Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli

Abstract

While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells.IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions than they are under aerobic conditions. Under anaerobic conditions, E. coli cells accumulate excess intracellular copper, which specifically targets iron-sulfur proteins by blocking iron-sulfur cluster biogenesis. Since iron-sulfur proteins are involved in diverse and vital physiological processes, inhibition of ironsulfur cluster biogenesis by copper disrupts multiple cellular functions and ultimately inhibits cell growth. The results from this study illustrate a new interplay between intracellular copper toxicity and iron-sulfur cluster biogenesis in bacterial cells under anaerobic conditions.

Copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to bypass of penicillin-binding proteins

“…Copper is an essential cofactor in many organisms, driving processes such as respiration and photosynthesis. However, aquate (or free) copper ions are extremely toxic even at low concentration (in the micromolar range) due to their high affinity for the active site of iron-cluster-containing metallo-enzymes (11,12). When misloaded with copper ions, these enzymes produce highly toxic reactive oxygen species.…”

“…Additionally, copper generates superoxide and other reactive oxygen species in the presence of molecular oxygen, causing damage to the cell membrane through lipid peroxidation (9,10). Copper can outcompete and replace other metals from their binding sites in metallo-proteins, such as the iron-sulfur protein fumarase A, isopropylmalate isomerase, and 6-phosphogluconate dehydratase in E. coli (11,12). It is likely that copper has other yet unknown targets, for example in the bacterial cell envelope.…”

Mitochondrial Ferredoxin Determines Vulnerability of Cells to Copper Excess

“…Toxicity of this essential micronutrient can arise from defects in homeostasis or environmental exposures ( Bandmann et al., 2015 , Pena et al., 1999 , Renwick, 2006 ). Previously described molecular targets of Cu toxicity include cytochrome c biogenesis ( Durand et al., 2015 ), oxidative damage to cell constituents such as membrane lipids ( Howlett and Avery, 1997 ), nucleotide synthesis ( Johnson et al., 2015 ), and FeS-protein integrity or biogenesis ( Alhebshi et al., 2012 , Brancaccio et al., 2017 , Foster et al., 2014 , Macomber and Imlay, 2009 , Tan et al., 2017 ). Here, Yah1 proved to be a Cu-sensitive weak point of the FeS-cluster biogenesis/delivery pathway in yeast and, as expected for a key target ( Avery, 2011 ), disabling the protein produced Cu-sensitive phenotypes while overexpression of Yah1 or human Fdx2 conferred resistance.…”

“…FeS clusters are also susceptible to displacement of Fe by metals such as Cu, Ag, and Hg ( Brancaccio et al., 2017 , Macomber and Imlay, 2009 , Tan et al., 2017 , Xu and Imlay, 2012 ). To date, most studies of ROS- and/or Cu-sensitive FeS proteins have focused on (conditionally-) nonessential FeS proteins such as aconitase, isopropylmalate isomerase, and fumarase ( Foster et al., 2014 , Jang and Imlay, 2007 , Jang and Imlay, 2010 , Macomber and Imlay, 2009 , Tan et al., 2017 ). Work with yeast showed that Cu stress also impairs function of the essential FeS protein Rli1 (ABCE1) ( Alhebshi et al., 2012 ).…”

Development of a Sensitive Escherichia coli Bioreporter Without Antibiotic Markers for Detecting Bioavailable Copper in Water Environments

“…It also has adverse effects on male fertility (Wirth and Mijal, 2010). In recent years, studies show that excessive copper may disrupt the labile [4Fe-4S] clusters in dehydrogenases (Macomber and Imlay, 2009) and block ironsulfur cluster biogenesis in Escherichia coli (Fung et al, 2013;Tan et al, 2017) and Bacillus subtilis (Chillappagari et al, 2010).…”