{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T10:01:52Z","timestamp":1768903312150,"version":"3.49.0"},"reference-count":50,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2011,8,11]],"date-time":"2011-08-11T00:00:00Z","timestamp":1313020800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>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 &lt; 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\u20130.05 mg\/L) and highest EC50 values for aniline (1,300\u20131,700 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 &lt; 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 &lt; 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 &gt;30 \u00b0C 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.<\/jats:p>","DOI":"10.3390\/s110807865","type":"journal-article","created":{"date-parts":[[2011,8,11]],"date-time":"2011-08-11T11:29:45Z","timestamp":1313062185000},"page":"7865-7878","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":62,"title":["LuxCDABE\u2014Transformed Constitutively Bioluminescent Escherichia coli for Toxicity Screening: Comparison with Naturally Luminous Vibrio fischeri"],"prefix":"10.3390","volume":"11","author":[{"given":"Imbi","family":"Kurvet","sequence":"first","affiliation":[{"name":"Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia"},{"name":"Department of Food Processing, Tallinn University of Technology, Ehitajate tee 5, Tallinn 19086, Estonia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3451-7211","authenticated-orcid":false,"given":"Angela","family":"Ivask","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Olesja","family":"Bondarenko","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia"},{"name":"Department of Gene Technology, Tallinn University of Technology, Ehitajate tee 5, Tallinn 19086, Estonia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mariliis","family":"Sihtm\u00e4e","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia"},{"name":"Department of Chemical Engineering, Tallinn University of Technology, Ehitajate tee 5, Tallinn 19086, Estonia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4944-4900","authenticated-orcid":false,"given":"Anne","family":"Kahru","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2011,8,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/S0378-4274(01)00495-7","article-title":"In vitro toxicology methods: Impact on regulation from technical and scientific advancements","volume":"127","author":"Carere","year":"2002","journal-title":"Toxicol. Lett"},{"key":"ref_2","unstructured":"Russel, WMS, and Burch, RL (1959). The Principles of Humane Experimental Technique, Methuen."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1515","DOI":"10.1289\/ehp.1002336","article-title":"The Salmonella mutagenicity assay: The stethoscope of genetic toxicology for the 21st century","volume":"118","author":"Claxton","year":"2010","journal-title":"Environ. Health Perspect"},{"key":"ref_4","first-page":"29","article-title":"Use of the luminescent bacterial system for the rapid assessment of aquatic toxicity","volume":"20","author":"Bulich","year":"1981","journal-title":"ISA Trans"},{"key":"ref_5","unstructured":"Kaiser, KLE, and Devillers, J (1994). Ecotoxicity of Chemicals to Photobacterium Phosphoreum, Handbooks of ecotoxicological data; Gordon and Breach Science Publishers S.A."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2823","DOI":"10.1099\/ijs.0.65081-0","article-title":"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","volume":"57","author":"Urbanczyk","year":"2007","journal-title":"Int. J. Syst. Evol. Microbiol"},{"key":"ref_7","unstructured":"Sperelakis, N (2001). Cell Physiology Source Book, Academic Press."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1128\/mr.55.1.123-142.1991","article-title":"Molecular biology of bacterial bioluminescence","volume":"55","author":"Meighen","year":"1991","journal-title":"Microbiol. Rev"},{"key":"ref_9","unstructured":"DeLuca, MA (1978). Methods in Enzymology, Academic Press."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1177\/026119299302100216","article-title":"In vitro toxicity testing using marine luminescent bacteria Photobacterium phosphoreum: The Biotox\u2122 test","volume":"21","author":"Kahru","year":"1993","journal-title":"ATLA-Altern. Lab. Anim"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1177\/026119299402200303","article-title":"Toxicity of 39 MEIC chemicals to bioluminescent photobacteria (the Biotox\u2122 test): Correlation with other test systems","volume":"22","author":"Kahru","year":"1994","journal-title":"ATLA-Altern. Lab. Anim"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1897\/03-59","article-title":"Toxicity testing of 16 priority polycyclic aromatic hydrocarbons using Lumistox","volume":"23","author":"Loibner","year":"2004","journal-title":"Environ. Toxicol. Chem"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1412","DOI":"10.1016\/j.tiv.2008.02.011","article-title":"Kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles and colored\/turbid samples","volume":"22","author":"Mortimer","year":"2008","journal-title":"Toxicol. In