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 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 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 computational process with the help of several programs (e.g., Amplicon, CODEHOP, DEFOG, DePiCt, HYDEN, MAD-DPD, PhiSiGns, and Primaclade). While identifying sequence regions to target is essential for primer development, it is also important to ensure the candidate primers do not retrieve undesired amplicon products, such as off-target gene sequences. This is of particular concern with primers that contain degenerate nucleotides. Validation of developed primer sets has largely been ignored in the development of computational tools for biology, and to address this gap we have developed a computer program that makes use of publicly available metagenomes to aid in primer set validation. The De-MetaST-BLAST software package allows for input of degenerate primer sequences, acts as a metagenome search tool, and is paired with NCBI’s BLAST server. Key steps in the software are conversion of inputted primer sequences and the reference database into a four-digit binary code, rapid identification and binning of in silico amplicons via bitwise searching, and assigning the top ten BLAST hits of each in silico amplicon with NCBI’s nr protein database. We have made this program publicly available as freeware, which can be executed on any modern Macintosh, Unix, or Windows operating system. An example of De-MetaST-BLAST’s utility will be shown in the context of designing and validating a novel primer set.

8B. Heat Shock Analysis of the Association Between Glucose-sensing and Pheromone-sensing GPCR pathways David Lee Haskins, Jennifer Brigati, Stephen Wright In Saccharomyces cerevisiae , common Baker’s yeast, the G protein-coupled receptors (GPCR) Gpr1p and Ste2p/Ste3p, control a low-affinity, glucose-sensing pathway and the pheromone-sensing pathway. These two pathways are the only known GPCR pathways active in yeast, and this makes them a useful model for study. Previous research has suggested that there are connections between these two signaling pathways in S. cerevisiae . To further explore these pathways, experiments were performed to determine if the low-affinity glucose-sensing pathway is dependent on any components of the pheromone-sensing pathway. An important characteristic of the glucose-sensing pathway is that the cell is known to display increased sensitivity to heat shock when exposed to high levels of glucose after a period of starvation. Heat shock assays were performed on strains of S. cerevisiae lacking various components of the pheromone-sensing pathway. The results suggest that deletion of genes in the pheromone-sensing pathway had an impact on the functionality of the glucose-sensing pathway. BY4741 (Mat α) strains lacking Gpa1p, Sst2p, Ste20p, and Arr4p displayed a 10 to 24-fold increase in relative survival compared to wild-type cells when heat shocked. In contrast, Mat a deletion strains lacking Sst2p, Ste18p, and Ste20p exhibited a 3 to 65-fold increase in relative survival when compared to wild-type cells. The results support the hypothesis that the components of the pheromone-sensing pathway and glucose- sensing pathways interact.

9A. Genome Assembly of an Agricultural Soil Bacillus Isolate Higgins, S.A. 1, Sanford, R.A. 2, Chee-Sanford, J. 3, Welsh, A. 2, Konstantinidis, K.T. 4, Löffler, F.L. 1,5,6 1 Department of Microbiology, University of Tennessee, Knoxville, TN 37996 2 Department of Geology, University of Illinois Urbana-Champaign, Urbana, IL 61801 3 Department of Natural Resources and Environmental Sciences, University of Illinois Urbana- Champaign, Urbana, IL 61801 4 Department of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332 5 Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN 37996 6 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 The nitrogen cycle is one of earth’s most disrupted biogeochemical cycles. Increased reliance on agricultural fertilizer has led to significant increases of fixed, biologically-active nitrogen (BN) into terrestrial and aquatic systems. Inputs of BN may alter microbial activity responsible for cycling nitrogenous compounds in these systems, but further investigation is required to identify environmental factors controlling microbial nitrogen cycling and the diversity of the microbial community carrying out these processes. In order to better understand the functional diversity of microorganisms involved in nitrogen cycling, data were collected on two heterogeneous agricultural soils in Illinois and DNA samples extracted from soil cores taken along a transect within both fields. Several bacterial isolates have been cultured from these field sites and DNA sequencing performed using Illumina Hi-Seq technology at Oak Ridge National Laboratories. Draft genome assembly was conducted using an established hybrid assembly protocol with Velvet and Newbler, producing a draft assembly of 16 contigs totaling 3.9 Mb with an average GC content of 44 %. Analysis of ribosomal RNA genes suggests Bacillus sp. strain ET is closely related to Bacillus subtilis subsp. spezizenii TU-10-B and W23. This relationship was also supported by a hierarchical clustering analysis method on COG functional gene categories between 36 Bacillus genomes. Future sequencing and assembly of other cultured isolates from these soils will complement on-going metagenomic analyses and generate testable hypotheses for wet lab research.

10B. Long term impacts of a genetically engineered microorganism (GEM) and polyaromatic hydrocarbons (PAHs) on soil microbial communities Xiaoci Ji 1, Steven A. Ripp 2, Alice C. Layton 2, Gary S. Sayler 2, and Jennifer M. DeBruyn 1 1. Department of Biosystems Engineering & Soil Science, University of Tennessee 2. Center for Environmental Biotechnology, University of Tennessee 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 structure in the lysimeters. In 2010, 14 years after experiment initiation, replicate soil cores were taken from each lysimeter. Soil bacterial community structures were determined by 454 pyrosequencing of 16S rRNA gene amplicons. Even though PAHs fell below detectable concentrations within the first couple years of the lysimeter experiment, +PAH lysimeters showed significantly higher soil organic matter content (1.30 ± 0.23%) than -PAH lysimeters (0.81 ± 0.08%). 16S libraries reveal that there was a change in the bacterial community structure in +PAH compared to -PAH lysimeters: 9.59% of OTUs (operational taxonomic units) were shared between +PAH lysimeters while only 4.08% were shared between +PAH and -PAH lysimeters. Multivariate ordination and cluster analysis of ΘYC distances indicate that communities fell into three clusters: lysimeter 1 and 2 (both +PAH +HK44); lysimeter 4 (+PAH +HK44) and 6 (+PAH -HK44); and lysimeter 3 and 5 (both - PAH -HK44). The addition of PAHs appears to be more influential than the introduction of a GEM on bacterial community structures over the long term.

11A. Evolution of Quasispecies in HIV-I subtype C infection Elizabeth Johnson and Vitaly Ganusov If the HIV-1 subtype C genome is averaged across its population, a general "consensus" sequence emerges. It is believed that this consensus sequence represents the hypothetical "most fit" form of the virus. An actual population of virus, however, often deviates from this consensus sequence as it attempts to evade immune pressure. Many of these accumulated mutations revert back to consensus once the viral population establishes in a new host. A set of differential equations along with a partial differential equation Markov type diffusion system has been constructed to predict the kinetics and qualitative behavior of these reversions in the Gag protein of HIV under varying regimes of immune pressure.

