Culture Conditions Influence Iridescence in Marine Microbes

Author: J Livingston
Mentor: Dr. George O’Toole, Dr. Dianne Newman,
Co-Mentor: Lynn Kee
Editor: Grace Xiong

Introduction

Microbiology is the study of microorganisms: how they grow, where they are found, and how they interact with each other and their environment.^1,2 The study of microbiology allows researchers to develop a better understanding of the basic processes of life and has led to breakthroughs in medicine, agriculture, and industry.^1,2 Some of the most important discoveries in the field of biology have been made by studying and isolating seemingly insignificant microbes. For example, the widely used technique of PCR (Polymerase Chain Reaction) was developed by studying the extremophilic bacterium Thermus aquaticus,³ isolated from hot springs in Yellowstone National Park.  The discovery of CRISPR-Cas9, a revolutionary gene-editing mechanism, was made while working with bacteria in industrial yogurt making.⁴.

Iridescence is a widespread phenomenon in the natural world, appearing in animals ranging from fish to butterflies.^5,6,7 However, it is poorly characterized in single-cell organisms like bacteria, particularly because of historical misunderstandings of the term.⁸ Some older publications use “iridescence” interchangeably with “fluorescence,” although they are distinct phenomena.⁸ While fluorescence is the emission of light, iridescence is the interaction of reflected light waves that result in different observed colors based on the angle of view or illumination.^7,8 In the past decade, work has been done to characterize and define bacterial iridescence. Different categories of iridescence have been defined, including rainbow, metallic, and “glitter-like.”⁷

Bacterial iridescence, as described in marine bacteria of the family Flavobacteriaceae, results from photonic crystals formed by periodically ordered cell populations in colony biofilms.⁹ Bacterial iridescence has also been linked to gliding motility, which is a form of movement used by bacteria to move across a surface without a propulsive structure.^10,11 While previous studies described and characterized iridescence in members of the genera Flavobacteria and Cellulophaga,^{5,6,8,9,10,12} iridescence in the Tenacibaculum genus had not been described well. Recent work at the Marine Biological Laboratory (Woods Hole, MA) has begun to decipher both the physical and genetic basis of iridescence in Tenacibaculum.^{13,14,15,16,17}

The biological function of iridescence in bacteria remains unknown. Some hypothesize that the structure that gives rise to iridescence may give the bacteria a selective advantage in the dynamic marine environment in which they live. Iridescent bacteria are found in rocky shore environments that are characterized by significant fluctuations in temperature, dryness, and salinity, so these iridescent structures might help them survive in changing environmental conditions.  It is also possible that the iridescence itself is an unimportant characteristic associated with an important biological structure, only by coincidence appearing as iridescence when the bacteria is grown in the lab. To better understand the functions of biological iridescence, we isolated and classified several new strains of iridescent Tenacibaculum, and characterized their iridescence in different culture conditions. By defining the conditions that promote iridescence, we hope to uncover the biological significance of this phenomenon.

Results and Methods:

To begin this project, we isolated iridescent bacteria from seawater samples collected between the rocks in a jetty at Stony Beach in Woods Hole, MA. We serially diluted each sample and spread them onto seawater complete (SWC) agar plates. Each plate was then incubated overnight at 30 ˚C and grew many different bacterial colony morphologies.

Iridescent colonies were isolated from these plates by using plastic inoculation loop to pick up the colony of interest and drag it on a new plate of SWC. This was repeated until the microbial growth appeared the same, indicating that the isolates were genetically identical. The iridescent microbes resembled those used in an ongoing project, which have been identified as Tenacibaculum discolor. We named this first strain CA-IR1 (Figure 1). Following this isolation, more samples were taken from Stoney Beach and three more strains of iridescent microbes were isolated using the same procedure, named CA-IR2, CA-IR3, and CA-IR4. We determined that all the strains of iridescent microbes were rod-shaped and between 1-6 microns in length by viewing the cultures under a microscope at 100X magnification.

To identify the genus and species of each iridescent strain, we sequenced the 16S ribosomal RNA gene and compared the sequences of our isolated strains with those of known bacteria. 16S sequences were obtained using colony PCR, in which a colony of the strain was boiled to lyse the cells, then the resulting lysate was used as a template in a PCR reaction designed to amplify the 16S rRNA sequence, and the PCR product was sent for Sanger sequencing. We used sequence similarity to determine the taxonomic relationship between our newly isolated strains and known strains using the NCBI (National Center for Biotechnology Information) 16S ribosomal RNA database. Additionally, genomic DNA of CA-IR1 was extracted, sequenced, and annotated, and the 16S sequence was extracted from the full genome. CA-IR1, CA-IR3, and CA-IR4 were identified as most similar to Tenacibaculum mesophilum strain “NBRC 16307,” indicating that these strains are closely related, despite being isolated from different environmental samples. A definitive identity for CA-IR2 was not obtained. In addition to the four strains isolated, in this study four strains (NH, RLM, LK1, and EP2) were used from previous projects (Figure 2).

