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Title: Advancing the study of intramembrane metalloproteases using two models from Bacillus subtilis
Creator: Parrell, Daniel D.
Date: 2019
Collection: Electronic Theses & Dissertations
Description: In recent years the field of intramembrane proteolysis has generated great discoveries about the unique process by which proteins are cut within cell membranes. The intramembrane metalloprotease (IMMP) family of intramembrane proteases has provided a number of excellent models for study in bacteria. The PDZ domain-containing IMMPs, RseP (Escherichia coli) and RasP (Bacillus subtilis) are the best understood IMMPs, and their study has have laid the groundwork for studying the role of IMMPs in...
Show moreIn recent years the field of intramembrane proteolysis has generated great discoveries about the unique process by which proteins are cut within cell membranes. The intramembrane metalloprotease (IMMP) family of intramembrane proteases has provided a number of excellent models for study in bacteria. The PDZ domain-containing IMMPs, RseP (Escherichia coli) and RasP (Bacillus subtilis) are the best understood IMMPs, and their study has have laid the groundwork for studying the role of IMMPs in a number of important bacterial pathogens. Work described in this dissertation has advanced the study of RasP. Better methods for heterologous expression of RasP and its substrates in E. coli are presented. Site-1 cleavage of the RasP substrate RsiW was required, while the substrate FtsL was cleaved directly in E. coli. Importantly, RasP is only the third IMMP to be purified and have in vitro activity demonstrated on a substrate (RsiW). Surprisingly, FtsL was not cleaved in vitro. The work also advanced knowledge about the cystathione beta synthase (CBS) domain-containing IMMP, SpoIVFB. IMMPs with CBS domains use ATP binding to regulate activity. To test for physiological conditions that change the ATP concentration, a luciferase-based ATP sensor was developed for B. subtilis. ATP levels change significantly during sporulation and in response to channels or "feeding tubes" present in B. subtilis cells during endospore formation. Interestingly, two different treatments that artificially lower ATP levels to the same extent affected processing of the substrate (Pro-sigmaK) differently, suggesting other adenine nucleotides may bind the CBS domain. Indeed, an assay for conformational change upon ATP binding demonstrated that AMP may modulate SpoIVFB conformational changes. Critical residues of the CBS domain were identified by substitutional analysis in E. coli and B. subtilis, and additional residues of interest were identified in a suppressor screen. Outstanding questions and future directions related to these projects are presented.
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Title: Enzyme electrocatalysis in mediated bioelectrodes
Creator: Chakraborty, Deboleena
Date: 2010
Collection: Electronic Theses & Dissertations
Description: Enzymatic biofuel cells utilize the unique activity and selectivity of enzymes to convert chemical energy directly to electrical energy, and have the potential to be miniaturized for small-scale power devices. This research program seeks to study the characteristics, limitations, and potential for improvement of a mediated laccase-catalyzed electrode for the reduction of oxygen, focusing on the role of mediator redox potential in the catalytic mechanism. The bio-cathode system studied...
Show moreEnzymatic biofuel cells utilize the unique activity and selectivity of enzymes to convert chemical energy directly to electrical energy, and have the potential to be miniaturized for small-scale power devices. This research program seeks to study the characteristics, limitations, and potential for improvement of a mediated laccase-catalyzed electrode for the reduction of oxygen, focusing on the role of mediator redox potential in the catalytic mechanism. The bio-cathode system studied utilizes purified laccase from Trametes versicolor mediated by osmium (Os) centered redox polymers. The results of these studies can enable design of mediated electrodes for biofuel cell applications.Exposure of fuel cell cathodes to fuels like methanol can reduce fuel efficiency and cell voltage via competitive reactions, and can foul the cathode catalyst. Introduction of selective electrocatalysts such as enzymes can solve these problems, and introduces the possibility of a mixed-feed fuel cell system with reduced complexity. The effect of redox potential of a redox hydrogel mediator on the performance of the mediated bio-cathode under varying alcohol concentrations is described. This study demonstrates that the selectivity of mediated laccase oxygen cathodes can facilitate high methanol feed concentration as compared to conventional direct methanol fuel cells and under certain optimum operating conditions, the enzyme might serve as a better cathode catalyst in presence of contaminants like methanol than the conventional Pt/Ru catalysts. A non-competitive inhibition model is proposed to describe the influence of methanol on laccase-catalyzed oxygen reduction kinetics. Methanol replaces water in the enzyme and thereby affects the electron transfer environment near the enzyme active site.In collaboration with Northeastern University, we employ X-ray absorption techniques to characterize the oxygen reduction mechanism of an immobilized laccase while the electrode is operated in situ. The overall goal of this project is to map the oxygen reduction reaction mechanism by a mediated laccase electrode as a function of mediator redox potential, applied electrode potential and presence/absence of substrate (O2). We have successfully detected active Cu sites (in micro-molar concentration) and identified key relationships between oxidation state and mediator redox potential in the presence and absence of oxygen. Our collaborators at Northeastern University have applied the powerful Δμ technique to determine the exact configuration of oxygen attachment to the Cu active sites and to identify the intermediates of the oxygen reduction reaction for a mediated biocathode. Electron-conducting redox hydrogels electrically connect the redox centers of enzymes to electrodes, enabling multi-layer activation and higher current density output. The physicochemical state of these redox polymers and their electron transport mechanism depends on the swelling behavior of these hydrogels in an ionic media. We have fabricated and characterized homogeneous sub-micron sized thin redox hydrogel films with great precision and great repeatability. We have estimated the transport and kinetic parameters for mediated enzymatic systems with precision to have a better understanding of the reaction mechanisms in these complex systems.
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Title: Disentangling the branched respiratory chain of Shewanella oneidensis MR-1
Creator: Duhl, Kody Lee
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Bacteria possess a wide range of metabolic pathways, allowing them to adapt to an array of environmental changes. Focusing on these different metabolic pathways allows us to observe how bacteria catabolize substrate or use anabolic pathways to generate biomass. A more in-depth look shows that many of these pathways are redundant, meaning a single organism can conduct the same overall reactions differing only by the types of enzymes or intermediates used. Overlapping pathways are common in...
Show moreBacteria possess a wide range of metabolic pathways, allowing them to adapt to an array of environmental changes. Focusing on these different metabolic pathways allows us to observe how bacteria catabolize substrate or use anabolic pathways to generate biomass. A more in-depth look shows that many of these pathways are redundant, meaning a single organism can conduct the same overall reactions differing only by the types of enzymes or intermediates used. Overlapping pathways are common in bacteria and have become a focal point of metabolism research to determine the advantages of conserving redundant pathways throughout evolution. The metal reducing bacterium Shewanella oneidensis MR-1 is a practical model organism for metabolic studies, as it has substantial branching within its respiratory pathways. In this work, we focused on the extensive electron transport chain (ETC) of S. oneidensis MR-1 to understand the importance of seemingly redundant respiratory complexes and their functions during aerobic growth. The S. oneidensis MR-1 genome encodes four different NADH dehydrogenases (NDHs): a proton-pumping Type I NDH (Nuo), two sodium-pumping NDHs (Nqr1 and Nqr2), and one type II ‘uncoupling NDH (Ndh). NDHs oxidize NADH to move electrons into the ETC and generate ion-motive force that drives ATP synthesis, active transport, and motility. We determined that either Nuo or Nqr1 was required for aerobic growth in minimal medium. The presence of theoretically redundant complexes (Nqr2 and Ndh) did not rescue cell growth. Further, we determined that knocking out NDHs led to the inability to properly oxidize NADH. NADH build up inhibited the tricarboxylic acid cycle causing an amino acid synthesis defect and inhibiting growth of the S. oneidensis strain lacking Nuo and Nqr1. Recently, bacterial metabolic models have been developed to explain the use of energetically inefficient pathways during fast growth. Two standout models postulate that energetically inefficient pathways are used to reduce a cell’s proteome cost by eliminating thermodynamic barriers or to reduce dependence on the ETC as cells grow larger. We sought to uncover if these models applied to the respiratory chain of S. oneidensis MR-1 during aerobic growth, as the ETC can vary in energetic efficiency based on the combination of NDH and terminal oxidase used. Our findings indicate that the models apply to S. oneidensis MR-1 in the context of overflow metabolism during growth at higher growth rates, while the structuring of the ETC was not in agreement. Most importantly, determined that both carbon metabolism and the ETC were restructured for adaptive growth under differing conditions. As carbon metabolism became less efficient at faster growth rates, the NDH step of the ETC became more efficient, using complexes with higher coupling efficiencies.