Vitro"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1177\/026119290102900317","article-title":"Study of the environmental hazard caused by the oil-shale industry solid waste","volume":"29","author":"Maloveryan","year":"2001","journal-title":"ATLA-Altern. Lab. Anim"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1016\/S0956-053X(02)00009-0","article-title":"Ecotoxicological assessment of leachates from MSWI bottom ashes","volume":"22","author":"Lapa","year":"2002","journal-title":"Waste Manag"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1016\/S0045-6535(01)00086-8","article-title":"Toxicity evaluation of reactive dyestuff, auxiliaries and selected effluents in textile finishing industry to luminescent bacteria Vibrio fisheri","volume":"46","author":"Wang","year":"2002","journal-title":"Chemosphere"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/S0166-445X(02)00132-7","article-title":"Ecotoxicological study of Lithuanian and Estonian wastewaters: Selection of the biotests and correspondence between toxicity and chemical-based indices","volume":"63","author":"Manusadzianas","year":"2003","journal-title":"Aquat. Toxicol"},{"key":"ref_18","first-page":"583","article-title":"Correlations of Vibrio fischeri bacteria test data with bioassay data for other organisms","volume":"106","author":"Kaiser","year":"1998","journal-title":"Environ. Health Perspect"},{"key":"ref_19","first-page":"302","article-title":"Ecotoxicological tests in non-ecotoxicological research: Contribution to the three Rs. Use of luminescent photobacteria for evaluating the toxicity of 47 MEIC reference chemicals","volume":"23","author":"Kahru","year":"2006","journal-title":"ALTEX"},{"key":"ref_20","unstructured":"ISO (2010). Water quality\u2014Kinetic Determination of the Inhibitory Effects of Sediment, Other Solids and Coloured Samples on the Light Emission of Vibrio fischeri (Kinetic Luminescent Bacteria Test)\u2014 ISO 21338:2010, International Organization for Standardization."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1016\/S0045-6535(00)00579-8","article-title":"Automated color correction method for Vibrio fischeri toxicity test. Comparison of standard and kinetic assays","volume":"45","author":"Lappalainen","year":"2001","journal-title":"Chemosphere"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e12441","DOI":"10.1371\/journal.pone.0012441","article-title":"Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line","volume":"27","author":"Close","year":"2010","journal-title":"PLoS One"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1072","DOI":"10.1016\/S0006-291X(02)02052-1","article-title":"Generation of thermostable monomeric luciferases from Photorhabdus luminescens","volume":"296","author":"Saviranta","year":"2002","journal-title":"Biochem. Biophys. Res. Commun"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1910","DOI":"10.1016\/j.chemosphere.2006.01.026","article-title":"Analysis of bioavailable phenols from natural samples by recombinant luminescent bacterial sensors","volume":"64","author":"Ivask","year":"2006","journal-title":"Chemosphere"},{"key":"ref_25","first-page":"9","article-title":"A suite of recombinant luminescent bacterial strains for the quantification of bioavailable heavy metals and toxicity testing","volume":"8","author":"Ivask","year":"2009","journal-title":"BMC Biotechnol"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2601","DOI":"10.1128\/aem.55.10.2601-2606.1989","article-title":"Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound","volume":"55","author":"Colepicolo","year":"1989","journal-title":"Appl. Environ. Microbiol"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3594","DOI":"10.1128\/IAI.68.6.3594-3600.2000","article-title":"Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct","volume":"68","author":"Francis","year":"2000","journal-title":"Infect. Immun"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e10777","DOI":"10.1371\/journal.pone.0010777","article-title":"Optimisation of bioluminescent reporters for use with mycobacteria","volume":"24","author":"Andreu","year":"2010","journal-title":"PLoS One"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1652","DOI":"10.1038\/nprot.2007.203","article-title":"Use of luminescent bacteria for rapid screening and characterization of short cationic antimicrobial peptides synthesized on cellulose using peptide array technology","volume":"2","author":"Hilpert","year":"2007","journal-title":"Nat. Protoc"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"588","DOI":"10.1007\/s00248-009-9626-5","article-title":"Effects of rhamnolipids from Pseudomonas aeruginosa DS10\u2013129 on luminescent bacteria: Toxicity and modulation of cadmium bioavailability","volume":"59","author":"Bondarenko","year":"2010","journal-title":"Microb. Ecol"},{"key":"ref_31","unstructured":"Stanley, PE, and Scoggins, BA (1974). Liquid Scintillation Counting: Recent Developments, Academic Press."},{"key":"ref_32","unstructured":"Sambrook, J, Fritsch, EF, and Maniatis, T (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. [2nd ed]."