12B. Predicting the protective role of vaccine-induced CD8 T cells during malaria infection of mice Reka K. Kelemen 1, Vitaly V. Ganusov 1,2 1. Graduate Program of Genome Science and Technology, University of Tennessee, Knoxville 2. Department of Microbiology, University of Tennessee, Knoxville, TN 37996 Malaria in humans is caused by eukaryotic parasitic protists in the Plasmodium genus and spread by mosquitos in tropical and subtropical areas of the world. The infection is the result of the immune system’s unsuccessful clearance of hepatocytes (liver cells) infected by the pathogen. If malaria specific CD8 T cells in immunized mice do not detect a hepatocyte infected by a malaria sporozoite during the liver stage of the disease, the pathogen will enter the blood and the host will experience the symptoms of blood stage malaria. In this project we developed several mathematical models of control of pathogen replication during the liver stage. In our model, we calculate the probability of clearing the infection as a function of malaria-specific T cell abundance in the blood and liver of mice that have been vaccinated against malaria. Using experimental data from our UTK collaborator we estimate several important parameters such as the rate at which infected hepatocytes are cleared by the CD8 T cell response, the effective number of infected hepatocytes, and the level of malaria-specific CD8 T cells required for a 95% probability of protection from blood-stage malaria in mice. This project is being extended to model differences in the infection dynamics due to factors such as Plasmodium species and infection dose.

13A. Clarification of interactions between two GPCRs in Saccharomyces cerevisiae Elisabeth Klouda, Jennifer Brigati, David Willhite, Katie Selcer, Stephen Wright Although the entire genome of Saccharomyces cerevisiae has been sequenced, approximately 60% of the genes have no known function. An even higher percentage of genes are not fully understood. Two such genes are Gpr1 and Ste2, the products of which, Gpr1p and Ste2p, are the only G-protein coupled receptors (GPCRs) in the S. cerevisiae genome. Previous studies have shown that deletion of Gpr1p has effects on the activity of the Ste2p-mediated pheromone-sensing pathway. Such effects can easily be seen in a growth inhibition assay, where deletion of Gpr1p results in reducedsensitivity to α factor. In addition, gene induction assays, carried out with cell strains containing a FUS1-lacZ construct, indicate that transcription of genes related to mating is downregulated in cells lacking Gpr1p. The exact nature of the interdependency exhibited by Gpr1p and Ste2p has not yet been elucidated, but this study suggests that the two proteins form a heterodimer. This hypothesis is heavily supported in mammals, where heterodimers such as GABA and beta-adrenergic receptors are non-functional in monomer form. Intriguingly, an overwhelming amount of Ste2p can make up for the functional difference seen in cells lacking Gpr1p, suggesting that Ste2p could dimerize (non-preferentially) with itself. This study provides evidence for the hypothesis that Gpr1p and Ste2p form a heterodimer and are most functional in this form. This hypothesis is supported by both growth inhibition and gene induction data that suggest the pheromone-sensing pathway is not as responsive to α factor in the absence of Gpr1p. In addition, and perhaps most importantly, co-immunoprecipitation experiments indicate that Ste2p can be pulled down with Gpr1p. The insensitivity shown by the growth inhibition and gene induction data, and protein complex formation indicated by the co-immunoprecipitation experiments, suggest that Gpr1p and Ste2p could form a heterodimer.

14B. Ethylene Affects the Phototactic Response in Cyanobacteria Randy Lacey and Brad Binder Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville In plants, ethylene acts as hormone that regulates many physiological processes. Perception of ethylene is mediated by a family of receptors that bind it and elicit downstream responses. As evolutionary ancestors to modern plants, various cyanobacterial organisms have genes that show sequence homology to the ethylene-binding domain of the ethylene receptors in Arabidopsis thaliana . Indeed, in radioactive ethylene binding assays these organisms do bind ethylene, and as shown in Synechocystis sp. PCC 6803, when the potential ethylene-binding domain is removed, ethylene-binding capacity is eliminated. Here, I seek to elucidate the physiological role of ethylene in the unicellular cyanobacteria, Synechocystis . In Synechocystis , slr1212 encodes a multidomain protein, SynETR, which has been shown to bind ethylene. While little is known about this protein, it does show structural similarity to proteins involved in phototaxis of this organism. Phototaxis is the directed movement toward or away from a light stimulus. It is hypothesized that ethylene regulates phototaxis in Synechocystis via SynETR. By use of phototaxis assays we have shown that ethylene affects the rate of phototaxis. Additionally, by knocking out slr1212, the rate of phototaxis is also affected. I also seek to bring to light the biochemical nature of ethylene binding in SynETR by mutaganizing specific residues in the ethylene-binding domain. This will further show the relationship of this protein to the ethylene receptors we see in modern plants.

15A. Short term direct current exposure increases caspase activity in human colon cancer cells Danielle E. Large, Nathan Tolbert, Gary S. Sayler, James T. Fleming Successful clinical electrochemical treatment of solid tumors has been demonstrated in skin, lung, liver pancreas, and breast tissue with differing electric field strengths and/or current densities [1]. The particular effects observed and the mechanism invoked to explain these effects vary depending on the type of electric current used (DC vs AC), field strength, electrode design and electrode composition. Recently treatment of tumor cells with DC has been shown to induce apoptosis in human leukemic [2] and oral mucosa cancer cells [3]. We set out to investigate the anti-proliferative and apoptotic effects of direct electric current exposure on colon cancer cells SW480 using a sophisticated electrochemical approach. Our experimental system uses precisely microfabricated platinum electrodes on silica chips in a three electrode configuration interfaced with an electrochemical potentiostat. Cells were grown on electrode chips using RPMI media contained within rubber gasket frames. Indium tin oxide (ITO) transparent slide placed on the surface of the frames functioned as the counter electrode. Cells were exposed for 300 s in triplicate to a DC field strength of 1.6-2.3 V/cm with current densities ranging from 0.05 -5 µA/cm 2. After 24 h the cells were tested for: 1) cell viability using a tetrazolium/formazan assay and 2) apoptosis using a caspase 3/7 assay. Caspase activities increased as much as 5 times over control cells at levels below those that adversely affect cell viability. In addition, we conclude that the observed decrease in cell viability with increasing current is due to necrosis instead of apoptosis. Electrochemical treatment may be a useful alternative cancer therapy for tumors that don’t respond to chemotherapy or are surgically difficult.

16B. Differences between free-living and particle associated bacterial communities during two photoautotrophic bloom events in the southern Pacific Ocean. Gary R. LeCleir 1, Jennifer M. DeBruyn 2, Elizabeth W. Maas 3, Philip W Boyd 4 and Steven W. Wilhelm 1 1. Department of Microbiology, The University of Tennessee, Knoxville, TN, USA. 2. Biosystems Engineering and Soil Science, The University of Tennessee, Knoxville, TN, USA 3. National Institute of Water and Atmospheric Research, Wellington, New Zealand 4. National Institute of Water and Atmospheric Research Centre for Chemical and Physical Oceanography, University of Otago, Dunedin, New Zealand Bacteria are important mineralizers of marine detrital material. As they are often challenged by different carbon sources, we wondered if changes in the input (i.e., biological origins) to marine heterotrophic bacterial communities would change the resulting community structure. Using newly developed marine sediment traps designed to collect and incubate microbial communities (RESPIRE traps), we collected and incubated marine aggregates/ particles in the southern Pacific Ocean from separate diatom and cyanobacterial bloom events in-situ. Using 454 pyrosequencing, we determined the phylogenetic affiliation of the microbes growing on aggregates exported from these bloom events. Water column samples were also collected and sequenced for comparison between the particle associated and free-living bacterial communities. Dramatic differences were found between the water column and sediment trap bacteria. These differences were consistent in traps incubated with particles collected from either bloom type. Pelagibacter sp., Flavobacters and Actinobacters generally dominated water column samples, while sediment trap samples contained members of the Roseobacter clade of the marine α-Proteobacteria in high abundance. Our findings indicated that rapid changes occurred to the microbial community associated with aggregates from either bloom type. Changes within the particle-associated community occurred within the first 24 h of collection. There was little change to the bacterial assemblage after the initial 24 h incubation period. The most abundant early colonizer was a Sulfitobacter sp. that appeared to reach peak abundance within the first 24 hrs. This study provides further evidence that Roseobacters are rapid colonizers of marine aggregates and that colonization can occur on very short time scales.

