Asymmetric distribution of type IV pili triggered by directional light in unicellular cyanobacteria

Abstract

The type IV pili (T4P) system is a supermolecular machine observed in prokaryotes. Cells repeat the cycle of T4P extension, surface attachment, and retraction to drive twitching motility. Although the properties of T4P as a motor have been scrutinized with biophysics techniques, the mechanism of regulation remains unclear. Here we provided the framework of the T4P dynamics at the single-cell level in Synechocystis sp. PCC6803, which can recognize light direction. We demonstrated that the dynamics was detected by fluorescent beads under an optical microscope and controlled by blue light that induces negative phototaxis; extension and retraction of T4P was activated at the forward side of lateral illumination to move away from the light source. Additionally, we directly visualized each pilus by fluorescent labeling, allowing us to quantify their asymmetric distribution. Finally, quantitative analyses of cell tracking indicated that T4P was generated uniformly within 0.2 min after blue-light exposure, and within the next 1 min the activation became asymmetric along the light axis to achieve directional cell motility; this process was mediated by the photo-sensing protein, PixD. This sequential process provides clues toward a general regulation mechanism of T4P system, which might be essentially common between archaella and other secretion apparatuses.

Keywords: Synechocystis sp. PCC6803; fluorescence; phototaxis; signal transduction; twitching motility.

Fig. 1.

Negative phototaxis visualized under an optical microscope at the single-cell level. (A) Schematics of the T4P-based twitching motility. A cell moves via a cycle of extension, attachment, and retraction of T4P. These three processes are indicated by red arrows or circle, respectively, in A. (B) Diagram of the experimental setup. Lateral and vertical lights with a wavelength of 488 nm were separately irradiated onto the specimen on the sample stage. This setup enables quantification of the phototactic response of each cell movement. (C) Bright-field image of Synechocystis sp. PCC6803 and their moving trajectories for 240 s (color lines) on a glass substrate coated with 0.007% collodion. The blue arrow in the Inset represents the direction of light propagation. (Scale bar, 30 μm.) (D) Rose plots under lateral (Left), vertical (Middle), and no illumination (Right) of blue light against the sample stage (n = 200 cells). The number of cells moving more than 3 μm min⁻¹ was counted. (E) Schematics of types of illumination in D. Thick arrows in the Insets and thin arrows represent the direction of light propagation and movement of cells, respectively. The fluence rate was 1,200 μmol m⁻² s⁻¹ from lateral or vertical illumination. BS, beam splitter; DM, dichroic mirror; Obj, objective lens.

Fig. S1.

Effect of collodion coating on negative phototaxis. (A) Bright-field image of cells and their moving trajectories for 180 s (color lines) on a glass substrate coated with collodion. The concentration of collodion was presented at the upper right part of each image. The blue arrow in the Inset represents the direction of light propagation. (Scale bar, 30 μm.) (B) Rose plots under lateral illumination against the sample stage (n = 200 cells). Cell was subjected to the lateral light from the left side (270° in the image) at a fluence rate of 1,200 μmol m⁻² s⁻¹. The number of cells moving more than 3 μm min⁻¹ was counted.

Fig. 2.

Visualization of T4P dynamics through fluorescent beads. (A) (Top) schematics of the observation. The thick arrow in the Inset represents the direction of light propagation. Blue and red thin arrows represent the retraction and extension of pili, respectively. The light-illuminated region is shown in pale blue. (Bottom Left) merged image of the cell (red) and fluorescent beads (green). The Inset represents the direction of light propagation, which follows the axis perpendicular to the observation plane. (Bottom Right) averaged intensity profile of the beads in the micrograph (n = 20 cells). (B) Schematic, micrograph, and profile of the beads’ distribution when light was laterally illuminated. The micrograph is shown with the same color codes and visualization as at Bottom Left in A. The blue arrow in the Inset in both A and B represents the direction of light propagation. (C) Same sets as in B when lights were partially illuminated in the right half of the cell from the bottom as shown in the schematic. Partially covered marks in the Insets represent the localized illumination. (Scale bars, 1 μm in A and B, also applies to C.) (D) Tracking of beads: (Left, Middle, and Right) the results under vertical, lateral, and partial illumination, respectively, in D–F. Rounds at the center indicate the cell position. Blue and red colors code the movements toward the cell and the movements away from the cell, respectively, in D–F. Beads were visualized by vertical or lateral blue-light illumination. (E) Time courses of beads under different illuminations. (F) Histograms of the speed of displacement of beads. The average and SD were plotted (n = 100 in 15 cells). (G) (Left and Middle) histograms of the angle distribution of beads’ displacement, looking from the center of cells under vertical and lateral illumination, respectively. (Right) Schematic of the definition of the angle θ.

Fig. S2.

Preference of accumulation of beads on the cell surface. (A) Merged image of the cell (red) and fluorescent beads (green). The cell was immobilized onto the glass surface. (Top) Sulfate beads. Cells were chemically fixed with glutaraldehyde before adding beads. (Middle) Carboxylated beads. (Bottom) BSA-coated carboxylated beads. Beads were visualized by mercury lamp after chemical fixation of the cell with glutaraldehyde. (Scale bar, 1 μm.) (B) Averaged intensity profile of the beads in the micrograph (n = 20 cells).

