Na(+)-driven flagellar motor of Vibrio

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

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

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

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

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

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 analysis of conserved polar residues in Vc-NhaD, Na+/H+ antiporter of Vibrio cholerae

Vc-NhaD is a Na(+)/H(+) antiporter from Vibrio cholerae with a sharp maximum of activity at pH approximately 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na(+)-dependent H(+) transfer in inside-out membrane vesicles. Substitutions T157A and S428A caused a significant increase of apparent K(m) values for alkali cations, with the K(m) for Li(+) elevated more than that for Na(+), indicating that Thr-157 and Ser-428 are involved in alkali cation binding/translocation. Of six conserved His residues, mutation of only His-93 and His-210 affected the Na(+)(Li(+))/H(+) antiport, resulting in an acidic shift of its pH profile, whereas H93A also caused a 7-fold increase of apparent K(m) for Na(+) without affecting the K(m) for Li(+). These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations.     

Thiosulfate reduction in Salmonella enterica is driven by the proton motive force

Thiosulfate respiration in Salmonella enterica serovar Typhimurium is catalyzed by the membrane-bound enzyme thiosulfate reductase. Experiments with quinone biosynthesis mutants show that menaquinol is the sole electron donor to thiosulfate reductase. However, the reduction of thiosulfate by menaquinol is highly endergonic under standard conditions (ΔE°' = -328 mV). Thiosulfate reductase activity was found to depend on the proton motive force (PMF) across the cytoplasmic membrane. A structural model for thiosulfate reductase suggests that the PMF drives endergonic electron flow within the enzyme by a reverse loop mechanism. Thiosulfate reductase was able to catalyze the combined oxidation of sulfide and sulfite to thiosulfate in a reverse of the physiological reaction. In contrast to the forward reaction the exergonic thiosulfate-forming reaction was PMF independent. Electron transfer from formate to thiosulfate in whole cells occurs predominantly by intraspecies hydrogen transfer.     

Riboflavin is an active redox cofactor in the Na+-pumping NADH: quinone oxidoreductase (Na+-NQR) from Vibrio cholerae

Here we present new evidence that riboflavin is present as one of four flavins in Na+-NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na+-NQR.     

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.     

Purification of flagellar cores of Vibrio cholerae.

A procedure is described for the purification of the cores of flagella sheared from Vibrio cholerae. V. cholerae is a monotrichous organism whose flagellar core (FC) is enclosed within a sheath. The purification procedure consists of several cycles of differential centrifugation and cesium chloride density-gradient ultracentrifugation in the presence of a neutral detergent, Triton X-100. Purity of the FC preparations is assessed by electron microscopy, polyacrylamide gel electrophoresis, and chemical analysis. The purified FC preparations are devoid of flagellar sheaths and free from detectable cell wall and cytoplasmic contamination. Antibody prepared in rabbits against purified FC reacts with the flagellum of intact V. cholerae or purified FC as seen by ferritin-labeled antibody studies. Purified FC is composed of a single protein subunit with an estimated molecular weight of 45,000 g/mol and a density of about 1.3 g/cm3.     

The sodium cycle. III. Vibrio alginolyticus resembles Vibrio cholerae and some other vibriones by flagellar motor and ribosomal 5S-RNA structures

An electron microscopic study of the basal bodies of the Vibrio albinolyticus flagellum revealed a four-disc structure. The diameters of the two discs localized closer to the cytoplasmic membrane proved to be about 2-fold shorter than those of the two others. In this respect the basal body of V. alginolyticus resembles very much that of V. cholerae described by Ferris and co-workers. The sequence of the V. alginolyticus ribosomal 5S-RNA showed that it is similar to those of V. cholerae, V. harveyi and some other vibriones. On the basis of the 5S-RNA sequences, a dendrogram of prokaryotes is presented. It confirmed the suggestion that V. alginolyticus is a typical representative of Vibrionaceae rather than a 'monster' greatly differing from other vibriones. Possible evolutionary relation of various bacterial species possessing the primary Na+ pumps is discussed.     

Quantification of flagellar motor stator dynamics through in vivo proton‐motive force control

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane‐bound stator complexes. We used the light‐driven proton pump proteorhodopsin (pR) to control the proton‐motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s^?1. Using GFP‐tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s^?1.     

Sucrose uptake is driven by the Na+ electrochemical potential in the marine bacterium Vibrio alginolyticus.

Na+ was found to be essential for the accumulation of sucrose by Vibrio alginolyticus. Sucrose uptake was completely inhibited by the addition of proton conductor at neutral pH, but not at alkaline pH, where the primary electrogenic Na+ pump generates the Na+ electrochemical gradient. We therefore conclude that sucrose transport is driven by the electrochemical potential of Na+ in this organism.     

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.     

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.     

Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae

The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na⁺ symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter’s functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na⁺-dependent transporter with a preference for C₄- and C₅-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate’s dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na⁺ ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism.