Life Sciences Commons^™

Quantitative Characterization Of Proteins And Post-Translational Modifications In Complex Proteomes Using High-Resolution Mass Spectrometry-Based Proteomics, Zhou Li

Doctoral Dissertations

Mass spectrometry-based proteomics is focused on identifying the entire suite of proteins and their post-translational modifications (PTMs) in a cell, organism, or community. In particular, quantitative proteomics measures abundance changes of thousands of proteins among multiple samples and provides network-level insight into how biological systems respond to environmental perturbations. Various quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling. This dissertation starts with systematic comparison of these three methods, and shows that isobaric chemical labeling provides accurate, precise, and reproducible quantification for thousands of proteins. Based on these results, we applied this approach to characterizing …

Urea As A Nitrogen Source For Microcystis Aeruginosa, Bernard Shafer Belisle

Masters Theses

Over the last decade, Lake Erie has experienced annual harmful algal blooms events dominated by the toxic cyanobacterium Microcystis aeruginosa. It is still unclear what causes Microcystis blooms to occur, but there is broad agreement that eutrophication of freshwater systems from anthropogenic sources (urban, industrial, etc.), has led to their proliferation. In particular, the organic compound urea has been implicated as an important source of anthropogenic nitrogen, due to its increased use in agricultural practices. Currently, urea constitutes more than 50% of the nitrogen used for agricultural fertilizer globally, and its usage has increased more than a 100-fold over …

Wood Decomposition In A Warmer World, Emily Elizabeth Austin

Doctoral Dissertations

Climatic warming is altering species distributions and ecosystem functions across the globe. Wood is an important carbon pool and the fungal communities in wood are relatively simple compared to those in soil. These factors make decomposing wood an ideal system for exploring the influence of decomposer community on the response of decomposition to warming. My research has focused on the effects of warming wood decomposition rates and wood decomposing communities. Using field and lab- based manipulative experiments and field observations I explore the influence of tree species, wood decomposition stage, geography and warming on fungal community structure and activity. In …

The Performance Of Bacterial Phytosensing Transgenic Tobacco Under Field Conditions, Michael Harrison Fethe

Masters Theses

Currently the platforms for wide-area detection of environmental contamination are limited. Therefore, there is interest in developing new platforms, especially for use in crop plants to detect and report the presence of biotic and abiotic stress agents. A biosensor uses a biological organism or substrate to detect the presence of an elicitor (i.e., heavy metal, TNT, or bacteria). The foundational groundwork to create biosensors in transgenic plants exists. The creation of bacterial phytosensing transgenic tobacco containing an orange fluorescent protein (OFP) reporter driven by synthetic pathogen-inducible promoters provides a fluorescent signal when infected with phytopathogens for earlier detection in the …

Environmental Constraints On Cyanomyophage Abundance In The Subtropical Pacific Ocean, Tiana Maria Pimentel

Masters Theses

Viruses are abundant in the world’s oceans and are thought to be important participants in marine biogeochemical cycling. Of these viruses, cyanophages are considered especially important because they infect and lyse cyanobacteria, which are some of the main primary producers in marine environments. Cyanophages are thought to influence the abundance and diversity of cyanobacterial populations and impart significant mortality, thereby affecting primary productivity and microbial community structure. Despite their ecological relevance, little is known about how environmental factors shape cyanophage abundance and diversity over large temporal and spatial scales. To address this gap in knowledge, seawater samples were collected during …

Genetic And Ecological Characterization Of Indigoidine Production By Phaeobacter Sp. Strain Y4i, William Nathan Cude

Doctoral Dissertations

The Roseobacter clade is a widely distributed, abundant, and biogeochemically active lineage of marine alpha-proteobacteria. Members of the Roseobacter lineage are prolific surface colonizers in marine coastal environments, and antimicrobial secondary metabolite production has been hypothesized to provide a competitive advantage in colonization. In this work, Phaeobacter sp. strain Y4I was found to produce the water soluble, blue pigment indigoidine via a nonribosomal peptide synthase-based biosynthetic pathway encoded by a novel series of genetically linked genes, termed igiBCDFE. Comparison of wildtype, non-pigmented, and hyper-pigmented Y4I insertional mutants demonstrated a perfect correlation between indigoidine production and the inhibition of Vibrio …

Ecology And Physiology Of Aerobic Aromatic Catabolism In Roseobacters, Christopher Adam Gulvik

