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Title: The evolution of complexity and robustness in small populations
Creator: LaBar, Thomas
Date: 2018
Collection: Electronic Theses & Dissertations
Description: "A central goal of evolutionary biology is to understand a population's evolutionary trajectory from fundamental population-level characteristics. The mathematical framework of population genetics provides the tools to make these predictions. And while population genetics provides a well-studied framework to understand how adaptation and neutral evolution quantitatively alter population fitness, less attention has been paid to using population genetics to predict qualitative evolutionary...
Show more"A central goal of evolutionary biology is to understand a population's evolutionary trajectory from fundamental population-level characteristics. The mathematical framework of population genetics provides the tools to make these predictions. And while population genetics provides a well-studied framework to understand how adaptation and neutral evolution quantitatively alter population fitness, less attention has been paid to using population genetics to predict qualitative evolutionary outcomes. For instance, do different populations evolve alternative genetic mechanisms to encode similar phenotypic traits, and if so, which processes lead to these differences? This dissertation investigates the role of population size in altering the qualitative outcome of evolution. It is difficult to experimentally investigate qualitative evolutionary outcomes, especially in small populations, due to the time required for novel evolutionary features to appear. To get around this constraint, I use digital experimental evolution. While digital evolution experiments lack aspects of biological realism, in some regards they are the only methodology that can approach the complexity of biological systems while maintaining the ease of analysis present in mathematical models. Digital evolution experiments can never prove that certain evolutionary trajectories occur in biological populations, but they can suggest hypotheses to test in more realistic model systems. First, I explore the role of population size in determining the evolution of both genomic and phenotypic complexity. Previous hypotheses have argued that small population size may lead to increases in complexity and I test aspects of those hypotheses here. Second, I introduce the novel concept of 'drift robustness' and argue that drift robustness is a strong factor in the evolution of small populations. Finally, I end with a project on the role of genome size in enhancing the extinction risk of small populations. I conclude with a broader discussion of the consequences of this research, some limitations of the results, and some ideas for future research."--Page ii.
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Title: Suicide, signals, and symbionts : evolving cooperation in agent-based systems
Creator: Vostinar, Anya E.
Date: 2017
Collection: Electronic Theses & Dissertations
Description: Cooperation is ubiquitous in nature despite the constant pressure for organisms to cheat by receiving a benefit from cooperators, while not cooperating themselves. The continued evolution and persistence of countless forms of cooperation is a central topic in evolutionary theory. Extensive research has been done on the theoretical dynamics of cooperation through game theory and the natural examples of cooperation. However, it remains difficult to understand thoroughly the evolution of many...
Show moreCooperation is ubiquitous in nature despite the constant pressure for organisms to cheat by receiving a benefit from cooperators, while not cooperating themselves. The continued evolution and persistence of countless forms of cooperation is a central topic in evolutionary theory. Extensive research has been done on the theoretical dynamics of cooperation through game theory and the natural examples of cooperation. However, it remains difficult to understand thoroughly the evolution of many cooperative systems, due in part to the ancient origins of these systems and the long time scales required to see cooperation evolve in any natural populations. I have systematically analyzed the evolution of three broad types of cooperation: programmed cell death, quorum sensing, and mutualisms (cooperation across species). I have provided evidence that programmed cell death can originate due to kin selection. I have also created two new systems to enable the extensive exploration of factors that affect the evolution of public goods cooperation and mutualism. Using these systems, I determine the effects of environmental factors on the evolution of public goods cooperation and mutualism. By uniting the expansive theoretical work on these forms of cooperation with a fully-controlled experimental system, I contributed to our understanding of how these forms of cooperation can emerge and be maintained in industrial and medical applications that rely on bacterial cooperation.
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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: Hijacking the cell : how bacteriophage Sf6 uses Shigella flexneri outer membrane proteins for infection
Creator: Hubbs, Natalia Barbara
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Viral infections cause problems worldwide and result in a multitude of human diseases ranging in severity from influenza to HIV. Most viruses infect their respective host cells by attaching to a receptor, ejecting their genomes, and replicating via the host cell machinery. The step of attachment and entry requires precise docking to a designated location on the surface of the cell followed by conformational changes that result in viral genome transfer into the cell. However, the molecular...
Show more"Viral infections cause problems worldwide and result in a multitude of human diseases ranging in severity from influenza to HIV. Most viruses infect their respective host cells by attaching to a receptor, ejecting their genomes, and replicating via the host cell machinery. The step of attachment and entry requires precise docking to a designated location on the surface of the cell followed by conformational changes that result in viral genome transfer into the cell. However, the molecular mechanisms that drive receptor binding have not been elucidated for the vast majority of viruses. Because viral protein structures and mechanisms are conserved across families of viruses, Sf6 and its host Shigella flexneri, may be used as a model system to provide insight into understanding viral attachment to host cells. Sf6 uses lipopolysaccharide (LPS) as a primary (1o ) receptor for an initial reversible, interaction, and it requires a secondary (2o ) irreversible receptor to commit to infection. Both outer membrane proteins A and C (OmpA and OmpC) may serve as secondary receptors for Sf6, although OmpA is slightly preferred. Here, we investigate how bacteriophage Sf6 utilizes OmpA and OmpC for infection. First, we identified that the surface loops of OmpA are important for Sf6 infection. Using a combination of in vivo and in vitro approaches including, but not limited to, phage plaque assays, site-directed mutagenesis, circular dichroism spectroscopy, and in vitro genome ejection assays we characterized which residues in the surface loops of OmpA are responsible for productive Sf6 infection. We showed that individual amino acid substitutions have a range of effects implicating some locations in the loops as more important than others for infection. Next, we used BioLayer Interferometry (BLI), an optical biosensing technique, to determine binding affinities of Sf6 to OmpA and single substitution variants. We immobilized whole virions and determined the kinetic parameters of Sf6 to various OmpAs to be fast-on and slow-off. The binding affinity of Sf6 to S. flexneri OmpA is in the low nM range. We also show that Sf6 binds to five variant OmpAs and the resultant kinetic parameters vary only slightly. These kinetic data suggest that Sf6:Omp receptor recognition is not solely based on kinetics, but potentially on the ability of the Omp to induce the correct conformational changes in the virion which result in translocation of the DNA. Finally, we purified OmpC and using a limited proteolysis approach, we obtained trypsin resistant and functional trimeric OmpC. The resultant OmpC, in combination with LPS, causes Sf6 genome ejection in vitro, but at a lower efficiency and rate than with OmpA. Taken together, the data presented in this dissertation shed light on how Sf6 interacts with its secondary receptors, OmpA and OmpC, an important aspect of host recognition, and provide new insights into Podoviridae attachment."--Pages ii-iii.
