title: Electron Paramagnetic Resonance (EPR) Spectroscopy to Detect Reactive Oxygen Species inStaphylococcus aureus.
abstract: Under aerobic conditions,Staphylococcus aureus(S. aureus) primarily metabolizes glucose to acetic acid. Although normallyS. aureusis 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 inS. aureusundergoing 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 forS. aureus, the methods described here should be easily adapted for other bacterial species. This protocol is adapted from Thomaset al.(2014) and Thomaset al.(2010).
authors.affiliation: Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, USA.
authors.forename: Vinai Chittezham
authors.initials: VC
authors.lastname: Thomas
authors.affiliation: Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, USA.
authors.forename: Sujata S
authors.initials: SS
authors.lastname: Chaudhari
authors.affiliation: Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, USA.
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Jones
authors.affiliation: Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Zimmerman
authors.affiliation: Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, USA.
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Bayles