Light Production of Vibrio fischeri

Microbial Physiology University of Santo Tomas

Paner, Crisencio Casamorin, Jovy Ann M. Miranda, Abigail Reyes, Sherwin

Vibrio fischeri
Gram negative Bioluminescent, facultative, aerobic and curved rods, 1-3 microns long Polarly and peritrichously flagellated. Has symbiotic relationship with the bobtail squid, angler fish and flashlight fish.

Vibrio fischeri
Oxidase positive Cell wall consists of an outer membrane containing lipopolysaccharides, periplasmic space with peptidoglycan layer, and an inner cytoplasmic membrane. Has the protein, lux operon Can be found in the guts of marine animals and on the surface of decomposing fish Can be found living freely as marine snow in fecal pellets,

Light Production
V. 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

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

Objectives
To 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.

Methodology
Preparation of Cultures: 1000 ml Broth
5.0g Tryptone 2.5g Yeast extract 30g NaCl 0.3g NH4Cl 0.3g MgSO4 0.01g FeCl3 1g CaCO3 3g K2HPO4 23.5g Sodium glycerophosphate

Buffered Sea water per liter:

30g NaCl 2g Na2HPO4 5% Sodium -glycephosphate

Methodology
Make 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.

Methodology
For the test tubes in rack:
1. 4ml broth, 1ml cell suspension 2. 4ml SW, 1ml cell suspension in SW 3. 4ml of SW, 0.05ml of 5 % glucerophosphate and 1ml cells

Results and Discussions

V 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

Summary
Bacterial 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.

Summary
Differs in a number of properties Growing conditions Nutritional requirements Growth on temperature

Summary
Luciferase Rod Shape Gram Negative Flagellated Facultative anaerobes

Summary
Despite 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.

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