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/0022-2836(80)90283-1","article-title":"Analysis of gene control signals by DNA fusion and cloning in Escherichia coli","volume":"138","author":"Casadaban","year":"1980","journal-title":"J. Mol. Biol"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1910","DOI":"10.1016\/j.chemosphere.2006.01.026","article-title":"Analysis of bioavailable phenols from natural samples by recombinant luminescent bacterial sensors","volume":"64","author":"Ivask","year":"2006","journal-title":"Chemosphere"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1134\/S0003683808030113","article-title":"Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors","volume":"44","author":"Deryabin","year":"2008","journal-title":"Appl. Biochem. Microbiol"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.jphotobiol.2010.06.006","article-title":"Improved detection of toxic chemicals by Photobacterium phosphoreum using modified Boss medium","volume":"101","author":"Hassan","year":"2010","journal-title":"J. Photochem. Photobiol. B"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1002\/(SICI)1521-3838(199804)17:02<131::AID-QSAR131>3.0.CO;2-L","article-title":"Quantitative structure-activity relationships of chemicals acting by non-polar narcosis\u2014theoretical considerations","volume":"17","author":"Zhao","year":"1998","journal-title":"Quant. Struct.-Act. Relat"},{"key":"ref_38","unstructured":"Vindimian, E Available online: http:\/\/eric.vindimian.9online.fr\/ (accessed on 5 July 2011)."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1007\/s002530051457","article-title":"Microbial heavy metal resistance. Molecular biology and utilization for biotechnology process","volume":"51","author":"Nies","year":"1999","journal-title":"Appl. Microbiol. Biotechnol"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1016\/0045-6535(92)90280-5","article-title":"Classifying environmental pollutants. 1: Structure-activity relationships for prediction of aquatic toxicity","volume":"25","author":"Verhaar","year":"1992","journal-title":"Chemosphere"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1002\/tox.2540030402","article-title":"Comparison of three rapid toxicity test procedures: Microtox, polytox, and activated sludge respiration inhibition","volume":"3","author":"Elnabarawy","year":"1988","journal-title":"Toxic. Assess"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.chemosphere.2010.06.059","article-title":"Aquatic multi-species acute toxicity of (chlorinated) anilines: Experimental versus predicted data","volume":"81","author":"Dom","year":"2010","journal-title":"Chemosphere"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/BF00027781","article-title":"Luminescent bacteria toxicity assay in the study of mercury speciation","volume":"188\/189","author":"Ribo","year":"1989","journal-title":"Hydrobiologia"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"3651","DOI":"10.1016\/j.watres.2004.05.022","article-title":"Toxicity of metals and metal mixtures: Analysis of concentration and time dependence for zinc and copper","volume":"38","author":"Utgikar","year":"2004","journal-title":"Water Res"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"566","DOI":"10.1007\/s0021663540566","article-title":"Toxicity of cadmium species on luminescent bacteria","volume":"354","author":"Villaescusa","year":"1996","journal-title":"Fresenius\u2019 J. Anal. Chem"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/S0167-7012(02)00155-0","article-title":"Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes","volume":"52","author":"Denich","year":"2003","journal-title":"J. Microbiol. Methods"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1038\/nrmicro1839","article-title":"Membrane lipid homeostasis in bacteria","volume":"6","author":"Zhang","year":"2008","journal-title":"Nat. Rev. Microbiol"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1006\/eesa.1999.1849","article-title":"Sensitivity and significance of luminescent bacteria in chronic toxicity testing based on growth and bioluminescence","volume":"45","author":"Gellert","year":"2000","journal-title":"Ecotoxicol. Environ. Saf"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1007\/s00253-001-0852-0","article-title":"Assessment of heavy metal bioavailability using Escherichia coli zntAp::lux and copAp::lux-based biosensors","volume":"57","author":"Riether","year":"2001","journal-title":"Appl. Microbiol. Biotechnol"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1016\/j.biortech.2007.01.041","article-title":"Physiological and toxicological characterization of an engineered whole-cell biosensor","volume":"99","author":"Chinalia","year":"2008","journal-title":"Bioresour. Technol"}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/11\/8\/7865\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:57:03Z","timestamp":1760219823000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/11\/8\/7865"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2011,8,11]]},"references-count":50,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2011,8]]}},"alternative-id":["s110807865"],"URL":"https:\/\/doi.org\/10.3390\/s110807865","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2011,8,11]]}}}