Asymmetric Distribution of Type IV Pili Triggered by Directional Light in Unicellular Cyanobacteria

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

Daisuke Nakanea and Takayuki Nishizakaa,1

aDepartment of Physics, Gakushuin University, Tokyo 171-8588, Japan

Edited by Caroline S. Harwood, University of Washington, Seattle, WA, and approved May 10, 2017 (received for review February 15, 2017)

The type IV pili (T4P) system is a supermolecular machine observed coccoid cell transmits the signals to the T4P machinery to achieve in prokaryotes. Cells repeat the cycle of T4P extension, surface directional cell motility. attachment, and retraction to drive twitching motility. Although the In this study, we directly visualized the dynamics of T4P under properties of T4P as a motor have been scrutinized with biophysics an optical microscope and showed that they were controlled by the techniques, the mechanism of regulation remains unclear. Here we blue-light exposure that induced the negative phototaxis. We provided the framework of the T4P dynamics at the single-cell level provide the direct evidence that the local difference of light in- in Synechocystis sp. PCC6803, which can recognize light direction. tensity in the cell induces the asymmetric activation of T4P to We demonstrated that the dynamics was detected by fluorescent achieve directional cell motility. The blue-light receptor, PixD, beads under an optical microscope and controlled by blue light that mediates the suppression of the T4P dynamics in the opposite induces negative phototaxis; extension and retraction of T4P was region to maintain the above activation. These findings highlight activated at the forward side of lateral illumination to move away the light-signal processing system in cyanobacteria, which regu- – from the light source. Additionally, we directly visualized each pilus lates T4P dynamics to navigate cells in a certain direction (18 20). by fluorescent labeling, allowing us to quantify their asymmetric This concept is in stark contrast to the mechanism of chemotaxis distribution. Finally, quantitative analyses of cell tracking indicated in bacteria, which changes the interval between random swimming that T4P was generated uniformly within 0.2 min after blue-light and tumbling triggered by repellants (21). exposure, and within the next 1 min the activation became asym- Results metric along the light axis to achieve directional cell motility; this process was mediated by the photo-sensing protein, PixD. This se- Observation of Phototaxis at the Single-Cell Level. Phototaxis of MICROBIOLOGY quential process provides clues toward a general regulation mech- bacteria has been visualized as colony migration on an agar plate anism of T4P system, which might be essentially common between (14, 22) or as the trajectory of cells (15, 18). To observe a detailed archaella and other secretion apparatuses. trajectory of negative phototaxis at the single-cell level, we con- structed an optical setup that allowed us to simultaneously illu- minate specimens on a glass substrate both laterally and vertically Synechocystis sp. PCC6803 | twitching motility | phototaxis | (Fig. 1B). Blue light with a wavelength of 488 nm was chosen to signal transduction | fluorescence induce negative phototaxis, and the stimulus of light intensity was − − adjusted in the range of 10–10,000 μmol m 2 s 1 (photon flux den- ype IV pili (T4P) are fascinating supermolecular machines sity in the visible light wavelength) on the sample plane, which was Tthat drive twitching motility, protein secretion, and DNA calibrated using the intensity of fluorescent beads (see Materials and uptake in prokaryotes (1). Twitching motility is now widely ac- Methods for more detail). The position of the cell was visualized by cepted as a form of bacterial translocation involving repetition of green light from a halogen lamp with a band-pass filter (532/40 nm) − − the cycle of extension and retraction of the pili (2, 3) (Fig. 1A), with a fluence rate of 1 μmol m 2 s 1, which was confirmed to have which is powered by assembly and disassembly ATPase at the base of helical pilus fibers (4). The mechanical response of a single Significance pilus has been scrutinized in great detail using biophysical ap- proaches such as optical tweezers methodology in Neisseria Type IV pili (T4P) are cell-surface appendages observed in pro- – gonorrheae (3, 5 7), although the dynamic properties resulting karyotes that perform critical functions in cell motility, surface from the response to various environmental signals remain less adhesion, virulence, and biofilm formation. Although the archi- understood than those of bacterial flagella. T4P is also evolu- tecture of T4P has already been determined, the dynamics tionarily and structurally related to flagella in archaea, which have resulting from the response to various environmental signals recently been designated archaella (8–11). Although newly de- remain unclear. Here we demonstrated the sequential process of veloped techniques enable us to examine the dynamic properties T4P dynamics from stimulus to taxis at the single-cell level in a of the machinery, little is known about the regulation mechanism model cyanobacterium, which can recognize light direction. We of T4P because tens of components cooperate to orchestrate the directly visualized that T4P filaments dominantly appeared from dynamics of both the archaella and T4P system. Such complexity the side of the cell opposite the illumination. This asymmetric hampers the design of an experimental setup with quantitative and activation is regulated on a timescale of minutes, and the process reproducible evaluations. was transitioned between three sequential phases. These find- To address how the T4P system is activated or repressed by ings provide clues toward a general regulation mechanism of the environmental signals over a short timescale, we here used T4P system. Synechocystis sp. PCC6803, a model cyanobacteria (12–14). This species exhibits T4P-dependent twitching motility on surfaces such Author contributions: D.N. and T.N. designed research; D.N. performed research; D.N. as soft-agar plates at speeds of a few micrometers per minute, and analyzed data; and D.N. and T.N. wrote the paper. the cell motility direction is regulated both positively and nega- The authors declare no conflict of interest. tively along the light axis (15). The phototactic behavior is medi- This article is a PNAS Direct Submission. ated by the photo-sensing proteins and its two component systems, Freely available online through the PNAS open access option. and, particularly at the blue-light region of the spectrum, the cell 1To whom correspondence should be addressed. Email: takayuki.nishizaka@gakushuin. exhibits negative phototaxis (16). A blue-light receptor, PixD, and ac.jp. response regulator, PixE, are related to the regulation of cyano- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. bacterial phototaxis (17). However, it remains a mystery how the 1073/pnas.1702395114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1702395114 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 ABC D Condenser Lateral Vercal Dark 0 0 Type IV pili 75 300 30 Cell Lateral 50 20 20 light Cell Glass 25 10 10

