Professor Richard Berry D. Phil.

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.