PROJECT SUMMARYDespite having a dedicated copper export system copper has been broadly toxic to bacteria since antiquity.Copper toxicity is seen in practice where copper surfaces and tools significantly reduce nosocomial infections.Furthermore during host mediated nutritional immunity (sequestering essential metals while bombardingbacterial with toxic metals) innate immune cells kill engulfed targets using copper which is tightly regulatedwithin the host. However current research is limited regarding the overarching mechanisms of toxicity and thepathways used overcome copper stress that work in conjunction with the copper export system. Through thisproposal we seek to understand how bacteria evolved to interact with toxic metals and overcome the ensuingstress. Furthermore we seek to gather a new cohort of bacterial therapeutic targets. For our model system weare using Streptococcus pneumoniae a global burden for which the vaccine only covers ~25% of known strains.Thus far using microarray data we obtained comparing the wild type S. pneumoniae strain TIGR4 and the copperexport protein deficient strain under copper stress and an assortment of techniques we have validated severalpathways and their corresponding mechanisms. These pathways include copper poisoning nucleotide synthesiszinc being synergistically toxic to bacteria with copper and sugar import being vital to mediation of coppertoxicity. We will focus our studies around the copper repressor protein CopY as we have data showing it not onlycontrols the copper export system but several other systems necessary to overcome copper stress. We willcharacterize these CopY-controlled pathways as well as structurally characterize CopY screen CopY for smallmolecule therapeutics that keep it bound to DNA and examine CopYs interactions with DNA and proteins. Wewill expound upon our current findings explore new avenues of how copper affects S. pneumoniae and applyour findings to other toxic metals and bacteria in hopes to elucidate the orchestrated bacterial response to metalstress.