Doctoral Dissertations

Roseobacters are an abundant and trophically versatile lineage of marine bacteria that are especially dominant in coastal salt marshes. Central to understanding of how members of the Roseobacter clade contribute to biogeochemical cycling in the world’s oceans is how these bacteria physiologically respond to mixtures of usable growth substrates present in their environmental niches. A prior study provided evidence that bacterioplankton transcripts most closely related to the Roseobacter Sagitulla stellata E-37 are among the most abundant in coastal systems for biogeochemically significant processes of N, P, and S cycling. Thus, this strain was used throughout this dissertation as an environmentally …

Genetic Analysis Of Bacterial Gene Variations In Sulfidic Springs And The Influence On Geochemistry, Brendan Joseph Headd

Doctoral Dissertations

Culture-independent methods have revolutionized environmental microbiology and geomicrobiology studies and next-generation sequencing and metagenomics techniques continue to reveal the vast genetic diversity of microorganisms. But, these approaches provide comparatively little quantitative information about the roles that naturally occurring microbial gene variations play in local biogeochemical cycling. The goal of this study was to identify how the physical distribution and genetic diversity of microbial genes within a habitat impact environmental geochemistry by examining the biogeography of 16S rRNA genes and bacterial sulfur oxidation (Sox) genes in terrestrial sulfidic springs. 16S rRNA gene pyrosequences were obtained from microbial mats inhabiting eight sulfidic …

Validating Approaches For Studying Microbial Diversity To Characterize Communities From Roots Of Populus Deltoides, Migun Shakya

Doctoral Dissertations

Microbial (archaeal, bacterial, and fungal) communities associated with plant roots are central to its health, survival, and growth. However, a robust understanding of root microbiota and the factors that govern their community structure and dynamics have remained elusive, especially in mature perennial plants from natural settings. Although the advent of Next Generation Sequencing (NGS) technologies have changed the scale of microbial ecological studies by enabling exhaustive characterization of microbial communities, the accuracy of taxonomic and quantitative inferences are affected by multiple experimental and computational steps and lack of knowledge of the true ecological diversity. To test for inaccuracies and biases, …

Factors Influencing Diarrheal Pathogen Presence In Tubewells Of Bangladesh, Kati Anne Ayers

Masters Theses

Diarrheal disease pathogens remain a major concern in developing countries as rotavirus is the leading cause of hospitalization of young children worldwide. A recent study has shown shallow groundwater in rural Bangladesh to be contaminated with bacterial and viral pathogens, but found no correlation between rotavirus and any fecal indicator or environmental parameter during the monsoon season of July, 2009. The objectives of this thesis were to examine the non-relationship between pathogens and fecal indicators, as well as to improve the understanding of the seasonal transport of viral pathogens, especially rotavirus, in shallow, sandy aquifers of Bangladesh. This was achieved …

Quorum Sensing And Metabolism In Marine Environments, Amanda May

Doctoral Dissertations

Quorum sensing (QS) is a phenomenon that allows bacteria to communicate with each other. Small molecules known as autoinducers are synthesized and released by bacteria, and once enough members of the community are around to ensure survival, i.e. quorum, a phenotype, e.g. bioluminescence, is expressed. There are two types of QS molecules, intra- and inter-species.

S-4,5-Dihydroxy-2,3-pentanedione (DPD) is a byproduct of the activated methyl cycle which recycles methionine. This has led to the discussion as to whether DPD is a metabolic byproduct or is the interspecies signal as proposed previously. The detection and quantitation of DPD however, has not …

Long Term Impacts Of A Genetically Engineered Microorganism (Gem) And Polycyclic Aromatic Hydrocarbons (Pahs) On Soil Bacterial Communities, Xiaoci Ji

Masters Theses

Microbes capable of polyaromatic hydrocarbon (PAH) biodegradation can be used to remediate soils contaminated with these persistent pollutants. To monitor in situ PAH-biodegradation, the bioluminescent bio-reporter Pseudomonas fluorescens HK44, containing a lux luminescent gene cassette inserted into its naphthalene degradation operon, was released into PAH-contaminated soil in lysimeters in 1996. Three treatments were imposed: strain HK44 mixed with PAH-contaminated soil (PAH+, HK44+; n=3); strain HK44 mixed with uncontaminated soil (PAH–, HK44+; n=2) and PAH-contaminated soil alone (PAH+, HK44–; n=1). The objective of this study was to assess the long term impacts of these treatments on the indigenous soil bacterial community …