Extension 90 90 Stage 0 90 0 0 Count of cell 270 270 270 Vercal light Obj Aach BS E 180 180 180 DM

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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 μmmin 1 was counted. (E) Schematics of types of illumination in D. Thick arrows in the Insets and thin arrows represent the di- − − rection 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.

no effect on motility (Fig. 1 D and E, Right). When a blue light this setup (Fig. 1). However, when light was partially illuminated − − with 1,200 μmol m 2 s 1, which is half of direct sunlight only at the right side of the cell, beads were accumulated only in − − (∼2,000 μmol m 2 s 1), was shone on cells from the side, most the illuminated region (Fig. 2C). These observations could be cells escaped from the direction of the light propagation on the explained by the microoptics effect of a cell, as previously reported glass coated with collodion (Fig. 1C and Movie S1). The effect of (18) (Fig. S3)(seeDiscussion). the collodion coating is shown in Fig. S1; a 0.007% solution of In the above three different illumination setups, the displace- collodion was used because cells clearly showed negative photo- ment of beads induced by T4P was accurately tracked at the taxis under this condition. The rose plot, a round histogram that single-pilus level (Fig. 2D). Most beads were retracted toward intuitively and simultaneously presents the number of occurrences cells, but we also observed the movement of beads away from the and direction, clearly indicates that the lateral light induced neg- cell body (red lines in Fig. 2 D and E). This particular movement ative phototaxis (Fig. 1 D and E, Left), whereas cells move ran- was attributable to the elongation of a single pilus, and its speed domly under the vertical illumination (Fig. 1 D and E, Middle). was about one third compared with the retraction speed (Fig. 2F). These observations indicated that our experimental system suc- The retraction and extension speeds were not substantially altered cessfully quantified the negative phototaxis of this species at the in the three different illumination setups, suggesting that the il- single-cell level. Note that in the absence of blue-light exposure lumination condition of light in each setup was well regulated in (i.e., in the dark), cells did not show directional movement on the our observation system, although the direction of light propaga- glass (Fig. 1 D and E, Right), suggesting that Synechocystis at least tion was different, and so the asymmetric dynamics of T4P observed here may be a natural characteristic of this species. As requires light exposure as a trigger to activate migration. predicted from the results shown in Fig. 2 A and B, the angle distribution of the beads’ movement was broadly distributed under Direct Visualization of T4P Dynamics Through Fluorescent Beads. To the vertical illumination (Fig. 2G, Left), whereas it was asymmetric reveal the dynamics of T4P quantitatively, we fixed cells on the under the lateral illumination (Fig. 2G, Middle). glass substrate. The motility of cells was hindered on the glass surface coating with high concentration of collodion, and when Visualization of T4P Filaments as a Fluorescent Image. Although the the concentration of collodion reached 0.2% the cells ceased to accumulation of beads was quantified at the single-cell level (Fig. move entirely and remained immobilized on the surface (Fig. S1). 