Examination and clarification of bioluminescence in marine creatures

Examination and clarification of bio freshness in maritime creaturesIn rules of order to sequestrate bioluminescent b lickeria from marine strains, cardinal must fix a better understanding of the phenomena of biog menial. Bio lambency is a type of luminescence. The legerity that usu bothy occurs at low temperatures is c exclusivelyed luminesence 1. Chemiluminescence, fluorescence is all the other types of luminescence and should not be addled with bioluminescence.As the result of a given answer, venting of heat and mail civilizes place, this phenomenon is referred to as chemiluminescence or in other words, chemiluminescence refers to the waiver of brightness level in an exergonic reaction. For example, if deuce reactants namely A and B react, it results in the formation of product, with an stirred intermediate C and generation of strike.A + B C Products + empty-headedThis is how a chemical reaction takes place 1.When a substance that has absorbed clear or any(pr enominal) other radiation of different wavelength in the electromagnetic spectrum, an waiver of light takes place by that substance, this is referred to as fluorescence.In most cases, emitted light has a longer wavelength, and on that pointfore lower thrust, than the absorbed radiation which has a high energy 1.In simple language, bioluminescence is the emission of light from living organisms. single bath alike describe bioluminescence as chemiluminescence in living organisms. pull ahead clarifications regarding the types of luminescence tail end be carried out with the help of an experiment that involves the enjoyment of refulgence or light sticks. A solution of luminol in DMSO, atomic number 11 hydroxide pellets, an aqueous solution of fluorescent dye and test renders. Luminol is a versatile chemical that exhibits chemiluminescence, with a striking dingy glow, when manifold with an capture oxidizing means 1 2.Glow sticks ar used to demonstrate the effect of tempe rature on the rates of chemicalreactions. The glow sticks contain two chemicals that argon mixed when the glass tobacco pipe on theinside is broken. This initiates a chemical reaction that gives off light. Higher the reactiontemperature, faster is the reaction, and more intense the chemiluminescence. reply ratesincrease about two times for every 10C rise in temperature 2.The luminol experiment demonstrates chemiluminescence and fluorescence. Luminol isoxidized (with molecular group O) in the front line of sodium hydroxide pellets. On shaking the test tube (containing luminol and sodium hydroxide pellets), oxygen is introduced into the solution. Hence chemiluminescence stops when the test tube is set up aside 2.When a fluorescent dye is added to the solution, the dye absorbs the light emitted by the luminol and re-emits light at a longer wavelength, changing the colouring material, so explaining the phenomena of fluorescence 2.Bioluminescence is the emission of light observ ed in living organisms. Apart from bioluminescence, there ar two other kinds of light emission that may take place from a living organism. These include(I)Photosynthetic delayed light emission. It is a weak red light which is emitted by all atomic number 19 plants and algae. This intensity is so low that one stop buoynot see it, though it can be measured 3.(II)Ultraweak light emission this occurs in all organisms. It is repayable to unhomogeneous processes, mostly (but not always) involving molecular oxygen. It is regarded as a by-effect of metabolous activity, but doesnt have a biological function. It cannot be seen 3.2. BioluminescenceThis is the best know biological luminescence phenomena, mostly because it can be observed victimization ones eyes merely. The bioluminescence occurs among a variety of organisms ranging from bacteria, dinoflagellates, protozoa, sponges, mollusks, echinoderms, insects and fish. The majority of bioluminescent species live in the sea, although t here argon also many sublunar bioluminescent insects, specially the beetles. It has been estimated that 60-80% of the fishes in the sibylline sea atomic number 18 bioluminescent 3.(i) jellyfish(ii) lightfish(iii) fungus kingdom(iv) beetle flesh 2.1 The above pictures show bioluminescence in variety of organisms.The bioluminescent bacteria mainly falls under triple genera namely Photobacterium, vibrio, andPhotorhabdus. Species inside the genus Photobacterium and vibrion generally exist in marine environment whereas the terrestrial species belong to the genus Photorhabdus. Species within thePhotobacteriumgenus be generally light organ symbionts of marine animals, whereas theVibrio species exist as free-living forms as well as symbionts in the sea 4.The luminescence of these microorganisms should not be confused with the server organisms. Many fish and molluscs species which have been regarded as bioluminescent organisms have been shown to glow by the light of symbiotic bacte ria 3. The bacteria forms a