AUTHOR DETAILS – JATIN KUMAR, 4th YEAR, B.TECH (BIOTECHNOLOGY), RPCAU, PUSA

As the sun sets on the horizon, glimpses of natures spectacular  phenomenon of glittering and sparkling light appears in the ecosystem by which any one can be easily fascinated by its beauty, shown by variety of terrestrial, marine and some fresh water organisms which we often called bioluminescence, one can easily see in the twilight. In the dark where life is hard to sustain, these species have evolved by much brighter response without burning an ounce of fossil fuels, and hence are called Living light, evolved as a gift of glow.

Fireflies are the most commonly found luminous organisms in the terrestrial ecosystem that mesmerizes us with their marvellous yellow blinking light in their abdomen; they do so to attract mates. Few species of fungi (mushroom) growing on decaying woods, are also among the terrestrial luminescent organism that glows consistently in night, called foxfire, and the only known land snail to show bioluminescence is Quantula striata, native to the tropics of Southeast Asia. Though the distributions of luminescent organisms are predominant in deep down marine ecosystems where sunlight is less or unable to penetrate, and have adapted to their environment by making their own light. However thousands of colonies of dinoflagellates, a type of plankton, on the surface of oceans creates a bluish-green colour that sparkles at night like a thousand of stars glittering on the surface of earth. Few luminescent organisms (Latia neritoides) also illuminate the fresh water ecosystems.

This phenomenon of emitting light by living organism through their metabolic reactions is called bioluminescence, also known as chemiluminescence. It is a“cold light”that means less than 20% of light generate thermal radiation or heat. These luminescences are generally used for ecological interactions, like to hunt prey, defend against predators, find mates and execute other vital activities. It is an active process that requires energy to show luminescence. Unlike the bioluminescence of the deep, the second kind found in shallower water that doesn’t produce light of its own, rather it absorbs light from an outside source in the tissue or structure, and then re-emits at different wavelengths, producing different colours, called biofluorescence and is a passive process. These processes of emitting light are a shining example of Darwin’s theory of evolution.

Over the last century, this light producing reaction has been discovered scattered across the entire natural world, both above the surface and beneath the waves, that’s how readily available these ingredients are. Actual chemical differ from creature to creature but the basic mechanism of fuel and spark is the same. The reaction is so common that it’s evolved independently in different branches of the evolutionary tree, more than 40 separate times.

In 1885, a French biologist working on a firefly species deciphered the chemistry responsible for their magical glow. He determined it’s the result of a reaction between two chemicals, luciferin, the light bearer that acts as a fuel and a luciferase enzyme, that sparks the fuel in the presence of oxygen molecules. This reaction results in the formation of oxyluciferin and excess amount of energy, this energy takes the form of light.

Biochemical reaction of
bioluminescence. Oxidation of luciferin under presence of luciferase enzyme and ATP.

There are five different luciferins that are responsible for the luminescence by most of the known bioluminescent organisms. They are –

  1. Bacterial luciferin (a derivative of riboflavin)
  2. Coelenterazine (extremely common, and found in several species)
  3. Dinoflagellate luciferin (similar to the chlorophyll structure)
  4. Firefly luciferin (requires ATP for luminescence)
  5. Vargulin (found in Ostracods).

While fluorescent proteins such as GFP (in jellyfishes) and lumazine protein (in Photobacterium phosphoreum) are also known to be involved in intense luminescence.

Bioluminescence by an organism can be shown in various patterns, like –

  1. Flashes (Fireflies, Squids, Odontosyllis)
  2. Continuous glow (bacteria, fungi)
  3. Blinking (bacteria)
  4. Wavy (Sea pansy, Renilla)
  5. Switch on and off system (Angler fish)

Interestingly some bioluminescent organisms such as Ophiuroids and earthworms show luminescence only in presence of some stimulating chemicals like KCl.

The luminescences emitted by different bioluminescent organisms are ranges from 400nm to 700nm in wavelength. And these organisms also show various colour patterns in their luminescence, like green (Odontosyllis phosphorea, Mycenafera), yellow (Fireflies, bacteria), red (Phrixothrix hirtus, Malacosteus niger), blue (bacteria, jellyfish), and pink (Photonectes waitti). These colour patterns are result of the different arrangements of luciferin molecules. Whereas few organisms can glow in more than one colour, the railroad worm (larva of a beetle), where its head glows red while the body glows green. And this is due to expression of different luciferases, differently. Yet the chemistry of luminescence of many bioluminescent organisms is still remains unknown.