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Title: A rapid assay to detect antibiotic resistance with novel 3D printed pharmacokinetic/pharmacodynamic technologies
Creator: Heller, Andrew A.
Date: 2019
Collection: Electronic Theses & Dissertations
Description: "The over-prescription and misuse of antibiotics has led some bacterial strains to become resistant to one, multiple, or all currently available antibiotics. In order to treat an antibiotic resistant bacterial infection, a novel antibiotic or therapeutic is required. Up until the last few years, there had been a trend of fewer new antibiotics due to pharmaceutical companies not pursuing antibiotic development. The continuous threat of antibiotic resistance and the lack of antibiotic research...
Show more"The over-prescription and misuse of antibiotics has led some bacterial strains to become resistant to one, multiple, or all currently available antibiotics. In order to treat an antibiotic resistant bacterial infection, a novel antibiotic or therapeutic is required. Up until the last few years, there had been a trend of fewer new antibiotics due to pharmaceutical companies not pursuing antibiotic development. The continuous threat of antibiotic resistance and the lack of antibiotic research led to the National Action Plan, which called for novel, rapid diagnostic tools and new therapeutics to combat antibiotic resistance. The main reason for the lack of interest in antibiotic development is that drug development now cost a pharmaceutical greater than $2.5 billion and can take over 10 years. In addition, only 10.4% of drugs that enter clinical trials eventually are approved. One of the main causes of this low success rate is that the drugs do not have the same pharmacokinetics (PK) or pharmacodynamics (PD) as the drugs did in in vitro and in vivo animal models. These differences in PK/PD can lead to safety and efficacy concerns in humans. In this dissertation, this issue is combated with new technologies for antibiotic resistance identification. A rapid, static susceptibility assay was created in order monitor the growth of a bacterial culture by measuring the extracellular ATP/OD600, which in a healthy culture should increase to a maximum during early logarithmic growth phase and then decrease. Adding an antibiotic to a growing culture after this ATP/OD600 maximum led to an increase in the ATP/OD600, while a healthy culture decreased leading a statistical difference (alpha = 0.05) in 20--60 minutes after adding the antibiotic. This increase in the ATP/OD600 was due to the antibiotic's ability to effectively kill the bacteria by lysing leading to the OD600 remaining stable and extracellular ATP levels to increase. This trend was not seen when an antibiotic that the bacteria were resistant to was added. This procedure could also determine which antibiotic is killing the most bacteria in a mixed bacterial culture. The above procedure was adapted to be dynamic in order to expose the bacteria to a PK curve similar to that seen in a human so more clinically-relevant PD data could be measured. This was achieved by creating a fluidic, two compartment model that was 3D printed, which utilized porous membrane inserts that were created by novel 3D printed procedures to incorporate the membranes into the 3D printing structure. The device was characterized using fluorescein (332.31 g/mol) due to having similar properties to the antibiotic, levofloxacin (361.37 g/mol). The devices were impervious to leaking and were successful in replicating PK curves for an oral, intermittent intravenous (IV), and continuous IV administration. Replacing the fluorescein solution with a levofloxacin solution in growth media, bacteria were able to be exposed to an oral levofloxacin PK curve (C max = 12.4 +/- 3.0 microM; tmax = 1 hour; half-life = 5.2 +/- 0.5 hours). A kanamycin-resistant strain of Escherichia coli was determined to have a statistical difference in the ATP/OD600 when exposed to a levofloxacin concentration of 3.5 +/- 1.3 microM in the secondary compartment while a chloramphenicol-resistant strain of Bacillus subtilis showed a statistical difference at a concentration of 4.8 +/- 1.8 microM."--Pages ii-iii.
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Title: CHARACTERIZATION OF BIOFILM FORMATION AND SILICA RESPONSE BY THE FILAMENTOUS FUNGUS FUSARIUM GRAMINEARUM, A PLANT PATHOGEN
Creator: Shay, Rebecca
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Fusarium graminearum is a filamentous fungus that is the primary causal agent of the disease Fusarium head blight (FHB) on cereal crops. Understanding details of pathogen biology, especially those that are directly related to infection, is vital to control of disease. While much is known about how F. graminearum initiates disease into its host plants, there are still knowledge gaps related to plant signals sensed by the fungus, and mechanisms that increase success in infection. To better...
Show moreFusarium graminearum is a filamentous fungus that is the primary causal agent of the disease Fusarium head blight (FHB) on cereal crops. Understanding details of pathogen biology, especially those that are directly related to infection, is vital to control of disease. While much is known about how F. graminearum initiates disease into its host plants, there are still knowledge gaps related to plant signals sensed by the fungus, and mechanisms that increase success in infection. To better understand early infection, I have focused on two aspects of the plant-pathogen interaction. Aquaporins are channel proteins that bring small molecules dissolved in water into cells. In F. graminearum, aquaporins are important in growth, development, and spore formation, and are related to how silica is utilized by cells. Silica is an important component of cereal crops, and silica-rich cells provide infection points for F. graminearum. Biofilms are three-dimensional formations important to many microbes for protection from adverse environmental conditions. Biofilms have primarily been studied in single-celled organisms, but there is a growing body of work on filamentous fungal biofilms. I found that biofilm formation is initiated in vitro with the adhesion of propagules to a surface, followed by growth of the structures and development of an extracellular matrix, then dispersal of propagules and senescence of biofilms. I have profiled the transcriptome of biofilm formation over time, and characterized genes significant to this process. Knockouts of these genes produced altered biofilm formations, especially in matrix composition. My work has identified novel early infection characteristics in F. graminearum, which will provide new targets for control.
