The hallmark of the physiology of Geobacter bacteria is their ability to couple their oxidative metabolism to the respiration of a broad spectrum of metals, including mineral phases such as FeIII and MnIV oxides. In nature, these metal oxides often coprecipitate with or adsorb various metal species such as the micronutrient Co, which is released into the environment during the reductive dissolution of the metal oxide minerals by Geobacter. Once released, the divalent species of Co (CoII) may be assimilated by Geobacter cells to synthesize cobamides for syntrophic partners. Potentially toxic concentrations of CoII are predicted to be solubilized from the metal oxides, exerting selective pressure on Geobacter and syntrophic partners to detoxify their local environment in order to remain viable and metabolically active. In this dissertation, I investigated the mechanisms that allow the model representative Geobacter sulfurreducens to tolerate CoII exposure and the role that its complex respiratory chains have in the reductive detoxification of the metal. Consistent with adaptation through regular exposure to CoII, G. sulfurreducens expresses a complex network of detoxification pathways to mitigate metal intoxication. In chapter 2, I demonstrate that this transcriptional response enables G. sulfurreducens to survive CoII concentrations typically used to enrich metal-resistant microorganisms. Metal-stressed cells removed 25 μM of CoII from culture supernatants and accumulated Co nanoparticles on the cell surface. This result suggested that extracellular mineralization plays a role in the detoxification response. Among the most highly upregulated genes during CoII treatment were cell envelope c-type cytochromes (CbcBA) involved in electron transfer to low-potential metal acceptors, which could provide a path for the reductive mineralization of the metal on the outer surface. In chapter 3, I investigated the role of CbcBA in CoII detoxification using a mutant carrying a deletion in the cbcBA genes and a genetically complemented strain. The studies identified roles for CbcBA in the acclimation of cells to CoII stress, but compensatory effects were often observed that minimized the impact of the cytochrome defect in metal detoxification. Notably, loss of the cytochrome pathway stimulated vesiculation even in the absence of CoII, a phenotype associated with increased membrane fluidity and permeability. As a result, more CoII permeated into resting cells lacking the cytochrome pathway than the wild type cells, which slowed down their growth recovery once transferred to metal-free growth medium. Hence, the CbcBA cytochrome pathway is part of a complex cellular response that contributes to CoII detoxification in G. sulfurreducens. In addition to having numerous cytochromes for the extracellular reduction of metals, G. sulfurreducens also assembles conductive pili decorated with metal traps that can bind CoII and, at a low but biologically relevant potential, reduce it to Co0 nanoparticles. In chapter 4, I investigated a biological role for the pili in CoII detoxification via the extracellular reduction and precipitation of the metal. The study showed that cells that dynamically extend and retract their pili have a growth advantage in the presence of CoII. Furthermore, piliated cells accumulate metal nanoparticles along the filaments, a phenotype previously associated with the ability of the pili to reductively precipitate metals extracellularly to prevent them from traversing the outer membrane. These results indicate that the conductive pili play a major role in the mineralization of CoII and that this reaction is critical to avoid metal intoxication. In chapter 5, I show that even subtle changes in the formulation of the buffer used for CoII reduction assays with resting cells affects their recovery in metal-free medium. The results emphasize the need to carefully optimize the reaction conditions to study CoII detoxification reactions in vitro and reduce the variability typically associated with cells undergoing metal intoxication. The last chapter summarizes the major conclusions of this work and describes future research directions that can expand understanding of the adaptive responses used by Geobacter bacteria to respire metals as a cellular protective mechanism. The ecological impacts of these reactions and applications in biotechnology are discussed.
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