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Summary: The MotA/B complex acts as the stator of the proton-driven bacterial flagellar motor. Proton translocation through the stator complex is efficiently coupled with torque generation by the stator–rotor interactions. In Salmonella enterica serovar Typhimurium, the highly conserved Pro173 residue of MotA is close to the absolutely conserved Asp33 residue of MotB, which is believed to be a proton-binding site. Pro173 is postulated to be involved in coupling proton influx to torque generation. However, it remains unknown what critical function Pro173 carries out. Here, we characterize the motility and the torque–speed relation of the flagellar motor of the slow motile motA(P173A) mutant of Salmonella. Stall torque produced by the mutant motor was at the wild-type level, indicating that neither the number of stators in the motor nor the rotor–stator interaction is affected by the P173A substitution. In agreement with this, the motA(P173A) allele exerted a strong dominant-negative effect on wild-type motility. In contrast, high-speed rotation at low load was significantly impaired by the mutation, suggesting that the maximum rate of torque generation cycle is severely limited. Simulation of the torque–speed curve by a simple kinetic model indicated that the mutation reduces the rate of conformational changes of the MotA/B complex that switches the exposure of Asp33 to the outside and the inside of the cell, thereby slowing down the mechanochemical reaction cycle. Based on these results, we propose that Pro173 plays an important role in facilitating the conformational dynamics of the stator complex for rapid proton translocation and torque generation cycle. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]