Soil Microbial Community Succession During Cadaver Decomposition, Kelly Lynn Cobaugh

Masters Theses

Microbes play critical roles in nutrient cycling in terrestrial ecosystems. In particular, microbial decomposition of organic matter is a key step in carbon and nutrient cycling, linking above-ground and below-ground pools. It is well known that the microbial community changes in structure and function following the introduction of organic matter into a terrestrial system. The decomposition of plant litter has been extensively investigated but the decomposition of animal-derived organic matter has often been overlooked. The unique characteristics of animal input are hypothesized to dictate a distinct decomposition process. This study examined the microbial community responsible for decomposition of animal-derived organic …

Revealing The Ecological Role Of Gemmatimonadetes Through Cultivation And Molecular Analysis Of Agricultural Soils, Mariam Naomi Fawaz

Masters Theses

Bacteria belonging to phylum Gemmatimonadetes are frequently detected in a variety of environments using culture-independent methods. Despite their ubiquity and prevalence, almost nothing is known about their physiology or ecology because so few strains have been isolated. The first objective of this study was to determine the distribution of Gemmatimonadetes within soil aggregates and the response of the relative abundance of Gemmatimonadetes to dry/wet cycling and soil management. The second objective was to analyze the effects of soil management, aggregate size, and atmospheric conditions on cultivability of Gemmatimonadetes. Universal and Gemmatimonadetes-specific 16S rRNA gene primers were used to …

Exploring Anaerobic Reductive Dechlorination At Low Ph Environments, Yi Yang

Masters Theses

Chlorinated aliphatic hydrocarbons (CAHs), such as tetrachloroethene (PCE) and trichloroethene (TCE), are ubiquitously pollutants in aquifer sediments and groundwater due to their heavy usage in industry and inappropriate disposal in the last century. Among about 1300 NPL (National Priorities List) sites, PCE and TCE are the two most frequently detected hazardous contaminants.

Engineered bioremediation, including biostimulation and bioaugmentation, is a promising technology to clean those PCE and/or TCE contaminated sites. However, in many contaminated groundwater systems and hazardous waste sites, pH can be lower than 5 to 6. And release of HCl (strong acid) from anaerobic reductive dechlorination may lower …

Sulfate Reducing Communities In Aquifer Systems Can Be Reliably Stimulated By Addition Of Complex Nutrients, Matthew Bryan Scholz

Doctoral Dissertations

The disseration presented below is the summation of research into the potential roles of microbial communities associated with aquifers of Bangladesh contaminated with naturally occuring arsenic. These investigations also included experimental microcosm experiments to assess the role of nutrients supplementation of complex carbon sources (molasses), and inorganic sulfate (MgSO4), on both the solubility of arsenic to determine the feasibility of this method for the goal of performing in situ bioremediation. Community structure and functional gene profiling was performed on all samples, as well as detection of community shifts following amendments predicted to encourage the growth of sulfate reducting microorganisms (SRM). …

Diversity And Activity Of Roseobacters And Roseophage, Charles Ryan Budinoff

Doctoral Dissertations

Bacteria of the Roseobacter lineage are dominant bacterioplankton in coastal systems and contribute significantly to secondary production in oceanic environments. Generalities of Roseobacter ecology, diversity, and distributions are known, but the intraspecific differences between species and their dynamics over short temporal periods is not well understood. Bacteriophage that infect Roseobacters (‘roseophage’) have the potential to shunt secondary production into the dissolved carbon pool and through the process of infection alter Roseobacter physiology. Despite their significance, little effort was made prior to the onset of this study to characterize roseophage. Using culture dependent and independent approaches, I describe the diversity and …

The Effects Of Nutrient Limitation And Cyanophage On Heterotrophic Microbial Diversity, Claire Elyse Campbell

Masters Theses

Marine viruses are critically important in the regulation of biogeochemical cycles and host microbial communities. In this study, we tested whether the indirect effects of virus predation on a phototroph (i.e., Synechococcus) affected the composition of co-occurring heterotrophic bacteria under nitrogen and phosphorus limitation in long-term chemostat experiments. Using 454 Titanium barcoded pyrosequencing of the 16S rRNA gene, microbial diversity and technical (i.e., sequencing) reproducibility were assessed for nine individual chemostats across five different time points. A total of 325,142 reads were obtained; 194,778 high-quality, non-cyanobacterial sequences were assigned to 110 OTUs. Our results show high reproducibility …

