Digital Commons Network^™

Development Of Protein Display Systems And Genetic Tools For Spore-Forming Bacteria, Erin Drufva

Doctoral Dissertations

One major area of synthetic biology is to engineer microbial cells and subcellular systems for diverse applications including biosynthesis, biocatalysis, therapeutics, drug delivery, and bioremediation. For most applications, robust cellular systems are preferred for longer activity half-life and resistance to harsh environments. Two projects related to robust cellular systems involving Gram-positive bacteria are presented in this work. One is to develop thermostable genetic reporters for Geobacilli species and the other is to display an enzyme on the Bacillus subtilis spore surface to enhance its robustness and present an alternative to purified enzymes for industrial applications.

Bacillus subtilis and Geobacillus thermoglucosidans …

Microbial Community And Vibrio Parahaemolyticus Population Dynamics In Relayed Oysters, Michael Anthony Taylor

Doctoral Dissertations

The CDC estimates that 45,000 people are sickened each year by foodborne Vibrio parahaemolyticus in the United States. Filter-feeding bivalve shellfish, such as oysters, are routinely inhabited by human pathogenic V. parahaemolyticus and there currently is not a contaminant management process that effectively reduces concentrations of V. parahaemolyticus in oysters. The transplanting of V. parahaemolyticus -laden oysters to an area with low concentrations or no V. parahaemolyticus, called oyster relay, is one reduction strategy that holds promise for treating live oysters. A key consideration for effective strategies to reduce Vibrio spp. in shellfish is the influence of microbiota in natural …

Molecular Mechanisms Of Salt And Osmotic Stress Tolerance In Frankia Strains Isolated From Casuarina Trees, Rediet Oshone

Doctoral Dissertations

Globally, 20% of total cultivated and 33% of irrigated agricultural lands are affected by high salinity. By 2050, more than 50% of the arable land will be salinized. The hyper-ionic and hyper-osmotic stresses associated with salt-affected soils threaten the ability of cells to maintain optimal turgor pressure and intracellular ionic concentration for growth and functioning. The nitrogen-fixing soil actinobacterium Frankia shows marked variability in its tolerance to salinity. When in a symbiotic association with actinorhizal plants, Frankia enhances the tolerance of the plants to a range of abiotic stresses, including salinity. The Casuarina-Frankia association has been used to reclaim salt …

The Rate, Spectrum And Effects Of Spontaneous Mutation In Bacteria With Multiple Chromosomes, Marcus M. Dillon

Doctoral Dissertations

Despite their essentiality for evolutionary change and role in many diseases, spontaneous mutations remain understudied because of both biological and technical barriers. Prokaryotic mutation biases are especially understudied and no studies have been conducted on bacteria with multiple chromosomes, leaving major gaps in our understanding of the role of genome content and structure on mutation. The application of mutation accumulation lines to whole-genome sequencing offers the opportunity to study spontaneous mutations in a wide range of prokaryotic organisms. Here, we present a genome-wide view of molecular mutation rates and spectra in Burkholderia cenocepacia, Vibrio fischeri, and Vibrio cholerae, three bacterial …

Causes And Consequences Of Diversity Within Experimental Biofilms Of Pseudomonas Aeruginosa, Kenneth Mark Flynn

Doctoral Dissertations

Currently, we do not understand how much biodiversity may be maintained in any environment, especially not in the structured environments of biofilms, which constitute the dominant mode of microbial life. Although maintenance is associated with the complex spatial structure of biofilm architecture and resulting ecological opportunities, how both the abiotic and biotic environment defines available niches remains poorly understood. Here, we experimentally evolved three replicate populations founded by Pseudomonas aeruginosa strain PA14 for 540 generations under conditions favoring a regular cycle of biofilm formation and dispersal. Utilizing a variety of laboratory and genomic sequencing approaches, we characterize the population genetic …

Interactions Of Shiga-Like Toxin-2 (Stx-2) From Escherichia Coli O157:H7 And The Bcl-2 Family Of Proteins During Host Cell Programmed Cell Death, Lia K. Jeffrey

Doctoral Dissertations

Stx-2 is a major contributor to the pathogenesis of Escherichia coli 0157:H7. Prior reports suggest that Stx-2 increases necrosis and apoptosis of a variety of host cells including those of endothelial origin as well as immune cells such as neutrophils (156). However, the role Stx-2 plays in delayed apoptosis of neutrophils is not fully understood given that previous studies have shown conflicting results (118, 51). The process of apoptosis is mediated by the Bcl-2 protein family (2, 46, 226). The purpose of this research was to define the molecular mechanisms of Stx-2 and Bcl-2 protein family interactions. These studies examined …

