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 produced by commensal bacteria, dramatically reduced C. jejuni colonization in chickens and are being directed toward on-farm control of this pathogen to protect public health. As an important strategy to evade killing by potential peptide antibiotics and by host innate defense, AMP resistance mechanisms in C. jejuni are critical to understand, but are still unknown. In this dissertation, molecular basis of Campylobacter resistance to polymyxin B, the anti-Campylobacter bacteriocins (BCNs), and a chicken host defense AMP (fowlicidin-1) was comprehensively examined using both in vitro and in vivo systems. Although polymyxin B has been successfully used as a model peptide to study AMP resistance in other Gram-negative bacteria, functional genomics examination in this study suggested that polymyxin B is not a good surrogate to study Campylobacter resistance to physiologically relevant AMPs. Campylobacter only developed low-level BCN resistance with low frequency in vitro and in vivo; the acquired BCN resistance was not stable in Campylobacter. Genomic examination of two BCN resistant mutants using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic and acquired resistance of Campylobacter to the BCNs. Random transposon mutagenesis and targeted site-directed mutagenesis identified four genes (cbrR, tig, cjaB, and cj1583c) involved in Campylobacter resistance to fowlicidin-1. These genes were also required for optimal colonization of Campylobacter in chickens. Together, the findings from this dissertation revealed uniqueness and complexity of AMP resistance in Campylobacter and will enable us to develop more sustainable peptide antibiotics and novel intervention strategies to prevent and control Campylobacter infections in humans and animal reservoirs. Key words: Campylobacter, antimicrobial peptide resistance, polymyxin B, bacteriocins, fowlicidins

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 grasses, its diverse metabolism is important for its survival in the competitive rhizospheric environment. The recently sequenced genome of strain Sp245 provided a basis for the proteome studies accomplished in this work. After initial mass spectrometer parameter optimization studies, the expressed proteomes of two strains of Azospirillum brasilense, Sp7 and Sp245, grown under both nitrogen fixing and optimal growth (non nitrogen fixing) conditions were analyzed using a bottom up proteomics methodology. Further proteome studies were conducted with A. brasilense strain Sp7 in order to ascertain the effect of one chemotaxis operon, termed Che1. In this study, proteomic surveys were conducted on two bacterial derivative strains, created earlier, which lacked either a forward signaling pathway or an adaptation pathway. The proteomic surveys conducted in this work provide a foundation for further biochemical investigations. In order to facilitate further investigation and a movement into functional proteomics, a set of destination vectors was created that contain a variety of tandem affinity tags. The addition of tandem affinity tags to a protein allow for generic purification schemes, and can facilitate future studies to investigate proteins of interest discovered in the first expression proteomic surveys of A. brasilense. Taken together, this dissertation provides a valuable data set for investigation into the physiology of A. brasilense and further provides biochemical tools for analysis of the functional protein interactions of A. brasilense cells.

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 friend Nichole has been there to offer love and support throughout my entire undergraduate and graduate degrees. It’s difficult to imagine making it this far without her (and that’s not just because she paid the bills). Abstract Characteristic transcriptional biomarkers have been identified for microbial cultures exposed to 2, 4, 6-trinitrotoluene (TNT), 2, 6-dinitrotoluene (DNT), or triacetone-triperoxide (TATP). This study describes the generation of expression profiles for exposure to each compound, the functional significance of each response, and the identification of the characteristic alterations in gene expression associated with exposure to each compound. Expression profiles were generated from a total of three different candidate organisms: Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida. Common to all three organisms, TNT exposure resulted in increased expression of genes involved in toxin resistance and drug efflux systems. The S.cerevisiae and E.coli expression profiles were both characterized by increased expression of genes involved in iron-sulfur cluster assembly, sulfur containing amino acids, sulfate transport and assimilation and the metabolism of nitrogen compounds. Only E.coli and Saccharomyces were used to generate DNT induced expression profiles; both profiles exhibited high degrees of similarity with each organism’s respective TNT profiles. This was especially true of the E.coli profile where 25 of the 30 alterations were also observed after exposure to TNT. A computational discriminant functional analysis was performed to identify characteristic biomarkers for each exposure. For each compound a set of transcriptional biomarkers (10 or less) was developed. An additional set of biomarkers was developed encompassing both TNT and DNT exposure. These sets of genes serve as a transcriptional fingerprint for exposure to each respective compound. The sensitivity and specificity of each transcriptional fingerprint is sufficient to correctly identify exposure to energetic materials against a background of non-energetic compound exposures. This study makes several novel contributions to the greater body of scientific knowledge: • This is the first documented study of the interactions of TATP in any biological system. • This is the first comprehensive gene expression study of the TNT response by P. putida, E.coli or E.coli. • This is the first application of computational class prediction in the development of biomarkers for exposure to energetic materials

