2 0 0 8 M e e t i n g

O c t o b e r 1 0 , 2 0 0 8

Department of Biology

Frank Family Science Center, Guilford College, Greensboro, NC

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Schedule

Preliminaries

8:00

RegistrationPoster and talk set-upCoffee breakAward Committees meeting/organization

8:45 Melanie Lee-Brown &Adrienne Israel Welcome & Introductory comments

Session 1 : Melanie Lee-Brown, Chair

9:00 Kristina Brzoza-Lewis Roles of MyD88 and type I IFN signaling in Listeria induced DC maturation

9:15 Kendra Hitz Expression of ahpCD and katE is coordinately regulated by OxyR and Irr in Brucella abortus 2308

9:30 Neelima SukumarImmunization with Bordetella Colonization Factor A (BcfA) Pro-tects Mice against Respiratory Challenge with Bordetella bron-chiseptica.

9:45 Ossie Dyson Establishing a Link between Cellular Attachment and KSHV Reacti-vation

10:00 Poster session 1 (unattended)Coffee break

Session 2 : Daniel Williams, Chair

10:45 Kenny Kuo Role of SsrA after transcription blockage induced by protein-DNA crosslinks

11:00 Adam Kulp Independent mechanisms of envelope stress relief via bacterial outer membrane vesicles

11:15 Alice Lee The Use of Archaeal Extremophile Genes to Dampen ROS Signaling in Plant Systems

11:30 Soo Chan Lee Microsporidia evolved from ancestral sexual fungi

11:45 Eric AndersonA B. abortus mutant lacking a homolog of the rhizobial iron regu-lator RirA displays altered iron acquisition in vitro and significant attenuation in experimentally infected mice

12:00 Lunch

12:45 Poster session 2 (Even numbered poster should be attended)

Session 3 : Kathy Zarilla, Chair

1:30 Ann Mathysse Binding of Escherichia coli O157:H7 to cut lettuce leaves.

1:45 MD Motaleb Motility and chemotaxis in the pathogenesis of Lyme disease

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Schedule

2:00 Deborah Threadgill The General System of Protein Glycosylation in Campylobacter Species

2:15 Margaret KanipesNC Invitational Talk Analysis of Campylobacter Lipooligosaccharides

2:50 Intermission

Plenary session : Kathy Zarilla, Chair

3:00 Eugene MadsenASM Branch Lecture

Bioremediation Demystified: Microbial roles and molecular in-sights

4:00 Poster session 3 (Odd numbered poster should be attended)Coffee break

Postscript

4:45 Kathy Zarilla Concluding remarksAwards

5:00 Jim Brown Business meetingOfficer election

5:30 Adjournment

Meeting Sponsors

The American Society for Microbiology

Guilford College(Melanie Lee-Brown)

Nikon Instruments(Larry Kordon)

Fisher Scientific(Tim Stewart)

ISC BioExpress(Rob Blackman)

Association of Southeastern Biologists

(Tim Atkinson)

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Abstracts (talks)

1.1) Roles of MyD88 and type I IFN signaling in Listeria induced DC maturation

Kristina L. Brzoza-Lewis, Curtis J. Henry, Jason M. Grayson, & Elizabeth M. HiltboldDepartment of Microbiology and Immunology, Wake Forest University School of Medicine

Dendritic cell maturation is an important step in the development of protective immunity to the intra-cellular bacterial pathogen, Listeria monocytogenes (L.m.). We have previously demonstrated that L.m. cytosolic entry is necessary to induce full DC maturation as measured by the upregulation of costimulatory molecules and secretion of cytokines. This enhanced DC maturation resulted in optimal priming of naive T cells. Both the MyD88 and type I IFN signaling pathways are vital in the generation of a response to Listeria due to the multiple TLR ligands present on an intact bacterium as well as the large amounts of IFN- that are produced upon cytosolic entry. Therefore, in order to address the regulation of L.m.-induced DC maturation, we generated DC from the bone marrow of both MyD88-/- and IFNAR-/- mice and infected them with L.m. We ascertained that MyD88 is important for the pro-duction of cytokines while signaling through IFNAR is required for the upregulation of costimulatory molecules in response to L.m. Therefore, we have observed a differential regulation of Listeria-induced DC maturation responses using an in vitro culture system. In order to determine if the same pattern of regulation of DC maturation induced by Listeria could also be observed in vivo, we infected WT, MyD88-/-, and IFNAR-/- mice with Listeria and assessed the maturation of splenic DC. We ob-served a decrease in cytokine production by DC isolated from MyD88-/- mice in agreement with our in vitro findings. However, in contrast to our in vitro findings, DC isolated from L.m.-infected IFNAR-/- mice were able to upregulate costimulatory molecules. We therefore propose that in IFNAR-/- mice, exogenous factors produced by other cell types induce the upregulation of costimulatory molecules on splenic DC. However, L.m.-induced DC cytokine production is regulated in vitro and in vivo by the TLR pathway.

1.2) Expression of ahpCD and katE is coordinately regulated by OxyR and Irr in Brucella abortus 2308

Kendra Hitz and R. Martin Roop IIDepartment of Microbiology and Immunology, East Carolina University

Brucella abortus is a facultative intracellular pathogen that causes abortion and infertility in cattle and a chronic, relapsing febrile illness in humans. The key to this bacterium’s virulence is its capacity to survive and replicate for prolonged periods in host macrophages. In order to maintain this chronic in-tracellular infection, the brucellae must successfully resist exposure to the toxic reactive oxygen in-termediates (ROIs) generated by the oxidative burst of host macrophages. Because they generate their energy by respiration, these bacteria these bacteria must also protect themselves from ROIs generated as a byproduct of aerobic metabolism. Phenotypic analysis of defined B. abortus mutants in vitro indi-cates that the periplasmic catalase (KatE) and a cytoplasmic peroxiredoxin (AhpCD) work in concert to protect B. abortus 2308 from oxidative stress. Both of these antioxidants detoxify hydrogen peroxide, but they exhibit their maximum activity at different physiological levels of this reactive oxygen spe-cies. Accordingly, experimental evidence suggests that B. abortus 2308 coordinates the expression of katE and ahpCD in response to cellular hydrogen peroxide levels, and that this coordinate expression is modulated by the transcriptional regulators OxyR and Irr. Current studies are aimed at determining the specific mechanisms by which OxyR and Irr control katE and ahpCD expression in B. abortus 2308 and defining the specific contributions of katE and ahpCD to virulence in the mouse model.

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1.3) Immunization with Bordetella Colonization Factor A (BcfA) Protects Mice against Respiratory Chal-lenge with Bordetella bronchiseptica.

Neelima Sukumar, Cheraton F. Love, Matt S. Conover, Nancy D. Kock, Purnima Dubey and Rajendar De-ora Department of Microbiology and Immunology, Wake Forest University Baptist Medical Center

Bordetella colonization factor A (BcfA) is an outermembrane immunogenic protein and is critical for efficient colonization of the murine respiratory tract. These properties of BcfA prompted us to examine its use in generating a protective immune response against B. bronchiseptica. Vaccination of mice with BcfA induced protective immunity against subsequent challenge with B. bronchiseptica in a mouse model of intranasal infection. Mice immunized with BcfA demonstrated reduced pathology in the lungs and harbored lower bacterial burdens in the respiratory tract. Immunization with BcfA led to the gen-eration of BcfA-specific antibodies in both the serum and lungs and passive immunization experiments led to the reduction of B. bronchiseptica in the trachea and the lungs. These results suggest that pro-tection after immunization with BcfA is mediated in part by antibodies against BcfA. To further investi-gate the mechanism of BcfA-induced immune clearance, we examined the role of neutrophils and macrophages. Our results demonstrate that neutrophils are critical for anti-BcfA antibody-mediated clearance and that opsonization with anti-BcfA serum enhances phagocytosis of B. bronchiseptica by murine macrophages. We show that immunization with BcfA results in the production of IFN-Î and sub-classes of IgG antibodies that are consistent with the induction of a Th1 type immune response. Our findings suggest that mechanism of BcfA-mediated immunity involves humoral and cellular responses. Finally, we demonstrate that the expression of BcfA is conserved among multiple clinical isolates of B. bronchiseptica. These results corroborate the importance of BcfA as a potential vaccine candidate and provide a model for the development of cell-free vaccines against B. bronchiseptica.

1.4) Establishing a Link between Cellular Attachment and KSHV Reactivation

Ossie F. Dyson and Shaw M. AkulaDepartment of Microbiology and Immunology, East Carolina University

Kaposi’s sarcoma-associated herpesvirus (KSHV) undergoes two stages of infection, latent and lytic, as a means of efficiently evading the host immune system. Reactivation of KSHV latency allows for the production of viral progeny followed by disease spreading and cell death. The cell cycle plays a major role in this process as cells in S phase provide the most suitable environment for virus reactivation. The current study aims to further understand the importance of the cell cycle in KSHV pathogenesis by identifying the expression pattern of several cell surface receptors during different phases of the cell cycle. Specifically, we identified integrins to be substantially elevated during S phase in epithelial cells, fibroblasts, and KSHV-infected primary effusion lymphoma (PEL) cells. KSHV-infected PEL cells utilize integrins to attach to extracellular matrix proteins. Additionally, in S phase cells supporting virus reactivation, the viral protein gB was shown to mediate attachment by binding to receptors on the surface of endothelial cells. This attachment may lead enhanced angiogenesis, an important factor regulating tumorigenesis, and spread of KSHV diseases. Taken together, we present evidence demon-strating the ability of KSHV infected cells to preferentially use virally encoded gB over cellular proteins ( ) to adhere to the endothelium depending on the state of viral replication.

