We show that in vitro toxicity assay based on inhibition of the bioluminescence of recombinant Escherichia coli encoding thermostable luciferase from Photorhabdus luminescens is a versatile alternative to Vibrio fischeri MicrotoxTM test. Performance of two luxCDABE-transformed E. coli MC1061 constructs (pDNlux) and (pSLlux) otherwise identical, but having 100-fold different background luminescence was compared with the performance of V. fischeri. The microplate luminometer and a kinetic Flash-Assay test format was used that differently from Microtox test is also applicable for high throughput analysis. Toxic effects (30-s till 30-min EC50) of four heavy metals (Zn, Cd, Hg, Cu) and three organic chemicals (aniline, 3,5-dichloroaniline and 3,5-dichlorophenol) were studied. Both E. coli strains had comparable sensitivity and the respective 30-min EC50 values highly correlated (log-log R2 = 0.99; p < 0.01) showing that the sensitivity of the recombinant bacteria towards chemicals analyzed did not depend on the bioluminescence level of the recombinant cells. The most toxic chemical for all used bacterial strains (E. coli, V. fischeri) was mercury whereas the lowest EC50 values for Hg (0.04–0.05 mg/L) and highest EC50 values for aniline (1,300–1,700 mg/L) were observed for E. coli strains. Despite of that, toxicity results obtained with both E. coli strains (pSLlux and pDNlux) significantly correlated with V. fischeri results (log-log R2 = 0.70/0.75; p < 0.05/0.01). The use of amino acids (0.25%) and glucose (0.05%)-supplemented M9 medium instead of leucine-supplemented saline significantly (p < 0.05) reduced the apparent toxicity of heavy metals to both E. coli strains up to three orders of magnitude, but had little or no complexing effect on organic compounds. Thus, P. luminescens luxCDABE-transformed E. coli strains can be successfully used for the acute toxicity screening of various types of organic chemicals and heavy metals and can replace V. fischeri in certain cases where the thermostability of luciferase >30 °C is crucial. The kinetic Flash Assay test format of the bioluminescence inhibition assay facilitates high throughput analysis. The assay medium, especially in case of testing heavy metals should be a compromise: optimal for the viability/luminescence of the recombinant test strain and of minimum complexing potential.
References
[1]
Carere, A; Stammati, A; Zucco, F. In vitro toxicology methods: Impact on regulation from technical and scientific advancements. Toxicol. Lett 2002, 127, 153–160.
[2]
Russel, WMS; Burch, RL. The Principles of Humane Experimental Technique; Methuen: London, UK, 1959.
[3]
Claxton, LD; Umbuzeiro, GD; Demarini, DM. The Salmonella mutagenicity assay: The stethoscope of genetic toxicology for the 21st century. Environ. Health Perspect 2010, 118, 1515–1522.
[4]
Bulich, AA; Isenberg, DL. Use of the luminescent bacterial system for the rapid assessment of aquatic toxicity. ISA Trans 1981, 20, 29–33.
[5]
Kaiser, KLE; Devillers, J. Ecotoxicity of Chemicals to Photobacterium Phosphoreum; Handbooks of ecotoxicological data; Gordon and Breach Science Publishers S.A: Amsterdam, The Netherlands, 1994; Volume 2.
[6]
Urbanczyk, H; Ast, JC; Higgins, MJ; Carson, J; Dunlap, PV. Reclassification of Vibrio fischeri, Vibrio logei, Vibrio salmonicida and Vibrio wodanis as Aliivibrio fischeri gen. nov., comb. nov., Aliivibrio logei comb. nov., Aliivibrio salmonicida comb. nov. and Aliivibrio wodanis comb. nov. Int. J. Syst. Evol. Microbiol 2007, 57, 2823–2829.
[7]
Hastings, JW. Bioluminescence. In Cell Physiology Source Book; Sperelakis, N, Ed.; Academic Press: New York, NY, USA, 2001.
Hastings, JW. Bacterial bioluminescence: An overview. In Methods in Enzymology; DeLuca, MA, Ed.; Academic Press: New York, NY, USA, 1978; pp. 125–135.
[10]
Kahru, A. In vitro toxicity testing using marine luminescent bacteria Photobacterium phosphoreum: The Biotox？ test. ATLA-Altern. Lab. Anim 1993, 21, 210–215.