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Title: Meta-analyses of gene expression in age-dependent diseases
Creator: Rogers, Lavida Rashida Kenera
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Physiological changes with age such as immune system decline and brain aging cause an increased risk for diseases. Age-related diseases are of major concern especially in the elderly population due to there being an increase in the average lifespan. This dissertation explores neurodegenerative and respiratory diseases and how gene expression varies due to disease status, age tissue and sex.Alzheimer's disease (AD) has been categorized by the Centers for Disease Control and Prevention (CDC) as...
Show morePhysiological changes with age such as immune system decline and brain aging cause an increased risk for diseases. Age-related diseases are of major concern especially in the elderly population due to there being an increase in the average lifespan. This dissertation explores neurodegenerative and respiratory diseases and how gene expression varies due to disease status, age tissue and sex.Alzheimer's disease (AD) has been categorized by the Centers for Disease Control and Prevention (CDC) as the 6th leading cause of death in the United States. AD is a significant health-care burden because of its increased occurrence (specifically in the elderly population), and the lack of effective treatments and preventive methods. AD targets neuronal function and can cause neuronal loss due to the buildup of amyloid-beta plaques and intracellular neurofibrillary tangles.The respiratory disease, chronic obstructive pulmonary disease (COPD), was classified by the Centers for Disease Control and Prevention in 2014 as the 3rd leading cause of death in the United States. The main cause of COPD is exposure to tobacco smoke and air pollutants. In addition to exploring genetic variation due to disease state, sex and age we also explored the role of smoking status on expression profiles.Additionally, the respiratory infections, influenza and pneumonia affect thousands of people worldwide. Young children, elderly and immunocompromised individuals are at higher risk for being infected by the influenza virus and Streptococcus pneumoniae. Host responses to these pathogens and vaccinations vary by the state of one's immune system.This dissertation includes multiple meta-analyses to assess genetic variation in Alzheimer's disease, COPD and Influenza, and an assessment of pneumococcal disease and aging. To identify significant differentially expressed genes we ran an analysis of variance with a linear model with disease state, age, sex, tissue, smoking status and study as effects that also included binaryinteractions.Our meta-analysis approach effectively combined multiple publicly available microarray datasetsto identify gene expression differences across diseases including full age, sex, smoking status and tissue type considerations. Our findings provide potential gene and pathway associations that can be targeted to improve treatment and prevention of diseases.
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Title: Reducing iron on a knife's edge - genomic and mechanistic studies of (hyper)thermophilic dissimilatory iron-reducing bacteria and archaea
Creator: Manzella, Michael P.
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Some members of the Bacteria and the Archaea have the rare ability to transfer electrons beyond the surface of their cells. This extracellular electron transfer permits the reduction of otherwise inaccessible electron acceptors such as insoluble Fe(III) oxides. The mechanisms that enable this ability have direct implications for geochemical cycles today and for life on early Earth. The physical settings present on early Earth, hot and influenced primarily by hydrothermal activity, can be...
Show moreSome members of the Bacteria and the Archaea have the rare ability to transfer electrons beyond the surface of their cells. This extracellular electron transfer permits the reduction of otherwise inaccessible electron acceptors such as insoluble Fe(III) oxides. The mechanisms that enable this ability have direct implications for geochemical cycles today and for life on early Earth. The physical settings present on early Earth, hot and influenced primarily by hydrothermal activity, can be found in rare sites on modern Earth. These sites most often surround deep-sea hydrothermal vents. Organisms present at these sites thrive, in most cases, absent of the sun’s influence. Instead of primary production from photosynthetic microorganisms, these communities rest on the shoulders of chemoautolithotrophic bacteria and archaea. Two of these organisms, the thermophilic bacterium Geothermobacter ehrlichii and the hyperthermophilic archaeon Geoglobus ahangari were selected to undergo genome and physiological characterization to determine how they interact with the abundant insoluble Fe(III) oxides found at hydrothermal vents. Both genomes were sequenced and, while only the genome of G. ahangari was complete, this permitted identification of critical components for iron respiration. In addition, mechanistic studies were performed on G. ahangari to elucidate a direct-contact mechanism of iron reduction. Finally, the extracellular filaments from these microorganisms were characterized and the more abundant filaments, in both organisms, were found to be conductive. These are the first examples of nanowires discovered outside of the mesophilic bacteria. In addition, the phylogenetic and geographic diversity between these isolates suggests that microbial nanowires are more widespread than previously thought.
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Title: The role of the second messenger cyclic di-GMP in light-dependent responses in cyanobacteria
Creator: Agostoni, Marco
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Second messengers are intracellular substances regulated by specific external stimuli globally known as first messengers. Cells rely on second messengers to generate rapid responses to environmental changes and the importance of their roles is becoming increasingly realized in cellular signaling research. Cyanobacteria are photooxygenic bacteria that inhabit most of Earth's environments. The ability of cyanobacteria to survive in ecologically diverse habitats is due to their capacity to adapt...