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2.1) Role of SsrA after transcription blockage induced by protein-DNA crosslinks

H. Kenny Kuo, Kenneth N. KreuzerDepartment of Biochemistry, Duke University Medical Center

5-azacytidine (aza-C) and its derivatives are effective anticancer drugs for leukemia treatment. The primary inhibitory effect of aza-C is prohibition of cytosine methylation which results in methyltransferase-DNA crosslinks at cytosine methylation sites. These DNA-protein crosslinks (DPCs) can cause chromosomal rearrangements and contribute to cytotoxicity, but cells can apparently repair at least some of the DNA damage caused by DPCs. To investigate the repair mechanisms of this type of DNA damage, we performed a transposon mutagenesis screen and isolated mutants that are hypersensi-tive to aza-C using an E. coli model system. We found transposon insertion into an interesting gene, ssrA, which is involved in the co-translational quality control system for truncated and miscoding mRNAs. SsrA is a specialized RNA that functions as both tRNA and mRNA when a ribosome is stuck on a broken mRNA or mRNA without a stop codon. SsrA releases the stalled ribosomes and results in tagging the aberrant polypeptides for degradation. The hypersensitivity of the ssrA mutant strongly suggests that this protein quality control system becomes critical in the presence of aza-C. Overproduction of methyltransferase is required for aza-C hypersensitivity in ssrA mutant suggesting that DPC formation is involved. In addition, Western blot analysis revealed that the methyltransferase level was similar in the mutant and the wildtype cells. One attractive model is that methyltransferase-DNA crosslinks induced by aza-C cause transcription blockage, which in turn blocks translating ribosomes. The ssrA pathway may then be essential to release the stalled ribosomes, perhaps the transcription complex by some mechanism. To test this hypothesis, we analyzed sensitivity to streptolydigin, an antibiotic that blocks transcription elongation. The ssrA mutant is indeed 3-fold more sensitive to streptolydigin. These re-sults strongly suggest that ssrA is directly involved in rescuing transcriptional-translation blockage after DPC formation or other transcriptional blockage events.

2.2) Independent mechanisms of envelope stress relief via bacterial outer membrane vesicles

Adam J. Kulp, Amanda J. McBroom, & Meta KuehnDepartment of Biochemistry, Duke University

Outer membrane vesicles (OMVs) are proteoliposomes, 20-200 nm in diameter, that are formed when a portion of the Gram-negative bacterial outer membrane buds and releases from the cell. OMVs consist of outer membrane proteins and lipids encapsulating periplasmic material, however the content of OMVs does not directly reflect the content of the respective bacterial envelope compartments. Altered protein content in the OMVs suggests a mechanism exists to enrich particular components during OMV formation. Vesiculation is a novel envelope stress response pathway that is upregulated when bacteria experience environmental stress and appears to aid in the removal of misfolded proteins from the bac-terial envelope. In fact, it has been shown that overproduction of OMVs enhances survival of Escheri-chia coli in adverse growth conditions. We are interested in uncovering the mechanism of vesiculation and OMV cargo packaging and how these processes are triggered in stressed cells. We have studied the amount and content of OMVs produced by E. coli overexpressing engineered mimics of misfolded enve-lope proteins. These proteins are fusions between periplasmic proteins and the C-terminus of an outer membrane protein. We found that particular misfolded protein mimics were concentrated in OMVs compared to housekeeping proteins. In this case, misfolded cargo are efficiently exported without ne-cessitating increased vesiculation, although in some cases, hypervesiculation also occurred. Fusion constructs that were not preferentially packaged into vesicles induced a strong increase in vesicula-tion. In this case, hypervesiculation alone was triggered as the envelope stress relief outlet. We ob-served differences in complexes formed by the mimics in the envelope and in the OMVs, and we antici-pate that further research into these differences will reveal the molecular basis of these different OMV export strategies. Our data suggest that preferential cargo inclusion into OMVs and OMV overproduction are independent mechanisms that serve to remove misfolded, toxic proteins from the Gram-negative bacterial envelope.

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2.3) The Use of Archaeal Extremophile Genes to Dampen ROS Signaling in Plant Systems

Alice M. Lee, Mikyoung Lee Ji, Yang Ju Im, Amy Grunden and Wendy BossDepartment of Microbiology, North Carolina State University

Reactive oxygen species, ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals, are pro-duced as part of basal metabolism and in response to environmental cues. The accumulation of ROS can damage living organisms. Plants, like other aerobic organisms, have ROS metabolizing enzymes such as superoxide dismutase (SOD), catalase and peroxidase to metabolize superoxide and hydrogen peroxide to prevent the buildup of toxic free radicals. Studies have shown that increasing the produc-tion of plant ROS metabolizing enzymes will enhance plant tolerance to high intensity light and envi-ronmental stresses. These experiments to genetically regulate ROS show great promise and provide proof of concept that genetically regulating ROS levels can be beneficial to plant growth and develop-ment. However, these studies have also shown that increased plant survival is limited by the tempera-ture range and localization of the enzymes produced and the need for compensatory mechanisms to reduce secondary oxygen species. Thus, in this study, we have introduced into plants a more effective and efficient ROS metabolizing enzyme, superoxide reductase, SOR, that reduces the level of superox-ide in the cytosol and does not increase endogenous hydrogen peroxide. Superoxide reductase (SOR) is part of a pathway in the hyperthermophilic archaeon, Pyrococcus furiosus, that is involved in reactive oxygen species (ROS) detoxification. SOR reduces superoxide, but unlike SOD, does not produce oxy-gen. It is extremely stable with a functional temperature range of 4-100°C, and since it is not found in plants, should not be regulated either transcriptionally or posttranscriptionally in the same manner as SOD. This may aid plants in the relief of oxidative stress that occurs during extreme environmental conditions. Our hypothesis is that we can use genes from extremophilic organisms to improve plant survival by dampening ROS. Previously, we have shown that an archaeal gene can be expressed in plants to produce a functional protein in NT-1 tobacco cells and then in the model plant system Arabi-dopsis thaliana. Experiments demonstrated that SOR transgenic Arabidopsis plants do not have in-creased endogenous ROS, and are more heat and drought tolerant. Future studies are aimed at trans-ferring this technology to crop plants which are constantly exposed to environmental stresses.

2.4) Microsporidia evolved from ancestral sexual fungi

Soo Chan Lee, Nicolas Corradi, Edmond J. Byrnes III, Santiago Torres-Martinez, Fred S. Dietrich, Patrick J. Keeling, and Joseph HeitmanDepartment of Molecular Genetics and Microbiology, Duke University Medical Center

Microsporidia are obligate intracellular eukaryotic pathogens that primarily infect animal cells. Over 1,200 species are known, of which a dozen infect humans. Previous studies have suggested the micro-sporidia might share a common ancestor with fungi. However, the exact nature of this relationship is unclear due to the unusual nature of microsporidial genomes. Not only do they encode a greatly re-duced set of genes for comparison, but the gene sequences are also highly divergent, interfering with phylogenetic analyses. As a consequence, it is presently unclear whether microsporidia evolved from a specific fungal lineage, the identity of that lineage, or even whether microsporidia are a sister group to all fungi. Here we present evidence to address this controversial question that is independent of phy-logenetic reconstruction, but rather based on genome structure. It has previously been shown that the zygomycete Phycomyces blakesleeanus contains a sex locus encoding a high mobility group (HMG) tran-scription factor flanked by a triose phosphate transporter (TPT) and an RNA helicase. We now show that the genomic context of this sex locus is highly conserved and syntenic within three zygomycete ge-nomes, but not in ascomycete, basidiomycete, or chytridiomycete genomes. Strikingly, two microspo-ridians, Encephalitozoon cuniculi and Enterocytozoon bieneusi, harbor a conserved and syntenic ge-nomic locus with TPT, HMG, and RNA helicase genes and the TPT and HMG genes are similarly arranged in a third microsporidian, Antonospora locustae. Genome wide analyses of synteny reveal other loci where microsporidia and zygomycetes are conserved to the exclusion of all other fungi. These findings support the hypothesis that microsporidia are fungi that descend from a zygomycete ancestor, and sug-gest the microsporidia may have retained elements of an ancestral sexual cycle.