[11]
Kahru, A; Borchardt, B. Toxicity of 39 MEIC chemicals to bioluminescent photobacteria (the Biotox？ test): Correlation with other test systems. ATLA-Altern. Lab. Anim 1994, 22, 147–160.
[12]
Loibner, AP; Szolar, OHJ; Braun, R; Hirmann, D. Toxicity testing of 16 priority polycyclic aromatic hydrocarbons using Lumistox. Environ. Toxicol. Chem 2004, 23, 557–564.
[13]
Mortimer, M; Kasemets, K; Kurvet, I; Heinlaan, M; Kahru, A. Kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles and colored/turbid samples. . In Vitro 2008, 22, 1412–1417.
[14]
P？llumaa, L; Maloveryan, A; Trapido, M; Sillak, H; Kahru, A. Study of the environmental hazard caused by the oil-shale industry solid waste. ATLA-Altern. Lab. Anim 2001, 29, 259–267.
Wang, C; Yediler, A; Lienert, D; Wang, Z; Kettrup, A. Toxicity evaluation of reactive dyestuff, auxiliaries and selected effluents in textile finishing industry to luminescent bacteria Vibrio fisheri. Chemosphere 2002, 46, 339–344.
[17]
Manusadzianas, L; Balkelyte, L; Sadauskas, K; Blinova, I; P？llumaa, L; Kahru, A. Ecotoxicological study of Lithuanian and Estonian wastewaters: Selection of the biotests and correspondence between toxicity and chemical-based indices. Aquat. Toxicol 2003, 63, 27–41.
[18]
Kaiser, KL. Correlations of Vibrio fischeri bacteria test data with bioassay data for other organisms. Environ. Health Perspect 1998, 106, 583–591.
[19]
Kahru, A. Ecotoxicological tests in non-ecotoxicological research: Contribution to the three Rs. Use of luminescent photobacteria for evaluating the toxicity of 47 MEIC reference chemicals. ALTEX 2006, 23, 302–308.
[20]
ISO. Water quality—Kinetic Determination of the Inhibitory Effects of Sediment, Other Solids and Coloured Samples on the Light Emission of Vibrio fischeri (Kinetic Luminescent Bacteria Test)— ISO 21338:2010; International Organization for Standardization: Geneva, Switzerland, 2010.
[21]
Lappalainen, J; Juvonen, R; Nurmi, J; Karp, M. Automated color correction method for Vibrio fischeri toxicity test. Comparison of standard and kinetic assays. Chemosphere 2001, 45, 635–641.
[22]
Close, DM; Patterson, SS; Ripp, S; Baek, SJ; Sanseverino, J; Sayler, GS. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line. PLoS One 2010, 27, e12441.
[23]
Westerlund-Karlsson, A; Saviranta, P; Karp, M. Generation of thermostable monomeric luciferases from Photorhabdus luminescens. Biochem. Biophys. Res. Commun 2002, 296, 1072–1076.
[24]
Leedj？rv, A; Ivask, A; Virta, M; Kahru, A. Analysis of bioavailable phenols from natural samples by recombinant luminescent bacterial sensors. Chemosphere 2006, 64, 1910–1919.
[25]
Ivask, A; R？lova, T; Kahru, A. A suite of recombinant luminescent bacterial strains for the quantification of bioavailable heavy metals and toxicity testing. BMC Biotechnol 2009, 8, 9–41.
[26]
Colepicolo, P; Cho, KW; Poinar, GO; Hastings, JW. Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. Appl. Environ. Microbiol 1989, 55, 2601–2606.
[27]
Francis, KP; Joh, D; Bellinger-Kawahara, C; Hawkinson, MJ; Purchio, TF; Contag, PR. Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct. Infect. Immun 2000, 68, 3594–3600.
[28]
Andreu, N; Zelmer, A; Fletcher, T; Elkington, PT; Ward, TH; Ripoll, J; Parish, T; Bancroft, GJ; Schaible, U; Robertson, BD; et al. Optimisation of bioluminescent reporters for use with mycobacteria. PLoS One 2010, 24, e10777.
[29]
Hilpert, K; Hancock, RE. Use of luminescent bacteria for rapid screening and characterization of short cationic antimicrobial peptides synthesized on cellulose using peptide array technology. Nat. Protoc 2007, 2, 1652–1660.
