A Molecular Mechanism of Bacterial Flagellar Motor Switching

The high-resolution structures of nearly all the proteins that comprise the bacterial flagellar motor switch complex have been solved; yet a clear picture of the switching mechanism has not emerged. Here, we used NMR to characterize the interaction modes and solution properties of a number of these proteins, including several soluble fragments of the flagellar motor proteins FliM and FliG, and the response-regulator CheY. We find that activated CheY, the switch signal, binds to a previously unidentified region of FliM, adjacent to the FliM-FliM interface. We also find that activated CheY and FliG bind with mutual exclusivity to this site on FliM, because their respective binding surfaces partially overlap. These data support a model of CheY-driven motor switching wherein the binding of activated CheY to FliM displaces the carboxy-terminal domain of FliG (FliG_C) from FliM, modulating the FliG_C-MotA interaction, and causing the motor to switch rotational sense as required for chemotaxis.     

Adaptive Remodelling by FliN in the Bacterial Rotary Motor

Sensory adaptation in the Escherichia coli chemosensory pathway has been the subject of interest for decades, with investigation focusing on the receptors that process extracellular inputs. Recent studies demonstrate that the flagellar motors responsible for cell locomotion also play a role, adding or subtracting FliM subunits to maximise sensitivity to pathway signals. It is difficult to reconcile this FliM remodelling with the observation that partner FliN subunits are relatively static fixtures in the motor. By fusing a fluorescent protein internally to FliN, we show that there is in fact significant FliN remodelling. The kinetics and stoichiometry of FliN in steady state and in adapting motors are investigated and found to match the behaviour of FliM in all respects except for timescale where FliN rates are about 4 times slower. We notice that motor adaptation is slower in the presence of the fluorescent protein, indicating a possible source for the difference. The behaviour of FliM and FliN is consistent with a kinetic and stoichiometric model that contradicts the traditional view of a packed, rigid motor architecture.     

Visualization of functional rotor proteins of the bacterial flagellar motor in the cell membrane

The bacterial flagellar motor is a rotary motor driven by the electrochemical potentials of specific ions across the cell membrane. Direct interactions between the rotor protein FliG and the stator protein MotA are thought to generate the rotational torque. Here, we used total internal reflection fluorescent microscopy to observe the localization of green fluorescent protein (GFP)-fused FliG in Escherichia coli cells. We identified three types of fluorescent punctate signals: immobile dots, mobile dots that exhibited simple diffusion, and mobile dots that exhibited restricted diffusion. When GFP-FliG was expressed in a DeltafliG background, most of the cells were not mobile. When the cells were tethered to a glass side, however, rotating cells were commonly observed and a single fluorescent dot was always observed at the rotational center of the tethered cell. These fluorescent dots were likely positions at which functional GFP-FliG had been incorporated into a flagellar motor. Our results suggest that flagellar basal bodies diffuse in the cytoplasmic membrane until the axial structure and/or other structures assemble.     

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.     

Thermal and Solvent-Isotope Effects on the Flagellar Rotary Motor near Zero Load

In Escherichia coli, the behavior of the flagellar rotary motor near zero load can be studied by scattering light from nanogold spheres attached to proximal hooks of cells lacking flagellar filaments. We used this method to monitor changes in speed when cells were subjected to changes in temperature or shifted from a medium made with H₂O to one made with D₂O. In H₂O, the speed increased with temperature in a near-exponential manner, with an activation enthalpy of 52 ± 4 kJ/mol (12.0 ± 1.0 kcal/mol). In D₂O, the speed increased in a similar manner, with an activation enthalpy of 50 ± 4 kJ/mol. The speed in H₂O was higher than that in D₂O by a factor of 1.53 ± 0.14. We performed comparison studies of variations in temperature and solvent isotope, using motors operating at high loads. The variations were small, consistent with previous observations. The implications of these results for proton translocation are discussed.     