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2.5) A B. abortus mutant lacking a homolog of the rhizobial iron regulator RirA displays altered iron acquisition in vitro and significant attenuation in experimentally infected mice

Eric S. Anderson, James T. Paulley, Jennifer M. Gaines, Daniel W. Martin, Jonathan D. Todd*, Andrew W. B. Johnston*, & R. Martin Roop IIDepartment of Microbiology and Immunology, ECU School of Medicine, *School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

The ferric iron uptake regulator (Fur) is responsible for the iron-dependent regulation of siderophore biosynthesis genes in the majority of prokaryotes. However, in Brucella abortus, iron-dependent regu-lation of siderophore biosynthesis has been shown to be Fur-independent. This phenomenon of iron-responsive, but Fur-independent regulation has been noted in another member of the alpha-proteobacteria. In the plant symbiont, Rhizobium leguminosarum the rhizobial iron regulator (RirA) serves as the global regulator of iron-responsive genes. A R. leguminosarum rirA mutant demonstrates constitutive, iron-independent expression of a number of iron-responsive operons including those in-volved in heme uptake (hmuPSTUV), vicibactin siderophore uptake (fhuA and fhuCDB) and vicibactin siderophore biosynthesis (vbsC, vbsGSO and vbsADL). The Brucella abortus 2308 genome contains an open reading frame (BAB2_0678) predicted to encode a protein with 79% identity to RirA, prompting the investigation of this gene for a possible role in the regulation of Brucella iron uptake. An isogenic rirA deletion mutant was generated in B. abortus 2308 via allelic exchange and analyzed for a role in the iron-dependent regulation of siderophore biosynthesis. Although the rirA mutant showed wild-type repression of siderophore production under iron-replete conditions, loss of RirA resulted in a growth defect in iron-restricted medium, increased sensitivity to chelator and a decreased ability to use heme as an iron source in vitro, suggesting that RirA may serve as a regulator for other iron-responsive genes in B. abortus. In B. abortus, transport of heme across the outer membrane is mediated by the Brucella heme uptake protein, BhuA. To examine the contribution of RirA to the regulation of the bhuA gene, a bhuA::lacZ transcriptional reporter was evaluated in a rirA- background. Preliminary data indi-cate that RirA serves as a transcriptional activator of bhuA during growth in iron-deplete medium. Ad-ditionally, previous studies have demonstrated that BhuA is essential for virulence in the murine model of chronic infection, and consistent with a role for RirA as an activator of bhuA, a rirA mutant also dis-plays significant attenuation in both cultured murine macrophages and BALB/c mice. Studies are cur-rently underway to further evaluate this relationship and identify other potential members of the RirA regulon.

3.1) Binding of Escherichia coli O157:H7 to cut lettuce leaves.

Ann G. Matthysse and Rachel B. SmithDepartment of Biology, University of North Carolina

Transmission of pathogenic E. coli by vegetable produce has become an increasing problem in recent years. These bacteria are able to bind tightly to plant surfaces and are not easily removed by washing with water. The binding of E. coli to cut edges of lettuce leaves was rapid. More than 103 pathogenic bacteria bound to each leaf within the first five minutes of incubation of cut leaves in water containing the bacteria. After the initial rapid binding, additional bacteria bound at a much slower rate. By 3 days, there were about 106 bacteria bound per leaf. When the initial binding of bacteria carrying a gfp gene was observed in the fluorescence microscope, the majority of the bacteria bound were found at the cut end of the central vein. Few bacteria bound to the intact surface of the leaf, although some bacteria were seen in the stomata. After 3 days the bacteria had entered the leaf and significant num-bers were seen in the spaces between the cells of the leaf mesophyll. At 4o C, binding to the cut edge of lettuce leaves was reduced slightly, while bacterial entry to the inside of the leaf was reduced markedly. Mutants of the bacteria unable to make the polysaccharides poly- -1, 6-N-acetyl-D-glucosamine (PGA), cellulose, or colanic acid were all able to bind to cut leaves but the number of bac-teria bound at later times was reduced in these mutants. This rapid binding was specific for E. coli. Both laboratory K12 and pathogenic O157 strains bound. Binding of A. tumefaciens and S. meliloti was much less and much slower than binding of E. coli.

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3.2) MOTILITY AND CHEMOTAXIS IN THE PATHOGENESIS OF LYME DISEASE

-M. Motaleb1, P. Stewart2, A. Bestor2, P. Rosa2 and N. Charon3

1Dept. of Microbiology & Immunology, East Carolina University2 Dept. of Human Bacterial Pathogenesis, Rocky Mountain labs, NIH, Montana3Dept. of Microbiology, Immunology & Cell Biology, West Virginia University

Borrelia burgdorferi is the causative agent of Lyme disease. It is the most prevalent arthropod borne infection in the United States with 23,305 reported cases on 2005. The disease is a multiple-systemic disorder with various clinical manifestations including erythema migrans rash, arthritis, musculoskeletal and neurological manifestations.

B. burgdorferi exists in nature in an enzootic cycle. Ixodes scapularis ticks (commonly known as deer ticks) acquire the infection when they feed on an infected host, mainly rodents. During subsequent tick feeding, which lasts for several days, B. burgdorferi migrate from the tick midgut, pass through the salivary glands, and are then transmitted to the mammal through the saliva. B. burgdorferi is highly invasive. After being deposited in the skin following a tick bite, the spirochetes can invade many tissues including the joints, heart, and nervous system. Motil-ity and chemotaxis are likely involved in this process.

Motility and chemotaxis are critical for bacterial survival and adaptation in diverse environ-mental conditions. In several species of bacteria, motility and chemotaxis have been shown to be associated with the disease process. Results obtained using B. burgdorferi with mutations in key motility and chemotaxis genes also indicate that these activities are required for the pathogenesis of Lyme disease. These results may be useful in identifying a unique molecule or pathway (e.g., chemotaxis signaling pathway) in the organism which may lead to the develop-ment of a novel pharmacological agent to treat/prevent Lyme disease.

3.3) The General System of Protein Glycosylation in Campylobacter Species

Deborah S. Threadgill, Michael J. LaGier, Jason M. Andrus, Ian M. Carroll, and Angelika JaehrigDepartment of Genetics, University of North Carolina

Previously, a general system for protein glycosylation was reported for Campylobacter jejuni. Using lectins with defined carbohydrate specificities, we have confirmed and extended the original findings regarding C. jejuni glycoprotein production. In particular, we show that the system of general protein glycosylation although present in all strains of C. jejuni analyzed, is not necessarily active to the same level in every strain. Additionally, environmental regulators such as growth temperature and media composition impact the level of lectin reactivity. The general protein glycosylation system also exists in other Campylobacters as well as C. jejuni, and can be monitored through the use of N-acetylgalactosamine specific lectins. The importance of the general glycosylation pathway to lifestyle and pathogenesis of Campylobacter species is currently under investigation.

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The North Carolina Invitational speaker will be Margaret Kanipes, Associate Professor in the Department of Chemis-try at NC A&T State University (NCAT). Dr. Kanipes earned her Bachelor of Science in Chemistry from North Carolina A&T State University and her doctoral degree in Biological Sciences from Carnegie Mellon University. She did postdoctoral training in the Department of Biochemistry in the School of Medicine at Duke University. Before joining the faculty at NCAT, she was an Assistant Professor of Biological Sciences at Fayetteville State University. She currently holds the position of Associate Professor of Chemistry at North Carolina Agricultural and Technical State University. Research in her lab focuses on three aspects of pathogenesis by Campylobacter species: li-pooligosaccharide core diversity in C. coli, genotyping/strain identification of C. jejuni, and lipooligosaccharide assembly in C. jejuni.

Analysis of Campylobacter Lipooligosaccharides

Campylobacter is the most important cause of gastroenteritis in the United States and a frequent cause of traveler’s diarrhea in developing countries. A small but significant number of Campylobac-ter infections have also been linked to the development of Guillain-Barre Syndrome (GBS). It is be-lieved that GBS is triggered as a result of molecular mimicry between the sialic acid sugar residues on the outer core of the Campylobacter glycolipid, lipooligosaccharide (LOS) and the carbohydrate moiety of human gangliosides. LOS has been shown to be one of many virulence factors involved in important processes associated with Campylobacter-mediated infection and GBS. Little attention has been given for studies on the biosynthesis of the core oligosaccharide of LOS and its role in pathogenesis in the food-borne pathogen, Campylobacter. Therefore, we wanted to understand the assembly of the Campylobacter LOS and the role that structural diversity of the LOS, which exists among various Campylobacter species, plays in pathogenesis. To better understand the function of the LOS and what role it may play in the development of bacterial gastroenteritis and GBS, we initi-ated a study of the isolation of mutants involved in the biosynthesis of the core oligosaccharide of LOS from the Campylobacter strain 81-176. We have found that there is no major defect in invasion in any of the LOS biosynthetic mutants, except for the deepest rough, waaC. Furthermore, the LOS core of the waaF mutant defines the minimal structure for effective invasion and/or intracellular survival of C. jejuni 81-176. These studies have important implications for the role of LOS in the pathogenesis of Campylobacter-mediated infection.

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The Keynote address will be given by Eugene Madsen, Asso-ciate Professor in the Section of Microbiology at Cornell University. Dr. Madsen was trained at the undergraduate level with bachelor's degrees in Chemistry (University of California at Santa Cruz) and soil science (Oregon State University). For his graduate education, he studied Rhizobium ecology in soil (MS) and metabolism of organic pollutants (Ph.D.) at Cornell University. Interest in field work led Madsen to a postdoctoral fellowship at Rutgers University where he studied transforma-tion of iron and sulfur in fresh water streams. Interests in an-aerobic metabolism of groundwater pollutants led to a post-doctoral appointment at Penn State University. A desire to explore the northwest led Madsen to an appointment as Senior Microbiologist at an environmental restoration firm in Bozeman Montana. After a year in industry Madsen accepted an appointment at Cornell that led to his current position. His ongoing research projects have objectives that include char-acterizing soil and subsurface (ground water) microorganisms and their activities, use of molecular biology in discerning mechanisms of metabolic adaptation to pollutant compounds, understanding geochemical and physiological characteristics which both prevent and foster microbial activity, and develop-ing a rapport with the biogeochemistry of field sites so that realism is an integral part of conceptual advancements in en-vironmental microbiology.