symbiotic relationship with the host organism as it is provided with a nutrient abounding environment for its evolution and the host organism has the benefit of camouflage and aegis from its predator. virtually of the bioluminescent bacteria are obligate symbionts that fulfill their nutritional requirements only from the host, hence they cannot be grown in the laboratory as they cannot be separated from the host organism 4. Apart from sharing a symbiotic relationship with the host organisms, some of the bioluminescent bacteria are also parasitic in nature, for example, the species in the genus Photobacterium and Vibrio infect the male person crustaceans whereas the species in Photorhabdus genus infect terrestrial insects such as caterpillars with nematodes playacting as an intermediate host for the bacteria. Majority of the bioluminescent bacteria act on the control surface of the marine organisms act as non-specific parasites. The bacterium that r esides in the mainstay of some marine organisms such as crustaceans produces chitinase (an enzyme) that facilitates the decomposition of chitin which is present in their exoskeleton.The different species of bioluminescent bacteria differ from each other in a number of properties including the optimal growing conditions i.e. the nutritional requirements and optimal ripening temperature, and the reaction kinetics of the enzyme luciferase involved in light generation.However, the morphology of all bioluminescent bacteria is the same i.e. they are rod-shaped, gram-negative microorganisms with flagella facilitating motion. Bioluminescent bacteria are also capable of growth when the supply of molecular oxygen is limited therefore they are also examples of facultative anaerobes. Despite the physiological diversity among different species of bioluminescent bacteria, all these microorganisms utilize passing homologic biochemical machineries to produce light. The onset and the energy outp ut of this light-producing molecular machinery are tightly regulated under a central signaling passage 4.2.1 Bioluminescence by calamarysLight-emission by most of the marine organisms belongs in the drab and ballparklight spectrum.This is delinquent to two reasons, firstly because the blue-green light (wavelength near 470 nm) transmits farthest in water, and secondly because most of the organisms are sensitive only to blue light, lacking pigments for the visualization of longer or shorter wavelengths1. calamari transports the pretension of the light emitted i.e. either blue or green light depending on its surrounding temperature. In case of squids, it produces green light when swimming in warm water and blue light in low temperature water 5. During the day, the squid resides in the deep waters rather than on surface waters. The sunlight that falls on the deep waters has been filtered with only blue light remaining. The squid matches this seeming by turning on its blue pho tophores (photophores are light producing tissues). During the iniquity, the squid is present on the shallow water. The corn liquor at shallow depths has not been filtered to a greater extent, as a result both blue and green light remains. The squid matches this color by turning on both of its green and blue photophores 5.Fig 2.1.1 The picture shows squids bioluminescence 52.2 Advantages of Bioluminescence at that place are four main advantages attributed to bioluminescence Camouflage, attraction, repulsion, and communication.CamouflageSome squids by development the phenomena of bioluminescence defend themselves against predators by producing light (a soft glow) on their ventral surface to match the light coming from above and qualification their straw man indetectable to the potential predators(just as a darker dorsal surface makes aquatic organisms difficult to detect from above. Some can also change the color of their luminescence to match moonlight or sunlight. This is ref erred to as counterillumination 1.AttractionBioluminescence is also used as to attract object by several deep sea fish, such as the anglerfish. A dangling appendage or a light-emitting rod that extends from the head of the fish that carries the bioluminescent bacteria attracts humbled animals to the front of its mouth.Fig 2.2.1 Anglerfish lures its prey by using bioluminescence 4.The biscuit cutter shark also uses bioluminescence for luring its prey. A fine patch on its underbelly remains dark and tends to appear as a small fish to large predatory fish like tuna. When these fish such as tuna try to consume the small fish, they themselves become prey for the the shark. Dinoflagellates have an interesting twist on this mechanism. When a predator of plankton is sense through motion in the water, the dinoflagellate luminesces. This in turn attracts even larger predators, which thusly consume the would-be predator of the dinoflagellate. The attraction of mates in fireflies during th e mating season is another proposed mechanism of bioluminescent action. This is done by periodic flashing