in the complex, we introduced the mutation P172C, which has been shown to cross-link PomA molecules, into tandem PomA dimers (PomA approximately PomA). These mutant dimers showed a dominant-negative effect. DTT could restore the function of PomA approximately P172C and P172C approximately P172C, but not P172C approximately PomA. Interdimer and intradimer cross-linked products were observed; the interdimer cross-linked products could be assembled with PomB. The formation of the interdimer cross-link suggests that the channel complex of the Na(+)-driven flagellar motor is composed of two units of a complex consisting of two PomA and one PomB, and that they might interact with each other via not only PomA but also PomB.     

Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na⁺-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na⁺). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at ∼ 2200 pN nm) from stall up to a “knee” speed of ∼ 420 Hz, and then falls linearly with speed, extrapolating to zero torque at ∼ 910 Hz. Motors containing one to five stators generated ∼ 200 pN nm per stator at speeds up to ∼ 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to ∼ 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to ∼ 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.     

Na+-driven flagellar motors of an alkalophilic Bacillus strain YN-1

Flagellar motors of some alkalophilic Bacillus strains have been suggested to be powered by the electrochemical potential gradient of Na+, namely the (formula: see text) (Hirota, N., Kitada, M., and Imae, Y. (1981) FEBS Lett. 132, 278-280). In the present study, we quantitatively measured the (formula: see text) and motility of one of the strains, YN-1. Swimming speed of YN-1 cells increased linearly with a logarithmic increase of Na+ concentration in the medium up to 100 mM. The intracellular Na+ concentration and the membrane potential of the cell were about 30 mM and -170 mV, respectively, and stayed constant irrespective of Na+ concentration in the medium. Thus, the swimming speed changed as a function of the chemical potential difference of Na+ across the cell membrane. When the membrane potential of YN-1 cells was decreased by a combination of valinomycin and various concentrations of K+ in the medium, the swimming speed of the cells decreased linearly and reached zero at around -90 mV. Under the condition, the intracellular Na+ concentration stayed constant. Thus, the membrane potential was also a determinant of the swimming speed. Furthermore, the chemical potential of Na+ and the membrane potential were found to be equivalent as the energy source for motility. Therefore, it is concluded that the (formula: see text) is the energy source for the flagellar motors of YN-1 cells. Threshold value of the (formula: see text) for motility was about -100 mV.     

Requirements for conversion of the Na(+)-driven flagellar motor of Vibrio cholerae to the H(+)-driven motor of Escherichia coli

Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H(+) or Na(+) into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions in pomA, pomB, motX, or motY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complemented fliG-null strains of the parent species but not fliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB, motX, and motY became weakly motile when the E. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins of V. cholerae and that the hybrid motors are driven by the proton motive force.     

Cell-free synthesis of the torque-generating membrane proteins, PomA and PomB, of the Na+-driven flagellar motor in Vibrio alginolyticus

Flagellar motor proteins, PomA and PomB, are essential for converting the sodium motive force into rotational energy in the Na(+)-driven flagella motor of Vibrio alginolyticus. PomA and PomB, which are cytoplasmic membrane proteins, together comprise the stator complex of the motor and form a Na(+) channel. We tried to synthesize PomA and PomB by using the cell-free protein synthesis system, PURESYSTEM. We succeeded in doing so in the presence of liposomes, and showed an interaction between them using the pull-down assay. It seems likely that the proteins are inserted into liposomes and assembled spontaneously. The N-terminal region of in vitro synthesized PomB appeared to be lost, but this problem was suppressed by fusing GFP to the N-terminus of PomB or by mutagenesis at Pro-11 or Pro-12. A structural change of the N-terminal region of PomB by these modifications may prevent cleavage during protein synthesis in PURESYSTEM. The mutations did not affect the functioning of the motor. Using this system, biochemical analysis of PomA and PomB can be performed easily and efficiently.     

Roles of charged residues of rotor and stator in flagellar rotation: comparative study using H+-driven and Na+-driven motors in Escherichia coli

In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na+-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.     