2), the essence of the dynamics of T4P was still unresolved: i.e., we Notably, in the presence of polystyrene beads in the aqueous detected the extension and retraction only after the beads at- solution, the immobilized cells were able to retract the beads tached to T4P with the above experimental setup. To gain further from the solution to the cell surface (Fig. 2A and Movies S2 and insight into the regulation mechanism, we attempted to visualize S3). This observation suggests that the cells nonspecifically attached pilus filaments with a fluorescent marker. We found that con- on the glass surface and thus were immobilized, but the ability of ventional labeling with fluorescent chemicals, such as FITC suc- pili to extend, catch targets, and retract them remained intact. As a cinimidyl ester (2), did not work in Synechocystis.Instead,pilus result, beads were accumulated on cells. However, cells treated by filaments were sufficiently visualized with labeling by avidin con- the chemical fixation with glutaraldehyde did not show such accu- jugated with FITC (Fig. S4A). Several other types of biotin- mulation (Fig. S2). Beads stabilized by sulfate charges were effec- binding proteins, such as streptavidin or neutravidin conjugated tively captured by pili, but neither carboxylated nor BSA-coated with FITC, were not applicable (Fig. S4B), indicating that the beads were accumulated (Fig. S2), suggesting the specificity of the avidin-specific labeling of pili is caused by the glycosylated region tip of a single T4P filament. The beads accumulated on the cell of avidin (23). It is already known that the cell has two morpho- surface were not released during our observation time, and thus the logically distinct pilus types, named thick and thin pili (13). To activity of T4P was directly visualized as the distribution of fluo- check which pilus filament is labeled with FITC–avidin, we applied rescence intensity of beads accumulated on the surface (Fig. 2A). this method to the GT strain, which is known to have nonmotile In Fig. 2A, light was illuminated vertically onto the sample phenotype (24). We found that the cells have low fluorescent plane. In contrast, when light was laterally illuminated, the distri- signal as a filament (Fig. S5A), and the intensity was much less bution of beads became not symmetric but asymmetric: beads were than that in the PCC-P strain, a standard strain in this paper (Fig. accumulated to the forward side of the cell along the lateral blue S4 A and B). It is consistent with the EM observation: the PCC-P light (Fig. 2B and Movies S4 and S5). The asymmetric accumu- strain had thick pili as long filaments with a diameter of 8 nm and lation suggests that T4P filaments located around the right side of more than 2 μminlength(Fig. S4 C and D), corresponding with the cell can drive rightward motion, as in Fig. 1C, because lateral the morphotype of T4P, as previously described (13), but the illumination induces negative phototaxis toward the right side in number of thick pili in the GT strain was much less (Fig. S5B).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1702395114 Nakane and Nishizaka Downloaded by guest on September 30, 2021 Vercal Lateral Paral D 3 3 3 A (μm) 0 0 0 y posion Adding beads -3 -3 -3 Vercal 1.5 -3 0 3 -3 0 3 -3 0 3 x posion (μm) 1.0 E 4 4 4 0.5 (μm) 0.0 2 2 2 Relave intensity Relave -2 -1 0 1 2 Posion (μm)

Distance Distance 0 0 0 B 1.5 Lateral F 0246810 0246810Time (2-s grid) 0246810 1.0 40 40 40 −0.81㼼0.14 −0.73㼼0.21 −0.86㼼0.12 0.5 0.13 0.39 0.14 20 0.24㼼 20 㼼 20 0.33㼼0.13 0.0 Relave intensity Relave

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-2 -1 0 1 2 Counts Paral light ←Light→ Posion (μm) 0 0 Cell center MICROBIOLOGY 180 Moving trace 0 09018027090 180 270 63 0 0 09018027090 180 270 63 0 of beads Angle (θ) Angle (θ)

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 μminA and B, also applies to C.) (D)Trackingofbeads:(Left, Middle,andRight) 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.Beadswere 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.