[30]
Bondarenko, O; Rahman, PK; Rahman, TJ; Kahru, A; Ivask, A. Effects of rhamnolipids from Pseudomonas aeruginosa DS10–129 on luminescent bacteria: Toxicity and modulation of cadmium bioavailability. Microb. Ecol 2010, 59, 588–600.
[31]
Lee, J; Murphy, CL; Faini, GJ; Baucom, TL. Bacterial bioluminescence and its application to analytical procedures. In Liquid Scintillation Counting: Recent Developments; Stanley, PE, Scoggins, BA, Eds.; Academic Press: New York, NY, USA, 1974.
[32]
Sambrook, J; Fritsch, EF; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed ed.; Cold Spring Harbor Laboratory Press: New York, NY, USA, 1989.
[33]
Casadaban, MJ; Cohen, SN. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol 1980, 138, 179–207.
[34]
Leedj？rv, A; Ivask, A; Virta, M; Kahru, A. Analysis of bioavailable phenols from natural samples by recombinant luminescent bacterial sensors. Chemosphere 2006, 64, 1910–1919.
[35]
Deryabin, DG; Aleshina, ES. Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors. Appl. Biochem. Microbiol 2008, 44, 292–296.
[36]
Hassan, SH; Oh, SE. Improved detection of toxic chemicals by Photobacterium phosphoreum using modified Boss medium. J. Photochem. Photobiol. B 2010, 101, 16–21.
Vindimian, E. MSExcel macro REGTOX EV7.0.5.xls. 2005, Available online: http://eric.vindimian.9online.fr/ (accessed on 5 July 2011).
[39]
Nies, DH. Microbial heavy metal resistance. Molecular biology and utilization for biotechnology process. Appl. Microbiol. Biotechnol 1999, 51, 730–750.
[40]
Verhaar, HJM; van Leeuwen, CJ; Hermens, JLM. Classifying environmental pollutants. 1: Structure-activity relationships for prediction of aquatic toxicity. Chemosphere 1992, 25, 471–491.
[41]
Elnabarawy, MT; Robideau, RR; Beach, SA. Comparison of three rapid toxicity test procedures: Microtox, polytox, and activated sludge respiration inhibition. Toxic. Assess 1988, 3, 361–370.
[42]
Dom, N; Knapen, D; Benoot, D; Nobels, I; Blust, R. Aquatic multi-species acute toxicity of (chlorinated) anilines: Experimental versus predicted data. Chemosphere 2010, 81, 177–186.
[43]
Ribo, JM; Yang, JE; Huang, PM. Luminescent bacteria toxicity assay in the study of mercury speciation. Hydrobiologia 1989, 188/189, 155–162.
[44]
Utgikar, VP; Chaudhary, N; Koeniger, A; Tabak, HH; Haines, JR; Govind, R. Toxicity of metals and metal mixtures: Analysis of concentration and time dependence for zinc and copper. Water Res 2004, 38, 3651–3658.
[45]
Villaescusa, I; Pilar, M; Hosta, C; Martinez, M; Murat, JC. Toxicity of cadmium species on luminescent bacteria. Fresenius’ J. Anal. Chem 1996, 354, 566–570.
[46]
Denich, TJ; Beaudette, LA; Lee, H; Trevors, JT. Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes. J. Microbiol. Methods 2003, 52, 149–182.
[47]
Zhang, YM; Rock, CO. Membrane lipid homeostasis in bacteria. Nat. Rev. Microbiol 2008, 6, 222–233.
[48]
Gellert, G. Sensitivity and significance of luminescent bacteria in chronic toxicity testing based on growth and bioluminescence. Ecotoxicol. Environ. Saf 2000, 45, 87–91.
[49]
Riether, KB; Dollard, MA; Billard, P. Assessment of heavy metal bioavailability using Escherichia coli zntAp::lux and copAp::lux-based biosensors. Appl. Microbiol. Biotechnol 2001, 57, 712–716.
[50]
Chinalia, FA; Paton, GI; Killham, KS. Physiological and toxicological characterization of an engineered whole-cell biosensor. Bioresour. Technol 2008, 99, 714–721.
