title: Exploring the redox balance inside gram-negative bacteria with redox-sensitive GFP.
abstract: 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.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4; Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Joris
authors.initials: J
authors.lastname: van der Heijden
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
authors.forename: Stefanie L
authors.initials: SL
authors.lastname: Vogt
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Reynolds
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4; Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Jorge
authors.initials: J
authors.lastname: Peña-Díaz
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
authors.forename: Audrey
authors.initials: A
authors.lastname: Tupin
authors.affiliation: Laboratoire de Chimie Bactérienne, CNRS, Institut de Microbiologie de la Méditerranée, Marseille, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Aussel
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4; Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address: bfinlay@interchange.ubc.ca.
authors.forename: B Brett
authors.initials: BB
authors.lastname: Finlay
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D003470:Culture Media; D005456:Fluorescent Dyes / Q000737:chemistry*; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010544:Peroxidases / Q000378:metabolism; D017967:Zinc Compounds / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002712:Chlorides; D003470:Culture Media; D005456:Fluorescent Dyes; D017967:Zinc Compounds; D049452:Green Fluorescent Proteins; C016837:zinc chloride; D006861:Hydrogen Peroxide; D010544:Peroxidases; D002374:Catalase