The relationship between flagellar motor dynamics and the proton motive force

Thesis

The bacterial flagellar motor is one of the few rotary motors found in nature, and an excellent example of a complex molecular machine. Flagellar motors in the model organism Escherichia coli are products of the coordinated expression of ∼50 different genes. The E. coli flagellar motor is powered by the proton-motive force (pmf), an electrochemical gradient across the cell membrane. Motor torque is gen- erated by proton flow through membrane-embedded stator units which bind to the basal body of the motor.

This thesis aimed to investigate the relationship between the pmf and the flag- ellar motor. A novel pmf control system was developed, based on the light-driven proton pump proteorhodopsin (pR). This system enabled pmf -dependent changes in motor behaviour to be precisely monitored in vivo. Expression of pR in E. coli was shown to be sufficient to drive the flagellar motor at wild-type speeds. Using the pR-based pmf control system, the motor was shown to respond to changes in pmf on a timescale of milliseconds. Surprisingly, motor speed increase was observed when pmf was increased above the physiological norm. Reduction of pmf to low levels enabled individual steps in motor rotation to be observed.

Motor response to loss of pmf was investigated. Motors were shown to exhibit a two-stage speed decrease after disruption of pmf , with motor speed falling to ∼20 % of its initial value within milliseconds, reaching a complete stop after 1 s. Extended periods of pmf loss was shown to lead to disengagement of stators from the motor, with motor speed increasing in a stepwise fashion after pmf restoration.

The integrity of the motor at different pmf levels was investigated by using TIRF microscopy to directly image positioning of fluorescently tagged motor components. The stator protein MotB was shown to physically leave and rejoin the motor after pmf disruption and restoration, with MotB dispersal following motor stop.

Authors: Tipping, M; Murray Tipping

• Publication date: 2011
• DOI: 10.5287/ora-aobbpqev2
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: Oxford University, UK
• Language: English
• Keywords: bioenergetics; biophysics; flagellar motor
• Subjects: Molecular biophysics (biochemistry)