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Browsing by Subject "Shewanella"

Now showing 1 - 8 of 8
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    Development and application of variable strength expression vectors in Shewanella oneidensis MR-1
    (2014-12) Harris, Audrey Jean
    A suite of expression vectors was designed and constructed with modular promoter and ribosome binding site sequences resulting in a wide range of protein expression levels in S. oneidensis MR-1. To allow IPTG induction regardless of host background, a subsequent set of vectors was constructed containing lacIq, the E. coli gene encoding lac operon repressor protein. The practical application of these inducible plasmids in S. oneidensis MR-1 was demonstrated by driving variable expression of genes involved in riboflavin biosynthesis and flavin adenosine dinucleotide transport across the inner membrane in order to increase extracellular flavin levels and thus extracellular respiration rates. This study demonstrates the benefit of being able to temporally control select gene expression and amplitude of expression by using a set of predesigned vectors. The modularity of this system will enable researchers to easily exchange alternative promoters or ribosome binding sites in order to modify protein expression for custom applications.
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    Dissimilatory metal reduction is marked by unexpected physiological and genetic complexity: EET taxis in Shewanella and the diverse and rapidly evolving Geobacter cytochrome pool
    (2023-10) Starwalt-Lee, Ruth
    Dissimilatory metal reduction by Shewanella and Geobacter species is more than just respiration with novel electron acceptors. In addition to the physical transfer of electrons out of the cell, EET In these organisms is supported by complex physiological and genetic features that are in the early stages of investigation.In S. oneidensis EET and motility are connected by taxis to extracellular electron acceptors and in particular, electrodes. In Geobacter and, to a lesser extent, Shewanella species, MHC genes are subject to higher mutation rates than the average gene in the cell. Neither taxis to electrodes nor increased mutation rates in MHC genes are understood, underscoring the fact that we have a long way to go in investigating the biology of dissimilatory metal reducers.
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    Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-mediated Counter-selection
    (2019-06) Domenech Corts, Anna
    Shewanella are invaluable hosts for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals, to create microbial fuel cells and for understanding extracellular electron transfer. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. In this dissertation, I describe development of simple and efficient genome editing tools for precise and site-directed mutagenesis of Shewanella. First, In Chapter I, I review recent advances in synthetic biology that accelerate the study and engineering of bacterial phenotypes. In chapter II, I show the development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of up to ~108 transformants/µg DNA and which is adaptable to other strains. Using this method for DNA transfer, in chapter III, I characterize a new phage recombinase, W3 Beta from Shewanella sp. W3-18-1 and show its utility for in vivo genome engineering (recombineering) using linear single-stranded DNA oligonucleotides. In my experiments the W3 Beta recombinase gives an efficiency of ~5% recombinants among total viable cells. In addition, I show the functionality of this new system in S. amazonensis, a strain with few genetic studies but of interest given its higher temperature range for growth and wide range of carbon sources utilized. In chapter IV, I demonstrate use of the CRISPR/Cas9 system as a counter-selection to isolate recombinants. When coupled to recombineering, this counter-selection results in an extremely high efficiency of >90% among total surviving cells, regardless of the gene or strain modified. This efficiency allows isolation of several different types of mutations made with recombineering, and even allows identification of rare recombinants that form independently of W3 Beta expression. This is the first effective and simple strategy for recombination with markerless mutations in Shewanella. With synthesized single-stranded DNA as substrates for homologous recombination and CRISPR/Cas9 as a counter-selection, this new system provides a rapid, scalable, versatile and scarless tool that will accelerate progress in Shewanella genomic engineering. Finally, I conclude in Chapter V with an overview of the challenges and future directions of the technologies demonstrated here, discussing possible advancements that could further enhance the study of Shewanella.
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    Electrochemical analysis of Shewanella oneidensis engineered to bind gold electrodes.
    (2011-03) Kane, Aunica L.
    Three-electrode bioreactors can be utilized to examine the mechanisms involved in electron flow from bacteria to insoluble electron acceptors and allow these processes to be analyzed quantitatively. As an electrode, gold is an ideal surface to study the electrophysiology occurring during extracellular respiration; yet previous research has shown that Shewanella is resistant to colonization on gold surfaces. Therefore, the goal of this work was to direct adhesion of Shewanella oneidensis to gold surfaces via cell surface display of a modified E. coli outer membrane protein, LamB, and a gold-binding peptide (5rGBP) to encourage microbe-electrode interaction, improve whole-cell biocatalytic systems, and increase overall current production. Expression of LamB-5rGBP increased the affinity of Shewanella for gold surfaces, but also led to the displacement of certain outer membrane components required for extracellular electron transport. Displacement of these outer membrane proteins decreased the rate at which Shewanella was able to reduce both insoluble iron and riboflavin. Expression of LamB-5rGBP, although effectively increasing attachment to gold, did not greatly increase current production in gold-electrode bioreactors.
