Applied Microbiology and Biotechnology

, Volume 64, Issue 1, pp 46–52 | Cite as

An Escherichia coli biosensor capable of detecting both genotoxic and oxidative damage

  • R. J. Mitchell
  • M. B. GuEmail author
Original Paper


A two-plasmid dual reporter Escherichia coli biosensor was developed using the genes for bacterial bioluminescence and a mutant of the green fluorescent protein, GFPuv4. To achieve this, the two plasmids, which were derivatives of pBR322 and pACYC184, had compatible origins of replication and different antibiotic selection markers: ampicillin and tetracycline. The parent strains DK1 and ACRG43, each carrying a single plasmid with one of the fusion genes (strain DK1 harboring a fusion of the katG promoter to the lux operon while in ACRG43, the recA promoter was fused with the GFP gene), were responsive to oxidative and DNA damage, respectively, resulting in higher bioluminescence or fluorescence under the relevant toxic conditions. The responses of the dual sensor strain, DUAL22, to various toxicants, e.g., mitomycin C, N-methyl-N-nitro-N-nitrosoguanidine, hydrogen peroxide and cadmium chloride, were characterized and compared with the responses of the parent strains to the same chemicals. Finally, several chemical mixtures that cause various stress responses were tested to demonstrate the ability of this biosensor to detect specific stress responses within a multiple toxicity environment.


Green Fluorescent Protein Maximum Slope Cadmium Chloride Genotoxic Damage Bacterial Luciferase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors would like to thank Dr. F. Marincs for pLITE201, Dr. J.M. Thomson for pMECA and Dr. Robert LaRossa for pkatG::LuxCDABE, pRecA::LuxCDABE and pDEW201, and New England Biolabs for providing pACYC184. Robert would also like to thank the Lord Jesus for giving him the chance to do what he loves. This research was supported by the National Research Laboratory (2001 NRL) program of the Korea Institute of Science and Technology Evaluation and Planning (Project No. M10104000094–01J000004100). We are grateful for the support.


