Regulation of MntH by a Dual Mn(II)- and Fe(II)- Dependent Transcriptional Repressor (DR2539) in Deinococcus radiodurans Hongxing Sun1., Mingfeng Li1., Guangzhi Xu 1 , Huan Chen2,3, Jiandong Jiao1, Bing Tian1, Liangyan Wang1*, Yuejin Hua1* 1 Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China, 2 Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine & Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China, 3 Zhejiang Institute of Microbiology, Zhejiang Province, Hangzhou, China Abstract The high intracellular Mn/Fe ratio observed within the bacteria Deinococcus radiodurans may contribute to its remarkable resistance to environmental stresses. We isolated DR2539, a novel regulator of intracellular Mn/Fe homeostasis in D. radiodurans. Electrophoretic gel mobility shift assays (EMSAs) revealed that DR2539 binds specifically to the promoter of the manganese acquisition transporter (MntH) gene, and that DR0865, the only Fur homologue in D. radiodurans, cannot bind to the promoter of mntH, but it can bind to the promoter of another manganese acquisition transporter, MntABC. b- galactosidase expression analysis indicated that DR2539 acts as a manganese- and iron-dependent transcriptional repressor. Further sequence alignment analysis revealed that DR2539 has evolved some special characteristics. Site-directed mutagenesis suggested that His98 plays an important role in the activities of DR2539, and further protein-DNA binding activity assays showed that the activity of H98Y mutants decreased dramatically relative to wild type DR2539. Our study suggests that D. radiodurans has evolved a very efficient manganese regulation mechanism that involves its high intracellular Mn/Fe ratio and permits resistance to extreme conditions. Citation: Sun H, Li M, Xu G, Chen H, Jiao J, et al. (2012) Regulation of MntH by a Dual Mn(II)- and Fe(II)-Dependent Transcriptional Repressor (DR2539) in Deinococcus radiodurans. PLoS ONE 7(4): e35057. doi:10.1371/journal.pone.0035057 Editor: John R. Battista, Louisiana State University and A & M College, United States of America Received November 26, 2011; Accepted March 8, 2012; Published April 16, 2012 Copyright: 2012 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from National Natural Science Foundation of China (30830006, 31170079, 31000045, 31100058, 31070080), a major scientific and technological project for significant new drug creation from the Ministry of Health of China (2009ZXJ09001-034), a major project for genetically modified organisms breeding (2009ZX08009-075B) and a grant of Special Fund for Agroscientific Research in the Public Interest from the Ministry of Agriculture of China (201103007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yjhua@zju.edu.cn (YH); liangyanwang@zju.edu.cn (LW) . These authors contributed equally to this work. Introduction Deinococcus radiodurans belongs to the Deinococcaceae family of bacteria. These bacteria are characterized by extreme resistance to ionizing radiation (IR), ultraviolet (UV) radiation, oxidative stress, and desiccation [1–3]. To evaluate its robust viability in extreme environments, extensive studies on its ability to repair its DNA have being carried out [4,5]. However, more and more evidence supports the view that proteins, especially those involved in DNA repair and replication, must be protected from damage by radiation toxicity before DNA repair can begin. The antioxidants present in D. radiodurans seem to play critical roles in cell survival during irradiation. Recent studies have demonstrated that Mn- dependent antioxidant mechanism can protect proteins from reactive oxygen species (ROS)-induced damage in many species, including D. radiodurans [6–8]. D. radiodurans contains approxi- mately 100 times more Mn(II) than Escherichia coli when grown in a defined minimal medium. This suggests that the regulation of Mn(II) homeostasis in D. radiodurans may be more complicated than in E. coli [9]. So far, two types of bacterial high-affinity manganese acquisition systems have been identified, the natural resistance- associated macrophage protein (Nramp) transporter and the ATP- binding cassette (ABC) transporters [10,11]. Both Nramp and ABC-type acquisition system homolog have been found in D. radiodurans [12,13]. Nramp is a ubiquitous proton-coupled divalent metal ion transporter, and bacterial homologues have been reported in Mycobacterium tuberculosis, Bacillus subtilis, Escherichia coli, and Salmonella enteric serover Typhimurium [10,14–16]. The primary role of bacterial Nramp homologues is to transport Mn(II), so it was renamed MntH (manganese transport, H- dependent) [10,11,14]. Manganese is an essential enzyme cofactor. It protects cells from oxidative damage. However, it can be toxic at high concentrations, and so the expression of MntH must be strictly regulated [6,17]. The regulation of mntH genes in response to Mn(II) availability is mediated by the manganese transport regulator (MntR), among other regulators, including Fe(II)-sensing ferric uptake repressor (Fur) and the peroxide-sensing OxyR factor [10,16,18,19]. MntR is a diphtheria toxin repressor (DtxR) homologue. It represses the transcription of genes encoding manganese acquisition systems, such as mntH and mntABCD in B. subtilis. Unlike other DtxR family members, which can sense both Mn(II) and Fe(II), the MntRs in B. subtilis, E. coli, and S. aureus PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35057