Following the isolation and identification of our iridescent strains, we used light microscopy to analyze the effect of several culture conditions on iridescence to better understand its biological significance. Images were taken daily of colonies grown in different culture conditions using light microscopy at 7.5X magnification, and visual iridescence was recorded subjectively, recording the apparent amount of color change and the level of brightness of iridescence for each colony. The goal of these experiments was to identify which conditions had a visible effect on iridescence, either promoting, inhibiting, or changing the color and type of iridescence. The culture conditions that we varied were pH, agar concentration, salinity, and nutrient availability.

1. pH

pH was observed to affect the growth rates of the colonies, with no growth occurring on SWC agar at pH 5 and slower growth occurring at pH 6 and 9 than at pH 7 and 8. Iridescence was observed across all conditions in which growth occurred, indicating that pH does not affect iridescence (data not shown).

1.  Agar Concentration

The concentration of agar in the medium was used to define the firmness of the growth substrate, with greater concentrations corresponding to greater firmness. Low agar concentrations inhibited iridescence (Figure 3), indicating that a solid surface is required for the strains to display iridescence. Growth was inhibited at higher agar concentrations, with colonies appearing thinner while displaying intense iridescence even earlier than the other conditions, possibly indicating that the lower cell density allowed the cells to form a regular pattern more easily. Distinct colony morphologies, with winding patterns and a rough texture, were observed at 0.5% and 0.7% agar concentrations compared to the control 1.5% agar concentration. In this condition, limited iridescence was observed, but was difficult to discern on clear agar, so the experiment was repeated with black ink added to the medium to add contrast. With the addition of black ink we observed iridescence after four days of growth in all agar concentrations except 0.25% and we continued to see the distinct colony morphologies at 0.5% and 0.7% agar. Different strains responded in different ways to the limited agar concentration conditions. The most striking response was by strain NH on 0.5% agar (Figure 4), which spread to a much larger colony size than on any other media type, overtaking the other colonies on the plate. Both NH and LK1 displayed this spreading morphology at 0.5% agar. This could indicate that LK1 and NH have more efficient gliding motility than the other strains, allowing them to move through the agar faster.

1. Salinity

We tested the effect of salinity on iridescence through two related experiments. First, we tested the effect of high salinity by growing spot colonies on black SWC with different amounts of added NaCl. Second, we tested the effect of low salinity by growing spot colonies on clear SWC made with a diluted seawater base (SWB). High salinity (≥ 40 g/L NaCl) appeared to inhibit growth, but did not appear to inhibit iridescence beyond the effects of the low growth levels. The color of the iridescent colonies was shifted from green to red at higher salinities than the control, but this may have also been due to the inhibited growth. Lower salinity appeared to inhibit iridescence, as iridescence was observed to be limited and dull on SWC made with 25% SWB. This may indicate that a threshold salinity is required for iridescence or growth. The lower salinity medium was less solid than that of the control, possibly contributing to the lower levels of iridescence. A minimum salinity is required for Tenacibaculum growth, as no growth was observed when strains were plated on media with no salts or with salt levels meant to replicate fresh water. What iridescence did appear on SWC made with 25% SWB appeared to be less green and more red and was much less “glitter-like”  than colonies on the control.

1. Nutrient Availability

We tested the effect of nutrient availability on iridescence by growing colonies on several different types of media (Figure 5). Strains were grown on SWC, a nutrient-limited form of SWC called SWC-GC (for growth curve), SWB without added carbon sources, marine agar (MA), black marine agar (BMA), and artificial seawater (ASW), supplemented with yeast (ASW-YE), tryptone, (ASW-T), and a combination of yeast, tryptone, and additional salts (ASW-CYT). We expected to see similar colony morphologies and levels of iridescence in all the complete media conditions (i.e. SWC, ASW-CYT, and MA) so we were surprised to see different colony morphologies, colors of iridescence, and even types of iridescence (“glitter-like” and dull)  in the different conditions. ASW-based media yielded colonies with a winding, wave-like morphology, similar to the morphology displayed by CA-IR1 and CA-IR2 in 0.5% agar concentration SWC. Interestingly, no glitter was observed in the first 4 days in any of the colonies on ASW-YE, even though an intense color change was observed. This suggests that tryptone may be responsible for the development of the “glitter-like” bright reflection typically seen in the iridescent colonies. This is supported by the fact that the ASWT condition had the most intense iridescence after 4 days, being so bright that the “glitter-like” reflection was visible when the lamp was pointed away. Colonies on MA appeared more red than those on the other types of media, and were smaller in size. Colonies on MA also had consistent, bright, “glitter-like” iridescence across the entire colony, without any dark spots or patterns of reflection. Colonies on ASW without added carbon sources took much longer to grow than other conditions, but developed after a week and color change was visible. Significant growth was not observed on SWB. Strains on SWC-GC appeared more green than on standard SWC in the first two days and developed iridescence as intense as standard SWC after a week.