Characterization Of The Prokaryotic Community Associated With The Giant Barrel Sponge, Xestospongia Muta Across The Caribbean, Cara L. Fiore

Doctoral Dissertations

Sponges have long been known to be ecologically important members of the benthic fauna on coral reefs. Recently, it has been shown that sponges, and their symbiotic microbes, are also important contributors to the nitrogen biogeochemistry of coral reefs. Here, I investigate the ecology and physiology of the microbial community associated the ecologically dominant sponge, Xestospongia muta. A natural experiment was conducted with X. muta form three different locations (Florida Keys, USA; Lee Stocking Island, Bahamas, and Little Cayman, Cayman Islands) to compare nitrogen cycling and prokaryotic community composition. The dissolved inorganic nitrogen (DIN) fluxes of sponges were studied using …

Calcium Homeostasis In Escherichia Coli, Muhammad Arif

Doctoral Dissertations

While the role of calcium ions as secondary chemical messengers has been well described in eukaryotic cells, little is known about calcium homeostasis in bacteria at the physiological and molecular level. No calcium influx gene has been identified so far. This dissertation focuses on calcium regulation of gene expression by (i) determining the effects of elevated and depleted calcium levels on global gene expression in wild-type cells (MG1655), (ii) employing transposon mutants (calcium-sensitive) and mutants that are defective in calcium transport, and (iii) performing quantitative analysis of a specific subset of 15 genes to elucidate their possible involvement in calcium …

Effects Of Nitrogen-Fixing Symbiotic Cyanobacteria On The Microbial Ecology Of The Coral, Montastraea Cavernosa, Jessica K. Jarett

Doctoral Dissertations

Corals form the physical structure of coral reefs, one of the most ecologically and economically important ecosystems in the world. The abundant and broadly distributed Caribbean coral Montastraea cavernosa forms a symbiosis with intracellular nitrogen-fixing cyanobacteria in some, but not all colonies, which make up approximately 30% of the population and display a characteristic orange fluorescence. Diverse and functionally important microbial communities of dinoflagellates, bacteria, Archaea, viruses, fungi, and other organisms are also associated with corals and together with the host compose what is termed the coral holobiont. Whether the cyanobacteria are mutualists, commensals, or parasites, and their effects on …

Character Displacement In Burkholderia Biofilm Communities And The Effect Of Adaptive History On The Evolution Of Niche Breadth, Crystal Nicole Ellis

Doctoral Dissertations

Ecologists agree that biodiversity is essential for maintaining highly productive and stable ecosystems, yet the mechanisms that generate and preserve diversity are unknown in many habitats. Competition for shared resources may cause selection to favor niche specialization, which reduces competition and reinforces diversity. If the environment is significantly altered, the ecological history of the remaining colonizer may influence fitness and capacity for diversification in new environments. In many cases, specialists have limited adaptive potential due to trade-offs generated by niche-specific adaptation; however, generalists may be adaptable in many habitats, which would make them good pioneer species for colonization. We investigated …

Existing Regulatory Circuitries Govern Backbone And Acquired Host Association Factors In The Human Pathogen Vibrio Parahaemolyticus, Jennifer C. Mahoney

Doctoral Dissertations

Vibrio parahaemolyticus is a poorly characterized human gastrointestinal pathogen whose virulence mechanisms are not well understood. Though closely related to Vibrio cholerae, V. parahaemolyticus infections are inflammatory and utilize virulence traits that are unique from the Cholera toxins yet remain poorly characterized. Vibrio spp. in general share an extensive core genome dedicated to environmental survival and unique, often horizontally acquired, gene content that is reserved for species specific lifestyles. This diversity has resulted in a genus of highly specialized bacteria partaking in dramatically different lifestyles ranging from symbiosis to pathogenesis. We propose that a comparative genomic and transcriptomic analysis of …

Experimental Evolution Of Burkholderia Cenocepacia Biofilm Populations, Steffen R. Poltak

Doctoral Dissertations

Many biofilm populations are known for their exceptional biodiversity but the relative contributions of the forces that could promote this diversity are poorly understood. This uncertainty pervades in the well-established communities found on many natural surfaces and in long-term, chronic infections. Here we describe the parallel evolution of cooperative communities derived from a clone of Burkholderia cenocepacia during approximately 1500 generations of biofilm selection. This long-term evolution was enabled by a new experimental method that selects for daily cycles of colonization, biofilm assembly, and dispersal. Each of six replicate biofilm populations underwent a common pattern of adaptive morphological diversification, in …