in their abdomens to attract the potential mates 1. horrorCertain small crustaceans also use bioluminescent chemical mixtures. A cloud of luminescence is emitted, which confuses and then repels a potential predator while the crustacean escapes to safety. This is also shown in some squids 1.CommunicationBioluminescence also t signers a direct role in communication between bacteria. It promotes the symbiotic inductance of bacteria into host species, and sometimes also plays a role in colony aggregation 1.2.3 Biochemistry of the Bioluminescence ReactionAs mentioned earlier, bioluminescence is defined as emission of light by living organisms arising from exothermic or exergonic chemical reactions. It is due to the substrate-enzyme interlacing of luciferin-luciferase within the cytoplasm of the cell. Luciferin refers to any light-emitting compound whereas luciferase is an enzyme. The luciferin-luciferase complex differs among species.In 1887, a scientist named Raphal Dubois isolated light producing chemicals from thepiddock, which is a clam that sash in the burrow. He discovered that on placing theclam in cold water, light was seen in the water, that glowed for several transactions, indicatingthat a light producing chemical was sublimateed from the lolly tissues. He also observed thatif he make a hot-water distill from another clam and added this to the original cold-waterextract, he could reactivate the light reaction. Dubois called his hot-water extract luciferin andthe cold-water extract luciferase. The reaction produces a molecule that is in an electronically activated state. later on(prenominal) the molecule gives off energy, it goes back to the intellect state and a photon of light is released 2.bacterial luciferase is the main enzyme that is used in the phenomena of bioluminescence. Apart from the function of luciferase, there are certain ot her enzymes that supply and regenerate the substrates of luciferase. In bacteria the scene of the genes related to bioluminescence are en cipherd by an operon called the lux operon.The lux operon is a 9 kilobase fragment that controls bioluminescence through the catalyzation of the enzyme luciferase. The lux operon has a cognise gene sequence of luxCDAB(F)E, where lux A and lux B code for the components of luciferase, and the lux CDE codes for a fatty acid reductase complex that makes the fatty acids obligatory for the luciferase mechanism. lux C codes for the enzyme acyl-reductase, lux D codes for acyl-transferase, and lux E makes the proteins inevitable for the enzyme acyl-protein synthetase. Apart from these genes, there are two more genes namely luxR and luxI that play an important role in the regulation of the operon 1. Other genes includingluxF,luxG, andluxH, whose functions are incomplete clearly defined nor apparently necessary for bioluminescence are also engraft in someluxoperons 4.Fig 2.3.1The arrangement of luxCDABE operon 4Luciferase is a heterodimer consisting of two different polypeptide reachs- of import and beta (molecular mass 40 kDa and 37 kDa, respectively, and encoded by theluxA andluxB genes, respectively). The active site is located within the alpha-beta fractional monetary unit. Absence of beta subunit leads to light emission of a weaker intensity. Studies have shown that the crystal structure of V. harveyi luciferase interacts and forms complex stick to patterns between several side chains and backbone amides of the alpha and beta subunits. Studies also reveal that the function of the beta subunit is to act as a supporting scaffold by assisting in the conformational change of the subunit during the catalysis 4.Fig 2.3.2 Bacterial luciferase structure 4.Fig 2.3.3 The angular box highlights the inter-subunit interactions (ionic attractions, hydrogen bonds, hydrophobic interactions) that play an important role in the assembly of bacterial luciferase enzyme 4.Bacterial luciferase uses reduced flavin mononucleotide (FMNH2), molecular oxygen, and long chain fatty aldehyde as substrates. During the reaction, the oxidation of FMNH2and aldehyde concomitant takes place along with the decrement of molecular oxygen and emission of energy, which is released as blue/green light ( MAX 490 nm). The energy level of the photon that was produced when the delirious electron on the flavin chromophore returns to the ground state is indicated by the characteristic color. Studies have shown that point mutations at the flavin chromophores binding site brings about a change in the color emission spectrum of bacterial bioluminescence, indicating that the distinctive emission color depends not only on the chromophore, but also on the electronic nature of the chromophore-binding microenvironment in luciferase. divagation from bacterial luciferase, some luminescent bacteria also carry fluorescent proteins to distinguish themselv es from other strains by modulating the emission color 4.For continuous light emission, constant supply of the substrates should be maintained by the enzymes coded by