Show moreSecond messengers are intracellular substances regulated by specific external stimuli globally known as first messengers. Cells rely on second messengers to generate rapid responses to environmental changes and the importance of their roles is becoming increasingly realized in cellular signaling research. Cyanobacteria are photooxygenic bacteria that inhabit most of Earth's environments. The ability of cyanobacteria to survive in ecologically diverse habitats is due to their capacity to adapt and respond to environmental changes. Chapter 1 will review known second messenger-controlled physiological processes in cyanobacteria. Second messengers used in these systems include the element calcium (Ca2+), the nucleotide-based guanosine tetraphosphate or pentaphosphate (ppGpp or pppGpp, represented as (p)ppGpp), the cyclic adenosine 3′,5′-monophosphate (cAMP), the c-di-GMP, the cyclic guanosine 3′,5′-monophosphate (cGMP), and the cyclic dimeric AMP (c-di-AMP), and the gaseous nitric oxide (NO). The discussion will focus on processes central to cyanobacteria such as nitrogen fixation, light perception, photosynthesis-related processes, and gliding motility. Chapter 2 will confirm that many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. I will also compare intracellular levels of c-di-GMP in Synechocystis sp. PCC 6803 and Fremyella diplosiphon under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo. Chapter 3 will demonstrate that biofilm formation, cellular aggregation or flocculation, and cellularbuoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 and F. diplosiphon. In this chapter, I present data on Synechocystis and F. diplosiphon transformed with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae and Escherichia coli, respectively. Engineering of these strains allowed modulation of intracellular c-di-GMP levels. Chapter 4 will confirm the role of c-di-GMP in F. diplosiphon in regulating pigments. I show that c-di-GMP is involved in light signalling networks and correctly tunes the pigments of cells under changes to ambient light. In chapter 5, I conclude by addressing future research trajectories needed to better understand the signaling networks and cross talk in the signaling pathways of these molecules in cyanobacteria. Second messengers have significant potential to be adapted as technological tools and possible novel and practical applications are highlighted based on current understanding of these molecules and the signaling networks that they control.
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Title: Defining the role of ballast water in the transport of viruses in aquatic environments through metagenomic approaches
Creator: Kim, Yiseul
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Global shipping activities transport 12 billion tons of water across regions each year. This so called ballast water contains a variety of biological materials and has been considered to transfer non−native species between biomes, resulting in potential ecological, economic, and public health problems in major ports worldwide. Despite the large amount of ballast water transported around the globe and its negative impact on native ecosystems, relatively little attention has been paid to viral...
Show moreGlobal shipping activities transport 12 billion tons of water across regions each year. This so called ballast water contains a variety of biological materials and has been considered to transfer non−native species between biomes, resulting in potential ecological, economic, and public health problems in major ports worldwide. Despite the large amount of ballast water transported around the globe and its negative impact on native ecosystems, relatively little attention has been paid to viral invasions via ballast water due to technical challenges in detecting the wide range of viruses. The limitations of virus discovery using traditional approaches can now be overcome with the emergence of metagenomics, which enables unprecedented views of viral diversity and functions. This dissertation brought together environmental virology, metagenomics, and bioinformatics for the first time in order to examine taxonomic composition and diversity of viruses in ballast and harbor waters collected from a freshwater system, and to investigate global transport of viruses through ballast water and effect of engineered, management, and environmental parameters associated with ballast water on ocean viruses. Viral communities in ballast water in the Great Lakes were examined due to the long history of non−native species invasions in this region of the world. Five ballast and three harbor waters were collected from the Port of Duluth−Superior on May 2013. Bioinformatics analyses of over 550 million Illumina reads showed that the viral sequences had mostly no homologs in the public database, indicating that our knowledge about viral diversity is still very limited. Among the sequences homologous to known viruses (22.3 ± 6.2%), ballast and harbor waters contained a diversity of viruses, which were largely dominated by double–stranded (ds) DNA phages, including Myoviridae, Podoviridae, and Siphoviridae. Along with these phage families, viruses that could infect a broad range of hosts, including archaea, fungi, invertebrate, plant, protist, and vertebrate, some of which are highly pathogenic to fish and shrimp, were present at different levels in the viral metagenomes (viromes). Comparative virome analyses showed that viromes were distinct among the Great Lakes and formed a specific group of temperate freshwater viromes, separate from viromes associated with marine environments and engineered freshwater systems.The scope of this research was expanded to examine viral communities in marine environments. Sixteen ballast and eight harbor waters were collected from the Port of Los Angeles/Long Beach and the Port of Singapore from March through May 2014. Bioinformatics analyses of 3.8 billion Illumina reads revealed that taxonomic profile of the sequences homologous to known viruses (30.6 ± 0.03%) was similar to that observed in the Great Lakes viromes, which were largely dominated by dsDNA phages. Moreover, this research was able to detect sequences most similar to viruses infecting human, fish, and shrimp, which are related to significant public health problems or direct economic impact. Variations in virome composition of ballast and harbor waters were found between geographic locations, suggesting that the movement of ballast water across the global shipping network transports the ocean viromes. Importantly, this research showed that virus richness (type of viruses) in ballast water was not governed by engineered or management variables but by conditions of local environment where viruses arise from showing associations with latittude. Outcomes of the present research represent the most detailed characterization to date of viruses in ballast water, defining the role of ballast water in the transport of freshwater and ocean viromes and an increased risk of exposure of aquatic fauna and flora to viruses. The present findings emphasize the need for implementing ballast water discharge limits for viruses and treatment. More research is needed on host population structure to better understand the impact of the transport of viruses between biomes.
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Title: Composition of gut microbiota affects C. jejuni-mediated inflammation and autoimmunity in murine models
Creator: Brooks, Phillip Tremaine
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Campylobacter jejuni is the leading antecedent infection to the acute peripheral neuropathy Guillain-Barre Syndrome (GBS). GBS is debilitating, often causes paralysis, and can require several months or more for recovery. Most concerning is that GBS patients are frequently left with long-term neurologic disabilities. Because a vaccine for Campylobacter is lacking there are no viable approaches for preventing this form of GBS. Currently, therapeutic approaches for GBS include plasma exchange...
