Flagellar Rotary Motor Research Articles

Electron Tomography and Molecular Modeling Study of Chemoreceptor Organization

The movement of bacteria in response to external stimuli represents a paradigm of broad general interest for the understanding of mechanisms underlying signal transduction across cell membranes. Bacterial chemoreceptors respond to changes in concentration of extracellular ligands by undergoing conformational changes that initiate a series of signaling events, leading ultimately to regulation of flagellar motor rotation. Atomic structures for several domains of chemoreceptors, including the periplasmic ligand-binding domain, the cytoplasmic signaling domain and the HAMP domain are available, but the molecular architectures of an intact receptor dimer, or the functionally relevant trimer-of-dimer configuration have remained elusive. Here, we have used cryo-electron tomography combined with 3D averaging to determine the in situ 3D structure of receptor assemblies in bacterial cells that have been engineered to overproduce only the receptor for serine chemotaxis, Tsr, and lacking all other chemotaxis receptors and signaling components. We identified two major conformations of the chemotaxis receptors. Through comparative modeling and map-constrained molecular dynamics simulations, we obtained the assembly structures of tsr organized in a two dimensional array. We show that receptors are organized in trimer-of-dimer conformations with peripasmic domain, HAMP domain, and signal traction domain transiting between conformations. It is suggested that the position of the ligand binding domain and the HAMP domain play a pivotal role in mediating signal transduction across the cell membrane.

Following the Behavior of the Flagellar Rotary Motor Near Zero Load

At room temperature at stall, the flagellar motor of the bacterium Escherichia coli exerts a torque of ~1300 pN nm. At zero external load, it spins ~330 Hz. A robust method for studying the motor near zero load is reviewed here.

3P-123 pHイメージングシステムを用いた細胞内pHとべん毛モーター回転の同時計測(分子モーター,第47回日本生物物理学会年会)

3P-123 pHイメージングシステムを用いた細胞内pHとべん毛モーター回転の同時計測(分子モーター,第47回日本生物物理学会年会)

Shear Stress Transmission Model for the Flagellar Rotary Motor

Most bacteria that swim are propelled by flagellar filaments, which are driven by a rotary motor powered by proton flux. The mechanism of the flagellar motor is discussed by reforming the model proposed by the present authors in 2005. It is shown that the mean strength of Coulomb field produced by a proton passing the channel is very strong in the Mot assembly so that the Mot assembly can be a shear force generator and induce the flagellar rotation. The model gives clear calculation results in agreement with experimental observations, e g., for the charasteristic torque-velocity relationship of the flagellar rotation.

Flagellar Rotation in the Archaeon Halobacterium salinarum Depends on ATP

Halobacterium salinarum swims with the help of a polarly inserted flagellar bundle. In energized cells, the flagellar motors rotate continuously, occasionally switching the rotational sense. Starving cells become immotile as the energy level drops. Presumably, there is a threshold of energy required for flagellar rotation. When starved, immotile cells are energized by exposure to light, the speed of flagellar rotation increases gradually to its steady state over several minutes. Since the light-driven proton pump bacteriorhodopsin energizes the cell membrane to the maximal level within a fraction of a second, the delay in reaching the maximal swimming speed suggests that the halobacterial flagellar motor may not be driven directly by proton motive force. Swimming cells, which obtain their energy exclusively through light-driven proton pumping, become immotile within 20 min when treated with N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton translocating ATP synthase. However, flagellar motility in DCCD-treated cells can be restored by the addition of L-arginine, which serves as a fermentative energy source and restores the cytoplasmic ATP level in the presence of DCCD. This suggests that flagellar motor rotation depends on ATP, and this is confirmed by the observation that motility is increased strongly by L-arginine at zero proton motive force levels. The flagellar motor may be driven either by ATP directly or by an ATP-generated ion gradient that is not coupled directly to the proton gradient or the proton motive force of the cell.

Resurrection of the flagellar rotary motor near zero load

Flagellated bacteria, such as Escherichia coli, are propelled by helical flagellar filaments, each driven at its base by a reversible rotary motor, powered by a transmembrane proton flux. Torque is generated by the interaction of stator proteins, MotA and MotB, with a rotor protein FliG. The physiology of the motor has been studied extensively in the regime of relatively high load and low speed, where it appears to operate close to thermodynamic equilibrium. Here, we describe an assay that allows systematic study of the motor near zero load, where proton translocation and movement of mechanical components are rate limiting. Sixty-nanometer-diameter gold spheres were attached to hooks of cells lacking flagellar filaments, and light scattered from a sphere was monitored at the image plane of a microscope through a small pinhole. Paralyzed motors of cells carrying a motA point mutation were resurrected at 23 degrees C by expression of wild-type MotA, and speeds jumped from zero to a maximum value ( approximately 300 Hz) in one step. Thus, near zero load, the speed of the motor is independent of the number of torque-generating units. Evidently, the units act independently (they do not interfere with one another), and there are no intervals during which a second unit can add to the speed generated by the first (the duty ratio is close to 1).

