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Light Production of Vibrio fischeriMicrobial PhysiologyUniversity of Santo Tomas

Paner, CrisencioCasamorin, Jovy Ann M.Miranda, AbigailReyes, Sherwin

1Vibrio fischeriGram negativeBioluminescent, facultative, aerobic and curved rods, 1-3 microns longPolarly and peritrichously flagellated.Has symbiotic relationship with the bobtail squid,angler fish and flashlight fish.

2Vibrio fischeriOxidase positiveCell wall consists of an outer membrane containing lipopolysaccharides, periplasmic space with peptidoglycan layer, and an inner cytoplasmic membrane.Has the protein, lux operonCan be found in the guts of marine animals and on the surface of decomposing fishCan be found living freely as marine snow in fecal pellets,

3Light ProductionV. fischeri produces light in a chemical reaction as follows:FMNH2 + RCHO + O2 --> FMN + RCOOH + H2O + hv(490nm)The reaction is oxidized by an enzyme called LUCIFERASE

4Light ProductionLight is produced when the organisms are present at high cell densities. They undergo a mechanism called Q uorum sensing.

5ObjectivesTo be able analyze the process using the reaction of inhibitors of cellular processes, provision, and absence of metabolites,; and the provision of oxygen to whole cells.

6MethodologyPreparation of Cultures:1000 ml Broth5.0g Tryptone2.5g Yeast extract30g NaCl0.3g NH4Cl0.3g MgSO40.01g FeCl31g CaCO33g K2HPO423.5g Sodium glycerophosphateBuffered Sea water per liter:30g NaCl2g Na2HPO45% Sodium -glycephosphate

MethodologyMake a liter of 1000ml broth. Place 400 ml in each of the two 32 oz. prescription bottles and add 6.0g powdered agar;Autoclave together with the remaining 200ml of broth.Streak one of the plates with the organism (V. fischeri) culture and pick a bright luminous colony and establish a broth culture in one text tube a week before the experiment.Two days before the experiment, inoculate each of the agar surfaces in the 32oz. Bottles with 1ml of the liquid culture and incubate.Wash the cells off the agar with SW and sediment the cells in a centrifuge (3000 x g).Re suspend the cells in 50ml of SW.

MethodologyFor the test tubes in rack:4ml broth, 1ml cell suspension4ml SW, 1ml cell suspension in SW4ml of SW, 0.05ml of 5 % -glucerophosphate and 1ml cells

Results and DiscussionsV ibrio fisheri can grow and produced light on the photobacterium agar

The light produced by the organism is viable until the 3rd day of plating

Results and Discussions

The luminous bacteria do not emit light on the photobacterium broth

On sea water the organism can still emit light

SummaryBacterial luciferase is the enzyme that catalyzes light emission at the heart of bacterial bioluminescence. However, the catalytic machinery involved in continuous light production in luminous bacteria includes not only bacterial luciferase, but also the enzymes that supply and regenerate the substrates of bacterial luciferase.SummaryDiffers in a number of propertiesGrowing conditionsNutritional requirementsGrowth on temperature

SummaryLuciferaseRod ShapeGram NegativeFlagellatedFacultative anaerobesSummaryDespite the physiological diversity among different species of luminous bacteria, all luminescent microorganisms utilize highly homologous biochemical machineries to produce light. The onset and the energy output of this light-producing molecular machinery are tightly regulated under a central signaling pathway. Thank You for listening