Decision letter: Cyanobacteria use micro-optics to sense light direction

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Bacterial phototaxis was first recognized over a century ago, but the method by which such small cells can sense the direction of illumination has remained puzzling. The unicellular cyanobacterium Synechocystis sp. PCC 6803 moves with Type IV pili and measures light intensity and color with a range of photoreceptors. Here, we show that individual Synechocystis cells do not respond to a spatiotemporal gradient in light intensity, but rather they directly and accurately sense the position of a light source. We show that directional light sensing is possible because Synechocystis cells act as spherical microlenses, allowing the cell to see a light source and move towards it. A high-resolution image of the light source is focused on the edge of the cell opposite to the source, triggering movement away from the focused spot. Spherical cyanobacteria are probably the world's smallest and oldest example of a camera eye. https://doi.org/10.7554/eLife.12620.001 eLife digest Cyanobacteria are blue-green bacteria that are abundant in the environment. Cyanobacteria in the oceans are among the world's most important oxygen producers and carbon dioxide consumers. Synechocystis is a spherical single-celled cyanobacterium that measures about three thousandths of a millimetre across. Because Synechocystis needs sunlight to produce energy, it is important for it to find places where the light is neither too weak nor too strong. Unlike some bacteria, Synechocystis can't swim, but it can crawl across surfaces. It uses this ability to move to places where the light conditions are better. It was already known that Synechocystis cells move towards a light source that is shone at them from one side, which implies that the cyanobacteria can "see" where the light is. But how can such a tiny cell accurately detect where light is coming from? Schuergers et al. tracked how Synechocystis moved in response to different light conditions, and found that the secret of "vision" in these cyanobacteria is that the cells act as tiny spherical lenses. When a light is shone at the cell, an image of the light source is focused at the opposite edge of the cell. Light-detecting molecules called photoreceptors respond to the focused image of the light source, and this provides the information needed to steer the cell towards the light. Although the details are different, and although a Synechocystis cell is in terms of volume about 500 billion times smaller than a human eyeball, vision in Synechocystis actually works by principles similar to vision in humans. Schuergers et al.'s findings open plenty of further questions, as other types of bacteria may also act as tiny lenses. More also remains to be learnt about how the cyanobacteria process visual information. https://doi.org/10.7554/eLife.12620.002 Introduction Many prokaryotes move directionally in response to a chemical or physical stimulus. However, it is generally assumed that bacteria are too small for direct sensing of a concentration gradient across the cell: instead they probe changes in stimulus concentration over time, as in the classic paradigm of flagella-mediated chemotaxis in Escherichia coli (reviewed by Wadhams and Armitage, 2004). When moving through a spatial concentration gradient of an attractant, E. coli cells experience temporal concentration changes, which they sense by employing a biochemical memory that directs a "biased random walk". Swimming along a straight path (run) alternates with random changes of direction (tumble). Tumbles become less frequent when cells sense a temporal increase in attractant concentration, introducing a bias to movement up a concentration gradient (Berg and Brown, 1972). For phototrophic prokaryotes, light is the main source of energy but also potentially harmful, depending on intensity and wavelength. Unsurprisingly, many phototrophs can alter their movement in response to the light environment (reviewed in Häder, 1987). Bacterial phototaxis was first noted in 1883 (Engelmann, 1883) and has been characterized in free-swimming phototrophs including purple bacteria and Halobacterium spp. (Hildebrand and Dencher, 1975; Alam and Oesterhelt, 1984). Cyanobacteria, which are oxygenic phototrophs, do not swim with flagella. Instead, various species exhibit "twitching" or "gliding" motility over moist surfaces (Pringsheim, 1968). This movement can be directed towards a light source, thus constituting true phototaxis (Choi et al., 1999; Bhaya, 2004; Yoshihara and Ikeuchi, 2004). The model unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) has spherical cells about 3 µm in diameter and moves using Type IV pili (T4P) (Bhaya et al., 2000; Yoshihara et al., 2001). The location of the T4P extension motor PilB1 implies that pili are extended at the leading edge of the cell, and therefore that movement is generated by pilus retraction (Schuergers et al., 2015; Wilde and Mullineaux, 2015), as has been shown in other bacteria (Merz et al., 2000). It has recently been established that the motility