Yoshiyuki Sowa

Bacterial flagellar motor

The bacterial flagellar motor is a reversible rotary nano-machine, about 45 nm in diameter, embedded in the bacterial cell envelope. It is powered by the flux of H+ or Na+ ions across the cytoplasmic membrane driven by an electrochemical gradient, the proton-motive force or the sodium-motive force. Each motor rotates a helical filament at several hundreds of revolutions per second (hertz). In many species, the motor switches direction stochastically, with the switching rates controlled by a network of sensory and signalling proteins. The bacterial flagellar motor was confirmed as a rotary motor in the early 1970s, the first direct observation of the function of a single molecular motor. However, because of the large size and complexity of the motor, much remains to be discovered, in particular, the structural details of the torque-generating mechanism. This review outlines what has been learned about the structure and function of the motor using a combination of genetics, single-molecule and biophysical techniques, with a focus on recent results and single-molecule techniques.

Direct observation of steps in rotation of the bacterial flagellar motor

The bacterial flagellar motor is a rotary molecular machine that rotates the helical filaments that propel many species of swimming bacteria. The rotor is a set of rings up to 45 nm in diameter in the cytoplasmic membrane; the stator contains about ten torque-generating units anchored to the cell wall at the perimeter of the rotor. The free-energy source for the motor is an inward-directed electrochemical gradient of ions across the cytoplasmic membrane, the protonmotive force or sodium-motive force for H+-driven and Na+-driven motors, respectively. Here we demonstrate a stepping motion of a Na+-driven chimaeric flagellar motor in Escherichia coli at low sodium-motive force and with controlled expression of a small number of torque-generating units. We observe 26 steps per revolution, which is consistent with the periodicity of the ring of FliG protein, the proposed site of torque generation on the rotor. Backwards steps despite the absence of the flagellar switching protein CheY indicate a small change in free energy per step, similar to that of a single ion transit.

Torque-speed relationship of the Na+-driven flagellar motor of Vibrio alginolyticus.

The torque-speed relationship of the Na(+)-driven flagellar motor of Vibrio alginolyticus was investigated. The rotation rate of the motor was measured by following the position of a bead, attached to a flagellar filament, using optical nanometry. In the presence of 50mM NaCl, the generated torque was relatively constant ( approximately 3800pNnm) at lower speeds (speeds up to approximately 300Hz) and then decreased steeply, similar to the H(+)-driven flagellar motor of Escherichia coli. When the external NaCl concentration was varied, the generated torque of the flagellar motor was changed over a wide range of speeds. This result could be reproduced using a simple kinetic model, which takes into consideration the association and dissociation of Na(+) onto the motor. These results imply that for a complete understanding of the mechanism of flagellar rotation it is essential to consider both the electrochemical gradient and the absolute concentration of the coupling ion.

The Systematic Substitutions Around the Conserved Charged Residues of the Cytoplasmic Loop of Na+-driven Flagellar Motor Component PomA

PomA, a homolog of MotA in the H+-driven flagellar motor, is an essential component for torque generation in the Na+-driven flagellar motor. Previous studies suggested that two charged residues, R90 and E98, which are in the single cytoplasmic loop of MotA, are directly involved in this process. These residues are conserved in PomA of Vibrio alginolyticus as R88 and E96, respectively. To explore the role of these charged residues in the Na+-driven motor, we replaced them with other amino acids. However, unlike in the H+-driven motor, both of the single and the double PomA mutants were functional. Several other positively and negatively charged residues near R88 and E96, namely K89, E97 and E99, were neutralized. Motility was retained in a strain producing the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein. The swimming speed of the AAQQQ strain was as fast as that of the wild-type PomA strain, but the direction of motor rotation was abnormally counterclockwise-biased. We could, however, isolate non-motile or poorly motile mutants when certain charged residues in PomA were reversed or neutralized. The charged residues at positions 88-99 of PomA may not be essential for torque generation in the Na+-driven motor and might play a role in motor function different from that of the equivalent residues of the H+-driven motor.

