An insufficient knowledge of the biological mechanisms of contaminant transformation often limits the performance of in situ subsurface bioremediation and long-term stewardship strategies. The in situ stimulation of Fe(III) oxide reduction by Geobacter bacteria leads to the concomitant precipitation of U(VI) from groundwater. However, the biological mechanism behind this reaction has remained elusive. Because Fe(III) oxide reduction requires the expression of conductive pili in Geobacter, we also evaluated their contribution to uranium reduction. In chapter 2 of my dissertation I demonstrate a previously unrecognized role for Geobacter pili in the extracellular reduction of uranium and its essential function as a catalytic and protective cellular mechanism.The expression of pili by Geobacter also promotes cell aggregation and biofilm formation. Recent work has shown that Geobacter cells transition from planktonic to biofilm physiologies during the active phase of U reduction in the subsurface. Despite these findings, the contribution of Geobacter biofilms to uranium removal and reduction has not been investigated. In chapter 3 of my dissertation I demonstrate that multilayer biofilms are able to reduce and tolerate substantially more U than planktonic cells for prolonged periods of time, making them an attractive option for the development of permeable biobarriers for U bioremediation. I also demonstrate the role of pili as a primary U reductase in the biofilm.To gain further insight into how biofilms transform U, in chapter 4 of my dissertation I screened a library of transposon-insertion mutants and identified mutants with biofilm defects. This study confirmed the role of Geobacter pili in biofilm formation, and identified other genes encoding cell envelope and electron transport components that had not previously been implicated in biofilm development. These molecular markers can be used to predict and monitor the physiological state of Geobacter bacteria during the in situ bioremediation of U.Previous work, including the prior chapters of my dissertation, has highlighted the importance of the cell envelope and its components for the survival of Geobacter in the subsurface. However, little is known regarding the regulation of the cell envelope. Thus, I investigated the role of the Geobacter's ECF sigma factor, RpoE. In the last chapter of my dissertation, I show that RpoE is required for response to cell envelope stress, as well as the regulation of Geobacter's extracellular electron transfer pathways. This highlights the functional specialization that RpoE has undergone to control the adaptive responses that enable Geobacter bacteria to survive in the environment, and links my findings to the physiology of Geobacter in the subsurface.
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