The steady-state counterclockwise/clockwise ratio of bacterial flagellar motors is regulated by protonmotive force

Abstract

A new aspect of the regulation of the bacterial flagellar motor is described. Protonmotive force, i.e. proton electrochemical potential across the cell membrane, not only drives motility, but was found to exert a regulatory effect on the switching of the motor between counterclockwise and clockwise senses. When motor speed was reduced by reducing protonmotive force (but not when speed was reduced by increasing external load), the motor spent a higher fraction of time in counter-clockwise rotation, although the speed in both senses remained equal; at approximately 80% of the maximum motor speed under any given external load condition, no detectable clockwise rotation remained in most cells, i.e. the motor no longer reversed and so cells swam without tumbling. The altered state of the motor remained as long as the state of low protonmotive force remained. Therefore the phenomenon was not a transient tactic response, although it did modify the response to chemotactic stimulation. The regulation of counter-clockwise xxx clockwise switching by protonmotive force appears to be rather general, having been demonstrated in Salmonella, Escherichia coli and Bacillus subtilis. A moderate reduction in protonmotive force from its normal level in actively metabolizing cells did not affect motor reversibility. Motor speed was also unaffected under these conditions (Khan & Macnab, following paper). The existence of a saturation regime with regard to both speed and reversibility suggests that the regulation of motor switching may involve the actual protons driving the motor. All general chemotaxis mutants tested, including those known to be defective in the chemotactic methylation system, were susceptible to protonmotive force regulation in a manner consistent with their normal steady-state counter-clockwise/clockwise ratio. Even mutants that appear “counterclockwise only” could be made more so upon lowering protonmotive force, in that their capacity for clockwise tactic responses was impaired. Since responses to attractants and repellents do not involve an alteration in speed, we conclude that protonmotive force and chemotactic regulatory inputs to the motor operate in parallel. the clockwise/counterclockwise ratio showed an increasing exponential dependence on motor speed. This was a result of both an increased counterclockwise→clockwise switching probability and a decreased clockwise→counterclockwise switching probability. These probabilities were found to be inversely related, so that their product was roughly independent of motor speed. An examination of the pattern of clockwise/counterclockwise switching in tactically stimulated cells and in chemotactic mutants suggests that this is a fundamental property of the motor, which may indicate that switching events in both directions are under the same controlling mechanism. We suggest that suppression of tumbling in circumstances of metabolic deprivation might have survival value by permitting a more rapid dispersal of a population from a region of scarcity of food or oxygen, even when no local gradient is present.