17A. Influence of oxidative stress and temperature on the growth and physiology of Prochlorococcus Lanying Ma, Erik Zinser University of Tennnessee Cyanobacteria of the genus Prochlorococcus are the smallest phytoplankton in the ocean. Hydrogen peroxide (HOOH) is generated in seawater by the photooxidation of dissolved organic carbon (DOC) by sunlight, and is highly toxic to Prochlorococcus. It has been shown that co-occurring heterotrophs such as Alteromonas sp. facilitate the growth of Prochlorococcus in the sunlit ocean by scavenging HOOH. Temperature is also a major influence on Prochlorococcus abundance and distribution in the ocean, and studies in other cyanobacteria suggest that HOOH and temperature extremes act synergistically to limit growth. We will present our study on the interaction of HOOH and temperature on the growth of Prochlorococcus . We confirmed that the growth rate of several Prochlorococcus strains was higher in the presence of heterotrophic bacteria, which decreased the concentration of HOOH to around zero. We discovered that the optimum temperature for each strain did not vary as a function of HOOH concentration.. However, we observed a synergistic effect of low temperature and high HOOH on the growth of one Prochlorococcus strain (MED4), which was eliminated in the presence of heterotrophic bacteria. The ecological implications of this synergy will be discussed.

18B. Genetic characterization of vanillin catabolism in the salt marsh bacterium Sagittula stellata E-37 Robbie M. Martin, Ashley M. Frank, Christopher A. Gulvik, Mary K. Hadden, Alison Buchan Department of Microbiology, University of Tennessee, Knoxville, TN Lignin, the most abundant aromatic polymer on earth, is a structurally heterogeneous and highly recalcitrant component of vascular plant material. In nature, lignin degradation is mediated by the concerted efforts of fungi and bacteria. Fungi typically play critical primary roles in converting lignin to a complex mixture of low molecular weight aromatic compounds that can be readily used by bacteria harboring the requisite catabolic pathways. Vanillin is one of the most prevalent aromatic compounds resulting from fungal degradation of lignin and the lignolytic salt marsh bacterium Sagittula stellata E-37 is capable of using this compound as a sole source of carbon and energy. However, this bacterium lacks homologs for vanillin catabolism genes identified in other microbes, suggesting the employment of a novel metabolic pathway. In this study, we aim to identify the genes encoding the enzymes mediating vanillin transformations in E-37 using a random transposon mutagenesis approach. A 6,000-member mini-Tn5 transposon library was created and screened for mutants deficient in vanillin catabolism. This initial screen identified a number of mutants which, following confirmation of the phenotype, will be subjected to arbitrary PCR to identify the location of the Tn5 insertion. These preliminary findings are laying the foundation for an improved understanding of microbial processes that are central to the global carbon cycling.

19A. Exploration of Microbial U(VI) Reduction Mechanisms and Associated U Fractionation Jenny Merryfield 2 and Frank E. Löffler 1,2,3 1 Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN 37996, 2 Department of Microbiology, University of Tennessee, Knoxville, TN 37996, 3 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 Contaminants, such as Uranium (U), are at risk of spreading through subsurface water at many contaminated sites in the United States. One possible method to immobilize U at these sites is to harness the metabolism of resident microorganisms that reduce U. Many microbes can use soluble, hexavalent uranium (U-VI) as an electron acceptor and reduce it to insoluble, tetravalent uranium (U-IV), which does not travel as readily through the subsurface. Stimulating these metabolic processes is a promising approach to limit the spread of U contamination. There is a need to understand in greater detail the pathways involving U reduction. The goals of this research are to elucidate the mechanisms and determine the rates by which microorganisms reduce U, and measure the change in ratios of 238 U and 235 U (i.e. isotopic fractionation) that follows U reduction. Initial experiments with resting cells measured unique enrichment factors for different microorganisms suggesting that different mechanisms and pathways are involved. We hypothesize that Anaeromyxobacter and Geobacter species use c-type cytochromes to reduce U and as a result, cause isotopic fractionation in the remaining U(VI). c-type cytochromes have been implicated in electron transfer to metals and radionuclides, and genome sequencing identified the respective genes in several metal reducing bacteria. A deeper understanding of fractionation and reduction mechanisms will lay the groundwork for developing molecular tools for monitoring U-reducing microorganisms and U states in U-impacted environments.

20B. Antibiotic resistance among cultured lactic acid bacteria from bioethanol fermentation facilities across the United States Colin A. Murphree, E. Patrick Heist, and Luke A. Moe The nascent bioethanol industry in the US is on track to triple production capacity in the next decade, owing to a federal mandate that stipulates biofuels added to gasoline must reach 36 billion gallons by the year 2022. Rapid growth in the industry has led to a collection of unforeseen issues with production— chief among these is the issue of bacterial contamination of the yeast-based fermentation (referred to as bacterial bloom). Bioethanol producers combat this through prophylactic addition of antibiotics to fermentations. This has led to a notable increase in antibiotic resistance among the most prevalent class of bacteria isolated from these facilities: the lactic acid bacteria (LAB). The LAB comprise taxonomically diverse clade that also includes human commensals, opportunistic pathogens, food starters, and probiotic additives. We have phylogenetically typed 32 antibiotic resistant LAB isolates from 7 different bioethanol fermentation facilities across the US. We have determined antibiotic resistance levels and identified the mechanisms and genes used by these isolates to resist the activity of the antibiotics used in the fermentation vessels (penicillin, erythromycin, virginiamycin). Each of the isolates displays resistance to at least one of the antibiotics; the majority show resistance to more than one class. Mechanisms for resistance and implications for evolution and transfer of resistance will be discussed.

