NC ASM 2009
North Carolina American Society for Microbiology
2009 Meeting
North Carolina Central University
October 3, 2009
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NC ASM 2009
Schedule
Preliminaries
8:00
Registration
Poster and talk set-up
Coffee break
Award Committees meeting/organization
8:45
Daniel Williams
Welcome & Introductory comments
Hazell Reed (Vice Chancellor of Research & Graduate Education)
Saundra Delauder (Assoc. Dean, College of Science & Technology)
Session 1 : Daniel Williams, Chair
9:00
Kristin E.D. Weimer
Coinfection with Haemophilus influenzae and Streptococcus
pneumoniae alters pneumococcal disease progression by promoting stable biofilm formation
9:15
Matthew S. Byrd
Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production
9:30
Benjamin Mudrak
Comparison of mutations affecting secretion of heat-labile enterotoxin and cholera toxin.
9:45
Ann G. Matthysse
Interaction of E. coli O157 with cut lettuce leaves
10:00
Poster session 1 (unattended)
Coffee break
Session 2 : Kathy Zarilla, Chair
10:45
Ine Jorgensen
The Chlamydial Protease CPAF Targets a Subset of Early Effector
Proteins
11:00
Hector A. Saka
The Fat and the Ugly: A Proteomics Approach to Dissect Lipid
Droplet-Chlamydia Interactions
11:15
Eric Anderson
The B. abortus Irr in required for iron-responsive regulation of
the gene encoding the heme transporter BhuA
11:30
Jennifer M. Gaines
The twisted knot of Hfq-dependent regulation of sodC in Brucella abortus 2308
11:45
Clayton C. Caswell
Role of the RNA chaperone Hfq in expression of the genes encoding the type IV secretion machinery of Brucella abortus 2308
12:00
Lunch
12:45
Poster session 2 (Even numbered poster should be attended)
Session 3 : Marty Roop, Chair
1:30
Erin McElvania TeKippe
The inflammasome adaptor ASC is important for granuloma formation and host defense in chronic Mycobacterium tuberculosis
infection
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NC ASM 2009
Schedule
1:45
Hsun-Cheng Su
Genes that modulate the rapid rise of Ciprofloxacin resistance in
P. aeruginosa
2:00
James R. Fuller
Regulation of the Staphylococcus aureus NO·-Stress Response
2:15
Frank Scholle
NC Invitational Talk
Flavivirus-Toll-like receptor Interactions
2:50
Intermission
Plenary session : Daniel Williams, Chair
3:00
Mark Goulian
ASM Branch Lecture
Perturbing, Imaging, Modeling, and Evolving Two-Component
Signaling Systems in E. coli
4:00
Poster session 3 (Odd numbered poster should be attended)
Coffee break
Postscript
4:45
Daniel Williams
Concluding remarks
Awards
5:00
Jim Brown
Business meeting
Officer election
5:30
Adjournment
Meeting Sponsors
The American
Society for Microbiology
North Carolina
Central University
Association of
Southeastern Biologists
Fisher Scientific
(Russell Salisbury)
ISC BioExpress
(Rob Blackman)
VWR
(Erica Vo)
Nikon
(Kurt Neumann)
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NC ASM 2009
Abstracts (talks)
1.1) Coinfection with Haemophilus influenzae and Streptococcus pneumoniae alters pneumococcal disease progression by promoting stable biofilm formation
Kristin E. D. Weimer, Chelsie E. Armbruster, Bing Pang, Richard A. Juneau, and W. Edward Swords
Department of Microbiology and Immunology, Wake Forest University School of Medicine
Otitis media (OM) is the number one reason for pediatric office visits, new antibiotic prescriptions and
surgery in children. The two most commonly isolated pathogens from otitis media infections are Streptococcus pneumoniae (Spn) and Haemophilus influenzae (Hi). Current data indicate that most OM infections involve simultaneous infection with multiple organisms, and thus there is a pressing need for a
better understanding of the impact of polymicrobial infection on the establishment, progression and
severity of OM disease. In this study, we used a well established chinchilla model of experimental otitis
media to test the impact of polymicrobial infection with Hi and Spn. Following transbullar infection,
stable biofilm communities containing Hi and Spn were formed and persisted for up to 21 d postinfection. Biofilms removed from coinfected animals contained both bacterial species. Notably, the
incidence of fatal systemic pneumococcal infection was dramatically reduced in the coinfected animals
as compared to animals infected with pneumococcus alone. Moreover, the percentage of translucent
pneumococcal colonies recovered from coinfected animals was significantly increased as compared to
animals infected with pneumococci alone, in which the majority of the population gradually shifted to
a predominantly opaque population. This finding is notable in that the phase variation from translucent (low capsule levels) to opaque (high capsule levels) colony type is a key step in the establishment
of systemic pneumococcal infection. Comparison of biofilm formation by Spn alone and in the presence of Hi using in vitro biofilm models recapitulated these findings, as pneumococcal biofilm formation was significantly increased in the presence of Hi. Therefore, we conclude that coinfection with Hi
moderates pneumococcal infection by promoting the persistence of translucent pneumococcal variants
within surface-attached biofilm communities. These results have great significance in understanding
the clinical course of pneumococcal infection, which in patients is usually a localized infection as opposed to the septic infections observed in most animal models.
