The Mechanism of Copper Toxicity in Escherichia Coli

133 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.The goal of this study was to determine the mechanism of copper toxicity. Copper is toxic to both eukaryotes and prokaryotes and is the causative agent of Indian childhood cirrhosis, Tyrolean infantile cirrhosis, and Wilson disease. Studies in eukaryotic systems demonstrated that copper-toxified cells had elevated levels of DNA damage and oxidative stress. Therefore, the mechanism of copper toxicity was hypothesized to be mediated by oxidative DNA damage through a Fenton-like reaction. In contrast to this hypothesis, copper-stressed Escherichia coli sustained less oxidative DNA damage than unsupplemented cells. The levels of hydroxyl radical formation between copper-stressed and unsupplemented cells were similar, demonstrating that copper did not prevent iron-mediated oxidative DNA damage by blocking the Fenton reaction. Therefore, hydroxyl radicals must be formed away from the DNA. We hypothesize that copper-stress leads to the removal of iron from the DNA. The mechanism by which copper-stress causes iron to be displaced from the DNA is unknown. This study demonstrated that copper-mediated oxidative DNA damage does not occur and therefore cannot be the primary mechanism of copper toxicity.While copper did not catalyze oxidative DNA damage, this result does not rule out the possibility that copper toxicity is mediated by oxidative stress. To test this hypothesis, the primary target of copper toxicity was determined and whether copper toxicity was dependent on the presence of oxygen. Copper-stressed cells failed to grow in the absence of branched-chain amino acids. This was due to damage to a family of iron-sulfur cluster dehydratases. Damage to these iron-sulfur cluster dehydratases occurred in both the presence and absence of oxygen, therefore, copper toxicity is not mediated through oxidative stress. Copper damaged iron-sulfur cluster dehydratases by displacing iron from the cluster, presumably by interacting with the sulfide atoms of the cluster. This study demonstrated that a primary toxicity of copper is the oxygen-independent inactivation of iron-sulfur cluster dehydratases.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD