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.     

Deletion analysis of MotA and MotB, components of the force-generating unit in the flagellar motor of Salmonella

MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.     

Conformational change in the stator of the bacterial flagellar motor.

MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor. To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32. Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments. The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes. It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque. Influences of key residues of MotA on conformation were also examined. Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes. We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.     

Arrangement of core membrane segments in the MotA/MotB proton-channel complex of Escherichia coli

The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.     

Co-overproduction and localization of the Escherichia coli motility proteins motA and motB.

The motility genes motA and motB of Escherichia coli were placed under control of the Serratia marcescens trp promoter. After induction with beta-indoleacrylic acid, the levels of MotA and MotB rose over about a 3-h period, reaching plateau levels approximately 50-fold higher than wild-type levels. Both overproduced proteins inserted into the cytoplasmic membrane. Growth and motility were essentially normal, suggesting that although the motor is a proton-conducting device, MotA and MotB together do not constitute a major proton leak. Derivative plasmids which maintained an intact version of motB but had the motA coding region deleted in various ways were constructed. With these, the levels of MotB were much lower, reaching a peak within 30 min after induction and declining thereafter; pulse-chase measurements indicated that a contributing factor was MotB degradation. The low levels of MotB occurred even with an in-frame internal deletion of motA, whose translational initiation and termination sites were intact, suggesting that it is the MotA protein, rather than the process of MotA synthesis, that is important for MotB stability. Termination at the usual site of overlap with the start of motB (ATGA) was not an absolute requirement for MotB synthesis but did result in higher rates of synthesis than when translation of motA information terminated prematurely. Even in the total absence of MotA, the MotB that was synthesized was found exclusively in the cytoplasmic membrane fraction. In wild-type cells, MotA was estimated by immunoprecipitation to be in about fourfold excess over MotB; a previous estimate of 600 +/- 250 copies of MotA per cell then yielded an estimate of 150 +/- 70 copies of MotB per cell.     

Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor

MotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C-terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP-MotB and MotA-mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP-MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild-type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA-mCherry was observed to colocalize with GFP-FliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotA(C) ), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotA(C) with FliG is required for the efficient assembly of the stators around the rotor.     

Function of Proline Residues of MotA in Torque Generation by the Flagellar Motor of Escherichia coli

Bacterial flagellar motors obtain energy for rotation from the membrane gradient of protons or, in some species, sodium ions. The molecular mechanism of flagellar rotation is not understood. MotA and MotB are integral membrane proteins that function in proton conduction and are believed to form the stator of the motor. Previous mutational studies identified two conserved proline residues in MotA (Pro 173 and Pro 222 in the protein from Escherichia coli) and a conserved aspartic acid residue in MotB (Asp 32) that are important for function. Asp 32 of MotB probably forms part of the proton path through the motor. To learn more about the roles of the conserved proline residues of MotA, we examined motor function in Pro 173 and Pro 222 mutants, making measurements of torque at high load, speed at low and intermediate loads, and solvent-isotope effects (D2O versus H2O). Proton conduction by wild-type and mutant MotA-MotB channels was also assayed, by a growth defect that occurs upon overexpression. Several different mutations of Pro 173 reduced the torque of the motor under high load, and a few prevented motor rotation but still allowed proton flow through the MotA-MotB channels. These and other properties of the mutants suggest that Pro 173 has a pivotal role in coupling proton flow to motor rotation and is positioned in the channel near Asp 32 of MotB. Replacements of Pro 222 abolished function in all assays and were strongly dominant. Certain Pro 222 mutant proteins prevented swimming almost completely when expressed at moderate levels in wild-type cells. This dominance might be caused by rotor-stator jamming, because it was weaker when FliG carried a mutation believed to increase rotor-stator clearance. We propose a mechanism for torque generation, in which specific functions are suggested for the proline residues of MotA and Asp32 of MotB.     

Regulated underexpression and overexpression of the FliN protein of Escherichia coli and evidence for an interaction between FliN and FliM in the flagellar motor.

The FliN protein of Escherichia coli is essential for the assembly and function of flagella. Here, we report the effects of regulated underexpression and overexpression of FliN in a fliN null strain. Cells that lack the FliN protein do not make flagella. When FliN is underexpressed, cells produce relatively few flagella and those made are defective, rotating at subnormal, rapidly varying speeds. These results are similar to what was seen previously when the flagellar protein FliM was underexpressed and unlike what was seen when the motility proteins MotA and MotB were underexpressed. Overexpression of FliN impairs motility and flagellation, as has been reported previously for FliM, but when FliN and FliM are co-overexpressed, motility is much less impaired. This and additional evidence presented indicate that FliM and FliN are associated in the flagellar motor, in a structure distinct from the MotA/MotB torque generators. A recent study showed that FliN might be involved in the export of flagellar components during assembly (A. P. Vogler, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). We show here that approximately 50 amino acid residues from the amino terminus of FliN are dispensable for function and that the remaining, essential part of FliN has sequence similarity to a part of Spa33, a protein that functions in transmembrane export in Shigella flexneri. Thus, FliN might function primarily in flagellar export, rather than in torque generation, as has sometimes been supposed.     

Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane.

The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein. Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorporate the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body.     

The Escherichia coli MotAB proton channel unplugged

The MotA and MotB proteins of Escherichia coli serve two functions. The MotA4MotB2 complex attaches to the cell wall via MotB to form the stator of the flagellar motor. The complex also couples the flow of hydrogen ions across the cell membrane to movement of the rotor. The TM3 and TM4 transmembrane helices of MotA and the single TM of MotB comprise the proton channel, which is inactive until the complex assembles into a motor. Here, we identify a segment of the MotB protein that acts as a plug to prevent premature proton flow. The plug is in the periplasm just C-terminal to the MotB TM. It consists of an amphipathic alpha helix flanked by Pro52 and Pro65. When MotA is over-expressed with MotB deleted for residues 51-70, a massive influx of protons acidifies the cytoplasm without significantly depleting the proton motive force. Either that acidification or some sequela thereof, such as potassium or water efflux from the cells, inhibits growth. The Pro residues and Ile58, Tyr61, and Phe62 are essential for plug function. Cys-substituted MotB proteins form a disulfide bond between the two plugs that hold the channels open, and the plugs function intrans within the MotA4MotB2 complex. We present a model in which the MotA4MotB2 complex forms in the bulk membrane. Before association with a motor, we propose the plugs insert into the cell membrane parallel with its periplasmic face and interfere with channel formation. When a complex incorporates into a motor, the plugs leave the membrane and associate with each other via their hydrophobic faces to hold the proton channel open.     

Solubilization and purification of the MotA/MotB complex of Escherichia coli

Bacterial flagella are driven at their base by a rotary motor fueled by the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which function together to conduct ions across the membrane and couple ion flow to rotation. An invariant aspartate residue in MotB (Asp32 in the protein of E. coli) is essential for rotation and appears to have a direct role in proton conduction. A recent study showed that changes at Asp32 in MotB cause a conformational change in the complex, as evidenced by altered patterns of protease susceptibility of MotA [Kojima, S., and Blair, D. F. (2001) Biochemistry 40 (43), 13041-13050]. It was proposed that protonation/deprotonation of Asp32 might regulate a conformational change in the stator that acts as the powerstroke to drive rotation of the rotor. Biochemical studies of the MotA/MotB complex have been hampered by the absence of a suitable assay for its integrity in detergent solution. Here, we have studied the behavior of the MotA/MotB complex in a variety of detergents, making use of the protease-susceptibility assay to monitor its integrity. Among about 25 detergents tested, a few were found to solubilize the proteins effectively while preserving certain conformational properties characteristic of an intact complex. The detergent dodecylphosphocholine, or DPC, proved especially effective. MotA/MotB complexes purified in DPC migrate with an apparent size of approximately 300 kDa in gel-filtration columns, and retain the Asp32-modulated conformational differences seen in membranes. (35)S-radiolabeling showed that MotA and MotB are present in a 2:1 ratio in the complex. Purified MotA/MotB complexes should enable in vitro study of the proton-induced conformational change and other aspects of stator function.     

Targeted disulfide cross-linking of the MotB protein of Escherichia coli: evidence for two H(+) channels in the stator Complex.

Bacterial flagella are turned by rotary motors that obtain energy from the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes that contain multiple copies of each protein. The complexes conduct ions across the membrane, and couple ion flow to motor rotation by a mechanism that appears to involve conformational changes [Kojima, S., and Blair, D. F. (2001) Biochemistry 40, 13041-13050]. Structural information on the MotA/MotB complex is very limited. MotA has four membrane-spanning segments, and MotB has one. We have begun a targeted disulfide-cross-linking study to probe the arrangement of membrane segments in the MotA/MotB complex, beginning with the single membrane segment of MotB. Cys residues were introduced in 21 consecutive positions in the segment, and disulfide cross-linking was studied in MotA/MotB complexes either in membranes or detergent solution. Most of the Cys-substituted MotB proteins formed disulfide-linked dimers in significant yield upon oxidation. The yield of dimer varied regularly with the position of the Cys substitution, following the pattern expected for a parallel, symmetric dimer of alpha-helices. In a structural model based on the cross-linking experiments, critical Asp32 residues that are believed to facilitate proton movement are positioned on separate surfaces of the MotB dimer and so probably function within two distinct proton channels. Regions accessible to solvent were mapped by measuring the reactivity of introduced Cys residues toward N-ethyl maleimide and a charged methanethiosulfonate reagent. Positions near the middle of the segment were inaccessible to sulhydryl reagents. Positions within 6-8 residues of either end, which includes residues around Asp32, were accessible.     

Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor

MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton flow with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of Salmonella enterica serovar Typhimurium MotB consisting of residues 78 through 309 (MotB(C)). MotB(C) significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotB(C)(Delta197-210), showed a significantly reduced inhibitory effect. Wild-type MotB(C) and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotB(C)-His(6) by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotB(C) heterodimer formation in the periplasm. However, the monomeric mutant variant MotB(C)(Delta197-210) did not bind to MotB, suggesting that MotB(C) is directly involved in stator assembly. We propose that the MotB(C) dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.     