Based on these observations, we conclude that we successfully Bottom; see also Figs. S6 and S7). These observations suggest that established a method to visualize T4P of Synechocystis under an PixD is responsible for the asymmetric distribution of T4P via an optical microscope. unknown mechanism, which suppresses the extension of T4P from Here we applied the labeling procedure in Fig. S4A and directly the light-illuminated side. quantified the effect of the lateral illumination on the extension of Finally, we reproduced the experiment in Fig. 2B under local- T4P (Fig. 3). As expected from the behavior of the accumulation ized illumination without beads and visualized the dynamics of of beads, T4P filaments dominantly appeared from the side of the T4P at the single-cell level. In the wild type, T4P were extended cell opposite the illumination (Fig. 3A). This observation was only in the illuminated region (Fig. 4A). In contrast, T4P appeared consistent with the idea that the probability of the tip of a pilus uniformly when the cell body of the ΔpixD mutant was partially binding to the target, in the case of a fluorescent bead in Fig. 2, is subjected to light (Fig. 4B). Quantification measurements clearly presumably stochastic, and negative phototaxis is mainly driven by revealed the above tendency (Fig. 4C), indicating that a partial the regulated asymmetric distribution of T4P filaments over the illumination was sufficient to trigger the extension of T4P from all cell surface (Fig. 3B). regions (Fig. 4D). The role of PixD is presumably to suppress the The next question raised here is how the asymmetric distribu- extension in unilluminated areas, which is consistent with the tion of T4P is regulated by the blue-light illumination. To evaluate scenario deduced from the results in Fig. 3. Such asymmetric the effect of the light, we applied a mutant that lacks PixD, a blue- distribution of T4P may be associated with the pilus assembly light–sensing protein (15, 17). Unexpectedly, the T4P was gener- motor, PilB1, because the region of T4P production has a similar ated in the ΔpixD mutant (Fig. 3C). The number of T4P increased dimension to the “crescent” localization of PilB1 (25). with the increase in the power of light in both the wild type and ΔpixD mutant in a Michaelis–Menten manner (Fig. 3D, Top). The Three Phases in the Realization of Phototaxis. The results shown in length of pili was similar in both samples (Fig. 3D, Middle), within Figs. 2, 3, and 4 suggest the possibility that the role of PixD is not a range of 2–4 μm on average, indicating that PixD is not required to activate but rather to suppress T4P extension to achieve neg- to induce the extension of T4P; i.e., there should be other pho- ative phototaxis. If this were the case, this suppression process toreceptors. Notably, the distribution of T4P in the ΔpixD mutant could be quantitatively detected via the twitching motility of the was more uniform than that in the wild type (Figs. 3C and 3D, cell body triggered by blue-light exposure. To test this hypothesis,

Nakane and Nishizaka PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 WT ∆pixD 0 A BD8 8

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0 0 Pilus number/cell Immobilized cell 6 10 100 1000 10000 6 180 (μm) 0 0 pixD C 24 WT 24 ∆ 3 3 xy-plane 16 0 16 Pilus length Base point Pilus length 0 0 8 8 1.0-2.5 (μm) 10 100 1000 10000 90 90 θ 90 2.5-4.0 0 0 100 100 Count of pilus of Count 270 270 270 4.0-5.5 5.5-7.0 (%) 50 50 Cell center 7.0- 180 Single pilus Rao 0 0 180 180 10 100 1000 10000 10 100 1000 10000 Photon ﬂux density (μmol m−2 s−1)

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 (see 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 ΔpixD mutant (Right, diamonds). The average and SD from 20 cells were plotted. Dashed lines represent the fitting of Michaelis–Menten kinetics.

we precisely analyzed both the directed motion of the wild type which is the direction of light propagation. In the wild type, the and the relatively random motion of the ΔpixD mutant under directed movement was barely observed for the first 1 min in most lateral illumination of blue light (Figs. 5 and S8 and Movies S1 and cells (Fig. 5B, Left). This tendency was clearly visible in the average S6). The traces clearly showed the negative phototaxis in the wild of traces (Fig. 5C, Left) because the net displacement was almost type, whereas the ΔpixD mutant wiggled (Fig. 5A, Left and Mid- zero for the first 1 min. dle). No substantial motility appeared without exposure (Fig. 5A, To address the question of how the cell movement transitions to Right). We plotted displacements of 50 samples along the x axis, the directed movement, we plotted the mean square displacement

WT ∆pixD AC0 Paral D blue light 8 8 0 WT 24

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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 (see 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)multiplecells.(Scalebar,5μm.) (B) Three sets of images similar to the Top in A of the ΔpixD mutant.(Scalebar,2μm.) (C) Rose plots of the distribution of T4P in the wild type (Top)andΔpixD 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 ΔpixD 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.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1702395114 Nakane and Nishizaka Downloaded by guest on September 30, 2021 pixD NO NO ADWT ∆ WT pixD (Dark) WT ∆ ) 2 (μm > (80-μm grid) 2 x < posion 0 1,000 2,000 3,000 y 05 01 0501 Time (min) Time (min) x posion (80-μm grid) E B ON ON 80 ) 2 03060 03060

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posion -40 x 0 200 400 -101234-101234 -80 Time (min) Time (min) 05 01 0 5 01 0501 Time (min) Time (min) Time (min) 1st phase 2nd phase 3rd phase C ON ON 80 F T4P dynamics Acvaon Acvaon Suppression 40 (μm)

> 0 x < Direconal -40 Diﬀusive

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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) MICROBIOLOGY 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.InC–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.

