Abstract
Under aerobic conditions, Staphylococcus aureus (S. aureus) primarily metabolizes glucose to acetic acid. Although normally S. aureus is able to re-utilize acetate as a carbon source following glucose exhaustion, significantly high levels of acetate in the culture media may not only be growth inhibitory but also potentiates cell death in stationary phase cultures by a mechanism dependent on cytoplasmic acidification. One consequence of acetic acid toxicity is the production of reactive oxygen species (ROS). The present protocol describes the detection of ROS in S. aureus undergoing cell death by electron paramagnetic resonance (EPR) spectroscopy. Using 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) as a cell permeable spin probe, we demonstrate the detection of various oxygen radicals generated by bacteria. Although standardized for S. aureus, the methods described here should be easily adapted for other bacterial species. This protocol is adapted from Thomas et al. (2014) and Thomas et al. (2010).
Keywords: EPR, Staphylococcus aureus, Reactive oxygen species, Spectroscopy
Materials and Reagents
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Representative data
A B Figure 1. EPR detection of S. aureus ROS production. A. Following inoculation of S. aureus to an initial OD600 of 0.06, cultures were incubated aerobically (flask: volume ratio= 10:1, 250 rpm). Culture samples for EPR analysis were withdrawn at 3 h, 24 h and 72 h of bacterial growth. B. S. aureus culture samples (72 h) were treated with either 400 units of SOD, 20 mM DMTU, or vehicle (wild-type, WT) before addition of the spin probe, CMH. The basal extent of CMH autoxidation in KDD buffer (blue line) was determined as control.
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Acknowledgments
This work was funded by NIH grant nos. R01-A1038901 (KWB), PO1-AI083211 (KWB), R01- HL103942 (MCZ), and American Heart Association postdoctoral fellowship 12POST12080155 (VCT). The EPR spectroscopy core is supported, in part, by a NIH Center of Biomedical Research Excellence (COBRE) grant (1P30GM103335-01) awarded to the University of Nebraska's Redox Biology Center. This protocol is adapted from Thomas et al. (2014) and Thomas et al. (2010).
References
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