Professor Richard Berry D. Phil.

Abstract:

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.

Abstract:

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na(+)-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na(+)). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at approximately 2200 pN nm) from stall up to a "knee" speed of approximately 420 Hz, and then falls linearly with speed, extrapolating to zero torque at approximately 910 Hz. Motors containing one to five stators generated approximately 200 pN nm per stator at speeds up to approximately 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to approximately 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to approximately 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.

Abstract:

This chapter summarizes the current understanding on the structure and function of the bacterial flagellar motor using a combination of genetics, single molecule, and biophysical techniques, with a focus on recent results and single molecule techniques. The bacterial flagellar motor is a reversible rotary nanomachine, 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. In many species, the motor switches direction stochastically, with the switching rates controlled by a network of sensory and signaling proteins. The bacterial flagellar motor was confirmed as a rotary motor in 1974 through tethered-cell experiments, the first direct observation of the function of a single molecular motor. However, due to the large size and complexity of the motor, much remains to be discovered, particularly the structural details of the torque-generating mechanism. The complex assembly pathway and requirement to anchor stators to the cell wall and locate them in an energized membrane have so far precluded the powerful in vitroreconstitution assays that have revealed so much about the other ATP-driven molecular motors in the past decade or two. Nonetheless, a great deal has been learned about the flagellar motor, including considerable recent progress in the application of single molecule techniques. This chapter summarizes the historical background and recent advances in the field. To observe the faster rotation of the motor when driving smaller loads, a variety of single molecule techniques have been used to visualize the rotating filaments of stuck or swimming cells including conventional dark field (DF), laser DF, differential interference contrast (DIC), fluorescence microscopy, back-focal-plane interferometry, and high-speed fluorescence microscopy.