1.2) Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal
overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production
Matthew S. Byrd, Irina Sadovskaya, Evgueny Vinogradov, Haiping Lu, April B. Sprinkle, Stephen H. Richardson, Luyan Ma, Brad Ralston, Matthew R. Parsek, Erin M. Anderson, Joseph S. Lam, and Daniel J.
Wozniak
Department of Microbiology and Immunology, Wake Forest University School of Medicine
Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc
pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide
synthesis locus (psl), is required for biofilm formation in non-mucoid strains that do not rely on alginate
as the principal biofilm polysaccharide. Mutants were constructed containing a deletion in the psl promoter region, a complete deletion of the operon, or unmarked, nonpolar deletions in individual psl
genes. Eleven genes, pslACDEFGHIJKL, proved to be required for Psl synthesis and surface attachment,
while four psl genes, pslB, pslM, pslN, and pslO were found not necessary for Psl production. We also
present the first structural analysis of the psl-dependent polysaccharide, which consists of a repeating
pentasaccharide containing D-mannose, D-glucose and L-rhamnose. In addition, we identified the sugar
nucleotide precursors involved in Psl generation and demonstrated the requirement for GDP-Dmannose, UDP-D-glucose and dTDP-L-rhamnose in Psl production and surface attachment. Finally, genetic analyses revealed that the A-band LPS enzyme WbpW restored Psl production in a pslB mutant
and PslB promoted A-band LPS synthesis in a wbpW mutant, indicating functional redundancy and overlapping roles for these two bifunctional enzymes. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and
pathogenesis of P. aeruginosa.
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1.3) Comparison of mutations affecting secretion of heat-labile enterotoxin and cholera toxin.
Benjamin Mudrak and Meta J. Kuehn
Department of Molecular Genetics & Microbiology, Duke University
Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae secrete highly homologous protein toxins
known as heat-labile enterotoxin (LT) and cholera toxin (CT), respectively. Each of these toxins consists of a single catalytically active A subunit and a ring of five B subunits mediating the binding properties of the toxins. Holotoxin formation occurs spontaneously in the periplasm, after which the assembled toxin is exported from the cell through the type II secretion system. The pentamer of B subunits
is responsible for the secretion of each toxin, and to date, one mutation affecting the secretion of CT
(E11K) has been reported. We have identified three mutations that reduce the efficiency of LT secretion from ETEC. One mutation, L25E, appears to have global effects on the folding of the toxin. In
contrast, another mutation, Q3K, reduces levels of secreted toxin to approximately half that of wildtype, with a corresponding increase in periplasmic levels of the mutant. Third, as with CT, the E11K
mutation reduces LT secretion from ETEC. Conversely, while the E11K mutation reduces secretion of
CT from V. cholerae in our system, as in a previous study, the Q3K mutation appears to have little effect on CT secretion. Thus, different residues appear to identify LT and CT as type II secretion substrates. Further mutational analysis determined that less drastic Q3A and E11A mutations in LT each
supported wild-type secretion, while secretion of an E11A CT mutant remained impaired in V. cholerae,
implying that mutation of key residues is tolerated to differing extents in the two species. Mutant LT B
pentamers were poorly expressed in V. cholerae and led to growth defects, but all CT mutants were
expressed and secreted at wild-type levels in ETEC. These results indicate that ETEC secretion system
can accommodate mutations in CT that it cannot tolerate in LT. Our analysis of the mutations affecting
the secretion of CT and LT may lead to discovery of the portions of the type II system involved in substrate recognition and the domains within these substrates that are identified during secretion.