(MSD) of all cells (Fig. 5 D, Left,andE, Left). Remarkably, the Discussion average of MSD linearly increased with time from 0.2 to 1.0 min We demonstrated that the mechanism of taxis in Synechocystis is after the light exposure. This finding was more notable in the substantially different from the mechanism of motility in well- magnified view (Fig. 5E, Inset), proving that the twitching motility studied bacteria such as Salmonella (19, 20). Flagellated bacteria was activated randomly with an apparent diffusion coefficient of induce directional net displacement by a change in the frequency μ 2 1.2 m /s, which is good agreement with the scaling behavior of of the transition between tumbling and swimming (21). This the motility in other bacteria (26, 27). The start point of the strategy is reasonable, considering that the direction of the parabolic line indicates the phase transition to directed twitching swimming in bacteria is fixed to the long axis of an ellipsoidal cell motility as a result of the negative phototaxis. Δ body; i.e., cells can only move on the axis along which flagella In the pixD mutant, the behavior of cells was different from protrude. In contrast, T4P can extend from all directions of the that in the wild type with respect to directed or random motion. body, and thus cells can move in any direction. Therefore, the The raw traces were fluctuating, and the net displacement was taxis in Synechocystis is achieved by an asymmetric distribution of zero (Fig. 5B, Middle). The linearity of MSD ensured that the T4P in the cell body, although cells are fundamentally sphere- twitching motility of the mutant was also activated, and the slope shaped, symmetric in rotation, and not polarized. coincided with that of the wild type (Fig. 5 D, Right,andE, Right). We visualized that the cell has the asymmetric distribution of However, no parabolic region appeared for the ΔpixD mutant, and MSD linearly increased for 10 min. T4P to move away from directional blue light (Fig. 3). Our data Our interpretation of the results in Fig. 5 is that the negative also suggest that the above polarization is triggered by the local phototaxis accompanies transitions between three phases (Fig. 5F). difference of blue-light intensity, and the T4P extension was First, cells are exposed to light, but a delay is needed before activated at the region where the intensity is higher (Fig. 4). These observations appear to have conflicting results because starting the wiggling motion within 0.2 min; this is the first phase. “ ” The delay is comparable to the time required for the extension of blue light would be expected to work as a repellent. However, T4P filaments, a speed of 0.3 μm/s to be 3 μm in length (Figs. 2 and this could be explained by the microoptics effect of the cell, as 3). In the second phase, pili uniformly appear from all directions of previously reported (18). When a single cell is exposed to colli- the cell surface, and so random twitching motility is activated both mated light, the cell condenses the light into the opposite side of inthewildtypeandΔpixD mutant. The random movement in the the light source because the cell body works as an optics with a wild type is terminated by PixD within 1 min after the exposure, by diffraction index higher than water. This model was proposed to suppression of the extension of T4P filaments only at the unil- explain positive phototaxis (18), and we extended the model to luminated region of the cell surface (Figs. 2, 3, and 4). Finally, in negative phototaxis based on the result of direct visualization of the third phase, Synechocystis drives the motion only by un- T4P and its dynamics. The fact that cell bodies function as an inhibited pili, which directs the cell body away from the light il- optics was directly confirmed in Chlamydomonas reinhardtii (28). lumination. These transitions between phases are the essence of The strategy seems reasonable because a microorganism with a negative phototaxis in this species, and other bacteria having size on the order of the visible wavelength cannot produce any photoreceptors might share a common mechanism. structure to shield itself from light.

