SignificanceThe bacterial flagellar motor is a rotary nano-machine that is driven by an electrochemical ion gradient across the cytoplasmic membrane, either H+or Na+ions. NaturalEscherichia colicells have only H+-driven motors. We... more
SignificanceThe bacterial flagellar motor is a rotary nano-machine that is driven by an electrochemical ion gradient across the cytoplasmic membrane, either H+or Na+ions. NaturalEscherichia colicells have only H+-driven motors. We demonstrate a genetically engineered hybrid-fuel flagellar motor inE. colithat runs on both types of ion gradient, H+and Na+. The hybrid motors switch between the two types of ion automatically and dynamically in response to external conditions, by swapping the stator components that determine their ion selectivity. These hybrid motors combine biological components with specific functions from different organisms that do not naturally coexist but demonstrate that natural hybrid motors could exist in other species.
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The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in Escherichia coli ,... more
The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in Escherichia coli , because the P ring was not assembled in a dsbB strain that was defective for disulfide bond formation in periplasmic proteins. We, however, found that the two Cys residues of FlgI are not conserved in other bacterial species. We then assessed the role of this intramolecular disulfide bond in FlgI. A Cys-eliminated FlgI derivative formed a P ring that complemented the flagellation defect of our Δ flgI strain when it was overproduced, suggesting that disulfide bond formation in FlgI is not absolutely required for P-ring assembly. The levels of the mature forms of the FlgI derivatives were significantly lower than that of wild-type FlgI, although the precursor protein levels were unchanged. Moreover, the FlgI derivatives were more susceptible to degradation...
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The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to... more
The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to C...
Research Interests: Biophysics, Thermodynamics, Bacteriology, Biology, Membrane Proteins, and 14 moreMedicine, Biological Sciences, Escherichia coli, Movement, Flagella, Temperature, Molecular motor proteins, Chemotaxis, Rotation, Protein Binding, Hydrostatic Pressure, Molecular Motor, Biomechanical Phenomena, and Medical and Health Sciences
The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The Δ flhF mutant makes no flagellum, whereas the Δ flhFG... more
The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The Δ flhF mutant makes no flagellum, whereas the Δ flhFG double-deletion mutant usually lacks a flagellum. However, the Δ flhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the Δ flhFG strain (Δ flhFG -sup). The suppressor strain forms peritrichous flagella in the majority of cells. We identified candidate suppressor mutations by comparing the genome sequence of the parental strain, VIO5, with the genome sequences of the suppressor strains. Two mutations were mapped to a gene, named sflA ( s uppressor of Δ fl hFG ), at the VEA003730 locus of the Vibrio sp. strain EX25 genome. This gene is specific for Vibrio species and is predicted to encode a transmembrane protein with a DnaJ domain. When the wild-type gene was introduced int...
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Research Interests: Biophysics, Bacteriology, Biology, Medicine, Biological Sciences, and 14 moreMotility, Movement, Flagella, Molecular motor proteins, D-Aspartic Acid, Phenotype, Vibrio, Sodium, Amino Acid Sequence, Site-directed Mutagenesis, Sodium Chloride, Molecular Sequence Data, sodium channels, and Medical and Health Sciences
Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four-transmembrane regions and a cytoplasmic domain of approximately 100 residues which interacts... more
Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four-transmembrane regions and a cytoplasmic domain of approximately 100 residues which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The three-dimensional structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66, and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to ...
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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... more
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...
Research Interests: Bacteriology, Biology, Medicine, Biological Sciences, Rhodobacter sphaeroides, and 15 moreFlagella, Molecular motor proteins, Chemotaxis, Vibrio, Sodium, Torque, Amino Acid Sequence, Hydrogen-Ion Concentration, Vibrio alginolyticus, Protons, Molecular Sequence Data, Amiloride, sodium channels, Medical and Health Sciences, and flagellum
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The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in... more
The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel. MotX was proposed to be a component of a sodium channel. Here we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions. These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene, suggestin...