Morphology, function and isolation of halobacterial flagella

Halobacterium halobium has right-handed helical flagella. During the logarithmic phase of growth, cells are predominantly monopolar, whereas in the stationary phase they are mostly bipolarly flagellated. The flagellar bundle consists of several filaments. Halobacteria swim forward by clockwise and backwards by counterclockwise rotation of their flagella. The flagellar bundle does not fly apart when the sense of rotation changes. In addition to the flagella attached to the cells, large amounts of loose flagella, which aggregate into thick super-flagella, can be observed at all phases of growth. During stationary phase, the production of these super-flagella, which are generally 10 to 20 times longer than the cell body, is significantly higher. Dissociation and association by high temperature and differential centrifugation allow the isolation of pure flagella. Three different protein bands, of 23,500, 26,500 and 31,500 apparent molecular weights, are seen on sodium dodecyl sulphate/polyacrylamide gels. Antibodies against halobacterial flagella were produced in chicken; these antibodies interact with the flagella even in 4 M-NaCl. Rotation of tethered cells demonstrates that Halobacteria move due to the rotation of the flagella.     

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.     

Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.     

Effect of Intracellular pH on the Torque-Speed Relationship of Bacterial Proton-Driven Flagellar Motor

Bacterial flagella responsible for motility are driven by rotary motors powered by the electrochemical potential difference of specific ions across the cytoplasmic membrane. The stator of proton-driven flagellar motor converts proton influx into mechanical work. However, the energy conversion mechanism remains unclear. Here, we show that the motor is sensitive to intracellular proton concentration for high-speed rotation at low load, which was considerably impaired by lowering intracellular pH, while zero-speed torque was not affected. The change in extracellular pH did not show any effect. These results suggest that a high intracellular proton concentration decreases the rate of proton translocation and therefore that of the mechanochemical reaction cycle of the motor but not the actual torque generation step within the cycle by the stator-rotor interactions.     

Switching of Bacterial Flagellar Motors Is Triggered by Mutant FliG

Binding of the chemotaxis response regulator CheY-P promotes switching between rotational states in flagellar motors of the bacterium Escherichia coli. Here, we induced switching in the absence of CheY-P by introducing copies of a mutant FliG locked in the clockwise (CW) conformation (FliG^CW). The composition of the mixed FliG ring was estimated via fluorescence imaging, and the probability of CW rotation (CW_bias) was determined from the rotation of tethered cells. The results were interpreted in the framework of a 1D Ising model. The data could be fit by assuming that mutant subunits are more stable in the CW conformation than in the counterclockwise conformation. We found that CW_bias varies depending on the spatial arrangement of the assembled subunits in the FliG ring. This offers a possible explanation for a previous observation of hysteresis in the switch function in analogous mixed FliM motors—in motors containing identical fractions of mutant FliM^CW in otherwise wild-type motors, the CW_bias differed depending on whether mutant subunits were expressed in strains with native motors or native subunits were expressed in strains with mutant motors.     

Structure of Flagellar Motor Proteins in Complex Allows for Insights into Motor Structure and Switching

The flagellar motor is one type of propulsion device of motile bacteria. The cytoplasmic ring (C-ring) of the motor interacts with the stator to generate torque in clockwise and counterclockwise directions. The C-ring is composed of three proteins, FliM, FliN, and FliG. Together they form the “switch complex” and regulate switching and torque generation. Here we report the crystal structure of the middle domain of FliM in complex with the middle and C-terminal domains of FliG that shows the interaction surface and orientations of the proteins. In the complex, FliG assumes a compact conformation in which the middle and C-terminal domains (FliG_MC) collapse and stack together similarly to the recently published structure of a mutant of FliG_MC with a clockwise rotational bias. This intramolecular stacking of the domains is distinct from the intermolecular stacking seen in other structures of FliG. We fit the complex structure into the three-dimensional reconstructions of the motor and propose that the cytoplasmic ring is assembled from 34 FliG and FliM molecules in a 1:1 fashion.     