Factors Regulating The Production Of Stx-2 In Escherichia Coli O157:H7, Kate P. Stefani

Doctoral Dissertations

The severity of Escherichia coli O157:H7 disease is due in part to a major virulence factor produced by the microbe, the shiga-like toxin 2 (Stx-2). Antibiotic treatment to reduce pathogen numbers is controversial, as it is thought that antibiotics may increase the levels of Stx-2 released from the pathogen. Currently, recommended treatment for E. coli O157:H7 is palliative The purpose of this study was to examine three critical factors potentially important to disease outcomes, and to determine their effect on expression of the stx2 gene and on release of Stx-2 from the pathogen. Those factors selected for study were: (i) …

Evaluation Of An Enhanced Magneto-Chemical Process For The Removal Of Pathogens In Wastewater, Christine N. Wright

Doctoral Dissertations

New wastewater treatment technologies are a necessity as a result of increasingly stringent discharge standards, particularly concerning viruses. The traditional approach to wastewater treatment consists of biological treatment, such as trickling filters, biofilms, and activated sludge. Although these biological processes have been an adequate means of wastewater treatment, they are intrinsically limited by their biological nature. A magneto-chemical process, known as CoMag(TM), has been developed to improve coagulation and solids separation and expand upon conventional wastewater treatment techniques.

The research objective of this project was to develop and evaluate a bench-scale model of the CoMag(TM) process for the removal of …

Role Of The Biofilm In Metals Accumulation By Fucoid Algae And Their Surrounding Rock Surfaces, Joan Lydia Beskenis

Doctoral Dissertations

Both inanimate and animate intertidal surfaces including Fucus and Ascophyllum develop a biofilm that can bind metals contributed by non-point and point sources of pollution. The biofilm also functions as a habitat for the many organisms, i.e. bacteria, diatoms and other algal cells that are part of the biofilm and contribute to the polysaccharide matrix. Examinations of the biofilm were quantified by polysaccharide analysis using a spectrophotometer. Biofilm samples were recovered from both intertidal rock surfaces and the surface of the surrounding fucoid algae to determine if they bind Cu and Zn. Locations in Kittery, Maine differed by the amount …

The Development Of Tools To Allow Genetic And Genomic Analysis Of Frankia, Tania Rawnsley Spenlinhauer

Doctoral Dissertations

Frankia, a nitrogen-fixing actinomycete, forms a symbiotic association with a variety of woody dicotyledonous plants. The lack of standard genetic tools for this important bacterium has hindered studies on the molecular biology of this symbiosis. We are interested in developing tools for the genetic analysis of Frankia physiology and its interactions with its host plants. Our approach has focused on the development of a physical and genetic map of the Frankia chromosome by macrorestriction analysis. Agarose-embedded chromosomal DNA plugs were prepared from three Frankia isolates: Eul1c, EAN1pec and Ccl3. The genome sizes of the three strains were determined by pulse-field …

Genetics And Physiology Of Motility By Photorhabdus Spp, Brandye A. Michaels

Doctoral Dissertations

Photorhabdus is a bacterial symbiont of soil nematodes and a lethal pathogen of insects. Many pathogenic or symbiotic bacteria utilize various methods of motility to reach favorable conditions, colonize a host, or have motility genes that also regulate virulence expression. It is not known how motility is regulated, or how it may confer an advantage, in the complex life cycle of Photorhabdus..

We characterized motility in Photorhabdus and found that the bacterium was motile both by swimming (movement in liquid) and swarming (movement on surfaces) under appropriate conditions. Both types of motility utilized the same peritrichous flagella and shared genetic …

Evaluation Of Bacteriophage And Viral Persistence During Alkaline Stabilization In Sludge And Biosolids Intended For Land Application, Jacqueline Joy Brabants

Doctoral Dissertations

The use of lime to reduce or eliminate pathogen content is a cost-effective treatment method currently being employed in many Class B biosolids production plants in the United States. A bench scale model of lime stabilization was designed to evaluate pathogen persistence. The survivability of poliovirus type 1, fecal coliforms, Salmonella, adenovirus type 5, rotavirus Wa, and the male-specific bacteriophage MS-2 was evaluated under lime stabilization conditions in various matrices. Salmonella and fecal coliforms were evaluated at 28°C and poliovirus and MS-2 were evaluated at both 28°C and 4°C for survivability under lime stabilization conditions in a sludge matrix. All …

