The

results shown represent the average and standard errors of at least three experiments. Western blot analysis of recombinant Y. pestis HSP inhibition topoisomerase I expression Exponential phase cultures were treated with indicated concentration of arabinose for 2 or 2.5 h. Cells were collected by centrifugation from volumes based on OD600 and resuspended in SDS gel sample buffer before boiling for 5 min and SDS page for total protein analysis. The coomassie blue stained gel was examined to confirm equal loading. For improved control of equal Selonsertib chemical structure loading in experiments using minimal media, total soluble proteins were prepared and quantitated by the BioRad Dc protein assay. Mouse monoclonal antibodies against E. coli topoisomerase I were used in Western blot analysis to detect the highly homologous Y. pestis topoisomerase I. Partially degraded Y. pestis topoisomerase I (YpTOP*) was also detected. Hydroxyl radicals formation assay BW27784 transformed with selleck vector or pInter was grown to exponential phase in LB before treatment with 250 ng/ml norfloxacin, or left untreated as control. After the indicated time, hydroxyl radicals were measured with the fluorescent reporter dye, 3′(p-hydroxyphenyl) fluorescence (HPF) in a FACScan flow cytometer

(Becton Dickinson) [13]. Conclusions We demonstrated that titration of the E. coli transcription factors FNR and PurR by plasmid clones with the transcription factor binding sites can confer resistance to cell killing

mediated by mutant topoisomerase I cleavage complex and norfloxacin acting on DNA gyrase. Our study showed that perturbation of the global regulator FNR and PurR Cyclin-dependent kinase 3 function as well as increase in purine nucleotide availability, could affect the oxidative damage cell death pathway initiated by topoisomerase cleavage complex. The metabolic state of the cell is likely to be an important factor for the bactericidal outcome in this cell death pathway. Acknowledgements We acknowledge NBRP-E. coli at NIG and the Yale E. coli Genetic Stock Center for providing strains. This study was funded by NIH grant R01AI069313 to Yuk-Ching Tse-Dinh. References 1. Schoeffler AJ, Berger JM: DNA topoisomerases: harnessing and constraining energy to govern chromosome topology. Q Rev Biophys 2008,41(1):41–101.PubMedCrossRef 2. Liu LF: DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 1989, 58:351–375.PubMedCrossRef 3. Bernard P, Couturier M: Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J Mol Biol 1992,226(3):735–745.PubMedCrossRef 4. Hooper DC: Quinolone mode of action. Drugs 1995,49(Suppl 2):10–15.PubMedCrossRef 5. Drlica K: Mechanism of fluoroquinolone action. Curr Opin Microbiol 1999,2(5):504–508.PubMedCrossRef 6. Goswami M, Mangoli SH, Jawali N: Involvement of reactive oxygen species in the action of ciprofloxacin against Escherichia coli . Antimicrob Agents Chemother 2006,50(3):949–954.PubMedCrossRef 7.