Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in response to co-oxidation of chloroform


Transcriptional fusions with gfp driven by the promoter region of mbla (NE2571) in pPRO/mbla4 and clpB (NE2402) in pPRO/clpb7 were used to transform the ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718). The two genes were chosen because their transcript levels were found at much higher levels in N. europaea in response to oxidation of chloroform and chloromethane. In N. europaea transformed with pPRO/mbla4, green fluorescent protein (GFP)-dependent fluorescence increased from 3- to 18-fold above control levels in response to increasing chloroform concentrations (7 to 28 μM), and from 8- to 10-fold in response to increasing hydrogen peroxide concentrations (2.5–7.5 mM). The GFP-dependent fluorescence of N. europaea transformed with pPRO/clpb7 also showed an increase of 6- to 10-fold in response to chloroform (28–100 μM) but did not respond to H2O2. Our data provide proof of concept that biosensors can be fabricated in ammonia-oxidizing bacteria using “sentinel” genes that up-regulate in response to stress caused either by co-oxidation of chlorinated solvents or by the presence of H2O2. The fabricated biosensors had a consistent concentration-dependent response to chloroform; however, these did not respond to other chlorinated compounds that cause similar cellular stress.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Albano CR, Randers-Eichhorn L, Bentley WE, Rao G (1998) Green fluorescent protein as a real time quantitative reporter of heterologous protein production. Biotechnol Prog 14:351–354

  2. Applegate BM, Kehrmeyer SR, Sayler GS (1998) A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethybenzene, and xylene (BTEX) sensing. Appl Environ Microbiol 64:2730–2735

  3. Bedard C, Knowles R (1989) Physiology, biochemistry, and specific inhibitors of CH4, NH4 +, and CO oxidation by methanotrophs and nitrifiers. Microbiol Rev 53:68–84

  4. Bolton EK, Sayler GS, Nivens DE, Rochelle JM, Ripp S, Simpson ML (2002) Integrated CMOS photodetectors and signal processing for very low-level chemical sensing with the bioluminescent bioreporter integrated circuit. Sens Actuators B Chem 85:179–185

  5. Booth IR, Ferguson GP, Miller S, Li C, Gunasekera B, Kinghorn S (2003) Bacterial production of methylglyoxal: a survival strategy or death by misadventure? Biochem Soc Trans 31:1406–1408

  6. Brandt KK, Pedersen A, Sorensen J (2002) Solid-phase contact assay that uses a lux-marked Nitrosomonas europaea reporter strain to estimate toxicity of bioavailable linear alkylbenzene sulfonate in soil. Appl Environ Microbiol 68:3502–3508

  7. Capestany CA, Tribble GD, Maeda K, Demuth DR, Lamont RJ (2008) Role of the Clp system in stress tolerance, biofilm formation, and intracellular invasion in Porphyromonas gingivalis. J Bacteriol 190:1436–1446

  8. Cha HJ, Dalal NG, Bentley WE (2004) In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris. Biotechnol Lett 26:1157–1162

  9. Chatterjee I, Becker P, Grundmeier M, other authors (2005) Staphylococcus aureus ClpC is required for stress resistance, aconitase activity, growth recovery, and death. J Bacteriol 187:4488–4496

  10. Ely RL, Williamson KJ, Hyman MR, Arp DJ (1999) Cometabolism of chlorinated solvents by nitrifying bacteria: kinetics, substrate interactions, toxicity effects, and bacterial response. Biotechnol Bioeng 63:756

  11. Franke GC, Dobinsky S, Mack D, other authors (2007) Expression and functional characterization of gfpmut3.1 and its unstable variants in Staphylococcus epidermidis. J Microbiol Methods 71:123–132

  12. Gvakharia BO, Permina EA, Gelfand MS, Bottomley PJ, Sayavedra-Soto LA, Arp DJ (2007) Global transcriptional response of Nitrosomonas europaea to chloroform and chloromethane. Appl Environ Microbiol 73:3440–3445

  13. Hansen LH, Sorensen SJ (2000) Versatile biosensor vectors for detection and quantification of mercury. FEMS Microbiol Lett 193:123–127

