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Poster Abstracts (Alphabetical by Author)

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Poster Abstracts (Alphabetical by Author) Poster Abstracts (alphabetical by author) 1A. Phage Targeted Bioluminescent Sensing of Enterohemorrhagic Escherichia coli Clara Beasley, Pat Jegier, Abby Smartt, Alice Layton, Gary Sayler, and Steven Ripp The University of Tennessee Center for Environmental Biotechnology E. coli O157:H7 and other members of the Shiga toxin producing (STEC) group of pathogenic bacteria cumulatively account for an estimated 250,000 human infections each year. Early detection and diagnosis of STEC infection is critical towards proper health care management. In addressing this need, we have genetically engineered a bacteriophage, PP01, for bioluminescent monitoring of viable shiga toxin producing E. coli in food and liquid matrices. To establish assay specificity, over 35 serotypes of infectious E. coli obtained from a variety of sources that included dairy, food, human and wastewater isolates were tested. The assay was performed by combining E. coli cells with PP01 bacteriophages harboring the Vibrio fischeri quorum sensing associated luxI gene. The consequent production of acylhomoserine lactone molecules after productive phage/host infections was linked to the emission of bioluminescence from integrated signaling reporter cells in high-throughput 96-well microtiter plate assays. We found the PP01 lux reporter bacteriophage to be highly specific for O157 serotypes with infection of only a few other E. coli serotypes, including O103 and O129. Although serotype O129 has been implicated in few human infections, serotype O103 in combination with O157 comprise nearly an estimated 57% of all STEC-related illnesses, therefore substantiating phage PP01 as a promising candidate for food and waterborne pathogenic E. coli surveillance. 2B. Is Urea a Driver For Microcystis Blooms In Lake Erie? B. Shafer Belisle, Morgan M. Steffen, Helena L. Pound and Steven W. Wilhelm Department of Microbiology, University of Tennessee, Knoxville, TN Microcystis aeruginosa is a single-celled blue green alga, or cyanobacterium, that grows as colonies in freshwater systems around the world. Microcystis can proliferate to form dense blooms that lead to large-scale deterioration of aquatic habitats and potential serious public health issues. Subpopulations of these cyanobacteria can produce multiple toxins, including microcystin, a potent hepatotoxin. These blooms occur in the greatest density in waters that are affected by eutrophication, a process by which bodies of water receive excess nutrients. Agricultural runoff is often responsible for an influx of nutrients, such as nitrogen and phosphorus, which have been shown previously to play important roles in bloom proliferation. Urea is an organic form of nitrogen frequently used as fertilizer, resulting in urea being a common nitrogen load to freshwater environments. Multiple cyanobacteria, including Microcystis spp ., possess the urease enzyme, which hydrolyzes urea into ammonia and carbon dioxide. To identify the potential role of urea in driving blooms of Microcystis in Lake Erie, urease enzymatic activity was measured at multiple stations in the lake using the indophenol method. Preliminary field and laboratory data suggest up-regulation of the urease enzyme activity in cultures and field samples of Microcystis spp. Subsequent research of nitrogen utilization by algae species may contribute to future mitigation of bloom events in aquatic systems such as Lake Erie and Lake Taihu. 3A. Detection of Entamoeba gingivalis in dental samples C. Crawford, S. Fox, M. B. Farone Periodontal disease is an important inflammatory disease that is poorly understood. The purpose of this study is to determine the prevalence of the organism, Entamoeba gingivalis , in dental samples from periodontal pockets. E. gingivialis has only been found in patients who have periodontitis or gingivitis, and littleknown about this organism. Through the use of Giemsa and trichrome staining, cytospin slides of patient samples were examined for the organism. We also performed DNA extraction and PCR to detect E. gingivalis in the samples. While no organisms have been identified from the samples, from the 20 samples collected, 5 (%25) tested positive by PCR for E. gingivalis. While these results are encouraging, we are continuing to examine patient samples for the prescence of this organism. 