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.     

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.     

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.     

Concerted effects of amino acid substitutions in conserved charged residues and other residues in the cytoplasmic domain of PomA, a stator component of Na+-driven flagella

PomA is a membrane protein that is one of the essential components of the sodium-driven flagellar motor in Vibrio species. The cytoplasmic charged residues of Escherichia coli MotA, which is a PomA homolog, are believed to be required for the interaction of MotA with the C-terminal region of FliG. It was previously shown that a PomA variant with neutral substitutions in the conserved charged residues (R88A, K89A, E96Q, E97Q, and E99Q; AAQQQ) was functional. In the present study, five other conserved charged residues were replaced with neutral amino acids in the AAQQQ PomA protein. These additional substitutions did not affect the function of PomA. However, strains expressing the AAQQQ PomA variant with either an L131F or a T132M substitution, neither of which affected motor function alone, exhibited a temperature-sensitive (TS) motility phenotype. The double substitutions R88A or E96Q together with L131F were sufficient for the TS phenotype. The motility of the PomA TS mutants immediately ceased upon a temperature shift from 20 to 42 degrees C and was restored to the original level approximately 10 min after the temperature was returned to 20 degrees C. It is believed that PomA forms a channel complex with PomB. The complex formation of TS PomA and PomB did not seem to be affected by temperature. Suppressor mutations of the TS phenotype were mapped in the cytoplasmic boundaries of the transmembrane segments of PomA. We suggest that the cytoplasmic surface of PomA is changed by the amino acid substitutions and that the interaction of this surface with the FliG C-terminal region is temperature sensitive.     

Functional Transfer of an Essential Aspartate for the Ion-binding Site in the Stator Proteins of the Bacterial Flagellar Motor

Rotation of the bacterial flagellar motor exploits the electrochemical potential of the coupling ion (H⁺ or Na⁺) as its energy source. In the marine bacterium Vibrio alginolyticus, the stator complex is composed of PomA and PomB, and conducts Na⁺ across the cytoplasmic membrane to generate rotation. The transmembrane (TM) region of PomB, which forms the Na⁺-conduction pathway together with TM3 and TM4 of PomA, has a highly conserved aspartate residue (Asp24) that is essential for flagellar rotation. This residue contributes to the Na⁺-binding site. However, it is not clear whether residues other than Asp24 are involved in binding the coupling ion. We examined the possibility that loss of the negative charge of Asp24 can be suppressed by introduction of negatively charged residues in TM3 or TM4 of PomA. The motility defect associated with the D24N substitution in PomB could be rescued only by a N194D substitution in PomA. This result suggests that there must be a negatively charged ion-binding pocket in the stator complex but that the presence of a negatively charged residue at position 24 of PomB is not essential. A tandemly fused PomA dimer containing the N194D mutation either in its N-terminal or C-terminal half with PomB-D24N was functional, suggesting that PomB-D24N can form an ion-binding pocket with either subunit of PomA dimer. The findings obtained in this study provide important clues to the mechanism of ion binding in the stator complex.     

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.     

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.     

Interaction of the C-Terminal Tail of FliF with FliG from the Na+-Driven Flagellar Motor of Vibrio alginolyticus

Rotation of the polar flagellum of Vibrio alginolyticus is driven by a Na⁺-type flagellar motor. FliG, one of the essential rotor proteins located at the upper rim of the C ring, binds to the membrane-embedded MS ring. The MS ring is composed of a single membrane protein, FliF, and serves as a foundation for flagellar assembly. Unexpectedly, about half of the Vibrio FliF protein produced at high levels in Escherichia coli was found in the soluble fraction. Soluble FliF purifies as an oligomer of ∼700 kDa, as judged by analytical size exclusion chromatography. By using fluorescence correlation spectroscopy, an interaction between a soluble FliF multimer and FliG was detected. This binding was weakened by a series of deletions at the C-terminal end of FliF and was nearly eliminated by a 24-residue deletion or a point mutation at a highly conserved tryptophan residue (W575). Mutations in FliF that caused a defect in FliF-FliG binding abolish flagellation and therefore confer a nonmotile phenotype. As data from in vitro binding assays using the soluble FliF multimer correlate with data from in vivo functional analyses, we conclude that the C-terminal region of the soluble form of FliF retains the ability to bind FliG. Our study confirms that the C-terminal tail of FliF provides the binding site for FliG and is thus required for flagellation in Vibrio, as reported for other species. This is the first report of detection of the FliF-FliG interaction in the Na⁺-driven flagellar motor, both in vivo and in vitro.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.     