Show moreCampylobacter jejuni is the leading antecedent infection to the acute peripheral neuropathy Guillain-Barre Syndrome (GBS). GBS is debilitating, often causes paralysis, and can require several months or more for recovery. Most concerning is that GBS patients are frequently left with long-term neurologic disabilities. Because a vaccine for Campylobacter is lacking there are no viable approaches for preventing this form of GBS. Currently, therapeutic approaches for GBS include plasma exchange and intravenous immunoglobulin but they require specialized equipment, pose significant financial burden, and produce mixed results. These strategies lack a strong rationale because GBS is poorly defined mechanistically. While new working mouse models of GBS may lead to alternative therapies, confirmation of C. jejuni’s specific role in precipitating GBS and the mechanism(s) through which this occurs remain elusive. Thus far, evidence gathered from murine models demonstrates that multiple factors influence C. jejuni pathogenesis, including host genetics and C. jejuni genetics, particularly the genetic plasticity of this pathogen. Notably, the gut microbiota can modulate C. jejuni colonization- and colitis- resistance; however, its role in modulating C. jejuni-triggered autoimmunity remains unknown. The overarching goal of this study is to determine if the composition of gut microbiota affects C. jejuni-triggered autoimmunity in murine models. The chapters of this thesis present the following data addressing this goal; mice infected with antimicrobial resistant Campylobacter strains from Guillain-Barré syndrome patients produced severe colitis and type 2 autoimmune responses when their microbiota were depleted by antibiotics. Furthermore, we demonstrated that transplanted human fecal microbiota alters the immune response to Campylobacter jejuni infection in C57BL/6 mice, potentially increasing the risk of autoimmune sequelae. Finally, comparative genomic analysis of passaged C. jejuni populations revealed genetic variation in multidrug transporter genes cmeB and cmeR in Campylobacter jejuni populations from antibiotic treated mice. CmeR regulates expression of C. jejuni cell surface molecules, again potentially impacting the risk of autoimmune sequelae. Taken together, our results demonstrate that composition of gut microbiota is a critical determinant of inflammatory and autoimmune outcomes in C. jejuni murine models.
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Title: Genome evolution of Campylobacter jejuni during experimental adaptation
Creator: Jerome, John Paul
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Campylobacter jejuni is a leading cause of foodborne bacterial enteritis in humans. An important reservoir for C. jejuni is in chickens, but it has been shown to colonize a large host range. Passage through a mouse model of campylobacteriosis resulted in a hypervirulent phenotype in mice for C. jejuni strain NCTC11168. After analyzing the wild-type and mouse-adapted variants by phenotype assays, expression microarray, pulse-field gel...
Show moreCampylobacter jejuni is a leading cause of foodborne bacterial enteritis in humans. An important reservoir for C. jejuni is in chickens, but it has been shown to colonize a large host range. Passage through a mouse model of campylobacteriosis resulted in a hypervirulent phenotype in mice for C. jejuni strain NCTC11168. After analyzing the wild-type and mouse-adapted variants by phenotype assays, expression microarray, pulse-field gel electrophoresis and whole genome sequencing we discovered that the genetic changes in the mouse-adapted variant were confined to thirteen hypermutable regions of DNA in contingency loci. We also show that specific contingency loci changes occurred in parallel during mouse infection when reisolates from multiple mice were analyzed. Furthermore, a mathematical model that considers contingency loci mutation rates and patterns does not explain the observed changes. Taken together, this is the first experimental evidence that contingency loci play a role in the rapid genetic adaptation of C. jejuni to a host, which results in increased virulence. In contrast to the observed virulence increase by serial host passage, we showed that C. jejuni rapidly loses an essential host colonization determinant during adaptive laboratory evolution. Passage in broth culture selected for flagellar motility deficient C. jejuni cells in parallel for five independently evolved lines. Moreover, the loss of motility occurred by two genetic mechanisms: reversible and irreversible. Reversible loss of motility occurred early during broth adaptation, followed by irreversible motility loss in the majority of cells by the end of the experiment. Whole genome sequencing implicated diverse mutation events that resulted in the loss of gene expression necessary for flagellar biosynthesis. Furthermore, reversible mutations in homopolymeric DNA tracts of adenine/thymine residues, and irreversible types of mutation such as gene deletion, were discovered in the broth-evolved populations. In all evolved lines, an alternative sigma factor necessary for flagellar structural gene expression was removed from the genome. Overall, this dissertation contains the first accounts of C. jejuni experimental evolution. The results provide insight into the biological importance of reversible mutations in homopolymeric DNA tracts, and provide a basis for future studies of C. jejuni evolvability.
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Title: Bacterial community assembly and stability on the surface of the Lake sturgeon (Acipenser fulvescens) eggs
Creator: Angoshtari, Roshan
Date: 2018
Collection: Electronic Theses & Dissertations
Description: High egg and larvae mortality represents a significant problem in aquaculture. Scientific evidence for a variety of fish, shrimp and shellfish species support the hypothesis that fish-microbe interactions are the major factors determining high levels of egg mortality. As such, in order to address this problem systematically, it is crucial to extend the knowledge of egg-associated microbial communities and develop a detailed understanding of their potential probiotic effects on the wellbeing...