2S3-4 高圧力によるべん毛モーターの回転運動変調(2S3 超分子ナノマシン細菌べん毛の機能に迫る,第46回日本生物物理学会年会)

2S3-4 高圧力によるべん毛モーターの回転運動変調(2S3 超分子ナノマシン細菌べん毛の機能に迫る,第46回日本生物物理学会年会)

PH sensing in bacterial chemotaxis.

Bacteria are able to sense a broad range of chemical and energetic stimuli and modulate their swimming behaviour to migrate to more favourable environments. Signal transduction in bacterial chemotaxis is mediated by a two-component system composed of a protein histidine kinase, CheA, and a response regulator, CheY. The phosphorylated response regulator, P approximately CheY, binds to a protein at the flagellar motor, FliM, to cause reversals in flagellar motor rotation. The level of P approximately CheY is controlled by the activity of the kinase CheA, which is in turn regulated by membrane receptors at the cell surface. Membrane receptors such as the aspartate receptor, Tar, are composed of two distinct regions: an extracellular sensing domain that binds stimulatory ligands, aspartate in the case of Tar; and an intracellular signalling domain that forms a complex with the protein kinase CheA. What is the mechanism of transmembrane signalling? How does aspartate binding to the sensing domain at the outside surface of the membrane translate into a change in kinase activity at the membrane cytosol interface? Recent results suggest that the mechanism depends on perturbations in lateral packing within an extensive array of receptors localized to patches at the cell poles. Receptor patching appears to depend on higher-order associations with the kinase CheA as well as an adaptor protein, CheW. It is difficult to assess the locus of pH effects within the context of even a simple signal transduction system like that involved in bacterial chemotaxis. Previous results with mutant strains have indicated that the serine receptor, Tsr, is critical for pH sensing, but in vitro results do not support such a straightforward interpretation of the genetic data.

Proposed model for the flagellar rotary motor

Flagellated bacteria swim by rotating helical filaments driven by motors embedded in the cell wall and cytoplasmic membrane. A model is proposed to explain the mechanism of the motor. The protons passing through the channels induce a strong electric field in Mot molecules. This field originates an impulse force to cause the flagellar rotation if the following conditions are fulfilled: (a) Mot molecules have a spontaneous electric polarization. (b) The lipid bilayers are viscoelastic. (c) There is a delay of deformation in response to stress in Mot molecules. The conclusions driven from the model are in agreement with the following experimental observations, denoting the flagellar rotation velocity as ω. (1) The torque is practically constant independent of ω from 0 to a critical value ω cr and then decreases sharply. (2) When ω is smaller than ω cr, the torque varies little with temperature. (3) The critical velocity ω cr shifts to lower speed at lower temperatures. (4) Where ω is larger than ω cr, declining of the torque steepens at lower temperatures. (5) When ω is smaller than ω cr, one revolution of the flagellar rotation consists of a constant number of steps. (6) When ω is smaller than ω cr, ω is proportional to the transmembrane potential difference. (7) The stator produces constant torque even when the stator is rotated relative to the rotor by external forces. (8) How the flagellar rotation velocity changes when the direction of the proton passage is reversed. (9) The motor has a switch that reverses the sense of the flagelllar rotation with the same absolute value of torque.

Chemo-sensitivity and reliability of flagellar rotary motor in a MEMS microfluidic actuation system

This paper presents a new MEMS microfluidic actuation mechanism using tethered, rotating bacterial flagella. In nature, bacteria swim with flagellar filaments driven by rotary motors embedded in the cell envelope. When a flagellum tethers down to a surface, the entire cell body counter-rotates around that flagellum. This distinctive rotary motion can be utilized to perform various mechanical functions in microfluidic systems, such as pumping, mixing, and/or valving. Most bacterial flagellar motors respond to changes in the acidity of their surroundings in a stochastic fashion. Our study shows that this sensitivity can be predicted through a probabilistic mathematical model and can be used to control their rotational behavior as actuators. Our model incorporates nanoscale physics into continuum level simulations using the Fokker–Planck Equation (FPE). The predictions of the model show a good agreement with experiments. Non-pathogenic, unidirectional Escherichia coli motors were utilized to experimentally validate the predictions of this model. In addition, their long-term performance as well as survivability was evaluated. The reliability of the flagellar motors, which can be influenced by time and environmental conditions, is critical for their practical application as actuators. The average rotational speed of bacterial motors was shown to decay over time with a half-life of 55 h in a closed system without additional nutrient feeding. The average lifetime of the entire population in the system was 34 h while a small percentage of cells lived for 168 h.

