Vrabioiu, A. M. | Search bioRxiv

bioRxiv2025

The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

The bacterial flagellar motor drives bacterial swimming and chemotaxis by rotating helical flagellar filaments. When Escherichia coli navigates chemical gradients, the motor switches from counterclockwise (CCW) during forward swimming to clockwise (CW) during direction-changing tumbles. The motor responds indirectly to extracellular chemosensory input to membrane-bound chemoreceptors using an intervening intracellular signaling pathway. How the motor responds to its direct input signal - the diffusible messenger CheY-P - remains poorly understood. Steady-state motor measurements have been modeled as an allosteric switch between CCW/CW states that depend on mean CheY-P levels. Allosteric models have suggested that as many as 20 CheY-P molecules can be bound to the motor when it switches rotational direction. But steady-state models cannot predict the sensitivity of the motor to dynamic changes in CheY-P that essentially modulate chemotactic behavior. We present an optogenetic reagent that precisely controls the direct dynamical input signal to the motor. We designed a "caged" molecule, Opto-CheY, that is transiently activated by photon absorption. We find that activation and binding of 1-3 CheY-P molecules is sufficient to switch the motor from the CCW to CW state. The sensitivity of the motor to small changes in CheY-P occupancy helps resolve a long-standing paradox about the high sensitivity of the chemotactic response to external sensory input. Optogenetic biochemistry by light-activated uncaging of signal molecules is a new strategy to dissect information-processing in the living cell. Significance StatementMotile bacteria swim to better environments by modulating the rotation of the bacterial flagellar motor. How this motor responds to intracellular signaling activity is poorly understood. The physiologically-relevant response of the motor is to transient activation of intracellular signaling molecules on the sub-second time scale of bacterial decision-making. Here, we report the first optogenetic probe that targets in vivo the output module of the the chemotactic network. We demonstrate that the motor has high dynamical sensitivity to the binding of single intracellular signaling molecules. This solves a long-standing problem of high sensitivity and signal amplification in the bacterial chemotactic response.

biophysics↗

bioRxiv2025

Torque-generating units of the bacterial flagellar motor are rotary motors

E. coli swims using helical flagellar filaments driven at their base by a rotary motor. Torque-generating stator units drive the bacterial flagellar motor (BFM) by transmitting mechanical power to a cytoplasmic rotor, the C-ring. Each stator unit is a proton-conducting heteromer. A central dimer of two MotB proteins anchor to the cell wall. A surrounding pentamer of five MotA proteins transmit mechanical power to the C-ring. This asymmetrical 5:2 structure is consistent with rotation as the mechanism of torque generation. Here, we test the hypothesis that the MotA5MotB2 stator units are rotary motors themselves and interact with the rotor like intermeshed gearwheels, where rotation of the C-ring is directly coupled to MotA5 rotation around the MotB2. We used in vivo polarized photo-bleaching microscopy. When a subset of fluorescent domains inside a multimer is rapidly photo-bleached by a strong pulse of polarized light, the induced polarization-dependent fluorescence of unbleached domains becomes a reporter of angular orientation. We applied polarized photo-bleaching microscopy to tethered cells rotating by single flagellar motors. We probed fluorescently-labeled MotA pentamer and MotB dimer calibrated to motor rotation. The MotB dimer rotates at the same angular speed as the cell body, consistent with its anchor to the cell wall. The MotA pentamer rotates [~]6.2x faster than the flagellar motor, revealing the gear ratio between stator and rotor. Significance StatementBacteria swim by rotating rigid helical flagellar filaments. Here, we find that the torque-generating unit that drives flagellar rotation is itself a rotary motor. Each torque-generating unit is a heteromeric macromolecular machine - a pentamer of MotA subunits that surround a dimer of proton-conducting MotB subunits. Torque is generated as the MotA spins around MotB. The MotA pentamer interacts with rotor of the flagellar motor in a manner resembling intermeshing gearwheels. The bacterial flagellar motor is driven by the first set of enmeshed gearwheels that has been described in any living cell.

microbiology↗