Bioremediation Demystified: Microbial roles and molecular insights

Bioremediation is a term with many connotations.  This seminar is designed to provide a clear over-view of what bioremediation is, what it can accomplish, and how quickly.  Broad concepts will be presented that are supported by the speaker’s direct experiences in working at contaminated field sites.  Field assays and physiological, geochemical, and molecular biological evidence for bioreme-diation will be emphasized. The presentation will be organized as follows: (i) Background and defini-tions pertinent to biodegradation and bioremediation; (ii) Field experience with Natural Attenuation in groundwater:  physiological clues and mechanisms; (iii) Stable isotope probing to identify the ac-tive populations; and (iv) Insights from genomics.

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Dr. Madsen’s plenary lecture is supported by the ASM Branch Lectureship Program. The ASMBL program, formerly known as the Waksman Foundation for Microbiology Lectures Program, allows ASM branches to secure outstanding lecturers for their scientific meetings. The program has been operating for over 40 years, and lecturers continue to enhance scientific meetings at the local level.

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Poster presentations

1 Robert Bastidas Tor signaling regulates cell-cell adhesion in Candida albicans

2 Suzanna Brauer Peptide-based probe capture of Mn oxides and associated bacteria at Loihi Seamount, Hawaii

3 John Bucci Use of T-RFLP Analysis to Categorize the Microbial Diversity Found in Native Bivalve Streams in a North Carolina River Basin

4 Edmond Byrnes III Australia to North America: Implications for the global spread of Cryp-tococcus gattii based on a select VGI genotype

5 Catherine Clodfel-ter

Analyzing ‘donuts’ for flagellar force production trends during regen-eration in Chlamydomonas reinhardtti

6 Anthony DevineTracking Microbial Populations in Swine Waste Lagoons in Eastern North Carolina Using Terminal Restriction Fragment Length Polymorphism Analysis

7 Jimmy Gosse Nanoporous Latex Coatings Containing Rhodopseudomonas palustris Pigment Mutants Spatially Distributed for Hydrogen Production

8 Kristin Johnson Bacterial Community Analysis of a High Altitude Wetland Disturbed by Iron and Manganese

9 Rosemary Le Characterization of an A. salmonicida bacteriophage isolated from a NC trout population

10 Charles Li Investigation of the sex locus of a basal fungus, Mucor circinelloides

11 Zachary Parker Quantification of polyamines and NspC gene expression in Vibrio chol-erae

12 Amanda Redfern &Caitlin Baber

The Isolation and Identification of a Bacterium that causes bent neck in Roses

13 Matt Schrenk Deep Biosphere Research at East Carolina University

14 Amanda Tarnowicz Characterization of the Role of Two Genes in the Binding of Agrobacte-rium tumefaciens to Plant Surfaces

15 Nicholas Walter Elucidation of Polyamine Synthesis and Transport Sytems in Vibrio chol-erae

16 Latoya M. Mitchell Differential Responses of Dendritic Cell Subsets to the Intracellular Bac-terial Pathogen, Listeria monocytogenes

17 Marianna Feretzaki A Molecular Dissection of the Cryptococcus Unisexual Reproduction Cas-cade

18 Evan A. Menscher A Brucella abortus Fur homolog regulates manganese dependent expres-sion of the divalent cation transporter mntH.

19 Michele K.H.Malotky

Elucidation of possible target receptors for the Estrogenic Endocrine Disrupting Chemical (EEDC) Bisphenol A in the soil nematode, Caenor-habitis elegans.

20 Keisha Findley Evolution of MAT and Sexual Reproduction in the Cryptococcus Species Complex

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Abstracts (posters)

1) Tor signaling regulates cell-cell adhesion in Candida albicans

Robert J. Bastidas, Maria E. Cardenas and Joseph HeitmanDepartment of Molecular Genetics and Microbiology, Duke University

Coordinated growth and interactions of eukaryotic cells in response to nutrient availability, growth fac-tors, and environmental stimuli are essential for survival. The rapamycin-sensitive Tor1 protein kinase, which is conserved from yeasts to humans, participates in a signaling pathway central to cellular nutri-ent responses. To gain insight into Tor mediated processes in human fungal pathogens, we have charac-terized Tor signaling in Candida albicans. Global transcriptional profiling revealed evolutionary con-served roles for Tor1 in regulating the expression of genes involved in nitrogen starvation response and ribosome biogenesis. Interestingly, we found that in C. albicans Tor1 plays a novel role in regulating the expression of several cell wall and hyphal specific genes, including adhesins and their transcrip-tional repressors NRG1 and TUP1. In accord with this transcriptional profile, rapamycin induced exten-sive cellular aggregation in an adhesin-dependent fashion. Moreover, adhesin gene induction and cellu-lar aggregation of rapamycin-treated cells were strongly dependent on the transactivators Bcr1 and Efg1. These findings support models in which Tor1 negatively controls cellular adhesion by governing the activities of Bcr1 and Efg1. Taken together these results provide evidence that Tor1 mediated cel-lular adhesion might be broadly conserved among eukaryotic organisms.

2) Peptide-based probe capture of Mn oxides and associated bacteria at Loihi Seamount, Hawaii

Brauer SL(1), and Tebo, BM(2)(1)Department of Biology, Appalachian State University, Boone, NC, and (2)Department of Environmen-tal & Biomolecular Systems, Oregon Health & Science University, Portland, OR

Many of the geochemical transformations occurring in the deep-sea are microbially mediated, and phy-logenetic analyses alone are insufficient to determine what organisms are involved in specific biogeo-chemical processes. This study aimed to identify the microorganisms associated with Mn oxide forma-tion and biomineralization in environmental samples using a peptide probe specific for Mn oxides pro-duced by bacteria. A dodecapeptide with affinity to Mn oxide material was selected using phage display screening. This peptide probe was coupled to magnetic beads and used to select for Mn oxide particles and associated bacteria in microbial mats from Loihi Seamount. Results demonstrated that significantly more material hybridized the probe-coated magnetic beads, compared to controls with no beads, or with beads only and no probe. Phylogenetic analysis of the bacterial community found in the Mn-probe captured material revealed an abundance of alpha-Proteobacteria, relative to material captured in a control experiment with no probe. The difference in abundance of alpha-Proteobacteria was deter-mined to be statistically significant using the Libcompare analysis on the Ribosomal Database Project webpage. These analyses have identified putative new Mn oxidizers among the Rhodobacter/Roseobacter clade. Results are supported by the recent isolation of a new Mn oxidizer in the Rhodobacter/Roseobacter clade, whose 16S rRNA gene sequence has 94% identity to one of the 16S rRNA gene sequences in the Mn probe captured library.

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3) Use of T-RFLP Analysis to Categorize the Microbial Diversity Found in Native Bivalve Streams in a North Carolina River Basin

J. P. Bucci, A. J. Szempruch, & J. F. LevineDepartment of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University

Habitat degradation and sedimentation have contributed to the decline of native freshwater bivalves. Identification of microbial communities has been limited by conventional culture-dependent microbio-logical methods. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis produces highly reproducible results from spatially relevant environmental samples. The objective of this study was to examine the use of T-RFLP to characterize the microbial diversity of stream sediments across sites with different land use (i.e., agricultural, urban and forested) in a North Carolina River Basin. Comparative analysis of the bacterial diversity in surficial freshwater sediment collected during the summer of 2007-08 was performed by using 16S rDNA sequences coupled with T-RFLP analysis. Stream study sites were characterized by surface water nitrate concentrations and stable isotope analysis of dissolved inorganic nitrate (15N) The Shannon Weaver Index of species diversity from the classified sequences was higher for sediment collected from the forested (4.22; n=94) sites when compared to the agricultural (3.86; n=63) and urban (3.98; n=58) sites. Class level analyses identified the presence of Bacteroidetes (i.e., Actino, Alpha-, Beta-, and Gamma-proteobacteria), which is consistent with what is found in freshwa-ter lake sediment. Preliminary phylotype analyses showed a higher representation of Gamma-proteobacteria from the agricultural and urban (32-36%) compared to the forested sites (27%). Further analysis demonstrated that Betaproteobacteria were the dominant group represented from the agricul-tural (19%) compared to the forested (9.9%) and urban (7.6%) sites. Family level differences showed that Bacillaceae, Burkholderiaceae, and Mycobacteriaceae were most abundant across all sites with Pseudomonadaceae representing a dominant portion from the agricultural site. This study has demon-strated that T-RFLP analysis can be used to show marked changes in the microbial community diversity available to bivalve consumers between forested, agricultural and urban sites. Subsequent analyses of unclassified bacterium may link the influence of site-specific nutrient factors with ammonia-oxidizing bacteria.