A partial atomic structure for the flagellar hook of Salmonella typhimurium

The axial proteins of the bacterial flagellum function as a drive shaft, universal joint, and propeller driven by the flagellar rotary motor; they also form the putative protein export channel. The N- and C-terminal sequences of the eight axial proteins were predicted to form interlocking alpha-domains generating an axial tube. We report on an approximately 1-nm resolution map of the hook from Salmonella typhimurium, which reveals such a tube made from interdigitated, 1-nm rod-like densities similar to those seen in maps of the filament. Atomic models for the two outer domains of the hook subunit were docked into the corresponding outermost features of the map. The N and C termini of the hook subunit fragment are positioned next to each other and face toward the axis of the hook. The placement of these termini would permit the residues missing in the fragment to form the rod-like features that form the core domain of the hook. We also fit the hook atomic model to an approximately 2-nm resolution map of the hook from Caulobacter crescentus. The hook protein sequence from C. crescentus is largely homologous to that of S. typhimurium except for a large insertion (20 kDa). According to difference maps and our fitting, this insertion is found on the outer surface of the hook, consistent with our modeling of the hook.

The Mechanism of the Ion-Driven Flagellar Motor Rotation: Where Is the Rotational Force Generated?

Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions, H+ or Na+, across the membrane. The force is generated by the interaction between a stator and a rotor of the motor which is located at the base of flagellum. A mechanical model of the H+-driven motor rotation by electrostatic interactions between the stator protein, MotA, and the rotor protein, FliG, has been presented. In this review, we summarize recent studies of the Na+-driven motor and discuss the molecular mechanism of the rotation comparing with the H+-driven motor.

Homologies Between Proteins of Borrelia burgdorferi and Thyroid Autoantigens

Subclinical exposure to microbic antigens that share amino acid sequence homology with self antigens might trigger autoimmune diseases in genetically predisposed individuals via molecular mimicry. Genetic predisposition to Graves' disease (GD) or Hashimoto's thyroiditis (HT) is conferred by HLA loci DR3 or DR5, respectively. Yersinia enterocolitica (YE) outer proteins (YOPs) are candidate triggers based on the high prevalence of serum antibodies (Ab) against YOPs in autoimmune thyroid diseases (AITD) and reactivity of these Ab with hTSH-R, suggesting homology between YOPs and hTSH-R. We have reported previously that the spirochete Borrelia burgdorferi (Bb) could be another trigger. We have explored further the homology of hTSH-R with YE and Bb. Using the Basic Local Alignment Search Tool (BLAST), we found four matches with YE and five matches with Bb . Residues 22-272, 186-330, 319-363 and 684-749 of hTSH-R matched YopM, Ysp, exopolygalacturonase and SpyA of YE (identity 23-31%, similarity 40-48%). Residues 112-205, 127-150, 141-260, 299-383 and 620-697 of hTSH-R matched outer surface protein A, flagellar motor rotation protein A, two hypothetical proteins (BBG02 and BBJ08) and DNA recombinase/ATP dependent helicase of Borrelia (identity 27-50%, similarity 40-75%). Interestingly, the above hTSH-R regions coincide with (or include) known human T-cell epitopes: aa 52-71, 140-176, 240-270, 340-380 and 441-661. Our data strengthen the hypothesis of Bb and YE as environmental triggers of AITD in genetically predisposed persons through molecular mimicry mechanisms.

The speed of the flagellar rotary motor of Escherichia coli varies linearly with protonmotive force.

A protonmotive force (pmf) across the cell's inner membrane powers the flagellar rotary motor of Escherichia coli. Speed is known to be proportional to pmf when viscous loads are heavy. Here we show that speed also is proportional to pmf when viscous loads are light. Two motors on the same bacterium were monitored as the cell was slowly deenergized. The first motor rotated the entire cell body (a heavy load), while the second motor rotated a small latex bead (a light load). The first motor rotated slowly and provided a measure of the cell's pmf. The second motor rotated rapidly and was compared with the first, to give the speed-pmf relation for light loads. Experiments were done at 24.0 degrees C and 16.2 degrees C, with initial speeds indicating operation well into the high-speed, low-torque regime. Speed was found to be proportional to pmf over the entire (accessible) dynamic range (0-270 Hz). If the passage of a fixed number of protons carries the motor through each revolution, i.e., if the motor is tightly coupled, a linear speed-pmf relation is expected close to stall, where the work done against the viscous load matches the energy dissipated in proton flow. A linear relation is expected at high speeds if proton translocation is rate-limiting and involves multiple steps, a model that also applies to simple proton channels. The present work shows that a linear relation is true more generally, providing an additional constraint on possible motor mechanisms.

