Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis

Abstract

Exposure to hydrogen peroxide (H(2)O(2)) and other reactive oxygen species is a universal feature of life in an aerobic environment. Bacteria express enzymes to detoxify H(2)O(2) and to repair the resulting damage, and their synthesis is typically regulated by redox-sensing transcription factors. The best characterized bacterial peroxide-sensors are Escherichia coli OxyR and Bacillus subtilis PerR. Analysis of their regulons has revealed that, in addition to inducible detoxification enzymes, adaptation to H(2)O(2) is mediated by modifications of metal ion homeostasis. Analogous adaptations appear to be present in other bacteria as here reviewed for Deinococcus radiodurans, Neisseria gonorrhoeae, Streptococcus pyogenes, and Bradyrhizobium japonicum. As a general theme, peroxide stress elicits changes in cytosolic metal distribution with the net effect of reducing the damage caused by reactive ferrous iron. Iron levels are reduced by repression of uptake, sequestration in storage proteins, and incorporation into metalloenzymes. In addition, peroxide-inducible transporters elevate cytosolic levels of Mn(II) and/or Zn(II) that can displace ferrous iron from sensitive targets. Although bacteria differ significantly in the detailed mechanisms employed to modulate cytosolic metal levels, a high Mn:Fe ratio has emerged as one key correlate of reactive oxygen species resistance.

FIG. 1.

General mechanisms for protection against oxidative stress. (A) To detoxify hydrogen peroxide (H₂O₂), many bacteria upregulate the expression of peroxidases and catalases. (B) H₂O₂ reacts rapidly with ferrous iron, generating hydroxyl radical, hydroxide anion, and oxidized ferric iron (Fenton reaction). The hydroxyl radical can then subsequently damage DNA and oxidize proteins. To protect against these toxic effects, bacteria may (C) decrease intracellular iron levels by decreasing Fe import and sequestering free iron through the upregulation of Dps. (D) Bacteria may also increase intracellular levels of Mn(II) and/or Zn(II) through increased import. (E) Mn(II) and Zn(II) can competitively inhibit the damaging reactions catalyzed by Fe(II) and H₂O₂.

FIG. 2.

The OxyR and ferric uptake regulator (Fur) regulons in Escherichia coli. (A) Upon exposure to H₂O₂, an intramolecular disulfide bond forms within each OxyR protomer, resulting in the activation of OxyR. Oxidized OxyR activates the sequestration of Fe (Dps), the detoxification of H₂O₂ (AhpC, KatG), and the import of Mn(II) (MntH). (B) OxyR also activates the transcription of the OxyS sRNA, resulting in the reduction of carbon utilization pathways and thus reducing endogenous H₂O₂ production. In addition, OxyR upregulates the synthesis of Fur. In Fe-rich conditions, (C) Fur represses additional Fe uptake and (D) indirectly activates some iron-containing proteins (including Fe-superoxide dismutase [SOD] and bacterioferritin [Bfr]) via the RyhB sRNA or, in the case of the FtnA iron storage protein, by reversing H-NS-mediated silencing (63). In these conditions, the Isc Fe-S cluster biogenesis machinery is upregulated. Fur:Fe is sensitive to H₂O₂ and can be inactivated upon exposure. (E) However, in Fe-limited and/or oxidative stress conditions, transcription of the second Fe-S cluster assembly machinery, Suf, is favored.

FIG. 3.

The PerR, Fur, and MntR regulons in Bacillus subtilis. (A) H₂O₂ oxidizes PerR:Fe, resulting in the derepression of peroxide detoxifying enzymes (KatA and AhpCF), an Fe-sequestration protein (MrgA), heme biosynthesis enzymes, and Zn(II) uptake. Although PerR:Mn can also repress these same genes, this form of PerR is relatively insensitive to oxidation by peroxide. Therefore, in low Fe but high Mn conditions, the PerR regulon is not derepressed upon exposure to peroxides. (B) Only PerR:Mn, not PerR:Fe, appears to be responsible for the repression of Fur. (C) In Fe-limited conditions, iron uptake is derepressed by Fur. (D) Production of low priority Fe-enzymes is decreased by the FsrA sRNA when Fe is limiting. (E) Independent of Fur and PerR activity, MntR represses Mn(II) uptake when Mn(II) is sufficient.

