DNase I-treated and purified total RNA sample, combined with 3 mg of random hexamers. The Real-time PCR amplification used SYBR Premix Ex TaqTM (TaKaRa Biotechnology (Dalian) Co, Ltd, China) following the manufacturer’s instructions. All assays were performed using the STRATAGENE Mx3005PTM Real-time detection. Site-directed mutagenesis of dr2539 and complementation assays of the dr2539 mutant Site-directed mutagenesis of dr2539 was performed as described previously [31]. Briefly, dr2539 was amplified by PCR using the primer pair MRF/MRR (Table S2) and cloned into pMD-18 simple vector. It was amplified using M126F/M126R (Table S2) as a primer and pMD-18-dr2539 as a template. Amplified vector was treated with the enzyme DpnI, which cleaves only when its recognition site is methylated. Following digestion, the DNA fragments were cloned into the pMD-18 simple vector. The D126A site mutation was confirmed by DNA sequencing. The confirmed pMD18-D126A was digested with NdeI and BamHI and cloned into pRADK [32]. It was treated with the same enzymes to yield the plasmid vector pD126A. Identical methods were used to construct pH98Y using primers M98F/M98R (Table S2). Plasmid vectors (pRKR, pD126A, pH98Y) were transferred into the dr2539 mutant as described previously [4]. In order to confirm the identities of the complementation strains, the genome of the complementation strain was extracted and PCR was conducted using the genome as a template and primer pair PradF/ PradR (Table S2). The PCR product was sent for commercial sequencing. Manganese-sensitive assay Complementation strains were cultured in TGY until OD600<1.0. Cells were collected and diluted with PBS. Cell suspensions (10 mL) were dripped on plates with different concentrations of Mn(II). (A plate without Mn(II) was used as a control.) Plates were cultured for 3 days at 30uC. The results were captured using a scanner. For plate counting, 104 and 105 cells were inoculated on plates containing different concentrations of Mn(II) (Plates without Mn(II) were used as controls). The number of clones was determined after 3 days at 30uC. References 1. Battista JR, Earl AM, Park MJ (1999) Why is Deinococcus radiodurans so resistant to ionizing radiation? Trends Microbiol 7: 362–365. 2. Rainey FA, Nobre MF, Schumann P, Stackebrandt E, da Costa MS (1997) Phylogenetic diversity of the deinococci as determined by 16S ribosomal DNA sequence comparison. Int J Syst Bacteriol 47: 510–514. 3. Daly MJ (2011) Death by protein damage in irradiated cells. DNA Repair (Amst) 11: 12–21. 4. Hua Y, Narumi I, Gao G, Tian B, Satoh K, et al. (2003) PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem Biophys Res Commun 306: 354–360. 5. Kim JI, Sharma AK, Abbott SN, Wood EA, Dwyer DW, et al. (2002) RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization. J Bacteriol 184: 1649–1660. 6. Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, et al. (2007) Protein Oxidation Implicated as the Primary Determinant of Bacterial Radioresistance. PLoS Biol 5: e92. 7. Sun H, Xu G, Zhan H, Chen H, Sun Z, et al. (2010) Identification and evaluation of the role of the manganese efflux protein in Deinococcus radiodurans. BMC Microbiology 10: 319. 8. Daly MJ, Gaidamakova EK, Matrosova VY, Kiang JG, Fukumoto R, et al. (2010) Small-Molecule Antioxidant Proteome-Shields in Deinococcus radio- durans. PLoS One 5. 9. Leibowitz PJ, Schwartzberg LS, Bruce AK (1976) The in vivo association of manganese with the chromosome of Micrococcus radiodurans. Photochem Photobiol 23: 45–50. 10. Kehres DG, Zaharik ML, Finlay BB, Maguire ME (2000) The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese A Novel MntH Repressor in D. radiodurans Supporting Information Figure S1 Overexpression and purification of the DR2539, H98Y mutant and DR0865 in E.coli. (A) Schematic of the construction of the IF-dr2539 fusion expression vector. (B) DR2539 was expressed as a fusion protein with solubility partner IF, and purified following digestion by TEV protease (1%) and Ni-NTA chromatography. Samples were analyzed by SDS-PAGE (12% acrylamide). Lane 1, Fusion expressed DR2539. Lane 2, Fusion expressed DR2539 treated with TEV. Lane 3, Purified DR2539 protein. Lane 4, Purified IF protein which was expressed by pETIF. MW, molecular size markers. (C) and (D) H98Y mutant of DR2539 and DR0865 proteins analyzed by SDS-PAGE (12% acrylamide). (TIF) Figure S2 DR2539 binds to dr1709 promoter depending on the present of inverted repeat region and Mn(II). (A) dr1709 promoter (p1709a) incubated with DR2539 in the presence of 25 mM Mn(II). (B) p1709b was incubated with DR2539 in the presence of 100 nM protein and 1 mM EDTA. (C) dr1709 promoter (p1709b) incubated with DR0865 in the presence of 25 mM Mn(II). (TIF) Strains and plasmids used in this study. Primers used in this study. Table S1 (DOC) Table S2 (DOC) Acknowledgments We would like to thank Dr. David Waugh (National Cancer Institute, U.S.) for his kind gift of pRK793 for TEV protease expression. Author Contributions Conceived and designed the experiments: YJH LYW HXS MFL. Performed the experiments: HXS MFL. Analyzed the data: HXS MFL GZX. Contributed reagents/materials/analysis tools: LYW HC JDJ BT. Wrote the paper: HXS MFL. Purified the proteins needed in this work and did the site mutants: HXS GZX JDJ. transporters involved in the response to reactive oxygen. Mol Microbiol 36: 1085–1100. 11. Makui H, Roig E, Cole ST, Helmann JD, Gros P, et al. (2000) Identification of the Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter. Mol Microbiol 35: 1065–1078. 12. Makarova KS, Omelchenko MV, Gaidamakova EK, Matrosova VY, Vasilenko A, et al. (2007) Deinococcus geothermalis: the pool of extreme radiation resistance genes shrinks. PLoS One 2: e955. 13. Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, et al. (2004) Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance. Science 306: 1025–1028. 14. Agranoff D, Monahan IM, Mangan JA, Butcher PD, Krishna S (1999) Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family. J Exp Med 190: 717–724. 15. Qian Y, Lee JH, Holmes RK (2002) Identification of a DtxR-regulated operon that is essential for siderophore-dependent iron uptake in Corynebacterium diphtheriae. J Bacteriol 184: 4846–4856. 16. Que Q, Helmann JD (2000) Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Mol Microbiol 35: 1454–1468. 17. Blasius M, Shevelev I, Jolivet E, Sommer S, Hubscher U (2006) DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 39R59 exonuclease activity involved in radioresistance. Mol Microbiol 60: 165–176. 18. Patzer SI, Hantke K (2001) Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli. J Bacteriol 183: 4806–4813. PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35057