Browsing by Subject "Geobacter"
Now showing 1 - 7 of 7
- Results Per Page
- Sort Options
Item 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, RuthDissimilatory 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.Item Electron Transfer Through The Outer Membrane Of Geobacter Sulfurreducens(2018-05) Jimenez, FernandaMicrobial metabolism represents a rich source of catalytic tools available for biotechnological applications. Microbial metal reduction is no exception, as it represents a mechanism for transferring electrons stored within nonreactive organic compounds to inorganic redox-active compounds. Despite the usefulness of organisms capable of metal reduction, not enough is known about their electron transfer pathway to be able to engineer them for real-world applications. Specifically, how electrons cross cell membranes to be available on the extracellular surface where they can react with metals or electrodes remains one of the least well-studied aspects of extracellular electron transfer. In this thesis, a compilation of studies into extracellular electron transfer pathways of the model organism Geobacter sulfurreducens, and the characteristics of current-producing biofilms produced by this species uncovers fundamental steps and bottlenecks in this metabolic strategy. Through isotopic label incorporation, the first spatially resolved direct measurement of anabolic activity in G. sulfurreducens biofilms was obtained, concluding that metabolic activity is constrained to the layers within the first few microns from the electrode surface. Combinatorial deletion of putative outer membrane electron conduit gene clusters followed by analysis of the ability of this mutant collection to reduce different extracellular substrates showed that several electron conduit gene clusters are necessary in tandem during metal reduction, while only a previously unstudied gene cluster extABCD was necessary for wild type levels of electrode reduction. Within this mutant collection, the strain lacking all studied gene clusters except extABCD (extABCD+), reached higher current densities with faster doubling times than wild type. The extABCD+ strain was found to form biofilms containing 30% more cells than wild type at the electrode:biofilm interface, where isotopic label incorporation showed 38% higher anabolic activity per cell in extABCD+ biofilms compared to wild type. Thus, by focusing on the fundamental physiology of extracellular electron transfer, evidence for substrate-specific electron transfer pathways were found, and a strain with a streamlined pathway for increased electrode reduction could be constructed. The findings in this work suggest a route to engineering organisms with metal- or electrode-specific reduction pathways, and enhancing electron transfer rates in these systems.Item From functional metagenomics to unique synthetic expression strategies in iron-reducing bacteria.(2012-05) Gonzalez, Tanhia DenysCellulose 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.Item Identification of extracellular matrix components essential for a conductive Geobacter sulfurreducens biofilm(2011-11) Rollefson, Janet BethElectron transfer from Geobacter sulfurreducens cells to electrodes or metal oxides requires proper expression and localization of redox-active proteins as well as attachment mechanisms that interface bacteria with surfaces and other cells. Type IV pili and c-type cytochromes have long been considered important components of this conductive network. In this work, a large-scale mutagenesis of G. sulfurreducens was performed and mutants were screened for extracellular electron transfer and attachment phenotypes, identifying new genes essential for a conductive Geobacter network. A mutant defective in polysaccharide export to the extracellular matrix (Δ1501, ΔxapD::kan) was identified based on its altered surface attachment. Characterization of this mutant revealed the importance of extracellular polysaccharides for proper attachment and anchoring of the external c-type cytochromes necessary for a conductive biofilm network. Furthermore, decreased polysaccharide content was found in commonly studied cytochrome and type IV pili mutants, with defects in cell to cell and cell to surface attachment correlating with levels of extracellular polysaccharides. The extracellular matrix of G. sulfurreducens is therefore a complex network of polysaccharides, type IV pili, and c-type cytochromes. Disruption of any one of these extracellular components alters overall matrix properties and impedes extracellular electron transfer and attachment.Item Microbial Interactions: From Microbes to Metals(2016-01) Kane, AunicaMicrobial 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.Item Redox Potential Controls Electron Transfer Through The Inner Membrane Of Geobacter Sulfurreducens(2021-08) Joshi, KomalHarnessing energy for growth and survival is universal to all living forms. Bacteria are constantly faced with changing environment forcing them to quickly adapt to the conditions to gain maximum energy available. Acquisition of energy involves transfer of electrons from substrate that gets oxidized to the reduction of electron acceptors. Microorganisms performing extracellular electron transfer have evolved to couple oxidation of electron donors to the reduction of electron acceptors present outside the cell using a chain of redox active proteins. Geobacter sulfurreducens is one such model organism for studying extracellular electron transfer, providing unique opportunities for the development of bioelectronic devices and sensors. Despite the usefulness of G. sulfurreducens extracellular electron transfer ability in biotechnological applications, the complete electron transfer pathway still remains unknown. The factors regulating the electron transfer between different cytochromes, as well as the specific utilization of different cytochromes in energy conservation is one of the lesser studied aspects of G. sulfurreducens physiology. The work presented in this thesis includes discovery and characterization of an inner membrane cytochrome complex, CbcBA essential for respiration of electron acceptors near the thermodynamic limit of acetate respiration (< -0.21 V vs. Standard Hydrogen Electrode (SHE)). A \sigma^{54}–dependent transcription factor, BccR controlling the expression of CbcBA was also characterized. Other inner membrane cytochromes involved in redox dependent electron transfer, ImcH, and CbcL are constitutively expressed. Using genetic and electrochemical approaches, CbcL was found to function as a redox dependent switch showing oxidative inactivation above redox potentials of -0.1 V vs. SHE. Using specific mutants lacking one or more inner membrane cytochromes, cellular yields were measured corroborating earlier reported data that the ImcH-dependent electron transfer pathway supported the highest cellular yield, while the CbcL-dependent pathway supported much lower cell yields. The CbcBA-dependent pathway could not support growth under conditions tested, but was found to be needed for survival under low electron acceptor conditions. Expressing fluorescent proteins in specific inner membrane cytochrome mutants allowed studying metabolic heterogeneity of G. sulfurreducens biofilms visualized using confocal microscopy. At high redox potentials (+0.24 V vs. SHE), G. sulfurreducens utilizes ImcH-dependent pathway in cells closest to the electrode, and CbcL-dependent pathway in cells beyond 10 µm from the electrode surface. At low redox potentials (-0.13 V vs. SHE), only the CbcL-dependent pathway is utilized. The findings reported in this thesis, suggests a route for building biosensors for redox sensing.Item Redox potential dependent respiration by Geobacter sulfurreducens(2015-10) Levar, CalebGeobacter sulfurreducens is a Gram negative δ-proteobacteria with the ability to couple the internal oxidation of a carbon and electron donor with the external reduction of extracellular electron acceptors. Extracellular electron acceptors utilized by G. sulfurreducens include insoluble Fe(III)- and Mn(IV)-oxides, electrodes poised at accepting potentials, and a variety of soluble acceptors including humic acids and chelated metals. These substrates exist over a redox potential window greater than 0.5 V, suggesting that respiratory flexibility to efficiently take advantage of electron acceptors with different redox potentials would be a useful trait. The data presented within this thesis demonstrate that G. sulfurreducens uses multiple electron transfer pathways for the reduction of extracellular electron acceptors of different redox potentials.