title: Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor.
abstract: BACKGROUND
  Molecular oxygen (O2) is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues.
  
  
  RESULTS
  We developed a genetically encoded Förster resonance energy transfer (FRET)-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen) consists of the yellow fluorescent protein (YFP) that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP). Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation.
  
  
  CONCLUSIONS
  FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (patho)physiological processes in living cells.
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Juelich Research Center, Wilhelm-Johnen-Straße, D-52425 Juelich, Germany.
authors.forename: Janko
authors.initials: J
authors.lastname: Potzkei
authors.forename: Martin
authors.initials: M
authors.lastname: Kunze
authors.forename: Thomas
authors.initials: T
authors.lastname: Drepper
authors.forename: Thomas
authors.initials: T
authors.lastname: Gensch
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D008164:Luminescent Proteins; D010100:Oxygen