A Novel MntH Repressor in D. radiodurans Figure 3. DR0865 binds to the promoter of MntABC in an ion-dependent manner. (A) and (B) DR0865 binding to p2523 and p2284 as the concentration of DR0865 increased. (C) Wild-type R1, dr2539 mutant (Ddr2539), and dr0865 mutant (Ddr0865) were cultured on TGY plates overlaid with filter discs saturated with 1 M solution MnCl2. (D) The zone of inhibition was measured from edge of disc after three days. *, P,0.05. Data represent the means6deviations of three independent experiments. doi:10.1371/journal.pone.0035057.g003 sequencing (Invitrogen Corporation). The pMD18-T-IF plasmid was digested with NdeI and EcoRI and ligated to pET-29b digested with the same enzymes to form pETIF. The pMD18-T-dr2539 vector was digested with EcoRI and HindIII and ligated to pETIF (digested with the same enzymes) to yield pET-29b-IF-dr2539 (pIFMR), as shown in Fig. S1. Overexpression and purification of DR2539 Wild-type DR2539 was overexpressed in E. coli strain BL21 (DE3) (pLysS) transformed with pIFMR. Typically, 500 ml of LB medium was inoculated with 5 mL of a clone expressing DR2539 and cultured until OD600 was <0.6. Then 200 mM IPTG was added and the cultures were incubated for another 4 h. Cells were harvested by centrifugation and resuspended in 20 mL of lysis Figure 4. Sequence alignment of the metal binding sites of DR2539 with other DxtR/MntR family members. ScaR (Streptococcus gordonii), SloR (Streptococcus suis), LCAS (Lactobacillus casei), SirR (Corynebacterium glutamicum), TroR (Treponema pallidum), TroR (Treponema denticola), IdeR (Mycobacterium tuberculosis), DtxR (Corynebacterium diptheriae), DR2539 (Deinococcus radiodurans), MntR (Staphylococcus aureus), MntR (Escherichia coli), and MntR (Bacillus subtilis). The sequences were aligned using the CLUSTAL W software. Residues shaded with black represent metal-binding sites that have been studied while residues shaded with grey represent predicted metal binding sites. doi:10.1371/journal.pone.0035057.g004 PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35057