Systematic and synthetic biology insights into copper homeostasis in Escherichia coli

Copper serves as an essential trace element in biological systems through its association with metalloenzymes. Two major regulatory systems (cueR and cus) participate in the transcriptional regulation of copper homeostasis genes, yet critical mechanisms remain poorly understood. We systematically investigated E. coli's copper response using systems and synthetic biology approaches, revealing more detailed mechanisms. Our results suggested that the cus system specifically responds to Cu⁺ but not Cu²⁺, with Cu²⁺ to Cu⁺ reduction occurring primarily in the cytoplasm. Cu⁺ in the periplasmic space mainly originates from CopA-mediated export through the cytoplasm. We discovered that CusR exhibits signal crosstalk, causing baseline expression, which can be regulated by optimal CusS concentrations. CusS functions as both a kinase and a phosphatase depending on Cu⁺ presence. We quantified E. coli's response to various extracellular Cu²⁺ concentrations, ultimately clarifying the relationship between the two copper ion response systems. These findings provide comprehensive insights into bacterial copper homeostasis mechanisms.