Date of Award

6-30-2016

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

First Advisor

F. Wayne Outten

Abstract

Various transition metals are essential to all forms of life, and are only required in trace amounts. But this dependence comes as a double-edged sword. All organisms must maintain a careful intracellular quota that does not traverse outside an acceptable range. One transition metal in particular is nickel. The importance of this transition metal has been debated widely and its function varies greatly between organisms, including bacteria. However, the adverse effects caused by over exposure to this metal have been the center of much experimentation in recent years. Still, the mechanisms of nickel toxicity and the subsequent effects on cellular health, particularly the stability of the iron metallome, in bacteria remains poorly understood. The overall aim of these studies was to further elucidate the effects of nickel toxicity on the overall state of iron homeostasis during the lag phase of growth, using Escherichia coli as the model organism. We therefore developed a growth scheme that forced cells pre-adapted to growth on glucose to alter their central carbon metabolism to accommodate growth on gluconate. This shift allotted an additional stress on the iron metallome, given that the 6-phosphogluconate dehydratase enzyme vital to gluconate metabolism requires a [4Fe-4S] cluster for proper function. Our data demonstrated that the activity of this enzyme is absent in the presence of nickel, and thereby inhibited growth on gluconate during nickel exposure. ICP-MS and EPR analyses further confirmed nickel exposure during the lag phase inhibited iron uptake, and several genes central to iron uptake and Fe-S cluster synthesis were expressed through the lag phase. Finally, wild type cells were observed to ultimately adapt to the nickel stress and grow to stationary phase. It was determined that these nickel treated cells developed a new phenotype that was resistant to nickel toxicity. The ferric reductase YqjH was linked to the development of this nickel resistance. A nickelhypersensitive DyqjH mutant, however, did not develop resistance to nickel toxicity as the wild type strain had done. Finally, YqjH has been linked to both iron and nickel homeostasis, suggesting a possible role for YqjH during nickel stress.

Rights

© 2016, Geoffrey Tuttle Ford

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