of a filamentous cyanobacterium is also T4P-dependent, suggesting that this form of motility is widespread in cyanobacteria (Khayatan et al., 2015). One likely exception is marine Synechococcus spp., which swims and exhibits chemotaxis without obvious surface appendages (Willey and Waterbury, 1989; Ehlers and Oster, 2012) apart from short spicules found in one of the motile Synechococcus strains (Samuel et al., 2001). Synechocystis T4P-dependent phototaxis can be observed microscopically at the single cell level and macroscopically through the migration of cell colonies. Genetic studies have identified a number of photoreceptors that influence phototactic behavior under different light regimes (Bhaya, 2004). While Synechocystis harbors signal transduction systems for pilus biogenesis that are homologous to the chemotaxis system in E. coli, it lacks the CheR methyltransferase and the CheB methylesterase that are required in most chemotactic bacteria to sense temporal changes in attractant concentration (Wuichet and Zhulin, 2010). This suggests a different mode of directional control. Previous studies of Synechocystis single cell phototaxis (Choi et al., 1999; Chau et al., 2015) have not addressed the question of how an individual cell might be able to perceive the direction of illumination. Here, we establish that individual Synechocystis cells can directly and accurately perceive the position of a unidirectional light source, and control their motility so as to move towards it. We then show that Synechocystis cells act as microlenses, and that the light intensity gradient across the cell due to this lensing effect is far greater than the effects of shading due to light absorption or reflection. Finally, we use highly-localized laser excitation to show that specific excitation of one side of the cell triggers movement away from the light, indicating that positive phototaxis results from movement away from an image of the light source focused on the opposite side of the cell. Essentially, the cell acts as a microscopic eyeball. Results Synechocystis phototaxis is based on direct light perception, rather than a biased random walk Individual Synechocystis cells moving in two dimensions on an agarose surface in response to different light regimes were tracked microscopically to determine whether single cells are capable of direct perception of the position of a light source. First, we tested the response of cells to a light intensity gradient projected onto the surface from the microscope condenser (Figure 1a), using a gradient of white light from 0–20 µmol photons m–2 s–1, an intensity range that is relevant for positive phototaxis (Choi et al., 1999; Bhaya, 2004; Chau et al., 2015). However, the cells moved randomly without any significant directional bias (Figure 1b; Video 1). If Synechocystis phototaxis were based on a biochemical memory like E. coli chemotaxis, cells would perceive temporal changes in light intensity as they move across the surface through the light gradient and would then accumulate in regions of optimal light intensity. This did not occur (Figure 1b, Video 1). By contrast, when cells were illuminated by a unidirectional light source (RGB illumination at 10 µmol photons m–2 s–1) at an angle oblique to the surface (Figure 1a), the majority of motile cells switched direction within about 1 min, and then moved directly towards the light source (Figure 1c,d,f; Video 1). Under illumination from two equal-intensity orthogonal light sources, the majority of cells moved towards a point midway between the two light sources (Figure 1e; Video 1). These behaviors are not consistent with a run-and-tumble mechanism or any kind of biased random walk. In accord with previous studies (Choi et al., 1999; Chau et al., 2015), we conclude that individual cells can directly and accurately perceive the position of a light source and control their motility accordingly. Figure 1 Download asset Open asset Movement of individual Synechocystis cells under different light regimes. Displacement over a 5 min time-frame was measured 1 min after the onset of illumination. The mean resultant length from a Rayleigh test (r) and the number of tracked cells (n) are shown. See also Video 1. All data were obtained with the same cells from a single continuous experiment. (a) Schematic diagram to illustrate the optical set-up for a light gradient projected onto the agar surface (as in b) and oblique directional illumination (as in c–e). (b) Cells moving in a white light gradient from 0–20 µmol photons m–2 s–1 over a 165 µm interval from the top to the bottom of the plot, showing no significant directional bias. (c) Illumination from an oblique RGB LED light source from the right, at intensity 10 µmol photons m–2 s–1. (d) Illumination from a similar light source placed orthogonally to that in (b). (e) Simultaneous illumination from both oblique light sources. (f) Correlation of cell movement with light direction, as a function of time after applying the light. The shading indicates the time window plotted in b–e. The y-axis shows the mean resultant length from a Rayleigh test (r) where 0 indicates random displacements and 1 indicates maximal clustering in the direction of illumination. LED, light emitting diode. https://doi.org/10.7554/eLife.12620.