1.4) Interaction of E. coli O157 with cut lettuce leaves
Ann G. Matthysse
Department of Biology, UNC-Chapel Hill
In recent years there have been several outbreaks of food-borne illness caused by Escherichia coli
O157:H7 carried on plant surfaces, particularly sprouts and lettuce and spinach leaves. E. coli O157 and
K12 were retained rapidly by cut leaves. Significant numbers of bacteria were retained by the cut
edge after 5 min. exposure. The number of bacteria retained increased slowly over the next 3 days.
Bacteria also entered the leaves in the region 4-5 cm above the cut edge. They appear to gain access
to the interior of the leaf in this region through the stomata. The increase in the number of bacteria
retained by the leaves after the first day appeared to be due to growth of bacteria already associated
with the leaves rather than continued recruitment from the solution.
E. coli was retained by cut
leaves at a higher initial rate than Agrobacterium tumefaciens or Sinorhizobium meliloti. A comparison of the retention of E. coli by cut leaves and sprouts of several plant species showed that the time
course and characteristics of the interaction were largely determined by the plant tissue rather than
the species. The rapid retention of bacteria and their presence inside the leaf pose difficult problems
for control of E. coli transmitted by cut leaves.
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2.1) The Chlamydial Protease CPAF Targets a Subset of Early Effector Proteins
Ine Jorgensen, Vishar Amin and Raphael Valdivia
Department of Molecular Genetics and Microbiology, Duke University
The intracellular bacterial pathogen Chlamydia trachomatis modulates a range of host cellular functions
by delivering effector proteins across the membrane of the pathogen-containing vacuole (inclusion)(1).
The secreted Chlamydial Protease-Like Activity Factor (CPAF) is synthesized late in infection and targets
multiple mammalian proteins for degradation to downregulate MHC expression (2), inhibit apoptosis (3)
and re-arrange the host cytoskeleton (4). Given the broad substrate specificity of CPAF, we hypothesized
that this protease may also target chlamydial proteins secreted during infection. We screened 325 GFPtagged chlamydial proteins (1/3 of entire genome) for sensitivity to proteolysis. The library is specific for
ORFs of unknown function and putative secreted proteins. This screen identified 24 ORFs that were either processed or degraded after incubation with infected cell cytosols. At least five ORFs were confirmed as substrates of CPAF - Ct005, Ct115, Ct116, Ct233, and Ct813. Purified versions of these proteins
were processed into distinct cleavage fragments or completely degraded after incubation with recombinant CPAF in an in vitro cleavage assay. All five CPAF substrates- Ct005, Ct115 (IncD), Ct116 (IncE) and
Ct233 (IncC)- contain a hydrophobic domain common to chlamydial proteins that associate with the inclusion membrane (Incs). Interestingly, these five proteins belong to a subclass of Incs that are expressed
during the very early stages of infection, and are either confirmed or predicted targets of type III secretion. We generated antibodies against Ct005, IncD and IncC and determined that they are expressed during early stages of infection and localize to the inclusion membrane. These findings suggest a novel role
for CPAF in regulating secreted bacterial proteins in the host cytoplasm.
The targeted degradation of
these early proteins late in infection may serve many purposes: 1) eliminate effectors that are only required at early stages of infection, 2) preclude toxic effects from the over accumulation of early effectors, 3) orchestrate a programmed dismantling of the inclusion at end of the infectious cycle to facilitate
bacterial dissemination. Alternatively, CPAF may prevent the development of secondary inclusion in previously infected cells, by degrading effectors important to the initial development of the inclusion.