the Lux operon.In addition to bacterial bioluminescence, all the other biological luminescence systems (such as fireflies, coelenterates and dinoflagellates) also utilize molecular oxygen as the oxidizing agent in their luminescence biochemistry, and the processes involved in the reduction of the molecular oxygen serves as an energy sink, draining the reducing power of the substrates. High energy unstable intermediates are formed that dissipate the potential energy of the excited chromophore in the form of light. In this regard, molecular oxygen can be considered to serve as a key to unleash the energy deposited in FMNH2and fatty aldehyde for bacterial bioluminescence 4.Fig 2.3.4 The pathway 4For example, in case of fireflies luciferin reacts with oxygen, with luciferase acting as an enzyme aided by cofactors such as c alcium ions, olibanum emitting light.2.4 Quorum signal detectionThe definition of quorum sensing states that it is a type of decision fashioning process used by decentralized groups to coordinate behavior 1. From the biological aspect, there are many species of bacteria such as Vibrio fischeri, Escherichia coli, Salmonella enterica, Pseudomonas aeroginosa that use quorum sensing to coordinate their gene expression concord to the local density of their population. It was first discovered in Vibrio fischeri 1.Since Vibrio fischeri uses quorum sensing, it constantly produces signaling molecules called as autoinducers. These bacteria have a sensory receptor that recognizes these signaling molecules. When the autoinducers bind to these receptors, it results in the transcription of certain genes, including those for inducer synthesis. There are less chances of the bacterium recognizing its own signaling molecules, hence for the energizing of gene transcription, the cell must also en counter signaling molecules from the local environment. Autoinducers and inducers are interchangeably used. If there is less number of same types of bacteria present in the local environment, then the concentration of the inducer decreases to zero(a) thus inactivating the gene transcription. But if the population of the bacteria increases, the concentration of the autoinducers increases, thereby resulting in the activation of gene transcription, thus causing bioluminescence. Therfore, quorum sensing plays a very important role in the regulation of luxCDAB(F)E expression in bioluminescent bacteria 1 4 .Fig 2.4.1 Chemical structure of the autoinducers of bioluminescent bacteria 4The autoinducer is a metabolic product that diffuses easily across the cellular membrane 4.Fig 2.4.2 The fig. shows the role played by an autoinducer in the mechanism of quorum sensing 4.Marine bioluminescent bacteria that is not present as a symbiont (free living bacteria) does not emit light. This is becau se for the emission of light, accumulation of autoinducers is necessary and this is viable only in a nutrient rich environment which is provided to the symbiotic bacteria 4.2.5 Applications of bioluminescenceOne of the major applications of bioluminescence is the development of biosensors. A biosensor is a finesse that detects, records, and transmits information regarding a physiological change or the presence of various chemical or biological materials in the environment. Some bacteria have been designed that gives off a detectable signal when in presence of a pollutant (e.g. toluene) that it likes to consume 6.In name of using the phenomena of bioluminescence, efforts are universe made to engineer agricultural plants that show luminescence when need lacrimation 1.As the primary function of bacterial luciferase is to catalyze the emission of light, this characteristic together with generation of the aldehyde substrate by fatty acid reductase can be successfully produced in ot her bacteria, by the transfer of theluxCDABE genes, which replace nonluminescent bacteria into light emitters 4.Fig 2.5.1 The insertion of the foreignluxCDABE structural genes into the organism such as E. coli confers the organism the expertness to emit light 4.The ability of the non-luminescent bacteria to emit light by means of recombinant DNA applied science has provided researchers an easy alternative to measure and detect the growth and living conditions of bacteria. The phenomena of bacterial bioluminescence are used in the detection of pathogenic bacteria in human food sources. By culturing a food sample in the presence of a recombinant bacteriophage (vector) carrying theluxCDABE insert, one can pronto go over the contamination by bacteria in the food source. In addition, the light emitting property of theluxCDABE genes has been employed as a reporter of gene expression for studying regulatory controls involved in affecting the efficiency of ribonucleic acid polymerase in initiation and transcription at different promoters. Then theluxCDABE genes are under the control of an environmentally regulated promoter (e.g., promoters whose efficiency is highly sensitive to the level of mercury, arsenic, or