21A. Engineering Photosystem I for Enhanced Electron Transport Rates In Vitro for Applied Photosynthesis Khoa Nguyen 1,2 , Natalie Myers 3, & Barry D. Bruce 1,2 1Department of Biochemistry, Cellular, and Molecular Biology 2NSF STAIR (Sustainable Technology through Advanced Interdisciplinary Research) IGERT, University of Tennessee, Knoxville, TN 37996, USA 3School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA Photosystem I (PSI) has been shown to be a robust photoactive nanoparticle capable of generating both hydrogen and electricity in vitro . These processes both derive their electrons from soluble metalloproteins, such as plastocyanin and cytochrome c, which re-reduce the oxidized special pair (P700) of PSI. Much of what we know about this process has been via the studying of model single cell organisms such as cyanobacteria. Cyanobacteria are important for carbon fixation and oxygen production and also offer a powerful and facile genetic system. Moreover, as oxygenic organisms they utilize the Z- scheme employing both PSII and PSI resembling that found in plants. Currently, the thermophilic cyanobacterium, Thermosynechococcus elongates BP-1 is extensively studied due to its sequenced genome, genetic transformability, and the recent success in crystallizing many protein complexes, including PSI and PSII. My project is to engineer the PSI’s surface chemistry. For example, we are making complementary changes on both PSI and cytochrome c 6 surfaces so that they interact quicker in solution. Specifically, we have made a psaF modification to enhance docking of cytochrome c 6 to the lumenal surface of PSI. These mutations will be functionally characterized through electrochemical and photochemical assays. The electron transfer rate can be tested using flash photolysis and analyzing the re- reduction profile of P700. The kinetics of this electron transport step is being investigated as a function of cytochrome content, temperature, pH, ionic strength, and post selective mutagenesis. These results will be discussed in light of our understanding of how Cyt c 6 and PSI interact.

22B. Isolation of bacteria capable of degrading a potent algal toxin Helena L. Pound, Lauren E. Krausfeldt, Gary R. LeCleir and Steven W. Wilhelm. Department of Microbiology, The University of Tennessee, Knoxville, TN. USA. Cyanobacterial harmful algal blooms occur worldwide and cause detrimental effects on aquatic ecosystems, thus impacting local biology and human economics. Blooms of the colonial cyanobacteria Microcystis spp. can produce toxins that pose threats to human health and limit water supply potability. For example microcystin-LR (MCLR), a hepatotoxin produced by Microcystis spp. and several other cyanobacterial genera, has been shown to cause liver damage. Importantly there are no current means of removing this toxin from aqueous environments or potable/recreational waters. Heterotrophic bacteria that co-occur with blooms have, however, been found to degrade MCLR in nature, using it as a carbon source for growth. To identify and isolate bacteria capable of degrading MCLR, whole water samples were taken from Lake Erie and Lake Tai ( Taihu ), China: both locations are known to have annual Microcystis blooms. Using BioLog MT2 plate assays, we performed initial screenings to isolate bacteria capable of degrading MCLR. Growth at various MCLR concentrations was analyzed spectrophotometrically and subsequently using flow cytometry. Seven bacterial strains were isolated and identified as MCLR degraders. Sequence analyses of 16S rDNA genes has been used to identify these bacteria. This study represents an important first step in the development of an effective biodigestor for these cyanotoxins.

23A. Viral 2A Peptide-Mediated Single Vector Polycistronic Expression of the Bacterial Bioluminescence Gene Cassette ( luxCDABEfrp ) in Human Cells Sarah Price 1, Tingting Xu 1, 2 , Dan Close 3, Gary Sayler 1, 2, 3 1Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 2Department of Microbiology, University of Tennessee, Knoxville, TN 3The Joint Institute for Biological Sciences, Oak Ridge National Laboratory, Oak Ridge, TN The simultaneous expression of multiple exogenous genes in eukaryotic cells is complicated relative to their prokaryotic counterparts due to fundamental differences in the genetic architecture and gene expression mechanisms between the two domains. Unlike in prokaryotes, where polycistronic expression of adjacent genes from one promoter is common, gene expression in eukaryotic cells normally requires that each gene be regulated by its own promoter. While several strategies have been developed to facilitate polycistronic gene expression in eukaryotes, thus far concurrent expression beyond five genes has not been demonstrated. It is reported here that through the use of divergent viral 2A elements, it is possible to polycistronically express up to six genes under the regulation of a single promoter in human cells. Successful expression of the full bacterial bioluminescence gene cassette ( luxCDABEfrp ) was demonstrated by production of a bioluminescent phenotype in cells harboring the 2A-mediated polycistronic vector. This single polycistronic vector system was also shown to improve bioluminescence production compared to a similar multi-promoter-based expression system. Quantitative RT-PCR analysis revealed that mRNA levels of the lux genes decreased distal to the promoter, suggesting a reduced transcription efficiency and/or increased RNA instability during the translational stage of expression. Since previous work has demonstrated that FMNH 2 is a major limiting reagent in the bioluminescent reaction in human cells, future work will focus on re-ordering the polycistronic lux vector system to bring the FMN reductase gene, frp , proximal to the promoter to enhance its expression for improved bioluminescent production.

24B. Antarctic Subglacial Sediment Visualization Techniques and its Application to the Subglacial Access Research Drilling (WISSARD) Project Alicia M. Purcell 1, Hector F. Castro 1, Keith Prater 2, John Biggerstaff 2, and Jill Mikucki 1 1Department of Microbiology, University of Tennessee, Knoxville, TN 2Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN is covered by two ice sheets (thickness ranging ~ 0.02 and 4.5 km). We now know extensive aquatic environments, including large lakes, exist below this ice cover. WISSARD is a US initiative to drill into Whillans, below the Whillans Ice Stream (WIS) in . The WISSARD project aims to study the lake biogeochemistry and hydrogeology and its implication to ice sheet stability, glacial hydrology, and microbiology. It is likely that microbial life exists below WIS because viable organisms have been found in other Antarctic subglacial environments including sediments from below the Kamb Ice Stream, ice cores taken above Subglacial , and in a brine outflow from below the Taylor Glacier. Subglacial Lake Whillans is unique from these other systems in that its water drains and refills periodically, flushing the lake and its contents. A key research objective of the WISSARD project is to obtain samples from the water column and sediments of Subglacial Lake Whillans and to determine the diversity of life in these samples. Visualizing microbial cells in sediment samples is challenging because cells are often masked by sediment particles and nonspecific binding of fluorescent stains to sediment particles. Here we used epi- fluorescent microscopy to compare two nucleic acid stains, SYBR Gold and Sytox Green (Life Technologies, Carlsbad, CA) for the enumeration of microbial cells in sediments. Studies have estimated that cell counts in subglacial sediments are likely to be ranged 2-4×10 5 cells g -1. Cell abundance within the McMurdo Dry Valleys ranges from 5 ×10 5 to 4 ×10 9 cells g -1. Our ‘model’ sediment sample was collected from subglacial debris at the terminus of the Taylor Glacier, McMurdo Dry Valleys, Victoria Land, Antarctica. This sample has an abundance of 2.1 ×10 5 cells g -1 of dry sediment. These methods are being optimized for Subglacial Lake Whillans samples, which will be collected during the 2012-13 austral summer field season. Nucleic acid staining in sediment as well as fluorescent in situ hybridization are starting points for enumerating cells, measuring biomass, visualizing phylogenetic diversity, and finding relationships between microorganisms and sediment particles in this unique microbial environment. Antarctic subglacial environments are largely unexplored and could be home to novel forms of microbial life.

