Oxford Molecular Motors

Abstract:

We demonstrate the use of a laser trap (‘optical tweezers’) and back-focal-plane position detector to measure rapid rotation in aqueous solution of single particles with sizes in the vicinity of 1 μm. Two types of rotation were measured: electrorotation of polystyrene microspheres and rotation of the flagellar motor of the bacterium Vibrio alginolyticus. In both cases, speeds in excess of 1000 Hz (rev s⁻¹) were measured. Polystyrene beads of diameter about 1 μm labelled with smaller beads were held at the centre of a microelectrode array by the optical tweezers. Electrorotation of the labelled beads was induced by applying a rotating electric field to the solution using microelectrodes. Electrorotation spectra were obtained by varying the frequency of the applied field and analysed to obtain the surface conductance of the beads. Single cells of V. alginolyticus were trapped and rotation of the polar sodium-driven flagellar motor was measured. Cells rotated more rapidly in media containing higher concentrations of Na⁺, and photodamage caused by the trap was considerably less when the suspending medium did not contain oxygen. The technique allows single-speed measurements to be made in less than a second and separate particles can be measured at a rate of several per minute. © 2003 Taylor & Francis Group, LLC.

Abstract:

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.