Exploring the redox balance inside gram-negative bacteria with redox-sensitive GFP

• Published in: Free radical biology & medicine 2016-02, Vol.91, p.34-44
• Main Authors: van der Heijden, Joris, Vogt, Stefanie L., Reynolds, Lisa A., Peña-Díaz, Jorge, Tupin, Audrey, Aussel, Laurent, Finlay, B. Brett
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; Bacterial Proteins - metabolism; Bacteriology; Biochemistry, Molecular Biology; Catalase - metabolism; Cellular Biology; Chlorides - pharmacology; Culture Media; Environmental Sciences; Fluorescent Dyes - chemistry; Genetics; Gram-Negative Bacteria - drug effects; Gram-Negative Bacteria - metabolism; Green Fluorescent Proteins - chemistry; Hydrogen Peroxide - metabolism; Hydrogen-Ion Concentration; Immunology; Life Sciences; Microbial Sensitivity Tests; Microbial Viability - drug effects; Microbiology and Parasitology; Oxidation-Reduction; Oxidative Stress; Peroxidases - metabolism; Zinc Compounds - pharmacology
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2015.11.029

Aerobic bacteria are continuously fighting potential oxidative stress due to endogenous and exogenous reactive oxygen species (ROS). To achieve this goal, bacteria possess a wide array of defenses and stress responses including detoxifying enzymes like catalases and peroxidases; however until now, the dynamics of the intra-bacterial redox balance remained poorly understood. Herein, we used redox-sensitive GFP (roGFP2) inside a variety of gram-negative bacteria to study real-time redox dynamics immediately after a challenge with hydrogen peroxide. Using this biosensor, we determined the individual contributions of catalases and peroxidases and found that each enzyme contributes more to rapid detoxification or to prolonged catalytic activity. We also found that the total catalytic power is affected by environmental conditions. Additionally, using a Salmonella strain that is devoid of detoxifying enzymes, we examined endogenous ROS production. By measuring endogenous ROS production, we assessed the role of oxidative stress in toxicity of heavy metals and antibiotics. We found that exposure to nickel induced significant oxidative stress whereas cobalt (which was previously implicated to induce oxidative stress) did not induce ROS formation. Since a turbulent debate evolves around oxidative stress as a general killing mechanism by antibiotics (aminoglycosides, fluoroquinolones and β-lactams), we measured oxidative stress in bacteria that were challenged with these antibiotics. Our results revealed that antibiotics do not induce ROS formation in bacteria thereby disputing a role for oxidative stress as a general killing mechanism. Together, our results expose how the intra-bacterial redox balance in individual microorganisms is affected by environmental conditions and encounters with stress-inducing compounds. These findings demonstrate the significant potential of roGFP2 as a redox biosensor in gram-negative bacteria to investigate redox dynamics under a variety of circumstances. •Redox-sensitive GFP can be used in numerous bacteria to determine redox dynamics.•Bacteria possess substantially different abilities to detoxify hydrogen peroxide.•The Michaelis Menten kinetics of detoxifying enzymes can be determined using roGFP2.•Nickel and zinc ions each contribute to oxidative stress in a different manner.•Antibiotics do not induce oxidative stress inside Salmonella.