The conserved charged residues of the C-terminal region of FliG, a rotor component of the Na+-driven flagellar motor

FliG is an essential component of the flagellar motor and functions in flagellar assembly, torque generation and regulation of the direction of flagellar rotation. The five charged residues important for the rotation of the flagellar motor were identified in Escherichiacoli FliG (FliG(E)). These residues are clustered in the C terminus and are all conserved in FliG(V) of the Na(+)-driven motor of Vibrioalginolyticus (Lys284, Arg301, Asp308, Asp309 and Arg317). To investigate the roles of these charged residues in the Na(+)-driven motor, we cloned the VibriofliG gene and introduced single or multiple substitutions into the corresponding positions in FliG(V). FliG(V) with double Ala replacements in all possible combinations at these five conserved positions still retained significant motile ability, although some of the mutations completely eliminated the function of FliG(E). All of the triple mutants constructed in this study also remained motile. These results suggest that the important charged residues may be located in different places and the conserved charged residues are not so important for the Na(+)-driven flagellar motor of Vibrio. The chimeric FliG protein (FliG(VE)), composed of the N-terminal domain from V.alginolyticus and the C-terminal domain from E.coli, functions in Vibrio cells. The mutations of the charge residues of the C-terminal region in FliG(VE) affected swarming ability as in E.coli. Both the FliG(V) and the FliG(VE) proteins with the triple mutation were more susceptible to proteolysis than proteins without the mutation, suggesting that their conformations were altered.     

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.     

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.     

Intracellular Na+ kinetically interferes with the rotation of the Na(+)-driven flagellar motors of Vibrio alginolyticus

To understand the mechanism of Na+ movement through the force-generating units of the Na(+)-driven flagellar motors of Vibrio alginolyticus, the effect of intracellular Na+ concentration on motor rotation was investigated. Control cells containing about 50 mM Na+ showed good motility even at 10 mM Na+ in the medium, i.e. in the absence of an inwardly directed Na+ gradient. In contrast, Na(+)-loaded cells containing about 400 mM Na+ showed very poor motility at 500 mM Na+ in the medium, i.e. even in the presence of an inwardly directed Na+ gradient. The membrane potential of the cells, which is a major driving force for the motor under these conditions, was not detectably altered, and consistently with this, Na(+)-coupled sucrose transport was only partly reduced in the Na(+)-loaded cells. Motility of the Na(+)-loaded cells was restored by decreasing the intracellular Na+ concentration, and the rate of restoration of motility correlated with the rate of the Na+ decrease. These results indicate that the absolute concentration of the intracellular Na+ is a determinant of the rotation rate of the Na(+)-driven flagellar motors of V. alginolyticus. A simple explanation for this phenomenon is that the force-generating unit of the motor has an intracellular Na(+)-binding site, at which the intracellular Na+ kinetically interferes with the rate of Na+ influx for motor rotation.     

The systematic substitutions around the conserved charged residues of the cytoplasmic loop of Na+-driven flagellar motor component PomA

PomA, a homolog of MotA in the H+-driven flagellar motor, is an essential component for torque generation in the Na+-driven flagellar motor. Previous studies suggested that two charged residues, R90 and E98, which are in the single cytoplasmic loop of MotA, are directly involved in this process. These residues are conserved in PomA of Vibrio alginolyticus as R88 and E96, respectively. To explore the role of these charged residues in the Na+-driven motor, we replaced them with other amino acids. However, unlike in the H+-driven motor, both of the single and the double PomA mutants were functional. Several other positively and negatively charged residues near R88 and E96, namely K89, E97 and E99, were neutralized. Motility was retained in a strain producing the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein. The swimming speed of the AAQQQ strain was as fast as that of the wild-type PomA strain, but the direction of motor rotation was abnormally counterclockwise-biased. We could, however, isolate non-motile or poorly motile mutants when certain charged residues in PomA were reversed or neutralized. The charged residues at positions 88-99 of PomA may not be essential for torque generation in the Na+-driven motor and might play a role in motor function different from that of the equivalent residues of the H+-driven motor.     

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.     

Study of the torque of the bacterial flagellar motor using a rotating electric field.

