title: LuxCDABE--transformed constitutively bioluminescent Escherichia coli for toxicity screening: comparison with naturally luminous Vibrio fischeri.
abstract: 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 Microtox™ 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 EC(50)) 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 EC(50) values highly correlated (log-log R(2) = 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 EC(50) values for Hg (0.04-0.05 mg/L) and highest EC(50) 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 R(2) = 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.
authors.affiliation: Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia. imbi.kurvet@kbfi.ee
authors.forename: Imbi
authors.initials: I
authors.lastname: Kurvet
authors.forename: Angela
authors.initials: A
authors.lastname: Ivask
authors.forename: Olesja
authors.initials: O
authors.lastname: Bondarenko
authors.forename: Mariliis
authors.initials: M
authors.lastname: Sihtmäe
authors.forename: Anne
authors.initials: A
authors.lastname: Kahru
mesh_terms: D000217:Acyltransferases / Q000378:metabolism; D048908:Aliivibrio fischeri / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D049449:Luminescence; D008628:Mercury / Q000737:chemistry; D019216:Metals, Heavy; D053758:Nanoparticles; D010088:Oxidoreductases / Q000378:metabolism; D020637:Photorhabdus / Q000378:metabolism*; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D019216:Metals, Heavy; D010088:Oxidoreductases; C055198:LuxC protein, Bacteria; D000217:Acyltransferases; C065928:luxD protein, Bacteria; D008628:Mercury