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Title: Systematic analysis of the signal responsive gene regulatory network governing Myxococcus xanthus development
Creator: Saha, Shreya
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Studies of signal-induced gene expression in bacteria have contributed to understanding of how bacteria cope with environmental stress. As an extensively studied model, Myxococcus xanthus provides fascinating insights into how changes at the level of gene expression enable which bacteria to survive environmental insults such as nutrient limitation. Upon starvation M. xanthus cells glide into aggregates and form mounds that mature into fruiting bodies as some cells form spores. Previously, our...
Show moreStudies of signal-induced gene expression in bacteria have contributed to understanding of how bacteria cope with environmental stress. As an extensively studied model, Myxococcus xanthus provides fascinating insights into how changes at the level of gene expression enable which bacteria to survive environmental insults such as nutrient limitation. Upon starvation M. xanthus cells glide into aggregates and form mounds that mature into fruiting bodies as some cells form spores. Previously, our group defined 24-30 h poststarvation as the critical period for commitment to spore formation, when cells commit to form spores despite perturbation of the starvation signal by nutrient addition. The process of multicellular development that culminates in sporulation is governed by a network of signal-responsive transcription factors that integrate signals for starvation and cellular alignment. In this dissertation I present the first systematic approach to elucidate the network dynamics during the commitment period.In the network, MrpC is a starvation-responsive transcription factor, whereas FruA is a transcription factor that responds to cellular alignment conveyed by C-signaling. Transcription of fruA is dependent on MrpC binding, and FruA activity is proposed to be posttranslationally regulated by C-signaling, although the mechanism is unknown. FruA and MrpC cooperatively regulate transcription of the dev operon. My systematic analysis of the network dynamics supported a model in which posttranslational activation of FruA by C-signaling is critical for dev transcription and for commitment to spore formation. Similar to dev, MrpC and C-signal-activated FruA combinatorially controlled transcription of the late-acting fadIJ operon involved in spore metabolism. Regulation of late-acting operons implicated in spore coat biogenesis (exoA-I, nfsA-H, MXAN_3259-MXAN_3263) was discovered to be under complex control by MrpC and FruA. My evidence suggests that transcription of these operons depends at least in part on a C-signal-dependent switch from negative regulation by unactivated FruA to positive regulation by activated FruA during the period leading up to and including commitment to sporulation. MrpC negatively regulated exo and MXAN_3259 during mound formation, but positively regulated nfs. During commitment to sporulation, MrpC continued to positively regulate nfs, switched to positive regulation of MXAN_3259, and continued to negatively regulate exo. A third transcription factor, Nla6, appeared to be a positive regulator of all the late genes. We propose that in combination with regulation by Nla6, differential regulation by FruA in response to C-signaling and by MrpC controls late gene expression to ensure that spore resistance and surface characteristics meet environmental demands.
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Title: Dropping acid makes you see stars : Samba virus as a model system for studying giant virus genome release
Creator: Schrad, Jason Robert
Date: 2019
Collection: Electronic Theses & Dissertations
Description: "As their name implies, giant viruses (GV) are viruses of immense size. These viruses tend to have capsids larger than 300 nm and genomes that encode for over 1000 open reading frames. These viruses dwarf more common viruses, such as the human rhinovirus (common cold) that has a particle size of 30 nm and encodes for only 11 proteins. Some GV genomes even contain introns, a feature not typically associated with viruses as they were thought to have evolved towards simplicity. The discovery of...
Show more"As their name implies, giant viruses (GV) are viruses of immense size. These viruses tend to have capsids larger than 300 nm and genomes that encode for over 1000 open reading frames. These viruses dwarf more common viruses, such as the human rhinovirus (common cold) that has a particle size of 30 nm and encodes for only 11 proteins. Some GV genomes even contain introns, a feature not typically associated with viruses as they were thought to have evolved towards simplicity. The discovery of these viruses challenged the canonical view of the virus as a small and simple biological entity and has cast some doubt on our current understanding of the definitions of life. GV have been isolated from every continent on the planet, yet most share several conserved structural features. These conserved features include an internal lipid membrane that contains the dsDNA genome as well as a seal complex that closes the capsid prior to genome release. In icosahedral GV (Mimivirus-like GV), this seal complex sits atop the capsid at one specific vertex, the stargate vertex, which opens to facilitate genome release. The mechanisms that trigger release of the seal complex in vivo remain unknown. To fill some of the gaps in our knowledge of the GV life cycle, I have developed an in vitro system for studying GV genome release using Samba virus (SMBV), an icosahedral GV isolated from a tributary of the Amazon River in Brazil. First, I developed a method to visualize SMBV using cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and scanning electron microscopy (SEM). I then investigated the molecular forces responsible for maintaining the structural integrity of the SMBV external seal complex, treating SMBV particles with conditions known to disrupt viral capsids. Following each treatment, we determined the percentage of open SMBV particles, looking for conditions that induced a marked increase in open SMBV capsids. Both low pH (at or below pH 3) and high temperature (100 °C) triggered an increase in open SMBV particles, suggesting that electrostatic interactions and entropy, respectively, play a role in maintaining the structural integrity of the SMBV external seal complex. The role of these forces in maintaining external seal complex integrity is conserved throughout the icosahedral GV as three other GV shared similar structural responses to these conditions.Following low pH treatment small cracks appear in the GV capsid, mimicking the initiation of the genome release process and facilitating release of infection-related proteins. I separated the released proteins from the remaining capsid via centrifugation and analyzed the two populations via differential mass spectrometry. Through these analyses we identified 3030300 proteins that are released from SMBV and/or Tupanvirus soda lake, a GV isolated from an alkaline lake in Brazil, capsids during the initial stages of the infection process. These findings provide some of the first molecular information on the GV genome release process and hint at what triggers this process in vivo. This work also provides the first in vitro system capable of mimicking stages of the GV infection process, paving the way for future structural and biochemical studies of the GV life cycle."--Pages ii-iii.
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Title: ENVIRONMENTAL DRIVERS AND EVOLUTIONARY CONSEQUENCES OF HORIZONTAL GENE TRANSFER IN SOIL BACTERIA
Creator: Kittredge, Heather
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Horizontal gene transfer (HGT) is a driving force in bacterial evolution and could drive rapid adaptation in bacterial communities. Natural transformation is one mechanism of HGT that allows bacteria to pick up extracellular DNA (eDNA) from the environment and integrate it into their genome. But the rate of HGT in natural environments, and the role this process plays in facilitating rapid adaptation remains unknown. As climate change threatens the stability of environments worldwide,...
