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.     

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 Function of the Na+-Driven Flagellum of Vibrio cholerae Is Determined by Osmolality and pH

Vibrio cholerae is motile by its polar flagellum, which is driven by a Na⁺-conducting motor. The stators of the motor, composed of four PomA and two PomB subunits, provide access for Na⁺ to the torque-generating unit of the motor. To characterize the Na⁺ pathway formed by the PomAB complex, we studied the influence of chloride salts (chaotropic, Na⁺, and K⁺) and pH on the motility of V. cholerae. Motility decreased at elevated pH but increased if a chaotropic chloride salt was added, which rules out a direct Na⁺ and H⁺ competition in the process of binding to the conserved PomB D23 residue. Cells expressing the PomB S26A/T or D42N variants lost motility at low Na⁺ concentrations but regained motility in the presence of 170 mM chloride. Both PomA and PomB were modified by N,N′-dicyclohexylcarbodiimide (DCCD), indicating the presence of protonated carboxyl groups in the hydrophobic regions of the two proteins. Na⁺ did not protect PomA and PomB from this modification. Our study shows that both osmolality and pH have an influence on the function of the flagellum from V. cholerae. We propose that D23, S26, and D42 of PomB are part of an ion-conducting pathway formed by the PomAB stator complex.     

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.     

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.     

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.     

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.     

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.     

Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes

A motor protein complex of the bacterial flagellum, PomA/B from Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB, and converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog called MotA/B, which utilizes proton instead of sodium ion. PomB and MotB have a peptidoglycan-binding motif in their C-terminal region, and therefore PomA/B and MotA/B are regarded as the stator. Energy filtering electron cryomicroscopy enhanced the image contrast of the proteins reconstituted into liposomes and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides. Image analysis combined with gold labeling and deletion of the peptidoglycan-binding motif revealed that the longer one, approximately 70 A long, is likely to correspond to the periplasmic domain, and the other, about half size, to the cytoplasmic domain.     

Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na+-Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus

In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na⁺-driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na⁺-driven motor than in the MotA-FliG interactions in the H⁺-driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na⁺-driven motor seem to be more complex than those previously proposed in the H⁺-driven motor.     

Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation

The bacterial flagellar motor is driven by an ion flux through a channel called MotAB in Escherichia coli or Salmonella and PomAB in Vibrio alginolyticus. PomAB is composed of two transmembrane (TM) components, PomA and PomB, and converts a sodium ion flux to rotation of the flagellum. Its homolog, MotAB, utilizes protons instead of sodium ions. PomB/MotB has a peptidoglycan (PG)-binding motif in the periplasmic domain, allowing it to function as the stator by being anchored to the PG layer. To generate torque, PomAB/MotAB is thought to undergo a conformational change triggered by the ion flux and to interact directly with FliG, a component of the rotor. Here, we present the first three-dimensional structure of this torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the TM and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a "plugged" conformation, which would prevent ions leaking through the channel. We propose a model for how PomAB units are placed around the flagellar basal body to function as torque generators.     

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.     

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.     

Sodium-dependent dynamic assembly of membrane complexes in sodium-driven flagellar motors

The bacterial flagellar motor is driven by the electrochemical potential of specific ions, H⁺ or Na⁺. The motor consists of a rotor and stator, and their interaction generates rotation. The stator, which is composed of PomA and PomB in the Na⁺ motor of Vibrio alginolyticus , is thought to be a torque generator converting the energy of ion flux into mechanical power. We found that specific mutations in PomB, including D24N, F33C and S248F, which caused motility defects, affected the assembly of stator complexes into the polar flagellar motor using green fluorescent protein-fused stator proteins. D24 of PomB is the predicted Na⁺-binding site. Furthermore, we demonstrated that the coupling ion, Na⁺, is required for stator assembly and that phenamil (an inhibitor of the Na⁺-driven motor) inhibited the assembly. Carbonyl cyanide m -chlorophenylhydrazone, which is a proton ionophore that collapses the sodium motive force in this organism at neutral pH, also inhibited the assembly. Thus we conclude that the process of Na⁺ influx through the channel, including Na⁺ binding, is essential for the assembly of the stator complex to the flagellar motor as well as for torque generation.     

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.     

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.     

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.     

Interactions of MotX with MotY and with the PomA/PomB sodium ion channel complex of the Vibrio alginolyticus polar flagellum

Rotation of the sodium ion-driven polar flagellum of Vibrio alginolyticus requires the inner membrane sodium ion channel complex PomA/PomB and the outer membrane components MotX and MotY. None of the detergents used in this study were able to solubilize MotX when it was expressed alone. However, when co-expressed with MotY, MotX was solubilized by some detergents. The change in the solubility of MotX suggests that MotY interacts with MotX. In agreement with this, a pull-down assay showed the association of MotY with MotX. Solubilized MotX and MotY eluted in the void volume from a gel-filtration column, suggesting that MotX and MotY form a large oligomeric structure(s). In the absence of MotY, MotX affected membrane localization of the PomA/PomB complex and of PomB alone but not of PomA alone, suggesting an interaction between MotX and PomB. We propose that MotX exhibits multiple interactions with the other motor components, first with MotY for its localization to the outer membrane and then with the PomA/PomB complex through PomB for the motor 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.     

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.