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. 2009 May;72(4):844-58.
doi: 10.1111/j.1365-2958.2009.06699.x. Epub 2009 Apr 21.

Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli

Adil Anjem  1 , Shery Varghese, James A Imlay
Affiliations

Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli

Adil Anjem et al. Mol Microbiol. 2009 May.

Abstract

Very little manganese is imported into Escherichia coli under routine growth conditions: the import system is weakly expressed, the manganese content is low, and a manganese-dependent enzyme is not correctly metallated. Mutants that lack MntH, the importer, grow at wild-type rates, indicating that manganese plays no critical role. However, MntH supports the growth of iron-deficient cells, suggesting that manganese can substitute for iron in activating at least some metalloenzymes. MntH is also strongly induced when cells are stressed by hydrogen peroxide. This adaptation is essential, as E. coli cannot tolerate peroxide stress if mntH is deleted. Other workers have observed that manganese improves the ability of a variety of microbes to tolerate oxidative stress, and the prevailing hypothesis is that manganese does so by chemically scavenging hydrogen peroxide and/or superoxide. We found that manganese does not protect peroxide-stressed cells by scavenging peroxide. Instead, the beneficial effects of manganese correlate with its ability to metallate mononuclear enzymes. Because iron-loaded enzymes are vulnerable to the Fenton reaction, the substitution of manganese may prevent protein damage. Accordingly, during H2O2 stress, mutants that cannot import manganese and/or are unable to sequester iron suffer high rates of protein oxidation.

