The Function of Bioluminescence in the Squid-Vibrio Symbiosis
Bioluminescence plays the most important role in the functioning of the Squid-Vibrio symbiosis. Without it, the squid would not have protection from predators at night, which would decrease their fitness. The light emitted from the v. fischeri shows a strong correlation with the overhead light intensity coming from the moon. The squid controls the bacterial luminescence two ways. First, the tissues that line the light organ act to ensure that the light is directed out of the animal’s ventral surface and also to diffuse the luminescence and second, because bioluminescence results from the oxidation of substrates, the squid withholds oxygen which limits the amount of luminescence (McFall-Ngai, 2008).


What is Bioluminescence?
Bioluminescence, a form of luminescence, is the production of light by a living organism. In the squid-vibrio symbiosis, the light emission comes from the bacteria, vibrio fischeri. Luminescence is produced by the activity of the bacterial enzyme, luciferase, as well as it’s substrate, luciferin. Luciferins are light emitting molecules that react with oxygen. Without oxygen, this light reaction will not take place. Luciferase enzymes catalyze the reaction. Luciferase is a heterodimer composed of two polypeptides, alpa and beta. The alpha subunit is encoded by the luxA gene whereas the beta subunit is encoded by the luxB gene. This reaction involves the oxidation of a reduced riboflavin phosphate and an aldehyde. The result is the emission of a blue-green light. “The reduced flavin, FMNH2, bound to the enzyme, reacts with 02 to form a 4a-peroxyflavin.This complex interacts with aldehyde to form a highly stable intermediate, which decays slowly, resulting in the emission of light along with the oxidation of the substrates” (Meighen, 1991).

FMNH2 + RCHO + 02 FMN + H20 + RCOOH + hv (490 nm)

This light-emitting reaction is highly specific for FMNH2. Any change to the structure of this molecule will significantly decrease the activity, and therefore reduce the amount of light that will be produced.Substrates must be supplied regularly to bacterial luciferase in order to emit light for a prolonged period of time.The enzymes that are necessary to replenish the substrate are coded by the luxCDE gene.


Genetics of Bioluminescence
A very complex signal transduction system induces the expression of bioluminescence. Bioluminescence is controlled by the lux system, also called the lux operon. The lux operon codes for proteins needed for bioluminescence. Five lux genes, luxCDABE, that have been identified as active in the emission of light are located on the lux operon.

Lux gene
Function
luxA
Code for luciferase
luxB
Code for luciferase
luxC
code for the reductase and transferase polypeptides of the fatty acid reductase
luxD
code for the reductase and transferase polypeptides of the fatty acid reductase
luxE
Codes for the synthetase enzyme
Table1: The functions of the luxCDABE

Two regulatory genes, LuxR and LuxI, are located upstream of the luxC gene on the lux operon. These two genes have to do with the function of the autoinducer, which is required for the induction of luminescence.
Lux gene
Function
LuxI
Production of the autoinducer
LuxR
Receptor for autoinducer
Table 2: The functions of luxI and luxR




http://www.photobiology.info/Lin_files/Fig25.png
http://www.photobiology.info/Lin_files/Fig25.png

The start site of transcription was identified by S1nuclease mapping, and was determined to be located between luxI and luxR.



How are the lux genes regulated?

Quorum sensing is a widespread regulatory mechanism in which gene expression of v. fischeri is dependent on cell density.

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“The term quorum sensing describes the ability of a microorganism to recognize and respond to other microorganisms in a population by detecting the concentration of self-produced intercellular molecules commonly known as autoinducers” (Qin et al, 2007). For v. fischeri, it is specifically acyl homoserine lactone-mediated quorum sensing. Quorum sensing creates a positive feedback loop based on the production of an autoinducer. LuxI and luxR genes are known to play important role in this process. “The LuxI acyl-HSL synthase and the LuxR transcriptional activator constitute the quorum-sensing system, which controls the lux operon directly”(Qin et al, 2007). Induction of luminescence is brought about at an early stage by the production of an autoinducer, called 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). As stated previously, the luxI gene is responsible for synthesizing the autoinducer molecule. The specificity for this autoinducer is very high and is species specific. At early stages of growth, the autoinducer is produced at low levels. As the autoinducer accumulates, it will bind to inactive LuxR. LuxR is the receptor for the autoinducer and is a quorum sensing regulator. The C-terminal domain (CTD) of LuxR has a helix-turn-helix motif and binds to a region of DNA termed the lux box, which is 20 bp long and has a dyad symmetry centered at a position 42.5 bp upstream of the start site for the lux operon"(Qin et al, 2007). Upon binding of the autoinducer to LuxR, the LuxR-HSL complex will become activated and bind to this lux box.This initiates the transcription of the lux operon, which will proceed to the right of the binding site. This will result in the transcription of luxA and luxB genes, which will produce luciferase, as well as the transcription of luxI gene, which will produce more autoinducer, as well as activation of bioluminescence.



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The Positive feedback loop


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