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    From functional metagenomics to unique synthetic expression strategies in iron-reducing bacteria.
    (2012-05) Gonzalez, Tanhia Denys
    Cellulose and hemicellulose are renewable sources of fermentable sugars. The use of fermentable sugars for the production of alternative energy sources (i.e. ethanol, butanol, etc.) is an attractive solution to alleviate the shortage and high prices of petroleum. Cellulases and hemicellulases are the two groups of glycosyl hydrolases responsible for breaking down the polysaccharide component of biomass into their respective sugar moieties. The enzymatic hydrolysis of cellulose and hemicellulose has relied on enzymes originally produced by culturable organisms. This thesis describes the use of metagenomics coupled to high-throughput screening techniques to identify glycosyl hydrolases originally encoded by uncultured organisms. The findings of this thesis include the identification and biochemical characterization of a unique endoglucanase. Besides catalyzing the hydrolysis of soluble and insoluble cellulosic substrates, this endoglucanase exhibited a domain architecture that has not been previously reported in the literature. This thesis also describes two different strategies to engineer the surface of (Fe+3)-reducing bacteria. These expression systems are a valuable tool for studying the cellular respiration of Geobacter and Shewanella. Furthermore, they have practical applications in the area of whole-cell biocatalysis in microbial fuel cells. The first strategy involved using an autodisplay system to engineer the cell envelope of Geobacter and Shewanella. The autodisplay system translocated a functional β-galactosidase enzyme to the cell envelope of G. sulfurreducens and S. oneidensis. Furthermore, this system proved to be an effective tool for catalyzing reactions in electrochemical cells using biofilms of G. sulfurreducens cells. The second strategy exploited the use of in-frame fusions with the c-type cytochrome OmcZ to translocate a recombinant protein to the outer membrane and extracellular matrix of Geobacter sulfurreducens. This is the first time that the c-type cytochrome OmcZ has been used to engineer biofilms of Geobacter sulfurreducens.
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    Interactions with Iron: Ferrous Iron Transport and Resistance in Shewanella oneidensis strain MR-1
    (2017-01) Bennett, Brittany
    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.
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    Life, Death, And Coexistence: Exploring And Manipulating The Respiratory Lifestyle Of Shewanella Oneidensis
    (2019-08) Kees, Eric
    In their natural environment, microbes often exist in stressful, suboptimal, ever-changing conditions and have evolved innumerable and varied successful strategies for managing stresses and thriving in flux. Microbial ecosystems are defined not only by specialized members occupying defined narrow niches, but also members that move between niches and exist in a mode of constant opportunism. One such opportunistic group of organisms are those belonging to the genus Shewanella which are largely defined by their respiratory versatility. A particularly well-studied member of this genus, S. oneidensis, is the most versatile respiratory organism described to date, and is a model organism for extracellular electron transfer. This thesis explores the respiratory lifestyle of S. oniedensis primarily through the lens of cell physiology and competitive fitness under optimal growth conditions and those that yield catastrophic death. The second chapter of this thesis is a study in cofactor acquisition by a key respiratory enzyme in S. oneidensis MR-1. The periplasmic protein FccA is both a fumarate reductase and an electron carrier protein for extracellular electron transfer, that requires a flavin cofactor for its function. Through genetic manipulation, growth experiments, and biochemical experiments, we found that for S. oneidensis, self-secretion of flavins comes at minimal metabolic cost and is required for periplasmic flavoprotein cofactor acquisition. The third chapter is a probing of natural and engineered factors that enable survival under respiratory stress. Shewanella is considered an obligate respiring organism, and when placed under conditions in which respiration cannot normally function, it experiences massive loss in viability accompanied by cell lysis. Despite, 99-99.99% of cells undergoing death in this condition, many persist. This study leveraged synthetic proton motive force supplementation, which affords enhanced survival of S. oneidensis, to profile the innate strategies used to survive under respiratory stress. The key finding of this study is that the sodium motive force plays a key role in survival of S. oneidensis under respiratory stress, even when survival is enhanced by proton motive force supplementation. The fourth chapter of this thesis is a series of competition experiments reframing a central paradigm of the competitive exclusion principle: that two organisms occupying the same niche cannot coexist. This principle states that in this scenario any organism with a reproductive advantage will ultimately overtake a population. This study demonstrated that two engineered strains of S. oneidensis utilizing the same medium with the same food and nutrient sources, but growing a vastly different rates, can remain at stable frequencies when grown attached to an electrode as the sole sink of electrons in respiration. The primary reason for this stability is that the original parent population remains actively growing on a surface to which daughtered cells are constantly removed. While the scenario we have engineered to “break” the competitive exclusion principle could be considered a form of niche differentiation, it demonstrates an effective strategy to combat strain degeneration and contamination in industrial fermentation, by allowing a selected population of less competitively fit individuals to act indefinitely as a progenitor population. The work in this thesis brings special attention to the adaptation towards an obligate respiratory lifestyle in S. oneidenisis. The systems it has evolved to secrete flavins as both respiratory cofactors and intermediates and the marked death it experiences under respiratory stress together emphasize adaptations in Shewanella to thrive in redox stratified environments, supported by the wide variety of respiratory nodes it can utilize. Finally, this work highlights the utility of S. oneidensis as a test platform for ideas, with potential benefits for biotechnological and industrial applications.