Show moreHigh egg and larvae mortality represents a significant problem in aquaculture. Scientific evidence for a variety of fish, shrimp and shellfish species support the hypothesis that fish-microbe interactions are the major factors determining high levels of egg mortality. As such, in order to address this problem systematically, it is crucial to extend the knowledge of egg-associated microbial communities and develop a detailed understanding of their potential probiotic effects on the wellbeing of fish eggs. In this dissertation we present the results of several studies related to development of bacterial communities on the surface of Lake Sturgeon eggs. Throughout this work our perspective on bacterial species associated with Sturgeon eggs is to treat them as an integral part of the egg-bacteria symbiotic relationship with the focus on manipulating bacterial communities in order to decrease egg mortality. First, we discuss the results of in vitro biofilm formation assays for six bacterial species previously isolated from the surface of healthy Lake Sturgeon (Acipebser fulvesens) eggs. The goal of this study was to understand how these bacterial species interact with each other when present in the same environment. We used a crystal violet assay, resazurin assay and Terminal Restriction Fragment Length Polymorphisms (T-RFLP) to analyze biofilm biomass formation, biofilm metabolic activity, and changes in the abundance of each isolate in double-species biofilms, respectively. Our results indicated that one of these isolates, Brevundimonas F16, produces a robust biofilm in vitro. Furthermore, biofilm formation increases significantly in mixed cultures of Brevundimonas-Hydrogenophaga and Brevundimonas-Acidovorax. However, biofilm formation decreased in mixed cultures that included Pseudomonas C22. Next, we describe the results of the study on how established biofilms of egg isolates interact with the river water microbiome. Our goal in this experiment was to measure the susceptibility to secondary colonization of a preexisting biofilm due to exposure to the river water. We measured the compositional stability of biofilms and identified specific river genera that invade or are recruited by preexisting biofilms. In this study, we were able to detect both highly resilient and weak biofilms, specific exclusions and recruitments of river populations by established biofilms of egg isolates, and apparent enhancements of biofilm development. Finally, we present the results of in vivo studies in the hatchery. Here we investigated the effect of early use of monosaccharaides and potential probiotics on assembly and stability of Lake Sturgeon egg-associated bacterial communities. Our results indicate that both monosaccharaides and bacterial treatments during early developmental stages of the egg could change the early egg-associated bacterial communities. Moreover, some members of Pseudomonas and Rheinheimera genera could be egg mutualists, protecting the eggs against pathogenic fungi and bacteria such as certain species from the genera Aeromonas and Flavobacterium.
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Title: Inhibitors of mycobacterium tuberculosis DosRST signaling and persistence
Creator: Zheng, Huiqing
Date: 2017
Collection: Electronic Theses & Dissertations
Description: Tuberculosis (TB) is one of the most deadly infectious diseases in human history and is caused by the bacterium Mycobacterium tuberculosis (Mtb). Current TB therapy requires 6-9 months of treatment with four different antibiotics, including isoniazid, rifampin, ethambutol and pyrazinamide. However, due to the long course of TB therapy and the evolution of drug-resistant Mtb strains, first-line anti-mycobacterial drugs are not sufficient to control the TB epidemic. Therefore, it is urgent to...
Show moreTuberculosis (TB) is one of the most deadly infectious diseases in human history and is caused by the bacterium Mycobacterium tuberculosis (Mtb). Current TB therapy requires 6-9 months of treatment with four different antibiotics, including isoniazid, rifampin, ethambutol and pyrazinamide. However, due to the long course of TB therapy and the evolution of drug-resistant Mtb strains, first-line anti-mycobacterial drugs are not sufficient to control the TB epidemic. Therefore, it is urgent to develop new drugs with novel targets to shorten the course of therapy, control the spread of drug-resistant TB and eradicate this deadly disease. In response to host immune cues, Mtb modulates its metabolism to establish a state of low metabolic activity called non-replicating persistence (NRP). During NRP, Mtb can remain viable in the host without causing disease symptoms, a state known as latent TB. DosRST is a two-component regulatory system that plays an essential role to establish and maintain NRP in Mtb. It is induced by host immune stimuli, such as hypoxia, carbon monoxide and nitric oxide, through the histidine kinase sensors DosS and DosT. The response regulator DosR regulates about 50 genes in the dormancy regulon. NRP bacilli are problematic for two reasons: 1) they are insensitive to several anti-mycobacterial agents and drive the long course of TB therapy; and, 2) they can resuscitate for growth once the immune system weakens, thereby serving as a source for reactivation of disease and infectious transmission of the Mtb. Therefore, inhibiting the DosRST pathway may help reduce the population of NRP bacteria during infection and thus function to reduce drug tolerance and shorten TB treatment. This dissertation presents a whole-cell phenotypic high-throughput screen of a ~540,000 compound small-molecule library. The screen employed a DosR-dependent, hypoxia-inducible fluorescent reporter strain, CDC1551(hspX::GFP), and successfully identified six distinct, novel chemical inhibitors of DosRST signaling, named HC101A-106A. Physiological and mechanistic studies were performed to characterize HC101-104 and HC106A. All five inhibitors are shown to inhibit genes of the DosRST regulon and persistence-associated physiologies, such as triacylglycerol accumulation. HC101A, HC102A, HC103A and HC106A also reduce Mtb survival when cultured under strongly hypoxic conditions. UV-visible spectroscopy studies show that HC101A (artemisinin) and HC106A target the heme group of sensor kinases DosS/T via distinct mechanisms. For example, artemisinin modulates the redox status of DosS/T and alkylates the heme to form artemisinin-heme adducts, whereas HC106A interacts with DosS heme in a similar manner to direct CO-heme or NO-heme interactions. In contrast, HC102A and HC103A do not target the heme group, but instead inhibit sensor kinase autophosphorylation activity. Electrophoretic mobility assays suggest that HC104A functions by directly inhibiting DosR DNA binding activity. Overall, this dissertation provides proof-of-concept that multiple components of the DosRST pathway can be targeted by small molecules to inhibit Mtb persistence and antibiotic tolerance. Additionally, this dissertation presents the discovery of a new chemical inhibitor, HC2091, that kills Mtb by targeting the mycolic acid transporter MmpL3. MmpL3 is an essential protein that functions to transport trehalose monomycolate across mycomembranes for trehalose dimycolate biosynthesis. HC2091 is bactericidal against Mtb in a dose- and time- dependent manner in vitro. It also has activity against Mtb inside of macrophages. Whole genome sequencing spontaneous mutants resistant to HC2091 identified five single nucleotide variants primarily located in the C-terminus of MmpL3, and HC2091-treated Mtb exhibits decreased mycolic acid synthesis, thus supporting that MmpL3 is the target of HC2091.