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Motility of Synechocystis cells under different illumination regimes. The video gives a schematic overview of the experimental set-up, followed by movement of cells in a projected light gradient, and with oblique illumination from two orthogonal directions, and then from both directions simultaneously. In each case, the raw video data is followed by the same movie clip with the tracks of cells superimposed. Time in minutes is indicated. https://doi.org/10.7554/eLife.12620.004 Shading is minimal in single Synechocystis cells Synechocystis cells contain a dense lamellar system of thylakoid membranes packed with photosynthetic complexes, and we initially postulated that light direction sensing depends on shading by the thylakoids, with the motility apparatus activated by brighter light at the illuminated side of the cell and deactivated at the shaded side of the cell. To assess the plausibility of this idea, we estimated the transmission spectrum of a single motile cell, scaling the absorption spectrum for a cell suspension according to the mean cellular pigment content. To ensure that pigment content was appropriate for phototactic cells, we used cells taken from moving colonies on motility plates (Figure 2). The transmission spectrum shows that even at peak absorption wavelengths, a single cell can absorb only about 20% of the photons that pass through it (Figure 2). Although local pigment concentrations within the cell are quite high (Figure 2), the very short optical path length means that light absorption by the cell is low. Note that our estimate assumes a homogeneous distribution of pigments within the thylakoid region of the cell: in reality, pigment clustering will tend to decrease the cell absorbance due to enhanced self-shading of pigments (Duysens, 1956). Thus, 20% represents a maximum estimate of the proportion of photons that can be absorbed, and the light intensity gradient across the cell due to shading must be almost negligible. The direct measurement of single cell absorption spectra is technically challenging, and we are aware of only one such measurement in the literature for a cyanobacterium. Sugiura and Itoh (2012) show that the peak absorbance for a single cell of Nostoc sp. is about 0.04. This corresponds to a peak absorption of about 10% of the photons that pass through the cell, which is even lower than our estimate for Synechocystis. Figure 2 Download asset Open asset Estimated transmission spectrum of a single motile Synechocystis cell. Estimate obtained by scaling and converting the absorption spectrum for a suspension of cells from a moving Synechocystis colony, as detailed in Materials and methods. Pigments (including chlorophyll: Chl and phycocyanin-coupled phycocyanobilin: PCB) are assumed to be evenly distributed within the thylakoid region as shown in the diagram, and the estimated transmission is for a narrow beam of light passing straight through the center of the cell, with an optical path length through the thylakoid region of 1 μm. The spectrum represents a minimum estimate for transmission through the cell, since the estimate assumes a homogeneous distribution of pigments within the thylakoid region. In reality, inhomogeneous distribution of membranes and pigments will tend to decrease absorption due to enhanced self-shading (Duysens, 1956). https://doi.org/10.7554/eLife.12620.005 Synechocystis cells act as microscopic spherical lenses Most light microscopy uses illumination orthogonal to the surface on which the sample rests, with the exception of dark-field microscopy, which uses oblique illumination from all sides. While investigating Synechocystis phototaxis, we observed cells instead with oblique illumination from one side only. Observations with this unusual illumination suggest a solution to the problem of directional light perception in Synechocystis. These images reveal that each cell acts as a microscopic spherical lens, focusing an intense light spot close to the opposite side of the cell from the light source and the direction of movement (Figure 3a). Images from two orthogonal light sources (as employed in one of the motility assays in Figure 1e and Video 1) are focused at different points on the cell periphery (Figure 3a), indicating that the cell can focus an image of its surroundings at the plasma membrane. Figure 3 with 1 supplement see all Download asset Open asset Micro-optic effects in cyanobacteria. (a) Synechocystis cells viewed with oblique illumination from the different directions shown, with enlarged image on the right. Scale-bars: 5 µm. (b) Images of periplasmic GFP fluorescence in Synechocystis torA-gfp (Spence et al., 2003). Left: two-channel confocal micrograph (GFP fluorescence in green; chlorophyll fluorescence in red) with GFP epifluorescence images of a single cell illuminated from the right, above and left (arrows indicate illumination direction; see Figure 3—figure supplement 1 for