A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

Abstract

Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stopstart flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides-using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27-28 discrete angles, locked in place by a relatively high torque, approximately 2-3 times its stall torque.

The effect of sampling rate on observed statistics in a correlated random walk

“…This model of motion is applicable to many species that are amenable to study using tracking methods. Examples include bacteria such as R. sphaeroides that reorient by stopping the rotation of their flagellum [31], flying insects that pause on landing sites [14] and ruminants that pause at foraging sites [4,17]. We demonstrated that the inclusion of stationary phases in the underlying motion leads to complex variation in the observed standard deviation of RASs, and suggested an explanation based on consideration of the evolution of the RAS pdf.…”

Osmotaxis in Escherichia coli through changes in motor speed

“…For BFM experiments flagellar filaments were truncated by passing a bacterial suspension through two syringes with narrow gauge needles (26G) connected with a plastic tube ('shearing device' [Bai et al, 2010, Pilizota et al, 2009). Subsequently, cells with truncated filaments were washed by centrifugation.…”

“…Subsequently, cells with truncated filaments were washed by centrifugation. Slides for BFM experiments were prepared as before [Bai et al, 2010, Pilizota et al, 2009] by layering two parallel strips of double sided sticky tape onto a microscope slide and covering them with a cover glass, forming a tunnel slide (SI Appendix Fig. 5) of approximate volume of ∼ 8 µl.…”

Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

“…The observation of such a fine stepping motion was made possible by reducing the number of functional stators in the sodium ion-driven chimeric flagellar motor and lowering sodium ion motive force to reduce the rotation rate to meet the spatial and temporal resolution of the measurement system (20). It has also been reported that the unidirectional motor of Rhodobacter sphaeroides stops at 27-28 discrete angles, locked in place by a relatively high torque (19). Interestingly, backward CW steps of the sodium ion-driven chimeric flagellar motor were also often observed even in the absence of CheY, but the step size (−10.3°) was slightly smaller than the forward CCW steps (13.8°) (20), suggesting an asymmetric mechanism of bidirectional rotation.…”

“…Recently, it has been reported that the c-di-GMP binding protein YcgR binds to MotA, FliG, and FliM in the presence of c-di-GMP, resulting not only in a reduced motor speed but also in the CCW bias in motor rotation (3,4). In Rhodobacter sphaeroides, the flagellar motor rotation is unidirectional, but CheY-P acts as a molecular brake to stop motor rotation to change the swimming direction (19).…”