2.2)The Fat and the Ugly: A Proteomics Approach to Dissect Lipid Droplet-Chlamydia Interactions
Hector A. Saka, J. Will Thompson, Laura G. Dubois, M. Arthur Moseley, and Raphael Valdivia
Department of Molecular Genetics & Microbiology, Duke University
The obligate intracellular pathogen Chlamydia trachomatis (CT) is the most common cause of sexually
transmitted infections and the leading cause of preventable blindness worldwide. CT infects epithelial
cells and multiplies within a membrane-bound vacuole or inclusion. Lipid droplets (LD), the main store of
neutral lipids in eukaryotic cells, constitute dynamic, ER-derived organelles consisting of a core of cholesterol esters or triacylglycerols, surrounded by a monolayer of phospholipids and LD-associated proteins.
Previous evidence from our laboratory shows that CT targets LDs, inducing an expansion of these organelles, their recruitment around the inclusion and ultimately their wholesale transport into the lumen of
the inclusion. These findings represent the first example of mammalian LD functions being co-opted by a
bacterial pathogen and highlights novel aspects of chlamydial cellular microbiology. To define the molecular basis for Chlamydia-LD interactions, LDs from infected and uninfected cells were purified, LDproteins extracted and analyzed by mass spectroscopy. A state of the art technique for proteomics analysis by bottom-up LC-MS/MS and LC-MSE (Waters nanoAcquity/Synapt-HDMS instrumentation) was used,
allowing for identification and absolute quantitation of proteins detected in the samples. 311 proteins
were identified in LDs, including most of the already recognized LD-associated proteins (i.e., the PAT
family, lipid metabolism-related proteins, Rab GTPases, chaperones and cytoskeleton proteins). A highly
restrictive algorithm allowed the identification and reliable quantification of a subset of 162 proteins in
all the processed samples (2 biological replicates, 6 runs each, for LD-uninfected, LD-20hpi and LD40hpi). These proteins (~90% of the samples total mass), were categorized based on protein function/
localization and changes in LD-protein levels from infected vs. uninfected cells were analyzed. In LDs
from Chlamydia-infected cells, lipid-metabolism proteins were increased whereas a large number of ER
chaperones, MHC class I and PDI were decreased. Considering that lipid acquisition is known to be essential for bacterial replication and that PDI and MHC class I are essential for optimal antigen processing/
presentation, we propose that CT targets LD to co-opt lipid transport and ER functions. Additionally, the
association of MHC-class I molecules to LDs supports a possible intersection between these organelles and
the immune response. This is to our knowledge, the first quantitative determination of these organelles’
proteome and the first comparative analysis of the LD proteome in the context of a bacterial infection.
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2.3) The B. abortus Irr in required for iron-responsive regulation of the gene encoding the heme transporter BhuA
Eric Anderson, James T. Paulley, Jennifer M. Gaines, David A. Martinson, Kendra R. Steele, & R. Martin
Roop II
Department of Microbiology & Immunology, East Carolina School of Medicine
Brucella abortus is a Gram-negative intracellular pathogen that causes abortion and infertility in its
natural bovine host. Incidental infection in humans results in a prolonged illness known as undulant
fever. As with most bacteria, iron is an essential micronutrient for B. abortus. This requirement for
iron presents a particular challenge for the brucellae, as in nature these bacteria are found exclusively
in association with mammalian hosts. Iron not incorporated into host tissues is sequestered by host
iron-binding proteins such as transferrin and lactoferrin in extracellular spaces and by ferritin with host
cells. This tight sequestration of iron in mammals serves to prevent iron toxicity in the host, as well as
limiting the availability of this nutrient to invading microbes. Previous studies have demonstrated that
heme represents an important iron source for the brucellae within their intracellular environment, and
this iron source is acquired via a TonB dependent outer membrane transporter, BhuA (Brucella heme
uptake protein A), whose corresponding gene in regulated in an iron-responsive manner. The ironresponsive regulator, Irr has been identified in a number of other members of the alphaproteobacteria, as both a transcriptional activator and repressor of genes required for the acquisition
and biosynthesis of heme. Here, we examine the role of the B. abortus Irr in controlling expression of
bhuA, showing that Irr activates expression of bhuA during iron limitation through direct interaction
with the bhuA promoter. Current studies are focused on defining the exact nature of this regulatory
link.
