exhibit pronounced selectivity for Mn(II) due to amino acid substitutions at the metal-binding site [20–23]. In D. radiodurans, dr1709 encodes the manganese transporter, which is believed to be responsible for its high Mn(II) accumulation in D. radiodurans [7–9,13]. Unfortunately, dr1709 could not be disrupted and its repression is still unknown [12]. dr2539 encodes a DtxR homologue, and previous studies have implied that it may regulate the intracellular Mn/Fe ratio [24]. However, which genes DR2539 regulates and the mechanism by which it regulates them remains to be investigated. In this study, we successfully expressed the DR2539 in vivo and analyzed its biochemical characteristics both in vivo and in vitro. We found that the transcription of dr1709 was strictly repressed by DR2539. Unexpectedly, the Fe(II)-induced repression of dr1709 was also found to be mediated by DR2539 but not by the Fur homologue DR0865. We demonstrated that DR2539 can be activated by both Fe(II) and Mn(II) in vitro and in vivo. Site-directed mutagenesis analysis showed that His98 plays an important role in the DNA binding activity of DR2539. Our study suggests that the metal binding mode of DR2539 may be different from what has been previously reported for the MntH repressor. It may represent a new subset of DtxR family proteins. Results Binding of DR2539 to the mntH (dr1709) promoter The MntH is a major component of the Mn(II) uptake system. It is involved in oxidative stress resistance in many bacteria [10]. A dr2539 mutant of D. radiodurans showed increased intracellular manganese concentration and enhanced oxidative resistance, suggesting that DR2539 may be involved in the regulation of MntH [24]. To determine whether DR2539 binds to the dr1709 promoter, we performed electrophoretic gel mobility shift assays (EMSA) in the presence of Mn(II). The dr1709 promoter was amplified by PCR from the genome of D. radiodurans strain R1 (Fig. 1A). MntRs in E. coli and S. enterica were found to regulate the mntH by binding to the reverted repeat region of mntH promoters. In D. radiodurans, a similar reverted repeat region was found in the dr1709 promoter. In order to confirm whether DR2539 binds to this region, we designed two pairs of primers: one pair was capable of generating dr1709 promoter DNA fragments containing reverted repeat regions (dr1709b), and the other pair of primers was capable of generating dr1709 promoter DNA fragments lacking the reverted repeat region (dr1709a) (Fig. 1A). As indicated by the gel shift, the DR2539 protein bound to the dr1709b promoter, and binding increased with protein concentration (Fig. 1B). In contrast, the DR2539 protein cannot bind to dr1709 promoters that lack inverted repeat regions (Fig. S2A). This suggests that the binding between DR2539 and dr1709 promoters depends on the presence of the inverted repeat region. The presence of EDTA in the EMSA reaction abolished binding to the dr1709 promoter, suggesting that Mn(II) is necessary for DR2539– DNA binding (Fig. S2B). These results indicate that DR2539 specifically binds to the mntH promoter of D. radiodurans. Effects of Mn(II) and Fe(II) on DR2539 in vitro Generally, the regulation of MntH genes involves iron-sensing by Fur regulatory proteins [18,23]. In D. radiodurans, dr0865 encodes the only Fur homologue. To investigate whether DR0865 might be involved in the regulation of the mntH gene, we expressed the DR0865 protein in vitro and performed DNA binding experiments. Unexpectedly, the EMSA results demonstrated that DR0865 cannot bind to the promoter of dr1709 (p1709b is the full length version of dr1709 promoter) (Fig. S2C). Current evidence A Novel MntH Repressor in D. radiodurans supports the idea that MntR is a strictly Mn(II)-specific metalloregulatory protein, and that the repression of MntH by Fe(II) is always mediated by Fur [20,21,25]. We therefore wondered whether DR2539 can respond to both Mn(II) and Fe(II) in vitro. We studied the binding efficiency of DR2539 to the dr1709b promoter using EMSA in the presence of varying concentrations of Mn(II) and Fe(II). As shown in Fig. 1C, the binding activity of DR2539 increased as the concentration of Mn(II) rose from 25 mM to 150 mM. The same response was observed when Mn(II) was replaced by Fe(II) (Fig. 1D), indicating that the activity of DR2539 can be regulated by both Mn(II) and Fe(II) in a concentration-dependent manner. Effects of Mn(II) and Fe(II) on DR2539 in vivo In order to confirm our results in vitro, the transcriptional regulation of the mntH promoter by different ions were tested by linking the dr1709 promoter to the b-galactosidase gene lacZ. As shown in Fig. 2A, the activity of b-galactosidase was repressed dramatically when Mn(II) or Fe(II) was included in the growth medium, but not when Ni(II), Cu(II), or Zn(II) was added in the absence of Mn(II) and Fe(II). This strongly suggests that the expression of D. radiodurans MntH is regulated by both Mn(II) and Fe(II) in vivo. A dr2539 mutant was constructed to investigate whether the MntH repression induced by Fe(II) was actually mediated by DR2539. The expression of lacZ was repressed when wild-type bacteria were treated with different concentrations of Mn(II) or Fe(II) (Fig. 2B). On the other hand, Mn(II)- and Fe(II)- dependent repression of b-galactosidase was abolished in the dr2539 mutant, indicating that Mn(II)/Fe(II)-dependent repression of MntH (dr1709) was mediated by DR2539 (Fig. 2C). Using Real-time PCR analysis, we compared the expression levels of dr1709 in the dr2539 mutant and wild-type strains. As shown in Fig. 2D, the mRNA of the wild-type strain decreased dramatically when the concentration of Mn(II) increased relative to that cultured in media without additional Mn(II) (indicating control). No difference was observed in dr2539 mutants, indicating that dr2539 encodes the dr1709 repressor. Binding of DR0865 to mntABC promoters Because DR0865 cannot regulate MntH transcription, we turned our attention to dr2523, dr2283, and dr2284, which encode the other putative Mn(II) acquisition transporters. The EMSA results indicate that DR0865 binds to the promoters of dr2523, dr2283, and dr2284 (dr2283 and dr2284 share one promoter), which encode the MntABC subunits in a manganese-dependant manner (Fig. 3A/3B). On the other hand, we found that DR2539 protein cannot bind to the promoters of dr2283, dr2284, or dr2523 (data not shown). The manganese resistance assay showed that the dr0865 mutant is comparable to the wild strain while the manganese resistance of the dr2539 mutant decreased significantly (Fig. 3C/3D). These results imply that the MntABC in D. radiodurans may not be involved in manganese transport. Therefore, DR2539 may be the most important regulator of intracellular manganese homeostasis in D. radiodurans. Effects of H98Y mutation on the metal-sensing function of DR2539 The protein sequences of various members of the bacterial MntRs/DtxR family were aligned to define those structure features of DR2539 that could confer dual Mn(II)/Fe(II)- dependent functional regulation. Sequence alignment analysis indicated that DR2539 possesses a conserved His98 that is also present in Mn(II)/Fe(II)-dependent DtxR but absent from the PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35057