Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

Abstract

BackgroundMolecular 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.ResultsWe 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.ConclusionsFluBO 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.

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

“…The steady-state concentration of CO within the cells of growing C. autoethanogenum cultures is predicted to be considerably lower than the O 2 concentrations of Ͻ10 M in cells of growing E. coli cultures (56). This prediction is based on the estimate that for the synthesis of 1 mol of ATP from ADP and phosphate at least 10 times more CO has to be oxidized to CO 2 in C. autoethanogenum than O 2 has to be reduced to H 2 O in E. coli, as deduced from the thermodynamics and ATP yields of the two energy metabolisms.…”

The photophysics of LOV-based fluorescent proteins — new tools for cell biology

“…Since the first description in 2007 8 LOV-based FPs were successfully applied in different hypoxic environments 13,14,[26][27][28][29] . Recently, a Förster resonance energy transfer (FRET) -based, genetically encoded biosensor called FluBO has been developed and characterized 30 . The oxygen biosensor consists of a FbFP as oxygen-independent FRET-donor domain and EYFP as oxygen-dependent FRET-acceptor domain.…”

Intravital microscopy of cancer: New insights into the spatiotemporal dynamics of the disease

“…Cell metabolism, which is deregulated in a number of cancers, can also be monitored using FRET biosensors. For instance, subcellular ATP FRET-biosensors are used to visualize intracellular energy transport following inhibition of glycolysis (7), while oxygen FRET-based biosensors can monitor intracellular oxygen transport and consumption (8). Wang and colleagues have developed a simultaneous multi-parametric analysis of distinct FRET biosensors including Src, RhoA and FAK and, thereby, demonstrated their interdependent regulation in response to mechanoreciprocity, which is known to drive tissue dysplasia and metastasis in a variety of cancers (9,10).…”