Assessment Of The Fate Of Cryptosporidium Parvum, Giardia Lamblia And Ascaris Lumbricoides In Class A Composting And Class B Lime Stabilization Biosolids Treatment Processes, Christine L. Bean

Doctoral Dissertations

The management of biosolids in the United States has become an issue of public health concern in regard to disposal. Congress created the Clean Water Act in 1972 to protect waters in the United States and as a result, no longer can human waste be dumped into oceans or rivers. Means of disposal of biosolids are focused on land application today. The practice of land application takes into consideration the classification of the biosolids material and management practices to control access to and growth of crops on the land. Biosolids are classified as either Class A or Class B depending …

Characterization Of Prokaryotic Diversity In A Chlorinated Solvent-Contaminated Bedrock Aquifer Using Denaturing Gradient Gel Electrophoresis, Ribosomal Dna Sequencing, And Real Time Pcr, Walid Naser

Doctoral Dissertations

A culture-independent molecular phylogenetic approach was implemented to investigate spatial and temporal variations in microbial diversity and abundance in a chlorinated solvent-contaminated bedrock aquifer. Core and groundwater samples were collected from several boreholes and included the following samples: (1) groundwater that was associated with the core, (2) open fractures and (3) partially mineralized sealed fractures (PMSF) exposed by striking the core with a surface-sterilized geology hammer. DNA representing the attached and unattached microbial communities was extracted from these samples, and the 16S rRNA gene was amplified with primers specific for Bacteria, Archaea, sulfate reducers, Geobacteraceae family, Dehalorespirers (Dehalococcoides sp. and …

Ompd But Not Ompc Is Involved In Adherence Of Salmonella Enterica Serovar Typhimurium To Human Cells, Bochiwe Hlezipi Hara-Kaonga

Doctoral Dissertations

Salmonella enterica serovar Typhimurium expresses three major outer membrane proteins: OmpC, OmpF, and OmpD. Conflicting reports exist regarding the role of OmpC and OmpD in the pathogenesis of this organism. This study investigated the role of OmpC and OmpD in adherence to human U937 macrophages and T84 intestinal epithelial cells. OmpC and ompD mutant strains were created by transposon mutagenesis utilizing P22-mediated transduction of Tn10 and Tn 5 insertions into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with PKH26-labeled T84 cells at various bacteria-to-cell ratios for 1 h at 37 C. Single and dual fluorescence of samples …

The Effect Of Freeze /Thaw Temperature Fluctuations On Microbial Metabolism Of Petroleum Hydrocarbon Contaminated Antarctic Soil, Conrad George Leszkiewicz

Doctoral Dissertations

Petroleum contaminated soil exists at McMurdo Station in Antarctica. These soils were contaminated with historic releases of JP-8 jet fuel. Over time, there does not appear to have been significant reduction in the petroleum concentrations in these soils. This lack of reduction has been attributed to the extremely cold Antarctic environment and the lack of available moisture.

Cold temperatures and/or lack of moisture may not be the factors inhibiting biodegradation. Soil temperatures can exceed 20 degrees centigrade (°C) during the austral summer and melt water becomes available. However, the soil temperatures have also been reported to fluctuate rapidly. Swings of …

The Physiological Role And Characterization Of Melanin Produced By Shewanella Algae Bry, Charles Edwin Turick

Doctoral Dissertations

Shewanella algae BrY is a Gram negative, facultative anaerobe isolated from the Great Bay Estuary of New Hampshire. This member of the gamma Proteobacteria demonstrates extensive versatility in the variety of terminal electron acceptors it uses for anaerobic respiration. Since the natural habitat of this non-fermenting organism is the oxiclanoxic interface of sediments, the ability to utilize various terminal electron acceptors contributes to its survival and growth. Among the terminal electron acceptors used by S. algae BrY are insoluble iron minerals. The transfer of electrons from a bacterial cell to an insoluble compound presents a strategic challenge for this organism. …

Detection Of Giardia Lamblia And Cryptosporidium Parvum In The Eastern Oyster (Crassostrea Virginica) Harvested From The Great Bay Estuary, Kim Marie Stowell

Doctoral Dissertations

The Great Bay Estuarine system is being impacted by point source and nonpoint source pollution that affect shellfish quality. Many outbreaks of human gastroenteritis are potentially caused by the consumption of raw or undercooked oysters in which the protozoan pathogens, Giardia lamblia and Cryptosporidium parvum are present. The development of a rapid and sensitive technique to detect infectious protozoa in oysters is a necessity.

