at a flow rate of 1.5 ml min21. Recombinant Fpx was eluted at approximately 0.14 M NaCl. The yellow-green-colored fractions were concentrated and desalted using a VIVASPIN 6 centrifugal filter device (Sartorius Stedim Biotech GmbH, Goettingen, Germany) and loaded onto a Mono Q HR 5/5 column (1 ml bed volume; GE Healthcare) equilibrated with buffer A. Proteins were eluted with a linear gradient of 0 to 0.5 M NaCl at a flow rate of 1.0 ml min21. Recombinant Fpx was eluted at approximately 0.06 M NaCl. Purified Fpx was stored at -80uC until use in a vial under anaerobic conditions by replacing the head space of cuvette with argon. N-terminal amino acid sequencing Recombinant Fpx was subjected to SDS-PAGE, followed by electroblotting at 1.6 mA/cm2 using the HorizBlot AE-6677P Semi-dry system (Atto, Tokyo, Japan) to a Sequi-BlotTM polyvinyl difluoride (PVDF) membrane (0.2 mm; Bio-Rad). The blotted protein was detected by Coomassie brilliant blue (CBB) staining. The N-terminal amino acid sequence of the detect protein was determined using a Procise 491HT protein sequencer (Applied Biosystems, Foster, CA, USA). Protein assay Protein concentrations were determined using a BCA Protein Assay kit (Pierce, Rockford, IL) with bovine serum albumin as the molecular standard. Gel filtration For the estimation of molecular mass, gel filtration was performed using a Superose 6 HR 10/30 column (GE Healthcare) equilibrated with buffer A containing 150 mM NaCl at flow rate of 0.5 ml min21. Chromatography was performed using the A¨KTA purifier system. Gel Filtration Standards (Bio-Rad) were used as molecular markers for the calibration. All measurements of standards and samples were performed in triplicate. Enzyme assays Peroxidase assays were routinely conducted anaerobically in a 200-ml reaction mixture containing 50 mM sodium phosphate [pH 6.0], 0.16 mM Fpx, 0.0037 mM FNR, 0.5 mM NADPH, and various concentrations of H2O2, t-BOOH, or CMOOH at 50uC for 20 min. The reaction was started by the addition of NADPH after a 1.5-min preincubation. The determination of hydroperox- ide concentrations was performed according to the report of Wolf et al. [40], with minor modification. Following the incubation period, 10-20 ml of the reaction mixtures were added to 1.5 ml FOX1 reagent (100 mM xylenol orange, 25 mM sulfuric acid, and 250 mM ammonium iron (II) sulfate) and then incubated at RT for 40 min, after which time color development was virtually completed. The remaining amount of hydroperoxide was monitored by measuring the absorbance at 560 nm. Due to the high sensitivity of the FOX1 detection reagent in the reaction mixtures incubated at a high temperature (70uC), the peroxidase assays were performed at 50uC and sorbitol was not included in the FOX1 reagent to minimize experimental error. A calibration curve was generated in each assay. FNR was purified as previously reported [41] and was stored at -80uC until use. SOD activity was detected using a SOD Assay Kit-WST (Dojindo Molecular Technologies) at 37uC according to the manufacturer’s instruc- tion. The rate of reduction of superoxide anion is linearly related to the xanthin oxidase activity, and is inhibited by SOD activity. The SOD activity was calculated based on the inhibition rate [42]. The rate of oxidation of reduced Fpx by O2 was determined using An Essential Antioxidant in H. thermophilus O2-free techniques. Completely reduced Fpx was prepared according to the report of Kawasaki et al [12]. Briefly, excess sodium dithionite was added to a Fpx solution (100 equiv. to Fpx), which was then desalted by passage through a PD-10 gel filtration column (Amersham, Japan) and eluted with 50 mM sodium phosphate buffer [pH 6.5] under nitrogen atmospheric conditions. O2-saturated buffer was prepared by purging with 100% O2 at 37uC for 15 min and sealing the buffer in an enclosed vial. A 1/5 volume of O2-saturated buffer (O2 concentration of 1065 mM at 37uC) was added to the eluted Fpx solution (48 mM final concentration), and the enzyme reaction was then conducted under the assumed condition of 213 mM dissolved O2. Homologous recombination of H. thermophilus Strain TK-6 was cultivated in a 500-mL shaking flask contain- ing 50 ml liquid medium on a shaker at 70uC under anaerobic conditions for 24 h. When the optical density at 540 nm of the culture reached 0.6, the cells were harvested by centrifugation from 2 ml of culture, resuspended in 20 mL of 100 mM CaCl2 solution, and then kept on ice for 30 min. Two mg of plasmid DNA (pFGD) in TE buffer was added to the suspension, which was further incubated on ice for 1 h. After a heat shock (70uC, 10 min), the cells were added to 500 mL liquid medium and cultivated in a 5-mL vial on a shaker at 70uC under anaerobic conditions for 6 h. The suspension was then spread on an inorganic solid medium plate containing 500 mg mL21 of kanamycin. The plates were placed in a desiccator and incubated under anaerobic conditions at 70uC for 7 days and gas-exchange was conducted daily. A single colony formed on a solid medium plate was inoculated into 3 ml liquid medium containing kanamycin in a test tube, which was then sealed with a butyl cap. Incubation with shaking was performed for 1–2 days until the medium became turbid. The disruption of the fpx gene was confirmed by determining the length of PCR-amplified DNA fragments containing the fpx and htk genes. Growth of H. thermophilus mutant under different oxic conditions H. thermophilus was precultivated in 10 ml inorganic medium in a 100-mL vial under aerobic, microaerobic or anaerobic conditions. As Dfpx was unable to grow under aerobic conditions, precultivation for aerobic conditions was performed anaerobically. The preculture was inoculated into 10 ml inorganic medium in a 100-mL vial, which was then incubated at 70uC with shaking under aerobic, microaerobic, and anaerobic conditions. Optical density at 540 nm was monitored at 2-h intervals. Initial optical densities at 540 nm were adjusted to 0.1 for the aerobic and anaerobic conditions, and to 0.05 for microaerobic conditions. Survival of H. thermophilus mutant in the presence of oxidative stress H. thermophilus was precultivated in 10 ml inorganic medium in a 100-mL vial under anaerobic conditions. The preculture was inoculated into 3 ml inorganic medium in a 30-mL test tube, which was then incubated at 70uC with shaking under anaerobic conditions in the presence of various concentrations of paraquat or H2O2. Optical density at 540 nm was monitored at 2-h intervals. Initial optical densities at 540 nm were adjusted to 0.5 to observe both bactericidal and growth profiles. Each optical density was calculated based on the measurements of duplicate samples, except the culture of Dfpx with 10 mM paraquat. PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34825