Fig. S3.

Effect of microoptics. (A) Schimatics of microoptics effect. (B) (Top) Optical field distribution calculated by FDTD method. Cell was illuminated with lateral light from the left side (270° in the image). The color indicates relative light intensity. Dashed line indicates the cell outline. (Scale bar, 1 μm.) (Bottom) intensity profile along the cell outlines from 270° to 90° in Top. (C) Fluorescent beads with the size of 0.2 μm located at the surface of the cell. (Top) Bright-field image. (Middle) Fluorescent image visualized by vertical illumination. (Bottom) Fluorescent image visualized by lateral illumination. Cross-mark indicates the center of the cell. (Scale bar, 2 μm.) (Right) Schematic illustration of the effect of microoptics. The blue arrow in the Inset in A–C represents the direction of light propagation. (D) Intensity profile of the beads. Left and Right profiles came from the two different beads indicated by the red and blue arrows in C. Gaussian fitting of the intensity was presented by a solid line. (Top) vertical illumination. (Bottom) lateral illumination. The beads were marked by the arrows in C.

Fig. S4.

Visualization of T4P filaments under an optical microscope. (A) (Left) magnified view of the bright-field image of the cell. (Scale bar, 2 μm.) (Middle) fluorescent micrograph labeled by FITC–avidin (see SI Materials and Methods for details). Cell was subjected to the vertical blue light at a fluence rate of 1,200 μmol m⁻² s⁻¹. Each T4P filament is clearly visualized. (Right) merged image of (Left) (red) and (Middle) (green). (B) Merged image of cells labeled by FITC–avidin (Top) and FITC–streptavidin (Bottom), respectively. (Scale bar, 5 μm.) (C) Image of a cell taken by the electron microscope with negative staining using ammonium molybdate. (Scale bar, 1 μm.) (D) Magnified image of the yellow boxed area in C. (Scale bar, 100 nm.)

Fig. S5.

Visualization of T4P filament in the GT strain. (A) Merged image of cells labeled by FITC–avidin. (Scale bar, 5 μm.) (B) Image of a cell taken by the electron microscope with negative staining using ammonium molybdate. (Scale bar, 1 μm.)

Fig. 3.

Distribution of T4P filaments in the wild type and ΔpixD mutant triggered by lateral illumination. (A) (Left) bright-field image of the cell, which was subjected to lateral light from the left side (270° in the image) at a fluence rate of 1,200 μmol m⁻² s⁻¹ (Materials and Methods for details). (Scale bar, 2 μm.) Middle: fluorescent micrograph. T4P were dominantly distributed around 90°. (Right) merged image of Left (red) and Middle (green). (B) Schematic showing the asymmetric tendency of the extension of T4P triggered by lateral light. The blue arrow in the Inset in both A and B represents the direction of light propagation. (C) Schematic of the definition of θ in rose plots (Left). Rose plot of the distribution of a T4P filament in the wild type (Middle), and the ΔpixD mutant (Right) (n = 20 cells). (D) Effects of lateral light intensity on the number of T4P per cell (Top), the pilus length (Middle), and the localization bias of pilus appearance (Bottom) as [(the number of pili in 0–180°)/(total number)] when light came from 270°, in the wild type (Left, circles) and ∆pixD mutant (Right, diamonds). The average and SD from 20 cells were plotted. Dashed lines represent the fitting of Michaelis–Menten kinetics.

Fig. S6.

Distribution of a single T4P triggered by lateral illumination in various light intensities. (A) Merged image of cell (red) and T4P (green). The cells of the wild type (Top) and ΔpixD mutant (Bottom) were subjected to the lateral light from the left side at various fluence rates, as presented in each image. The cells were subsequently chemically fixed by glutaraldehyde and labeled by FITC-avidin. The blue arrow in the Inset represents the direction of light propagation. (Scale bars, 5 μm.) (B) Rose plot of the distribution of a single T4P in the wild type (Top) and ΔpixD mutant (Bottom) (n = 20 cells). The definition of angle θ was the same in Fig. 3C. The fluence rate of the lateral illumination was presented in each graph.

Fig. S7.

Visualization of T4P dynamics through fluorescent beads in ΔpixD mutant. (A) Merged image of the cell (red) and fluorescent beads (green). Sulfate beads were visualized with a mercury lamp after chemical fixation of the cell with glutaraldehyde. (Inset) The direction of light propagation. (Top) Vertical. (Middle) lateral. (Bottom) partial. Partial blue light was applied to the right half of the cell, as shown in pale blue. The blue arrow in the Inset represents the direction of light propagation. (Scale bar, 1 μm.) (B) Averaged intensity profile of the beads in the micrograph (n = 20 cells). (C) Tracking of the beads under lateral illuminations. Rounds at the center indicate the cell and rough position of the cell surface: blue, movements toward the cell; red, movements away from the cell. Beads were visualized by vertical or lateral blue-light illumination. (D) Time courses of beads under lateral illuminations. The color codes of the lines are the same as in C. (E) Histogram of the speed of displacement of beads. The color codes of bars are the same as in C. Values in the graphs are the average± SD (n = 100 in 10 cells). (F) Histogram of the angle distribution of beads’ displacement looking from the center of cells under lateral illumination. The definition of angle θ was the same in Fig. 2G.

