Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

Abstract

The bacterial flagellar motor can rotate in both counterclockwise (CCW) and clockwise (CW) directions. It has been shown that the sodium ion-driven chimeric flagellar motor rotates with 26 steps per revolution, which corresponds to the number of FliG subunits that form part of the rotor ring, but the size of the backward step is smaller than the forward one. Here we report that the proton-driven flagellar motor of Salmonella also rotates with 26 steps per revolution but symmetrical in both CCW and CW directions with occasional smaller backward steps in both directions. Occasional shift in the stepping positions is also observed, suggesting the frequent exchange of stators in one of the 11-12 possible anchoring positions around the rotor. These observations indicate that the elementary process of torque generation by the cyclic association/dissociation of the stator with every FliG subunit along the circumference of the rotor is symmetric in CCW and CW rotation even though the structure of FliG is highly asymmetric and suggests a 180° rotation of a FliG domain for the rotor-stator interaction to reverse the direction of rotation.

Fig. 1.

Stepping rotation of the Salmonella motor in CCW and CW directions. (A), (B) Angular positions of the beads around the motor axis plotted against the time. (A) The beads attached to the flagellar filaments of MM3076iC (exclusively rotates CCW), and (D) those of SJW46 (rotates CCW and CW). The increase and the decrease in the angle correspond to CCW and CW rotation, respectively. Different colors correspond to the rotation of different motors of different cells. The bead positions were determined from images sampled at a frame rate of 2.4 kHz, and epifluorescence excitation was done using a dye laser (≈20 mW). A two-dimensional trajectory of a bead corresponding to one of the two angular curves is shown in the inset of (A), (D), respectively. (B), (E) Selected parts of the angular plots in (A), (D), respectively. Red lines are steps determined by a step-finding algorithm. (C), (F) Histograms of step angles obtained from CCW and CW rotation, respectively. Cells were grown with 25 μM IPTG for 4 h. All the measurements were carried out at an external pH 6.0 in the presence of 20 mM potassium benzoate at 23 °C.

Fig. 2.

Full 14° backward steps observed with motors of two different cells. (A) MM3076iC and (B) SJW46 motors. Arrowheads indicate switching events. The upward and downward displacements correspond to CCW and CW rotation, respectively.

Fig. 3.

Occasional shifts in the step positions of regular 14° interval. A set of horizontal lines in (B) is shifted from that in (A) by 9° to indicate the amount of shift in the second phase labeled with open arrowheads. Solid and open arrowheads indicate the step positions matching the horizontal lines in (A), (B), respectively.