Rotation of Bacterial Flagella at High Frequency

Flagellated bacteria propel themselves through their fluid environment by rotating one or more helical filaments which are driven at their base by a reversible motor powered by a protonmotive force. In some species many filaments join. together to form a single flagellar bundle during swimming. Past work has characterized the functional properties of the motor at low speed and high torque from studies of cells tethered by a single filament. This thesis describes work in which properties of the motor at high speed and low torque were investigated by studying free swimming cells. A method was developed for measuring the rotation rates of filaments in bundles of swimming cells. Images of cell bodies were projected onto the photocathode of a photomultiplier tube whose sensitivity is spatially inhomogeneous, and the power spectral density of the output was computed using the Fast Fourier Transform. Averages of many spectra revealed a peak at high frequency due to the vibration of the cell body by the rotating filaments. This method was analysed in detail by mechanical simulations and computer models. Techniques were also developed for following the rotation of single motors by attaching markers to sheared flagella. Most experiments were done with a motile strain of Streptococcus. At 22°C, filaments of this organism rotate at ca. 100 Hz relative to the cell body. Higher frequencies were seen in Escherichia coli at the same temperature, ca. 180 Hz. The relation between torque and speed of the flagellar motor at fixed protonmotive force was determined by varying the viscosity of the medium. Torque was found to drop linearly with increasing speed over the upper half of the speed range. A comparison with the torque generated by tethered cells suggests that linearity may hold over the entire speed range. This behavior is consistent with a scheme whereby the free energy available per proton is dissipated in a series of small steps. The bundle frequency of glycolysing Streptococcus was found to increase linearly with temperature, by a factor of 7 from 10°C to 42°C, corresponding to an increase in torque by a factor of 3. The protonmotive force did not vary by more than 10% from 16°C to 32°C. Conditions were found under which cells swam when artificially energized by a combination of transmembrane pH gradient and potassium diffusion potential. These cells exhibited a large deuterium isotope effect, their speed dropping by 30 - 50% in D20. Thus, proton transfer reactions appear to be limiting the rate of motor rotation in swimming cells.