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 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.

Effect of low-dose ionizing radiation on luminous marine bacteria: radiation hormesis and toxicity

“…Now the luminous bacteria are widely used as a bioassay to monitor toxicity of water solutions (Shao et al, 2012;Girotti et al, 2008), e.g., acute toxicity of wastewaters contaminated with metals (Qua et al, 2013), organic oxidizers (Wang et al, 2009), or compositions of explosive nitrated organic compounds (Yea et al, 2011). Genes of bacterial enzymes responsible for the generation of light (lux genes) can be cloned from a bioluminescent microorganism into organism that is not naturally bioluminescent; light output can be monitored to provide information on the metabolic state, quantity of cells, and toxicity of the environment (Morrisseya et al, 2013;Kurvet et al, 2011;Roda et al, 2009Roda et al, , 2004Girotti et al, 2008).…”

The Effect of Composition of Different Ecotoxicological Test Media on Free and Bioavailable Copper from CuSO4 and CuO Nanoparticles: Comparative Evidence from a Cu-Selective Electrode and a Cu-Biosensor

“…Kasemets, personal communication; 8-h growth inhibition test;f[18]; 30-min bioluminescence inhibition test;g[29]; 30-min bioluminescence inhibition assay with recombinant E. coli ;h[21]; 30-min bioluminescence inhibition test; AA—Cas-amino acids.…”

Cells of Escherichia coli are protected against severe chemical stress by co-habiting cell aggregates formed by Pseudomonas aeruginosa

“…For the third requirement, we utilized bacterial bioluminescence as a viability marker for E. coli. This energydependent luciferase-catalyzed reaction is a direct measure for the ability of cells to perform energy conservation and is being used as a certified test for water quality evaluation with the non-engineered bioluminescent bacterium Aliivibrio fischeri (EN ISO 11348-3), as well as for toxicity screenings with engineered E. coli strains (Kurvet et al 2011). In addition, bioluminescence can be easily monitored in vivo in biofilms and cell aggregates.…”