Nakane and Nishizaka PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 Here we elucidated the following sequence of events from and an optical table (RS-2000; Newport). The position of the cell was visualized by green light from a halogen lamp with a band-pass filter (FF01-531/40; stimulus to taxis in detail. First, blue-light stimulation induces − − the phototactic response as the uniform extension of T4P fila- Semrock) at a fluence rate of 1 μmol m 2 s 1, as needed. Projection of the ments. The localization of pilus assembly motor PilB1 may not image to the camera was made at 330 nm, 100 nm, and 67 nm per pixel. be completed at the specific part of the cell in this stage (25). Sequential images of cells were captured as 16-bit images with a CMOS camera Second, after 1-min delay, T4P dynamics is regulated to be under 1-s or 0.1-s resolution and converted into a sequential TIF file without asymmetric along the light axis through the photoreceptor PixD, any compression. All data were analyzed by ImageJ 1.48v (rsb.info.nih.gov/ij/) which mediates a suppression of the T4P dynamics in the region and its plugins, particle tracker and multitracker. where the intensity is relatively low (Fig. 5F and Fig. S9). Because PixD is known to form a complex with the response regulator, Blue-Light Illumination. Lateral and vertical lights with a wavelength of PixE, and the complex is disassembled by the illumination of blue 488 nm from a blue laser (OBIS 488 LS; Coherent) were separately irradiated onto the specimen on the sample stage of a microscope (Fig. S10A) (see SI light in vivo (17, 29, 30), it is conceivable that the 1-min delay Materials and Methods for details). observed in Fig. 5 originates from the above reaction, whereas the role of PixE to the suppression is not yet clear. Finally, the cell Phototaxis on Glass. All procedures were done at room temperature (RT). The produced directional cell motility as resultant negative phototaxis. cell culture was poured into a tunnel assembled by taping a coverslip (36). The Our experimental setup enables us to evaluate the regulation of coverslip was coated with 0.007% (vol/vol) collodion in isoamyl acetate T4P filament under an optical microscope on a timescale of minutes and air-dried before use. After incubation for 10 min in moderate light and thus could be an ideal tool to quantify the detail of phototactic (10 μmol m−2 s−1 photons), the sample chamber was set on the microscope stage. responses in Synechocystis (14, 15, 18, 31). We also visualized the The position of the cell was visualized by green light from a halogen lamp with a − − retraction of T4P by tracking a fluorescent bead that attached to the band-pass filter (FF01-531/40; Semrock) at a fluence rate of 1 μmol m 2 s 1,and edge of a single pilus. This assay could contribute to quantification the image was recorded at 1-s intervals. The cells were subjected to lateral or of the dynamic properties of T4P, which may be common in other vertical illumination by the blue laser (see SI Materials and Methods for details). bacteria such as Neisseria gonorrheae (6, 32) or Pseudomonas aeruginosa (33). The combination of our experimental system Beads’ Assay and Fluorescent Labeling of Pili. Fluorescent polystyrene beads of and mutants that lack other receptors or key proteins in various size 0.2 μm (Sulfate: F8848; Thermo Fisher) were 100 times diluted to be species will reveal more detailed mechanisms in the near future. 0.02% (wt/vol) in BG11 and used for beads’ assay. Avidin-FITC (Sigma) at the concentration of 0.035 mg/mL in BG-11 containing 2% BSA were used for Materials and Methods labeling (see SI Materials and Methods for details). Strains and Culture Conditions. The motile strains (PCC-P) of Synechocystis sp. PCC6803 and ΔpixD mutant (34, 35) were grown in BG-11 medium in ACKNOWLEDGMENTS. The authors thank S. Kojima for discussions that moderate light (10 μmol m−2 s−1 photons) at 30 degrees with shaking to an were critical in preparing the manuscript; and S. Masuda for supplying the Δ optical density of around 1.0 at 750 nm. wild type and pixD mutant of Synechocystis sp. PCC6803. This study was supported by a Grant-in-Aid for Scientific Research on Innovative Areas [“Fluctuation & Structure” of JP16H00808 (to T.N.), “Cilia & Centrosomes” Optical Microscopy and Data Analyses. Cells were visualized under an inverted of JP87003306 (to T.N.), and “Motility Machinery” of JP15H01329 (to D.N.)]; fluorescence microscope (IX83; Olympus) equipped with 20×,60×,and100× Grant JP24117002 (to T.N.) from the Ministry of Education, Culture, Sports, objective lenses (UPLAPO, 0.8 N.A., UPLAPO, 1.4 N.A., and UPLSAPO, 1.4 N.A.; Science, and Technology of Japan; and by Japan Society for the Promotion of Olympus), a filter set (FITC-5050A; Semrock), a CMOS camera (Zyla 4.2; Andor), Science KAKENHI [Grants JP16H06230 (to D.N.) and JP15H04364 (to T.N.)].

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