Show moreHorizontal gene transfer (HGT) is a driving force in bacterial evolution and could drive rapid adaptation in bacterial communities. Natural transformation is one mechanism of HGT that allows bacteria to pick up extracellular DNA (eDNA) from the environment and integrate it into their genome. But the rate of HGT in natural environments, and the role this process plays in facilitating rapid adaptation remains unknown. As climate change threatens the stability of environments worldwide, understanding how quickly bacteria can adapt to novel environments is essential. My dissertation research characterizes the environmental drivers and evolutionary consequences of natural transformation in a highly transformable model soil bacterium Pseudomonas stutzeri.Despite decades of research on understanding HGT at the molecular level, less is known about the ecological drivers of HGT. To understand the soil conditions relevant for transformation, I first measured eDNA in the field over a short-term drying rewetting disturbance (Ch. 2). I found that eDNA increased in response to the rewetting disturbance but quickly disappeared from soil, suggesting a small portion of this eDNA could be transformed by bacterial cells recovering from the disturbance. To test the efficiency of transformation under the conditions in which eDNA disappeared, I created a novel microcosm system for quantifying transformation in soil (Ch. 3). Here, I inoculated soil with live antibiotic-susceptible, and dead antibiotic-resistant P. stutzeri. I then tracked the evolution of antibiotic resistance over a range of soil conditions and eDNA concentrations. Transformation drove the evolution of antibiotic resistance across a wide range of soil moistures and increased in response to larger inputs of dead cells (eDNA source), with antibiotic resistance repeatedly appearing in antibiotic free soil. Despite the prevalence of transformation across bacterial species, the evolutionary origins and consequences of transformation are still largely unknown. Transformation presumably provides a fitness benefit in stressful or continuously changing environments, but few studies have quantified changes in transformation in response to adaptive evolution. Here, I evolved P. stutzeri at different salinities and tested how the growth rate and transformation efficiency changed in response to salt adaptation (Ch. 4). Overall, the growth rate increased in response to adaptation, but the transformation efficiency declined, with only ~50% of the evolved populations transforming eDNA at the end of experiment – as opposed to 100% of ancestral populations transforming eDNA. Overall, my dissertation research elucidates the factors driving transformation in soil, setting the stage for future experiments to scale up estimates of transformation to the whole community level. I find that transformation occurs under most soil conditions and allows genetic variants to arise at low frequencies in the absence of selection. I also report novel experimental evidence that transformation efficiency can change dramatically, and in a highly variable manner, over just ~330 generations. Taken together, this body of research highlights a role for transformation in many natural systems of ecological significance, and points to dead cells as an important but often overlooked source of genetic diversity.
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Title: EXAMINING INHIBITION AND SUBSTRATE INTERACTION OF SPOIVFB, AN INTRAMEMBRANE METALLOPROTEASE OF BACILLUS SUBTILIS
Creator: Olenic, Sandra D
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Intramembrane proteases (IPs) regulate diverse signaling pathways in all three domains of life by cleaving membrane-associated substrates. Currently, there are four known IP families, including intramembrane metalloproteases (IMMPs), which contain characteristic HEXXH and NPDG motifs that coordinate a zinc ion at the active site. The study of IMMPs, including RseP (Escherichia coli) and SpoIVFB (Bacillus subtilis) have provided insights into potential mechanisms for substrate interaction and...
Show moreIntramembrane proteases (IPs) regulate diverse signaling pathways in all three domains of life by cleaving membrane-associated substrates. Currently, there are four known IP families, including intramembrane metalloproteases (IMMPs), which contain characteristic HEXXH and NPDG motifs that coordinate a zinc ion at the active site. The study of IMMPs, including RseP (Escherichia coli) and SpoIVFB (Bacillus subtilis) have provided insights into potential mechanisms for substrate interaction and cleavage, which could guide efforts to develop inhibitors of IMMPs that regulate virulence in bacterial pathogens. Work described in this dissertation has advanced the knowledge of inhibition and substrate interaction of SpoIVFB, which cleaves Pro-σK during endosporulation. Improved methods are presented for heterologous production in E. coli of SpoIVFB, Pro-σK, and the inhibitory proteins BofA and SpoIVFA. Three conserved BofA residues (N48, N61, T64) in or near transmembrane segment (TMS) 2 are required for SpoIVFB inhibition. Cross-linking indicates that BofA TMS2 occupies the SpoIVFB active site region. Interestingly, BofA and SpoIVFA do not prevent interactions between portions of Pro-σK and SpoIVFB, so all four proteins can exist in a complex. A structural model of SpoIVFB with BofA and parts of SpoIVFA and Pro-σK was built using partial homology and constraints from cross-linking and co-evolutionary analyses. The model suggests that SpoIVFA stabilizes BofA in the SpoIVFB active site region and leads us to propose that BofA TMS2 sterically hinders access of the substrate. Our work has also advanced knowledge of interactions between Pro-σK and the broadly conserved SpoIVFB NPDG motif, which is located in a predicted short loop that interrupts TMS4 and faces the active site. Three highly conserved residues (N129, P132, P135) of SpoIVFB were found to be important for substrate interactions and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. More work is needed to fully understand how IMMPs interact with their substrates and whether the insights from BofA inhibition of SpoIVFB can be applied to other IMMPs. Outstanding questions and future directions related to these two projects are described.
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Title: Deconstructing the correlated nature of ancient and emergent traits : an evolutionary investigation of metabolism, morphology, and mortality
Creator: Grant, Nkrumah Alions
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Phenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have...
Show morePhenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have maintained - and even improved - the capacity of E. coli to grow in an anoxic environment despite 50,000 generations of relaxed selection for anaerobic growth. I present genomic evidence illustrating substantially more mutations have accumulated in anaerobic-specific genes and show parallel evolution at two genetic loci whose protein products regulate the aerobic-to-anaerobic metabolic switch. My findings reject the "if you don't use it, you lose it" notion underpinning relaxed selection and show modules with deep evolutionary roots can overlap more, hence making them harder to break. In chapter 2, I revisit previous work in the LTEE showing that the fitness increases measured for the 12 populations positively correlated with an increase in cell size. This finding was contrary to theory predicting smaller cells should have evolved. Sixty thousand generations have surpassed since that initial study, and new fitness data collected for the 12 populations show fitness has continued to increase over this period. Here, I asked whether cell size also continued to increase. To this end, I measured the size of cells for each of the 12 populations spanning 50,000 generations of evolution using a particle counter, microscopy, and machine learning. I show cell size has continued to increase and that it remains positively correlated with fitness. I also present several other observations including heterogeneity in cell shape and size, parallel mutations in cell-shape determining genes, and elevated cell death in the single LTEE population that evolved a novel metabolism - namely the ability to grow aerobically on citrate. This last observation formed the basis of my chapter 3 research where my collaborators and I fully examine the cell death finding and the associated genotypic and phenotypic consequences of the citrate metabolic innovation.
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Title: Reducing Water and Agrochemical Movement from Container Nursery Production Using Bioreactors and Irrigation Management
Creator: Abdi, Damon Edward
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Container crop production is an input intensive agricultural sector, oftentimes demanding frequent, typically daily irrigation, substantial fertilizer use, and multiple pesticide applications throughout the production cycle. The combination of these factors increases the risk for agrochemical movement in irrigation return flow (IRF). Over the course of two studies, a model nursery designed to collect surface and subsurface IRF was used to investigate water use and agrochemical movement in a...