Constraints On Primary Production In Lake Erie, Matthew Alan Saxton

Doctoral Dissertations

The Laurentian Great Lake, Lake Erie is an invaluable global resource and its watershed is home to over 11 million people. The pressures placed on the lake because of this high population caused Lake Erie to experience numerous environmental problems, including seasonal hypoxia and harmful algal blooms. While these topics have been widely studied in Lake Erie for over 40 years a more nuanced understanding of the interaction between phytoplankton and nutrient is needed to properly address the problems continuing to face the lake. In this study we combine classical limnological and cell growth experiments with modern molecular biological techniques …

Genomic And Molecular Analysis Of The Exopolysaccharide Production In The Bacterium Thauera Aminoaromatica Mz1t, Ke Jiang

Doctoral Dissertations

Thauera aminoaromatica MZ1T is an exopolysaccharide (EPS)-producing Gram negative bacterium isolated from the wastewater treatment plant of a major industrial chemical manufacturer as the causal agent for poor sludge dewatering. It shares common features with other known Thauera spp. (i.e. Thauera aromatica, and Thauera selenatis), being capable of degrading aromatic compounds anaerobically and using acetate and succinate as carbon sources. It is unique among the Thauera spp. in its production of abundant EPS which results in viscous bulking and poor sludge dewaterability. In this respect, it is similar to Azoarcus sp. EbN1 and BH72. Thaueran is the proposed …

The ‘Helper’ Phenotype: A Symbiotic Interaction Between Prochlorococcus And Hydrogen Peroxide Scavenging Microorganisms, James Jeffrey Morris

Doctoral Dissertations

The unicellular cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism throughout the temperate and tropical open oceans, but it is difficult to grow in pure cultures. We developed a system for rendering spontaneous streptomycin-resistant mutants of Prochlorococcus axenic by diluting them to extinction in the presence of “helper” heterotrophic bacteria, allowing them to grow to high cell concentrations, and then killing the helpers with streptomycin. Using axenic strains obtained in this fashion, we demonstrated that Prochlorococcus experiences a number of growth defects in dilute axenic culture, including reduced growth rate, inability to form colonies on solid media, and higher incidence …

Quantification And Ecological Perspectives On Cyanophage And Aquatic Viruses, Audrey Renee Matteson

Doctoral Dissertations

The field of viral ecology is still relatively new and many processes by which viruses influence hosts are still widely unknown. One problem is that there are few standardized techniques in virus ecology, making comparisons of data very difficult. To better understand the methodology, we first set out to make a video showing the process for the viral production assay using the “dilution and reoccurrence” method, which has become the standard to analyze production rates in aquatic ecosystems. Using this method, we also determined the production rates of viruses during a seasonal pelagic phytoplankton bloom during a cruise off the …

Functional Cloning And Characterization Of Antibiotic Resistance Genes From The Chicken Gut Microflora, Wei Zhou

Masters Theses

A recent study using human fecal samples in conjunction with a culture-independent approach revealed immense diversity of antibiotic resistance (AR) genes in the human gut microflora. We hypothesize that food animal gut microflora also contain diverse and novel AR genes which could contribute to the emergence and transmission of AR in pathogens important in animal and human health. To test this, we examined AR reservoir in chicken gut microflora using a metagenomic, functional cloning method. Total genomic DNA was extracted from individual cecal contents of two free range chickens and two conventionally raised chickens. The DNAs were physically sheered into …

Characterization Of The Extracellular Proteome Of A Natural Microbial Community With An Integrated Mass Spectrometric / Bioinformatic Approach, Brian Keith Erickson

Doctoral Dissertations

Proteomics comprises the identification and characterization of the complete suite of expressed proteins in a given cell, organism or community. The coupling of high performance liquid chromatography (LC) with high throughput mass spectrometry (MS) has provided the foundation for current proteomic progression. The transition from proteomic analysis of a single cultivated microbe to that of natural microbial assemblages has required significant advancement in technology and has provided greater biological understanding of microbial community diversity and function.