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

Significance Using new experimental methods, we measure the mechanical output of the bacterial flagellar motor, the rotary molecular machine that propels swimming bacteria, while varying both electrical and chemical components of the ion-motive force that drives it. We find that each independent torque-generating stator in the motor passes 37 ± 2 ions per revolution, at odds with previous indications of 26 or 52 ions. Fitting our data to theoretical models reveals the kinetics of the motor mechanism. Our thorough search of the multidimensional parameter space of generalized motor models, guided by experimental data, is an approach that may be widely applicable. The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque–speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque–speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a “powerstroke” in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration.

A simple backscattering microscope for fast tracking of biological molecules

Recent developments in techniques for observing single molecules under light microscopes have helped reveal the mechanisms by which molecular machines work. A wide range of markers can be used to detect molecules, from single fluorophores to micron sized markers, depending on the research interest. Here, we present a new and simple objective-type backscattering microscope to track gold nanoparticles with nanometer and microsecond resolution. The total noise of our system in a 55 kHz bandwidth is ~0.6 nm per axis, sufficient to measure molecular movement. We found our backscattering microscopy to be useful not only for in vitro but also for in vivo experiments because of lower background scattering from cells than in conventional dark-field microscopy. We demonstrate the application of this technique to measuring the motion of a biological rotary molecular motor, the bacterial flagellar motor, in live Escherichia coli cells.

Steps and bumps:precision extraction of discrete states of molecular machines

We report statistical time-series analysis tools providing improvements in the rapid, precision extraction of discrete state dynamics from time traces of experimental observations of molecular machines. By building physical knowledge and statistical innovations into analysis tools, we provide techniques for estimating discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of steplike rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, periodicities and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a step in the direction toward automated analysis of high-sample-rate, molecular-machine dynamics. Modular, open-source software that implements these techniques is provided.

Steps in the Bacterial Flagellar Motor

The bacterial flagellar motor is a highly efficient rotary machine used by many bacteria to propel themselves. It has recently been shown that at low speeds its rotation proceeds in steps. Here we propose a simple physical model, based on the storage of energy in protein springs, that accounts for this stepping behavior as a random walk in a tilted corrugated potential that combines torque and contact forces. We argue that the absolute angular position of the rotor is crucial for understanding step properties and show this hypothesis to be consistent with the available data, in particular the observation that backward steps are smaller on average than forward steps. We also predict a sublinear speed versus torque relationship for fixed load at low torque, and a peak in rotor diffusion as a function of torque. Our model provides a comprehensive framework for understanding and analyzing stepping behavior in the bacterial flagellar motor and proposes novel, testable predictions. More broadly, the storage of energy in protein springs by the flagellar motor may provide useful general insights into the design of highly efficient molecular machines.

Microscopic Analysis of Bacterial Motility at High Pressure

The bacterial flagellar motor is a molecular machine that converts an ion flux to the rotation of a helical flagellar filament. Counterclockwise rotation of the filaments allows them to join in a bundle and propel the cell forward. Loss of motility can be caused by environmental factors such as temperature, pH, and solvation. Hydrostatic pressure is also a physical inhibitor of bacterial motility, but the detailed mechanism of this inhibition is still unknown. Here, we developed a high-pressure microscope that enables us to acquire high-resolution microscopic images, regardless of applied pressures. We also characterized the pressure dependence of the motility of swimming Escherichia coli cells and the rotation of single flagellar motors. The fraction and speed of swimming cells decreased with increased pressure. At 80 MPa, all cells stopped swimming and simply diffused in solution. After the release of pressure, most cells immediately recovered their initial motility. Direct observation of the motility of single flagellar motors revealed that at 80 MPa, the motors generate torque that should be sufficient to join rotating filaments in a bundle. The discrepancy in the behavior of free swimming cells and individual motors could be due to the applied pressure inhibiting the formation of rotating filament bundles that can propel the cell body in an aqueous environment.

High Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli Flagellar Motor

The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20°C, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P.