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 name for EPS produced by MZ1T for research purpose. The focus of this research is to fully characterize the microorganism and identify and characterize the genes responsible for thaueran synthesis and export through bioinformatics, transposon mutagenesis, gene clone and expression, reverse transcriptase quantitative real time PCR, and genome sequencing and annotation. Ultimately, this knowledge will contribute to control of viscous bulking and sludge dewatering problems. However, a broader range of important environmental biotechnical processes may be forthcoming from understanding thaueran synthesis. They may include thaueran remedial solutions for heavy metal and radionuclide immobilization, anaerobic carbon channeling and sequestration, greenhouse gas mitigation through acetate incorporation into thaueran, and novel applications such as thaueran-mediated wound healing. Sequencing of MZ1T genome has been accomplished through collaboration with the Joint Genome Institute (JGI). The genome size is 4.5 Mbp, GC content is 68.3%, and there are 4,092 protein coding genes. Three putative thaueran gene clusters were found within the genome. One tight cluster with a size of 20.67kb encoding 14 genes may contain most necessary genes for thaueran formation and export. Another two clusters are loosely organized. Through transposon mutagenesis, mutants not producing abundant thaueran and not flocculating have been obtained and verified, and were further used in reverse transcriptase quantitative real time PCR to compare the differential expression levels of the presumable EPS genes among mutants, wild type MZ1T and under different growth conditions. The results indicated a correlation of the expression level of the test genes and the abundance of EPS.

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 and microscopy to more completely describe the aquatic microbial ecology of the lake. We used an oxalate rinse technique to examine the surface absorbed P pool of the toxic cyanobacterium Microcystis aeruginosa grown under a range of P conditions, as well as the general Lake Erie plankton assemblage. Our results suggest that while Microcystis is plastic in its cellular P needs, the ratio of intracellular to extracellular P remains stable across growth conditions. We describe the effect of the phosphonate herbicide glyphosate on the Lake Erie phytoplankton community using laboratory cell growth studies, field microcosm experiments and PCR amplification of a gene implicated in the breakdown of this compound from the environment. Results from these experiments suggest that the presence of glyphosate can affect community structure in multiple ways and may explain areas of unexplained phytoplankton diversity in coastal areas of Lake Erie. We also show heterotrophic bacteria are likely critical to the breakdown of glyphosate and further illustrate that understanding the context of the larger microbial community is critical to understanding the ecology of the constituent members of the community. Finally, we investigate the activity of the phytoplankton community in winter months with a focus on diatoms abundant in Lake Erie under the ice. We show these diatoms are active and that the winter bloom is a likely source of carbon important to seasonal hypoxia formation. Together, these studies significantly enrich our understanding of how phytoplankton influence important ecological processes in Lake Erie.

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 developed and applied. This has resulted in a deep characterization of the extracellular fraction of a community of microbes thriving in an acid mine drainage system. Among the notable features of this relatively low complexity community, they exist in a solution that is highly acidic (pH < 1) and hot (temperature > 40°C), with molar concentrations of metals. The extracellular fraction is of particular interest due to the potential to identify and characterize novel proteins that are critical for survival and interactions with the harsh environment. The following analyses have resulted in the specific identification and characterization of novel extracellular proteins. In order to more accurately identify which proteins are present in the extracellular space, a combined computational prediction and experimental identification of the extracellular fraction was performed. Among the hundreds of proteins identified, a highly abundant novel cytochrome was targeted and ultimately characterized through high performance MS. In order to achieve deep proteomic coverage of the extracellular fraction, a metal affinity based protein enrichment utilizing seven different metals was developed and employed resulting in novel protein identifications. A combined top down and bottom up analysis resulted in the characterization of the intact molecular forms of extracellular proteins, including the identification of post-translational modifications. Finally, in order to determine the effectiveness of current MS methodologies, a software package was designed to characterize the > 100,000 mass spectra collected during an MS experiment, revealing that specific optimizations in the LC, MS and protein sequence database have a significant impact on proteomic depth.

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 my research in 4 cattle-access (CA) and 4 no-access (NA) farm ponds on the University of Tennessee Plateau Research and Education Center from May 2005 – April 2006. Periphyton community composition and water quality were measured every 2 weeks using standard environmental monitoring procedures. I documented 181 new periphyton taxonomic records (7 phyla, 52 families, and 132 genera) in Cumberland County, Tennessee. Periphyton species richness was greatest in NA ponds. Mean biovolume of pollution-sensitive diatoms (e.g., Achnanthidium minutissimum, Cymbella sp., Eunotia sp., Fragilaria crotonensis and Tabellaria fenestrata) was greater in NA ponds. In contrast, pollution-tolerant diatoms (e.g., Gomphonema sp. and Navicula sp.) and non-diatoms (e.g., Oscillatoria sp. and Scenedesmus sp.) were more abundant in CA ponds. Turbidity, pH, conductivity, and concentrations of the total Kjeldahl nitrogen, total phosphorus, potassium, periphyton phosphorus, silicon, iron, magnesium and aluminum were greater in CA ponds. Thus, changes in water quality associated with cattle-access likely mediated changes in the periphyton community. Considering that changes in periphyton community composition can destabilize higher trophic levels, I recommend that cattle farmers take advantage of USDA conservation programs that provide funds for fencing cattle from watersheds and developing alternate water sources. My results also provide evidence that monitoring the periphyton community is a reliable technique to detect water pollution from cattle.