Research Interests: Bacteriology, Biology, Membrane Proteins, Medicine, Biological Sciences, and 15 moreSequence alignment, Swimming speed, Flagella, Marine Bacteria, Vibrio, Sodium, Sodium channel, Amino Acid Sequence, Base Sequence, Protons, Molecular Sequence Data, sodium channels, Restriction Mapping, Medical and Health Sciences, and flagellum
The flagellar motor of Vibrio alginolyticus is made of two parts: a stator consisting of proteins PomA and PomB, and a rotor whose main component is FliG. The interaction between FliG and PomA generates torque for flagellar rotation.... more
The flagellar motor of Vibrio alginolyticus is made of two parts: a stator consisting of proteins PomA and PomB, and a rotor whose main component is FliG. The interaction between FliG and PomA generates torque for flagellar rotation. Based on cross-linking experiments of double-Cys mutants of PomB, we previously proposed that a conformational change in the periplasmic region of PomB caused stator activation. Double-Cys mutants lost their motility due to an intramolecular disulfide bridge. In this study, we found that the addition of serine, a chemotactic attractant, to a PomB(L160C/I186C) mutant restored motility without cleaving the disulfide bridge. We speculate that serine changed the rotor (FliG) conformation, affecting rotational direction. Combined with the counterclockwise (CCW)-biased mutation FliG(G214S), motility of PomB(L160C/I186C) was restored without the addition of serine. Likewise, motility was restored without serine in Che(-) mutants, in either a CCW-locked or cloc...
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Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form... more
Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx, and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the trans...
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Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed... more
Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+). As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient b...
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The torque of the bacterial flagellum is generated by the rotor-stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor... more
The torque of the bacterial flagellum is generated by the rotor-stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomBC) at 2.0-Å resolution, and the structure suggests a conformational change in the N-terminal region of PomBC for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that t...
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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... more
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 nonmot...
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Flagellar motility is a key factor for bacterial survival and growth in fluctuating environments. The polar flagellum of a marine bacterium, Vibrio alginolyticus , is driven by sodium ion influx and rotates approximately six times faster... more
Flagellar motility is a key factor for bacterial survival and growth in fluctuating environments. The polar flagellum of a marine bacterium, Vibrio alginolyticus , is driven by sodium ion influx and rotates approximately six times faster than the proton-driven motor of Escherichia coli . The basal body of the sodium motor has two unique ring structures, the T ring and the H ring. These structures are essential for proper assembly of the stator unit into the basal body and to stabilize the motor. FlgT, which is a flagellar protein specific for Vibrio sp., is required to form and stabilize both ring structures. Here, we report the crystal structure of FlgT at 2.0-Å resolution. FlgT is composed of three domains, the N-terminal domain (FlgT-N), the middle domain (FlgT-M), and the C-terminal domain (FlgT-C). FlgT-M is similar to the N-terminal domain of TolB, and FlgT-C resembles the N-terminal domain of FliI and the α/β subunits of F 1 -ATPase. To elucidate the role of each domain, we p...
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SummaryRotation of the sodium‐driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium‐driven motor of Vibrio, have been believed to be... more
SummaryRotation of the sodium‐driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium‐driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N‐terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N‐terminal hydrophobic segments, the N‐termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor protein...
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SummaryThe 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... more
SummaryThe 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. Th...
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The polar flagella ofVibrio alginolyticushave sodium-driven motors, and four membrane proteins, PomA, PomB, MotX and MotY, are essential for torque generation of the motor. PomA and PomB are believed to form a sodium-conducting channel.... more
The polar flagella ofVibrio alginolyticushave sodium-driven motors, and four membrane proteins, PomA, PomB, MotX and MotY, are essential for torque generation of the motor. PomA and PomB are believed to form a sodium-conducting channel. This paper reports the purification of the motor complex by using sucrose monocaprate, a non-ionic detergent, to solubilize the complex. Plasmid pKJ301, which encodes intact PomA, and PomB tagged with a C-terminal hexahistidine that does not interfere with PomB function, was constructed. The membrane fraction of cells transformed with pKJ301 was solubilized with sucrose monocaprate, and the solubilized materials were applied to a Ni-NTA column. The imidazole eluate contained both PomA and PomB, which were further purified by anion-exchange chromatography. Gel-filtration chromatography was used to investigate the apparent molecular size of the complex; the PomA/PomB complex was eluted as approx. 900 kDa and PomB alone was eluted as approx. 260 kDa. Th...