Show moreContainer crop production is an input intensive agricultural sector, oftentimes demanding frequent, typically daily irrigation, substantial fertilizer use, and multiple pesticide applications throughout the production cycle. The combination of these factors increases the risk for agrochemical movement in irrigation return flow (IRF). Over the course of two studies, a model nursery designed to collect surface and subsurface IRF was used to investigate water use and agrochemical movement in a model nursery. An overhead control was compared to micro-irrigation (SS) and substrate volumetric moisture content (θ) sensor based overhead irrigation (OH) in the volume of water applied, volume of water lost to IRF, and associated fertilizer and pesticide content transported. Irrigating using OH and SS reduced the volume of irrigation applied by 49% and 78% compared to the control. Surface IRF was reduced by 80% using OH and was largely eliminated using SS; however, subsurface IRF was generally equivalent between the control and treatments. Surface IRF movement of nitrate and phosphate was reduced by 72% - 76% when irrigating using OH, and up to 98% when irrigating using SS. Pesticide mobility in irrigation return flow was reflective of pesticide physiochemical properties, with more soluble pesticides exhibiting greater movement than less soluble pesticides, particularly in subsurface IRF. OH reduced surface IRF movement of the 10 pesticides by 43-89%, while SS reduced surface IRF movement by 77-100%. There were typically no differences in subsurface IRF pesticide movement between the control and treatments. For all studied taxa (Cornus obliqua 'Powell Gardens', Cornus sericea 'Farrow', Hydrangea paniculata 'Limelight', Physocarpus opulifolius 'Seward', Rosa x'Meipeporia', Spiraea japonica 'SMNSJMFP', and Weigela florida 'Elvera') an equivalent growth index to the control was achieved when irrigating based on θ. Irrigation treatments were capable of producing an equivalent weight of shoot dry biomass for all taxa except C. obliqua, P. opulifolius, and S. japonica where the control was greater than all treatments. For the three species where root dry biomass was investigated (H. paniculata, R. x., and S. japonica), only S. japonica exhibited reduced root dry biomass under the OH and SS treatment compared to the control. Irrigating based on θ, regardless of the delivery method, can produce woody ornamental species of equivalent quality, while also reducing water use and agrochemical export in irrigation return flow; however, bioactive concentrations of agrochemicals may still be present. Woodchip bioreactors (WB) and adsorbent aggregate filters (AF) are treatment technologies that are capable of remediating or sequestering contaminants from IRF via biological and sorptive processes, provided a sufficient hydraulic retention time (HRT). A 72 hour HRT reduced over 99% of influent nitrate in WB and up to 87% of phosphate in AF; whereas, an HRT of 21 minutes was insufficient for nutrient remediation. An HRT of 21 minutes was effective in reducing the movement of bifenthrin, chlorpyrifos, and oxyfluorfen by 76%, 63%, and 31%, respectively, using WB. Microbial analysis of WB identified shifts in species composition when exposed to pesticides, enriching for a number of species within the Pseudomonas and Exiguobacterium genus, while decreasing the number and diversity of Bacillus species compared to the nutrient only control.
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Title: [FeFe]-hydrogenase substrate transport mechanisms and investigation of algal hydrogen metabolism
Creator: Cornish, Adam
Date: 2012
Collection: Electronic Theses & Dissertations
Description: The global population has recently exceeded 7 billion people and the demand for energy continues to expand as the number of industrialized countries grows. Currently, the energy economy is dominated by the utilization of polluting and non-renewable fossil fuels. Both the collection and use of petroleum-based fuels is destructive to the environment and is not sustainable over a long time-scale, which justifies the investigation into the development of renewable, alternative fuels. Of the...
Show moreThe global population has recently exceeded 7 billion people and the demand for energy continues to expand as the number of industrialized countries grows. Currently, the energy economy is dominated by the utilization of polluting and non-renewable fossil fuels. Both the collection and use of petroleum-based fuels is destructive to the environment and is not sustainable over a long time-scale, which justifies the investigation into the development of renewable, alternative fuels. Of the various fuels that have been proposed, molecular hydrogen (H₂) in particular holds great promise as a clean-burning fuel capable of supplementing the current energy economy, especially as the combustion of H₂ generates only water vapor as by-product. H₂ can be generated via a number of chemical processes, but current H₂ technologies either require fossil fuels as inputs or are energy-inefficient. The biological production of H₂, however, has garnered a great deal of interest because microorganisms are able to drive H₂ synthesis using energy derived from both light and dark fermentative metabolisms. This manner of production does not depend on mining non-renewable resources and microbes can be cultured at the industrial scale without competing with arable land needed for agriculture. H₂ evolution in these microorganisms is dependent on nitrogenases and/or hydrogenases, enzymes which utilize unique metal centers for catalysis. Hydrogenases have been of particular interest for industrial-scale H₂ production because these enzymes are found in a diverse array of organisms and require only protons and electrons as substrates. In particular, [FeFe]-hydrogenases have very high turnover numbers and catalysis can be coupled to photosynthesis. Unfortunately, these enzymes are inactivated by molecular oxygen (O₂), and a number of studies have therefore attempted to engineer O₂-tolerant hydrogenases. However, engineering enzymes to introduce optimal qualities has been impeded by an incomplete understanding of the overall reaction mechanism. Substrate (protons, electrons, and ₂) transport is essential to hydrogenase activity, yet relatively little information is available regarding the intraprotein transport of substrate in [FeFe]-hydrogenase. I focused my investigation on identifying and testing pathways important for substrate transport between the enzyme surface and the active site in the Clostridium pasteurianum [FeFe]-hydrogenase. I have elucidated a key pathway for proton transport and confirmed that two iron-sulfur clusters are essential in an electron transfer relay, contributing to the overall characterization of [FeFe]-hydrogenase activity. Green algae utilize [FeFe]-hydrogenases to catalyze H₂ production using reducing equivalents derived from photosynthesis and these enzymes are an integral component of anaerobic metabolism in these microalgae. I explored the H₂ production capabilities of a multicellular green alga, Volvox carteri, and characterized two hydrogenases likely responsible for this activity. In addition, a unique hydrogenase gene cluster discovered within the Volvox carteri genome provided interesting hints into the origin of [FeFe]-hydrogenase in green algae.
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Title: Physiological and ecological investigations of Clostridium difficile
Creator: Robinson, Catherine D.
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Disease caused by Clostridium difficile is currently the most prevalent nosocomial infection and leading cause of antibiotic-associated diarrhea. It is clear that the intestinal microbiota plays a role in preventing C. difficile infection in the absence of antibiotics; however, the mechanisms involved in this protective function are poorly understood. Since antibiotic administration is an inducing factor of C. difficile infection, treatment employing antibiotics often results in recurrent...
