Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor.
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ABSTRACT:
SUBMITTER: Nakamura S
PROVIDER: S-EPMC2955094 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual "power stroke." Specifically, we propose that ion-induced conformational changes about a proline "hinge" residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque-speed and speed-ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.
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Project description:The bacterial flagellar motor plays a crucial role in both bacterial locomotion and chemotaxis. Recent experiments reveal that the switching dynamics of the motor depend on the rotation speed of the motor, and thus the motor torque, nonmonotonically. Here we present a unified mathematical model which treats motor torque generation based on experimental torque-speed curves and the torque-dependent switching based on the conformational spread model. The model successfully reproduces the observed switching rate as a function of the rotation speed, and provides a generic physical explanation independent of most details. A stator affects the switching dynamics through two mechanisms: accelerating the conformational flipping rate of individual rotor-switching units, which contributes most when the stator works at a high torque and thus a low speed; and influencing a larger number of rotor-switching units within unit time, whose contribution is the greatest when the motor rotates at a high speed. Consequently, the switching rate shows a maximum at intermediate speed, where the above two mechanisms find an optimal output. The load-switching relation may serve as a mechanism for sensing the physical environment, similar to the chemotaxis mechanism for sensing the chemical environment. It may also coordinate the switch dynamics of motors within the same cell.
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Project description:The flagellar motor is a bidirectional rotary nanomachine used by many bacteria to sense and move through environments of varying complexity. The bidirectional rotation of the motor is governed by interactions between the inner membrane-associated stator units and the C-ring in the cytoplasm. In this review, we take a structural biology perspective to discuss the distinct conformations of the stator complex and the C-ring that regulate bacterial motility by switching rotational direction between the clockwise (CW) and counterclockwise (CCW) senses. We further contextualize recent in situ structural insights into the modulation of the stator units by accessory proteins, such as FliL, to generate full torque. The dynamic structural remodeling of the C-ring and stator complexes as well as their association with signaling and accessory molecules provide a mechanistic basis for how bacteria adjust motility to sense, move through, and survive in specific niches both outside and within host cells and tissues.
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Project description:Many swimming bacteria are propelled by flagellar filaments driven by a rotary motor. Each of these tiny motors can generate an impressive torque. The motor torque vs. speed relationship is considered one of the most important measurable characteristics of the motor and therefore is a major criterion for judging models proposed for the working mechanism. Here we give an explicit explanation for this torque-speed curve. The same physics also can explain certain puzzling properties of other motors.
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Project description:Multisubunit protein complexes are ubiquitous in biology and perform a plethora of essential functions. Most of the scientific literature treats such assemblies as static: their function is assumed to be independent of their manner of assembly, and their structure is assumed to remain intact until they are degraded. Recent observations of the bacterial flagellar motor, among others, bring these notions into question. The torque-generating stator units of the motor assemble and disassemble in response to changes in load. Here, we used electrorotation to drive tethered cells forward, which decreases motor load, and measured the resulting stator dynamics. No disassembly occurred while the torque remained high, but all of the stator units were released when the motor was spun near the zero-torque speed. When the electrorotation was turned off, so that the load was again high, stator units were recruited, increasing motor speed in a stepwise fashion. A model in which speed affects the binding rate and torque affects the free energy of bound stator units captures the observed torque-dependent stator assembly dynamics, providing a quantitative framework for the environmentally regulated self-assembly of a major macromolecular machine.
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Project description: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 ?m bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 ?m 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.
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Project description:The stator-complex in the bacterial flagellar motor is responsible for surface-sensing. It remodels in response to perturbations in viscous loads, recruiting additional stator-units as the load increases. Here, we tested a hypothesis that the amount of torque generated by each stator-unit modulates its association with the rotor. To do this, we measured stator-binding to the rotor in mutants in which motors reportedly develop lower torque compared to wildtype motors. First, we employed a strain lacking fliL. Contrary to earlier reports, measurements indicated that the torque generated by motors in the fliL strain was similar to that in the wildtype, at high loads. In these motors, stator-binding was unchanged. Next, experiments with a paralyzed strain indicated that the stator-binding was measurably weaker when motors were unable to generate torque. An analytical model was developed that incorporated an exponential dependence of the unit's dissociation rate on the force delivered to the rotor. The model provided accurate fits to measurements of stator-rotor binding over a wide range of loads. Based on these results, we propose that the binding of each stator-unit is enhanced by the force it develops. Furthermore, FliL does not play a significant role in motor function in E. coli.
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Project description:Many bacteria rotate their flagella both counterclockwise (CCW) and clockwise (CW) to achieve swimming toward attractants or away from repellents. Highly conserved charged residues are important for that motility, which suggests that electrostatic interactions are crucial for the rotor-stator function. It remains unclear if those residues contribute equally to rotation in the CCW and CW directions. To address this uncertainty, in this study, we expressed chimeric rotors and stators from Vibrio alginolyticus and Escherichia coli in E. coli, and measured the rotational speed of each motor in both directions using a tethered-cell assay. In wild-type cells, the rotational speeds in both directions were equal, as demonstrated previously. Some charge-neutralizing residue replacements in the stator decreased the rotational speed in both directions to the same extent. However, mutations in two charged residues in the rotor decreased the rotational speed only in the CCW direction. Subsequent analysis and previous results suggest that these amino acid residues are involved in supporting the conformation of the rotor, which is important for proper torque generation in the CCW direction.
| S-EPMC6079164 | biostudies-literature
Project description:The bacterial flagellar motor of Escherichia coli is a nanoscale rotary engine essential for bacterial propulsion. Studies on the power output of single motors rely on the measurement of motor torque and rotation under external load. Here, we investigate the use of magnetic tweezers, which in principle allow the application and active control of a calibrated load torque, to study single flagellar motors in Escherichia coli. We manipulate the external load on the motor by adjusting the magnetic field experienced by a magnetic bead linked to the motor, and we probe the motor's response. A simple model describes the average motor speed over the entire range of applied fields. We extract the motor torque at stall and find it to be similar to the motor torque at drag-limited speed. In addition, use of the magnetic tweezers allows us to force motor rotation in both forward and backward directions. We monitor the motor's performance before and after periods of forced rotation and observe no destructive effects on the motor. Our experiments show how magnetic tweezers can provide active and fast control of the external load while also exposing remaining challenges in calibration. Through their non-invasive character and straightforward parallelization, magnetic tweezers provide an attractive platform to study nanoscale rotary motors at the single-motor level.
| S-EPMC5339722 | biostudies-literature
Project description:The bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant of Salmonella The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-μm beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-μm beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly.IMPORTANCE The bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.
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