The maximum torque the bacterial flagellar motor generates, the stall torque, is a critical parameter that describes the motor energetics. As the motor operates in equilibrium near stall, from the stall torque one can determine how many protons each torque-generating unit (stator) of the motor passes per revolution and then test whether motor rotation and proton flux are tightly or loosely coupled, which has been controversial in recent years.

The bacterial flagellar motor is a nanometer-sized rotary motor that generates the torque to drive the rotation of the flagellar filament. The output torque is an important property of the motor. The motor rotation was usually monitored by attaching a μm-sized bead to a shortened flagellar filament, and the torque was extracted by calculating the torque due to the viscous drag of the medium on the bead rotation. We sought for an independent extraction of the torque from thermal fluctuation in the motor rotation using the fluctuation theorem (FT). However, we identified an overwhelming fluctuation beyond the thermal noise that precluded the use of FT. We further characterized the timescale and the amplitude of this fluctuation, finding that it was probably due to the stepping of the motor. The amplitude of torque fluctuation we characterized here provided new information on the torque-generating interaction potential curve.

The flagellar motor drives the rotation of flagellar filaments, propeling the swimming of flagellated bacteria. The maximum torque the motor generates, the stall torque, is a key characteristics of the motor function. Direct measurements of the stall torque carried out three decades ago suffered from large experimental uncertainties, and subsequently there were only indirect measurements. Here, we applied magnetic tweezer to directly measure the stall torque in E. coli. We precisely calibrated the torsional stiffness of the magnetic tweezer, and performed motor resurrection experiments at stall, accomplishing a precise determination of the stall torque per torque-generating unit (stator unit). From our measurements, each stator passes 2 protons per step, indicating a tight coupling between motor rotation and proton flux.

The output of the bacterial chemotaxis signaling pathway, the level of the intracellular regulator CheY-P, modulates the rotation direction of the flagellar motor, thereby regulating bacterial run-and-tumble behavior. The multiple flagellar motors on an E. coli cell are controlled by a common cytoplasmic pool of CheY-P. Fluctuation of the CheY-P level was thought to be able to coordinate the switching of multiple motors. Here, we measured the correlation of rotation directions between two motors on a cell, finding that it surprisingly exhibits two well separated timescales. We found that the slow timescale (∼6 s) can be explained by the slow fluctuation of the CheY-P level due to stochastic activity of the chemotactic adaptation enzymes, whereas the fast timescale (∼0.3 s) can be explained by the random pulse-like fluctuation of the CheY-P level, due probably to the activity of the chemoreceptor clusters. We extracted information on the properties of the fast CheY-P pulses based on the correlation measurements. The two well-separated timescales in the fluctuation of CheY-P level help to coordinate multiple motors on a cell and to enhance bacterial chemotactic performance.