Not all bioluminescent organisms synthesize their luciferin by their own; instead they absorb it through other organisms, either in the form of food or in a symbiotic relationship. Some species of midshipman fish get their luciferin through consuming “seed shrimps”. While marine animals like squid provide shelter to the bioluminescent bacteria in their light organs, where form a symbiotic relationship.

Also not all bioluminescent reactions involve luciferin and luciferase, some bioluminescence reaction lacks luciferase enzymes and involves a chemical called Photoprotein. Photoprotein combines with luciferin and oxygen along with an ion of calcium, to produce light. Scientists have recently discovered these photoproteins and are still studying their unusual properties. It was first studied in bioluminescent crystal jellies and the photoproteins found in them are called “green fluorescent protein” or GFP. These are now used as reporter genes. GFP reporter gene allows scientists to trace and monitor the activity of studied genes like its expression in a cell, or its interaction with other chemicals, usually by their fluorescence.

Natural gene construct of bacterial Luciferase

Proteins and genes involved in luminescence of such organisms can be used for medical and biotechnological applications, below is the list of those miscellaneous applications –

1. Green fluorescent protein (GFP) could be used to light-up living nerve cells and ultimately map the human brain. The inside of brain is a black box and is most amazing instrument in the entire earth and nothing else comes close to the ability of human brain and we don’t have even a very thin understanding of how they work. Putting proteins (GFP) into the nerve cells, so that it glows and we can interpret that information and find out how the brain is doing what it does.

2. Scientists are using fluorescent proteins to light-up the inner working of cells. The initial work began with GFP, isolated from jellyfish. GFP can be tagged to any protein and their movement can be watched within the cells in real time. To do this, scientists have taken the gene that codes for the green fluorescent protein and inserted into the cell, once the genetic construction are inside, the cells make the proteins. Now when scientists hit the organism with blue light, the target itself fluoresces as green. So this revolutionized our ability to see at the protein level inside the living cells.

3. GFP was a Nobel Prize winning discovery, bringing to light everything from the wake of cancer spread (by tagging GFP to malignant cells and monitoring their travel through blood vessels), to how the viruses infect and replicate within the host cell. Scientists attached GFP to virus resistant genes and inserted them into the DNA of animals like mice and cats, in this way they can even assess the potential cure for AIDS, lighting-up the entire creature in the process.

Biotechnological approach for various applications

4. Scientists have also manipulated GFP to fluoresce different colours, enabling them to tag different cells at once. Now they are in search of new fluorescent protein that could light-up beyond light spectrum, to study the human brain. Because human brain tissues are quite dense that none of the colours discovered so far that can easily pass through it, so neuroscientists have been looking for colours with longer wavelengths like far red and infrared that can. So RFP (red fluorescent protein) could be used as a probe to study human brain.

5. Scientists using genetic engineering fused the fluorescent proteins to other that are sensitive to voltage (the language of brain cells). They fused GFP with fruit flies brain and then inserted glowing voltage sensitive proteins into the flies. Now under special microscopes they watched, as the flies think, each time a neuron fires, the voltage changes and so does the intensity of glow.

6. Bioluminescent bacteria can be used to monitor water pollution. Toxins in polluted water interfere with the bacteria’s ability to produce light. As the pollution level of water goes up, the light becomes dimmer, so one can get a relative measure of toxicity, thus creating a whole-cell biosensor for detecting various toxicants. This technique provides a quick and cost-effective way to detect and monitor pollution.

7. Scientists have modified firefly’s genes in hope of creating bioluminescent trees that could one day light-up city streets and highways, thus reducing the need of electricity. Bioluminescent crops and other plants can be developed so that when they require water or nutrients, or when they are ready to be harvested, they could illuminate, reducing the cost of farmers and agribusiness. Another lab co-opted marine bacteria to produce an electricity free lamp. One company has made bioluminescence available to masses in the form of green fluorescent ice-cream.

8. Bioluminescent proteins and GFP are also used in bioluminescent imaging (BLI) to study the interaction of infectious microorganisms with living cells.

9. Phenomenon of interaction between luminescent bacteria i.e. quorum sensing, horizontal gene transfer of lux genes among luminescent and non-luminescent bacteria, acquirement of luciferins through food chain by some marine organisms (ex- Euphausiids feeds on zooplankton) are the interesting area of research.

Who know what the illuminating world awaits, but thanks to those alluring creatures of light, the future of at least one species, our own may be an enlighten one.

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