KV.Complementation of E. coli sodC null mutantL929 murine fibroblast cells persistently infected with C. burnetii, Nine Mile, (RSA493), were fixed and processed as described previously [36]. Cells were fixed with 0.2 picric acid, 1 glutaraldehyde, 4 paraformaldehyde, 0.5 mM CaCl2 in phosphate buffered saline (PBS, 140 mM NaCl2, 3 mM KCl2, 2 mM KPO4, 10 mM NaPO4), pH 7.4 for 3 hr at room temperature while turning end over end. Cells were spun at 10,000 x g for 10 min and the pellet was incubated for 1 hr PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28993237 at 4 after resuspension in 50 mM NH4Cl, 250 mM sucrose, PBS. Cells were then centrifuged at 10,0000 x g for 10 min. Ammonium chloride was removed by resuspending the pellet in 3.5 sucrose, 0.5 mM CaCl2 in PBS pH 7.4 overnight at 4 . Phosphate buffers were removed by washing 4 x 15 min with 0.1 M maleate buffer, with 3.5 sucrose, pH 6.5. Post fixation staining was carried out with 2 uranyl acetate in sucrose/maleate buffer, pH 6.0 for 2 hr at 0 protected from light. Dehydration and infiltration into LR White was carried out a room temperature in 45 min. steps of: 50 acetone, 70 acetone, 90 acetone, 1:1 100 ethanol/LR White, 3:7 100 ethanol/LR White, 100 LR White, then fresh LR White overnight, second change of fresh LR White before samples were enclosed in gelatin capsules (Electron Microscopy Scences, Hatfield, PA). Polymerization was carried out at 50 for 24 hr. Silver to gold sections were collected on 300 mesh U0126MedChemExpress U0126-EtOH nickel grids (Electron Microscopy Sciences). All staining was carried out in the BioWaveComplementation studies were carried out using an E. coli sodC mutant (AS454) previously demonstrated to be more sensitive to killing by exogenous H2O2 than the wild type parental strain (AN387) during early stationary phase [8]. To genetically complement the sodC mutant, plasmid pREB102 was electroporated into AS454. Transformants were selected on LB agar plates containing ampicillin (150 g/ml). AN387, AS454, and AS454 (pREB102) were grown in LB broth or LB broth containing ampicillin (100 g/ml) overnight at 37 on a shaker, subcultured into fresh media at a starting OD600 of 0.01. In order achieve expression of sodC, AS454 and AS454 (pREB102) were either induced or not induced with 2 arabinose 4 hr prior to H2O2 challenge. At 45 min intervals aliquots for H2O2 challenges were removed, diluted 1:10,000 in PBS, and challenged with 2 mM H2O2 for 30 min while shaking at 37 as previously described [8]. Survival was determined as the percentage of colony counts (cfu/mL) from surviving bacteria after H2O2 treatment and from untreated bacteria by plating on LB plates with or without ampicillin (150 g/ml).Competing interests The authors declare that they have no competing interests. Authors’ contributions REB designed and conducted cloning, expression, Western blot, SOD activity gel experiments, and E. coli sodC mutant rescue experiments, and drafted the manuscript. KS performed immunogold labeling and electron microscopy experiments. RB performed the DDC SOD inhibition experiments. JES Participated in the study design and helped to draft the manuscript. All authors have read and approved the final manuscript. Acknowledgements This work was supported by the National Institutes of Health grant A1037744 (James E Samuel) and 3M Non-tenured Faculty Grant (Robert E Brennan). We especially thank Dr. James Imlay, University of Illinois at Urbana-Champaign for generously providing the E. coli sodC mutant strain (AS454) and wild type parent.