9116 Nucleic Acids Research, 2012, Vol. 40, No. 18 We asked what chemical property of phosphorothioa- tion leads to the DNA ds breakage (Dnd phenotype), and whether this susceptibility to ds DNA cleavage makes the host bacterial cell sensitive or insensitive to oxidative environment. MATERIALS AND METHODS Reagents PT-containing dinucleotides d(GPSA) (PS indicates the phosphorus–sulfur bond) and dinucleotide d(GA) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Per- acetic acid (PAA) was purchased from Shanghai Habo Company or obtained by mixing 1 M acetic acid with 1 M hydrogen peroxide (H2O2) (1 M Stock), and keeping the mixture dark at room temperature for 72 h. PAA was then kept in the dark and stored at 4 C until use. PAA– TAE was made by mixing 1% 1 M Stock PAA with TAE buffer (40 mM tris base adjusted to pH 7.5 using acetic acid and 0.8 mM ethylenediaminetetraacetic acid (EDTA)). H–phosphonate modiﬁed d(GHA) dinucleotide was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and released from resin using Ammonium hydroxide. Oxidation of PT modiﬁed DNA Plasmid DNA was linearized using XhoI or EcoRI ac- cording to protocols provided by NEB Co., Ltd. Genomic or linearized plasmid DNAs was dissolved in 1 TAE to a concentration of 300 ng/ml. A total amount of 30 ng/ml of DNA were incubated in PAA–TAE or elec- trophoresis buffer (sampled at the anode after running for 2 h) for 20 min. DNA was then precipitated using propanol according to Sambrook (13). The precipitated DNA was resuspended in TAE buffer for agarose gel elec- trophoresis analysis. A similar protocol was used for the H2O2 and PAA– TAE oxidation of the dinucleotide. The dinucleotides d(GA) and d(GPSA) were dissolved to a concentration of 400 mM in water (stock solution). An amount of 20 mM of dinucleotides were incubated in PAA–TAE, 10 mM PAA or H2O2 for 30 min. The reaction products were analyzed directly using LC–MS or lyophilized and kept at 20 C until use. For the generation of the hydrogen–phosphonate di- nucleotide d(GHA), 20 mM d(GPSA) was incubated in 30 mM PAA supplemented with 40 mM acetic acid at room temperature for 30 min. The reaction products were then lyophilized and kept at 20 C until use. Liquid chromatography mass spectrometry analysis Liquid chromatography–mass spectrometry (HPLC/MS) analysis was routinely carried out on an Agilent 1100 series LC/MSD Trap system. Ten microliters of ﬁnal reaction sample was loaded on Agilent’s C18 reversed phase column (250 4.6 mm, 5 mm) and the absorbance was monitored at 254 and 210 nm. HPLC was carried out at a ﬂow rate of 0.3 ml/min at room temperature. Eluent A was 0.1% acetic acid in water, and eluent B was 0.1% acetic acid in acetonitrile. HPLC conditions used were 1–13% buffer B for 35.5 min, 13–30% buffer B for another 20 min and then 30–1% buffer B for 5 min. Drying gas ﬂow: 10 l/min; nebulizer pressure: 30 psi; drying gas temperature: 325 C; capillary voltage: 3200 V (using the positive detection mode and scanning between 50 and 900 m/z for mass spectrometer analysis). The LC-electrospray (ES)-quadrupole time of ﬂight (QTOF) (Agilent 1200 system, Agilent Co., CA) equipped with a 2.1- by 30-mm C18 reverse-phase column (Agilent Co., CA) was used for high resolution analysis of the reaction products. The ﬂow rate was set at 0.2 ml/min. HPLC was carried out using a two-step program starting with 98% solvent A (0.1% formic acid in water) and 2% solvent B (0.1% formic acid in acetoni- trile) to 20% solvent B over 20 min and with 20 solvent B to 95% solvent B for 5 min. Tandem MS (MS/MS) was performed in the target-dependent mode under these con- ditions: drying gas ﬂow: 8 ml/min; nebulizer pressure: 35 psi; drying gas temperature: 300 C; sheath gas tempera- ture: 300 C; sheath gas ﬂow: 7 ml/min; capillary, 3500 V; skimmer, 65 V; OCT RF V, 750 V; fragmentor, 170 V; ion polarity: positive, and the mass scanning range was set at m/z 50–1500. The Agilent 1200 series LC system equipped with YMC ODS-AQ reversed phase column (250 4.6 mm, 5 mm) was used for quantiﬁcation of H2O2 reduced by d(GPSA). Absorbance was measured at 254 and 210 nm. Eluent A contained 0.1% acetic acid in water, and eluent B contained 0.1% acetic acid in acetonitrile under this condition: 1–12% buffer B for 10 min, 12–40% buffer B for 5 min and 40% of 1% buffer B for 5 min. H2O2 calibration curve was prepared from UV254nm absorption peak areas and H2O2 concentrations from 10 to 100 mM. Under this condition, the UV254 absorption peak area shows collinearity with H2O2 concentration. The calibration curves for d(GPSA) and other analytes were prepared similarly. Bacterial growth curves and H2O2 treatment Salmonella enterica serovar Cerro 87 wild-type and the dptB-E gene mutants were cultured in Luria Broth medium at 37 C overnight. The cultures were then diluted using LB medium to 1 108 cfu in a volume of 100 ml in Greiner 96-well plates. H2O2 was then added to the ﬁnal concentration of 0, 2.2, 4.4 and 8.8 mM. The growth curves were monitored using Gen5 (Biotek) from Gene Co. Ltd. The cultures were kept shaking at 37 C. OD600 was monitored for 12 h at 10-min intervals. For the analysis of DNA ds cleavage caused by H2O2 oxidization, S. enterica serovar Cerro 87 strains were cultured in LB at 37 C overnight. The cultures were then diluted by LB medium to OD600 1.5, and H2O2 was added to a ﬁnal concentration of 176, 528, 704 and 880 mM. The cultures were kept at 37 C for 2 h shaking at 220 rpm. Total DNA was then extracted and analyzed using 1% agarose gel electrophoresis. Software Quantity One from Bio-Rad company was used for the gel densi- tometry measurements.