BIOLUMINESCENCE

SOIL MICROBIOLOGY

BIOL/CSES 4684

Bioluminescence is defined as "light emission by living organisms arising by exergonic chemical reactions" (309, Meighen).  Itis due to the substrate-enzyme complex of luciferin-luciferase within the cellular cytoplasm.  Luciferin refers to any light-emitting compound (the root lucifer means 'light-bringing' in Latin), and bacterial luciferase is an autoinducible enzyme.   Autoinducible means that above a certain concentration the luminescence operon encodes luciferase.  It is important to note that luciferin-luciferase complexes differ among species.

To the left and below is the generalized reaction that occurs in bioluminescent bacteria involving oxidation of a reduced flavin mononucleotide and a long-chain aliphatic aldehyde by molecular oxygen.  In this reaction luciferase acts as a mixed-function oxidase.

Bacterial luciferin is a reduced riboflavin phosphate, FMH₂.

The reaction requires the presence of free O₂ and different cofactors, NAD and FMN in bacteria.  Light emission is observed as blue-green light with a maximum light intensity at 490 nm, although wavelengths can range from 475 nm to 505 nm and to 530 nm in fungi. 

Bioluminescence is generated by various species.  The most familiar example is the firefly.  Certain microorganisms, fungi, jellyfish, insects (click beetle), algae (marine dinoflagellates), fish, and clams also bioluminesce. 

Terrestrial luminescent bacteria are primarily found in a symbiotic relationship with nematodes acting as a parasite for caterpillars.  Fungi can also light up the night.  Luminescent fungi produce a continuous (non-pulsing) light in the fruiting bodies and mycelium of some agarics.  Examples are Armillaria mellea and Mycena spp. 

There are molecular-based techniques that identify and quantify microbial populations in nature.  One method in particular is bioluminescence.  The purpose is to tag the target microbe with a biomarker that labels the cell with a unique phenotype that is easily detected.  In prokaryotes, the recombinant lux DNA can be transferred by transformation, transduction, and conjugation.  Depending on the bacteria, one method may be better than the others.  The luminescent system of X. luminescens may be the most suitable to transfer because it is a very stable lux system. 

Biomarkers are important in control and regulation of genetically engineered microbes (GEMs).  Until recently there has been no easy, cost-efficient method to detect, in a mixed sample, which organisms are modified and which are not.  Bioluminescence allows this to happen.

This is a KX Series camera from Apogee Instrument, Inc.

Following the gene probe methodology, the luxC-DABE structural genes are inserted into the microbe.  These genes are responsible for encoding bacterial luciferase.  Expression of the luciferase genes depend on many things, including the promoter proficiency, copy number of the recombinant lux DNA, stability and translation of the lux messenger RNA, and the folding and stability of luciferase. 

The microbe with the unique DNA sequence is selected.  It can be detected using charged couple device (CCD) imaging.  Once released into the soil environment the strain can be monitored by flow cytometry and/or luminometry. 

There are many advantages in using bioluminescence.  The assay is sensitive, rapid, and safe.  The linear relationship between the concentration of luciferase and the light signal occurs over a wide range.  The assay is low cost and convenient which are reasons for its rapid development in the scientific field.  There is also the possibility of in situ analysis in biological specimens.  Visualization and evaluation by image intensification are also advantages of bioluminescence. 

A notable advantage to bioluminescence is its wide use; there are many applications of bioluminescence.  It has been used (i) to detect low concentrations of dissolved oxygen, (ii) as a tool for mapping organism distribution patterns, and (iii) to quantify a correlation between the number of cells and the amount of ATP.  Bioluminescence has also been used in nucleic acid probe technology as a nonisotopic label. 

However, there are disadvantages of using bioluminescence.  First, light emission is a continuous process that shunts the respiratory pathway.  Second, luciferase is repressible by iron.

Govindjee, J. Amesz, and D.C. Fork. Light Emission by Plants and Bacteria. Academic Press, Inc.: New York, 1986. pp. 638.

Meighen, E. A. Bioluminescence, Bacterial. In: Encyclopedia of Microbiology, Volume 1, A-C. J. Lederberg, editor-in-chief. Academic Press, Inc.: New York, 1992. pp. 648.

Singleton, P. and D. Sainsbury. Dictionary of Microbiology & Molecular Biology, 2nd edition. John Wiley & Sons: New York, 1987. pp. 1019.

Van Dyke, K. Bioluminescence and Chemiluminescence: Instruments and Applications, Volume I. CRC Press, Inc.: Boca Raton, 1985. pp. 241.

Van Dyke, K. Bioluminescence and Chemiluminescence: Instruments and Applications, Volume II. CRC Press, Inc.: Boca Raton, 1985. pp. 276.

Biomarker Methods in Soil Using X. luminescens

Luminometry from Turner Designs

Harbor Branch Oceanographic Institution This site provides methods and equipment used in detection of marine bioluminescent populations.

Other Web Sites on Bioluminescence

World's Brightest Glowing Bay Enough dinoflagellates to read by.