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    Microbial Interactions: From Microbes to Metals
    (2016-01) Kane, Aunica
    Microbial communities are the major drivers of biochemical cycling and nutrient flux on the planet, yet despite their importance, the factors that influence and shape behavior and function of microbial ecosystems remain largely undefined. The knowledge gap existing for microbial communities stems partly from a focus of microbiologists on monoculture but also because studies of multispecies systems are impeded by their complexity and dynamic nature. Synthetic ecology, the engineering of rationally designed communities in well-defined environments, provides an innovative and robust approach to reduce the complexity inherent in natural systems and mimic microbial interaction in a controlled framework. Synthetic ecology was used to engineer a co-culture using two previously non-interacting bacteria, Shewanella oneidensis and Geobacter sulfurreducens, both organisms important for multiple applications in biotechnology. The S. oneidensis and G. sulfurreducens co-culture provided a model laboratory co-culture to study microbial interactions and revealed that genetic mutations in metabolic pathways can provide the foundation to initiate cooperation and syntrophic relationships in multispecies ecosystems. Syntrophy between S. oneidensis and G. sulfurreducens was studied further using three-electrode bioreactors. Both S. oneidensis and G. sulfurreducens are capable of respiring insoluble terminal electron acceptors, a process termed extracellular respiration. During extracellular respiration, electrons produced during oxidative metabolism are transferred across both membranes to the outer surface of the bacterial cell where they reduce terminal electron acceptors such as metal oxides. Extracellular respiration can be monitored in real time as current produced in bioreactors with electrodes serving as a proxy for metal oxides. The ability of both S. oneidensis and G. sulfurreducens to transfer electrons to their outer surface enabled the study of a process central to many syntrophic communities known as interspecies electron transfer – the transfer of reducing equivalents between organisms. Mutants in various electron transfer pathways revealed that interspecies electron transfer in an obligate S. oneidensis/G. sulfurreducens co-culture was mediated by soluble redox-active flavins secreted by Shewanella serving as electron shuttles between species. The second half of this thesis focuses on S. oneidensis metabolism and interactions of microbes with metals. Microbial transfer of electrons to metals has a large impact on biogeochemical cycles and can also be harnessed for biotechnology applications in bioenergy and bioremediation. In order to effectively engineer S. oneidensis for these applications, it is imperative to understand how Shewanella gains energy from the oxidation of electron donors and the efficiency of electron transfer to metals and electrodes. Work in Chapter 4 revealed formate oxidation to be a central strategy under anaerobic conditions for energy conservation through the generation of proton motive force in S. oneidensis. Work in Chapter 5 quantified the effect of hydrogen metabolism on electron transfer reactions in Shewanella three-electrode bioreactors. Deletion of the hydrogenase large subunits, hyaB and hydA, in Shewanella resulted in higher current density and coulombic efficiency in single-chamber three-electrode bioreactors by diverting electron flux to the anode instead of to hydrogen production. The final chapter of this thesis focused on harnessing microbial transformation of metals for bioremediation purposes. An engineered Escherichia coli strain containing a mercury resistance plasmid was constructed to facilitate the remediation of organic and ionic forms of mercury pollution. The engineered strain was then encapsulated using silica sol gel technology generating a bio-filtration material for use in bioremediation platforms. Work in this thesis highlights the importance of microbial interactions, both with other organisms and with metals in the environment. Comprehensive knowledge on microbial interactions is important not only for a better understanding of ecosystem function but can also be harnessed for biotechnology applications. Microbial interactions and transformation of metals shape the world around us and have also facilitated use and further engineering of microorganisms for bioenergy and bioremediation technologies.