  1. Aldsworth TG, Sharman RL, Dodd CE (1999) Bacterial suicide through stress. Cell Mol Life Sci 56:378–383PubMedGoogle Scholar
  2. Asad NR, Asad LM, Silva AB, Felzenszwalb I, Leitao AC (1998) Hydrogen peroxide effects in Escherichia coli cells. Acta Biochim Pol 45:677–690PubMedGoogle Scholar
  3. Belkin S, Smulski DR, Vollmer AC, Van Dyk TK, LaRossa RA (1996) Oxidative stress detection with Escherichia coli harboring a katG′::lux fusion. Appl Environ Microbiol 62:2252–2256PubMedGoogle Scholar
  4. Bruce D (1991) Regulation of bacterial oxidative stress genes. Annu Rev Genet 25:315–337PubMedGoogle Scholar
  5. Demple B, Harrison L (1994) Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem 63:915–948CrossRefPubMedGoogle Scholar
  6. Engebrecht J, Silverman M (1984) Identification of genes and gene products necessary for bacterial bioluminescence. Proc Natl Acad Sci USA 81:4154–4158PubMedGoogle Scholar
  7. Feige U, Morimoto RI, Yahara I, Polla BS (1996) Transcriptional regulators for oxidative stress inducible genes in prokaryotes. In: Stress-inducible cellular responses. Deutsche Bibliothek Cataloging-in-Publication Data, Deutsche Bibliothek, Frankfurt, pp 240–243Google Scholar
  8. Greenberg JT, Monach P, Chou JH, Josephy PD, Demple B (1990) Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. Proc Natl Acad Sci USA 87:6181–6185PubMedGoogle Scholar
  9. Hidalgo E, Nunoshiba T, Demple B (1994) soxRS oxidative stress regulon of Escherichia coli. Methods Mol Genet 3:325–339Google Scholar
  10. Ito Y, Suzuki M, Husimi Y (1999) A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation. Biochem Biophys Res Commun 264:556–560PubMedGoogle Scholar
  11. Justus T, Thomas SM (1999) Evaluation of transcriptional fusions with green fluorescent protein versus luciferase as reporters in bacterial mutagenicity tests. Mutagenesis 14:351–356PubMedGoogle Scholar
  12. Kenyon CJ, Brent R, Ptashne M, Walker GC (1982) Regulation of damage-inducible genes in Escherichia coli. J Mol Biol 160:445–457PubMedGoogle Scholar
  13. Kim BC, Gu MB (2003) A bioluminescent sensor for high throughput toxicity classification. Biosens Bioelectron 18:1015–1021PubMedGoogle Scholar
  14. Kim YH, Lee J, Ahn JY, Gu MB, Moon SH (2002) Enhanced degradation of an endocrine-disrupting chemical, butyl benzyl phthalate, by Fusarium oxysporum f. sp. pisi cutinase. Appl Environ Microbiol 68:4684–4688CrossRefPubMedGoogle Scholar
  15. Kostrzynska M, Leung KT, Lee H, Trevors JT (2002) Green fluorescent protein-based biosensor for detecting SOS-inducing activity of genotoxic compounds. J Microb Methods 48:43–51Google Scholar
  16. Kozubek S, Ogievertskaya MM, Krasavin EA, Drasil V, Soska J (1990) Investigation of the SOS response of Escherichia coli after γ irradiation by the means of the SOS chromotest. Mutat Res 230:1–7PubMedGoogle Scholar
  17. Li VS, Choi D, Tang M, Kohn H (1995) Structural requirements for Mitomycin C DNA bonding. Biochemistry 34:7120–7126PubMedGoogle Scholar
  18. Marincs F, White DW (1994) Immobilization of Escherichia coli expressing the lux genes of Xenorhabdus luminescens. Appl Environ Microbiol 60:3862–3863PubMedGoogle Scholar
  19. Meighen EA (1991) Molecular biology of bacterial bioluminescence. Microbiol Rev 55:123–142PubMedGoogle Scholar
  20. Meighen EA, Dunlap PV (1993) Physiological, biochemical and genetic control of bacterial bioluminescence. Adv Microb Physiol 34:2–58Google Scholar
  21. Millard JT, Spencer RJ, Hopkins PB (1998) Effect of nucleosome structure on DNA interstrand cross-linking reactions. Biochemistry 37:5211–5219PubMedGoogle Scholar
  22. Min J, Kim EJ, LaRossa RA, Gu MB (1999) Distinct responses of a recA::luxCDABE Escherichia coli strain to direct and indirect DNA damaging agents. Mutat Res 442:61–68PubMedGoogle Scholar
  23. Min J, Lee CW, Moon SH, LaRossa RA, Gu MB (2000) Detection of radiation effects using recombinant bioluminescent Escherichia coli strains. Radiat Environ Biophys 39:41–45PubMedGoogle Scholar
  24. Nunoshiba T, Nishioka H (1991) 'Rec-lac test' for detecting SOS-inducing activity of environmental genotoxic substances. Mutat Res 245: 71–77Google Scholar
  25. Ptitsynm LR, Horneck G, Komova O, Kozubek S, Krasavin EA, Bonev M, Rettberg P (1997) A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells. Appl Environ Microbiol 63:4377–4384PubMedGoogle Scholar
  26. Quillardet P, Hofnung M (1993) The SOS chromotest: a review. Mutat Res 297:235–279PubMedGoogle Scholar
  27. Quillardet P, Frelat G, Nguyen VD, Hofnung M (1989) Detection of ionizing radiations with the SOS chromotest, a bacterial short-term test for genotoxic agents. Mutat Res 216:251–257PubMedGoogle Scholar
  28. Sassanfar M, Roberts JW (1990) Nature of the SOS-inducing signal in Escherichia coli: the involvement of DNA replication. J Mol Biol 212:79–96PubMedGoogle Scholar
  29. Scheller F, Schmid RD (1991) Biosensors: fundamentals, technologies and applications. VCH, Weinheim, GermanyGoogle Scholar
  30. Stewart GSAB, Williams P (1992) lux genes and the applications of bacterial bioluminescence. J Genet Microbiol 138:1289–1300Google Scholar
  31. Storz G, Farr SB, Ames BN (1990) Bacterial defenses against oxidative stress. Trends Genet 6:363–368PubMedGoogle Scholar
  32. Thomson JM, Parrott WA (1998) pMECA: a cloning plasmid with 44 unique restriction sites that allows selection of recombinants based on colony size. Biotechniques 24:922–928PubMedGoogle Scholar
  33. Van Dyk TK, Belkin S, Vollmer AC, Reed TR, Smulski DR, LaRossa RA (1994) Fusions of Vibrio fischeri lux genes to Escherichia coli stress promoters: detection of environmental stress. In: Campbell AK, Kricka LJ, Stanley PE (eds) Bioluminescence and chemiluminescence: fundamentals and applied aspects. Wiley, Chichester, UK, pp 147–150Google Scholar
  34. Vogel EW, Asby J (1997) Structure-activity relationships: experimental approaches. In: Tardiff RG, Lohman PHM, Wogan GN (eds) Methods to assess DNA damage and repair: interspecies comparisons. Wiley, New York, pp 231–254Google Scholar
  35. Vollmer AC, Belkin S, Smulski DR, Van Dyk TK, LaRossa RA (1997) Detection of DNA damage by use of Escherichia coli carrying recA′::lux, uvrA′::lux, or alkA′::lux reporter plasmids. Appl Environ Microbiol 63:2566–2571PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.National Research Laboratory on Environmental Biotechnology, Department of Environmental Science and EngineeringKwangju Institute of Science and TechnologyKwangju South Korea

Personalised recommendations