Amiloride, a specific inhibitor for the Na+-driven flagellar motors of alkalophilic Bacillus

Na+-driven flagellar motors of alkalophilic Bacillus were found to be inhibited by amiloride, a potent inhibitor for many Na+-coupled systems. A concentration of 0.5 mM of amiloride completely inhibited motility but showed almost no effect on the membrane potential, the intracellular pH homeostasis, and the ATP content of the cells. Furthermore, the activity of a Na+-coupled amino acid transport system was reduced only by half by this concentration of amiloride. Thus, the inhibition of motility of alkalophilic Bacillus by amiloride was rather specific. The inhibition of motility produced by amiloride was restored by increasing Na+ concentrations in the medium. Kinetic analysis of the data revealed that the inhibition was competitive with respect to the concentration of Na+ in the medium. Therefore, it is quite logical to assume that amiloride inhibits the rotation of the Na+-driven flagellar motors of alkalophilic Bacillus by competing with Na+ at the force-generating site of the motor. Some amiloride analogs known to selectively inhibit Na+ channels were potent inhibitors for the flagellar motors, suggesting that the Na+-interacting site of the motors has some similarity to that of the Na+ channels.     

Forced rotation of Na+-driven flagellar motor in a coupling ion-free environment

Rotational characteristics of Na+-driven flagellar motor in the presence and absence of coupling ion were analyzed by electrorotation method. The motor rotated spontaneously in the presence of Na+, and the rotation accelerated or decelerated following the direction of the applied external torque. The spontaneous motor rotation was inhibited by removal of external Na+, however, the motor could be forcibly rotated by relatively small external torque applied by the electrorotation apparatus. The observed characteristic of the motor was completely different from that of ATP-driven motor systems, which form rigor bond when their energy source, ATP, is absent. The internal resistance of the flagellar motor increased significantly when the coupling ion could not access the inside of the motor, suggesting that the interaction between the rotor and the stator is changed by the binding of the coupling ion to the internal sites of the motor.     

Specific inhibition of the Na(+)-driven flagellar motors of alkalophilic Bacillus strains by the amiloride analog phenamil.

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus strains, was found to cause growth inhibition; therefore, the use of amiloride for the isolation of motility mutants was difficult. On the other hand, phenamil, an amiloride analog, inhibited motor rotation without affecting cell growth. A concentration of 50 microM phenamil completely inhibited the motility of strain RA-1 but showed no effect on the membrane potential, the intracellular pH, or Na(+)-coupled amino acid transport, which was consistent with the fact that there was no effect on cell growth. Kinetic analysis of the inhibition of motility by phenamil indicated that the inhibition was noncompetitive with Na+ in the medium. A motility mutant was isolated as a swarmer on a swarm agar plate containing 50 microM phenamil. The motility of the mutant showed an increased resistance to phenamil but normal sensitivity to amiloride. These results suggest that phenamil and amiloride interact at different sites on the motor. By examining various bacterial species, phenamil was found to be a specific and potent inhibitor for the Na(+)-driven flaggellar motors not only in various strains of alkalophilic Bacillus spp. but also in a marine Vibrio sp.     

Multimeric structure of PomA, a component of the Na+-driven polar flagellar motor of vibrio alginolyticus

Four integral membrane proteins, PomA, PomB, MotX, and MotY, are thought to be directly involved in torque generation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. Our previous study showed that PomA and PomB form a complex, which catalyzes sodium influx in response to a potassium diffusion potential. PomA forms a stable dimer when expressed in a PomB null mutant. To explore the possible functional dependence of PomA domains in adjacent subunits, we prepared a series of PomA dimer fusions containing different combinations of wild-type or mutant subunits. Introduction of the mutation P199L, which completely inactivates flagellar rotation, into either the first or the second half of the dimer abolished motility. The P199L mutation in monomeric PomA also altered the PomA-PomB interaction. PomA dimer with the P199L mutation even in one subunit also had no ability to interact with PomB, indicating that the both subunits in the dimer are required for the functional interaction between PomA and PomB. Flagellar rotation by wild-type PomA dimer was completely inactivated by phenamil, a sodium channel blocker. However, activity was retained in the presence of phenamil when either half of the dimer was replaced with a phenamil-resistant subunit, indicating that both subunits must bind phenamil for motility to be fully inhibited. These observations demonstrate that both halves of the PomA dimer function together to generate the torque for flagellar rotation.     

Intermolecular cross-linking between the periplasmic Loop3-4 regions of PomA, a component of the Na+-driven flagellar motor of Vibrio alginolyticus

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 C-terminal 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.