4) Australia to North America: Implications for the global spread of Cryptococcus gattii based on a se-lect VGI genotype

Edmond J. Byrnes III, Gary M. Cox, John R. Perfect, Dee A. Carter, and Joseph HeitmanDepartment of Molecular Genetics and Microbiology, Duke University

In 2007, the first confirmed case of Cryptococcus gattii was reported in the state of North Carolina, USA. The otherwise healthy HIV negative male patient presented with a large upper thigh cryptococ-coma in February, which was surgically removed and the patient started fluconazole treatment. In May of 2007, the patient presented to Duke University hospital emergency room with seizures. Magnetic resonance imaging revealed two large CNS lesions found to be cryptococcomas on brain biopsy. Cul-tures from both the leg and brain biopsies were subjected to analysis. Based on phenotypic and mo-lecular methods, both isolates were C. gattii, VGI molecular type, and distinct from the Vancouver Is-land outbreak isolates that are predominantly VGII. Based on genotyping, the leg and brain isolates appear identical but the two differ in mating fertility. Prior chest CT imaging had revealed small lung nodules indicating that C. gattii spores or desiccated yeast were likely inhaled into the lungs and dis-seminated. The patient’s travel history included a visit to the San Francisco, CA region in September through October of 2006, consistent with acquisition during this time period. Multi locus sequence typ-ing (MLST) analysis revealed that these isolates represent a specific VGI subtype that shares a common MLST genotype with a small group of environmental isolates from Renmark Australia, a single clinical isolate from Australia, and a single clinical isolate from the San Francisco area. In addition, while C. gattii has never been isolated environmentally from North Carolina, a VGII isolate was previously iso-lated from a Eucalyptus camaldulensis tree in San Francisco by other investigators. Our findings sup-port a model in which C. gattii VGI has traveled from Australia to California, possibly though an asso-ciation with its common host plant E. camaldulensis. We posit that this patient inhaled infectious pro-pagules in San Francisco and returned to present with disease in North Carolina. At the current time, the patient has subsequently done very well with no further seizures and his CNS lesions have de-creased in size on high-dose fluconazole. These studies provide insight into the origins and geographic dispersal of an emerging microbial pathogen.

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5) Analyzing ‘donuts’ for flagellar force production trends during regeneration in Chlamydomonas re-inhardtti

Catherine Clodfelter, Dr. John Yukich, Dr. Karen BerndDepartments of Physics and Biology, Davidson College

Several respiratory, digestive, and reproductive disorders result from dysfunctional cilia and flagella. Because the form, strength or frequency of flagella motion is abnormal in these disorders, biological functions performed by the cells utilizing flagella and cilia are debilitated. The causes of debilitation are not known as the internal mechanism for creating the flagella’s breast stroke-like motion is still not fully understood. This study uses Chlamydomonas reinhardtii flagella as a model system and reports standardization of PSD calibration, laser trapping and cell movement recording methods. When motion in a laser trap is viewed as a trace, natural cell rotation while swimming (caused by flagellar domi-nance) results in a spirograph-like ‘donut’ shape. Circle fit analysis programs were written to measure trends in force generation during flagellar regeneration. The percentage of ‘donuts’ per flagella length increases linearly, similar to the increase in length over regeneration time. However the flagel-lar force trend shows a significant, unexpected dip during flagella lengthening. Neither the flagella in-frastructure necessary to establish dominance nor the causes of the decrease in force are known, thus these data open many new research directions.

6) Tracking Microbial Populations in Swine Waste Lagoons in Eastern North Carolina Using Terminal Re-striction Fragment Length Polymorphism Analysis

Anthony A. Devine, J. Mark Rice, J. Christopher Ellis. and Amy M. GrundenDepartment of Microbiology, North Carolina State University

Gaining insights into complex microbial communities has relied primarily on 16S rDNA sequencing of pooled PCR reactions and utilizing more traditional microbiology techniques of plating, isolating and characterizing microorganisms. Only recently has TRFLP (Terminal Restriction Fragment Length Poly-morphism) analysis been used in place of these traditional methods for determining complex microbial communities. In this study, TRFLP analysis is used to determine the microbial communities in hog waste lagoon systems and how the populations change over the course of a year.

In this study a total of 9 finishing farms are being tracked over the course of a year (October 2007-August 2008) with each farm being sampled once each quarter. A total of three composite samples were collected for each farm, the samples were collected at the surface, 1 meter below the surface, and approximately 6 inches below the liquid/sludge interface. DNA was then isolated from each of these samples and 16S rDNA directed PCR was carried out on each sample. The PCR products were then purified and digested using a specific set of restriction enzymes. The detected fragments were then analyzed using a custom designed TRFLP fragment pattern database.

The data collected and analyzed so far shows that there are differences in the microbial populations found in both the two farms presented. Between these two farms Sandy Ridge (SR) has been identified as a ‘problem’ lagoon and Tator’s Pig Parlor (TPP) as a ‘healthy’ lagoon. TPP shows a higher number of patterns detected at all three sample sites compared to SR. This higher diversity can also be seen over time, with the TPP lagoon having a higher overall number of patterns detected between quarter one and quarter two compared to the SR lagoon. However both lagoons had a higher diversity of mi-croorganisms detected over the time of the two quarters analyzed which is indicative of the changing of the seasons between each quarter.

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7) Nanoporous Latex Coatings Containing Rhodopseudomonas palustris Pigment Mutants Spatially Dis-tributed for Hydrogen Production

Jimmy Gosse1, Brian Engel2, Federico Rey3, Caroline Harwood3, and Michael Flickinger41 Dept. of Biol. Agri. Eng. North Carolina State University, 2 Dept. of Biochem. Mol. Biol. and Biophys-ics. University of Minnesota, 3 Dept. of Microbiol. University of Washington, 4 Dept. of Microbiol. North Carolina State University

The hydrogen production rate from <70 um thick latex coatings containing non-growing Rhodopseudo-monas palustris CGA009 in the presence of acetate is stable for >3000 hours at 2 mmol H2 m-2 hr-1. Although this rate is significant it can be further improved by altering the photosynthetic capacity of Rps. palustris and thereby optimizing the coating for light absorption. It has been demonstrated in sus-pension photobioreactors that reduced pigment mutants of photosynthetic organisms have higher hy-drogen production rates than an equivalent density of wild type cells. Immobilization of these mutants in latex may result in an increase in the specific activity of the cells. A multi-layer coating allows for combining strains in defined spatial arrangements for the optimal utilization of light. Hydrogen pro-duction rates have been determined for monolayer latex coatings containing photosynthetic pigment mutants of Rps. palustris. Composite coatings of mutants were evaluated to investigate the affect of spatial arrangement (uniform mono-layer, mixed mono-layer, and multi-layer coatings) on hydrogen production specific activity (H2 production rate / coating thickness). Stable long term hydrogen pro-duction rates can be achieved for each system

8) Bacterial Community Analysis of a High Altitude Wetland Disturbed by Iron and Manganese

Kristin Johnson, Ece Karatan, Michael Windelspecht, Suzannah BrauerDepartment of Biology, Appalachian State University

Sorrento Wetland is a high altitude riparian wetland located in Boone, NC. The stream leading to the wetland contains several iron and manganese seeps, leading to high concentrations of these metals and their oxides at locations within the stream and wetland. Concentrations of iron are 20 fold higher at the seeps than at upstream reference sites and concentrations of manganese are 30 fold higher at the seeps than at upstream reference sites. Iron and manganese oxidation has been previously shown to limit the diversity of macroinvertebrates present, but has little effect on bacterial diversity. It has been hypothesized that the iron and manganese oxidation is a biologically independent process. My research aims to determine if iron and manganese oxidizing bacteria are located within the wetland stream and at upstream reference sites to assess whether this is the case. Preliminary tests using Leu-coberbelin Blue (LBB) to indicate the presence of manganese oxides show that manganese oxidizing bacteria are present at the seeps. Current research is in progress that will quantify numbers of man-ganese and iron oxidizing bacteria using an assay consisting of 96 well plates with dilutions of sediment and water from the seeps, wetland, and upstream reference sites using LBB and ferrozine respectively. Initial FISH results using Leptothrix specific probes indicate the presence of Leptothrix, an iron and manganese oxidizing bacteria, within the wetland. Current research is underway to test for the pres-ence of Leptothrix in the seeps, wetland, and upstream reference sites. Results of this work will elu-cidate the biological effects of high iron and manganese levels on bacterial communities.

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9) Characterization of an A. salmonicida bacteriophage isolated from a NC trout population

Rosemary Le, Melissa Cox, Ben Wallace, Sue CarsonBiotechnology Program, North Carolina State University

Salmonid furunculosis is a disease that is caused by the infectious bacterial agent, Aeromonas salmoni-cida subspecies salmonicida. It affects freshwater brown trout and rainbow trout, among other salmo-nid species worldwide, by causing kidney and boil-like surface lesions, severe septicemia, and acute mortality. A. salmonicida is also of interest because of its devastating effect on the salmonid fishing and hatcheries industries that cause major losses of populations due to infection. We were interested in obtaining bacteriophage (phage) against this organism for two possible applications: treatment, pre-vention, and control against salmonid furunculosis; and development of molecular tools for working with Aeromonas. We tested trout samples from various locations on the East Prong Roaring River, NC, and isolated live phage from a mixed sample of adult rainbow trout and brown trout. TEM showed phage with icosohedral heads and sheathed, contractile tails. Based on these images, we suspect that the phage are of the family Myoviridae.

