The speed of the flagellar rotary motor of Escherichia coli varies linearly with protonmotive force

Abstract

A protonmotive force (pmf) across the cell's inner membrane powers the flagellar rotary motor of Escherichia coli. Speed is known to be proportional to pmf when viscous loads are heavy. Here we show that speed also is proportional to pmf when viscous loads are light. Two motors on the same bacterium were monitored as the cell was slowly deenergized. The first motor rotated the entire cell body (a heavy load), while the second motor rotated a small latex bead (a light load). The first motor rotated slowly and provided a measure of the cell's pmf. The second motor rotated rapidly and was compared with the first, to give the speed-pmf relation for light loads. Experiments were done at 24.0°C and 16.2°C, with initial speeds indicating operation well into the high-speed, low-torque regime. Speed was found to be proportional to pmf over the entire (accessible) dynamic range (0 -270 Hz). If the passage of a fixed number of protons carries the motor through each revolution, i.e., if the motor is tightly coupled, a linear speed-pmf relation is expected close to stall, where the work done against the viscous load matches the energy dissipated in proton flow. A linear relation is expected at high speeds if proton translocation is rate-limiting and involves multiple steps, a model that also applies to simple proton channels. The present work shows that a linear relation is true more generally, providing an additional constraint on possible motor mechanisms.bacteria ͉ energetics ͉ molecular motors ͉ motility T he flagellar rotary motor of an Escherichia coli bacterium couples proton flux across the inner cell membrane to motor rotation. Protons move down an electrochemical gradient through specific MotA͞B protein complexes anchored to the cell wall. As a result, these complexes, known as torque-generating units, step along the rotor, causing it to rotate. At heavy viscous loads and low speeds, motor torque is approximately constant (and efficiencies are high), whereas at light viscous loads and high speeds, motor torque declines rapidly (and efficiencies become small). For a review of motor structure and function, see ref.1. Fung and Berg (2) found that when the motor operates in the low-speed regime near stall (Ͻ10 Hz), its speed is proportional to the voltage applied across the inner cell membrane. The experiments reported here extend these measurements well into the motor's high-speed regime. In this regime, the speedprotonmotive force (pmf) relation will depend explicitly on the mechanochemical cycle of the motor; therefore, its characterization puts new restrictions on possible motor mechanisms. MethodsCells of E. coli strain KAF95, a strain with sticky filaments that rotate exclusively counterclockwise (3), were inoculated from frozen stocks, grown in Luria broth [1% tryptone (Difco)͞0.5% yeast extract (Difco)͞0.5% NaCl] at 30°C until mid-exponential phase, and then sheared by using syringes with 26-gauge needles connected by 0.58-mm i.d. polyethylene tubing, producing cells with short flagellar stubs. These cells were...

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