Genetic manipulation of structural color in bacterial colonies

Abstract

Naturally occurring photonic structures are responsible for the bright and vivid coloration in a large variety of living organisms. Despite efforts to understand their biological functions, development, and complex optical response, little is known of the underlying genes involved in the development of these nanostructures in any domain of life. Here, we used Flavobacterium colonies as a model system to demonstrate that genes responsible for gliding motility, cell shape, the stringent response, and tRNA modification contribute to the optical appearance of the colony. By structural and optical analysis, we obtained a detailed correlation of how genetic modifications alter structural color in bacterial colonies. Understanding of genotype and phenotype relations in this system opens the way to genetic engineering of on-demand living optical materials, for use as paints and living sensors.

Keywords: Flavobacteria; disorder; genetics; self-organization; structural color.

Fig. 1.

Structural coloration of Flavobacterium IR1 WT and mutants. (A and B) Photographs of WT and three mutants, grown for 4 d, taken at two different angles to exhibit the colonies’ pronounced angle-dependent coloration. WT and M16 produce vivid coloration whereas M17 produces some coloration and M5 very little in comparison. (Scale bars in A and B, 1 cm.) (C–G) Mutants M22, M65, and M41 (left to right in all photographs) photographed from five different angles under the same illumination showing the variation in color after growth for 2 d. D is photographed from directly above, whereas the other observation angles are oblique. (Scale bars in C–G, 1 cm.)

Fig. 2.

Structural and optical investigations of IR1 strains. (A–D) SEM top view images of the four bacterium strains WT, M5, M16, and M17, at identical magnifications. (E–H) Scattered intensity for samples upon light illumination with an incident angle of $-{60}^{\circ }$. To visualize the specular reflections, the signals within the dashed lines are divided by 900, 200, 350, and 250, respectively, from Left to Right. In E, text annotations highlight the features caused by structural organization: confined intensity spots lying on a very weak line governed by the grating equation (stated in SI Appendix), as well as a more defined scattered line spanning the whole angular range and also crossing the diffraction spots.

Fig. 3.

Diagrams explaining the light scattering observed from colonies of IR1. (A) If the cells are arranged in a strictly periodic manner, they will reflect light only at certain angles, but with a very strong intensity as described by the grating equation (Materials and Methods). (B) The intense color selectivity is determined by the layered stacking of the bacteria, where interference, for example, reflects green light but transmits blue and red. (C) If this periodicity is disrupted, the grating effect is obscured, and less strong intensity of reflected light is observed, but over a broader angular range. (D) Top view of bacteria. Since the bacteria macroscopically do not have a preferred orientation with respect to in-plane rotation ($\phi$), the visual appearance is independent of rotation along this axis. For example, only the two encircled, aligned areas will contribute to the optical response in the observation plane perpendicular to their orientation.

Fig. 4.

Phenotyping and mapping of transposon insertions affecting motility and iridescence. (A) Comparison of iridescence of WT IR1 and mutants viewed under the microscope and cultured on ASWBC agar overnight at 22°C. M12–M23 have been color enhanced since they would otherwise appear black in comparison. (B) The rate of colony expansion of the WT and mutant strains quantified on ASWBC and ASWVLow plates. (C) Mapping of transposon insertions of mutants 12, 23, 147, and 149 within the sprC–F gene cluster of strain IR1, showing close homology with a similar operon from F. johnsoniae UW101. (D) Mapping of mutants 6 and 17 within the gldiA gene of IR1 and comparison with the region of the Flavobacterium F52 genome that contains homologous genes (black, a single copy in F52 and two in IR1). Genes marked in yellow are found in this region only for F52. Genes in red (eamA, a flotillin motif-containing gene, and nfeD) are found in this region only in IR1. Other colors indicate ORFs found in this region in common between the two bacteria, including a putative methionine tRNA ligase upstream and the cat chloramphenicol resistance gene downstream.

Fig. 5.

The effect of growth on algae and algal products directly and indirectly on IR1. (A) The effect of exposing IR1 to volatiles from the algae P. yezoensis and H. fusiforme. An ASWLow plate (assay plate) was inoculated in the center with a $10\mu \mathrm{l}$ aliquot containing ${10}^7$ cfu of WT IR1. This was placed facing a second plate (donor plate) containing salt agar without algae or with $10\mathrm{g}$ of the specified hydrated algae (either autoclaved or not). Plates were sealed with parafilm and incubated for 7 d under humidified conditions. (B) Example of fucoidan-triggered recovery of structural color. The entire 9-cm diameter agar plate was spread with IR1, which became intensely green across the whole plate after 24 h and then lost coloration completely after 7 d. Addition of $50\mathrm{mg}$ of powdered fucoidan to the center of the plate facilitated recovery of structural color after 1 d, as shown. (C) WT IR1 shown growing as a 6-mm diameter iridescent green colony on a $4\times 4$-${\mathrm{cm}}^2$ stack of P. yezoensis after $18\mathrm{h}$ at $20{}^{\circ }\mathrm{C}$. The algae were embedded in agarose with $1\%$ (wt/vol) KCl and nigrosin. (D) Culture of WT and IR1 mutants on 4-mm diameter stalks of H. fusiforme after $36\mathrm{h}$. (Scale bars in D, Left to Right, 1.5 mm.)