A Novel MntH Repressor in D. radiodurans Figure 2. Mn(II) and Fe(II) modulate the binding activity of DR2539 in vivo. (A) Effects of divalent metals (50 mM) on expression of pRAZH in D. radiodurans. Data shown are the means 6 standard deviations of three independent experiments. (B) Effects of Mn(II) (squares) and Fe(II) (circles) on the expression of pRAZH in wild-type samples expressing DR2539. (C) Effects of Mn(II) (squares) and Fe(II) (circles) on the expression of pRAZH in the dr2539 mutant. (D) Rea-time PCR analysis of the dr1709 gene expression using dr0089 as internal control gene. Longitudinal axes indicate the change fold of dr1709 mRNA relative to controls. Control cells were cultured in medium without Mn(II). *, P,0.05 relative to control. The data are the means 6 standard deviations of three independent experiments. doi:10.1371/journal.pone.0035057.g002 reported MntR, because His98 is not involved in the metal binding of these proteins. This shows that DR2539 may represent a new subset of the DtxR family. The disruption of the MntABC transporter could induce the accumulation of iron but could not change the manganese level in D. radiodurans [26]. Our results indicate that the DR0865 (Fur homologue) participates in the transcriptional regulation of the MntABC. The disruption of dr0865 did not affect the manganese resistance of D. radiodurans. Therefore, we speculate that MntABC in D. radiodurans may be responsible for the intracellular iron efflux. However, there is still some controversy as to whether such an iron efflux mechanism exists in bacteria [27]. The function of DR0865 still needs to be elucidated. Many studies have attempted to determine why MntR can selectively respond to Mn(II) while DtxR prefers to bind Fe(II). Here, we described a novel MntH repressor (DR2539) from D. radiodurans, and found it capable of responding to both Mn(II) and Fe(II). Moreover, the His98 in DR2539 is crucial to its activity. This is quite different from with results of previous reports on MntH repressors. However, crystallography analysis is still needed to confirm this and clarify the structure-function relationship. Materials and Methods Strains and media All trains and plasmids used in this study are listed in Table S1. D. radiodurans strains were cultured at 30uC on TGY medium (0.5% Bacto tryptone, 0.1% glucose, 0.3% Bacto yeast extract) with aeration or on TGY plates supplemented with 1.2% Bacto agar. E. coli strain DH5a was used for propagation of plasmids and grown at 37uC on LB medium with appropriate antibiotics. Construction of pET-29b-IF-dr2539 fusion expression vector The IF and dr2539 transcripts were cloned from E. coli using primers IFF/IFR and RFF/RFR (listed in Table S2). Then the PCR products were ligated separately into the pMD18-T simple vector (TaKaRa Biotechnology (Dalian) CO., Ltd, China) for PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35057