Direct observation of speed fluctuations of flagellar motor rotation at extremely low load close to zero

Abstract

The bacterial flagellar motor accommodates ten stator units around the rotor to produce large torque at high load. But when external load is low, some previous studies showed that a single stator unit can spin the rotor at the maximum speed, suggesting that the maximum speed does not depend on the number of active stator units, whereas others reported that the speed is also dependent on the stator number. To clarify these two controversial observations, much more precise measurements of motor rotation would be required at external load as close to zero as possible. Here, we constructed a Salmonella filament-less mutant that produces a rigid, straight, twice longer hook to efficiently label a 60 nm gold particle and analyzed flagellar motor dynamics at low load close to zero. The maximum motor speed was about 400 Hz. Large speed fluctuations and long pausing events were frequently observed, and they were suppressed by either over-expression of the MotAB stator complex or increase in the external load, suggesting that the number of active stator units in the motor largely fluctuates near zero load. We conclude that the lifetime of the active stator unit becomes much shorter when the motor operates near zero load. K E Y W O R D Sbacterial flagellar motor, duty ratio, hook, MotAB stator complex, torque generation

Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

“…We used the 60 nm gold-nanoparticle assay to get the more accurate flagellar rotation speed at lowered load using the previous protocol (30) with slight modification. Briefly, the SHU174 E. coli (∆motAmotB ∆fliC ∆cheY flgE+GSS+3Cys fliK2798) containing plasmids encoding stators was grown and washed with motility buffer [10 mM potassium-phosphate, 10 mM lactate-Na, 67 mM NaCl, 0.1 mM EDTA, 1% DMSO, pH 7.0] and prepared as a cell suspension.…”

Bacterial flagellar motor as a multimodal biosensor

“…To achieve even lower loads for studies of BFM mechanism, the bead assay was modified to include nanometer-sized gold particles (beads or rods) [59,58]. Given the nm-size of the load, such studies are performed in strain backgrounds that lack the filament protein encoding gene, and the beads are instead attached to the motor hook, a 50 nm in size structure that acts as a universal joint, but can be genetically modified to be longer and more rigid [59,58,60]. The attachment is achieved either using antipolyhook antibody [59,58], streptavidin-biotin link or a cystein introducing mutation [61,12,60].…”

“…Given the nm-size of the load, such studies are performed in strain backgrounds that lack the filament protein encoding gene, and the beads are instead attached to the motor hook, a 50 nm in size structure that acts as a universal joint, but can be genetically modified to be longer and more rigid [59,58,60]. The attachment is achieved either using antipolyhook antibody [59,58], streptavidin-biotin link or a cystein introducing mutation [61,12,60]. Depending on the viscous load, motor rotation frequency can vary from dozens to hundreds of Hz [4] and can be measured with high speed cameras [12], or back-focal-plane interferometry, where the rotating bead is placed in the focus of a heavily attenuated laser beam and the changes in the interference pattern are recorded with the quadrant photo-diode or position sensitive detector, Fig.…”

The Architectural Dynamics of the Bacterial Flagellar Motor Switch

“…Focal back-plane interferometry with high spatiotemporal resolution (1 • , 10 −3 s) recorded incomplete, intermediate switching events to extend the temporally resolved measurement of switch transitions [53]. More recently, gold nanospheres (d = 60 nm) conjugated to genetically engineered, rigid Salmonella flagellar hooks, imaged by dark-field and rapid (5000 Hz) CMOS cameras, have been exploited to measure motor rotation [54]. This study interpreted the large speed fluctuations recorded near the zero-torque speed (~400 Hz) that persisted over many revolutions as the association-dissociation of individual stator units.…”

“…Advances in switch physiology. (A) Time-resolved motor rotation: Schematic of a motor rotation assay with a nanosphere conjugated to the hook connector contiguous with the rod and basal body (reproduced from[54] with permission). (B) CW and CCW interval distributions: (i) Idealized time series of a motor alternating between CW and CCW rotation.…”