Show moreDisease caused by Clostridium difficile is currently the most prevalent nosocomial infection and leading cause of antibiotic-associated diarrhea. It is clear that the intestinal microbiota plays a role in preventing C. difficile infection in the absence of antibiotics; however, the mechanisms involved in this protective function are poorly understood. Since antibiotic administration is an inducing factor of C. difficile infection, treatment employing antibiotics often results in recurrent disease, yet it is still the primary line of treatment. Therefore, a central goal of research in this area is to better define the role of the intestinal microbiota in suppression of disease, and ultimately develop alternative ways to prevent and treat C. difficile infection. In this thesis, I present a novel in vitro model that was developed to study complex fecal communities. This in vitro model is a continuous-culture system that utilizes arrays of small-volume reactors; it is unique in its simple set-up and high replication. We adapted this model to operate as a C. difficile infection model, where in vivo C. difficile invasion dynamics are replicated in that the fecal communities established in the reactors are resistant to C. difficile growth unless disrupted by antibiotic administration. We then go on to use this model to show that newly emerged, epidemic strains of C. difficile have a competitive fitness advantage when competed against non-epidemic strains. We also show this competitive advantage in vivo, using a mouse infection model. This result is exciting, as it suggests that physiological attributes of these strains, aside from classical virulence factors, contribute to their epidemic phenotype. Finally, the metabolic potential of C. difficile in regards to carbon source utilization is explored, and reveals that epidemic strains are able to grow more efficiently on trehalose, a disaccharide sugar. Moreover, preliminary in vivo mouse studies suggest that trehalose utilization plays a role in colonization. Therefore, the growth advantage conferred by this increased ability to utilize trehalose may contribute to the ecological fitness of these strains in vivo. The in vitro model developed and presented in this thesis could be used to study many aspects of C. difficile-microbiota interactions and has the potential to elucidate mechanisms that are important for in vivo resistance to establishment of disease. In addition, the metabolic investigations described provide insight into understanding the physiology of not only C. difficile as a whole, but also physiological attributes unique to epidemic strains. Ultimately, these types of ecological and physiological investigations will bring us closer to finding better ways to treat and prevent disease caused by C. difficile.
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Title: Defining the roles of the Vc2 riboswitch and tfoY in the c-di-GMP regulatory network of Vibrio cholerae
Creator: Pursley, Benjamin Richard
Date: 2016
Collection: Electronic Theses & Dissertations
Description: The second messenger cyclic dimeric guanosine-monophosphate (c-di-GMP) is a central regulator of many different cellular activities in the bacterial domain and it plays an especially important role in the lifestyle transition of Vibrio cholerae between the marine environment and human infection. One of the primary effectors of this signal, the Vc2 c-di-GMP-binding riboswitch of V. cholerae, has been heavily studied in vitro, yet it remains poorly understood in vivo. Riboswitches have been...
Show moreThe second messenger cyclic dimeric guanosine-monophosphate (c-di-GMP) is a central regulator of many different cellular activities in the bacterial domain and it plays an especially important role in the lifestyle transition of Vibrio cholerae between the marine environment and human infection. One of the primary effectors of this signal, the Vc2 c-di-GMP-binding riboswitch of V. cholerae, has been heavily studied in vitro, yet it remains poorly understood in vivo. Riboswitches have been traditionally characterized as cis-acting RNA elements that serve to regulate the gene expression of downstream coding sequences, but the relationship between the Vc2 element and its downstream gene, tfoY, is uncharacterized. In this work, we determine that tfoY is a vital component of the V. cholerae c-di-GMP program, specifically involved in motility, biofilm formation, and the direct genetic regulation of c-di-GMP metabolic enzymes. We also reveal that V. cholerae possesses both Vc2-dependent and Vc2-independent mechanisms for c-di-GMP regulation of tfoY expression. And finally, we present a novel paradigm for riboswitch and c-di-GMP gene regulation in which the stability and abundance of a small RNA is controlled by the ligand binding state of the Vc2 riboswitch aptamer domain.
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Title: Novel insights into sugar and succinate metabolism of Actinobacillus succinogenes from strains evolved for improved growth on lignocellulose hydrolysate sugars
Creator: McPherson, Nikolas Robin
Date: 2017
Collection: Electronic Theses & Dissertations
Description: A wide variety of industrially vital chemicals are currently produced from petroleum, using very well-refined processes and a large industrial infrastructure. However, petroleum processing has a number of hazardous and otherwise negative impacts on the environment, as well as on human health. The supply and price of oil into the future are uncertain as well, and supplementing oil with feedstocks from renewable sources can help extend the current supply of oil and eventually replace it....
Show moreA wide variety of industrially vital chemicals are currently produced from petroleum, using very well-refined processes and a large industrial infrastructure. However, petroleum processing has a number of hazardous and otherwise negative impacts on the environment, as well as on human health. The supply and price of oil into the future are uncertain as well, and supplementing oil with feedstocks from renewable sources can help extend the current supply of oil and eventually replace it. Succinate is a specialty chemical currently produced from maleic anhydride from petroleum processing. If bio-based succinate could compete with the cost of maleic anhydride, it could replace maleic anhydride in a $15 billion commodity chemical market, taking advantage of the existing chemical production infrastructure. A major potential feedstock source for conversion to succinate is lignocellulose from agricultural waste or from bioenergy crops. The bacterium Actinobacillus succinogenes is one of the best natural succinate producers and it grows on a wide variety of carbohydrates, including the major sugars in lignocellulose. A. succinogenes grows well on glucose, the most common sugar in lignocellulose, but does not grow as quickly on other lignocellulosic sugars.I have evolved strains of A. succinogenes to grow faster on xylose, the second most common lignocellulosic sugar, as well as on arabinose, galactose, and lignocellulose hydrolysates. Many of the evolved strains produce more succinate than the parental strain as well, even though the evolution process did not specifically select for succinate production. The evolved strains were resequenced to identify the mutations accumulated during evolution. RNA sequencing of the xylose-evolved strains helped identify changes in transcript levels and was used to refine our conclusions about the xylose-evolved (X) strains. I discovered that the genes that encode many glycolytic enzymes were upregulated in at least one X strain, several genes encoding succinate production enzymes were upregulated, while genes that encode enzymes that redirect fluxes from the succinate pathway to other fermentation products were downregulated. During the directed evolution process, I obtained a strain of A. succinogenes that can grow on galactose, a sugar that the base strain cannot use. The final evolved strain grew faster than the wild-type strain on xylose, arabinose, and lignocellulose hydrolysate, and could grow on galactose. I determined that A. succinogenes will co-consume glucose and xylose, but that xylose represses arabinose consumption. After directed evolution, though, arabinose represses xylose consumption. Finally, I used multiplex transformation to introduce mutations from the evolved strains into the wild-type strain. The first strain produced, using the xylose symporter mutation from a xylose-evolved strain, produced 40% more succinate than wild-type A. succinogenes, even though it grew at less than half the speed. In summary, I have evolved a set of A. succinogenes strains that grow faster on lignocellulose sugars and some have a higher succinate yield, I know the location and nature of their mutations and have RNA sequencing data for the xylose-evolved strains. I have conducted numerous additional experiments to characterize sugar consumption in A. succinogenes and what causes the evolved strains to be able to grow faster and produce more succinate. My results lay solid groundwork for future work with A. succinogenes and other bacteria being grown on sugars and synthesizing succinate.
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Title: Breaking biofilms : regulation of Type II secretion system in V. cholerae and the formation of the hyper-pseudopilus
Creator: Sloup, Rudolph E.
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is the causative agent of the human disease cholera, it resides in aquatic resevoirs and forms biofilms, which are closely associated communities of bacteria embedded in polysaccharides, DNA, and proteins. In V. cholerae biofilm formation is regulated by the second messenger molecule cyclic di-GMP (c-di-GMP). A genetic screen for promoters regulated by the c-di-GMP revealed a novel promoter (PepsG) in the eps operon encoding the V. cholerae Type 2 secretion system (T2SS). The...