2.4) The twisted knot of Hfq-dependent regulation of sodC in Brucella abortus 2308
Jennifer M. Gaines, Brian Tjaden, Brittany Carrol, John Baumgartner, Clayton Caswell, Eric Anderson,
and R. Martin Roop II
Department of Microbiology & Immunology, East Carolina School of Medicine
Brucella abortus is a Gram-negative pathogen that causes abortions in animals and a chronic debilitating disease in humans known as Malta fever. Brucella is well-adapted to evade the host immune system
and establish an intracellular niche within the host macrophage. One critical Brucella virulence determinant is the periplasmic superoxide dismutase SodC which detoxifies reactive oxygen species produced by the host macrophage. Two-dimensional gel analyses of B. abortus 2308 protein lysates showed
that optimal expression of sodC required the RNA chaperone Hfq. Hfq forms a homo-hexameric ring
that scaffolds the imperfect binding of a regulatory small RNA (sRNA) and target mRNA. This interaction
modulates gene expression by altering mRNA secondary structure and/or stability of the message. A
Brucella hfq mutant is highly attenuated in both the chronic mouse model of infection and in the ruminant host, stressing the importance of sRNA regulation in Brucella. The aim of this study is to identify
the regulatory link between Hfq, a sRNA(s), and sodC expression in B. abortus 2308. Preliminary studies
using a transcriptional sodC-lacZ reporter fusion and anti-sera to SodC indicate that Hfq plays an indirect role regulating sodC expression. Nested deletion studies using an inducible promoter fused to the
untranslated region and open reading frame of sodC identified a second sodC promoter within the first
18 nucleotides of the untranslated region. This second promoter is active only in the presence of Hfq.
These results indicate that Hfq and a sRNA modulate the expression of a sodC transcriptional regulator.
Current studies are focused to identify the Hfq dependent regulator of sodC.
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2.5) Role of the RNA chaperone Hfq in expression of the genes encoding the type IV secretion machinery of Brucella abortus 2308
Clayton C. Caswell, Jennifer M. Gaines, Eric S. Anderson and R. Martin Roop II
Department of Microbiology & Immunology, East Carolina School of Medicine
Brucella spp. naturally infect a variety of domesticated and wild animals leading to abortions and sterility. These bacteria are also capable of zoonotic infections resulting from human exposure to infected
animals and animal products. During the course of chronic infection, the brucellae reside within
macrophages where they replicate in a specialized compartment associated with the endoplasmic reticulum, and the ability of the brucellae to survive and replicate within macrophages is essential to
their virulence. The type IV secretion system encoded by the virB gene cluster (virB1-12) is required for
full virulence of the brucellae. Mutation of the virB genes leads to improper trafficking of Brucella
strains within macrophages, increased bacterial killing in these phagocytes, and attenuation in experimental animal models of infection. Preliminary studies in our laboratory have linked the RNA chaperone Hfq to wild-type expression of the virB system in B. abortus 2308. Two-dimensional gel analyses
revealed that VirB1 protein levels were decreased approximately 6-fold in a B. abortus hfq mutant
compared to the parental 2308 strain. virB1 promoter fusions to lacZ were constructed and electroporated into B. abortus 2308 and the isogenic hfq mutant, and β-galactosidase assays determined that
both transcription and translation of virB1 are significantly decreased in the hfq mutant compared to
the parent. These results confirm a role for Hfq in virB1 expression in B. abortus 2308 and have led to
the working hypothesis that Hfq regulation of virB1 is mediated through an intermediate transcriptional
regulator. Recent experiments in our laboratory indicate this intermediate regulator may be the
Brucella quorum-sensing regulator BlxR, which has been shown by other laboratories to activate transcription of virB1. A translational blxR-lacZ fusion was constructed, and it was shown that βgalactosidase activity was significantly reduced in the hfq mutant compared to wild-type B. abortus
2308. Current work is aimed at confirming the link between Hfq and the expression of blxR and virB1,
as well as identifying the small regulatory RNA that works in unison with Hfq to modulate the expression of blxR.