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.

Behavioral responses of Rhodobacter sphaeroides to linear gradients of the nutrients succinate and acetate.

Rhodobacter sphaeroides cells were tethered by their flagella and subjected to increasing and decreasing nutrient gradients. Using motion analysis, changes in flagellar motor rotation were measured and the responses of the cells to the chemotactic gradients were determined. The steepness and concentration ranges of increasing and decreasing gradients were varied, and the bacterial responses were measured. This allowed the limits of gradients that would invoke changes in flagellar behavior to be determined and thus predicts the nature of gradients that would evoke chemotaxis in the environment. The sensory threshold was measured at 30 nM, and the response showed saturation at 150 microM. The study determined that cells detected and responded to changing concentration rates as low as 1 nM/s for acetate and 5 nM/s for succinate. The complex sensory system of R. sphaeroides responded to both increasing and decreasing concentration gradients of attractant with different sensitivities. In addition, transition phases involving changes in the motor speed and the smoothness of motor rotation were found.

Solvent-Isotope and pH Effects on Flagellar Rotation in Escherichia coli

We studied changes in speed of the flagellar rotary motor of Escherichia coli when tethered cells or cells carrying small latex spheres on flagellar stubs were shifted from H 2O to D 2O or subjected to changes in external pH. In the high-torque, low-speed regime, solvent isotope effects were found to be small; in the low-torque, high-speed regime, they were large. The boundaries between these regimes were close to those found earlier in measurements of the torque-speed relationship of the flagellar rotary motor (Berg and Turner, 1993, Biophys. J. 65:2201–2216; Chen and Berg, 2000, Biophys. J., 78:1036–1041). This observation provides direct evidence that the decline in torque at high speed is due primarily to limits in rates of proton transfer. However, variations of speed (and torque) with shifts of external pH (from 4.7 to 8.8) were small for both regimes. Therefore, rates of proton transfer are not very dependent on external pH.

Constraints on models for the flagellar rotary motor

Most bacteria that swim are propelled by flagellar filaments, each driven at its base by a rotary motor embedded in the cell wall and cytoplasmic membrane. A motor is about 45 nm in diameter and made up of about 20 different kinds of parts. It is assembled from the inside out. It is powered by a proton (or in some species, a sodium-ion) flux. It steps at least 400 times per revolution. At low speeds and high torques, about 1000 protons are required per revolution, speed is proportional to protonmotive force, and torque varies little with temperature or hydrogen isotope. At high speeds and low torques, torque increases with temperature and is sensitive to hydrogen isotope. At room temperature, torque varies remarkably little with speed from about -100 Hz (the present limit of measurement) to about 200 Hz, and then it declines rapidly reaching zero at about 300 Hz. These are facts that motor models should explain. None of the existing models for the flagellar rotary motor completely do so.

Torque-Speed Relationship of the Flagellar Rotary Motor of Escherichia coli

The output of a rotary motor is characterized by its torque and speed. We measured the torque-speed relationship of the flagellar rotary motor of Escherichia coli by a new method. Small latex spheres were attached to flagellar stubs on cells fixed to the surface of a glass slide. The angular speeds of the spheres were monitored in a weak optical trap by back-focal-plane interferometry in solutions containing different concentrations of the viscous agent Ficoll. Plots of relative torque (viscosity × speed) versus speed were obtained over a wide dynamic range (up to speeds of ∼300 Hz) at three different temperatures, 22.7, 17.7, and 15.8°C. Results obtained earlier by electrorotation (Berg and Turner, 1993, Biophys. J. 65:2201–2216) were confirmed. The motor operates in two dynamic regimes. At 23°C, the torque is approximately constant up to a knee speed of nearly 200 Hz, and then it falls rapidly with speed to a zero-torque speed of ∼350 Hz. In the low-speed regime, torque is insensitive to changes in temperature. In the high-speed regime, it decreases markedly at lower temperature. These results are consistent with models in which torque is generated by a powerstroke mechanism (Berry and Berg, 1999, Biophys. J. 76:580–587).

Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor.

FliG, FliM, and FliN, key proteins for torque generation, are located in two rings. The first protein is in the M ring and the last two are in the C ring. The rotational symmetries of the C and M rings have been determined to be about 34 (this paper) and 26 (previous work), respectively. The mechanism proposed here depends on the symmetry mismatch between the rings: the C ring extends 34 levers, of which 26 can bind to the 26 equivalent sites on the M ring. The remaining 8 levers bind to proton-pore complexes (studs) to form 8 torque generators. Movement results from the swapping of stud-bound levers with M ring-bound levers. The model predicts that both the M and C rings rotate in the same direction but at different speeds.