other pollutants), the structurallux genes can function as a biosensor, whose expression allow for monitor the presence of toxic waste in the environment. In the pharmaceutical industry, genetically limited bacteria carrying the lux genes have been utilized to evaluate the efficiency of antibiotics in fighting against bacterial transmittances in mammals with animals such as mice, pigs, and monkeys serving as potential human models. In this testing procedure, the lesser the intensity of luminescence in the infected organs/tissues, the more efficient the antibiotics against bacterial infection therefore, bacterial bioluminescence serves as an indicator of bacterial growth allowing the proper dosages of antibiotics to be determined and effective treatment to be established 4.3. Laboratory experimentation3.1 Sample CollectionAfter the literature study, it was decided that squid will serve as a sample for this experiment as it is readily available in the U.A.E. fish market. A fresh catch was interpreted as a sample for this experiment. Since some of these microbes i.e. bioluminescent bacteria are also found in seawater, seawater sample from Sharjah was also amass for this experiment.3.2 Methodology for the isolation of bioluminescent bacteria from squidMaterials RequiredSquidLuminescent Broth (Appendix 1)Luminescent Agar (BOSS Medium) (pH=7.3) (Appendix 2) mathematical process1. The squid is put in a beaker and just enough 3.0% NaCl solution is added such that round 10-20% of the sample is above the level of the liquid as shown in fig 3.2.1. The NaCl solution preserves the squid by preventing any other microbic growth other than that of bioluminescent bacteria, as required.Fig 3.2.1 Squid hardened in a beaker containing NaCl solut ion.2. The flaskful is then unplowed for pensiveness in a cool dark manner (18-22C) and is observed at intervals up to 24 hours. The room is darkened totally such that the flask can be observed for aglow(predicate) areas on the sample. Sometimes the squid secretes ink that might hinder the view of luminous areas on the squid. In order to prevent this, the NaCl solution is changed when required.3. Four petriplates of Luminescent Agar (formula above) are streak from four different luminous areas on the squid. Forceps and craft knife are required and it is used one at a time in the burner for its sterilization. The knife and forceps are then cooled for a while. Squid is held with the forceps and its skin is thinly scraped of that shows luminescence with the tip of the knife. The scraped off skin is transferred on to a unfertilised inoculating loop for streaking on the plates.4. The plates are then kept for brooding in the cool room (18-22C) for 24 hours. (No more than 48 hours.)5. After observing luminous isolated colonies, these isolated colonies are individually streaky on to a new plate of Luminescent Agar and incubated as above.Fig 3.2.2 Streaked petriplates6. One or more of the more resplendent colonies is then chosen and streaky onto a slant of Luminescent Agar. The nutrient nutrient agar slants are incubated overnight or until luminescent growth is seen and then refrigerated.7. From the agar slants, flasks of Luminescent Broth are inoculated. The flasks are then lay in the shaking incubator for 10-15 hrs at 18-22C.8The flasks that show bioluminesence is then used for studying the growth curves and characterization of the bioluminescent bacteria. essence and InferenceNo luminous colonies were observed from the squid on the first attempt, even though the squid did show luminous areas on its body surface. The failure can be attributed to the fact that streaking was not carried out on the same day it showed luminescence.However, on the second attemp t, out of the four petriplates that were move with the skin of the squid, only one petriplate showed six luminous colonies.Fig 3.1.3 The above pictures are a telephone extension as to how colonies appear when placed in light (left picture) and dark (right picture) 10.The colonies that appeared during the escape of my experiment (only six in number) were not so densely live as observed in the pictures above.These six colonies were then streaked on six different petriplates containing Luminescent Agar. The picture below shows bioluminescence in the streaked petriplates.Fig 3.2.4 The picture below shows bioluminescence in the streaked petriplates.The agar slants were also prepared from the petriplates.The six flasks containing Luminescent Broth were then inoculated with husbandry from the agar slants. The flasks were then kept in the shaking incubator for 18-24 hrs. at room temperature.Out of the six flasks containing Luminescent Broth, only triple flasks showed microbial growth. The bacterial cultures were then used for growth curves.3.3 Methodology for the isolation of bioluminescent bacteria from seawater sampleMaterials Requiredseawater sample was collected from Sharjah.Seawater Complete Agar (Appendix 3)Procedure1. Seawater sample is collected in a clean