25A. Identification and characterization of enzymes involved in the metabolism of D-amino acids in the soil-dwelling bacterium Pseudomonas putida KT2440 Atanas Radkov and Luke Moe University of Kentucky Being the preferred for an immense variety and a vast number of organisms, the plant rhizosphere (the plant root-soil interface) is a remarkably complex ecological environment. Plant roots exude a nutrient-rich cocktail into the rhizosphere soil, this mixture includes sugars, amino acids, and phenolics. This selects for a rich population of microbial inhabitants. One well-characterized bacterial representative of rhizosphere communities is Pseudomonas putida KT2440. This particular species has been documented to inhabit the rhizosphere of different plants and has been established as a model organism for studying plant root colonization and plant-microbe communication. P. putida KT2440 has evolved a remarkable metabolic capacity enabling it to thrive in otherwise inhospitable environments. This includes the ability to metabolize both L- and D-amino acids, which is an underappreciated trait among bacteria. D-amino acids are stereoisomers of the proteinogenic L-amino acids, and have recently been shown to play major roles in bacterial physiology, including roles in cell wall modification, biofilm formation and disassembly, and spore germination. We aim to identify the genes encoding enzymes that act on D-amino acids, biochemically characterize the enzymes, and establish the pathways allowing this strain to metabolize D-amino acids. Genomic libraries of P. putida KT2440 were constructed in Leucine, Proline, Aspartate, Tyrosine, and Lysine amino acid E. coli auxotrophs. Each library was screened on minimal media containing the corresponding D-amino acid in order to determine the genomic regions that rescued the auxotrophic phenotype. Genes recovered from the screen were identified based on comparison with the annotated genome sequence. Three genes rescuing growth were cloned, verified, and their proteins were purified via Nickel/NTA affinity chromatography. These genes, annotated as putative racemases, were assayed for amino acid racemase activity against a collection of D- and L-amino acids. The products were derivatized with Marfey’s reagent in order to discriminate between the two enantiomers of chiral amino acids and were then analyzed by HPLC. The data we have collected so far indicate that one of the enzymes (annotated as an Alanine racemase) appears to be a broad spectrum racemase, exhibiting the highest activity on D-Lysine, followed by D-Arginine, D-Methionine, D-Serine, and D-Leucine; the second enzyme (also annotated as an Alanine racemase) appears to be a more specific racemase and shows most enzymatic activity when D-Alanine is used as a substrate. The enzyme annotated as Proline racemase has not shown activity on any of the amino acids tested to this point. In prospective, we would like to obtain a complete picture of the activity of the above racemases (all proteinogenic chiral L-amino acids and the corresponding D-enantiomers) as well as characterize additional racemases and other enzymes, identified from our screens.

26B. Use of 16S rRNA Deep Sequencing to Identify Bacteria Specific for Cattle Manure and Pathogens Alexandra Rogers, Erik Holweg, Spencer Cate, Alice Layton, Dan Williams, Gary Sayler The Center for Environmental Biotechnology The University of Tennessee, Knoxville Fecal contamination of waterways can lead to disease in humans and in rural areas cattle are often a primary source of fecal contamination. Bacterial pathogens of concern originating from beef and dairy cow manure include E. coli and Streptococcus. The objective of this research was to use high-throughput 454 amplicon DNA sequencing of dairy and beef manure samples to identify bacterial species found in these sample types. DNA extractions were performed using the FastDNA Kit followed by clean-up of these samples using the Qiagen Kit. PCR ampliflications using primers for bacterial 16S rDNA were performed followed by amplification using internal barcoded 16S rDNA 454 sequencing fusion primers. Following 454 sequencing, bioinformatic and statistical techniques were used to determine if a significant relationship exists between the types of bacteria found in these groups of samples. The sequences were evaluated using Ribosomal Database Project, Mothur and the Metagenomics RAST. Based on principal component analysis, each individual group of samples demonstrated clustering within its own group, e.g. beef versus dairy. The majority of beef and dairy manure samples contained 0.01 to 1.33% E. coli and 0.01 to 0.21% Streptococcus. In addition, other pathogens were found including Clostridium difficile and Camplyobacter. These results demonstrated that 454 amplicon sequencing of manure samples can be used to identify the types of bacteria present in each group of samples. Thus, amplicon sequencing analysis can lead to efficient identification of pathogens in fecal contaminated water.

27A. Physico-Chemical Characterization of Cryoturbated Soils from Barrow, Alaska Authors: Taniya Roy Chowdhury 1, Baohua Gu 2, Dwayne A. Elias 2, Tommy J. Phelps 2, Liyuan Liang 2 and David E. Graham 1 1Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 2Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN A central focus of the U.S. DOE Next-Generation Ecosystem Experiments (NGEE Arctic) is to advance understanding of processes and prediction of ecosystem-climate feedbacks in the rapidly changing ecosystems of the Arctic tundra through a combination of field and laboratory studies, and multiscale model simulation. This study presents the physico-chemical characteristics of one of the three sites representative of permafrost degradation – the low-centered polygons located at the Barrow Environmental Observatory, Barrow, Alaska. The low-centered polygons were further divided into three distinct microtopography, viz. trough, edge, and center. Intact soil cores from each microtopographic location were collected in April, 2012 and sectioned into organic horizon, active layer, and permafrost with average depths of 0 - 10, 10 - 45 and 45 - 80 cm respectively. Each horizon was homogenized for characterization and microcosm study of methane fluxes. Organic horizons had the lowest mean pH values (4.5) with no significant difference between the microtopographies. Mean gravimetric water -1 content ( ɵg= 8.4 gH 2O g dry soil) was significantly higher in the organic horizons of the trough and the highest mean organic carbon (43.5 %) was observed in the organic horizons at the center of the low- centered polygons. The C:N ratio ranged from 19 – 20 with no difference between the horizons and -1 microtopographic sites. Mean pH (5.2) and ɵg (3.65 gH 2O g dry soil) of the permafrost was higher than -1 those for active layer (pH= 4.93, ɵg=0.99 gH 2O g dry soil). Results from an in-depth physico-chemical characterization will facilitate our better understanding of the critical surface-subsurface processes like permafrost degradation, mechanisms and rates associated with organic carbon decomposition in Arctic soils.

37A. C60 fullerenes reduce mercury bioavailability in aqueous solutions Wen-Juan Shi, Jie Zhuang, Tingting Xu, Clara Beasley, Steven Ripp, Alice Layton, Fu-Min Menn, Gary Sayler Department of Biosystems Engineering and Soil Science, Institute for a Secure and Sustainable Environment, and the Center for Environmental Biotechnology, The University of Tennessee, Knoxville, and the Key Laboratory of Northwestern Water Resources and Ecological Environment, Xi’An University of Technology, Xi’An 710048, China The effects of C60 fullerenes on mercury bioavailability and sorption were investigated at different C60 dosages, reaction times, and pH ranges using the merR :: luxCDABE bioluminescent bioreporter Escherichia coli ARL1. The results demonstrated that the bioavailability of mercury (Hg 2+ ) decreased with increasing C60 dosage. Approximately 30% of aqueous mercury became biologically unavailable two hours after interaction with C60 at a mass ratio of C60 to mercury as low as 0.01. However, a further five-order increase of C60 concentration resulted in only a 20% additional decrease in bioavailability. If this reduction in bioluminescence output is attributable to mercury sorption on C60, each one-order increase of C60 concentration caused a decrease in the partitioning coefficient ( Kd) of mercury on C60 by 0.86-order. This relationship implies the presence of a small portion of high mercury-affinitive sites on C60. In addition, lowering the solution pH from 7.2 to 5.8 decreased mercury bioavailability due likely to the increase in mercury association with C60. These results suggest that C60 may be able to decrease the methylation of mercury (a more toxic form of mercury) while weakening the negative effect of C60 on biological cells.