10) Investigation of the sex locus of a basal fungus, Mucor circinelloides

Charles H. Li, Soo Chan Lee, Joseph HeitmanDepartment of Molecular Genetics and Microbiology, Duke University Medical Center

Mucor circinelloides is a zygomycete fungus that is an opportunistic pathogen with the potential to in-fect immunosuppressed, and occasionally immunocompetent individuals, resulting in life-threatening mucormycosis. M. circinelloides is a heterothallic fungus, forming a specialized sexual structure, the zygospore, when two mating type strains are co-cultured. The sex locus of a related zygomycete, Phy-comyces blakesleeanus, which governs and orchestrates sexual reproduction, was characterized to con-sist of a high mobility group (HMG) transcription factor gene flanked by a triose phosphate transporter (TPT) and an RNA helicase gene. The HMG domain proteins are designated SexP or SexM for the (+) and (-) strains respectively. The sex locus was investigated in three subspecies of M. circinelloides: M. cir-cinelloides f. lusitanicus, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides. Ge-nome search and sequence analysis was used to analyze the sex locus of the subspecies. We found the SexP and SexM genes are conserved and the sex synteny (TPT, sexP or sexM, RNA helicase) was also found in the different subspecies of M. circinelloides. These findings suggest that the sex locus is highly conserved in the M. circinelloides subspecies and may help further unravel the evolution of the sex loci and sex determination in basal fungi.

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11) Quantification of polyamines and NspC gene expression in Vibrio cholerae

Zachary M. Parker, Marcus W. McGinnis, Ece KaratanDepartment of Biology, Appalachian State University

The disease cholera is caused by the pathogenic Gram-negative proteobacterium Vibrio cholerae. The survival of V. cholerae depends greatly on its ability to create biofilms. Recent findings have shown that the polyamine norspermidine acts as an extracellular signal for V. cholerae to activate biofilm syn-thesis (Karatan et al., 2005). Also, in the gram negative bacterium Yersinia pestis the reduction of the intracellular amounts of the polyamine putrescine causes a reduction in biofilm (Patel et al., 2006). These two studies suggest that the polyamine synthesis and transport could play a crucial role in the regulation of biofilms in V. cholerae. In this work, our goal was to study the role of norspermidine syn-thesis and transport on biofilm formation in V. cholerae. Using high performance liquid chromatography and qRT-PCR techniques, we have quantified the intracellular levels of polyamines and nspC (a gene encoding a norspermidine synthesis enzyme) expression, respectively, in V. cholerae under various con-ditions. We have found that expression of the nspC gene from a multicopy plasmid leads to increased transcript levels; however, the intracellular levels of norspermidine do not increase, suggesting the presence of a post-transcriptional feedback inhibition mechanism. Furthermore, we have shown that V. cholerae is able to uptake norspermidine added to the culture medium; however, transport of norsper-midine into cell does not affect nspC transcript levels. Interestingly, norspermidine uptake also leads to a significant decrease in the levels of its precursor, diaminopropane. These results suggest that transport of norspermidine into the cell down regulates de novo norspermidine synthesis at the level of its precursors, but has not effect on transcription of nspC gene. Experiments are underway to elucidate the details of the regulatory systems which maintain cellular levels of norspermidine.

12) The Isolation and Identification of a Bacterium that causes bent neck in Roses

Amanda Redfern, Caitlin Baber, Ann MatthysseDepartment of Biology, University of North Carolina at Chapel Hill

In order to determine whether bacteria are a main contributor to bent necks in roses, three strains of unknown bacteria labeled (S102, H101, and H100) were isolated from the stem of roses with bent neck. Spontaneous nalidixic acid resistant mutants of each isolate were obtained and used to inoculate a group of roses. Two days after inoculation with H100, bent necks were observed. Bacterial harvests at lengths of 0-1 cm, 10-11 cm, and 20-21 cm from the bottom of the stem were performed and plated on nutrient agar containing nalidixic acid. In further measurements taken below and above the bent neck, a significantly greater number of bacteria were observed below the bent neck in roses whose water was inoculated with H100. Because fewer bacteria were observed above the bent neck, it was assumed that a vascular tissue blockage had occurred in a lower region of the stem, preventing the uptake of water. This vascular tissue blockage ultimately caused the bent neck of roses whose stem was exposed to sterile water inoculated with H100. While inoculation of vase water with H100 routinely produced roses with bent neck, the inoculation of vase water with S102 and H101 had no effect on the occur-rence of bent neck. In order to confirm the location of the vascular tissue blockage below the bent neck, individual bent neck roses were removed from the inoculated water and placed in sterile water containing red dye. After two hours, the location of the red dye was recorded and bacterial harvests were performed. Additional measurements were also taken a centimeter directly below and directly above the termination of the red dye. A significantly larger number of bacteria were found in the red dye region than directly above it, suggesting vascular tissue blockage at the same point in the stem as the end of the observed red dye. This vascular tissue blockage, caused by the unknown bacteria, was the main contributor to bent neck and eventual death of roses.

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13) Deep Biosphere Research at East Carolina University

Matthew O. SchrenkDepartment of Biology, East Carolina University

A vast proportion of Earth’s biosphere is hosted with the pores and fractures of the subsurface. Subsur-face microbial populations comprise a majority of prokaryotic cells on Earth and play important roles in controlling the oxidation state of the crust and modulating the flux of carbon and energy between deep and surface environments. Recent estimates indicate that at least 50% of the prokaryotes (0.25 - 2.5 — 10^30 cells) on Earth may reside in subsurface habitats , and that they may be reservoir of between 56 and 303 Pg of Carbon, a number equivalent to that of plant life at the Earth’s surface. Furthermore, prokaryotes typically contain 10-fold more Phosphorous and Nitrogen than do plants, and therefore this substantial subsurface microbial biosphere may be an enormous reservoir of nutrients as well. Little is known, however, about the physiology of microorganisms pertinent to the deep subsurface biosphere and how they are specifically adapted to the high pressures, high temperatures, and low energy fluxes characteristic of subsurface habitats. This presentation describes several initiatives in the Department of Biology at East Carolina University which seek to elucidate the physiology, activities, and ecology of microorganisms which reside in the deep biosphere. Four research foci will be highlighted, including; 1.) Structural and biochemical studies of biofilms in extreme environments, 2.) Studies into the physio-logical effects of high hydrostatic pressure, 3.) Functional genomics studies of ‘dark energy’ microbial ecosystems, independent of sunlight, and 4.) Novel in situ experimentation approaches to tap into the functional diversity of uncultured microorganisms. This presentation will describe progress being made in the field of deep biosphere science and opportunities for future students and collaborators.

14) Characterization of the Role of Two Genes in the Binding of Agrobacterium tumefaciens to Plant Surfaces

Amanda TarnowiczDepartment of Biology, University of North Carolina at Chapel Hill

Agrobacterium tumefaciens causes the formation of crown gall tumors in higher plants. Tumor forma-tion results from the transfer of DNA (T-DNA) from the bacterium to the host cell. Mutants of A. tume-faciens which fail to bind to plant cells and are therefore avirulent have been isolated. However, none of these mutations directly affects molecules involved in binding. We approached the problem of iden-tifying genes whose products are directly involved in binding by testing genes of the sequenced A. tu-mefaciens genome that are predicted by homology to have characteristics suggesting their possible in-volvement. Several genes were identified by blast homology as conserved in the agrobacteria and as encoding possible surface-exposed calcium-binding proteins. Two of these genes, Atu4190 and Atu2707, were studied. These genes were cloned in pBluescriptKS- behind the lac promoter and introduced into non-binding Escherichia coli K12 ER2267. Compared to wild type, ER2267 carrying either A. tumefaci-ens gene demonstrated increased binding to alfalfa sprouts. ER2267 pAtu2707 consistently bound in higher numbers than ER2267 pAtu4190, but the latter demonstrated a higher degree of aggregation. Light microscopy confirmed these results. C58 insertion mutants were constructed for these genes and were named C58 4190ALT and C58 2707ALT. These mutants were tested for virulence on and binding to Rutgers tomato and Bryophyllum daigremontiana plants. Both mutants were avirulent on both tomato and B. daigremontiana. Under the microscope, C58 4190ALT failed to bind to B. daigremontiana cut root ends and root hairs. This mutant bound to tomato root hairs, and its ability to bind to tomato cut root ends has yet to be determined. C58 2707ALT bound to B. daigremontiana cut root ends and root hairs. This mutant also bound to tomato root hairs but failed to bind to cut root ends. It appears that the gene Atu4190 is involved in allowing A. tumefaciens to bind to plants, for the mutant strain C58 4190ALT failed to bind to, and produce tumors on, B. daigremontiana cut root ends and wound sites, respectively. It also appears that Atu2707 is involved in binding since the mutant C58 2707ALT failed to bind to, and produce tumors on, tomato.