Immunofluorescence has been employed as a technique for the detection of these organisms in drinking water. Cross reactivity with algae and the production of false positive results limits the usefulness of this technique in environmental studies. …

Characterization Of The Interactions Within The Ccr4 -Not Complex And The Interactions Between Adr1 And Tfiid Components, Yongli Bai

Doctoral Dissertations

The mechanisms of transcriptional regulation are well conserved from yeast to human cells. Most genes are regulated at the transcriptional level, particularly, at the transcription initiation step. In Saccharomyces cerevisiae at least one transcriptional activator, ADR1, and one transcription complex, CCR4-NOT, participate in the activation of the ADH2 gene. The core 1 x 106 dalton CCR4-NOT complex consists of CCR4, CAF1, the five NOT proteins (NOT1-NOT5), and three other uncharacterized proteins. Deletion of NOT3, NOT4, or NOT5 did not affect the association of CCR4 with CAF1 in the gel filtration analysis. In contrast, NOT2 inactivation caused partial destabilization of the …

The Role Of Cytokines In The Pathogenesis Of Experimental Legionella Pneumophila Infections, Corinna Mary Krinos

Doctoral Dissertations

Legionnaires' disease is an acute lobar pneumonia caused, primarily by the facultative intracellular pathogen Legionella pneumophila. This organism when inhaled by humans descends into the lower respiratory tract and parasitizes alveolar macrophages. L. pneumophila adhered to U-937 cells, A549 cells and peritoneal macrophages from A/J mice in an opsonin-independent fashion. Following attachment, the organism penetrated the cell membrane, replicated within these cells eventually inducing lysis. To better define the adhesion of L. pneumophila to host cells, an E. coli clone (LP 116), expressing the 25 kDa major outer membrane protein (MOMP) of L. pneumophila was used in binding studies. This …

The Development Of Rna Probe And Rt-Pcr Assays For The Detection Of Enteroviruses In Sludge, Amy Elisabeth Moore

Doctoral Dissertations

Many wastewater treatment plants generate more sludge than can be disposed of by conventional means. The United States Environmental Protection Agency (USEPA) has encouraged communities to dispose of sludge by land application. Sludge may contain enteric viruses that are known to survive for long periods of time in sludge-amended soil and can travel great distances, potentially contaminating surface and ground water.

Standard cell culture methods for the detection of enteric viruses are costly and results are not obtained for 30 or more days. The development of methods that provide results more quickly and with lower cost are needed.

A 32P …

Alternative Removal Methodologies For Environmental Waters, Shannon Patrick Hogan

Doctoral Dissertations

The Environmental Protection Agency (EPA) continues to establish criteria for the reduction of pathogens in environmental waters used for drinking water. The EPA also states criteria on the amount of chlorine by-products allowed in drinking water. Alternative removal methods that are used in conjunction with chlorine are needed to assure safe drinking water.

The Surface Water Treatment Rule (SWTR) states that a 99.99% reduction of viruses and a 99.9% reduction in Giardia lamblia cysts needs to be achieved by a treatment facility that uses surface water for the production of drinking water. The SWTR also states Maximum Contaminant Levels (MCL) …

Attachment Of Listeria Monocytogenes To Murine And Human Phagocytic Cells, Srinivas Maganti

Doctoral Dissertations

Listeria monocytogenes is a facultative intracellular pathogen responsible for meningoencephalitis, septicemia, and abortion in susceptible and immunocompromised individuals. Subsequent to colonization and penetration of the gut epithelium, the organism attaches to resident macrophages and replicates intracellularly, thus evading the humoral immune system of the infected host. The mechanism surrounding the precise interactions between L. monocytogenes and macrophages employs bacterial ligands and macrophage receptors which are highly complex and not well understood. The focus of the present study was to investigate the attachment of the organism to murine and human macrophages under opsonin-dependent and opsonin-independent conditions to better understand the pathobiology …

Molecular Characterization Of A Cytotoxic Porin Protein From Campylobacter Jejuni And Its Role In Campylobacteriosis, David John Bacon

Doctoral Dissertations

Campylobacter jejuni is a major cause of enteritis in developed and developing countries. A cytotoxic complex was isolated from organism-free filtrates of broth grown organisms by screening for activity in HEp-2 cells following high performance liquid chromatography using a combination of size exclusion and DEAE column chromatography. The toxin was found to coincide with a 45 kDa protein possessing an N-terminus sequence indicative of a bacterial outer membrane porin protein together with a high molecular weight carbohydrate which was determined to be lipopolysaccharide (LPS).

The carbohydrate portion of the LPS had reactivity for the lectins Galanthus nivalis agglutinin, Maakia amurensis …