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Figures

Fig 1
Fig 1
Unstressed cells do not depend upon manganese. A. In standard medium, manganese import is too slight to activate MnSOD. Where indicated, media were unsupplemented or were supplemented with 50 μM manganese, and cell extracts were assayed before and after in vitro demetallation/reactivation with manganese. Each strain lacks sodB, to permit assay of MnSOD. The strains were AA138 (mntH+), AA141 (ΔmntH), and AA141/pAA01 (ΔmntH/pmntH). B. Manganese import is unnecessary for rapid growth. Wild-type MG1655 cultured without (open circles) or with (closed circles) 50 μM manganese supplement, and ΔmntH (AA99, plus sign) without manganese.
Fig 1
Fig 1
Unstressed cells do not depend upon manganese. A. In standard medium, manganese import is too slight to activate MnSOD. Where indicated, media were unsupplemented or were supplemented with 50 μM manganese, and cell extracts were assayed before and after in vitro demetallation/reactivation with manganese. Each strain lacks sodB, to permit assay of MnSOD. The strains were AA138 (mntH+), AA141 (ΔmntH), and AA141/pAA01 (ΔmntH/pmntH). B. Manganese import is unnecessary for rapid growth. Wild-type MG1655 cultured without (open circles) or with (closed circles) 50 μM manganese supplement, and ΔmntH (AA99, plus sign) without manganese.
Fig 2
Fig 2
Total manganese concentration of cells, measured by ICP. Cells were grown in defined medium (glucose/amino acids) or, where indicated, LB medium. Data represents means of three independent cultures. The strains used were MG1655 (wild-type), AA99 (ΔmntH), LC106 (Hpx-) and AA30 (Hpx- ΔmntH).
Fig 3
Fig 3
The gene mntH is strongly induced in aerobically growing Hpx- cells. Cells bearing a mntH’-lacZ fusion were grown in anaerobic defined medium (glucose/amino acids) and aerated at time zero. At intervals β-galactosidase was assayed. This experiment is representative of four independent replicates. AA183 (wild-type, circles) and AA191 (Hpx-, squares).
Fig 4
Fig 4
Manganese import is essential for H2O2-stressed cells. Cells were grown in defined medium (glucose/amino acids). A. Cells were cultured anaerobically and then aerated at time zero. Where indicated, manganese (50 μM) was included in the culture medium. LC106 (Hpx-, open squares), SMV42 (Hpx- ΔmntH, open diamonds), SMV42 + MnCl2 (closed diamonds), SMV42/pAA01 (Hpx- ΔmntH/pmntH, closed triangles) and AA153 (Hpx- mntH(NI), open triangles). B. MnSOD protein is correctly metallated in H2O2-stressed cells, due to increased manganese import. Each strain lacks sodB, to permit assay of MnSOD. SMV32 (wild-type), SMV29 (Hpx-), AA145 (Hpx- ΔmntH).
Fig 4
Fig 4
Manganese import is essential for H2O2-stressed cells. Cells were grown in defined medium (glucose/amino acids). A. Cells were cultured anaerobically and then aerated at time zero. Where indicated, manganese (50 μM) was included in the culture medium. LC106 (Hpx-, open squares), SMV42 (Hpx- ΔmntH, open diamonds), SMV42 + MnCl2 (closed diamonds), SMV42/pAA01 (Hpx- ΔmntH/pmntH, closed triangles) and AA153 (Hpx- mntH(NI), open triangles). B. MnSOD protein is correctly metallated in H2O2-stressed cells, due to increased manganese import. Each strain lacks sodB, to permit assay of MnSOD. SMV32 (wild-type), SMV29 (Hpx-), AA145 (Hpx- ΔmntH).
Fig 5
Fig 5
Imported manganese does not scavenge H2O2. A. Manganese did not prevent H2O2 accumulation by Hpx- and Hpx- ΔmntH cells. Cells were grown anaerobically and aerated at time zero. At intervals the medium was assayed for accumulated H2O2. LC106 (Hpx-, squares) and SMV42 (Hpx- ΔmntH, diamonds). Filled symbols: 50 μM manganese was included in the growth medium. B. Manganese-supplemented cells did not exhibit significant H2O2-scavenging activity. H2O2 (1 μM) was added at time zero to cultures of log-phase cells. Filled symbols: 50 μM manganese was included in the growth medium. MG1655 (open circles), LC106 (Hpx-, squares), SMV42 (Hpx- ΔmntH, diamonds).
Fig 5
Fig 5
Imported manganese does not scavenge H2O2. A. Manganese did not prevent H2O2 accumulation by Hpx- and Hpx- ΔmntH cells. Cells were grown anaerobically and aerated at time zero. At intervals the medium was assayed for accumulated H2O2. LC106 (Hpx-, squares) and SMV42 (Hpx- ΔmntH, diamonds). Filled symbols: 50 μM manganese was included in the growth medium. B. Manganese-supplemented cells did not exhibit significant H2O2-scavenging activity. H2O2 (1 μM) was added at time zero to cultures of log-phase cells. Filled symbols: 50 μM manganese was included in the growth medium. MG1655 (open circles), LC106 (Hpx-, squares), SMV42 (Hpx- ΔmntH, diamonds).
Fig 6
Fig 6
The primary role of intracellular manganese is not to degrade O2-. A. Metallation of MnSOD is not the primary purpose of manganese import. Cells were precultured in anaerobic defined medium (glucose/amino acids) and then aerated at time zero. LC106 (Hpx-, squares), AA30 (Hpx- ΔmntH, diamonds) and SMV30 (Hpx- ΔsodA, X’s). B. Overproduction of MnSOD debilitates Hpx- ΔmntH cells. Anaerobic cells, with or without the pDT1.5 sodA over-expression plasmid, were aerated at time zero in defined medium (glucose/amino acids) containing 100 μM MnCl2. At the indicated time (arrow), manganese was removed. LC106 (Hpx-, open squares), LC106/pDT1.5 (closed squares) and SMV42/pDT1.5 (Hpx- ΔmntH, closed diamonds).
Fig 6
Fig 6
The primary role of intracellular manganese is not to degrade O2-. A. Metallation of MnSOD is not the primary purpose of manganese import. Cells were precultured in anaerobic defined medium (glucose/amino acids) and then aerated at time zero. LC106 (Hpx-, squares), AA30 (Hpx- ΔmntH, diamonds) and SMV30 (Hpx- ΔsodA, X’s). B. Overproduction of MnSOD debilitates Hpx- ΔmntH cells. Anaerobic cells, with or without the pDT1.5 sodA over-expression plasmid, were aerated at time zero in defined medium (glucose/amino acids) containing 100 μM MnCl2. At the indicated time (arrow), manganese was removed. LC106 (Hpx-, open squares), LC106/pDT1.5 (closed squares) and SMV42/pDT1.5 (Hpx- ΔmntH, closed diamonds).
Fig 7
Fig 7
The amount of manganese needed to protect Hpx- mutants matches the amount needed to metallate enzymes. AA145 (Hpx- ΔmntH) cells were precultured anaerobically in defined medium (glucose/amino acids) and then subcultured at time zero into aerobic defined medium (glucose/amino acids) containing the indicated concentration of manganese. Optical density was monitored continuously, and MnSOD activity was measured when the cultures reached an OD600 of 0.2. The lowest doubling time, and thus the highest growth rate, for each culture is denoted in the figure.
Fig 8
Fig 8
Manganese is required to rescue Hpx- cells from iron overload. A. Manganese supplementation rescues Hpx- Δdps cells. Cells growing in anaerobic defined medium (glucose/amino acids), +/- 50 μM MnCl2, were aerated at time zero. LC106 (Hpx-, open squares), SP66 (Hpx- Δdps, open diamonds), SP66 grown + MnCl2 (closed diamonds). B. The growth defect of Hpx- ΔmntH was suppressed by an iron-specific chelator. Cells (AA30) were grown in aerobic defined medium (glucose/amino acids) without MnCl2 (open diamonds), with 1 μM MnCl2 (dotted line, closed diamonds) or with 1 μM MnCl2 plus 0.1 mM deferoxamine (closed diamonds).
Fig 8
Fig 8
Manganese is required to rescue Hpx- cells from iron overload. A. Manganese supplementation rescues Hpx- Δdps cells. Cells growing in anaerobic defined medium (glucose/amino acids), +/- 50 μM MnCl2, were aerated at time zero. LC106 (Hpx-, open squares), SP66 (Hpx- Δdps, open diamonds), SP66 grown + MnCl2 (closed diamonds). B. The growth defect of Hpx- ΔmntH was suppressed by an iron-specific chelator. Cells (AA30) were grown in aerobic defined medium (glucose/amino acids) without MnCl2 (open diamonds), with 1 μM MnCl2 (dotted line, closed diamonds) or with 1 μM MnCl2 plus 0.1 mM deferoxamine (closed diamonds).
Fig 9
Fig 9
Imported manganese suppresses oxidative protein carbonylation. Cells were grown in aerobic defined medium (glucose/amino acids) +/- 50 μM MnCl2, and proteins were harvested at an OD600 of ~ 0.2. Proteins were derivatized and western blotted as described in experimental procedures. The arrow indicates a non-oxidative carbonylation that was detected even when cells were grown and derivatized anaerobically. The strains used were MG1655 (wild-type), AA99 (ΔmntH), LC106 (Hpx-), AA30 (Hpx- ΔmntH) and SP66 (Hpx- Δdps).
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