Helical flow of surface protein required for bacterial gliding motility

Abstract

Cells of Flavobacterium johnsoniae and of many other members of the phylum Bacteroidetes exhibit rapid gliding motility over surfaces by a unique mechanism. These cells do not have flagella or pili; instead, they rely on a novel motility apparatus composed of Gld and Spr proteins. SprB, a 669-kDa cell-surface adhesin, is required for efficient gliding. SprB was visualized by electron microscopy as thin 150-nm-long filaments extending from the cell surface. Fluorescence microscopy revealed movement of SprB proteins toward the poles of the cell at ∼2 μm/s. The fluorescent signals appeared to migrate around the pole and continue at the same speed toward the opposite pole along an apparent left-handed helical closed loop. Movement of SprB, and of cells, was rapidly and reversibly blocked by the addition of carbonyl cyanide m-chlorophenylhydrazone, which dissipates the proton gradient across the cytoplasmic membrane. In a gliding cell, some of the SprB protein appeared to attach to the substratum. The cell body moved forward and rotated with respect to this point of attachment. Upon reaching the rear of the cell, the attached SprB often was released from the substratum, and apparently recirculated to the front of the cell along a helical path. The results suggest a model for Flavobacterium gliding, supported by mathematical analysis, in which adhesins such as SprB are propelled along a closed helical loop track, generating rotation and translation of the cell body.

Keywords: cell motility; continuous track; immunofluorescence microscopy; left-handed helix; proton motive force.

Fig. 1.

SprB forms cell-surface filaments. (A) Negative staining of cells of wild-type F. johnsoniae UW101 and of the sprB deletion mutant CJ1922. Images were treated with a bandpass filter to visualize the surface features clearly. (Inset) Surface regions where the filaments were found are drawn schematically as black dots. (B) Magnified image of polar regions (dashed boxes in A) of wild-type and sprB deletion mutant cells. Yellow arrowheads indicate filamentous structures extending from the cell surface. (C) Negative staining of the SprB fraction partially purified from wild-type cells. The SprB fraction is the same as that used in Fig. S4A, lane 5. (D) Immunogold EM on the SprB fraction treated with antisera against SprB (Upper) and GldJ (Lower).

Fig. 2.

Helical loop-like motion of SprB. (A) Localization of SprB observed by epifluorescence microscopy. SprB was immunolabeled by antisera against SprB and fluorescent secondary antibody (Materials and Methods). In a translocating cell, the fluorescent signals were recorded at 0.1-s intervals for 2 s, colored from red (time 0) to blue (2 s), and integrated into a single image (Bottom). The images come from Movie S2. (B) Kymograph of SprB signals. The same cell as shown in A was used. The x-axis is the position of SprB signals with respect to the substratum (glass), and the y-axis is time. The orange arrow and dashed lines indicate the translocating direction of the cell and the approximate positions of cell poles, respectively. (C) Velocity of SprB movement and cell motility along the x-axis. SprB signals displaying well-separated foci were used for calculations. The velocities of 80 signals (Upper) and 13 cells (Lower) were integrated into the histograms.

Fig. 3.

Left-handed helical flow of SprB on the cell surface. (A) Location of SprB observed by TIRF microscopy (TIRFM). A cell translocating to the right was analyzed. Cell outline was visualized by simultaneous weak illumination using a halogen lamp. The SprB signals were colored from red to blue at 0.05-s intervals for 1.25 s and integrated into one image (Lower). The image is the view from the glass side. The images come from Movie S4. (B) Traces of typical SprB signals (Upper). Each signal was dotted with 0.05-s intervals. The same cell as shown in A was used. The moving direction of each trace is indicated by the arrows (Lower). (C) Tracking of typical signals. (Left) Montage of signals at 0.05-s intervals for 0.3 s. (Right) The images at 0.05 s and 0.25 s were colored magenta and green, respectively, and merged into a single image. Tracking of the signal is represented by the yellow arrow. (D) Velocity of SprB signals. The velocities of 72 signals were calculated from the x-axes of the fitting line and integrated into a histogram. (E) Angle of SprB signal traces. The angles of the fitting lines of the traces of 39 SprB signals moving from lower left to upper right with respect to the x-axis were measured and integrated into a histogram. SprB signals displaying well-separated foci were used for calculations.

Fig. 4.

Helical flow of SprB in a nontranslocating cell. (A) Localization of SprB observed by epifluorescence microscopy. Cells were immunolabeled with anti-SprB antiserum and fluorescent secondary antibody (Materials and Methods). The fluorescent signals were traced at 0.1-s intervals for 2 s and integrated into one image (Lower), with red indicating time 0 and blue indicating 2 s. The images come from Movie S5. (B) Localization of SprB observed by TIRFM. The SprB signals were colored from red to blue at 0.05-s intervals for 1 s and integrated into one image (Lower). The image is the view from the glass side. The images come from Movie S6. (C) Traces of typical SprB signals (Upper). Each signal was dotted with 0.05-s intervals for 1 s. The same cell as shown in B was used. The moving direction of each trace is indicated by the arrows (Lower). (D) Tracking of typical signals. Montage of signals at 0.1-s intervals for 0.3 s (Left). The images at 0 and 0.3 s were colored magenta and green, respectively, and merged into a single image (Right). Tracking of the signal is represented by the yellow arrow. (E) Velocity of SprB signals. The velocities of 61 signals were calculated from the x-axes of the fitting lines and integrated into a histogram. + and −, right and left directions of SprB motion, respectively. (F) Angle of SprB signal traces. The angles of the fitting lines with respect to the x-axis were measured and integrated into a histogram. SprB signals displaying well-separated foci were used for calculations.

Fig. 5.

Model of Flavobacterium gliding motility. (A) A nontranslocating cell. Adhesin SprB moves along the left-handed helical loop with a speed of υ₀. (B) A translocating cell on glass. SprB has two different states: SprB moving toward the front of the cell and SprB moving toward the rear of the cell. In a translocating cell, SprB moving toward the rear of the cell adheres to the surface, generating left-handed rotation and right-directed translocation of the cell. SprB moving toward the front of the cell apparently runs twice as fast with respect to the glass surface than SprB on a nontranslocating cell.