To enhance the capabilities of a mass spectrometric based proteomic analysis, an integrated approach combining bioinformatics with analytical preparations and experimental data collection was …

Mechanisms Of Antimicrobial Peptide Resistance In Campylobacter, Ky Van Hoang

Doctoral Dissertations

Campylobacter is the major bacterial cause of human gastroenteritis in the United States and other developed countries. Poultry are considered a main source of human Campylobacter infections. Thus, reduction of Campylobacter load in poultry is significant in food safety and public health. However, no effective measure is commercially available to prevent Campylobacter colonization in poultry to date. Antimicrobial peptides (AMPs) are short and bactericidal peptides widely present in intestine to limit bacterial infections. Recently, AMPs have been increasingly recognized as a novel class of antibiotics (peptide antibiotics) to control foodborne pathogens. Notably, several potent anti-Campylobacter bacteriocins, a group of AMPs …

An Analysis Of Global Gene Expression Resulting From Exposure To Energetic Materials, Vernon L. Mcintosh Jr.

Doctoral Dissertations

AN ANALYSIS OF GLOBAL GENE EXPRESSION RESULTING FROM EXPOSURE TO ENERGETIC MATERIALS

A Dissertation

Presented for the Doctor of Philosophy Degree

University of Tennessee, Knoxville

VERNON LASHAWN MCINTOSH JR.

August 2010

Dedication

This dissertation is dedicated to my family. My mother and father Debra and Vernon McIntosh instilled in me the respect for academic excellence and the drive maximize my potential. Early on, my younger brother Kyle started showing signs of a shared interest in biology thus my desire to be a positive role model for him kept me motivated. Last but certainly not least, my loving wife and best …

Cattle Access Affects Periphyton Community Structure In Tennessee Farm Ponds., Robert Gerald Middleton

Masters Theses

Cattle farming is vital to the economy of the United States. Frequently, cattle are given access to ponds and streams for water. The relative impacts of cattle access in natural water sources on the periphyton community have been rarely investigated. Periphyton is the basis of the aquatic food web, and community composition can serve as a bioindicator of pollution. Thus, my objectives were to quantify the effects of cattle access in aquatic lentic systems on periphyton community structure and biovolume, identify taxa that were associated with cattle access, and identify abiotic mechanisms that might be driving assemblage changes. I conducted …

Use Of Proteomics Tools To Investigate Protein Expression In Azospirillum Brasilense, Gurusahai K. Khalsa-Moyers

Doctoral Dissertations

Mass spectrometry based proteomics has emerged as a powerful methodology for investigating protein expression. “Bottom up” techniques in which proteins are first digested, and resulting peptides separated via multi-dimensional chromatography then analyzed via mass spectrometry provide a wide depth of coverage of expressed proteomes. This technique has been successfully and extensively used to survey protein expression (expression proteomics) and also to investigate proteins and their associated interacting partners in order to ascertain function of unknown proteins (functional proteomics). Azospirillum brasilense is a free-living diazotrophic soil bacteria, with world-wide significance as a plant-growth promoting bacteria. Living within the rhizosphere of cereal …

Agricultural Soil Bacteria; A Study Of Collection, Cultivation, And Lysogeny, Katherine Elizabeth Sides

Masters Theses

The aim of this research project was to test new collection and cultivation techniques that may increase the range of cultivable diversity of soil bacteria. Fortified BioSep beads were employed in situ to capture soil bacteria, and the success of the beads was analyzed using Phylochip microarray analysis. In the cultivation phase, three different media substrates and increased incubation period were evaluated for the ability to select novel or rare bacteria. Over 700 agricultural soil bacterial isolates were classified, including a rare Gemmatimonadetes sp., a rare Verrucomicrobia sp., several Acidobacteria sp., and many novel isolates. Land management, media, and incubation …

Biogeography And Genetic Diversity Of Toxin Producing Cyanobacteria In A Laurentian Great Lake, Johanna Maaria Rinta-Kanto

Doctoral Dissertations

The North American Great Lakes are a vital source on a global scale, as they hold ~18 % of the potable water resources on our planet. Cyanobacteria of the genus Microcystis are commonly found in fresh water environments around the world, and since the mid-1990s also in Lake Erie. The reasons for the success for these potentially toxic cyanobacteria in Lake Erie are not completely understood. In this study we have applied modern molecular tools to analyze field samples to provide an insight into the genotypic composition and diversity of the Microcystis community in the past and present day Lake …