Control of speed modulation (chemokinesis) in the unidirectional rotary motor of Sinorhizobium meliloti

Swimming cells of Sinorhizobium meliloti are driven by flagella that rotate only clockwise. They can modulate rotary speed (achieve chemokinesis) and reorient the swimming path by slowing flagellar rotation. The flagellar motor is energized by proton motive force, and torque is generated by electrostatic interactions at the rotor/stator (FliG/MotA-MotB) interface. Like the Escherichia coli flagellar motor that switches between counterclockwise and clockwise rotation, the S. meliloti rotary motor depends on electrostatic interactions between conserved charged residues, namely, Arg294 and Glu302 (FliG) and Arg90, Glu98 and Glu150 (MotA). Unlike in E. coli, however, Glu150 is essential for torque generation, whereas residues Arg90 and Glu98 are crucial for the chemotaxis-controlled variation of rotary speed. Substitutions of either Arg90 or Glu98 by charge-neutralizing residues or even by their smaller, charge-maintaining isologues, lysine and aspartate, resulted in top-speed flagellar rotation and decreased potential to slow down in response to tactic signalling (chemokinesis-defective mutants). The data infer a novel mechanism of flagellar speed control by electrostatic forces acting at the rotor/stator interface. These features have been integrated into a working model of the speed-modulating rotary 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.     

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.     

Hybrid Motor with H+- and Na+-Driven Components Can Rotate Vibrio Polar Flagella by Using Sodium Ions

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium Rhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembrane region of PomB. To examine whether the R. sphaeroides motor genes can function in place of the pomA and pomB genes of V. alginolyticus, we constructed plasmids including both motA and motB or motA alone and transformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand, pomB mutants were not restored to motility by any plasmid containing motA and/or motB. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of R. sphaeroides can generate torque by coupling with the sodium ion flux in place of PomA of V. alginolyticus.     

Motility Protein Complexes in the Bacterial Flagellar Motor

Among the many proteins needed for the assembly and function of bacterial flagella, only five have been suggested to be involved in torque generation. These are MotA, MotB, FliG, FliM and FliN. In this study, we have probed binding interactions among these proteins, by using protein fusions to glutathioneS-transferase or to oligo-histidine, in conjunction with co-isolation assays. The results show that FliG, FliM and FliN all bind to each other, and that each also self-associates. MotA and MotB also bind to each other, and MotA interacts, but only weakly, with FliG and FliM. Taken together with previous genetic, physiological and ultrastructural studies, these results provide strong support for the view that FliG, FliM and FliN function together in a complex on the rotor of the flagellar motor, whereas MotA and MotB form a distinct complex that functions as the stator. Torque generation in the flagellar motor is thus likely to involve interactions between these two protein complexes.     

Serine 26 in the PomB Subunit of the Flagellar Motor Is Essential for Hypermotility of Vibrio cholerae

Vibrio cholerae is motile by means of its single polar flagellum which is driven by the sodium-motive force. In the motor driving rotation of the flagellar filament, a stator complex consisting of subunits PomA and PomB converts the electrochemical sodium ion gradient into torque. Charged or polar residues within the membrane part of PomB could act as ligands for Na⁺, or stabilize a hydrogen bond network by interacting with water within the putative channel between PomA and PomB. By analyzing a large data set of individual tracks of swimming cells, we show that S26 located within the transmembrane helix of PomB is required to promote very fast swimming of V. cholerae. Loss of hypermotility was observed with the S26T variant of PomB at pH 7.0, but fast swimming was restored by decreasing the H⁺ concentration of the external medium. Our study identifies S26 as a second important residue besides D23 in the PomB channel. It is proposed that S26, together with D23 located in close proximity, is important to perturb the hydration shell of Na⁺ before its passage through a constriction within the stator channel.     