28B. Defining the Essential Community Supporting Dehalococcoides Reductive Dechlorination Activity Burcu Şim şir 1, Kirsti M. Ritalahti 2,3 and Frank E. Löffler 1,2,3 1 Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN 37996, 2 Department of Microbiology, University of Tennessee, Knoxville, TN 37996, 3 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 Microbial anaerobic reductive dechlorination plays a major role in the transformation and detoxification of chlorinated solvents. Dehalococcoides mccartyi strains, the keystone bacteria for chlorinated ethene detoxification, depend on community members to supply hydrogen and essential growth factors (e.g., vitamin B 12 ). Therefore, to meaningfully predict and stimulate reductive dechlorination activity for bioremediation strategies, it is necessary to monitor the dechlorinating populations and the relevant accessory community members. To explore key dechlorinators and their community interactions, enrichment cultures were derived from sediments from a chlorinated solvent-contaminated site (Third Creek, Knoxville, TN). Enrichment cultures were amended with lactate as electron donor and a chlorinated ethene or ethane (i.e., PCE, TCE, cDCE, VC, 1,1,1-TCA or 1,1-DCA). In addition, PCE- dechlorinating enrichment cultures were maintained with/without vitamin B 12 addition. Chlorinated ethenes were dechlorinated to ethene, 1,1,1-TCA and 1,1-DCA to chloroethane, and chloroform was dechlorinated to dichloromethane, which was further degraded. 454 pyrotag sequencing and quantitative real-time PCR (qPCR) characterized changes in community structure during the enrichment process and monitored the abundance of known dechlorinators (e.g., Dehalococcoides , Dehalogenimonas , Dehalobacter ). Results indicated that Dehalococcoides strains contributed up to 60% of the bacterial 16S rRNA genes in the enrichments maintained with chlorinated ethenes, while Dehalobacter strains dominated cultures amended with chlorinated ethanes and chloroform. Interestingly, Clostridium spp., Acetobacterium spp., and Sedimentbacter spp. were enriched in the enrichments amended with chlorinated ethenes and the Dehalococcoides 16S rRNA gene sequences found formed a distinct cluster, separate from the known groups. Molecular analysis suggested that stable consortia were established, and current efforts are focused in characterizing the specific functions of individual community members. The detailed characterization of the ecophysiologies of keystone dechlorinators aims to implement bioremediation practices with greater efficiency and predictable outcomes.

40B. Cold Acclimation: What Allows P. fluorescens HK44 to Function at Low Temperatures? Abby Smartt 1,2 , Alice Layton 1,2 , Elizabeth Fozo 1, Steven Ripp 1,2 , and Gary Sayler 1,2,3 1 Department of Microbiology, University of Tennessee, Knoxville 2 Center for Environmental Biotechnology, University of Tennessee, Knoxville 3 Joint Institute of Biological Sciences, Oak Ridge National Laboratory, Oak Ridge, TN

Pseudomonas fluorescens HK44 is a psychrotolerant rhizosphere organism, capable of growing at temperatures ranging from 4˚C to 32˚C. Rhizospheric P. fluorescens often support plant growth and have been shown to help plants adapt and survive cold weather. While past studies have examined the cold shock mechanisms of cold tolerant organisms, there is an overall lack of knowledge of the mechanisms that allow psychrotolerant organisms to thrive at low temperatures. Here we look at P. fluorescens HK44 as a model organism to explore growth rates across temperatures, as well as mine the genome for genes that may serve in cold acclimation. There are seven potential cold shock genes that have been identified within the HK44 genome, of which six are considered genes for major cold shock proteins. The surrounding genes are being investigated to determine their potential role in cold acclimation. These key genes will be observed for expression levels using RNAseq to determine if they have higher expression levels at low temperatures, providing insight into the mechanism(s) of cold acclimation in psychrotolerant organisms.

29A. Freshwater bloom metagenomics: what makes Microcystis bloom? M.M. Steffen 1, T.C. Effler 1, Z. Li 2, G.L. Boyer 3, L.J. Hauser 1,2,4 , S.W. Wilhelm 1 1. Department of Microbiology, University of Tennessee, Knoxville, tn, usa 2. Graduate School of Genome Sciences and Technology, University of Tennessee, Knoxville, tn, usa 3. Department of Chemistry, College of Environmental Science and Forestry, State University of New York, Syracuse, ny, usa 4. Oak Ridge National Laboratory, Oak Ridge, tn, usa Toxic blooms of cyanobacteria occur annually in important freshwater resources worldwide. Production of the hepatotoxin microcystin by members of the genus Microcystis and others is a major concern associated with these blooms. Although this organism has been identified as part of bloom communities on multiple continents, questions remain as to what factors drive large scale bloom formation. To establish the role of biotic factors in Microcystis bloom formation and success, shotgun sequencing via the 454 titanium chemistry platform was used to construct metagenomes from blooms in Lake Erie (USA/Canada), Lake Tai ( Taihu, China), and Grand Lake St Marys (OH, USA). Recruitment of Microcystis spp. reads was used to determine genomic differences between environmental Microcystis and the sequenced isolate used as a laboratory model ( M. aeruginosa NIES 843). Six metagenomic islands were identified in the recruitment plots generated from the Lake Erie and Taihu data sets, indicating regions in bloom-associated Microcystis spp. that may be genetically divergent from the lab isolate. Phylogenetic profiles established from each bloom community showed different community composition between the North American Lakes and Taihu . Despite differences in phylogenetic makeup, functional potential of the communities remained relatively static. This result represents a recent development in microbial ecology that suggests microbial community dynamics may be a reflection of function rather than phylogenetic makeup.