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Title: Investigating the role of Lactobacillus reuteri and the microbiota in bone health
Creator: Quach, Darin
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Osteoporosis is a disease characterized by low bone mass, which can subsequently lead to an increased risk of sustaining a bone fracture. With the advancements made in research and medicine in the past century, the average lifespan has increased substantially. However, this has also lead to an increase in the elderly population that is susceptible to age-related diseases such as osteoporosis. It is currently estimated that over 300 million people worldwide are impacted by osteoporosis. Taking...
Show moreOsteoporosis is a disease characterized by low bone mass, which can subsequently lead to an increased risk of sustaining a bone fracture. With the advancements made in research and medicine in the past century, the average lifespan has increased substantially. However, this has also lead to an increase in the elderly population that is susceptible to age-related diseases such as osteoporosis. It is currently estimated that over 300 million people worldwide are impacted by osteoporosis. Taking into consideration the side effects stemming from medications used to treat this illness, there has been an increase in research efforts to develop novel therapeutics for osteoporosis. One area of research that has garnered recent interest involves investigating the therapeutic potential of the gut microbiota in bone health. As a result, an overarching goal in this area of research revolves around identifying microbes that impact bone health and understanding the mechanisms mediating these responses. In this thesis, I present the beneficial use of the probiotic bacterium Lactobacillus reuteri (L. reuteri) in an in vivo murine model of osteoporosis mediated by estrogen deficiency. Using female Balb/c mice that are rendered estrogen deficient following ovariectomy, we demonstrated that supplementation with L. reuteri was capable of preventing bone loss. In addition to this, we identified that the process of osteoclastogenesis was down-regulated following L. reuteri treatment in these mice suggesting that this could be the mechanism by which L. reuteri confers its benefit on bone health. Osteoclasts are the main cell type responsible for bone resorption. Using an in vitro model of osteoclastogenesis, we demonstrated that cell-free conditioned medium (CCM) from L. reuteri inhibited the maturation of osteoclasts from macrophages. We further characterized this by demonstrating that L. reuteri CCM halted osteoclastogenesis at an intermediate stage characterized by fused polykaryons. Using an antagonist to the G protein coupled receptor GPR120, we decreased the ability of L. reuteri CCM to suppress osteoclastogenesis from 70% to 38% suggesting that L. reuteri is partially signaling through GPR120 to suppress osteoclastogenesis. Taking into account that GPR120 was a receptor for long chain fatty acids, we investigated the impact of lactobacillic acid (LA), a long chain fatty acid produced by L. reuteri, and observed that the suppression of osteoclastogenesis by L. reuteri involved the production of LA. Moreover, purified LA could suppress osteoclast formation in a dose dependent manner. To elucidate the effect of L. reuteri treatment on host cell physiology, we performed RNA sequencing at multiple time points during osteoclastogenesis. An analysis of the transcriptome data identified several pathways that were modulated following L. reuteri treatment. Further investigations indicated that NF-κB and p38 activation were impacted by L. reuteri in RAW264.7 cells. These sets of experiments have led to the identification of a possible effector molecule produced by L. reuteri and mechanism by which it acts to benefit bone health. In the last part of this thesis, I present an in vivo murine model using germ free (GF) mice to study the impact of the gut microbiota on bone health. By colonizing GF mice with microbiotas with different compositions, the goal was to identify specific microbes that could be impacting bone health in either a positive or negative manner. Interestingly enough, we discovered that the microbial communities that were introduced into the different groups of mice in our studies did not impact bone health in comparison to the GF control group. This was in stark contrast to existing literature that reported a deleterious effect in bone health following the introduction of a gut microbiota. This reinforced the fact that our knowledge remains limited in terms of understanding how the gut microbiota impacts bone health. Nevertheless, the discoveries stemming from these studies contribute to the growing body of work in this discipline and will guide future research that aims to uncover novel therapeutic options to combat osteoporosis.
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Title: Construction of a secretome mutant library of Lactobacillus reuteri
Creator: Ortiz Villalobos, Javiera
Date: 2013
Collection: Electronic Theses & Dissertations
Description: During the last 10 years increased scientific attention has been given to probiotics due to their promising therapeutic benefits in enteric diseases. In this sense, Lactobacillus reuteri 6475 has emerged as a potential probiotic strain that has demonstrated various probiotic therapeutic features. These characteristics include the ability of this strain to produce and secrete the antimicrobial compound reuterin and anti-inflammatory factors. Because the main probiotic effects...
Show moreDuring the last 10 years increased scientific attention has been given to probiotics due to their promising therapeutic benefits in enteric diseases. In this sense, Lactobacillus reuteri 6475 has emerged as a potential probiotic strain that has demonstrated various probiotic therapeutic features. These characteristics include the ability of this strain to produce and secrete the antimicrobial compound reuterin and anti-inflammatory factors. Because the main probiotic effects of L. reuteri 6475 that have been identified thus far are secreted, we were interested in developing a mutant library consisting of disruptions of genes that encode for secreted or cell-wall proteins. To complete this task we employed two different strategies: the identification of secretome protein sequences by predictive mathematical methods and the disruption of the protein coding sequences by single-stranded DNA recombineering. As a result, we have developed a mutant library of 127 secretome genes that would drive the elucidation of important L. reuteri probiotic mechanisms of action. To demonstrate the utility of this library, I screened all 127 mutants for the ability to produce reuterin and found 11 genes that increase reuterin secretion and 3 that eliminate reuterin secretion when disrupted. Future characterization of these genes will further elucidate the bacterial pathways that are critical for reuterin production.
