We developed a robust bead assay for studying flagellar motor behavior of Pseudomonas aeruginosa . Using this assay, we studied the dynamics of the two stator systems in the flagellar motor. We found that the two sets of stators function differently, with MotAB stators providing higher total torque, and MotCD stators ensuring more stable motor speed. The motors in wild-type cells adjust the stator compositions according to the environment, resulting in an optimal performance in environmental exploration compared to mutants with one set of stators. The bead assay we developed here can be further used to study P. aeruginosa chemotaxis at the level of single cell using the motor behavior as the chemotaxis output. Importance Cells of Pseudomonas aeruginosa possess a single polar flagellum, driven by a rotatory motor powered by two sets of torque-generating units (stators). We developed a robust bead assay for studying the behavior of the flagellar motor in P. aeruginosa , by attaching a microsphere to shortened flagellar filament and using it as an indicator of motor rotation. Using this assay, we revealed the dynamics of the two stator systems in the flagellar motor, and found that the motors in wild-type cells adjust the stator compositions according to the environment, resulting in an optimal performance in environmental exploration compared to mutants with one set of stators.

Significance The monotrichous Pseudomonas aeruginosa was usually thought to swim in a pattern of “run and reverse” (possibly with pauses in between), where straight runs alternated with reverses with angular changes of swimming direction near 180°. Here, by simultaneously tracking the cell swimming and the morphology of its flagellum, we discovered a swimming mode in P. aeruginosa —the wrap mode, during which the flagellar filament wrapped around the cell body and induced large fluctuation of the body orientation. The wrap mode randomized swimming direction, resulting in a broad distribution of angular changes over 0 to 180° with a peak near 90°. This allowed the bacterium to explore the environment more efficiently, which we confirmed by stochastic simulations of P. aeruginosa chemotaxis.

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In a crowded environment such as a bacterial swarm, cells frequently got jammed and came to a stop, but were able to escape the traps by backing up in their moving course with a head-to-tail change (a reversal). Reversals are essential for the expansion of a bacterial swarm. Reversal for a wildtype cell usually involved polymorphic transformation of the flagellar filaments induced by directional switching of the flagellar motors. Here we discovered a new way of reversal in cells without motor switching and characterized its mechanisms. We further found that this type of reversal was not limited to swarmer cells, but also occurred for cells grown in a bulk solution. Therefore, reversal was a general way of escaping when cells got jammed in their natural complex habitats. The new way of reversal we discovered here offered a general strategy for cells to escape traps and explore their environment.

FliL emerged as a modulator of flagellar motor function in several bacterial species, but its function in Pseudomonas aeruginosa was unknown. Here, by performing single-motor studies using a bead assay, we elucidated its effects on the flagellar motor in P. aeruginosa .