A rotary motor drives Flavobacterium gliding

Abstract

Cells of Flavobacterium johnsoniae, a rod-shaped bacterium devoid of pili or flagella, glide over glass at speeds of 2-4 μm/s [1]. Gliding is powered by a protonmotive force [2], but the machinery required for this motion is not known. Usually, cells move along straight paths, but sometimes they exhibit a reciprocal motion, attach near one pole and flip end over end, or rotate. This behavior is similar to that of a Cytophaga species described earlier [3]. Development of genetic tools for F. johnsoniae led to discovery of proteins involved in gliding [4]. These include the surface adhesin SprB that forms filaments about 160 nm long by 6 nm in diameter, which, when labeled with a fluorescent antibody [2] or a latex bead [5], are seen to move longitudinally down the length of a cell, occasionally shifting positions to the right or the left. Evidently, interaction of these filaments with a surface produces gliding. To learn more about the gliding motor, we sheared cells to reduce the number and size of SprB filaments and tethered cells to glass by adding anti-SprB antibody. Cells spun about fixed points, mostly counterclockwise, rotating at speeds of 1 Hz or more. The torques required to sustain such speeds were large, comparable to those generated by the flagellar rotary motor. However, we found that a gliding motor runs at constant speed rather than at constant torque. Now, there are three rotary motors powered by protonmotive force: the bacterial flagellar motor, the Fo ATP synthase, and the gliding motor.

Figure 1

Evidence for a rotary motor. (A) The F. johnsoniae adhesin SprB is present on the cell surface as ~160-nm long filaments. SprB was sheared off and anti-SprB antibody was used to tether F. johnsoniae to a glass surface. (B) The trajectory of the center of mass of a tethered cell plotted over 1000 frames with the center of rotation plotted as a black circle (C) The position of the center of rotation was averaged over 100 frames and plotted as black circles for a movie spanning 1200 frames; the drift of the center of rotation shown with a dotted line was negligible (< 5 nm). (D) Center of mass of a 0.5 μm polystyrene bead tethered onto a sheared cell was tracked over 2192 frames. (E) Speed of rotation is plotted in grey, average speed was calculated every 10 frames and plotted in black.

Figure 2

Speeds and torques recorded for 74 tethered cells. (A) 92% of cells rotated counterclockwise and 8% clockwise. Speed was calculated by recording cell rotation twice for 1-minute periods. Average speed for each recording was calculated. Error bars represent variation in speed of the same cell between the two recordings. Changes in direction of rotation were not seen. (B) Frequency distribution of pivot position for 74 cells normalized for an average cell length of 6 um. Most cells tethered at a distance of ~1 um from the cell pole. (C) Speeds ranged from 0.2–3 Hz, with a majority of cells rotating with a speed ~1 Hz. (D) The torque varied from ~200 to ~6000 pN nm with the majority of cells at a torque ~1000 pN nm. For torque calculations, see Materials and Methods.

Figure 3

Measured speeds and computed torques for cells in 0% and 8% Ficoll. (A) Speed at 0% and 8% Ficoll. (B) The ratio of speeds at 8% and 0% Ficoll are close to 1. (C) Torque at 0% and 8% Ficoll. (D) The ratio of torques at 8% and 0% Ficoll approximate the ratio of viscosities, 3.91.

Figure 4

Flavobacterium gliding model. A Flavobacterium cell with two gliding motors attached to a baseplate mounted on a looped track (bottom). Two SprB filaments are attached to the baseplate and move with it. If either of these filaments adheres to the substratum, the cell glides. Shearing shortens the filaments and disrupts the baseplate, so that each filament is driven by a different motor. If one filament adheres to the substratum, the cell body spins about the axis of the motor.