Imagine you could grow a glowstick on the side of your head. The Hawaiian Bobtail Squid doesn’t have to! If you’ve been paying any attention to the world of microbiology in the past few years, it’s likely you’ve heard of the bacteria Vibrio fischeri or at least will recognize its characteristic blue-green glow.
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| Light organ of Hawaiian Bobtail Squid depicted at left and bioluminescent Vibrio fischeri at right. Source and source. |
Through this bacteria, we’ve been learning about common aspects of bacterial life such as quorum sensing and, most popularly, symbiotic relationships like that between V. fischeri and the Hawaiian Bobtail Squid. This squid is a nocturnal hunter that has a light organ around the outside of its head thats specific purpose is containing tens of millions of V. fischeri. These bacteria elect to gather within the light organ as it provides them a continuous source of sugars and amino acids that help to power their bioluminescence. In return for temporary food and shelter, the bacteria lend their glow to the squid, allowing it to hunt at night without casting a shadow in the moonlight and more easily sneak up on its prey. Then in the morning before it tucks itself into the sand to sleep for the day, it will squeeze out a majority of the V. fischeri leaving only a few to multiply and refill the light organ by nightfall.
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| The Hawaiian Bobtail Squid returning to the ocean floor after expelling its personal colony of V. fischeri until they are needed again at night fall. Source. |
For this symbiotic relationship to work effectively, V. fischeri must create a complex biofilm that can act and form in a variety of ways. A biofilm is created when many single bacteria aggregate in an area and secrete polysaccharides–long chains of sugar units–cementing them together. Though we aren’t sure exactly why it is necessary in this situation, there are many benefits to bacteria in a biofilm. Some of these advantages include increased ability to communicate efficiently or simply making sure they can stay in a suitable environment without being swept away by something like water flow. Both of these reasons would be advantageous for a marine-dwelling bacteria like V. fischeri that relies on quorum sensing for its ability to glow. Biofilms also can provide protection against antibiotics, making them a clear target of interest for medical purposes.
V. fischeri’s production of biofilm is necessary for the success of this symbiotic relationship and was the focus of a study done by Alice Tischler and colleagues. This pathway is known to be controlled by the syp (symbiosis polysaccharides) and bcs (bacterial cellulose synthesis) genes. The syp genes create adhesion between cells while the bcs genes lead to adhesion between the cells and a surface.
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Process of biofilm formation. Bacteria will locate a preferable environment and attach to its surface. In the case of V. fischeri, the genes syp and bcs will encode production of extracellular polysaccharides, or adhesive building materials, that allow groups of attached bacteria to form a monolayer. Over time, these genes continue to be expressed and contribute to the production of a complex, three dimensional biofilm that helps the bacteria to remain stationary for as long as preferable conditions persist. Source.
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Each of these genes can be affected by a variety of regulators that will either assist or deter their transcription, and in turn, will determine whether or not a biofilm forms. Specifically, Tischler et al. investigate the role of calcium in biofilm formation, which is typically promoted by overexpression of a protein embedded in the cell membrane called RscS. RscS is a sensor kinase which means, when given the correct signal from outside the cell, it will dephosphorylate two ATP and transfer these phosphate groups to another molecule within the cell. This receiving compound will work as either an activator or inhibitor of transcription. Calcium is a common small signalling ion that is known to be a controlling factor in the biofilm growth for many species of bacteria, both as an activator and an inhibitor.
When RscS receives the proper enzymatic signal, it releases further signalling cascades in order to induce transcription. RscS is controlled by a downstream two-component signaling system consisting of SypF and SypG- the first being another signal kinase that will self-phosphorylate upon stimulation with a particular signal and the second being a secondary protein that will take on the phosphate group of the phosphorylated signal kinase. When phosphorylated and ready, SypG promotes the transcription of the RscS gene, eventually leading to normal biofilm production. Alternatively, the SypG response regulator can be inhibited by binK, another kinase, and biofilm production is prevented. To see a fully flushed out diagram of these regulatory systems, click here for access to the full Tischler paper.