3.1) The inflammasome adaptor ASC is important for granuloma formation and host defense in chronic
Mycobacterium tuberculosis infection
Erin McElvania TeKippe, Irving C. Allen, Paul D. Hulseberg, Matyas Sandor, Jenny P.-Y. Ting, and Miriam
Braunstein
Department Microbiology and Immunology, UNC-Chapel Hill
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is spread from person to person
via aerosol. It first infects resident macrophages within the alveolar spaces of the lung. The host goes
on to form granulomas which contain Mtb during the chronic phase of infection. The NLR gene family
mediates host immunity to various pathogenic stimuli. The majority of research focuses on acute infections while the relationship between NLRs and chronic infection has been relatively unexplored. We
study the role of the NLR protein, Nlrp3, and NLR adaptor protein ASC during infection with virulent
Mtb. We show that Mtb infection induces IL-1β secretion from human monocytes and mouse macrophages, and this secretion is dependent on the NLR inflammasome components ASC, caspase-1, and
Nlrp3. Mtb specifically stimulates the Nlrp3 inflammasome in macrophages, as IL-1β production is independent of the closely related inflammasome forming NLR, Nlrc4.
Surprisingly, in an in vivo study
of inflammasome components Nlrp3-/- and Casp-1-/- did not reveal an overt role for host protection
during chronic Mtb infection. In contrast to Nlrp3 and caspase-1, a significant role was observed for ASC
in protecting the host from Mtb infection, as shown by an abrupt decreased survival time of ASC-/mice during the chronic phase of infection. Decreased survival of ASC -/- animals is associated with a
reduced number of granulomas present in the lungs compared to wild type animals. Acid-fast staining
of infected mouse lungs reveal that ASC -/- animals contain bacteria in non-granulomatous tissue, indicating the mice are unable to contain bacteria within granulomas. In wild type mice this event is rare.
These data clearly show that the NLR adaptor ASC has an essential role in host defense during chronic
Mtb infection. In contrast, Nlrp3 and caspase-1 do not play a detectable role during Mtb infection.
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3.2) Genes that modulate the rapid rise of Ciprofloxacin resistance in P. aeruginosa
Hsun-Cheng Su, Kevin Ramkissoon, Janet Doolittle, Martha Clark, Matthew C. Wolfgang, and Morgan C.
Giddings
Department Microbiology and Immunology, UNC-Chapel Hill
Antimicrobial drug resistance has arisen in response to nearly every antibiotic developed, and the steps
leading to drug resistance remain mysterious. The opportunistic pathogen Pseudomonas aeruginosa is a
key etiological agent responsible for CF patients’ chronic lung infection. During repeated exposure to
antibiotic treatments with the drugs ciprofloxacin and tobramycin, significant increases in drug resistance quickly develop in P. aeruginosa. We have identified a two-stage process by which Pseudomonas
aeruginosa acquires drug resistance following exposure to ciprofloxacin in vitro. In Stage I, small cell
populations survive initial antibiotic exposure through non-heritable antibiotic tolerance, and in Stage
II, a population develops showing significant heritable increases in drug resistance. We compared naïve
wild-type P. aeruginosa populations to those that underwent short-term ciprofloxacin exposure, by applying multiplexed 2D-gel electrophoresis with both total protein stain and phosphoprotein-specific
stain, to identify proteins putatively associated with drug resistance. Cells from Stage II displayed two
up-regulated phosphoproteins, SSADH and MMSADH, and a highly conserved protein of unknown function. The two identified phosphoproteins are suggested by annotations to have roles in energy production and/or the production of NADH. Without functional copies of the genes encoding these proteins,
the development of Stage II heritable resistance to ciprofloxacin was strongly inhibited. Our results
suggest that Stage II heritable ciprofloxacin resistance occurs due to the modulation and ratcheting of
a pre-existing cellular pathway comprised of both known and unknown proteins, which appear to be
directly involved in mediating the first steps of antibiotic resistance.
3.3) Regulation of the Staphylococcus aureus NO-Stress Response
James R. Fuller and Anthony R. Richardson
Department Microbiology and Immunology, UNC-Chapel Hill
The ability to resist host innate immunity, including nitric oxide (NO) production, is central to the
pathogenesis of Staphylococcus aureus.