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 period each resulted in lineage specific preferences. The yeast fortified BioSep bead cultivation collection was significantly different from the bulk soil or acyl homoserine lactone (AHL) fortified bead cultivation collections, and there were lineage specific differences in all three collection types. Phylochip analysis showed a significant difference between bulk soil and all BioSep bead (water, yeast, or AHL fortified) communities based on microarray analysis of 16S rDNA. The yeast fortified BioSep bead community was richer in operational taxonomic units (OTU) than all others. The number of phyla determined by the Phylochip analysis was much higher than that seen in the overall cultivation collection. Prophage induction assays of 21 isolates were performed, using mitomycin C (mitC) and a mixture of six AHLs, to examine soil lysogenic phage-host interactions. The fraction induced by mitC was 29%, and 10% were induced by AHL. There was no correlation between induction and land management or host growth rate. This research showed that increases in cultivable diversity can be attained by the use of BioSep beads in the collection process, varying media substrates, and by extending incubation of inoculate cultures. Phylochip analysis, however, revealed that even with altered cultivation methods, there is still a wealth of soil bacterial diversity that remains to be cultivated from this site. We also found that AHLs impact the interactions between soil bacterial hosts and prophage.

4-Ethylphenol (4-EP) is a malodorant of swine waste and is derived from a component of lignin called p-coumaric acid (p-CA). The production of 4-EP from lignin in swine waste is untested. Additionally, the effect of Fe (III) on 4-EP levels is unknown. Four experiments were performed to determine if Lactobacillus sp. pep8 cultures, as well as enriched swine lagoon slurries, could liberate p-CA from lignin and convert p-CA to 4-EP. Furthermore, it was tested if the addition of Fe (III) influences the conversion of p- CA to 4-EP. Experiment 1 tested Lactobacillus sp. pep8 cultures to determine if the addition of 10 mM Fe (III) and 0.2% sulphite lignin to Lactobacillussp. pep8 cultures would stimulate production of 4-EP. Experiment 2 tested the effect of 0.2% sulphite lignin and 10 mM Fe (III) on 4-EP production in the presence of enriched swine lagoon slurries. On day 0 there was no detectable 4‐EP, for either 0.2% sulphite lignin addition or the 10 mmol l‐1 Fe (III) additions. Experiment 3 tested alternative forms of lignin, including 0.2% sulphite, indulin, or sigma lignin as potential source compounds for 4-EP production in enriched swine lagoon slurries. 4-EP produced in all three conditions are likely endogenous to the lagoon slurry additions. Experiment 4 was designed to measure the degradation of exogenous 4-EP with varying final concentrations of 4-EP in enriched swine lagoon slurries. Data in Figure 7 indicate immediate degradation of 4-EP by day 5, however, by day 7 synthesis of 4-EP occurred until day 14 where 4-EP levels remained in a steady state. Our results suggest that when both Lactobacillus sp. pep8 cultures and enriched swine lagoons are supplemented with p-CA, 4-EP is produced indicating that p-CA serves as a source of 4-EP. However, when supplemented with Fe (III) and/or sulphite, indulin, or sigma lignin, 4-EP production was not stimulated. This data indicates that, 4- EP production is not enhanced by the presence of Fe (III) in either Lactobacillus sp. pep8 cultures or in enriched swine lagoon slurries. Furthermore, lignin did not serve as a source of 4-EP.

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 period each resulted in lineage specific preferences. The yeast fortified BioSep bead cultivation collection was significantly different from the bulk soil or acyl homoserine lactone (AHL) fortified bead cultivation collections, and there were lineage specific differences in all three collection types. Phylochip analysis showed a significant difference between bulk soil and all BioSep bead (water, yeast, or AHL fortified) communities based on microarray analysis of 16S rDNA. The yeast fortified BioSep bead community was richer in operational taxonomic units (OTU) than all others. The number of phyla determined by the Phylochip analysis was much higher than that seen in the overall cultivation collection.Prophage induction assays of 21 isolates were performed, using mitomycin C (mitC) and a mixture of six AHLs, to examine soil lysogenic phage-host interactions. The fraction induced by mitC was 29%, and 10% were induced by AHL. There was no correlation between induction and land management or host growth rate. This research showed that increases in cultivable diversity can be attained by the use of BioSep beads in the collection process, varying media substrates, and by extending incubation of inoculate cultures. Phylochip analysis, however, revealed that even with altered cultivation methods, there is still a wealth of soil bacterial diversity that remains to be cultivated from this site. We also found that AHLs impact the interactions between soil bacterial hosts and prophage.