Now onto the role of calcium. In this experiment, calcium was added to growth media containing V. fischeri to investigate its effects on the biofilm produced in comparison with those produced by Rscs’ influence. In normal growth conditions, these RscS biofilms were noted to take on a distinctly wrinkled texture that were unable to form in shaking liquid cultures. When in the presence of calcium, however, V. fischeri in shaking liquid cultures were capable of forming a new and unexpected phenotype- a ring formation at the surface of the liquid, as well as a dense clump situated in the bottom of the tube.
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| Wrinkled biofilm phenotype produced by overproduction of Rscs in a stationary culture. From Tischler et al. |
When left to grow undisturbed for an extended period of time, long cellular tethers are seen attaching the clump and ring together, forming an even more complex 3D biofilm architecture!
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Clump and ring biofilm phenotype grown in a shaking liquid culture containing calcium. Images taken after longer periods of time depict growths between the ring and clump, forming a multifaceted biofilm. From Tischler et al.
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Tischler then conducts a secondary experiment to reveal the more intimate process of how this ring and clump phenotype forms, specifically investigating the interaction between calcium and the genes syp and bcs. This experiment focused on the use of binK mutants. BinK, the previously mentioned kinase, represses the activity of RscS and therefore also inhibits transcription of syp. This mutant strain lacks the gene necessary to make binK which would usually repress biofilm production. This mutation was done to clarify the specific regulatory effect that calcium has on the biofilm and remove the possibility of a false negative caused by binK. Following this logic, another mutant was produced that lacked both syp and binK, taking away one of the needed genes and the primary inhibitor. This strain, when activated by calcium alone, created a biofilm with only a ring above the liquid level and no cellular clump. When compared to a binK mutant which produced both components, ring and clump, it became clear that ring formation must be dependent on the bcs locus while cell clump formation depends on syp. This hypothesis was validated in a similar experiment in which a bcs binK mutant was produced and shown to lack ring formation while sustaining cell clump formation. A triple mutant lacking syp, bcs, and binK showed no biofilm production at all.
Calcium unveils an entirely new phenotype for V. fischeri biofilm formation that appears to be suitable, even preferable, for aquatic conditions as opposed to the static, wrinkled biofilm produced by RscS overproduction. Though RscS was shown to contribute to the biofilm formed by calcium, these little calcium ions are also capable of getting the ball rolling all on their own. Considering the symbiotic relationship between V. fischeri and the Hawaiian Bobtail Squid, as well as their aquatic environment, calcium may be an essential component to the success of this interspecies teamwork. While the Tischler paper focused solely on in vitro experiments (meaning that these biological pathways were studied outside of the squid and instead isolated into a laboratory setting), future studies of the relevance of calcium in vivo could yield valuable information about this symbiosis on a cellular level. In this same vein, continued investigation might help to determine specifically what piece of biofilm regulation is the direct target of calcium’s influence, a mystery which Tischler suggests remains largely uninvestigated.
About the Authors:
Arden Hegberg '20
Arden is a sophomore Biochemistry major on the pre-Health track. They're from Minneapolis, Minnesota and would be happy to try to explain what "oofda" and "hotdish" are if you're not from the Midwest. Arden is working in a lab studying the effects of restoration on microbial ecology and hopes to one day study microbes as a medical researcher.
Everett is a biochemistry major at Mount Holyoke College. They research immunopathology of Chlamydia trachomatis in the Lijek Lab here on campus. In their free time, Everett likes to hike, journal, and spend time on the green with friends.
Sources Cited:
DeLoney C.R., Bartley T.M., and Visick K.L. (2002) Role for Phosphoglucomutase in Vibrio fischeri-Euprymna scolopes Symbiosis. Journal of Bacteriology, 184(18). 5121-5129.
Lee K., Ruby E. (1994) Symbiotic Role of the Viable but Nonculturable State of Vibrio fischeri in Hawaiian Coastal Seawater. Applied and Environmental Microbiology, 61(1). 278-283.
Tischler A.H., Lie L., Thompson C.M., Visick K.L. (2018) Discovery of calcium as a biofilm-promoting signal for Vibrio fischeri reveals new phenotypes and underlying regulatory complexity. Journal of Bacteriology.
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