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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: Cyclic di-GMP dependent regulation in the plant pathogenic bacterium Erwinia amylovora
Creator: Castiblanco Mosos, Luisa F.
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Bacterial pathogenesis generally depends on the ability to effectively coordinate the synthesis and expression of pathogenicity and virulence factors in order to successfully colonize a host. In some cases, this involves a need to rapidly reprogram cellular behaviors in response to host defenses or based on spatial location in the host. This genetic regulation often involves small signaling molecules such as cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger synthesized...
Show moreBacterial pathogenesis generally depends on the ability to effectively coordinate the synthesis and expression of pathogenicity and virulence factors in order to successfully colonize a host. In some cases, this involves a need to rapidly reprogram cellular behaviors in response to host defenses or based on spatial location in the host. This genetic regulation often involves small signaling molecules such as cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger synthesized intracellularly and degraded by diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. This nucleotide signaling molecule is involved in the regulation of many cellular processes in numerous bacterial species, including the transition from motile to sessile lifestyle, virulence, biosynthesis of exopolysaccharides and adhesion structures, and cell differentiation. Signal transduction and phenotypic modulation is determined by binding of c-di-GMP to specific downstream receptor molecules.Erwinia amylovora uses motility and the hypertensive response and pathogenicity- (Hrp-) type III secretion system (T3SS) during initial phases of plant infection. Once inside the xylem, this pathogen aggregates due to the production of exopolysaccharides (EPS) and attachment structures forming a biofilm that ultimately plugs the xylem vessels. Although c-di-GMP has been shown to be an important intracellular signal in several plant pathogenic bacteria, the importance of this molecule in Erwinia amylovora has not been previously investigated. This doctoral research explores the role of c-di-GMP in the genetic regulation of key cellular processes associated with pathogenesis in E. amylovora. Five active DGC enzymes (EdcA, EdcB, EdcC, EdcD and EdcE) were identified in this bacterial pathogen. Phenotypic analyses demonstrated that c-di-GMP positively regulates the biosynthesis of both cellulose and amylovoran, positively regulates biofilm formation, and represses motility. Disease assays demonstrated that c-di-GMP negatively regulates virulence in these infection models.The exopolysaccharides amylovoran and levan and attachment structures such as fimbriae, type IV pili and curli, have been demonstrated to be critical for the formation of a mature biofilm in E. amylovora. The results presented in this work demonstrated that c-di-GMP binds to the receptor protein BcsA, the cellulose synthase subunit, and activates the biosynthesis of cellulose in E. amylovora at a post-translational level. In addition, using SEM and confocal microscopy, it was demonstrated that cellulose is a main component of the biofilms formed by E. amylovora in vitro and in the host. Gene overexpression and site-directed mutagenesis analyses, suggest that BcsZ, an endoglucanase, is also required for cyclic di-GMP activation of cellulose biosynthesis and biofilm formation.Further investigation into the mechanisms of c-di-GMP dependent regulation of virulence, with a special emphasis on the regulation of hrp-T3SS gene expression, revealed that high intracellular levels of c-di-GMP via DGC overexpression lead to a significant reduction in gene expression for hrpL, the hrp alternative sigma factor, and dspE. C-di-GMP binding assays suggest that HrpS, the σ54 dependent transcriptional regulator of hrpL, is a candidate receptor of c-di-GMP. Moreover, the results presented in this work indicate that HrpS can bind to other guanosine-containing signaling molecules including the c-di-GMP degradation product pGpG. This work provides an overview of some of the molecular mechanisms for c-di-GMP-dependent regulation on the main pathogenicity determinants in E. amylovora, in order to orchestrate pathogenesis and successfully cause disease.
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Title: Ecological effects on the evolution of cooperative behaviors
Creator: Connelly, Brian Dale
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Cooperative behaviors abound in nature and can be observed across the spectrum of life, from humans and primates to bacteria and other microorganisms. A deeper understanding of the forces that shape cooperation can offer key insights into how groups of organisms form and co-exist, how life transitioned to multicellularity, and account for the vast diversity present in ecosystems. This knowledge lends itself to a number of applications, such as understanding animal behavior and engineering...
Show moreCooperative behaviors abound in nature and can be observed across the spectrum of life, from humans and primates to bacteria and other microorganisms. A deeper understanding of the forces that shape cooperation can offer key insights into how groups of organisms form and co-exist, how life transitioned to multicellularity, and account for the vast diversity present in ecosystems. This knowledge lends itself to a number of applications, such as understanding animal behavior and engineering cooperative multi-agent systems, and may further help provide a fundamental basis for new industrial and medical treatments targeting communities of cooperating microorganisms.Although these behaviors are common, how evolution selected for and maintained them remains a difficult question for which several theories have been introduced. These theories, such as inclusive fitness and group selection, generally focus on the fitness costs and benefits of the behavior in question, and are often invoked to examine whether a trait with some predetermined costs and benefits could be maintained as an evolutionarily-stable strategy. Populations, however, do not exist and evolve in a vacuum. The environment in which they find themselves can play a critical role in shaping the types of adaptations that organisms accumulate, since one behavior may be highly beneficial in one environment, yet a hindrance in another. Ever-changing environments further complicate this picture, as maintaining a repertoire of behaviors for surviving in different environments is often costly. In addition to these environmental forces, the number and composition of other organisms with which individuals interact impose additional constraints. The combination of these factors results in significantly more complex dynamics.Using computational models and microbial populations, this dissertation examines several ways in which ecological factors can affect the evolution of cooperative behaviors. First, environmental disturbance is examined, in which a cooperative act enables organisms and their surrounding neighbors to survive a periodic kill event (population bottleneck) of varying severity. Resource availability is then studied, where populations must determine how much resource to allocate to cooperation. Finally, the effect that social structure, which define the patterns of interactions among the individuals in a population, is investigated.
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