FIG. 4.

The OxyR and DtxR regulons in Deinococcus radiodurans. (A) D. radiodurans OxyR has only one redox active cysteine residue. This cysteine residue is oxidized upon exposure to H₂O₂. OxyR may regulate Mn and Fe homeostasis; however, the details of this regulation are not yet clearly defined. (B) Catalase activity is regulated by three regulatory proteins: OxyR, DtxR, and a PerR/Fur homolog. This tight regulation may relate to the high level of catalase activity present in this bacterium. (C) In addition to OxyR, DtxR also regulates Mn and Fe import. Mn transport is repressed under conditions where Fe transport is induced. Direct effects are shown as solid lines and potentially indirect effects are shown as dotted lines.

FIG. 5.

The OxyR, PerR, and Fur regulons in Neisseria gonorrhoeae. (A) Oxidization of OxyR results in the upregulation of two H₂O₂ detoxifying enzymes, KatA and Prx. (B) Regulation of the oxidative stress response and Fe homeostasis are linked by the (potentially indirect) repression of OxyR by Fur:Fe. In addition, Fur may be inactivated by H₂O₂. In Fe-limited conditions, Fe uptake is derepressed and (C) Fe storage is repressed by the NrrF sRNA. NrrF is also responsible for the repression of an Fe-SOD. (D) In addition to OxyR, N. gonorrhoeae contains a PerR homolog that likely functions as a Zur (or possibly a Mur) regulatory protein. PerR is responsible for the repression of Mn and Zn uptake and a periplasmic peroxidase in Mn-replete (and possibly Zn-replete) conditions. Direct effects are shown as solid lines and potentially indirect effects are shown as dotted lines.

FIG. 6.

The PerR regulon in Streptococcus pyogenes. (A) Upon exposure to peroxides, oxidized PerR derepresses iron sequestration (Dps) and cation export (Zn and possibly Fe) via PmtA. Import of Fe, Zn, and Mn may also be decreased upon exposure to H₂O₂ via the downregulation of MtsABC. (B) The combined effect of these alterations of cation import is the activation of the zinc starvation response through AdcR. (C) Mn, Fe, and heme homeostasis are additionally controlled by MtsR in a metal-specific manner; either Fe- or Mn-bound MtsR can repress MtsABC, but only MtsR:Fe can repress heme import. Direct effects are shown as solid lines and potentially indirect effects are shown as dotted lines.

FIG. 7.

The Irr and Fur regulons of Bradyrhizobium japonicum. (A) OxyR does not appear to play a large role in the regulation of gene expression in B. japonicum. The oxidation of OxyR appears to upregulate the expression of catalase; however, this effect may be indirect, and appears to result from the derepression of katG by reduced OxyR. (B) Irr appears to play an important role in the coordination of the oxidative stress response with iron and heme homeostasis. In Fe-limited conditions, Irr is active as a regulatory protein. However, when Fe and heme are plentiful, Irr is degraded in a peroxide dependent manner. Irr is responsible for the regulation of heme transport and heme biosynthesis, inversely regulating import with synthesis. Irr also regulates Fe import and Fe storage. (C) In addition to heme directly regulating Irr activity, Fur:Fe represses the transcription of irr, preventing its production in conditions where Irr would be degraded. Fur may also regulate heme and iron homeostasis; however, these effects by Fur:Fe have not yet been shown to be direct (D). In addition to Fe, Fur also binds Mn. Fur:Mn appears to regulate Mn import and possibly heme biosynthesis. Direct effects are shown as solid lines and potentially indirect effects are shown as dotted lines.