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      A new short-term toxicity assay using Aspergillus awamori with recombinant aequorin gene

      , 1 , 4 , 2 , 3

      BMC Microbiology

      BioMed Central

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          Abstract

          Background

          Most currently available short-term toxicity assays are based on bacterial cells. Therefore there is a need for novel eukaryotic microbial bioassays that will be relevant to higher eukaryotes such as animals and plants. Ca 2+ is a universal intracellular signalling molecule found in all organisms from prokaryotes to highly specialized animal cells. In fungi calcium has been demonstrated to be involved in control of many important processes. The recombinant aequorin gene from the jellyfish Aequorea victoria responsible for the expression of the Ca 2+-sensitive aequorin photoprotein has been cloned in the filamentous fungus Aspergillus awamori. This has allowed real life monitoring of [Ca 2+] c changes in living fungal cells. When subjected to different physico-chemical stimuli fungal cells respond by transiently changing the concentration of free Ca 2+ in the cytosol ([Ca 2+] c) and the pattern of these changes (Ca 2+ signature) is specific to each particular stimulus. Therefore it was interesting to investigate whether different environmental toxicants would be able to affect the pattern of [Ca 2+] c changes in a reproducible and dose dependant manner.

          Results

          Toxicity bioassay has been developed to monitor changes [Ca 2+] c of the recombinant fungus in the presence of toxicants representing heavy metals – Cr 6+ and Zn 2+ and a phenolic polar narcotic -3,5-DCP. The fungus responds to toxicants by a decrease in the amplitude of [Ca 2+] c response to 5 mM external CaCl 2 and an increase in Ca 2+ final resting levels and recovery time.

          Conclusion

          A novel toxicity bioassay utilizing eukaryotic cells has been developed based on filamentous fungi transformed with the recombinant aequorin gene. A range of parameters characterising changes in [Ca 2+] c has been identified, e.g. Amplitude, Length of Transient, Final Resting Level and Recovery Time. These parameters can be used to determine the toxicity of a range of chemicals to eukaryotic cells in a 96-well microtitre plate method.

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          Most cited references 29

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          Bioluminescence.

          Bioluminescence has evolved independently many times; thus the responsible genes are unrelated in bacteria, unicellular algae, coelenterates, beetles, fishes, and others. Chemically, all involve exergonic reactions of molecular oxygen with different substrates (luciferins) and enzymes (luciferases), resulting in photons of visible light (approximately 50 kcal). In addition to the structure of luciferan, several factors determine the color of the emissions, such as the amino acid sequence of the luciferase (as in beetles, for example) or the presence of accessory proteins, notably GFP, discovered in coelenterates and now used as a reporter of gene expression and a cellular marker. The mechanisms used to control the intensity and kinetics of luminescence, often emitted as flashes, also vary. Bioluminescence is credited with the discovery of how some bacteria, luminous or not, sense their density and regulate specific genes by chemical communication, as in the fascinating example of symbiosis between luminous bacteria and squid.
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            Molecular biology of bacterial bioluminescence.

             E A Meighen (1991)
            The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
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              A convenient growth medium for Neurospora (Medium N)

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                Author and article information

                Journal
                BMC Microbiol
                BMC Microbiology
                BioMed Central (London )
                1471-2180
                2005
                2 July 2005
                : 5
                : 40
                Affiliations
                [1 ]Institute of Cell & Molecular Biology, The University of Edinburgh, King's Buildings, Edinburgh, EH9 3JL, UK
                [2 ]Pollution Research Unit, Napier University, Merchiston Campus, Edinburgh, EH10 5DT, UK; Presently, Surfactant Technologies Ltd., C/o Avecia Fine Chemicals,, Grangemouth, FK3 8XG, UK
                [3 ]Pollution Research Unit, Napier University, Merchiston Campus, Edinburgh, EH10 5DT, UK
                [4 ]LUTESS Ltd., Orchard Brae House,, Edinburgh EH4 2HG, UK
                1471-2180-5-40
                10.1186/1471-2180-5-40
                1177953
                15992407
                Copyright © 2005 Kozlova et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Methodology Article

                Microbiology & Virology

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