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15) Elucidation of Polyamine Synthesis and Transport Sytems in Vibrio cholerae

Walter N., Rutkovsky A., McGinnis M., Karatan E. Department of Biology, Appalachian State University

Polyamines are small aliphatic hydrocarbon molecules with two or more amine groups spaced through-out the molecule, which are present in virtually all living organisms. The intracellular levels of polya-mines are highly regulated and maintained by adjusting the rate of biosynthesis, degradation and ex-tracellular uptake. In prokaryotes polyamines have diverse functions including modulation of outer membrane proteins, protection from the toxic effects of reactive oxygen species and facilitation of the enhancement of translation of messenger RNA, In Vibrio cholerae, norspermidine, a triamine, acti-vates biofilm formation by binding a putative periplasmic receptor protein. Norspermidine is also one of the precursors of the V. cholerae siderophore vibriobactin. Despite the important role norspermidine plays in the physiology of V. cholerae, there is very little information on the putative polyamine syn-thesis and transport pathways and nothing known about the composition of the intracellular polyamine pool in this organism. In this work, we have studied the polyamine pools in V. cholerae in various ge-netic backgrounds under various conditions. Polyamines were extracted from the cells, derivatized by benzoylation, and quantified using high performance liquid chromatography. We have shown that V. cholerae contains 5 polyamines when grown in Luria-Bertani broth; namely, putrescine, diaminopro-pane, cadaverine, norspermidine, and spermidine. We have also shown that spermidine is not synthe-sized by V. cholerae, but is taken up from the culture medium. Uptake of spermidine was carried out by a homolog of PotD, the periplasmic binding protein of the ABC-type transport system for spermidine in Escherichia coli. We present evidence that of the two PotD homologs in V. cholerea, only one (PotD1) is responsible for both spermidine and norspermidine uptake and these two polyamines com-pete for the same binding protein. In conclusion, we have begun to characterize the polyamine syn-thesis and uptake systems of V. cholerae. Armed with this knowledge, we hope to be able to better understand the effect of polyamines in various aspects of V. cholerae physiology such as biofilm forma-tion and iron acquisition.

16) Differential Responses of Dendritic Cell Subsets to the Intracellular Bacterial Pathogen, Listeria monocytogenes

Latoya M. Mitchell, Curtis J. Henry, Jason M. Grayson PhD, Elizabeth M. Hiltbold PhD Department of Microbiology and Immunology, Wake Forest University School of Medicine

Infection with the food-borne bacterium Listeria monocytogenes (Lm) can pose serious complications for immunocompromised individuals such as pregnant women and the elderly. An intracellular patho-gen, Lm is taken up into vacuoles and via the hemolysin, Listeriolysin O, enters the host cytoplasm where it will replicate and spread to neighboring cells. While immunization with wt Lm is able to elicit a protective immune response, immunization with a vacuolar-retained strain of Lm does not provide protection. Dendritic cells (DC) are antigen presenting cells that bridge the innate and adaptive im-mune systems and are a vital component for clearance and protection against Lm infection. DC are divided into several subsets based on their function, location, and cell surface markers. Because of the role of DC in immunity against Lm and the various DC subsets, the goal of our work is determine the response of DC subsets to wt and vacuolar Lm infection. We have shown using an in vivo murine model of infection that splenic DC subsets show increased surface costimulatory molecule expression in response to wt Lm and to vacuolar Lm, although to a lesser extent. The peak of the response was at 24 hours post infection (hpi) and for two DC subsets, sustained until 72 hpi. Using in vitro bone marrow derived DC that are comparable to DC found in the spleen, we have shown that these DC subsets ma-ture to different levels in response to both wt and vacuolar Lm. Maturation was dependent on the TLR signaling adapter protein, MyD88, but not TRIF. Within the mixed cultures, we observed that the CD11b+ DC subset increased costimulatory molecule expression and cytokine secretion more than the B220+ DC subset. CD11b+ DC were also more heavily infected and more efficient at priming naive T cells than B220+ DC. In conclusion, DC subsets found in the spleen or derived in vitro from bone mar-row respond to both wt and vacuolar Lm. While the level of DC maturation responses differed between Lm strains thus impacting the activation of naive T cells, this work sheds light on the differential role individual DC may have in protective immunity.

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17) A Molecular Dissection of the Cryptococcus Unisexual Reproduction Cascade

Marianna Feretzaki, Joseph Heitman Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA

The basidiomycete Cryptococcus neoformans is a common global human pathogen. Human infection is thought to be caused by inhalation of spores from the environment. The small diameter of spores facili-tates alveolar penetration, and following germination the yeast disseminates hematogenously. In the laboratory, nutrient limitation and pheromones induce a dimorphic transition from yeast to hyphae via sexual reproduction. The traditional sexual cycle involves α and a opposite mating type cells. Mono-karyotic fruiting is a modified sexual cycle involving cells of the same mating type, usually α. Both forms of sexual reproduction lead to the formation of hyphae and basidia, where meiosis followed by multiple rounds of mitosis produces infectious basidiospores. The conserved MAP kinase and cAMP-protein kinase signaling cascades control mating; however, the pathway targets are largely unknown. Here we applied insertional mutagenesis to identify genes governing unisexual reproduction. A hyper-filamentous strain was subjected to T-DNA mutagenesis using Agrobacterium tumefaciens. 6,100 mu-tant strains (~1x genomic coverage) were generated, of which 225 exhibited filamentation defects on V8 media. These mutants were categorized based on phenotype: afilamentous (20), short filaments (111), hyperfilamentous (73), increased chlamydospore production (5) and dense, sparse filaments (9). Four mutants altered in pH or light responses were also obtained. Representative mutants from each group have been selected for further characterization. Our aim is to identify and characterize novel unisexual mating pathway components in Cryptococcus.

18) A Brucella abortus Fur homolog regulates manganese dependent expression of the divalent cation transporter mntH.

Evan A. Menscher, Eric S. Anderson, James T. Paulley, Michelle Wright-Valderas*, and R.M Roop II.

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina and *Department of Veterinary Pathobiology, Oklahoma State University Center for Vet-erinary Health Sciences, Stillwater, Oklahoma.

Sequestering divalent cations from the environment is essential for virulence and proper growth of Brucella abortus, which causes the zoonotic disease brucellosis. Brucella spp. are members of the alpha-proteobacteria family and express a number of genes specific for divalent cation transport. We identified BAB1_1460, a gene homologue of the E. coli mntH (H+-dependent manganese transporter), a member of the NRAMP-1 (Natural resistance-associated macrophage protein) family of proteins which transport divalent cations in a proton dependent manner. We constructed a Brucella abortus mntH knockout, and the resulting mutant, MWV15, is more sensitive to oxidative killing and shows attenuated growth in vivo. In this study, we have constructed lacZ transcriptional fusions to monitor expression of mntH by inserting 230 bp of mntH upstream sequence into the promoterless plasmid pMR15 creating pEAM1. We grew B. abortus containing pEAM1 in rich media over a range of Mn concentrations. In-creased Î -galactosidase activity was observed at low concentrations of Mn and this expression was re-pressed by higher concentrations of Mn, but was not repressed by Fe. B. abortus contains a homolog of the ferric ion regulator Fur. We moved pEAM1 into the fur knockout strain Fur2 and found no repres-sion of beta-galactosidase activity in the presence of either Mn or Fe. We conclude that Mn is the sig-nal that Fur uses to repress mntH expression. Based on the current data, we predict that Fur function-ally acts as a Mur (manganese uptake regulator) as is seen in Rhizobium leguminosarum. We plan to use biochemical and genetic methods to verify the role of Mur in Mn-specific regulation of mntH and identify sequences to which Mur binds to the mntH promoter region.

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19) Elucidation of possible target receptors for the Estrogenic Endocrine Disrupting Chemical (EEDC) Bisphenol A in the soil nematode, Caenorhabitis elegans.

Michele K.H.Malotky, Madeline Winters, Kendra K. Scudder and E. Katherine Miller Department of Biology, Guilford College

Endocrine Disrupting Chemicals are agents that interfere with normal endocrine signaling pathways. Numerous reports in invertebrate and vertebrate systems have identified aberrations in reproduction upon exposure to these compounds. In the current study, we are using Caenorhabitis elegans, a free-living soil nematode to identify the target receptor of an estrogenic endocrine disrupting chemical (EEDC), Bisphenol A (BisA). Although previous work in this model system has reported physical abnor-malities and decreases in fecundity upon exposure to BisA in a multigenerational bioassay, the target receptor of this compound has yet to be identified. We are currently in the process of creating a plas-mid construct that exogenously expresses a short sequence of the gene for a putative estrogenic recep-tor. This gene, nhr-14, has significant similarity to known estrogen receptors in other animals and has recently been identified as a target of estrogen signaling in C elegans. Upon transfection into E. coli, we plan to use RNA interference (RNAi) to assess the potential role of this gene product in the EDC ef-fects of BisA.

20) Evolution of MAT and Sexual Reproduction in the Cryptococcus Species Complex

Keisha Findley, James Fraser, Fred Dietrich and Joseph Heitman Department of Molecular Genetics and Microbiology Duke University

Sexual reproduction in the human fungal pathogen Cryptococcus neoformans involves a bipolar mating system in which a single >100 kb locus encodes pheromones, receptors and homeodomain transcription factors. In contrast, in many basidiomycetes a more complex system governs mating where two un-linked MAT loci play a role (tetrapolar). To elucidate the steps in Cryptococcus MAT locus evolution, we have employed a comparative genomics approach utilizing closely and distantly related species. To test our hypothesis that the Cryptococcus MAT locus evolved from an ancestral tetrapolar system, we have characterized MAT from two of the closest known species of C. neoformans, Tsuchiyea wingfieldii and Cryptococcus amylolentus. Our results reveal two large ~40 and ~70 kb gene clusters spanning the ho-meodomain (HD) and pheromone/receptor loci in T. wingfieldii. Two linked, divergently transcribed genes encoding orthologues of both the C. neoformans HD proteins, Sxi1a and Sxi2a, are arranged simi-larly to other tetrapolar mating systems, suggesting a recent loss of one or the other HD gene occurred in the Cryptococcus pathogenic species cluster. Additionally, the five genes predicted previously to be the most recent MAT locus acquisitions are linked to the pheromone/receptor MAT locus, and thus were linked prior to fusion of the two MAT loci. Pulsed field gel electrophoresis of whole chromosomes com-bined with Southern blot analysis demonstrates the two MAT subloci are unlinked and lie on different chromosomes, similar to tetrapolar mating systems. Comparative analysis of the C. amylolentus MAT assembly provides evidence that these genomic regions may be linked to mating type, and reveals key events that punctuated evolution of the Cryptococcus MAT locus. Additionally, studies are in progress to define how transitions in the mating-type locus contributed to the emergence of the Cryptococcus pathogenic species cluster.

