Color is in the Eye of the Bacteria-Beholder

by Janie
 

Figure 1. (A and B) Photographs of WT and three mutant colonies, taken at two different an­gles to demonstrate the an­gle-dependent co­loration. WT and M16 produce vivid coloration, M17 produces some coloration, and M5 pro­duces very little. (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 angle-depen­dent va­riation in color. D is photo­graphed from directly above, where­as the other ob­servation angles are oblique. (Scale bars in C–G, 1 cm.) Source

Pigmentation is a straight­forward color­ation: the color is the same from every view­ing angle. In contrast, struc­tural color­ation is in the eye of the beholder: the colors are de­pend­ent on the view­ing angle.
 

We prev­iously covered the knack of various bacteria for syn­thesiz­ing true blue pigments, which fre­quently serve de­fen­sive purposes. Flavobacteria turn out to have a penchant for struc­tural color­ation: they can assemble into 2D pho­ton­ic crystals, which are periodic assem­blages that reflect light only at certain angles. Flavobacterium strain Iridescent 1 (IR1) cells, isolated from a harbor in the Netherlands, ar­range into dense hex­agons that together inter­fere with light to appear a flashy green. Figure 1 confirms that they owe their vivid pizz­azz to struct­ural color­ation, since the appar­ent color of each colony changes with the view­ing angle!
 

This organiza­tion is no easy feat. Self-assembl­ing into these highly regular struc­tures consumes both genetic coding ca­pacity and meta­bolic re­sources – so why do it?
 

A study from 2018 by Johansen et al. discov­ered a link be­tween the pili-depen­dent gliding motility character­istic of Flavobacteria and struc­tural color­ation. IR1 mutants with defec­tive motility were unable to self-organize into periodic hexa­gonal structures and were conse­quently dull-colored. The authors then sifted out genes in­volved in struc­tural color­ation with a trans­poson mutagen­esis screen. The majority of these hits were involved in glid­ing motility, plus some with no prev­iously assigned func­tion.
 

Figure 2. Top panel: A Flavobacterium IR1 cells ar­range into 2D photonic crystals, which are periodic structures that reflect green light with strong intensity at cer­tain angles. B The cells in this case ap­pear green because they reflect green light but transmit blue and red. C If the or­derly periodicity is disrupted, the in­tensity of reflected green light is re­duced. Bottom panel: 1D, 2D, and 3D pho­­tonic crystals. Source (top), Source (bot­tom)

The presence of macro­algae also influences the bac­teria, which naturally degrade poly­mers from algae in their es­tu­arine environ­ments. In the 2018 study, the IR1 colonies adjusted their periodic struc­ture – and hence, their appar­ent color­ation – depend­ing on what algal polymer was avail­able. The presence of fucoidan, carra­geenan, or starch resulted in red, blue, and purple color­ation in add­ition to the normal green. The removal or add­ition of powdered fucoidan caused the loss or restor­ation, respec­tively, of struc­tural color­ation. Fucoidan again plays a curious role (we prev­iously covered other fucoidan-degrading bac­teria here).
 

A more recent study from 2020 by Hamidjaja et al. linked struc­tural color­ation to another trait of the IR1 Flavobacte­ria : pred­ation. Like their relative Flavobacterium johnson­iae, the IR1 cells can invade adjacent colonies and prey upon the cells in a contact-dependent manner. When the research­ers spotted IR1 and prey bacteria onto agar, IR1 sur­rounded and invaded the prey colonies, and the purple and red color­ation typical of growth on media contain­ing fucoidan shifted to a bright green. This indic­ated that IR1 was self-organizing into 2D pho­tonic crystals during pred­ation.
 

Figure 3. Inoculation of IR1 adjacent to B12 on agar supplemented with fu­coi­dan, showing the result 10 h after con­tact between the spreading colony of IR1 and the static mass of B12. IR1 surrounds the B12 colony (w) and creates breaches (x) in the thicker edge of the B12 colony. There is a shift in IR1 from dull pur­ple/ red structural coloration to green (y). Source

As expected, mutants that could not glide were unable to penetrate prey colonies. More surpris­ingly, mutants that could glide but were unable to produce struc­tural color­ation were also unable to penetrate prey colonies. So, the ability of the cells to organize into photonic crystals is required for the ability to engage in pred­atory behav­ior. This 2020 study marks the first clear-cut link be­tween such struc­tural color­ation and biolog­ical rele­vance in bacteria.
 

IR1 also appears to prefer to glide along edges – like those of a glass slide provided by research­ers, or the edges of prey colonies – rather than a flat expanse of agar. Track­ing along edges in this way may be a strategy for quickly sur­rounding prey. IR1 isn't picky about its food; the range of prey includes both Gram-positive and Gram-negative bac­teria as well as the yeast Candida albicans, but its appetite also isn't indiscrim­inate. While IR1 approached and sur­rounded other flavo­bacterial colonies as quickly as it did prey colonies, it did not prey on its kin, indi­cating that it prefers to act (sur­round other colonies) before think­ing (discern between self and other).
 

Figure 4. Images of PIR4 (P, white) ap­parently moving towards and degrading a colony of IR1 (IR1 SC green) after 30 and 48 h (left and right, respectively). Scale bar indicates 5 mm. Source

What of the flip­ped situation, in which the hunter becomes the hunted? The authors isolated Rhodococcus spp PIR4, which preys on IR1. When spot­ted on agar, PIR4 invaded and degraded both wild-type IR1 and IR1 def­icient in struc­tural color­ation. Organiz­ation of IR1 into pho­tonic crys­tals offered no protec­tion against pred­ation – evidently, a good offense isn't always the best defense.
 

IR1 isn't the only case of struc­tural color­ation in bacteria. This trick­ery of light has also been observed in other Flavobacteria such as Cellulophaga lytica, isolated from anemone. Struc­tural color­ation in all of these bac­teria is an intrin­sically multi­cellular behav­ior, as it is only pos­sible at the colony level and not in indi­vidual cells. Evi­dently color­ation has snuck into the multi­cellular pred­ator's tool­kit, whether in eukary­otes like tigers and vipers or in bac­teria.