container2. Two plates of SWC agar medium were then prepared.3. The two plates were then pipetted with 0.1 ml and 0.2 ml of seawater sample respectively.4. The samples were thoroughly spread over the surfaces of the plates with a L-shaped glass rod.5. The plates are then inverted after the samples have absorbed into the agar (about 5 minutes) and then kept for incubation at room temperature.6. The plates were then examined after 18-36 hours.7Result and InferenceThe plates did not show any luminous growth. This maybe because the sample that was collected was not from deep water as bioluminescent bacteria tends to be present in deep waters. Since no growth was observed, further steps involving the prep aration and inoculation of agar slants and luminescent broth could not be carried out.3.4 Bacterial Growth curve of the isolatesOut of the six flasks that contained Luminescent Broth, only ternion flasks showed microbial growth. The triplet flasks that showed microbial growth were then again inoculated into three flasks containing luminescent broth. Their O.D. ( visual density) values were measured after every 30 minutes (for 5 hrs) at 530 nm using UV-visible spectrophotometer. The initial O.D. value should be set at 0.05 so that there is sufficient bacterial culture in the broth. The values then helped us in determining the bacterial growth curves.Fig 3.4.1 UV-visible spectrophotometer 11Procedure1. The machine along with the monitor screen is turned on using the switch.2. The necessary adjustments are then made in the program.3. For auto zeroing the sample, the blank (broth in which are bacteria is growing) is placed in the cuvette. The cuvette is then placed in the holder.4. Th e O.D. values of all the three samples are measured after every 30 minutes for 5 hrs.5. The optical density vs. time graph is then plotted for all the three samples.Observation TableTable 3.4.1 Sample 1 quantify (in hrs.)O.D. values00.080.50.0910.121.50.1620.212.50.2830.383.50.540.714.50.9951.145.51.41Table 3.4.2 Sample 2 metre (in hrs.)O.D. values00.050.50.0610.081.50.1220.162.50.2130.253.50.3840.444.50.48Table 3.4.3 Sample 3 clock time (in hrs.)O.D.values00.130.50.1510.181.50.2320.32.50.3830.533.50.7141.044.51.1651.37Result and Inferencegraphical record 3.4.1 Bacterial growth curve of sample 1Graph 3.4.2 Bacterial growth curve of sample 2Graph 3.4.3 Bacterial growth curve of sample 3The bacterial growth curves of all the three samples suggest that the cultures are still in their exponential function phase. The 0.D .values should be measured for a much longer duration so that the stationary and the death phases can also be observed. The broth was kept overnight in the shaking incub ator at 18-22C. Next morning, only one of the samples showed bioluminescence indicating that the bacterial culture has grown to that level when the lux genes are switched on.Fig 3.4.2 The picture is a reference as to how a flask containing Luminescent Broth shows luminescent growth 6. The bioluminescence that was observed during my experiment was of low intensity.3.5 Luminescence (light emission intensity) curve studies on the isolatesFor the growth curve studies, agar slants were used to streak on to the petriplates, for the isolation of bioluminescent bacteria. The same set of agar slants were used to revive the culture. The revived culture was then streaked on to the luminescent agar petriplates to study the luminescence curve. However, contamination was observed in the petriplates, even though luminescent colonies were formed. Majority of the colonies that were formed were poster in shape and opaque with a dense material in the centre. Some of the colonies were circular and tra nslucent. These colonies were then again used for sub-culturing. Contamination was again observed in the petriplates. This might be attributed to some error in the methodology of streaking the petriplates. Finally, after five attempts, successful isolation of bioluminescent bacteria took place. These bacteria were then inoculated in the flasks containing luminescent broth. After an over night incubation, these flasks showed bioluminescence. These samples were then taken for measuring their light emission studies with the help of an autoanalyser. The luminescence is measured after every one hour. It is measured in terms of counts per second (cps). Meanwhile, the samples are kept in the shaking incubator.Fig 3.5.1 Perkin-Elmer Auto-analyzer 12Procedure1. The machine along with the monitor screen is turned on using the switch.2. The luminescence mode is then chosen.3. The wells in the microtitre plate containing the sample are then chosen in the protocol editor.4. The program is then s tarted.5. The luminescence of all the three samples is measured after every 1hour.5. The optical density, luminescence vs. time graph is then plotted for all the three samples.Observation TableTable 3.5.1 Bacterial Sample 1Time (hrs.)Cell Density(O.D.)Light emissionIntensity (cpu)00.07850.50.092610.11891.50.15520.21392.50.28263