A unique self-organization of bacterial sub-communities creates iridescence in Cellulophaga lytica colony biofilms

Abstract

Iridescent color appearances are widespread in nature. They arise from the interaction of light with micron- and submicron-sized physical structures spatially arranged with periodic geometry and are usually associated with bright angle-dependent hues. Iridescence has been reported for many animals and marine organisms. However, iridescence has not been well studied in bacteria. Recently, we reported a brilliant "pointillistic" iridescence in colony biofilms of marine Flavobacteria that exhibit gliding motility. The mechanism of their iridescence is unknown. Here, using a multi-disciplinary approach, we show that the cause of iridescence is a unique periodicity of the cell population in the colony biofilm. Cells are arranged together to form hexagonal photonic crystals. Our model highlights a novel pattern of self-organization in a bacterial biofilm. "Pointillistic" bacterial iridescence can be considered a new light-dependent phenomenon for the field of microbiology.

Figure 1. Observations under direct epi-illumination of Cellulophaga lytica CECT 8139’s colonies grown on several agar media (see also Supplementary Movies S1-S5).

C. lytica was aerobically grown for 24 h at 25 °C on (a) Marine Agar (MA), (b) MA supplemented with black ink (Paper Mate^© 1% v/v), (c) Cytophaga agar (CYT) or (d) Low Nutrient (LN) medium (See Methods for media compositions). In (1,2), macroscopic pictures were taken under oblique epi-illumination with a light angle of 67.5°. (1), standard isolation procedure. (2), streaking procedure using a thin 5 cm-linear streak for inoculation. In (3), optical digital microscopy (Keyence©, VHX-1000E) was used and pictures were taken at ×50 (a), ×100 (b,c) or ×200 (d) magnifications. To avoid specular reflections, the camera was oriented at a 60° angle from the Petri dish. In macroscopic pictures (1,2), the green iridescence appearance is dominant but blue and other colors (such as red and violet at the colony edges) are also observed. In (b), a little ink (1% v/v) was added to the culture medium in order to limit reflections of incident light into the agar at the time of observation. Gliding motility can be identified as the spreading zone from the colony center (for instance, see 2(a–c)). Bacterial agarolysis corresponds to the dark halo visible on colony edges. In the particular LN condition, C.lytica forms transparent colonies which appear green iridescent when moving the Petri dish and/or changing the illumination-observation angles (see also Supplementary Movie S3). In microscopic pictures (Keyence© microscopy), the iridescent “speckles” are well visible. Green speckles are dominant but yellow, orange, red, and violet “pointillistic” iridescences are easily observed at the colony edges.

Figure 2. Color variations at the edges of an iridescent C. lytica CECT 8139’s colony grown on marine agar (MA).

(a) Optical digital microscopy images (× 200) were taken at high (1), intermediate (2) and low (3) light incidence angles. (b) Processed image showing the iridescent pixels (superimposed in white to the original image) varying in color or luminance between images (1) to (2) and/or (1) to (3). (c) Separate processing of the upper and lower zones showing the iridescent pixels varying in color or luminance at the three incidence angles. In that case, iridescent pixels that appear or disappear in at least one image were not recorded. (d) Color profiles of the iridescent pixels (from (c)). Statistics were obtained by computing circular histograms on the Hue channel. Luminances were normalized from 0 to 1 (with 0.5 and 1 diameter values being shown on the circular histograms). As illustrated by the chromatic wheel, each color corresponds to an angle interval: red [0:30°], orange [30:45°], yellow [45:75°], green [75:120°], cyan [~180°], blue and magenta [~240–300°]. Percentages of each color were calculated to give a simplified view of the color profiles. (e) Distribution analysis of pointillist iridescent regions in the upper zone. The areas of iridescent pixels were computed within a regular grid. The resulting frequency plot is shown. (f) Statistic determination of “speckle” sizes within a C. lytica’s colony. Optical digital microscopy images were taken at × 100 (top image) and × 200 (image below) magnifications. In this example (a CYT-grown colony), a zone with several speckle colors was analyzed.

Figure 3. Spectrophotometry of iridescent and non-iridescent C. lytica colonies and TEM and Delaunay triangulations of the internal structures.

(a) Appearances of the colonies (streaking method on agar plate). (b) For spectrophotometry of assays, samples were illuminated at a fixed light angle of −70°. Scattered wavelengths from 300 nm to 850 nm were recorded at different detection angles from −65° to 70° with 2° angle step resolution. Results are shown using a color intensity scale for each scattered wavelength. Wavelength values for each color were as follows: UV, <400 nm; violet, 400 to 435 nm; blue, 435 to 490 nm; cyan, 490 to 520 nm; green, 520 to 560 nm; yellow, 560 to 590 nm; orange, 590 to 620 nm; red, 620 to 700 nm; and infrared, >700 nm. (c) Transmission Electron Microscopy (TEM) images of colony cross-sections obtained by using an adapted protocol (see Methods). (d) Extraction of periodic structures from TEM images with Delaunay triangulations. (e) Each histogram represents the frequency plot of the mean distance from the six nearest neighbours for all the cells in the image. Values are the associated mean distances from the six nearest neighbours and standard deviations.

Figure 4. TEM cross-section images of C. lytica CECT 8139 colonies under different iridescent conditions.

(a–e) C. lytica was aerobically grown for 24 h at 25 °C on Cytophaga agar (CYT). (f) C. lytica was aerobically grown for 24 h at 25 °C on Marine Agar (MA).

Figure 5. Comparison of experimental and modeled light scattering data and reflectance spectra.

(a) Schematic diagram of the model geometry. Two sets of scattering planes have been highlighted. (b) Modeled scattering pattern for a hexagonal-packed array with a periodicity of 320 nm. Data are shown (as in (c,d) using a color intensity scale for each scattered wavelength. (c) Experimental light scattering data. The sample was illuminated at an incident angle of −30°. (d) Modeled scattering pattern which comprises a weighted average of scattering patterns from many models with a range of geometric orientations. (e,f) Experimental and modeled reflectance spectra, calculated by summing optical scatter in the reflectance hemisphere. (e) Experimental (black line) and modeled (red line) reflectance data, calculated by summing the optical scatter plotted in (a) (experimental) and (d) (modeled). (f) Experimental reflectance data (as in (e)) overlaid with modeled reflectance, calculated using a multi-periodicity approach and a weighted average derived from the periodicity displayed in Fig. 3(d,e) and Supplementary Dataset S6.