Construction of a whole-cell gene reporter for the fluorescent bioassay of nitrate.

The development of a whole-cell fluorescence-based biosensor for nitrate is reported. The sensor is Escherichia coli transformed with a plasmid (pPNARGFP) in which the promoter and regulatory regions of the membrane-bound nitrate reductase narGHJI operon (Pnar) are fused to a gfp gene encoding green fluorescent protein (GFP). Pnar-gfp activity was measured at a range of nitrate concentrations using whole-cell GFP fluorescence. The bioassay conditions have been optimized so that the fluorescence intensity is proportional to the extracellular nitrate concentration. The developed bioassay has established that E. coli (pPNARGFP) can be used for the quantitative determination of nitrate in environmental waters without interference from other electron acceptors, e.g., nitrite, dimethyl sulfoxide, trimethylamine-N-oxide and fumerate, and azide, an inhibitor of redox-active proteins.

[1]  M. Winson,et al.  The construction and application of a lux‐based nitrate biosensor , 1997, Letters in applied microbiology.

[2]  Charles R. Martin,et al.  Nitrate Biosensor Based on the Ultrathin-Film Composite Membrane Concept , 1998 .

[3]  J. Demoss,et al.  Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals , 1987, Journal of bacteriology.

[4]  M. N. Hughes,et al.  Nitrite and nitrosyl compounds in food preservation. , 1999, Biochimica et biophysica acta.

[5]  S. Busby,et al.  Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K‐12 depends on DNA binding site arrangement , 1997, Molecular microbiology.

[6]  S. Cosnier,et al.  New electropolymerizable amphiphilic viologens for the immobilization and electrical wiring of a nitrate reductase , 1997 .

[7]  J. Guest,et al.  FNR and its role in oxygen-regulated gene expression in Escherichia coli. , 1990, FEMS microbiology reviews.

[8]  I. Willner,et al.  A Crosslinked Microperoxidase‐11 and Nitrate Reductase Monolayer on a Gold Electrode: An Integrated Electrically Contacted Electrode for the Bioelectrocatalyzed Reduction of NO3− , 1998 .

[9]  E. A. Garwood,et al.  Nitrate leaching from grassland , 1984, Nature.

[10]  Itamar Willner,et al.  Integration of a Reconstituted de Novo Synthesized Hemoprotein and Native Metalloproteins with Electrode Supports for Bioelectronic and Bioelectrocatalytic Applications , 1999 .

[11]  A. Fan,et al.  Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. , 1996, Regulatory toxicology and pharmacology : RTP.

[12]  G. Giovannoni,et al.  Urinary markers of disease activity in multiple sclerosis , 1998, Multiple sclerosis.

[13]  M. Kelm.  Nitric oxide metabolism and breakdown. , 1999, Biochimica et biophysica acta.

[14]  S. Yin,et al.  Use of a Green Fluorescent Protein-Based Reporter Fusion for Detection of Nitric Oxide Produced by Denitrifiers , 2003, Applied and Environmental Microbiology.

[15]  E. Sodergren,et al.  Roles of the narJ and narI gene products in the expression of nitrate reductase in Escherichia coli. , 1988, The Journal of biological chemistry.

[16]  V. Stewart,et al.  Discrimination between structurally related ligands nitrate and nitrite controls autokinase activity of the NarX transmembrane signal transducer of Escherichia coli K‐12 , 1997, Molecular microbiology.

[17]  J. Vincent,et al.  Nitric oxide production is enhanced in patients with heat stroke , 1999, Intensive Care Medicine.

[18]  R. Gunsalus,et al.  Activation of the Escherichia coli nitrate reductase (narGHJI) operon by NarL and Fnr requires integration host factor. , 1993, The Journal of biological chemistry.

[19]  D. A. Russell,et al.  Optical Biosensing of Nitrate Ions Using a Sol–Gel Immobilized Nitrate Reductase , 1997 .

[20]  R. Compton,et al.  Detection and determination of nitrate and nitrite: a review. , 2001, Talanta.

[21]  L. Kinzl,et al.  EARLY POSTTRAUMATIC INCREASE IN PRODUCTION OF NITRIC OXIDE IN HUMANS , 1998, Shock.

[22]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[23]  R D Schmid,et al.  Reporter gene bioassays in environmental analysis , 2000, Fresenius' journal of analytical chemistry.

[24]  C. Harris,et al.  Molecular epidemiology and the genetics of environmental cancer. , 1991, JAMA.

[25]  A. Tsezou,et al.  High nitrate content in drinking water: cytogenetic effects in exposed children. , 1996, Archives of environmental health.

[26]  J. Guest,et al.  FNR and its role in oxygen-regulated gene expression in Escherichia coli , 1990 .

[27]  L. Engstrand,et al.  Urinary nitrite: more than a marker of infection. , 1997, Urology.

[28]  S. Libes.  An introduction to marine biogeochemistry , 1992 .

[29]  J. Demoss,et al.  Phosphorylation and dephosphorylation catalyzed in vitro by purified components of the nitrate sensing system, NarX and NarL. , 1993, The Journal of biological chemistry.

[30]  J. Demoss,et al.  NarL‐phosphate must bind to multiple upstream sites to activate transcription the narG promoter of Escherichia coli , 1994, Molecular microbiology.

[31]  S. Busby,et al.  Definition of nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL , 1993, Molecular microbiology.

[32]  S A Glazier,et al.  Construction and characterization of nitrate reductase-based amperometric electrode and nitrate assay of fertilizers and drinking water. , 1998, Analytical chemistry.

[33]  P. Brimblecombe,et al.  Historical evidence for a dramatic increase in the nitrate component of acid rain , 1982, Nature.

[34]  J. Demoss,et al.  Location of sequences in the nar promoter of Escherichia coli required for regulation by Fnr and NarL. , 1988, The Journal of biological chemistry.

[35]  R. Brilli,et al.  Circulating methemoglobin and nitrite/nitrate concentrations as indicators of nitric oxide overproduction in critically ill children with septic shock. , 1997, Critical care medicine.

[36]  S. Lindow,et al.  An improved GFP cloning cassette designed for prokaryotic transcriptional fusions. , 1997, Gene.

[37]  G. Ramsay,et al.  Utility of wiring nitrate reductase by alkylpyrroleviologen-based redox polymers for electrochemical biosensor and bioreactor applications. , 1999, Analytical chemistry.

[38]  J B Kaneene,et al.  The effects of nitrate, nitrite and N-nitroso compounds on human health: a review. , 1993, Veterinary and human toxicology.

[39]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[40]  Rao Y. Surampalli,et al.  Nitrate Contamination of Groundwater: Sources and Potential Health Effects , 1992 .

[41]  Vaclav Smil,et al.  Global Population and the Nitrogen Cycle , 1997 .

[42]  I. Badr,et al.  Green fluorescent protein in the design of a living biosensing system for L-arabinose. , 1999, Analytical chemistry.

[43]  S. Cosnier,et al.  Amperometric detection of nitrate via a nitrate reductase immobilized and electrically wired at the electrode surface , 1994 .

[44]  G. Barrett,et al.  Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. , 2000, Chemical reviews.

[45]  V. Stewart,et al.  In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12. , 1992, Proceedings of the National Academy of Sciences of the United States of America.