A Novel MntH Repressor in D. radiodurans Figure 1. DR2539 binds to the MntH promoter DNA fragment in a Mn(II)- and Fe(II)-dependent manner. (A) Schematic of dr1709 promoter (p1709a and p1709b) DNA sequence region. The inverted repeat region is shown by the inverted arrows. (B) DR2539 binding to p1709b with increasing quantities of DR2539 and 25 mM Mn(II). (C) and (D) EMSA analysis was performed using DR2539 and p1709b with increasing concentration of Mn(II) or Fe(II). RBS, ribosome binding site; Start, transcription start codon. p1709a and p1709b sequence regions are underlined by straight lines and dashed lines, respectively. doi:10.1371/journal.pone.0035057.g001 MntH regulator (MntR). ScaR represents a new group of Mn(II)- responsive transcription factors, and its metal binding residues are conserved in DR2539 except Asp126 (Fig. 4). Site mutants H98Y and D126A were constructed to evaluate the metal binding mode of DR2539. These dr2539 mutants were reverted to dr2539 mutants. The manganese resistance of the dr2539 mutant complementation strain was examined in TGY plates supple- mented with 6 mM manganese. As shown in Fig. 5A, the manganese resistance of the D126A complementation strain is comparable to that of the wild-type dr2539 complementation strain. In contrast, the manganese resistance of H98Y comple- mentation strain decreased dramatically. To investigate the DNA binding activity of the H98Y mutant, the H98Y mutant protein was expressed in vitro, and the DNA binding activity was examined. The DNA binding activity of the H98Y mutant was found to be much weaker than that of wild type DR2539 at the same manganese concentration (Fig. 5B/5C). This shows that His98 may play a very important role in the function of DR2539 and that D126 may not be involved in the metal binding activity of DR2539. Discussion D. radiodurans contains more intracellular Mn(II) than other bacteria [9]. It has been suggested that the high Mn/Fe ratio in D. radiodurans facilitates the pronounced resistance to extreme oxidative stress observed in this species [8,13]. Intracellular Mn(II) is known to confer protection against oxidative stress and other forms of injury, including ionizing radiation [13]. We analyzed the genetic regulation of the MntH gene dr1709 to study manganese homeostasis in D. radiodurans. In E. coli, mntH transcription can be repressed either by the Mn(II)-dependent MntR or by the Fe(II)-dependent transcription- al regulator Fur [18]. The MntH of S. enterica has also been reported to be responsive to Fe(II) and the repression induced by Fe(II) was mainly mediated by Fur [23]. In contrast to previous studies, the gene dr0865 was here found to encode the only Fur homologue in D. radiodurans, but it cannot bind to dr1709 promoter. This indicates that Fe(II)-dependent regulation of MntH must be mediated by another Fe(II)-responsive transcrip- tional regulator. D. radiodurans DR2539 exhibited both Mn(II)- dependent and Fe(II)-dependent repression of dr1709. This explains the Fe(II)-dependence of MntH regulation in the absence of a functional Fur homologue. The activation of metalloregulators is closely correlated with their structures. We compared the protein sequences of DR2539 to DtxR homologues and found substantial structural and functional differences. The Glu11 that provides two metal binding ligands in the MntR binuclear metal binding site is substituted by a basic amino acid (Lys) in DR2539. The presence of a positive charge at this position suggests that DR2539 cannot form a regular MntR-type metal binding site (Fig. 4). In addition, the His98 and SH3-like domains that are conserved in DtxR homologues (but missing in other MntRs) are present in DR2539. In this paper, we observed that DR2539 responded to both Mn(II) and Fe(II), and the impact of H98Y and D126A on activity was assessed. The results indicate that the Mn(II) resistance of the C- H98Y complementation strain decreased significantly after exposure to Mn(II) and Fe(II), while the C- D126A strain was relatively resistant to Mn(II). A DNA binding assay implied that His98 is crucial to protein activity. This is different from previously PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35057