The Shift-Work Accident Rate is More Related to the Shift Type than to Shift Rotation

The current study investigated the accident rates across morning, late, and night shifts in rotating shift-workers employed in two different shift rotations at the same steel work factory. A retrospective analysis has been performed of accident data (N = 578) over a 5-year period (2003 through 2007) of 730 male shift-workers employed in either a clockwise (mean age of the workers 38.1 ± SD 9.8 years) or counterclockwise rotation (mean age 38.0 ± SD 10.1 years) with comparable work conditions. The overall accident rate across the 24-h day was not significantly different between clockwise and counterclockwise shift rotation. In both shift-work rotations, morning shifts as opposed to night shifts exhibited a significantly higher accident rate. There was no significant difference between late shifts and morning or night shifts in either shift rotation. The increased accident rate in the morning shift at this steel factory could be related to the early starting time of the shift and to this shift being more labor intensive in both shift rotations. These findings suggest that work-related factors must be considered in addition to shift-work schedules when investigating accident rates in rotating shift-workers.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

Measurement of the Internal Frictional Drag of the Bacterial Flagellar Motor by Fluctuation Analysis

The bacterial flagellar motor generates the torque that drives the rotation of bacterial flagellar filaments. The torque it generates depends sensitively on the frictional viscous drag on the motor, which includes the frictional viscous drag on the filaments (external load) and the internal frictional viscous drag on the rotor (internal load). The internal load was roughly estimated previously by modeling it as a sphere of a radius of 20 nm rotating in a lipid of viscosity of 100 cp but was never measured experimentally. Here, we measured the internal load by fluctuation analysis of the motor velocity traces. A similar approach should be applicable to other molecular motors.     

Functional Transfer of an Essential Aspartate for the Ion-binding Site in the Stator Proteins of the Bacterial Flagellar Motor

Rotation of the bacterial flagellar motor exploits the electrochemical potential of the coupling ion (H⁺ or Na⁺) as its energy source. In the marine bacterium Vibrio alginolyticus, the stator complex is composed of PomA and PomB, and conducts Na⁺ across the cytoplasmic membrane to generate rotation. The transmembrane (TM) region of PomB, which forms the Na⁺-conduction pathway together with TM3 and TM4 of PomA, has a highly conserved aspartate residue (Asp24) that is essential for flagellar rotation. This residue contributes to the Na⁺-binding site. However, it is not clear whether residues other than Asp24 are involved in binding the coupling ion. We examined the possibility that loss of the negative charge of Asp24 can be suppressed by introduction of negatively charged residues in TM3 or TM4 of PomA. The motility defect associated with the D24N substitution in PomB could be rescued only by a N194D substitution in PomA. This result suggests that there must be a negatively charged ion-binding pocket in the stator complex but that the presence of a negatively charged residue at position 24 of PomB is not essential. A tandemly fused PomA dimer containing the N194D mutation either in its N-terminal or C-terminal half with PomB-D24N was functional, suggesting that PomB-D24N can form an ion-binding pocket with either subunit of PomA dimer. The findings obtained in this study provide important clues to the mechanism of ion binding in the stator complex.     

Model Studies of the Dynamics of Bacterial Flagellar Motors

The bacterial flagellar motor is a rotary molecular machine that rotates the helical filaments that propel swimming bacteria. Extensive experimental and theoretical studies exist on the structure, assembly, energy input, power generation, and switching mechanism of the motor. In a previous article, we explained the general physics underneath the observed torque-speed curves with a simple two-state Fokker-Planck model. Here, we further analyze that model, showing that 1), the model predicts that the two components of the ion motive force can affect the motor dynamics differently, in agreement with latest experiments; 2), with explicit consideration of the stator spring, the model also explains the lack of dependence of the zero-load speed on stator number in the proton motor, as recently observed; and 3), the model reproduces the stepping behavior of the motor even with the existence of the stator springs and predicts the dwell-time distribution. The predicted stepping behavior of motors with two stators is discussed, and we suggest future experimental procedures for verification.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.