A Novel MntH Repressor in D. radiodurans Figure 5. His98 plays an important role in DNA binding activity of DR2539. (A) 10 ml cell dilution was dripped on the TGY plate to which 6 mM of Mn(II) had been added. The cells were cultured for 3 days. (B) H98Y mutant and wild-type DR2539 proteins were incubated with p1709b at different concentrations of Mn(II). (C) Quantification of the fluorescence intensity of binding bands was performed using ImageJ. *, P,0.05. doi:10.1371/journal.pone.0035057.g005 buffer (50 mM Tris-HCl (pH 8.0), 0.22 mg/ml lysozyme, 100 mM PMSF, and 10 mM DTT) and sonicated on ice. The lysate was centrifuged at 39,0006g for 30 min at 4uC. The clear supernatant containing the soluble protein was mixed with Ni-NTA slurry (QIAGEN) and rocked for 60 min at 4uC. The mixture was poured through a Ni-NTA column and washed with 50 ml of phosphate wash buffer (20 mM imidazole, 1 M NaCl, 10% glycerol, 50 mM Tris-HCl, pH 8.0). Purified His-IF-DR2539 protein was eluted with elution buffer (500 mM imidazole, 1 M NaCl, 10% glycerol, 50 mM Tris-HCl, pH 8.0). The imidazole and excess NaCl were removed by dialysis in 50 mM Tris-HCl, plus 300 mM NaCl and 10% glycerol. Then, the TEV protease, 5 mM EDTA and 0.1% DTT were added to the purified protein solution and digested overnight at 4uC. Imidazole (10 mM) was added to the digested protein suspension and the IF was removed by a Ni-NTA column. The protein was further purified with ion exchange chromatography and gel filtration chromatography. The DR0865 was purified using the same method. The purity of the proteins was analyzed with the SDS-PAGE (12% acrylamide) (Fig. S1). Electrophoretic mobility shift assay Electrophoretic mobility shift assays (EMSAs) were performed as previously described with some modifications [28]. Briefly, FITC-labeled promoters were amplified from the genome of D. radiodurans strain R1 with primer pairs MHF/MHRa, MHF/ MHRb, and MAF/MAR (listed in Table S2). The (NH4)2Fe(SO4) solution used for the DNA binding assay was prepared immediately before use to limit oxidation. The binding buffer contained 30 mM Tris-HCl (pH 6.8), 50 mM NaCl, and 5% glycerol. The DR2539 protein was incubated for 30 min at 4uC in EMSA binding buffer supplemented with 50 mg/mL calf thymus DNA, 50 mg/mL bovine serum albumin, Mn2+ or Fe2+ (at various concentrations), and 10 nM FITC-labeled promoter DNA frag- ments containing the promoter. After incubation, the reaction mixtures were analyzed on 5% nondenaturing polyacrylamide gels. The labeled DNA was detected using a LAS-3000 cooled CCD camera system (Fujifilm). Construction of promoter-lacZ transcriptional fusions and b-galactosidase assays D. radiodurans dr1709 gene promoter was amplified by PCR using the primer pair HPF/HPR (Table S2). The PCR products were ligated to pMD18-T simple vector for sequencing (Sango Biotech (Shanghai) Co., Ltd). Then, the dr1709 gene promoter was digested with BglII and SpeI and further ligated into pRADZ vector yielding pRAZH [29]. The pRAZH vector was transformed into the dr2539 mutant as described previously [4,24]. For b-galactosidase activity assays, cells were grown to OD600<1.0 and permeabilized with Triton X100 as described previously [30]. b-galactosidase activity was assayed using standard methods. Activity was expressed in Miller units, defined as 1,000 times the scattering-corrected OD420 per OD600 of cells per minute. Real-time quantitative PCR Real-time quantitative PCR was used to determine whether DR2539 modulates the transcription of dr1709. In short, cells were grown to OD600<0.2 and then 10 mM, 20 mM, or 30 mM MnCl2 was added. Cells were harvested by centrifugation at 4000 rpm at 4uC after the OD600 was 0.4–0.45. Total RNA was extracted from 200 ml of cell cultures using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA, U.S.) after liquid nitrogen grinding and then treated with 10 units of RNase free DNase I (Promega, Mannheim, Germany). First-strand cDNA synthesis was carried out in 20 ml reaction mixtures containing 1 mg of each PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35057