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. 2012;7(4):e35057.
doi: 10.1371/journal.pone.0035057. Epub 2012 Apr 16.

Regulation of MntH by a dual Mn(II)- and Fe(II)-dependent transcriptional repressor (DR2539) in Deinococcus radiodurans

Hongxing Sun  1 , Mingfeng Li, Guangzhi Xu, Huan Chen, Jiandong Jiao, Bing Tian, Liangyan Wang, Yuejin Hua
Affiliations

Regulation of MntH by a dual Mn(II)- and Fe(II)-dependent transcriptional repressor (DR2539) in Deinococcus radiodurans

Hongxing Sun et al. PLoS One. 2012.

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. β-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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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 µM 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.
Figure 2
Figure 2. Mn(II) and Fe(II) modulate the binding activity of DR2539 in vivo.
(A) Effects of divalent metals (50 µM) on expression of pRAZH in D. radiodurans. Data shown are the means ± 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 null 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 ± standard deviations of three independent experiments.
Figure 3
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 null mutant (Δ dr2539), and dr0865 null mutant (Δ dr0865) 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 means±deviations of three independent experiments.
Figure 4
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.
Figure 5
Figure 5. His98 plays an important role in DNA binding activity of DR2539.
(A) 10 µl cell dilution was dripped on the TGY plate to which 6 mM of Mn(II) had been added. The cells were cultured for 3 days. (B) H98Y mutant and wild-type DR2539 proteins were incubated with p1709b at different concentrations of Mn(II). (C) Quantification of the fluorescence intensity of binding bands was performed using ImageJ. *, P<0.05.
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