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

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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 Retracon Blue laser 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.
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