Absence of a barrier to backwards rotation of the bacterial flagellar motor demonstrated with optical tweezers

Abstract

A cell of the bacterium Escherichia coli was tethered covalently to a glass coverslip by a single f lagellum, and its rotation was stopped by using optical tweezers. The tweezers acted directly on the cell body or indirectly, via a trapped polystyrene bead. The torque generated by the f lagellar motor was determined by measuring the displacement of the laser beam on a quadrant photodiode. The coverslip was mounted on a computer-controlled piezo-electric stage that moved the tether point in a circle around the center of the trap so that the speed of rotation of the motor could be varied. The motor generated Ϸ4500 pN nm of torque at all angles, regardless of whether it was stalled, allowed to rotate very slowly forwards, or driven very slowly backwards. This argues against models of motor function in which rotation is tightly coupled to proton transit and back-transport of protons is severely limited.Many species of bacteria swim with the aid of helical flagella that are powered at their base by a rotary motor embedded in the cell envelope. The power source for the motor is an electrochemical gradient of ions across the cytoplasmic membrane, the motor being a device for coupling rotation to ionic flux. These ions are either H ϩ (protons) or, in alkalophilic or marine bacteria, Na ϩ . In Escherichia coli, the motor can rotate in either direction, and cells navigate toward regions rich in nutrients by controlling this direction (1). Flagellar rotation traditionally has been measured by using the ''tethered cell'' assay, in which a single flagellum is attached to the surface, causing the cell body to counter-rotate (2). Whereas the flagella of swimming E. coli rotate at over 100 Hz and are difficult to observe directly, tethered cells rotate at Ϸ10 Hz and are comparatively easy to monitor.Measurement of the rotation rates of tethered cells and of flagellar bundles in swimming cells (3) established that the motor generates more torque under conditions of high load and low speed (tethered cells, or cells swimming in highly viscous media) than at low load and high speed (cells swimming in normal aqueous media). This behavior is predicted by most models of the motor mechanism and cannot, therefore, be used to distinguish between them. The torque generated when the motor is forced to rotate against its natural direction provides more information and has been measured by using electrorotation (4, 5). The latter authors (5) found a barrier to backwards rotation, i.e., that approximately twice as much torque was needed to make cells rotate backwards as was sufficient to stop them. This is predicted by some models of the motor mechanism, in particular those that include a stage in the torque-generating cycle in which the rotor and stator are tightly bound to each other, but not by others, for example those that generate torque by long range electrostatic interactions. However, further work using electrorotation suggested that the barrier to backwards rotation might be an artefact of that technique (6) arising from ang...

Physics of Bacterial Morphogenesis

“…However, there are several well-known rotary motors: the bacterial flagellar motor powers the swimming motion of E. coli by converting chemical energy stored in the electrochemical gradient in the inner membrane to rotational motion (13,14,16,18,34,109,150,173). In this large motor assembly, 8 motor subunits (stators) operate together to turn a motor unit.…”

“…As the protein deforms, the assembly kinetics and enzymatic activities of the protein often change. This complex interplay is the origin of the observed cooperativity in many biological systems and underlies the mechanism of bacterial propulsion by the flagellar motor (13,14,16,18,34,109,150,173). It is also the basis of chemoreceptor performance and chemical gradient sensing (22,48,132,137,138,154,177).…”

Force and Velocity of Mycoplasma mobile Gliding

“…An optical tweezer produced by a 985-nm laser was used to trap beads on gliding cells with enough force to stall forward movement. The trap design, position measurement, and force calibration have been described previously (5). By measuring displacement of the bead from the trap center using back-focal-plane interferometry, the stall force could be calculated.…”

Optical tweezers for single cells

“…Bacterial cells containing locomotivespecific appendages such as flagella have been a model system for the measurement of force transduction and the motility of cells. A comprehensive review has been made by Berry & Berg (1997) and, recently, the bacterial rotor has been reported by Reid et al (2006) and Darnton et al (2007).…”