Fig. 4.

Distribution of T4P filaments in the wild type and ΔpixD mutant triggered by localized illumination. (A) (Top Left) magnified view of the bright-field image of a cell subjected to localized light from the bottom side (highlighted by pale blue) with a strength of 10,000 μmol m⁻² s⁻¹ (Materials and Methods for details). (Scale bar, 2 μm.) (Top Middle) fluorescent micrograph. T4P were dominantly distributed in the illuminated area, in this case the right half (0–180°) of the cell. (Top Right) merged image of Top Left (red) and Top Middle (green). (Bottom) multiple cells. (Scale bar, 5 μm.) (B) Three sets of images similar to the Top in A of the ΔpixD mutant. (Scale bar, 2 μm.) (C) Rose plots of the distribution of T4P in the wild type (Top) and ΔpixD mutant (Bottom) (n = 20 cells). (D) Effects of light intensity on the number of T4P per cell (Top), the pilus length (Middle), and the localization bias of pilus appearance (Bottom) as [(the number of pili in 0–180°)/(total number)] when the cells were illuminated only in the right-half region (0–180°), in the wild type (Left, circles), and ∆pixD mutant (Right, diamonds). The average and SD from 20 cells were plotted. Dashed lines represent the fitting of Michaelis–Menten kinetics. (E) Schematics of our observations in A and B. Red arrows represent the extension of T4P triggered by localized illumination. The thick blue arrow in the Inset represents the direction of light propagation. The thin pale blue arrows represent the region of the localized light illuminated in the right half of the cell.

Fig. 5.

Analyses of twitching motions of the wild type and ΔpixD mutant triggered by lateral illumination. (Left) The wild type. (Middle) ΔpixD mutant. (Right) wild type without the illumination. (A) xy plots where light propagation of the lateral illumination was set parallel to the x axis (n = 50 cells). The blue arrow in the Inset represents the direction of light propagation. (B) Raw data of the time course of x (n = 50). (C) The average of x (thick blue line). The SD at each time point was plotted as a cyan perpendicular bar. (D) MSD plots (thick blue line). The cyan curve represents a hyperbolic fitting. The orange line represents a linear fitting. (E) A magnified view of D. In C–E, the pink region is from 0.2 to 0.9 min after the exposure; blue arrows in C–E indicate the transition from random movement to directed movement; and red arrows in E indicate the transition where the motion starts after the exposure. (F) Schematics of the cell response triggered by lateral blue-light illumination. Blue and red thin arrows represent the retraction and extension of pili, respectively. ON, the starting point of the light exposure.

Fig. S8.

Analyses of twitching motions triggered by lateral illumination in various light intensities. (A) Time course of the average displacement of cells along the light propagation of the lateral illumination (n = 50 cells). The color indicates the fluence rate of lateral illumination. (Left) The wild type. (Right) ΔpixD mutant. (B) Average of MSD plots (n = 50 cells). The color codes are the same as in A. (C) Effect of lateral light intensity on the cell velocity. The average and SD of velocity along the light propagation was plotted (n = 50 cells). ON, the starting point of the light exposure.

Fig. S9.

Schematic illustration of PixD-dependent negative phototaxis. (A) PixD forms complex with the response regulator PixE, and the complex is disassembled by the illumination of blue light. (B) PixD presumably releases PixE at the forward side of light illumination, where the intensity is relatively high, and this release mediates a suppression of the T4P dynamics in the region where the intensity is relatively low. (C) T4P dynamics is activated at the higher-intensity side, allowing navigation of directed twitching motility. The blue arrow in the Inset in both B and C represents the direction of light propagation. The pale red and blue arrows in C represent the extension and retraction of T4P, respectively.

Fig. S10.

Experimental setup of light illumination on the microscope stage. (A) Diagram of lateral and vertical illuminations. Blue lasers with a wavelength of 488 nm were separately irradiated onto the specimen on the sample stage. (B) Diagram of partial illumination. The Ronchi-ruling, a constant-interval bar, was set at the conjugative position of sample plane to be a 2-μm interval on the sample stage. (C) Partial illumination of fluorescent beads. (Top) A 1-μm bead. (Bottom) A 2-μm bead. Partial blue light was applied to the right half of the cell, as shown in pale blue. (Scale bars, 2 μm.) (D) Calibration of blue-light intensity on the sample stage. Fluorescent intensity of beads with a size of 0.2 μm was linearly fitted by the vertical and lateral illumination. (E) Typical example of fluorescent intensity of beads in various blue-light intensities. (Left) vertical. (Right) lateral. (Scale bars, 0.5 μm.) (F) Diagram of mercury lamp. Accumulation of beads and FITC-labeled T4P was visualized by this setup. The blue arrow in the Inset in both D and E represents the direction of light propagation. BE, beam expander; BP, band-pass filter; BS, beam splitter; DM, dichroic mirror.