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. 2002 Jun;184(12):3151-8.
doi: 10.1128/JB.184.12.3151-3158.2002.

Regulation of Salmonella enterica serovar Typhimurium mntH transcription by H(2)O(2), Fe(2+), and Mn(2+)

David G Kehres  1 , Anu Janakiraman, James M Slauch, Michael E Maguire
Affiliations

Regulation of Salmonella enterica serovar Typhimurium mntH transcription by H(2)O(2), Fe(2+), and Mn(2+)

David G Kehres et al. J Bacteriol. 2002 Jun.

Abstract

MntH, a bacterial homolog of mammalian natural resistance associated macrophage protein 1 (Nramp1), is a primary transporter for Mn(2+) influx in Salmonella enterica serovar Typhimurium and Escherichia coli. S. enterica serovar Typhimurium MntH contributes to H(2)O(2) resistance and is important for full virulence. Consistent with its phenotype and function, mntH is regulated at the transcriptional level by both H(2)O(2) and substrate cation. We have now identified three trans-acting regulatory factors and the three corresponding cis-acting mntH promoter motifs that mediate this regulation. In the presence of hydrogen peroxide, mntH is activated by OxyR, acting through an OxyR-binding motif centered just upstream of the likely -35 RNA polymerase-binding site. In the presence of Fe(2+), mntH is repressed primarily by Fur, acting through a Fur-binding motif overlapping the -35 region. In the presence of Mn(2+), mntH is repressed primarily by the Salmonella equivalent of E. coli b0817, a distant homolog of the Bacillus subtilis manganese transport repressor, MntR, acting through an inverted-repeat motif located between the likely -10 polymerase binding site and the ribosome binding site. E. coli b0817 was recently shown to bind the identical inverted-repeat motif in the E. coli mntH promoter and hence has been renamed MntR (S. I. Patzer and K. Hantke, J. Bacteriol. 183:4806-4813, 2001). Using Deltafur, DeltamntR, and Deltafur DeltamntR mutant strains as well as mutations in the Fur- and MntR-binding motif elements, we found that Fe(2+) can also mediate repression through the Mn(2+) repressor MntR.

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Figures

FIG. 1.
FIG. 1.
Wild-type and mutant candidate cis-acting motifs in the mntH promoter. Proximal regions of the mntH promoters from S. enterica serovar Typhimurium (24), E. coli (24, 27), and Y. pestis (5) were aligned and compared to consensus motifs for OxyR (40) and Fur (2, 9). The S. enterica serovar Typhimurium and E. coli mntH promoters both have a candidate OxyR-binding site upstream of the predicted −35 region and a candidate Fur-binding site overlapping and extending downstream of the −35 region. Also highlighted is an inverted-repeat motif between the predicted −10 region and the ribosome binding site (RBS) of the mntH coding region. Of these three motifs, only a putative Fur-binding site is readily discernible in the Y. pestis mntH promoter. Below these motifs are lines indicating the substitutions made in the three cis-acting S. enterica serovar Typhimurium promoter mutants used in this study, pDGK261, pDGK262, and pDGK263. Identical nucleotides in the three promoter regions (asterisks), novel restriction sites introduced between motifs or within block substitutions (described in Materials and Methods) (bases in bold italic type), and isopositional sequences from the bacteriophage P22 Pant promoter (bases in lightface roman type) are indicated. As some substituted bases fortuitously matched those found in the wild-type S. enterica serovar Typhimurium mntH promoter, the bases actually changed in pDGK261, pDGK262, and pDGK263 are underlined. In the consensus OxyR-binding motif the bases shown in lowercase type are common but not completely conserved bases at these sites (38).
FIG. 2.
FIG. 2.
Repression of mntH transcription by transition metals. Wild-type reporter strain MM2507 (S. enterica serovar Typhimurium SL1344 bearing plasmid pMLZ104) was grown aerobically overnight at 37°C in M9-0.2% glucose-50 μg of ampicillin/ml containing the sulfate or chloride salts of various divalent first-row transition metals at a concentration of 10 or 100 μM as indicated. The β-galactosidase activity of saturated cultures (OD600 of 1.0 to 1.5 ) was normalized to that of a culture containing no added metal (about 300 Miller units). Results represent the means and standard errors of two to five independent experiments in each case.
FIG. 3.
FIG. 3.
Activation of mntH transcription by H2O2. Cultures were grown at 37°C in M9 with 0.2% glucose and 50 μg of ampicillin/ml to an OD600 of 0.3, when they were supplemented with 100 μM H2O2. β-Galactosidase activity was determined at different times following H2O2 challenge. The response of the wild-type strain/promoter combination (MM2507) is compared to that of a strain in which the OxyR-binding motif of the mntH::lacZYA reporter is mutated (MM2616) and to that of a strain in which the OxyR protein is mutated (MM2529). Data shown here are for cells grown microaerobically (see Materials and Methods). A similar pattern was seen in cells grown aerobically, except that maximal induced β-galactosidase activity was three- to fivefold lower for each strain. Results are the means and standard errors of three independent experiments.
FIG. 4.
FIG. 4.
Control of iron and manganese repression by Fur and MntR. Cultures were grown overnight in M9-0.2% glucose-50 μg of ampicillin/ml with no added metal, with 10 μM MnSO4, or with 10 μM FeSO4. For each strain the β-galactosidase activity of metal-challenged cultures was normalized to that of the same strain grown in the absence of added metal. Results are the means and standard errors of two to five independent experiments. (A) Fe2+ repression dose-response curves comparing the wild-type strain/promoter combination (MM2507) to strains bearing a mutated Fur protein (MM2646), a mutated Fur-binding motif (MM2617), or both (MM2762). (B) Mn2+ repression dose-response curves for the same four strains as in panel A. (C) Fe2+ repression dose-response curves comparing the wild-type strain/promoter combination (MM2507) to strains bearing a mutated MntR protein (MM2645), a mutated MntR-binding motif (MM2618), or both (MM2760). (D) Mn2+ repression dose-response curves for the same four strains as in panel C. (E) Fe2+ repression dose-response curves comparing the wild-type strain/promoter combination (MM2507) to strains bearing a mutated Fur protein and a mutated MntR protein (MM2654), a mutated Fur protein and a mutated MntR-binding motif (MM2763), or a mutated MntR protein and a mutated Fur-binding motif (MM2759). (F) Mn2+ repression dose-response curves for the same four strains as in panel E.
FIG. 5.
FIG. 5.
Occurrence of H2O2 activation and metal repression in the same culture. Three cultures of MM2507, containing wild-type forms of all three trans-acting factors and cis-acting motifs, were grown microaerobically at 37°C in M9-0.2% glucose-50 μg of ampicillin/ml with no added metal, with 10 μM MnSO4, or with 10 μM FeSO4. At an OD600 of 0.3 each culture was supplemented with 100 μM H2O2 and β-galactosidase activity was determined at different times following H2O2 challenge. The cultures were then allowed to grow overnight to saturation (OD600 of 1.0 to 1.5 ), and β-galactosidase activity was determined again. The data show that H2O2 activation still occurs even if 10 μM MnSO4 or 10 μM FeSO4 was present (A) and that manganese and iron repression still occurs even if the cells had been previously challenged with H2O2 (B). Note the different scales for β-galactosidase activity in the two panels.
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