the optical set-up). (c) GFP fluorescence profile around the cell circumference, extracted from the epifluorescence image in (b) with illumination from the right. The profile was taken in an anti-clockwise direction starting at the point nearest the light source. (d) Schematic illustration of the measurement by photolithography of near-field optical effects of a Synechocystis cell. (e) Height profile reconstructed from an AFM image of a photolithograph from the experiment illustrated in (d). (f) Finite difference time domain model of the light path (wavelength 365 nm) through a Synechocystis cell (illumination direction indicated by the arrow). The color scale indicates relative light intensity obtained by time-averaging the amplitude of the Poynting vector for the electromagnetic field. The wave patterns represent a snapshot of the oscillating electromagnetic field propagating through the model cell. GFP, green fluorescent protein. https://doi.org/10.7554/eLife.12620.006 The images in Figure 3a do not give a quantitative picture of the lensing effect since the focused light spot is observed very indirectly via light reflected from the agar surface. Therefore, to quantify the lensing effect, we first employed a Synechocystis mutant that accumulates green fluorescent protein (GFP) evenly distributed in the periplasm (Spence et al., 2003). Thus, GFP can be used as local reporter of light intensity at the cell periphery (Figure 3b) since GFP fluorescence will be proportional to the local excitation light intensity. GFP fluorescence images with oblique laser excitation show a spot of light sharply focused at the opposite edge of the cell. Profiles of GFP fluorescence around the cell perimeter (Figure 3c) indicate that the mean ratio of the light intensity at the center of this focused spot to the intensity at the front of the cell facing the light source was 4.1 ± 1.5 (mean and standard deviation, n = 13 cells from one representative experiment) and the observed mean full width at half maximum (FWHM) of the focused spot was 609 ± 30 nm (mean and standard deviation, n = 11 cells). Since the measured FWHM for the point-spread function of the microscope was 270 nm, the true FWHM for the focused spot of 488 nm light at the cell periphery can be estimated to be about 550 nm. This experiment shows that for 488 nm light, lensing effects leading to concentration of light at the opposite side of the cell from the light source are overwhelmingly greater than shading effects, which would lead to higher light intensity on the illuminated side of the cell. Shading effects at other wavelengths in the range from 400 to 750 nm could be only marginally greater than at 488 nm (Figure 2). The spatial resolution of the fluorescence measurement in Figure 3b is limited by the optical point-spread function of the microscope. Therefore, for higher-resolution measurement of near-field light perturbation by Synechocystis cells, we used a photolithographic method that gives a very high resolution image of the light pattern adjacent to the cell, although it does not provide such a direct and quantitative measurement of relative light intensity as the fluorescence imaging in Figure 3b. Synechocystis cells were adsorbed onto the surface of a photopolymer and collimated UV light (365 nm) was projected vertically down onto them (Figure 3d). The UV light induces cross-linking of the photopolymer. After development, the surface relief of the photopolymer gives a high-resolution replica of the light field around and beneath each cell, which we examined by atomic force microscopy (Figure 3E). Synechocystis cells produced distinctive near-field optical scattering patterns on the surface with a and intense peak beneath the center of each cell. We measured an FWHM of ± nm (mean and standard deviation, n = from at different across the representative image (Figure This experiment indicates that light is focused to a spot with diameter less than the wavelength. Figure also that light is at the side of the cell opposite to the light source in a similar to light in the with light (Figure the lensing effect over any shading effects with light (Figure To probe the physical for the lensing effect, we light perturbation by the cell with difference time domain a method that uses the electromagnetic without any This electromagnetic all observed effects to the of with by their with the which in this was a model of the cell as a with The method a of effects on are as and reflection. We found that the observed near-field light pattern (Figure could be accurately by an the cell as a with a diameter of 3 µm and a of (Figure with similar dimensions to Synechocystis have been shown to produce similar sharply focused light at the edge of the opposite to the light these are et al., et al., Synechocystis positive phototaxis is a response to excitation of one side of the cell The effects shown in Figure 3 produce intensity across Synechocystis cells that are opposite in and at times greater than from shading due to light absorption by the photosynthetic pigments Figure with Figure 2). Figure provide direct experimental that light