  14. Harms H, Wells MC, van der Meer JR (2006) Whole-cell living biosensors—are they ready for environmental application? Appl Microbiol Biotechnol 70:273–280

  15. Hommes NG, Sayavedra-Soto LA, Arp DJ (1996) Mutagenesis of hydroxylamine oxidoreductase in Nitrosomonas europaea by transformation and recombination. J Bacteriol 178:3710–3714

  16. Hyman MR, Russell SA, Ely RL, Williamson KJ, Arp DJ (1995) Inhibition, inactivation, and recovery of ammonia-oxidizing activity in cometabolism of trichloroethylene by Nitrosomonas europaea. Appl Environ Microbiol 61:1480–1487

  17. Iizumi T, Mizumoto M, Nakamura K (1998) A bioluminescence assay using Nitrosomonas europaea for rapid and sensitive detection of nitrification inhibitors. Appl Environ Microbiol 64:3656–3662

  18. Jan W, Damecour R, Quon H (1999) Substances used or formed during the wastewater treatment process. In National Pollutant Release Inventory Guidance Manual for the Wastewater Sector: Canadian Environmental Protection Act (

  19. Keener WK, Arp DJ (1993) Kinetic studies of ammonia monooxygenase inhibition in Nitrosomonas europaea by hydrocarbons and halogenated hydrocarbons in an optimized whole-cell assay. Appl Environ Microbiol 59:2501–2510

  20. Lu C, Bentley WE, Rao G (2004) A high-throughput approach to promoter study using green fluorescent protein. Biotechnol Prog 20:1634–1640

  21. Lu C, Albano CR, Bentley WE, Rao G (2005) Quantitative and kinetic study of oxidative stress regulons using green fluorescent protein. Biotechnol Bioeng 89:574–587

  22. Miller WG, Leveau JH, Lindow SE (2000) Improved gfp and inaZ broad-host-range promoter-probe vectors. Mol Plant Microbe Interact 13:1243–1250

  23. Park S, Ely RL (2008) Genome-wide transcriptional responses of Nitrosomonas europaea to zinc. Arch Microbiol 189:541–548

  24. Princic A, Mahne II, Megusar F, Paul EA, Tiedje JM (1998) Effects of pH and oxygen and ammonium concentrations on the community structure of nitrifying bacteria from wastewater. Appl Environ Microbiol 64:3584–3590

  25. Radniecki TS, Dolan ME, Semprini L (2008) Physiological and transcriptional responses of Nitrosomonas europaea to toluene and benzene inhibition. Environ Sci Technol 42:4093–4098

  26. Rasche ME, Hyman MR, Arp DJ (1991) Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: cometabolic inactivation of ammonia monooxygenase and substrate specificity. Appl Environ Microbiol 57:2986–2994

  27. Ron EZ (2007) Biosensing environmental pollution. Curr Opin Biotechnol 18:252–256

  28. Wei X, Yan T, Hommes NG, other authors (2006) Transcript profiles of Nitrosomonas europaea during growth and upon deprivation of ammonia and carbonate. FEMS Microbiol Lett 257:76–83

  29. Wood NJ, Sørensen J (2001) Catalase and superoxide dismutase activity in ammonia-oxidising bacteria. FEMS Microbiol Ecol 38:53–58

  30. Zellmeier S, Schumann W, Wiegert T (2006) Involvement of Clp protease activity in modulating the Bacillus subtilis sigmaw stress response. Mol Microbiol 61:1569–1582

Download references


This research was funded through an NSF biocomplexity grant number 0412711. The authors thank the laboratory of L.W. Lindow for the pPROBE-NT vector used in this work. We wish to also thank C. Bayne for the use of his laboratory facilities and R. Bender for his help. We thank S. Long for her technical assistance funded through an undergraduate internship from the Subsurface Biosphere Initiative at OSU.

Author information

Correspondence to Luis A. Sayavedra-Soto.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gvakharia, B.O., Bottomley, P.J., Arp, D.J. et al. Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in response to co-oxidation of chloroform. Appl Microbiol Biotechnol 82, 1179–1185 (2009).

Download citation


  • Nitrosomonas europaea
  • Biosensor
  • Green fluorescent protein
  • Chloroform
  • Hydrogen peroxide