Show moreVibrio cholerae is the causative agent of the human disease cholera, it resides in aquatic resevoirs and forms biofilms, which are closely associated communities of bacteria embedded in polysaccharides, DNA, and proteins. In V. cholerae biofilm formation is regulated by the second messenger molecule cyclic di-GMP (c-di-GMP). A genetic screen for promoters regulated by the c-di-GMP revealed a novel promoter (PepsG) in the eps operon encoding the V. cholerae Type 2 secretion system (T2SS). The T2SS, which exports proteins from the periplasm to the extracellular space, is phylogenetically related to Type 4 pili. The major pseudopilin is encoded by epsG which forms a short piston like structure necessary for secretion. I hypothesized that differential regulation of the eps operon extends the pseudopilin forming a structure called a hyper-pseudopilus outside the cell where it promotes biofilm development. In Chapter 2, I determined that the promoter upstream of the operon (PepsC1) is induced four fold by c-di-GMP and this induction is mediated by the c-di-GMP binding transcription factor VpsR directly. High levels of c-di-GMP were found to decrease the activity of extra cellular proteases secreted by the T2SS, however this effect was not a direct result of regulation of the T2SS as determined by mutation of the VpsR binding site in PepsC1. I was unable to establish a phenotype for the transcriptional control of the eps operon. This work establishes T2S as a new phenotype which is transcriptionally controlled by c-di-GMP and the biofilm associated transcription factor VpsR. In Chapter 3, I show that overexpression of epsG in a continuous flow cell system increased V. cholerae biofilms while a ΔepsG strain showed no biofilm formation. However, there was no change in activity of T2S dependent serine proteases while epsG was over expressed indicating increased biofilms is not likely due to increased secretion. Polyclonal antibody stained EpsG was also detectable on the surface of WT cells and long pseudopili were visualized with over expression of epsG. This evidence suggests the T2SS forms a hyper-pseudopilus important for biofilm formation. In Chapter 4, I present my work identifying novel anti-biofilm compounds. In 2011 Escherichia coli O104:H4 caused the deadliest E. coli outbreak in modern times resulting in 54 deaths and the highest rate of hemolytic uremic syndrome ever recorded. Subsequently, we showed a correlation between biofilm gene expression and virulence factor expression. I sought to identify small molecule compounds effective at inhibiting O104:H4 biofilms. I discovered at a concentration of 0.01% the nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to inhibit biofilm formation by 90% and 91% respectively. These compounds were able to disperse preformed biofilms. Treatment of mice infected with E. coli O104:H4 resulted in high bacterial loads and inflammation. While addition of PS80 in the drinking water of the mice did not reduce bacterial loads, it completely abolished inflammation symptoms. PS80 is an FDA approved compound, well studied and effective at low nanomolar concentrations that reduces symptoms of infection in mice. which establishes it as an excellent candidate for further study as an anti-infective agent with anti-biofilm capabilities
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Title: Integrated biological processes for conversion of AFEX(TM) pretreated biomass to ethanol
Creator: Jin, Mingjie
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Ethanol production from lignocellulosic biomass has gained much momentum due to its benefits to energy security, reduction of green house gas emission, as well as both environmental and social sustainability. The technology for lignocellulosic ethanol production, however, is not yet fully commercialized. The major issues impeding the cellulosic ethanol production in the biochemical route include the high enzyme loadings needed, long enzymatic hydrolysis time, slow xylose fermentation and low...
Show moreEthanol production from lignocellulosic biomass has gained much momentum due to its benefits to energy security, reduction of green house gas emission, as well as both environmental and social sustainability. The technology for lignocellulosic ethanol production, however, is not yet fully commercialized. The major issues impeding the cellulosic ethanol production in the biochemical route include the high enzyme loadings needed, long enzymatic hydrolysis time, slow xylose fermentation and low ethanol productivity, which result in a high production cost. Ammonia Fiber Expansion (AFEXTM) is a leading alkaline pretreatment. It provides a biomass substrate with high enzymatic digestibility and high fermentation potential. Previous studies on ethanol production from AFEXTM pretreated biomass focused on a separate enzymatic hydrolysis and fermentation process (SHF). However, integrated biological processes, such as simultaneous saccharification and co-fermentation (SSCF) and consolidated bioprocessing (CBP), are strongly believed to have lower cost compared to SHF. This dissertation work studies the integrated biological processes performed on AFEXTM pretreated biomass and resolves the aforementioned issues for biochemical production of cellulosic ethanol. The slow xylose fermentation issue in hydrolysate by Saccharomyces cerevisiae 424A (LNH-ST) was quantitatively studied. The xylose fermentation inhibition was not only from degradation products but also from ethanol and metabolites generated during glucose fermentation (Chapter II). Based on such understanding, a two-step SSCF process was developed, in which xylan was hydrolyzed and fermented ahead of glucan/glucose. As a result, xylose fermentation was greatly improved (Chapter III). Through the study of conventional SSCF on AFEXTM treated corn stover (CS), it was found that pre-hydrolysis to generate a certain amount of glucose was crucial for achieving a good result (Chapter IV). A high solids loading process can save much cost for ethanol production. However, ethanol yield decreased with increasing solids loading. From an economic point of view, 6% (w/w) glucan loading was the optimal solids loading during SSCF of AFEXTM CS (Chapter V). For improvement of productivity, a continuous SSCF process using multi-stage continuous stirred tank reactors (CSTRs) was developed based on the kinetic studies of the two reactions in SSCF (enzymatic hydrolysis and fermentation)(Chapter VI). Based on the fundamental understandings of the cellulosic ethanol production, a novel industrially-relevant integrated biological process was developed. This process shortened the biological processing time (including enzymatic hydrolysis and fermentation) from 11 days to around 2 days, reduced enzyme loading by more than 1/3 and enhanced ethanol productivity by 2-3 times (Chapter VII). Consolidated bioprocessing (CBP) eliminates the enzyme cost and is believed to be the ultimate low-cost industrial configuration for cellulosic ethanol production. CBP studies using Clostridium phytofermentans on AFEXTM CS at both low and high solids loadings showed promising results (Chapter VIII & IX).
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Title: Investigations into urease maturation and metal ion selectivity
Creator: Carter, Eric Lee
Date: 2011
Collection: Electronic Theses & Dissertations
Description: This dissertation consists of three projects that dissect the nature of urease maturation and metal ion selectivity. The first project examines the role of the UreD accessory protein during in vivo maturation of the nickel-containing urease from Klebsiella aerogenes. A translational fusion of the maltose binding protein with UreD (MBP-UreD) was generated and found to be soluble. The UreD domain of MBP-UreD bound nickel and zinc ions, formed complexes with ...