intensity is at the edge of the cell from the light source. This suggests that the for directional light perception by Synechocystis on the lensing of the cells, with positive phototaxis based on the cell moving away from the light spot focused at its We tested this by using a focused laser light spot et al., 2015) as an to one edge of the cell. Synechocystis cells moving directionally on an agarose surface towards a nm) light emitting light source were by fluorescence from the photosynthetic pigments by the LED light. of light at the periphery of the cell were observed under this illumination (Figure Video 2), showing that the focused light the thylakoid region. The moving cells were to a spot of nm laser light focused on the agarose surface (Figure Video 2). The intensity gradient at the edge of the laser spot was to ensure specific of one side of the cell to the light (Figure one edge of a cell the edge of the laser the cell direction to move away from the laser illumination (Figure Video 2). Cells did not the center of the intense laser but instead direction when the laser light intensity at the front edge of the cell the intensity of the light spot focused by the cell at its periphery by a of about as from the of fluorescence from the photosynthetic pigments (Figure In accord with our this shows that Synechocystis phototaxis is a response to excitation of one side of the cell. The data in Figure and Video 2 indicate that this response is with when the cells laser light that is than the focused light spot at the edge of the cell, they direction to move away from the laser light. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg of a laser spot on directional motility in Synechocystis. Cells are by fluorescence from the photosynthetic and are moving towards an oblique LED light at the bottom of the the focused light spot at the edge of each cell. The spot indicates the position of the and time in min is shown at the top LED, light emitting diode. Figure with 1 supplement see all Download asset Open asset by specific excitation of one edge of the cell. Synechocystis cells were moving in response to an oblique nm light source from the direction indicated by the and to a spot of nm laser light focused on the agarose surface. See Video 2 for the full data and Figure supplement 1 for the optical set-up and the intensity profile of the laser spot. (a) color image of fluorescence from the photosynthetic with the laser spot indicated by the The white indicate the cell and the white of the focused images of the light source at the edge of the cell. Cells the laser spot show excitation of the leading edge of the cell. (b) by with the edge of the laser spot. The indicate displacements of representative cells over time of and after to the laser spot. The mean of the tracks ± standard deviation, n = is shown. (c) intensity required to the path of Synechocystis of the of individual cells are shown over a 30 min time with a color scale indicating the ratio of laser intensity to the intensity of the phototactic LED light focused on the cell. color indicates tracks of cells in which by the laser by the light focused on the cell for phototaxis by at LED, light emitting Discussion light perception in Synechocystis depends on not shading we have shown that Synechocystis cells act as very spherical that focus a image of a light source at the opposite edge of the cell. This implies that positive phototaxis movement towards a light is actually as a response to the focused spot of light at periphery of the cell. We directly tested this by cells to a spot of laser light that was sharply focused to one edge of the cell (Figure Video 2). by our cells moved away from laser excitation that was only brighter than the focused image of the light source (Figure Video 2). This is the opposite of would be from a model for directional light perception, which would that light in this intensity range the control of motility in Synechocystis Synechocystis motility depends on the and retraction of T4P (Bhaya, which is by the motor and (Merz et al., 2000). We examined the of one of these motor in Synechocystis with (Schuergers et al., 2015). We were to a with but a in a was motile and capable of phototaxis, with lower than the (Schuergers et al., 2015). of in Synechocystis shows that it is in a of the plasma at one side of the cell, and that these can to other of the (Schuergers et al., 2015). For cells without directional random of the within a 5 min The direction of motility in Synechocystis with the position of the PilB1 indicating that direction is by the of probably in with other T4P such as (Schuergers et al., 2015). In both and show between the two cell on et al., signal transduction in Synechocystis It is that photoreceptors are in Synechocystis phototaxis, with light that absorb at different wavelengths and positive or phototaxis et al., 2004). In motility is by the et al., 2012) and and 1999; et al., However, the for a directional for positive phototaxis is known as in which the is show only phototaxis et al., 2000; et al., 2001). is a with two a domain and a domain with to the chemotaxis