No glitter was observed in the ASW-T condition after 24 hours or in the ASW-YE condition after four days, though a distinctive iridescent color change was observed in both. ASW-T had the strongest iridescence of any media after four days, to the point that color change and bright, “glitter-like” reflection was visible even when the lamp was not pointed at the colony. Different colony morphologies were observed depending on which salt mixture was used as a base. Colonies on marine agar were smaller than those in other conditions, had iridescence everywhere in the colony rather than having a dark spot in the middle, and were more red than green. Colonies on ASW-based media had an irregular, bumpy morphology similar to the morphology in .5% agar SWC. Strong iridescence was observed on SWC, SWC-GC, ASW, and ASW-T media. No significant growth was observed on SWB media, which was expected due to the lack of carbon sources.

Conclusions and Future Work:

In this work we investigated the effect of culture conditions on iridescence. The only culture conditions in which iridescence was inhibited were low salinity and low agar concentration, both of which had the effect of making the medium less solid. This suggests that iridescence in a colony biofilm requires that the biofilm grow on a more rigid surface. The iridescence observed on agar could be the result of the individual cell-cell interactions found in biofilms being maintained and repeated due to the unmoving solid surface. So what benefit do bacteria gain from growing in a highly ordered biofilm that appears iridescent? The structure might help the biofilm maintain its shape in the highly agitated waters of the marine environment where these bacterial live. While we know these bacteria form aggregates in liquid, they have not been observed displaying  iridescence in their natural environment, so it is possible that the iridescence does not appear in the wild.  Comparing the iridescent properties of biofilms grown in liquid to those grown on solid surfaces could provide insight into the relationship between the iridescence observed on agar and the behavior of these bacteria in their natural environment.

Our work supports prior findings of a relationship between gliding motility and iridescence, as we found that higher levels of iridescence were observed in broader colonies such as those that formed by NH on 0.5% agar. Future studies may include the difference between the strains with higher gliding motility and those with lower gliding motility, such as between the fast-spreading NH strain and the slower-spreading strain of the same species CA-IR1. A comparative genomic experiment could give us a better sense of the genes that regulate both gliding motility and iridescence in these strains.

The variation in color and level of iridescence among strains grown on different types of media is surprising and warrants further study, especially the lack of “glitter” in the ASW-YE condition. A broader experiment testing the effect of different carbon sources on iridescence should be performed to better understand this phenomenon. The different intensities and colors of the iridescence could be due to differences in the properties of the colony biofilms. Growing the bacteria on a wide range of media with different types or concentrations of carbon sources and taking TEM (Transmission Electron Microscopy) images of the resulting biofilms to assess the level of organization could give us a better idea of the effect of carbon sources and nutrient availability on iridescence. TEM allows for the imaging of the physical structure of the colonies on the level of individual cells, so the TEM experiments will provide a better idea of the physical underpinnings of the different forms of iridescence observed in Tenacibaculum biofilms.

We have shown that the appearance of iridescence is affected by changes in dryness, salinity, pH, temperature, and nutrient availability, so it is important to control for all of these things in future experiments to determine which change causes which effect. Ideally, this project should be repeated with the addition of time-point transcriptomics to determine whether changes in iridescence are caused by changes in gene expression. Iridescent colonies should also be analyzed using TEM, to determine what changes in the spatial configuration of the bacteria are causing the differences in iridescence between conditions. A strain-strain interaction condition should also be tested, as some strains have been observed to lose iridescence when mixed with another strain. Finally, a condition varying the growth surface will be essential to verify whether the iridescence in Tenacibaculum is plausibly able to occur in a marine environment, as to date it has only been observed on agar in a laboratory setting. Attempting to grow iridescent bacteria on fish carcasses, sea sponges, or seaweed will shed light on whether the iridescence we observe in laboratory conditions could reflect the strain’s behavior in the marine environment.

