Abstract

Transition metal ions (Zn(II), Cu(II)/(I), Fe(III)/(II), Mn(II)) are essential for life and participate in a wide range of biological functions. Cellular Zn(II) levels must be high enough to ensure that it can perform its essential roles. Yet, since Zn(II) binds to ligands with high avidity, excess Zn(II) can lead to protein mismetallation. The major targets of mismetallation, and the underlying causes of Zn(II) intoxication, are not well understood. Here, we use a forward genetic selection to identify targets of Zn(II) toxicity. In wild-type cells, in which Zn(II) efflux prevents intoxication of the cytoplasm, extracellular Zn(II) inhibits the electron transport chain due to the inactivation of the major aerobic cytochrome oxidase. This toxicity can be ameliorated by depression of an alternate oxidase or by mutations that restrict access of Zn(II) to the cell surface. Conversely, efflux deficient cells are sensitive to low levels of Zn(II) that do not inhibit the respiratory chain. Under these conditions, intracellular Zn(II) accumulates and leads to heme toxicity. Heme accumulation results from dysregulation of the regulon controlled by PerR, a metal-dependent repressor of peroxide stress genes. When metallated with Fe(II) or Mn(II), PerR represses both heme biosynthesis (hemAXCDBL operon) and the abundant heme protein catalase (katA). Metallation of PerR with Zn(II) disrupts this coordination, resulting in depression of heme biosynthesis but continued repression of catalase. Our results support a model in which excess heme partitions to the membrane and undergoes redox cycling catalyzed by reduced menaquinone thereby resulting in oxidative stress.

Highlights

  • 30% of proteins require a metal cofactor

  • Using a forward genetic approach in B. subtilis, we demonstrate that elevated levels of external Zn (II) inhibit the electron transport chain, whereas intracellular Zn(II) intoxication is due to dysregulation of heme biosynthesis

  • Our results suggest that in wild type cells, which are competent for export of Zn(II) from the cytosol, Zn(II) intoxication results from inactivation of the electron transport chain due to inhibition of the major aerobic cytochrome aa3 oxidase

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Summary

Introduction

30% of proteins require a metal cofactor. Unlike iron (Fe(II)), which can generate cell damaging hydroxyl radicals in the presence of hydrogen peroxide (Fenton reaction), Zn(II) is not redox reactive. Cellular Zn(II) is highly regulated at multiple levels: in Bacillus subtilis the total intracellular concentration at equilibrium is ~0.8 mM, and much of this is sequestered in metalloproteins. We constructed mutants in which each Rex-regulated gene was individually deleted in a wild-type or Δrex background. Deletion of cydABCD resulted in a Zn(II) sensitive phenotype in a wild-type background (Fig 1A), while there was no Zn(II) phenotype associated with deletion of any other member of the Rex regulon. Deletion of cydABCD in a Δrex background completely reversed the Zn(II) resistance phenotype of the Δrex mutant, consistent with the idea that derepression of cydABCD confers Zn(II) resistance (Fig 1B). Expression of the Rex regulon is derepressed under conditions of Zn(II) intoxication as measured by qRTPCR of cydA and ldh expression (Fig 2)

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