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.     

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.     

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.     

The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na-driven flagella and required for stator formation

The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na(+)-driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a DeltamotX or DeltamotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.     

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.     

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.     

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.     

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.     

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.     

Interaction of PomB with the third transmembrane segment of PomA in the Na+-driven polar flagellum of Vibrio alginolyticus

The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na(+)-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na(+) than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na(+) conduction to flagellar rotation.     

Mutations targeting the C-terminal domain of FliG can disrupt motor assembly in the Na(+)-driven flagella of Vibrio alginolyticus

The torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor-stator interactions. Here, we investigated stator assembly using a mutational approach of the Na⁺-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor-stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side.     

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.     

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.     

Assembly of motor proteins, PomA and PomB, in the Na+-driven stator of the flagellar motor

PomA and PomB are transmembrane proteins that form the stator complex in the sodium-driven flagellar motor of Vibrio alginolyticus and are believed to surround the rotor part of the flagellar motor. We constructed and observed green fluorescent protein (GFP) fusions of the stator proteins PomA and PomB in living cells to clarify how stator proteins are assembled and installed into the flagellar motor. We were able to demonstrate that GFP-PomA and GFP-PomB localized to a cell pole dependent on the presence of the polar flagellum. Localization of the GFP-fused stator proteins required their partner subunit, PomA or PomB, and the C-terminal domain of PomB, which has a peptidoglycan-binding motif. Each of the GFP-fused stator proteins was co-isolated with its partner subunit from detergent-solubilized membrane. From these lines of evidence, we have demonstrated that the stator proteins are incorporated into the flagellar motor as a PomA/PomB complex and are fixed to the cell wall via the C-terminal domain of PomB.     

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.     

Coupling ion specificity of chimeras between H(+)- and Na(+)-driven motor proteins, MotB and PomB, in Vibrio polar flagella

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.     

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.     

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.     

Deletion analysis of the carboxyl-terminal region of the PomB component of the vibrio alginolyticus polar flagellar motor

The stator of the sodium-driven flagellar motor of Vibrio alginolyticus is a membrane protein complex composed of four PomA and two PomB subunits. PomB has a peptidoglycan-binding motif in the C-terminal region. In this study, four kinds of PomB deletions in the C terminus were constructed. None of the deletion proteins restored motility of the DeltapomB strain. The PomA protein was coisolated with all of the PomB derivatives under detergent-solubilized conditions. Homotypic disulfide cross-linking of all of the deletion derivatives through naturally occurring Cys residues was detected. We conclude that the C-terminal region of PomB is essential for motor function but not for oligomerization of PomB with itself or PomA.     

Cloning and characterization of motX, a Vibrio alginolyticus sodium-driven flagellar motor gene.

Four motor proteins, MotX, MotY, PomA, and PomB, have been identified as constituents of the Na(+)-driven flagellum of Vibrio species. In this study, the complete motX gene was cloned from Vibrio alginolyticus and shown to complement three mot mutations, motX94, motX115, and motX119, as well as a V. parahaemolyticus motX mutant. The motX94 mutant contains a frameshift at Val86 of MotX, while the motX115 and motX119 mutations comprise substitutions of Ala146 to Val and Gln 194 to amber, respectively. When MotX was overexpressed in Vibrio cells, the amount of MotY detected in the membrane fraction increased, and vice versa, suggesting that MotX and MotY mutually stabilize each other by interacting at the membrane level. When a plasmid containing the motX gene was introduced into motY mutants NMB117 (motY117) and VIO542 (motY542), the mutations were suppressed. In contrast, motY could not cause the recovery of any swarm-defective motX mutants studied. Considering the above evidence, we propose that MotX is more directly involved than MotY in the mechanical functioning of the Na(+)-type flagellar motor, and that MotY may stabilize MotX to support its interaction with other Mot proteins.