Copper Stress Affects Iron Homeostasis by Destabilizing Iron-Sulfur Cluster Formation in Bacillus subtilis

Abstract

Copper and iron are essential elements for cellular growth. Although bacteria have to overcome limitations of these metals by affine and selective uptake, excessive amounts of both metals are toxic for the cells. Here we investigated the influences of copper stress on iron homeostasis in Bacillus subtilis, and we present evidence that copper excess leads to imbalances of intracellular iron metabolism by disturbing assembly of iron-sulfur cofactors. Connections between copper and iron homeostasis were initially observed in microarray studies showing upregulation of Fur-dependent genes under conditions of copper excess. This effect was found to be relieved in a csoR mutant showing constitutive copper efflux. In contrast, stronger Fur-dependent gene induction was found in a copper efflux-deficient copA mutant. A significant induction of the PerR regulon was not observed under copper stress, indicating that oxidative stress did not play a major role under these conditions. Intracellular iron and copper quantification revealed that the total iron content was stable during different states of copper excess or efflux and hence that global iron limitation did not account for copperdependent Fur derepression. Strikingly, the microarray data for copper stress revealed a broad effect on the expression of genes coding for iron-sulfur cluster biogenesis (suf genes) and associated pathways such as cysteine biosynthesis and genes coding for iron-sulfur cluster proteins. Since these effects suggested an interaction of copper and iron-sulfur cluster maturation, a mutant with a conditional mutation of sufU, encoding the essential iron-sulfur scaffold protein in B. subtilis, was assayed for copper sensitivity, and its growth was found to be highly susceptible to copper stress. Further, different intracellular levels of SufU were found to influence the strength of Fur-dependent gene expression. By investigating the influence of copper on cluster-loaded SufU in vitro, Cu(I) was found to destabilize the scaffolded cluster at submicromolar concentrations. Thus, by interfering with iron-sulfur cluster formation, copper stress leads to enhanced expression of cluster scaffold and target proteins as well as iron and sulfur acquisition pathways, suggesting a possible feedback strategy to reestablish cluster biogenesis.

Visualizing Biological Copper Storage: The Importance of Thiolate‐Coordinated Tetranuclear Clusters

“…The ability to store and protect Cu I ions in the cytosol is driven by the formation of thiolate‐coordinated tetranuclear clusters in both prokaryotes and eukaryotes, but is achieved with dramatically different protein structures. Paradoxically, the same facile cluster chemistry of Cu I is also responsible for toxicity by the displacement of iron at Cys‐bound 4Fe–4S clusters 27, 28, 29, 30. MTs have been suggested to contribute to a “chelation” rather than “compartmentalization” mechanism to combat copper toxicity in eukaryotes 27.…”

Copper-responsive gene regulation in bacteria

“…When in excess, however, copper is highly toxic to living cells, as it interacts with free proteinogenic thiol groups, destabilizes iron-sulfur cofactors, competes with other metals for protein binding sites, and possibly leads to formation of reactive oxygen species (Chillappagari et al, 2010;Hiniker et al, 2005;Macomber & Imlay, 2009). Remarkably, disturbance of copper homeostasis is thought to lead to human diseases such as Alzheimer's disease, Parkinson's disease, Wilson's disease and Menkes syndrome (Barnham & Bush, 2008;Lenartowicz et al, 2011;Lutsenko, 2010).…”

Copper chloride induces antioxidant gene expression but reduces ability to mediate H2O2 toxicity in Xanthomonas campestris

“…Increased intracellular Cu(I) ions destabilize ironsulfur clusters, leading to functionally inactive proteins. Genome-wide transcriptome analysis of Bacillus subtilis has demonstrated that numerous genes for iron-sulfur cluster-containing enzymes, including those involved in the electron transport chain, are upregulated during copper stress conditions (Chillappagari et al, 2010). It is likely that excess copper ions can cause ineffective electron transport, resulting in increased generation of superoxide anions.…”

“…A recent finding has shown that iron-sulfur clusters are primary targets for copper toxicity (Chillappagari et al, 2010;Macomber & Imlay, 2009). Increased intracellular Cu(I) ions destabilize ironsulfur clusters, leading to functionally inactive proteins.…”