Abstract
Recent advances in the design of genetically encoded redox biosensors, such as redox-sensitive GFP (roGFP) have facilitated the real-time imaging of the intracellular redox potential in eukaryotic cells at high sensitivity and at spatiotemporal resolution. To increase the specificity of roGFP2 for the interaction with the glutathione (GSH)/ glutathione disulfide (GSSG) redox couple, roGFP2 has been fused to glutaredoxin (Grx) to construct the Grx-roGFP2 biosensor. We have previously designed the related Brx-roGFP2 redox biosensor for dynamic measurement of the bacillithiol redox potential (EBSH) in the human pathogen Staphylococcus aureus. Here, we describe the detailed method for measurements of the oxidation degree (OxD) of the Brx-roGFP2 biosensor in S. aureus using the microplate reader. In particularly, we provide details for determination of the EBSH changes during the growth and after oxidative stress. For future biosensor applications at the single cell level, we recommend the design of genome-encoded roGFP2 biosensors enabling stable expression and fluorescence in bacteria.•Brx-roGFP2 is specific for measurements of the bacillithiol redox potential in Staphylococcus aureus cells•Control samples for fully reduced and oxidized states of Brx-roGFP2 are required for calibration during OxD measurements•Easy to measure fluorescence excitation intensities at the 405 and 488 nm excitation maxima using microplate readers
Highlights
To survive under oxidative stress, S. aureus utilizes the low-molecular-weight (LMW) thiol bacillithiol (BSH), which serves as glutathione (GSH) surrogate to maintain the intracellular redox balance [1]
Disulfide bond formation between Cys147 and Cys204 of roGFP2 leads to ratiometric changes of the fluorescence intensities at the 405 nm and 488 nm excitation maxima [3]
We have previously constructed a Brx-roGFP2 fused biosensor to monitor BSH redox potential (EBSH) changes during the growth, under oxidative stress and after antimicrobial treatments in the wild type and different mutant backgrounds that are impaired in redox homeostasis (Fig. 1) [6,7,8,9]
Summary
Method for measurement of bacillithiol redox potential changes using the Brx-roGFP2 redox biosensor in Staphylococcus aureus. We have previously designed the related Brx-roGFP2 redox biosensor for dynamic measurement of the bacillithiol redox potential (EBSH) in the human pathogen Staphylococcus aureus. We describe the detailed method for measurements of the oxidation degree (OxD) of the Brx-roGFP2 biosensor in S. aureus using the microplate reader. Brx-roGFP2 is specific for measurements of the bacillithiol redox potential in Staphylococcus aureus cells Control samples for fully reduced and oxidized states of Brx-roGFP2 are required for calibration during OxD measurements Easy to measure fluorescence excitation intensities at the 405 and 488 nm excitation maxima using microplate readers. Method name: Brx-roGFP2 biosensor measurement method Keywords: Genetically encoded roGFP2 biosensors, Microplate reader measurements, Bacillithiol, Staphylococcus aureus Article history: Received 7 January 2020; Accepted 17 April 2020; Available online 24 April 2020. Hardware and materials necessary to reproduce the method are described in the Methods details
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