Using a chemical genetic screen to enhance our understanding of the antimicrobial properties of copper.

The competitive toxic and stress inducing nature of copper necessitates systems that sequester and export this metal from the cytoplasm of bacterial cells. Several predicted mechanisms of toxicity include the production of reactive oxygen species, thiol depletion, DNA and iron-sulfur cluster disruption. Accompanying these mechanisms include pathways of homeostasis such as chelation, oxidation, and transport. Still, the mechanisms of copper resistance and sensitivity are not fully understood. Furthermore, studies fail to recognize that the response to copper is likely a result of numerous mechanisms, as in the case for homeostasis, in which proteins and enzymes work as a collective to maintain appropriate copper concentrations. In this study we used the Keio collection, an array of 3985 Escherichia coli mutants, each with a deleted non-essential gene, to gain a better understanding of prolonged copper exposure. In short, we recovered two copper homeostatic gene and genes involved in transporting and assembling to be involved in mediating prolonged copper stress under the conditions assessed. The gene coding for the protein TolC was uncovered as a sensitive hit and we demonstrated that tolC, an outer membrane efflux channel, is key in mitigating copper sensitivity. Additionally, the activity of tRNA processing was enriched and the deletion of several proteins involved in import generated copper tolerance. Lastly, key genes belonging to central carbon metabolism and nicotinamide adenine dinucleotide biosynthesis were uncovered as tolerant hits. Overall, this study shows that copper sensitivity and tolerance are a result of numerous mechanisms acting in combination within the cell.