4B. Dynamic activity and crosstalk modeling with the pheromone and glucose sensing pathways of Saccharomyces cerevisiae Zachary Duck, Maria Siopsis, Jennifer Brigati, Grant Willhite, and Stephen Wright G-protein coupled receptors have demonstrated remarkable potential for chemical control of cells and nearly half of the pharmaceutically treated ailments. By characterizing the mechanism, functions, and interactions of signaling proteins involved with the only two receptor activated G-protein signals in yeast, the complexity of crosstalk and regulation between signals is observed with significantly less noise and nonspecific activation in comparison to human cells. In order to provide a dynamic, global perspective on mechanisms in G- protein signaling and predict potential sites for crosstalk between the glucose and pheromone sensing pathways, we developed a computational approach for modeling independent signaling pathways subject to crosstalk. A system of differential rate laws specific to the mechanism of each protein demonstrates elaborate profiling of individual component responses, and an analysis of the sensitivity and robustness for each interaction allows for calculation of potential mechanistic probabilities. Our model is written in COPASI and utilizes the steady cellular state assumption to isolate mechanisms of gene specific amplification associated with accumulation or consumption of proteins and their complexes. Ste2p (pheromone receptor) and Gpr1p (low affinity glucose receptor) create an ideal example of crosstalk that regulates the need for energy production and storage in conjunction with the prolonged growth of the organism based on the local composition of the surroundings. 5A. What does it take to build a virus? Computational determination of nutrient stoichiometry based on genomic sequence T. Chad Effler, Alison Buchan, Steven W. Wilhelm Department of Microbiology, The University of Tennessee, Knoxville, TN. USA. With interest concerning the role(s) of marine viruses in global carbon cycles growing, we set out to determine how virus particles could contribute to nutrient pools by determining how much carbon, nitrogen and phosphorus an individual virus particle contains. Empirical determination of the elemental composition of viruses is technically challenging due to the difficulties associated with removing host macromolecular components from purified virus. Thus, we sought to employ a computational approach with a focus on a well-characterized virus for which genomic and structural data are known: bacteriophage T4. We began by constructing two lookup tables, one for nucleotides and one for amino acids that would determine the respective carbon, nitrogen, and phosphorus atoms within a virus particle based on both genomic and proteomic reconstructions. From the publically available genome and published estimates of structural protein abundances, we estimated a C:N:P ratio of 26:8:1 per virion, and an overall mass equivalent of 0.18 fg C per particle. This information will be discussed within the context of how marine virus particles may contribute to biogeochemical cycles. 6B. Detection of Prochlorococcus specific Cyanomyovirus P. Jackson Gainer, Alise J. Ponsero, Erik R. Zinser, and Steven W. Wilhelm Department of Microbiology, The University of Tennessee, Knoxville, TN. Viruses are pervasive in marine systems, and play a vital role in constraining microbial community structuring as well as in the cycling of nutrient elements. Within the marine viral population, T4-like bacteriophage have been noted to be the predominate cyanobacteria-infecting phage: these are often referred to as cyanomyophage. In the oligotrophic open ocean Prochlorococcus is the numerically dominant cyanobacteria, accounting for 25% of primary production in these areas, and as such, play an important role in global biogeochemical cycling. Therefore viruses, which are host-specific to this species, may play a major role in nutrient cycling on a global scale. To explore the dynamics of the relationship between Prochlorococcus and its viral counterparts we have developed primers that are specific to cyanomyophage which are limited to Prochlorococcus as a host. Using samples collected on a Pacific Ocean transect from Hawaii to San Diego, CA, we used these primers to detect this group of viruses. Data on the presence and diversity of this group will be presented and contrasted with a variety of environmental metadata, including temperature, nutrient profiles and cyanobacterial distributions in order to begin to shed some light on the factors that constrain or promote virus mediated-lysis of Prochlorococcus populations. 7A. Using Publicly Available Metagenomes and Clone Libraries to Validate Degenerate Primer Sets with De-MetaST-BLAST, a Freeware and Open Source Program Christopher A. Gulvik, T. Chad Effler, Reantha Pillay, Steven W. Wilhelm, and Alison Buchan The polymerase chain reaction (PCR) is among the most widely used techniques in the life sciences. Primer sets are continually being developed and refined based on the ever-increasing and publicly available nucleotide and protein sequences. Design of candidate primer sets to target sequences of interest can be an entirely automated
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