Intracellular Zn(II) Intoxication Leads to Dysregulation of the PerR Regulon Resulting in Heme Toxicity in Bacillus subtilis

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.

Fig 1. Derepression of the alternative cytochrome bd oxidase contributes to Zn(II) resistance.

(A and B) Susceptibility of WT and mutant strains to Zn(II) as assessed by disk diffusion assay. The data are expressed as the diameter of the zone of inhibition (ZOI) as measured in millimeters. The mean and standard error of three independent experiments is shown. Asterisks indicate significance as determined by a Student’s t-test (P<0.05). (C) Model of the contribution of the Rex regulon to Zn(II) resistance.

Fig 2. Zn(II) intoxication leads to derepression of the Rex regulon in wild type cells, and dysregulation of PerR in a Zn(II) efflux deficient mutant.

Gene expression levels of representative members of the Rex (cydA and ldh) and PerR regulons (hemA and katA) was monitored after exposure to Zn(II) (200 μM for WT, 50 μM for cadA czcD). Asterisks indicate significance as determined by a Student’s t-test (P<0.05).

Fig 3. Characterization of Zn(II) resistant suppressors isolated from a Zn(II) efflux deficient mutant.

Susceptibility of WT and isolated suppressor strains to Zn(II) as assessed by disk diffusion assay. The data are expressed as the diameter of the zone of inhibition (ZOI) as measured in millimeters. The mean and standard error of three independent experiments is shown. Asterisks indicate significance as determined by a Student’s t-test (P<0.05).

Fig 4. Menaquinone and heme biosynthesis contribute to intracellular Zn(II) intoxication.

Susceptibility of WT and mutant strains to Zn(II) as assessed by disk diffusion assay in the presence or absence of 10 μg/ml 1,4-dihydroxy-2-naphthoic acid or menaquinone. The data are expressed as the diameter of the zone of inhibition (ZOI) as measured in millimeters. The mean and standard error of three independent experiments is shown. Asterisks indicate significance as determined by a Student’s t-test (P<0.05) and ND indicates no significant difference.

Fig 5. Zn(II) intoxication leads to intracellular heme accumulation.

(A) Measurement of heme content of crude extract of cells treated with 50 μM Zn(II). Fluorescence emission from 400 to 500 nm was recorded after excitation at 380 nm. The peak fluorescence intensity at 450 is plotted. The mean and standard error of three independent experiments is shown. (B) Susceptibility of WT and heme monooxygenase mutant strains to Zn(II) as assessed by disk diffusion assay in the presence or absence of external supplementation of menaquinone. The data are expressed as the diameter of the zone of inhibition (ZOI) as measured in millimeters. The mean and standard error of three independent experiments is shown. (C) Catalase activity of wild type and a Zn(II) efflux defective mutant was monitored after a 10 minute exposure to Zn(II) (200 μM for WT, 50 μM for cadA czcD). The mean and standard error of three independent experiments is shown. Asterisks indicate significance as determined by a Student’s t-test (P<0.05) and ND indicates no significant difference.

Fig 6. Zn(II) intoxication leads to dysregulation of the PerR regulon.

(A) Dissociation of Mn-cofactored PerR (PerR:Zn,Mn) from DNA as monitored by fluorescence anisotropy. Anisotropy was determined with 100 nM DNA containing PerR binding sites from katA, hemA, and mrgA after the addition of 100 nM active PerR dimer and 10 μM MnCl₂. ZnCl₂ was titrated at the indicated concentrations. A representative data set is shown. (B) Model for dysregulation of the PerR regulon by Zn(II) intoxication. The structural Zn(II) atom in each monomer is represented by an orange circle and is required for protein folding and stability. The blue circles represent Zn(II) that can enter the cell and, in the absence of efflux, leads to mismetallation of PerR (converting the PerR:Zn,Mn form to a PerR:Zn,Zn form).