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Sensors 2011, 11(8), 7865-7878; doi:10.3390/s110807865

LuxCDABE—Transformed Constitutively Bioluminescent Escherichia coli for Toxicity Screening: Comparison with Naturally Luminous Vibrio fischeri

1
Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia
2
Department of Food Processing, Tallinn University of Technology, Ehitajate tee 5, Tallinn 19086, Estonia
3
Department of Gene Technology, Tallinn University of Technology, Ehitajate tee 5, Tallinn 19086, Estonia
4
Department of Chemical Engineering, Tallinn University of Technology, Ehitajate tee 5, Tallinn 19086, Estonia
*
Authors to whom correspondence should be addressed.
Received: 5 July 2011 / Revised: 4 August 2011 / Accepted: 8 August 2011 / Published: 11 August 2011
(This article belongs to the Special Issue Sensing of Toxic and Hazardous Metals in Various Environmental Media)
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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 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. View Full-Text
Keywords: bioluminescence; luxCDABE; toxicity; heavy metals; anilines; high throughput assay bioluminescence; luxCDABE; toxicity; heavy metals; anilines; high throughput assay
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MDPI and ACS Style

Kurvet, I.; Ivask, A.; Bondarenko, O.; Sihtmäe, M.; Kahru, A. LuxCDABE—Transformed Constitutively Bioluminescent Escherichia coli for Toxicity Screening: Comparison with Naturally Luminous Vibrio fischeri. Sensors 2011, 11, 7865-7878.

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