References

1. Madigan, Michael T., et al. Brock Biology of Microorganisms. 14th ed., Pearson, 2015.
2. Wainwright, M, and J Lederberg. “History of Microbiology.” Encyclopedia of microbiology 2 (1992): 419–437. Web.
3. Ishino, Sonoko, and Yoshizumi Ishino. “DNA Polymerases as Useful Reagents for Biotechnology – The History of Developmental Research in the Field.” Frontiers in Microbiology 5.AUG (2014): 1–8. Web.
4. Patrick, Hsu D., Lander S. Eric, and Feng Zhang. “Development and Applications of CRISPR-Cas9 for Genome Engineering Patrick.” Cell 157.6 (2014): 1262–1278. Web.
5. Kientz, Betty et al. “Glitter-Like Iridescence within the Bacteroidetes Especially Cellulophaga Spp.: Optical Properties and Correlation with Gliding Motility.” PLoS ONE 7.12 (2012): n. pag. Web.
6. Kientz, Betty, Pauline Marié, and Eric Rosenfeld. “Effect of Abiotic Factors on the Unique Glitter-like Iridescence of Cellulophaga Lytica.” FEMS Microbiology Letters 333.2 (2012): 101–108. Web.
7. Doucet, Stéphanie M., and Melissa G. Meadows. “Iridescence: A Functional Perspective.” Journal of the Royal Society Interface 6.SUPPL. 2 (2009): n. pag. Web.
8. Kientz, Betty et al. “Iridescence of a Marine Bacterium and Classification of Prokaryotic Structural Colors.” Applied and Environmental Microbiology 78.7 (2012): 2092–2099. Web.
9. Kientz, Betty et al. “A Unique Self-Organization of Bacterial Sub-Communities Creates Iridescence in Cellulophaga lytica Colony Biofilms.” Scientific Reports 6.July 2015 (2016): 1–11. Web.
10. Kientz, Betty et al. “Glitter-Like Iridescence within the Bacteroidetes Especially Cellulophaga spp.: Optical Properties and Correlation with Gliding Motility.” PLoS ONE 7.12 (2012): n. pag. Web.
11. Chapelais-Baron, Maylis et al. “Colony Analysis and Deep Learning Uncover 5-Hydroxyindole as an Inhibitor of Gliding Motility and Iridescence in Cellulophaga lytica.” Microbiology 164.3 (2018): 308–321. Web. 28 Sept. 2018.
12. Johansen, Villads Egede et al. “Genetic Manipulation of Structural Color in Bacterial Colonies.” Proceedings of the National Academy of Sciences 24 (2018): 201716214. Web.
13. Perry, E. “Pleomorphism and iridescence in Tenacibaculum geojense. Microb. Divers.” Course mini-project, (2017).
14. Mickol, R. “Making a Monster into a Mutant: Isolation and Analysis of a Tenacibaculum sp.” Microb. Divers. Course mini-project, 13 (2016).
15. Nguyen, C. “Shining the Light on the Structural Color of Tenacibaculum ectocooler.” Microb. Divers. Course mini-project, 15 (2017).
16. Herrera, N. “Characterization of the iridescence and motility mutants in Tenacibaculum sp. strain ECSM.” Microb. Divers. Course mini-project, (2017).
17. Kee, L. “Characterization of Gliding and Iridescent Mutant in marine Tenacibaculum discolor.” Microb. Divers. Course mini-project, (2016).

Further Reading:

For more information on the Microbial Diversity Program at MBL:

https://www.mbl.edu/education/courses/microbial-diversity/

For more information about the phenomenon of bacterial iridescence, a seminal paper that defined the terms used to describe prokaryotic structural color:

Kientz, Betty et al. “Iridescence of a Marine Bacterium and Classification of Prokaryotic Structural Colors.” Applied and Environmental Microbiology 78.7

For more information about the genetic basis of bacterial iridescence, a recent article uncovering the genes underlying structural color through a mutant analysis:

Johansen, Villads Egede et al. “Genetic Manipulation of Structural Color in Bacterial Colonies.” Proceedings of the National Academy of Sciences 24 (2018): 201716214.

For more information about the physical basis of bacterial iridescence, an article uncovering the structural components of biofilms that give rise to iridescence:

Kientz, Betty et al. “A Unique Self-Organization of Bacterial Sub-Communities Creates Iridescence in Cellulophaga lytica Colony Biofilms.” Scientific Reports 6.July 2015 (2016): 1–11.

Acknowledgements

I would like to thank my mentors, George O’Toole and Dianne Newman, for their help and instruction as well as for the opportunity, Lynn Kee, Callie Chappell, and Janet Sheung who all worked with me on bacterial iridescence, Rachel Whitaker, Scott Sanders, Gabriela Kovacikova, Kurt Hanselmann, Dominique Limoli, Sarah Guest, and all the rest of Microbial Diversity course faculty for their help, support and instruction, my fellow course assistants Rebecca Wipfler and Deaja Sanders for working with me and helping with my project, our course funding sources including the Simons Foundation, Promega, the Agouron Institute, the Gordon and Betty Moore Foundation, the Howard Hughes Medical Institute, NASA, the National Science Foundation, the U.S. Department of Energy, and Zeiss, and the MBL and the Caltech Student-Faculty Programs for providing me with this opportunity and with funding.