30B. Microbial Diversity on Weathered Cool Roof Materials Samantha Stewart, Karen Cheng, and Susan Pfiffner Center for Environmental Biotechnology, University of Tennessee, Knoxville Roofing industry standards require a three year long process of evaluating roofing materials prior to marketing, which in turn reduces both competiveness and efficacy of product development. Previous studies of roof materials have shown solar reflectance decreases significantly within the first two years of use, which suggests degradation to be caused in part by biofouling from microbial communities. This project can expand the knowledge of microbial diversity on roofing tiles in order to benefit the research and development of cool roof coating and materials. In continuation of earlier work, that demonstrated the importance of microbial and chemical elements in relation to roofing material, samples from various environmental roof materials at different geographic locations were extracted for genomic DNA and were cultured for the isolation of fungi and bacteria. Roofing samples with varying exposure time (4 to14 years) were collected from Pennsylvania, Tennessee, Arizona, Minnesota, and Florida. To generate a more comprehensive understanding of the microbial community membership, the community genomic DNA samples were subjected to 454 pyrosequencing to examine the prokaryotic diversity by targeting the bacterial V3 16S rRNA gene region. Florida samples had the highest abundance of Cyanobacteria, while Pennsylvania had the highest abundance of Protobacteria, and Tennessee had the highest abundance of Actinobacteria. To identify fungi that had been isolated from the samples, select fungal isolates were grown in potato dextrose broth and then extracted using the MP Bio FastDNA © Spin Kit for Soil. 18s PCR was performed prior to sample preparation for sequencing. Using LROR and ITSF1 primers, the most dominant fungal genus present across all samples were Hypocreasceae, Trichoderma, Ascomycete and Aspergillus. With identification of dominant fungi and the potential for symbiotic microbial relationships, a better understanding of the roof microbial communities can be developed to further examine the environmental conditions and nutritional requirements leading to biofouling on roofing materials. This knowledge can then be used for research and development of preventing such degradation on roofing materials.

31A. Continuous Monitoring of Hormonally Active Compounds in Effluents from Hallsdale-Powell Wastewater Treatment Facility at Knoxville, TN Jun Wang, Melanie Eldridge, Fu-min Menn, Gary Sayler Endocrine disruptive chemicals (EDCs) have drawn great public concern due to their harmful health impact on both human beings and animals. Wastewater treatment process is the major barrier in preventing EDCs’ release into aquatic environment. However, there is no established standard method for evaluating the performance of wastewater treatment in EDCs’ removal. Traditional wastewater treatment uses concentrated microorganisms (activated sludge) for organic compounds removal and secondary clarification for water-solid separation. While an emerging technology called membrane bioreactor (MBR) uses filter for water-solid separation, allowing higher density of biomass and greater loading capacity. Hallsdale-Powell Utility District (HPUD) at Knoxville, TN recently adopted the state-of-the-art MBR, running paralleled with its traditional activated sludge process. This setup provides great opportunity for comparing the performance of MBR and conventional activated sludge in EDCs removal. In this study, we performed a six months continuous monitoring on the effluent from HPUD using high throughput yeast bioluminescent assay. Effluent from both traditional activated sludge and MBR in HPUD were sampled each week and extracted using established solid phase extraction method. Extraction efficiency was evaluated using GC-MS. Standardized bioluminescent yeast-based bioassay was used to evaluate the estrogenic/androgenic equivalency of each sample. Our results showed that androgenic compounds are attenuated in HPUD to below-detection-limit level. Estrogenic compounds can be detected in effluents from both MBR and traditional activated sludge. Membrane bioreactor produced effluent with on average ~10 fold less estrogenic equivalency than that of the traditional activated sludge.

32B. Development of a synthetic pathway for alkane production in Saccharomyces cerevisiae James Webb 1, Dan Close 2, and Gary Sayler 1,2,3 1Department of Ecology and Evolutionary Biology, The University of Tennessee, Knoxville, TN 37996 2The Joint Institute for Biological Sciences, The University of Tennessee, Knoxville, TN 37996 3Department of Microbiology, The University of Tennessee, Knoxville, TN 37996 The United States is the largest petroleum consumer in the world, needing to import greater than 50% of its oil to meet usage demands. One solution for assuaging this importation burden has been the development biologically produced “drop-in-ready” hydrocarbon biofuels. These biohydrocarbon fuels can utilize existing infrastructure for storage and distribution and maintain a similar energy content to existing fuels, allowing for their direct use alongside traditional fossil fuels. However, major hurdles, such as a lack of manufacturing capabilities from industrially relevant organisms and the relatively difficult large-scale exploitation of organisms with natural biohydrocarbon production pathways, have prevented their large-scale adoption. To overcome these challenges we here demonstrate the proof-in- principle production of a biohydrocarbon fuel product from the industrially relevant yeast Saccharomyces cerevisiae. Biohydrocarbon production was established through the expression of a synthetic pathway consisting of two distinct units. An aldehyde production unit was established by expressing a modified version of the bacterial luciferase gene cassette, while the second unit, consisting of the Nostoc punctiforme aldehyde decarbonylase gene Npun R1711, converted the aldehyde to an alkane. Expression of this pathway in wild type S. cerevisiae resulted in approximately 10 µg/ml per unit OD 600 hydrocarbon production, with 7 µg/ml of the product retained within the yeast and 3 µg/ml excreted naturally into the media. Computational modeling of S. cerevisiae ’s metabolic pathways suggests that these production levels can be significantly enhanced through genetic alterations to increase the metabolic flux through this novel exogenous pathway.

33A. Effects of viral lysis on carbon cycling in marine microbial communities: assaying extracellular enzymes and tracking size fractions of released dissolved organic carbon Austen T. Webber, Jasmine P. Vazin, Andrew D. Steen and Steven W. Wilhelm. Department of Microbiology, The University of Tennessee, Knoxville, TN. USA. Oceanic microbial communities are important in the formation and recycling of dissolved organic carbon (DOC). Cell disruption due to viral lysis releases fresh cell biomass into the dissolved phase, which may serve as a carbon source for other organisms. This biomass may contain active enzymes that have the ability to hydrolyze existing DOC. Here we report the effects of viral lysis of two strains of bacteria, Sulfitobacter CB2047 and Vibrio alginolyticus isolate PWH3a, on the size fractions of DOC generated during cell lysis as well as on the activities of extracellular enzymes ( β-glucosidase and two peptidases, chymotrypsin, and leucyl aminopeptidase). Viral lysis of Vibrio PWH3a increased extracellular chymotrypsin activity substantially, implying that intracellular chymotrypsin was released into the bulk medium during viral lysis. In contrast, leucyl aminopeptidase activity decreased immediately after lysis, suggesting it is actively produced and released by growing cells in the absence of viruses. In parallel with the above work we are also assessing the effect of viral lysis on the fate of microbial biomass: this involves labeling microbial biomass with 14 C, and tracking the various size fractions of dissolved and particulate organic carbon that are generated during this process. Overall, our goal is to contribute to the understanding of the effect of viruses on extracellular enzyme activity and DOC composition and how these may shape the marine carbon cycle.

34B. Examining heterologous T cell suppression in Plasmodium infected mice Chelsi White, Nathan Schmidt Ph.D. University of Tennessee, Knoxville Malaria is a deadly disease caused by a parasite of the genus Plasmodium . This parasite infects approximately 300 million people annually, with 1 million of those infections being lethal. The parasite’s complex life cycle enables it to evade the immune system extremely well, making it difficult to develop an efficacious vaccine. The blood stage of the Plasmodium life cycle has been demonstrated to be immunosuppressive, which could potentially alter immune responses to other infectious agents or vaccines. At present, it is not known to what extent Plasmodium blood stage infections suppress immune responses to heterologous infections. To address this question we used two rodent-specific Plasmodium species, P. chabaudi and P. yoelii that cause chronic and acute infections, respectively , to determine to what extent they suppress T cell responses to a subsequent Listeria monocytogenes infection. We show here that both P. chabaudi and P. yoelii suppress L. monocytogenes -specific T cell responses. Immunosuppression is most dramatic during the initial two weeks of the Plasmodium infection. Curiously, while T cell responses to Plasmodium recover relatively quickly, even in the presence of high parasitemia, clearance of L. monocytogenes by the innate immune system remains suppressed for many weeks after resolution of a P. yoelii infection. These data suggest that Plasmodium blood stage infections differentially suppress innate and adaptive immune responses to L. monocytogenes . Given a recent recommendation that vaccines be administered, in malaria-endemic areas, even in the presence of clinical malaria, our data suggest this policy may not be beneficial especially for those that aim to induce T cell responses.

