The molecular basis of the synergistic toxicity of Ni and Cu, common environmental co-contaminants

Heavy metals are ubiquitous in the environment due to both natural processes and anthropogenic activities. Nickel (Ni) and copper (Cu) commonly co-occur in contaminated environments, yet most toxicity studies focus on individual metals. We investigated the combined toxicity of Ni and Cu in Escherichia coli using environmentally relevant concentrations of each. While each metal alone caused minimal growth inhibition, their combination was synergistically toxic. Transcriptomic and metabolomic analyses revealed unique alterations in gene expression and metabolites during the combined metal treatment. Key pathways uniquely impacted by the combined metal exposure included sulfur assimilation, cysteine biosynthesis, and the tricarboxylic acid cycle. Many of these responses appeared to be linked to dysregulation of iron-sulfur (Fe-S) cluster metabolism. We observed increased expression of the genes encoding ISC Fe-S cluster assembly machinery only during metal co-treatment. Growth experiments with deletion mutants confirmed that the ISC machinery was required for survival only under the combined metal stress. We also observed the activation of a sulfur starvation response during the combined metal stress that was consistent with increased sulfur demand for Fe-S cluster biosynthesis. Deletion of cysK, encoding cysteine synthase, impaired growth only under combined metal exposure. Because Fe-S clusters are universal across microbial taxa, the common co-occurrence of Ni and Cu in the environment represents a widespread and underrecognized threat to microbial life and the ecosystem processes they sustain. Our findings highlight the need to further assess the effects of metal mixtures, which can trigger emergent stress responses not yet predictable from single-metal exposures. IMPORTANCEMany environments are contaminated by metals. These metals are toxic to the microorganisms that inhabit these environments and carry out important ecosystem services. While much is known about bacterial responses to single metal stress, in most contaminated environments, metals typically exist as mixtures. Nickel (Ni) and copper (Cu) are common co-contaminants. We tested Ni and Cu in combination to shed light on the mechanism behind their synergistic toxicity in the model bacterium Escherichia coli K-12 using a novel, multi-omics approach. We found that the two metals in combination are likely disrupting iron-sulfur (Fe-S) clusters. Since Fe-S clusters are ubiquitous across microbial taxa and critical for microbial metabolism, this suggests that these two common co-contaminants may be toxic to diverse microorganisms.