Show moreThis dissertation consists of three projects that dissect the nature of urease maturation and metal ion selectivity. The first project examines the role of the UreD accessory protein during in vivo maturation of the nickel-containing urease from Klebsiella aerogenes. A translational fusion of the maltose binding protein with UreD (MBP-UreD) was generated and found to be soluble. The UreD domain of MBP-UreD bound nickel and zinc ions, formed complexes with (UreABC)₃, UreF, UreG, UreF plus UreG, and (UreABC)₃-UreF-UreG in vivo, and formed a complex with the UreF domain of the UreE-UreF fusion in vitro. MBP-UreD was shown to be a functional form of UreD as malE-ureD partially complemented for a ∆ureD urease cluster. The second project revealed several roles for the K. aerogenes urease structural subunit UreB during urease maturation. UreB was purified as a monomer and shown to spontaneously bind to isolated (UreAC)₃, forming (UreABC*)₃, while an N-terminal deletion mutant of UreB lacking the first 19 residues did not form the apoprotein complex. (UreABC*)₃ shared similar in vitro activation properties as urease apoprotein preformed in vivo, whereas exposure of a mixture of (UreAC)₃ and UreB∆1-19 to activation conditions led to negligible levels of active enzyme. Activity assays and metal analyses of various in vitro activated species demonstrated that UreB facilitates efficient incorporation of Ni2+ into the active site and protects the metal from chelators. Additional studies revealed that UreB interfaced with accessory proteins, and the N-terminus was critical for this process. Finally, UreB enhanced the stability of UreC against proteolytic cleavage by trypsin. The third project characterized a unique urease from Helicobacter mustelae, UreA2B2, which was shown to exhibit O₂-labile activity in whole cells. UreA2B2 was purified aerobically from its native host and found to contain ~ 2 iron per active site, or ~1 iron and 0.7 zinc when purified under anaerobic conditions. Anaerobically purified UreA2B2 was active, though highly O₂-labile, with its activity enhanced by EDTA and inhibited by acetohydroxamic acid or nickel ions. The inactive, oxidized enzyme was slowly reactivated by incubation with dithionite to levels approaching the wild-type enzyme accompanied by bleaching of its UV-visible spectrum, where the chromophore was consistent with μ-oxo bridged diferric atoms. Resonance Raman spectroscopy of this sample revealed bands at ~500 cm-1 and ~780 cm-1 that are characteristic of a Fe(III)-O-Fe(III) metallocenter. The ~500 cm-1 feature was sensitive to bulk solvent exchange with H₂18O or deuterium oxide, and both features were downshifted in the presence of urea. Protein purified aerobically from recombinant Escherichia coli grown in rich medium contained ~1 equivalent of iron and negligible levels of other metals, while E. coli cultured in minimal medium generated apoprotein with ~0.2 equivalents of iron and 0.0-0.2 zinc. Temperature-dependent circular dichroism measurements indicated that iron enhanced the thermal stability of UreA2B2. The apoprotein form of the enzyme was activated to levels representing ~20% of wild-type activity with ferrous ions and bicarbonate under anaerobic conditions. Lastly, the crystal structure of UreA2B2 was determined at 3.0 Å revealing an active site architecture nearly identical to that for nickel ureases. Numerous amino acid residue substitutions around the active site suggest that metal specificity for iron likely arises during the metal loading process.
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Title: Engineering Actinobacillus succinogenes for succinate production : a focus on succinate transporters and small RNAs
Creator: Joshi, Rajasi Virendra
Date: 2017
Collection: Electronic Theses & Dissertations
Description: An important aspect of any industrial scale bio-based production is the choice of biocatalyst used. Many commercially relevant microorganisms and industrial strains have been engineered to optimize the production of bio-based chemicals. One such chemical is succinate, listed as one of the top 12 building block chemicals from biomass by the US Department of Energy. Succinate is considered an important platform chemical as it has a number of applications and, most importantly, is a precursor to...
Show moreAn important aspect of any industrial scale bio-based production is the choice of biocatalyst used. Many commercially relevant microorganisms and industrial strains have been engineered to optimize the production of bio-based chemicals. One such chemical is succinate, listed as one of the top 12 building block chemicals from biomass by the US Department of Energy. Succinate is considered an important platform chemical as it has a number of applications and, most importantly, is a precursor to high-volume value-added commodity chemicals. Bio-based succinate is currently being produced at industrial scale levels using engineered microorganisms such as E. coli and S. cerevisiae. Actinobacillus succinogenes is one of the best natural succinate producers, which can grow on a wide variety of substrates, and, with the advance in genetic tools, can possibly be engineered for increased succinate production. Very few studies have focused on using succinate exporters as metabolic engineering targets for succinate production. Only a handful of studies have been carried out in E. coli and C. glutamicum with none whatsoever in A. succinogenes. With a combination of proteomics and transcriptomics we have identified candidate succinate transporters in A. succinogenes. Four of the top hits in our proteomics analysis were Asuc_1999, Asuc_0142, Asuc_2058 and Asuc_1990-91. To carefully tune the expression of these membrane proteins, we generated a library of promoters covering a large range of strengths below the strong, constitutive promoter (ppckA) we had been using. Some of the promoters were truncated versions of ppckA, and others were identified from our transcriptomics data. These promoters were tested using lacZ as the reporter gene in an A. succinogenes ∆lacZ background. Promoters ranged from ppckA as the highest down to pAsuc_0701 with a strength 209-fold lower than ppckA. The four succinate transporter candidates over-expressed under ppckA-92, a truncated version of ppckA, increased the succinate yield in glucose cultures compared to the control strain carrying the empty vector. Synthetic small RNAs (sRNAs) are another tool for metabolically engineering industrially relevant microorganisms. However, no sRNAs have been identified in A. succinogenes and only a few have been identified in other members of the Pasteurellaceae family. We identified sRNAs in A. succinogenes grown anaerobically on glucose and microaerobically on glycerol by RNA sequencing. We found 260 sRNAs in total, of which 39 were predicted by at least one of five computational programs. We validated 14 sRNAs identified from sequencing with RT-PCR. Additionally we identified probable Hfq-binding sRNAs through their Rho-independent terminators, a key feature of Hfq-binding sRNAs. Using additional characteristics of Hfqbinding sRNAs, we designed synthetic sRNAs targeting lacZ expression as a proof of concept. One plasmid-borne synthetic sRNA caused a 32% decrease in β-galactosidase activity in lactose- grown cultures. Using the same sRNAs scaffolds, we generated synthetic sRNAs that targeted the ackA and pta mRNAs to decrease the production of acetate, one of the major by-products of succinate production. One of the synthetic sRNAs targeting ackA caused a 14% decrease in the acetate yield of glucose-grown cultures. In summary, we have identified candidate succinate transporters and seen an increase in succinate production upon their overexpression. In the process, we have developed a promoter library for tunable expression of genes in A. succinogenes. We have also shown that sRNAs can be used as a tool for metabolic engineering in A. succinogenes, although additional studies are needed to make it more tunable and robust.
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Title: Understanding and improving respiratory succinate production from glycerol by Actinobacillus succinogenes
Creator: Schindler, Bryan David
Date: 2011
Collection: Electronic Theses & Dissertations
Description: Succinic acid tops the U.S. Department of Energy's list of value-added products from biomass, because it has the potential, if produced economically, to become the feedstock for a bulk chemical industry currently based on maleic anhydride, a petrochemical. In addition to the large market potential for succinate and its immediate derivatives, bio-based succinate production has the added environmental benefit of using CO2, a greenhouse gas, as a substrate. Actinobacillus succinogenes 130Z...
