Bacterial bioluminescence

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

B, bioluminescent bacterial system on nylon L, bioluminescent firefly system on nylon M, bioluminescent firefly system on methacrylate beads r-LM, recombinant firefly luciferase on methacrylate beads r-LN, recombinant firefly luciferase on nylon. 

Sun TS, Stahr HM. Evaluation and application of a bioluminescent bacterial genotox-icity test. fAOACInt. 1993 76(4) 893-898. 

Premkumar JR et al (2001) Antibody-based immobdization of bioluminescent bacterial sensor ceUs. Talanta 55(5) 1029-1038... 

CONSTRUCTION OF A NOVEL BIOLUMINESCENT BACTERIAL BIOSENSOR FOR REAL-TIME MONITORING OF CYTOTOXIC DRUGS ACTIVITY... 

Novel Bioluminescent Bacterial Biosensor for Real-Time Monitoring... 

Within the food industry, the use of self-bioluminescent bacterial reporters has been widespread and is well documented. In this study the use of self-bioluminescent food borne pathogens enables real time, non-destructive, in-situ monitoring of bacterial inactivation and recovery on food surfaces during and after heat treatment. 

Intensity of luminescence of cultures A. isolated in Krasnoyarsk area in 2007 was higher in comparison with the cultures from the Mushrooms Collection of BIN. We studied the effect of different concentrations (from 10 6 mg/mL to 1 mg/mL) of organic and inorganic toxicants on culture luminescence of 3 species of luminous fungi A. borealis, A. mellea and L. japonicus. It was shown that the mushroom A. mellea were more sensitive to action of toxicants than A. borealis and L. japonicus. The minimal concentration of benzoquinone resulted in a 21 % decrease in luminescence in comparison with the initial value, and a decrease in luminescence on 73 % was found at higher concentrations. The lowest concentration of copper ions which could be determined using this system was 10 5 mg/mL (19 % decrease in luminescence and a concentration 10"4 mg/mL caused a 33 % reduction of luminescence in comparison with the initial value) (Fig. 1,2). Lyophilized bioluminescent bacterial biosensors are popular nowadays because they are very sensitive even to micro-quantities of toxicants, and are very simple to... 

Push a 1-cm segment of intravenous catheter with its associated bioluminescent bacterial biofilm (e.g., Xen) through the incision into the tunnel (subcutaneous tunnel usually created is 1.5 cm in length). One catheter segment is inserted on each side of each animal. 

The use of bioluminescence has resulted in many different tests, including the use of naturally bioluminescent bacteria (27) aside from Microtox . The genes from the bacterium Vibrio fischeri, and other bioluminescent bacteria, have been cloned into bacteriophage (15) and into plasmids that are functional in a wide range of bacterial hosts, including E. coli and strains of Pseudomonas (3,21,22,29). Many different bacterial strains have been constructed with these plasmids and produce the lux or luc (fire-fly) genes, and, therefore, light, either under constitutive expression or by some method of induction. These bioluminescent bacterial strains have been used in... 

Bioluminescence can also be used as the basis for immunoassay. For example, bacterial luciferase has been used in a co-immobilized system to detect and quantify progesterone using a competitive immunoassay format (34), and other luciferase-based immunoassays have been used to quantify insulin, digoxin, biotin, and other clinically important analytes (35). 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Fig. 2.1 Mechanism of the bacterial bioluminescence reaction. The molecule of FMNH2 is deprotonated at N1 when bound to a luciferase molecule, which is then readily peroxidized at C4a to form Intermediate A. Intermediate A reacts with a fatty aldehyde (such as dodecanal and tetradecanal) to form Intermediate B. Intermediate B decomposes and yields the excited state of 4a-hydroxyflavin (Intermediate C) and a fatty acid. Light (Amax 490 nm) is emitted when the excited state of C falls to the ground state. The ground state C decomposes into FMN plus H2O. All the intermediates (A, B, and C) are luciferase-bound forms. The FMN formed can be reduced to FMNH2 in the presence of FMN reductase and NADH.

The reported quantum yields of the long-chain aldehydes in the luminescence reaction catalyzed by P. fischeri luciferase are 0.1 for dodecanal with the standard I (Lee, 1972) 0.13 for decanal with the standard I (McCapra and Hysert, 1973) and 0.15-0.16 for decanal, dodecanal and tetradecanal with the standard III (Shimomura et al., 1972). Thus, the quantum yield of long-chain aldehydes in the bacterial bioluminescence reaction appears to be in the range of 0.10-0.16. 

Overview. In the cultures of luminous bacteria, the bacterial cells are not luminous in their early stages of propagation. The formation of bioluminescence system is controlled by a substance called autoinducer that is produced by the cells of luminous bacteria. 

The autoinducer is a low molecular weight compound that is easily leached from the cells into the culture medium. By the propagation of bacterial cells, the concentration of the autoinducer in the medium increases. When the concentration reaches a certain threshold, the biosynthesis of bioluminescence system begins, and the bacteria become luminescent. The process is also called quorum sensing (Fuqua et al., 1994). 

Baldwin, T. O., et al. (1989). The complete nucleotide sequence of the lux regulon of Vibrio fischeri and the luxABN region of bacterial bioluminescence. ]. Biolumin. Chemilumin. 4 326-341. 

Balny, C., and Hastings, J. W. (1975). Fluorescence and bioluminescence of bacterial luciferase intermediates. Biochemistry 14 4719-4723. 

Eckstein, J. W., et al. (1990). A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-l. Proc. Natl. Acad. Sci. USA 87 1466-1470. 

Eckstein, J. W., Hastings, J. W., and Ghisla, S. (1993). Mechanism of bacterial bioluminescence. 4a,5-Dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics. Biochemistry 32 404 111. 

Eley, M., et al. (1970). Bacterial bioluminescence. Comparisons of bioluminescence emission spectra, the fluorescence of luciferase reaction mixtures, and the fluorescence of flavin cations. Biochemistry 9 2902-2908. 

Hastings, J. W. (1978). Bacterial and dinoflagellate luminescent systems. In Herring, P. J. (ed.), Bioluminescence in Action, pp. 129-170. Academic Press, London. 

Kurfuerst, M., Hastings, J. W., Ghisla, S., and Macheroux, P. (1984). Identification of the luciferase-bound flavin-4a-hydroxide as the primary emitter in the bacterial bioluminescence reaction. In Bray, R. C., et al. (eds.), Flavins Flavoproteins, Proc. Int. Symp. 8th, pp. 657-667. de Gruyter, Berlin. 

Lee, J. (1972). Bacterial bioluminescence. Quantum yields and stoichiometry of the reactants reduced flavin mononucleotide, dodecanal and oxygen, and of a product hydrogen peroxide. Biochemistry 11 3350-3359. 

Lee, J. (1993). Lumazine protein and the excitation mechanism in bacterial bioluminescence. Biophys. Chem. 48 149-158. 

Lee, J., O Kane, D. J., and Gibson, B. G. (1989). Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence. Biochemistry 28 4263-4271. 

---