Metal contamination is a widespread environmental threat in great need of sustainable remediation strategies. Electroactive (electricity-producing) bacteria, particularly those in the order Geobacterales, are ubiquitous in these environments and drive metal transformations that limit their mobility and spread. Studying these bacteria thus affords opportunities to understand cellular mechanisms for enhanced detoxification and harness the knowledge in environmental clean-up and metal recovery applications. The unifying feature of the physiology of Geobacterales and close relatives in the Desulfuromonadia class is their ability to discharge respiratory electrons to metals. This allows them to respire metal minerals, particularly bioreducible ironIII oxide minerals abundant in the Earth’s crust, from terrestrial soils and sediments to hot sediments near deep-sea hydrothermal vents. Their metal-reducing activities dissolve the minerals and release metal cations trapped in the solid phases, including metals of economic importance such as uranium, cobalt, and palladium. Despite the release of metal cations at concentrations toxic to most living organisms, Geobacterales grow in these environments and contribute to their reductive precipitation. The reductive dissolution of the ironIII oxides and the mineralization of solubilized metal cations require the assembly of conductive protein appendages (pili), which function as nanowires between the cell and the metals via electric discharges at dedicated surface motifs on the pili. This dissertation describes novel paths for enhanced metal detoxification in the laboratory representative Geobacter sulfurreducens and unique adaptations of the first described nanowire-producing, thermophilic relative, Geothermobacter ehrlichii, to metal cycling in deep-sea hydrothermal vents. Chapter 1 introduces the reader to the topic of metal contamination and the unique adaptive mechanisms used by Geobacterales and close relatives to detoxify toxic metals via their reductive mineralization by pilus nanowires. Chapter 2 describes gaps of knowledge in the field based on studies of uranium reduction in the model representative Geobacter sulfurreducens and identifies the cell’s rough lipopolysaccharide (LPS) as an overlooked mechanism to enhance metal immobilization in this organism. This chapter also identifies outer membrane vesiculation as a pathway for the detoxification of the LPS-bound uranium that can substitute for the nanowire pathways under certain laboratory conditions. Chapter 3 further investigates the environmental signals that modulate vesiculation in G. sulfurreducens. The study revealed that subtle and easily overlooked differences in the ionic strength of media formulations, but also the presence of reducible metal cations such as the uranyl cation, influence cell wall structure and modulate the formation of outer membrane vesicles. These cation-mediated cell wall dynamics play a critical role in the selection of pathways for metal detoxification, showcasing the complementarity of nanowires and vesiculation. This work also underscores the importance of media formulations in the reproducibility of metal reduction phenotypes in Geobacterales and the translational power of the laboratory studies in field applications. Chapter 4 expands metal detoxification studies beyond Geobacterales and into the thermophilic relative G. ehrlichii. Sequencing and closure of its genome enabled identification of conserved metal detoxification pathways, including rough LPS and conductive pili. Comparison of the G. ehrlichii pilus subunit (pilin) with those of the Geobacterales relatives revealed structural similarities and differences in aromatic density (a hallmark of conductive peptides). Furthermore, analysis of reported values of pilus conductivity in these bacteria identified a strong correlation between pilin aromatic density and resistivity of the assembled pilus. Lastly, divergent features at the G. ehrlichii pilin carboxy-terminus are consistent with a metal trap evolved to immobilize trivalent metal cations such as rare earth elements, which are enriched in hydrothermal vent fluids. These results suggest that conductive pili are a widespread metal detoxification strategy in Desulfuromonadia but also identify divergent features reflecting ecological adaptations that could be harnessed to reclaim an expansive catalog of economically important metals. Future directions to achieve this are described in more detail in Chapter 5 along with the main conclusions of the experimental work reported in this dissertation.
Read