Proton-conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in Salmonella

MotA and MotB form the proton-channel complex of the proton-driven bacterial flagellar motor. A plug segment of Escherichia coli MotB suppresses proton leakage through the MotA/B complex when it is not assembled into the motor. Using a ratiometric pH indicator protein, pHluorin, we show that the proton-conductivity of a Salmonella MotA/B complex not incorporated into the motor is two orders of magnitude lower than that of a complex that is incorporated and activated. This leakage is, however, significant enough to change the cytoplasmic pH to a level at which the chemotaxis signal transduction system responds.     

Membrane segment organization in the stator complex of the flagellar motor: implications for proton flow and proton-induced conformational change

MotA and MotB are membrane proteins that form the stator of the bacterial flagellar motor. Each motor contains several MotA 4MotB 2 complexes, which function independently to conduct protons across the membrane and couple proton flow to rotation. The mechanism of rotation is not understood in detail but is thought to involve conformational changes in the stator complexes driven by proton association/dissociation at a critical Asp residue of MotB (Asp 32 in the protein of Escherichia coli). MotA has four membrane segments and MotB has one. Previous studies using targeted disulfide cross-linking showed that the membrane segments of the two MotB subunits are together at the center of the complex, surrounded by the TM3 and TM4 segments of the four MotA subunits. Here, the cross-linking studies are extended to TM1 and TM2 of MotA, using Cys residues introduced in several positions in the segments. The observed patterns of disulfide cross-linking indicate that the TM2 segment is positioned between segments TM3 and TM4 of the same subunit, where it could contribute to the proton-channel-forming part of the structure. TM1 is at the interface between TM4 of its own subunit and the TM3 segment of another subunit, where it could stabilize the complex. A structural model based on the cross-linking results shows unobstructed pathways reaching from the periplasm to the Asp 32 residues near the inner ends of the MotB segments. The model indicates a close proximity for certain conserved, functionally important residues. The results are used to develop an explicit model for the proton-induced conformational change in the stator.     

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.     

Amplification of bacterial plasmids without blocking protein biosynthesis

The effect of amino acids (presence or absence from the growth media) and metal ions on the replication of Escherichia coli plasmids in rel A+ strains was studied. It was found that: (i) The absence of one amino acid from the growth media had no effect on the plasmid copy number in prototrophic E. coli strains: (ii) The presence of only one amino acid in artificial media free of amino acids had a negligible effect on the plasmid copy number for the amino acids Ala, Arg, Glu, His, Leu, Phe, Thr, Trp, and Tyr: (iii) The combination of Met and Thr caused a rise in pBR322 plasmid copy number up to 90-100 plasmid copies per cell: (iv) The Fe3+ concentration had an amplification effect on E. coli plasmids. The pBR322 plasmid copy number for media free of amino acids and supplemented with 0.2-0.4 mM FeCl3 was 60-80 plasmid copies per cell: (v) The combination of Fe3+ with certain amino acids (Ala, Arg, Glu, Leu, Thr, and Trp) leads to a dramatic increase in the plasmid copy number reaching 180-270 plasmid copies per cell for the plasmid pBR322 and 20-24 for the plasmid pR100.     

Molecular modeling of the bacterial outer membrane receptor energizer,ExbBD/TonB,based on homology with the flagellar motor,MotAB

The MotA/MotB proteins serve as the motor that drives bacterial flagellar rotation in response to the proton motive force (pmf). They have been shown to comprise a transmembrane proton pathway. The ExbB/ExbD/TonB protein complex serves to energize transport of iron siderophores and vitamin B12 across the outer membrane of the Gram-negative bacterial cell using the pmf. These two protein complexes have the same topology and are homologous. Based on molecular data for the MotA/MotB proteins, we propose simple three-dimensional channel structures for both MotA/MotB and ExbB/ExbD/TonB using modeling methods. Features of the derived channels are discussed, and two possible proton transfer pathways for the ExbBD/TonB system are proposed. These analyses provide a guide for molecular studies aimed at elucidating the mechanism by which chemiosmotic energy can be transferred either between two adjacent membranes to energize outer membrane transport or to the bacterial flagellum to generate torque.     

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

We have shown that a hybrid motor consisting of proton-type Rhodobacter sphaeroides MotA and sodium-type VIBRIO: alginolyticus PomB, MotX and MotY, can work as a sodium-driven motor in VIBRIO: cells. In this study, we tried to substitute the B subunits, which contain a putative ion-binding site in the transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N-terminal MotB and C-terminal PomB. MomB proteins, with the entire transmembrane region derived from the H(+)-type MotB, gave rise to an Na(+) motor with MotA. The other two MomB proteins, in which the junction sites were within the transmembrane region, also formed Na(+) motors with PomA, but were changed for Na(+) or Li(+) specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na(+)- and H(+)-type subunits and this can affect the ion specificity. This is the first report to have changed the specificity of the coupling ions in a bacterial flagellar motor.