Bacterial flagella are driven by a rotary motor that is energized by an electrochemical ion gradient across the cell membrane. In this study the torque generated by the flagellar motor was measured in tethered cells of a smooth-swimming Escherichia coli strain by using rotating electric fields to determine the relationship between the torque and speed over a wide range. By measuring the electric current applied to the sample cell and combining the data obtained at different viscosities, the torque of the flagellar motor was estimated up to 55 Hz, and also at negative rotation rates. By this method we have found that the torque of the flagellar motor linearly decreases with rotation rate from negative through positive rate of rotation. In addition, the dependence of torque upon temperature was also investigated. We showed that torque at the high speeds encountered in swimming cells had a much steeper dependence on temperature that at the low speeds encountered in tethered cells. From these results, the activation energy of the proton transfer reaction in the torque-generating unit was calculated to be about 7.0 x 10(-20) J.     

Roles of charged residues in the C-terminal region of PomA, a stator component of the Na+-driven flagellar motor

Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na(+)-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na(+)-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na(+)-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na(+)-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.     

The stall torque of the bacterial flagellar motor.

The bacterial flagellar motor couples the flow of protons across the cytoplasmic membrane to the rotation of a helical flagellar filament. Using tethered cells, we have measured the stall torque required to block this rotation and compared it with the torque of the running motor over a wide range of values of proton-motive force and pH. The stall torque and the running torque vary identically: both appear to saturate at large values of the proton-motive force and both decrease at low or high pH. This suggests that up to speeds of approximately 5 Hz the operation of the motor is not limited by the mobility of its internal components or the rates of proton transfer reactions coupled to flagellar rotation.     

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.     

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.     

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.     

The bidirectional polar and unidirectional lateral flagellar motors of Vibrio alginolyticus are controlled by a single CheY species

The bacterial flagellar motor is an elaborate molecular machine that converts ion-motive force into mechanical force (rotation). One of its remarkable features is its swift switching of the rotational direction or speed upon binding of the response regulator phospho-CheY, which causes the changes in swimming that achieve chemotaxis. Vibrio alginolyticus has dual flagellar systems: the Na(+)-driven polar flagellum (Pof) and the H(+)-driven lateral flagella (Laf), which are used for swimming in liquid and swarming over surfaces respectively. Here we show that both swimming and surface-swarming of V. alginolyticus involve chemotaxis and are regulated by a single CheY species. Some of the substitutions of CheY residues conserved in various bacteria have different effects on the Pof and Laf motors, implying that CheY interacts with the two motors differently. Furthermore, analyses of tethered cells revealed that their switching modes are different: the Laf motor rotates exclusively counterclockwise and is slowed down by CheY, whereas the Pof motor turns both counterclockwise and clockwise, and CheY controls its rotational direction.     

Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load

Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.     

Torque-speed relationship of the flagellar rotary motor of Escherichia coli

The output of a rotary motor is characterized by its torque and speed. We measured the torque-speed relationship of the flagellar rotary motor of Escherichia coli by a new method. Small latex spheres were attached to flagellar stubs on cells fixed to the surface of a glass slide. The angular speeds of the spheres were monitored in a weak optical trap by back-focal-plane interferometry in solutions containing different concentrations of the viscous agent Ficoll. Plots of relative torque (viscosity x speed) versus speed were obtained over a wide dynamic range (up to speeds of approximately 300 Hz) at three different temperatures, 22.7, 17.7, and 15.8 degrees C. Results obtained earlier by electrorotation (, Biophys. J. 65:2201-2216) were confirmed. The motor operates in two dynamic regimes. At 23 degrees C, the torque is approximately constant up to a knee speed of nearly 200 Hz, and then it falls rapidly with speed to a zero-torque speed of approximately 350 Hz. In the low-speed regime, torque is insensitive to changes in temperature. In the high-speed regime, it decreases markedly at lower temperature. These results are consistent with models in which torque is generated by a powerstroke mechanism (, Biophys. J. 76:580-587).     

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.     

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.     

Functional role of a conserved aspartic acid residue in the motor of the Na-driven flagellum from Vibrio cholerae

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H⁺ or Na⁺) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na⁺-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na⁺ to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H⁺-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.     

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.