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Research Interests: Molecular Biology, Biology, Medicine, Movement, Flagella, and 15 moreMolecular motor proteins, Green Fluorescent Protein, Sodium, Fluorescent Protein, Base Sequence, Cell Wall, Membrane Protein, Subcellular Fractions, Vibrio alginolyticus, GFP, Stator, Biochemistry and cell biology, Protein requirement, sodium channels, and flagellum
Research Interests: Biophysics, Molecular Biology, Biology, Immunohistochemistry, Medicine, and 15 moreFlagella, Liposomes, Molecular motor proteins, Liposome, Transfer Function, Sodium, Cryo Electron Microscopy, Protein Conformation, Lipid bilayers, Peptidoglycan, Protein Binding, Vibrio alginolyticus, Biochemistry and cell biology, sodium channels, and flagellum
Research Interests: Chemistry, Molecular Biology, Biology, Medicine, Mutation, and 14 moreMicrobial genetic and drug resistance, Flagella, Molecular motor proteins, Vibrio, Sodium, Protein Conformation, Amino Acid Sequence, Base Sequence, Site-directed Mutagenesis, Biochemistry and cell biology, Molecular Sequence Data, Amiloride, sodium channels, and binding sites
Research Interests: Biochemistry, Biology, Medicine, Mutation, Escherichia coli, and 12 moreMovement, Vibrio cholerae, Flagella, Protein Secondary Structure Prediction, Sodium, Protein Binding, Cytoplasm, Vibrio alginolyticus, Biochemistry and cell biology, sodium channels, conserved sequence , and Medical biochemistry and metabolomics
Research Interests: Biochemistry, Biology, Membrane Proteins, Medicine, Mutation, and 13 moreFlagella, Molecular motor proteins, Gene, Molecular cloning, Vibrio, Sodium, Amino Acid Sequence, Vibrio alginolyticus, Biochemistry and cell biology, Molecular Sequence Data, frameshift mutation, Medical biochemistry and metabolomics, and flagellum
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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... more
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...
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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... more
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 Δ pomB 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.
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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... more
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 immed...
Research Interests: Bacteriology, Biology, Medicine, Biological Sciences, Escherichia coli, and 15 moreMovement, Flagella, Temperature, Molecular motor proteins, Vibrio, Sodium, Motor Function, Amino Acid Sequence, Amino Acid Substitution Rates, Membrane Protein, Cytoplasm, Molecular Motor, Molecular Sequence Data, Medical and Health Sciences, and flagellum
The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na + -driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred... more
The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na + -driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.
Research Interests: Bacteriology, Biology, Membrane Proteins, Medicine, Protein Stability, and 13 moreBiological Sciences, Plasmids, Gram-negative bacteria, Vibrio, Sodium, Motor Function, Cysteine, Amino Acid Sequence, Site-directed Mutagenesis, Disulfide bond, Driving force, conserved sequence , and Medical and Health Sciences
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PomA is thought to be a component of the ion channel in the sodium-driven polar-flagellar motor of Vibrio alginolyticus . We have found that some cysteine substitutions in the periplasmic region of PomA result in a slow-motility... more
PomA is thought to be a component of the ion channel in the sodium-driven polar-flagellar motor of Vibrio alginolyticus . We have found that some cysteine substitutions in the periplasmic region of PomA result in a slow-motility phenotype, in which swarming and swimming speeds are reduced even in the presence of high concentrations of NaCl. Most of the mutants showed a sodium ion dependence similar to that of the wild type but with significantly reduced motility at all sodium ion concentrations. By contrast, motility of the D31C mutant showed a sharp dependence on NaCl concentration, with a threshold at 38 mM. The motor of the D31C mutant rotates stably, as monitored by laser dark-field microscopy, suggesting that the mutant PomA protein is assembled normally into the motor complex. Mutational studies of Asp31 suggest that, although this residue is not essential for motor rotation, a negative charge at this position contributes to optimal speed and/or efficiency of the motor.