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Presentation Awards

The Mary Poston Award was established to recognize the best paper given by a student at meetings of the NC Branch of the ASM. Mary Pos-ton was a longtime employee of Duke University who contributed much to the NC Branch and she was held in high esteem both by her col-leagues and by medical students. She contributed much to the NC Branch, including service as Branch Secretary-Treasurer from 1950 until her death in 1961. Many letters of appreciation have been written over the years by student recipients of the Mary Poston Award, commenting on the confidence the award gave them and on the importance of the competition for the award as part of their graduate training.

Last years winner:Elizabeth Waligora, Wake Forest University School of MedicineStructure-function analyses reveal a role for AmrZ in P. aeruginosa gene expression and pathogenesis.

The Thoyd Melton Award was established to recognize an outstanding oral presentation by a graduate student. At the time of his premature death on Nov. 22, 2000, Thoyd Melton was Associate Vice Chancellor for Academic Affairs and Dean of graduate studies at N.C. A&T State Uni-versity. Prior to this position, Dr. Melton was a member of NC State Uni-versity's Department of Microbiology and an Associate Dean of the Graduate School. Dr. Melton was very active in research and particularly in graduate education. In 1999, he received the William A. Hinton Re-search Training Award from ASM. This award honors an individual who has made significant contributions toward fostering the research train-ing of underrepresented minorities in microbiology.

Last years winner:Nathan Rigel, University of North Carolina at Chapel HillCharacterization of the SecA2 specialized secretion pathway of myco-bacteria.

The Best Poster award is open to anyone presenting a poster at the NC ASM meeting.

Last years winner:Alice Lee, NC State UniversityRecombinant Expression of Superoxide Reductase from Pyrococcus fu-riosus in Arabidopsis thaliana Enhances Heat and Drought Tolerance.

The Paul Phibbs Award is awarded for the best presentation by an un-dergraduate student at NC ASM Branch meetings.

Last years winner:Brittany Baffer & Amanda Tarnowicz, University of North Carolina at Chapel HillIsolation of Gene(s) Responsible for Binding of Agrobacterium tumefa-ciens to Plant Host Cells.

A check for $100 will be given for each of these awards at the conclusion of the meeting.

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Pre-registrants

# Name Home Email Position1 Jennifer Abelin Guilford jabelin@guilford.edu Undergraduate2 Sherrice Allen FSU svallen@uncfsu.edu Faculty3 Eric Anderson ECU andersone@ecu.edu Postdoc4 Ronald Ash ronpeeps@gmail.com5 Tim Atkinson ASB Tim.Atkinson@carolina.com Vendor/Exhibitor6 Caitlin Baber UNC-CH baber@email.unc.edu Undergraduate7 Robert Bastidas Duke rjb10@duke.edu Grad student8 Manish Bharadwaj WFU mbharada@wfubmc.edu Postdoc9 Suzanna Brauer ASU brauersl@appstate.edu Faculty10 Jamila Broadway NCCU Grad student11 James Brown NCSU james_brown@ncsu.edu Faculty12 Kristina Brzoza-Lewis WFU kbrzoza@wfubmc.edu Grad student13 John Bucci NCSU jpbucci@ncsu.edu Postdoc14 Edmond Byrnes III Duke edmond.byrnes@duke.edu Grad student15 Worth Calfee US EPA Calfee.Worth@epamail.epa.gov Postdoc16 Krystal Carpenter Guilford carpenterka@guilford.edu Undergraduate17 Sue Carson NCSU sue_carson@ncsu.edu Faculty18 Latarsha Casterlow Guilford casterlowln@guilford.edu Undergraduate19 Catherine Clodfelter Davidson caclodfelter@davidson.edu Undergraduate20 Matt Conover WFU Grad student21 Melissa Cox NCSU melissa_cox@ncsu.edu22 Ryan Davis Guilford rdavis3@guilford.edu Undergraduate23 Anthony Devine NCSU aadevine@ncsu.edu Grad student24 Ossie Dyson dysono@ecu.edu Grad student25 Marianna Feretzaki Duke mf55@duke.edu Grad student26 Keisha Findley Duke kmf13@duke.edu Grad student27 Claire Foxman Guilford cfoxman@guilford.edu Undergraduate28 Jimmy Gosse NCSU jlgosse@ncsu.edu Postdoc29 Amy Grunden NCSU amy_grunden@ncsu.edu Faculty30 Natan Harel Guilford nharel@guilford.edu Undergraduate31 Abigail Harris Guilford aharris@guilford.edu Undergraduate32 Joseph Heitman Duke heitm001@duke.edu Faculty33 Kendra Hitz ECU krh0918@ecu.edu Grad student34 Kristin Johnson ASU kw48775@appstate.edu Grad student35 Tonya Johnson Guilford johnsontl@guilford.edu Undergraduate36 Margaret Kanipes NC A&T mikanipe@ncat.edu Faculty37 Ece Karatan ASU karatane@appstate.edu Faculty38 Melissa Keller Guilford kellerml@guilford.edu Undergraduate39 Rushyannah Killens NCSU rrkillen@ncat.edu Grad student40 Heather Koehler Guilford Undergraduate41 Adam Kulp Duke ajk26@duke.edu Grad student42 Kenny Kuo Duke hk21@duke.edu Grad student43 Shannon Landvater NC A&T swlandva@ncat.edu Undergraduate44 Hatajai Lassiter NCCU halassit2005@hotmail.com Grad student

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# Name Home Email Position45 Rosemary Le NCSU rkle@ncsu.edu Undergraduate46 Alice Lee NCSU amlee3@ncsu.edu Grad student47 Soo Chan Lee Duke soochan.lee@duke.edu Postdoc48 Melanie Lee-Brown Guilford mleebro@guilford.edu Faculty49 Charles Li Duke charles.li@duke.edu Undergraduate50 Jed Litsey Guilford jlitsey@guilford.edu Undergraduate51 Leandro Lobo ECU lobol@ecu.edu Postdoc52 Gerry Luginbuhl NCSU gerry_luginbuhl@ncsu.edu Faculty53 Shaina Machlus Guilford smachlus@guilford.edu Undergraduate54 Kunapuli Madhusudhan Clean Earth Techkmadhusudhan@cleanearthtech.comProfessional55 Michele Malotky Guilford mmalotky@guilford.edu Faculty56 Cody Martin Guilford cmartin@guilford.edu Undergraduate57 Ann Matthysse UNC-CH ann_matthysse@unc.edu Faculty58 Evan Menscher ECU eam1007@ecu.edu Grad student59 Sonja Milek WFU smilek@wfubmc.edu Grad student60 Alexis Mincer Guilford amincer@guilford.edu Undergraduate61 Latoya Mitchell WFU lamitche@wfubmc.edu Grad student62 Caitlin Mocarski Guilford cmocarsk@guilford.edu Undergraduate63 Marvin Morales Guilford Undergraduate64 MD MOTALEB ECU motalebm@ecu.edu Faculty65 Zachary Parker ASU zp51609@appstate.edu Grad student66 Hanna Piolo Guilford hpioli@guilford.edu Undergraduate67 Rebecca Pittman Guilford rpittman@guilford.edu Undergraduate68 Amanda Redfern UNC-CH aredfern@email.unc.edu Undergraduate69 Marianela Rodriguez-Carres Duke marianela@notes.duke.edu Postdoc70 Amy Rothbard RTI arothbard@rti.org Professional71 Matt Schrenk ECU schrenkm@ecu.edu Faculty72 Tricia Schwartz RTI tschwartz@rti.org Professional73 Angie Shotwell ISC ashotwell@bioexpress.com Vendor/Exhibitor74 Jennifer Spack Guilford spackjm@guilford.edu Undergraduate75 Neelima Sukumar WFU nsukumar@wfubmc.edu Grad student76 Amanda Tarnowicz UNC-CH alynntar@email.unc.edu Undergraduate77 Deborah Threadgill UNC-CH Deborah_Threadgill@med.unc.edu Faculty78 Sherry Tove NCSU srtove@ncsu.edu79 Nicholas Walter ASU NW76557@appstate.edu Undergraduate80 Lindsay Watson Guilford watsonlm@guilford.edu Undergraduate81 Melissa Wilks Guilford wilkinsmk@guilford.edu Undergraduate82 Madeline Winters Guilford mwinters@guilford.edu Undergraduate83 Joe Wolf Peace jwolf@peace.edu Faculty84 Kathy Zarilla DTCC zarillak@durhamtech.edu Faculty

Meeting Organizing Committee

Melanie Lee-Brown (President), Guilford CollegeKathy Zarilla (President-elect), Durham Tech Community CollegeJames W. Brown (Secretary), NC State UniversityGeraldine Luginbuhl (Treasurer), NC State University

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