All living cells have requirements for metals, largely for the catalytic functions of metalloenzymes and other metal-containing proteins. However, metals become toxic to cells at higher concentrations. Therefore, it is imperative that organisms maintain intracellular metal concentrations within a viable range. As such, cells have many means through which to import, export, store, and detoxify metals, in order to fine-tune the intracellular concentration and reduce the toxicity of each. Iron is one of the most-used metals in metalloproteins, due to both its abundance in the Earth’s crust and its redox flexibility. Easily reduced to the ferrous state (Fe2+) or oxidized to ferric state (Fe3+), iron is widely used in enzymes involved in electron transfer, such as cytochromes, or redox sensing, such as transcription factors. The importance of iron is underscored by the large number of cellular processes that have been discovered in all domains of life that regulate the concentration and usage of iron. Multiple transport systems, for example, mediate the influx and efflux of both Fe2+ and Fe3+. Additionally, the redox flexibility of iron and its midrange redox potentials make iron a potential substrate for anaerobic respiration. Shewanella oneidensis strain MR-1 is a dissimilatory metal-reducing bacterium that lives in the redox transition zones of aquatic sediments. S. oneidensis produces numerous cytochromes that allow it to respire a wide variety of substrates, including extracellular, insoluble Fe3+ compounds, which are reduced to Fe2+. Fe2+ is much more soluble than Fe3+ in physiologically relevant conditions; therefore, S. oneidensis must contend with increasing local concentrations of soluble Fe2+ as it continues to respire Fe3+. How S. oneidensis interacts with Fe2+ and resists Fe2+ toxicity is the subject of this thesis. The second and third chapters of this thesis describe two newly discovered Fe2+ transport proteins in S. oneidensis. The first, which has been named FeoE (ferrous iron export), is an Fe2+ exporter that reduces the intracellular Fe2+ concentration during Fe3+ respiration by S. oneidensis. FeoE belongs to the Cation Diffusion Facilitator superfamily of divalent metal efflux proteins, which includes transporters of Cd2+, Co2+, Cu2+, Fe2+, Ni2+, and Zn2+. Studies presented in this dissertation demonstrate that FeoE is exclusively an Fe2+ exporter. The transporter described in Chapter 3, which was named FicI (ferrous iron and cobalt importer), is an Fe2+ and Co2+ importer. FicI belongs to the Magnesium Transporter-E (MgtE) family of Mg2+ and Co2+ importers; this is the first discovery of an MgtE protein that imports Fe2+ and not Mg2+. FicI appears to represent a secondary Fe2+ importer active at higher Fe2+ concentrations. FicI doesn’t require nucleotide hydrolysis for Fe2+ import, unlike the primary Fe2+ importer FeoB, therefore allowing the cell to conserve energy under high Fe2+ conditions. The fourth chapter in this thesis concerns the ATP-dependent protease ClpXP. ClpXP has previously been found to be involved in various cellular functions in several bacterial species, including releasing stalled proteins from ribosomes and the regulation of sigma factors, which influence the transcription of large groups of genes. The work presented in Chapter 4 shows that ClpXP is needed for the resistance of S. oneidensis to higher concentrations of Fe2+, which does not appear to involve previously described functions of ClpXP. Data presented in Chapter 4 indicate that ClpXP may target metalloproteins during Fe2+ stress, a finding that implicates high Fe2+ concentrations in protein mismetallation and misfolding. Supplementary Tables S1 and S2 contain transposon screen and protein-trapping results, respectively, relevant to this chapter. The work in this thesis expands the knowledge of the ways in which S. oneidensis interacts with Fe2+, including its uptake and efflux, and presents a potential mode of Fe2+ toxicity under anoxic conditions. As iron is an essential metal to most living organisms, and as there are many microorganisms living in metal-rich environments, the work presented here is relevant both to the study of S. oneidensis and to microbiology in general. The protein families discussed here are highly conserved among many microorganisms, and their newly discovered functions in S. oneidensis are likely to apply in others as well. More broadly, this work presents several widely-conserved proteins that have been repurposed or given added functions to meet the needs of an organism in order for it to thrive in a particular environmental niche, which reflects the adaptive nature of evolution.
University of Minnesota Ph.D. dissertation. January 2017. Major: Microbiology, Immunology and Cancer Biology. Advisor: Jeffrey Gralnick. 1 computer file (PDF); viii, 103 pages.
Interactions with Iron: Ferrous Iron Transport and Resistance in Shewanella oneidensis strain MR-1.
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