Research Interests: Biophysics, Bacteriology, Biology, Medicine, Biological Sciences, and 14 moreMotility, Movement, Flagella, Molecular motor proteins, D-Aspartic Acid, Phenotype, Vibrio, Sodium, Amino Acid Sequence, Site-directed Mutagenesis, Sodium Chloride, Molecular Sequence Data, sodium channels, and Medical and Health Sciences
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... more
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...
Research Interests: Bacteriology, Hydrogen, Biology, Locomotion, Membrane Proteins, and 15 moreComparative Study, Medicine, Biological Sciences, Mutation, Escherichia coli, Flagella, Molecular motor proteins, Protein Function, Protein Interaction, Sodium, Motor Function, Protons, Electrostatic interaction, Medical and Health Sciences, and flagellum
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... more
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 ea...
Research Interests: Bacteriology, Biology, Membrane Proteins, Medicine, Biological Sciences, and 12 moreMutation, Movement, Flagella, Sodium, Protein Conformation, Amino Acid Sequence, Protein Binding, Vibrio alginolyticus, Site-directed Mutagenesis, Molecular Sequence Data, sodium channels, and Medical and Health Sciences
Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR)... more
Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR) moiety, was constructed. High levels of expression were achieved in Escherichia coli (60–100 nmol/liter) and the enzyme was catalytically active with optimal activities achieved in the presence of the antioxidant, GSH.
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Vibrio species are Gram-negative rod-shaped bacteria that are ubiquitous and often highly motile in aqueous environments. Vibrio swimming motility is driven by a polar flagellum covered with a membranous sheath, but this sheathed... more
Vibrio species are Gram-negative rod-shaped bacteria that are ubiquitous and often highly motile in aqueous environments. Vibrio swimming motility is driven by a polar flagellum covered with a membranous sheath, but this sheathed flagellum is not well understood at the molecular level because of limited structural information. Here, we use Vibrio alginolyticus as a model system to study the sheathed flagellum in intact cells by combining cryoelectron tomography (cryo-ET) and subtomogram analysis with a genetic approach. We reveal striking differences between sheathed and unsheathed flagella in V. alginolyticus cells, including a novel ring-like structure at the bottom of the hook that is associated with major remodeling of the outer membrane and sheath formation. Using mutants defective in flagellar motor components, we defined a Vibrio-specific feature (also known as the T ring) as a distinctive periplasmic structure with 13-fold symmetry. The unique architecture of the T ring prov...
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Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H+ or Na+) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins... more
Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H+ or Na+) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na+-driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H+-type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na+ t...
Research Interests:
Research Interests:
Research Interests: Chemistry, Biological Chemistry, Medicine, Biological Sciences, Mutation, and 12 moreFlagella, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Vibrio, Sodium, Vibrio alginolyticus, Sodium Chloride, Dimerization, Cell Membrane, Amiloride, sodium channels, and Medical and Health Sciences
The sodium-driven motor consists of the products of at least four genes, pomA , pomB , motX , and motY , in Vibrio alginolyticus . PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four... more
The sodium-driven motor consists of the products of at least four genes, pomA , pomB , motX , and motY , in Vibrio alginolyticus . PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop 1-2 ) and at positions 167 to 180 between membrane segments 3 and 4 (loop 3-4 ). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutations in the periplasmic loops and all of the cytoplasmic mutations d...
Research Interests: Biochemistry, Bacteriology, Biology, Medicine, Biological Sciences, and 15 moreMutagenesis, Electroporation, Flagella, Biotin, Protein Secondary Structure Prediction, Vibrio, Sodium, Cysteine, Amino Acid Sequence, Amino Acid Substitution Rates, Vibrio alginolyticus, Maleimides, Membrane topology, Molecular Sequence Data, and Medical and Health Sciences
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... more
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 re...
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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... more
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 N...