Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence

The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR, sodA and sodB. We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H(2)O(2), and its very low capacity for organic hydroperoxides, thus extending MitoPY1′s specificity for H(2)O(2) in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) is more sensitive to organic peroxides than to H(2)O(2), confirming the lack of selectivity of H(2)DCF-DA to H(2)O(2). Treatment with organic peroxides and H(2)O(2) suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli, suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H(2)O(2) than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.