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.     

Function of Protonatable Residues in the Flagellar Motor of Escherichia coli: a Critical Role for Asp 32 of MotB

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.     

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.     

Isolation of Basal Bodies with C-Ring Components from the Na+-Driven Flagellar Motor of Vibrio alginolyticus

To investigate the Na⁺-driven flagellar motor of Vibrio alginolyticus, we attempted to isolate its C-ring structure. FliG but not FliM copurified with the basal bodies. FliM proteins may be easily dissociated from the basal body. We could detect FliG on the MS ring surface of the basal bodies.The basal body, which is the part of the rotor, is composed of four rings and a rod that penetrates them. Three of these rings, the L, P, and MS rings, are embedded in the outer membrane, peptidoglycan layer and in the inner membrane, respectively (1), while the C-ring of Salmonella species is attached to the cytoplasmic side of the basal body (3). The C-ring is composed of the proteins FliG, FliM, and FliN (25), and genetic evidence indicates that the C-ring is important for flagellar assembly, torque generation, and regulation of rotational direction (33, 34). FliG, 26 molecules of which are incorporated into the motor, appears to be the protein that is most directly involved in torque generation (15). Mutational analysis suggests that electrostatic interactions between conserved charged residues in the C-terminal domain of FliG and the cytoplasmic domain of MotA are important in torque generation (14), although this may not be the case for the Na⁺-type motor of Vibrio alginolyticus (32, 35, 36). FliM interacts with the chemotactic signaling protein CheY in its phosphorylated form (CheY-P) to regulate rotational direction (30). It has been reported that 33 to 35 copies of FliM assemble into a ring structure (28, 29). FliN contributes mostly to forming the C-ring structure (37). The crystal structure of FliN revealed a hydrophobic patch formed by several well-conserved hydrophobic residues (2). Mutational analysis showed that this patch is important for flagellar assembly and rotational switching (23, 24). The association state of FliN in solution was studied by analytical ultracentrifugation, which provided clues to the higher-level organization of the protein. Thermotoga maritima FliN exists primarily as a dimer in solution, and T. maritima FliN and FliM together formed a stable FliM₁-FliN₄ complex (2). The spatial distribution of these proteins in the C-ring of Salmonella species was investigated using three-dimensional reconstitution analysis with electron microscopy (28). However, the correct positioning has still not been clarified.The Na⁺-driven motor requires two additional proteins, MotX and MotY, for torque generation (19-21, 22). These proteins form a unique ring structure, the T ring, located below the LP ring in the polar flagellum of V. alginolyticus (9, 26). It has been suggested that MotX interacts with MotY and PomB (11, 27). Unlike peritrichously flagellated Escherichia coli and Salmonella species, V. alginolyticus has two different flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum is used for motility in low-viscosity environments such as seawater (18). As described above, it is driven by a Na⁺-type motor. However, in high-viscosity environments, such as the mucus-coated surfaces of fish bodies, cells induce numerous unsheathed lateral flagella that have H⁺-driven motors (7, 8). We have been focusing on the Na⁺-driven polar flagellar motor, since there are certain advantages to studying its mechanism of torque generation over the H⁺-type motor: sodium motive force can be easily manipulated by controlling the Na⁺ concentration in the medium, and motor rotation can be specifically inhibited using phenamil (10). Moreover, its rotation rate is surprisingly high, up to 1,700 rps (compared to ∼200 rps and ∼300 rps for Salmonella species flagella and E. coli flagella, respectively) (12, 16, 17).Although understanding the C-ring structure and function is essential for clarifying the mechanism of motor rotation, there is no information about the C-ring of the polar flagellar motor of Vibrio species or the flagella of any genus other than Salmonella. Since Vibrio species have all of the genes coding for C-ring components, we would expect its location to be on the cytoplasmic side of the MS ring, as in Salmonella species. In this study, we attempted to isolate the polar flagellar basal body with the C-ring attached and investigate whether it is organized similarly to the H⁺-driven flagellar motor of Salmonella enterica serovar Typhimurium.     

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.