35A. Evidence for an Association Between an Arthropod, an Endosymbiont and a Bacteriophage. DONNA WILLIAMS*, SEBASTIAN SANCHEZ, T. KEITH PHILIPS and RODNEY A. KING. Department of Biology, Western Kentucky University, Bowling Green, KY 42101. Wolbachia are gram-negative, maternally inherited, obligate intracellular symbionts that infect a wide variety of invertebrates including medical and hygienic pests. In arthropods, the infections cause a number of reproductive alterations including cytoplasmic incompatibility, male killing and feminization. It is estimated that 20-75% of all insects are infected with Wolbachia but evidence for Wolbachia in the beetle superfamily Bostrichoidea , has not been reported. To determine if members of the Bostrichoidea are infected with Wolbachia , we used polymerase chain reaction (PCR) to amplify selected Wolbachia genes. The analysis of the PCR reaction products by agarose gel electrophoresis showed that the beetles are infected with this bacterium. We used a similar analysis and found that the endosymboint is itself parasitized by a bacteriophage called WO. This unusual tripartite association has important implications for the evolution of Wolbachia and its arthropod host.

36B. Omics-driven analysis of nutrient metabolism in harmful brown tides Wurch LL; Bertrand E; Gobler CJ; Saito M, Walker E; Dyhrman ST Brown tides, caused by the phytoplankton Aureococcus anophagefferens form in bays and estuaries in the Eastern U.S as well as South Africa. Classified as a harmful algal bloom (HAB), brown tides degrade coastal ecosystems and adversely impact commercially important shellfish. Recent analysis of the A. anophagefferens CCMP 1984 genome revealed that this organism contains a large number of genes for nitrogen (N) and phosphorus (P) metabolism. Here, global transcript (N and P) and protein (P only) profiling were performed to screen for genes involved in N and P metabolism. Transcripts and proteins associated with P deficiency included a phosphate transporter (PTA3), a 5'-nucleotidase and an alkaline phosphatase, while transcripts associated with N deficiency included an ammonium transporter and a xantinie/uracil/vitaminC permease (XUV). Additional studies using targeted gene expression have confirmed that PTA3 and XUV expression are tightly linked to P and N availability, respectively. Analysis of these genes in natural populations reveal that P deficiency may be playing a previously unforeseen role during early stages of brown tide formation.

38B. Anaerobic Reductive Dechlorination in Low pH Environments Yi Yang 1, Tyler Marcet 2, Natalie Capiro 2, Kurt D. Pennell 2, Frank E. Löffler 1,3,4 1Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN 2Department of Civil and Environmental Engineering, Tufts University, MA 3Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 4Department of Microbiology, University of Tennessee, Knoxville, TN 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. Engineered bioremediation, including biostimulation and bioaugmentation, is a promising technology to clean those PCE and/or TCE contaminated sites. However, during bioremediating the contaminated sites, release of HCl (strong acid) from anaerobic reductive dechlorination may lower the pH of groundwater. Besides, another main source of acidity comes from the fermentation of additive electron donors such as alcohols, organic acids and etc. Decreasing pH has been proved to be detrimental to the microbes that dechlorinated PCE or TCE. We intended to enrich and isolate microorganisms, which can perform anaerobic reductive dechlorination at low pH environments, by establishing microcosms, which will be beneficial to in situ bioremediation. We also screened some existing cultures for dechlorinating activity at low pH and determined the pH tolerance of consortium BDI, which had been successfully, applied for in situ bioremediation. Besides, this study investigated and explored the effects of solids on BDI consortium under low pH conditions. Generally, various dechlorinating pure cultures and consortium BDI show highest dechlorination rates and extent at circumneutral pH. Only Sulfurospirillum multivorans among tested cultures dechlorinated PCE to cDCE at pH 5.5. The screening efforts suggest that microbes capable of dechlorination below pH 5.5 are not common. It was observed that solids play an important role for enhancing microbial activities under low pH conditions.

39A. Temporal Gene Expression Profile in Mice Infected with Distinct lineages of Toxoplasma gondii Li Yu, Rachel D. Hill, Julia S. Gouffon, Chunlei Su Department of microbiology, The University of Tennessee, Knoxville, TN 37996-0845 Type I, II and III lineages of T. gondii have distinct virulence phenotypes in mice, with Type I being highly virulent and killing mice in two weeks, whereas Type II and III being less virulent and establishing chronic infection. However, the knowledge of host response to these different lineages is limited to in vitro model and limited sampling time. To define transcriptional patterns and biological processes that characterize the host response to different lineages of T. gondii , a time course of transcriptional profiles in mice were investigated using DNA microarray. For each of the three T. gondii lineaages, 500 tachyzoites were prepared in cell culture and injected to CD-1 outbred mice by peritoneal injection. At days 1, 2, 3, 4, 5 and 7 post infections, cells from mouse peritoneal cavity were collected to isolate RNA for gene transcription analysis using Affymetrix mouse arrays. The data was processed using Partek software. The results showed that the number of differentially expressed genes (≥2-fold ) increased gradually from day 1 to day 5 post infection in Type I, II, and III infections, and the number of down regulated genes was greater than that of up-regulated genes. However, at day 7 post infection, the number of differentially expressed genes decreased dramatically, and the number of up-regulated genes was greater than that of down-regulated genes in all three infection group. From day 1 to day 3, the numbers of differentially expressed genes in Type II and III infection were similar, and were greater than that in Type I infection. However, at day 4 and day 5, Type I and II infection had the similar number of differentially expressed genes, which are greater than that in Type III infection. At day 7, Type I infection owned the largest number of 2 fold change genes, while the number in Type II infection was the lowest. Gene ontology (GO) analysis showed the differentially expressed genes were involved in many biological processes. Cell cycle, mitosis, immune response, inflammatory response, cell division, cellular response to interferon- beta, innate immune response, DNA replication, and cellular response to interferon-gamma were top function groups affected. Type II and III infection shared similar time course expression pattern in these function groups, while Type I displayed an unique expression pattern. To confirm the reliability of microarray data, four up regulated genes and one down regulated gene at five different time points were selected for quantitative RT-PCR analysis. The quantitative RT-PCR expression data showed strong correlation with the microarray expression data (R 2 =0.796). Results of this study revealed dynamic changes of host responses to three distinct strains of T. gondii . Currently we are carrying out network analysis to reveal major cause-effect relationship among a large number of transcripts to identify genes that control the outcomes of infection in host.