Show moreSuccinic acid tops the U.S. Department of Energy's list of value-added products from biomass, because it has the potential, if produced economically, to become the feedstock for a bulk chemical industry currently based on maleic anhydride, a petrochemical. In addition to the large market potential for succinate and its immediate derivatives, bio-based succinate production has the added environmental benefit of using CO2, a greenhouse gas, as a substrate. Actinobacillus succinogenes 130Z naturally produces among the highest levels of succinate from a variety of inexpensive carbon substrates. Previous reports of A. succinogenes's metabolic capabilities mainly used glucose as a feedstock and provided insight into several key factors controlling succinate production. Conversely, little is known about how A. succinogenes metabolizes glycerol, a waste product of biodiesel manufacture and an inexpensive feedstock with potential application in bio-based succinate production. As suggested by our manual annotation of its genome, A. succinogenes cannot ferment glycerol in defined minimal medium but it can metabolize glycerol by aerobic or anaerobic respiration. We investigated A. succinogenes's glycerol metabolism in a variety of respiratory conditions by comparing growth, metabolite production, and in vitro activity of terminal oxidoreductases. Under conditions of nitrate-respiration and fully aerobic respiration, acetate was the primary acid produced from glycerol. However, succinate was the primary product of dimethyl sulfoxide-respiring cultures and cultures grown in microaerobic conditions. The highest succinate yield observed was 0.69 mol succinate/mol glycerol (69% of the maximum theoretical yield) under microaerobic conditions. We also show that A. succinogenes can grow and produce succinate on partially refined glycerols obtained directly from biodiesel manufacture. We used recently developed genetic tools to create knockout mutants of A. succinogenes. The gene knockout strategy uses natural transformation to introduce linearized DNA into the cells. The isocitrate dehydrogenase gene (icd) from Escherichia coli was used as a selection marker, enabling positive selection of recombination events based on the glutamate auxotrophy of A. succinogenes. After successful deletion of the target gene, we employed the Saccharomyces cerevisiae flippase recombinase to remove the icd marker, enabling its re-use. With the aim of increasing succinate yields, the A. succinogenes pflB gene (encoding pyruvate formate-lyase, PFL) was targeted for deletion. Strain ÄpflB produced higher succinate yields than strain 130Z (0.85 mol/mol glycerol) under microaerobic conditions.In summary, in optimized respiratory conditions, A. succinogenes can conserve most of the reducing power available in glycerol for succinate production. The increased understanding of A. succinogenes's glycerol metabolism, combined with new genetic tools, sets the stage for future strain and process development towards a highly productive and economic glycerol-to-succinate conversion process.
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Title: Nanoscale studies of metal reduction by microbial biocatalysts using in vitro biomimetic platforms
Creator: Awate, Bhushan Prabhakar
Date: 2014
Collection: Electronic Theses & Dissertations
Description: NANOSCALE STUDIES OF METAL REDUCTION BY MICROBIAL BIOCATALYSTS USING IN VITRO BIOMIMETIC PLATFORMSMetal-reducing bacteria like Geobacter sulfurreducens use cytochrome proteins to reductively precipitate water-soluble uranium salts. However, the mechanism by which the cytochromes achieve multistep electron transfer to extracellular metals is not yet understood. Previous studies of cytochromes' role in electron transfer have involved a genetic approach, in which...
Show moreNANOSCALE STUDIES OF METAL REDUCTION BY MICROBIAL BIOCATALYSTS USING IN VITRO BIOMIMETIC PLATFORMSMetal-reducing bacteria like Geobacter sulfurreducens use cytochrome proteins to reductively precipitate water-soluble uranium salts. However, the mechanism by which the cytochromes achieve multistep electron transfer to extracellular metals is not yet understood. Previous studies of cytochromes' role in electron transfer have involved a genetic approach, in which specific cytochromes are either deleted or overexpressed. However, results of these genetic studies are difficult to interpret, because mutation of one gene can cause multiple phenotypic changes, resulting in complex alterations of the cell's electron-transfer machinery. These limitations can be bypassed using a biomimetic approach, in which Geobacter cytochromes are assembled into nanostructured interfaces that mimic the cell envelope and electron-carrier machinery.In this study, we heterologously expressed some of Geobacter's most abundant and conserved cytochromes in Escherichia coli. We then used these cytochromes to fabricate nanostructured biomimetic interfaces that mimicked Geobacter's double-membrane cellenvelope. A self-assembled monolayer of alkanethiols on a gold electrode mimicked the inner membrane; an aqueous layer containing PpcA (a periplasmic cytochrome) mimicked the periplasmic space; and a synthetic bilayer lipid membrane containing OmcB (an outer membrane cytochrome) mimicked the outer membrane. Cytochrome-mediated electron transfer from the gold electrode to soluble metal salts was characterized using cyclic voltammetry. The PpcA was found to transfer electrons to U(VI) more rapidly than to other soluble electron acceptors, consistent with the observation that U(VI) is reductively precipitated in Geobacter's periplasm. Spectroelectrochemical characterization of PpcA and OmcB demonstrated for the first time electron transfer between these two proteins, suggesting that they may be redox partners in Geobacter's electron transport chain.Fabrication of electrochemically active nanostructured bioelectronic interfaces that mimic Geobacter's double-layered cell envelope establishes a new experimental platform with which to characterize Geobacter's electron transfer machinery. Addition of more Geobacter components will make the interface more realistic and enable hypotheses about electron-transfer mechanisms to be systematically tested. An improved understanding of Geobacter's ability to reduce metals may lead to new technologies for in situ reductive immobilization of uranium and other toxic metals.
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Title: Scanning probe studies of the pilus nanowires in Geobacter sulfurreducens
Creator: Veazey, Joshua P.
Date: 2011
Collection: Electronic Theses & Dissertations
Description: In microbial organisms like bacteria, pili (singular: pilus) are filament-like appendages that are nanometers in diameter and microns long. The sizes and structures of the different types of pili found in nature are adapted to serve one of many distinct functions for the organism from which they come. The pili expressed by the bacterium Geobacter sulfurreducens act as electrically conductive nanowires that provide conduits for electrons to leave the cell during its...
Show moreIn microbial organisms like bacteria, pili (singular: pilus) are filament-like appendages that are nanometers in diameter and microns long. The sizes and structures of the different types of pili found in nature are adapted to serve one of many distinct functions for the organism from which they come. The pili expressed by the bacterium Geobacter sulfurreducens act as electrically conductive nanowires that provide conduits for electrons to leave the cell during its respiratory cycle. Biological experiments have suggested that long range electron transfer across micron distances may proceed along the protein matrix, rather than by metalcofactors (metal atoms bound to the protein). Protein conductivity across such distances would require a novel transport mechanism. In an effort to elucidate this mechanism, our lab has used two electronically sensitive scanning probe techniques: Scanning Tunneling Microscopy (STM) and Conductive Probe Atomic Force Microscopy (CP-AFM).I employed the high resolution imaging and electronic sensitivity of STM to resolve the molecular sub-structure and local electronic density of states (LDOS) at different points above pili from purified preparations, deposited onto a conducting substrate. The significantand stable tunneling currents achieved for biologically relevant voltages, in the absence of metal cofactors, demonstrated conduction between tip and substrate via the protein matrix.We observed periodicity of roughly 10 nm and 2.5 nm in topographs of the pili. In our acquisition of LDOS, we observed gap-like asymmetric energy spectra that were dependent upon the location of the tip above the pilus, suggestive of easier current flow out of oneside of the cylindrical pilus and into the opposite side. Voltage-dependent STM imaging, which also contains information about the LDOS at each pixel, was consistent with this interpretation. The asymmetry in spectra observed on one pilus edge had a slightly larger magnitude than the other edge, by a factor of 1.3. The width of the gap-like feature was roughly 1 V.For direct observation of the long range electron transport, we developed a method whereby a conductive AFM tip measured current flowing to a surface electrode via the longitudinal axis of a deposited pilus. These samples also lacked embedded metal atoms. We achieved a proof-of-principle measurement of conductivity across a 200 nm distance. The upper bound of the resistance at this distance was 40 megaohms, suggesting a preliminary upper bound on the longitudinal resistivity of 0.4 Ohm-cm, comparable to that of the basal plan of graphite. I will place these results in context with continuing work in our lab to optimize the protocols for reproducible deposition quality on the electrodes.
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