Visualization of NO3−/NO2− Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System

• Published in: The Journal of biological chemistry Vol. 291; no. 5; pp. 2260 - 2269
• Main Authors: Hidaka, Masafumi, Gotoh, Aina, Shimizu, Taiki, Minamisawa, Kiwamu, Imamura, Hiromi, Uchida, Takafumi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29.01.2016; American Society for Biochemistry and Molecular Biology
• Subjects: bacterial signal transduction; Binding Sites; Biosensing Techniques; biosensor; Bradyrhizobium; Denitrification; fluorescence resonance energy transfer (FRET); Fluorescence Resonance Energy Transfer - methods; Gene Expression Regulation; HeLa Cells; Humans; in vivo imaging; Mutation; Nitrates - chemistry; Nitric Oxide; Nitrites - chemistry; Nitrogen - chemistry; nitrogen metabolism; NO3−/NO2− in physiology; Plant Roots - microbiology; Protein Interaction Mapping; Protein Structure and Folding; protein-protein interaction; Rhizobium; Signal Transduction; NO3−/NO2− in physiology; in vivo imaging; biosensor; fluorescence resonance energy transfer (FRET); nitrogen metabolism; bacterial signal transduction; protein-protein interaction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.687632

Nitrate (NO3−) and nitrite (NO2−) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO3−/NO2− exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO3−/NO2− for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO3−/NO2− in physiology), by employing NO3−/NO2−-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO3− and NO2−, and its FRET signal is changed in response to NO3−/NO2− in the micromolar range. Furthermore, both an increase and decrease in cellular NO3− concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO3−/NO2− dynamics in various organisms, including plants and animals.