S. aureus NO-resistance is comprised of two components:
enzymatic detoxification and metabolic adaptation. The NO-dependent induction of the S. aureus flavohemoprotein, Hmp, allows for the conversion of NO to nitrate. However, residual NO still inhibits S.
aureus respiration necessitating the induction of fermentative genes including ldh1. hmp and ldh1 are
divergently transcribed in S. aureus, a genetic organization not found in other staphylococci. As of yet
the mechanism behind the response of the hmp-ldh1 locus to NO is unknown. Here, we show that the
NO-dependent induction of ldh1 is mediated through Rex, a redox sensing transcriptional regulator that
responds to the levels of cellular NADH. The 572 bp hmp-ldh1 intergenic region contains two conserved
repeat motifs (TGTGANNNNNNTCACA) residing upstream of the ldh1 promoter. Bioinformatic analyses
identified these same motifs in the 5’-untranslated regions of sixteen genes in S. aureus: yycF, adhE,
SACOL0166, pflB, ldh1, SACOL0235, SACOL0301, SACOL0660, ald1, srrA, SACOL2006, lctP, nirR, SACOL2491, ddh, and arcA. Many of these genes are required for fermentative metabolism, and are induced by NO-exposure in a Rex-dependent fashion. In contrast to ldh1, the NO-dependent induction of
hmp is independent of Rex. The two-component signal transduction system SrrAB is required for full
hmp expression, yet a NO-response is still evident in a srrABrex mutant. Thus, the coordinate regulation of S. aureus NO-detoxification and metabolic adaptation involves a complex network of multiple
regulons, including, but not limited to SrrAB and Rex.
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NC ASM 2009
The North Carolina Invitational speaker will be Frank
Scholle, Assistant Professor in the Department of Microbiology at NC State University. Frank Scholle was born and raised
in a little-known town in southern Germany. During a foreign
exchange year at the University of North Carolina Chapel Hill,
he joined the laboratory of Dr. Nancy Raab-Traub at UNCChapel Hill, studying contributions of Epstein-Barr virus gene
expression to development of nasopharyngeal carcinoma. Following completion of his Ph.D., he relocated to Galveston,
Texas to study hepatitis C virus biology with Dr. Stanley Lemon
and followed that by switching his interest to acute viral infections, working with Dr. Peter Mason on West Nile virus-host
interactions, which remains his research focus today. Anxious
to escape the Texas heat, flooding and mosquitoes, Frank
happily moved back to North Carolina and joined the department of Microbiology at NC State University in July 2005.
Flavivirus-Toll-like receptor Interactions
Frank Scholle, Department of Microbiology, NC State University
Members of the family Flaviviridae are important human pathogens. Like many other viruses, flaviviruses have evolved complex mechanisms to interact with the host immune response. The earliest
immune responses to infection involve components of the innate immune system, starting with the
recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors
(PRRs), such as toll-like receptors (TLRs), and intracellular RNA helicases, such as RIG-I and Mda-5.
PAMP recognition results in activation of specific signal transduction pathways that result in production of proinflammatory cytokines, such as type I interferon (IFN), TNF alpha and others. Type I interferon is one of the most important mediators of the early immune response. Mice deficient in IFN
signaling are more susceptible to flaviviruses and pretreatment of mice with type I IFN can prevent
flavivirus disease. One of the major pathways of IFN synthesis in viral infection in vivo is the stimulation of plasmacytoid dendritic cells (pDCs) via TLRs 7, 8 or 9. We are investigating the mechanism
of flavivirus recognition by these TLRs and our data indicates that mosquito-derived flaviviruses are
unable to stimulate TLR7 and 8 while viruses grown in mammalian cells strongly stimulate TLR7 and
8. This stimulation does not require active replication. In addition to this activation of the innate
immune response WNV, and more specifically its NS1 protein, is able to interfere with signal transduction from TLRs that are localized to endosomes. NS1 is a glycoprotein secreted to high levels
during flavivirus infection that can be endocytosed by several cell types. We are investigating the
mechanisms of NS1 inhibition of TLR signaling with particular emphasis on a potential role for secreted NS1 in this process.
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NC ASM 2009
The Keynote address will be given by Mark Goulian, Edmund J. and Louise W. Kahn Endowed Term Associate Professor of Biology, in the Department of Biology at the University of Pennsylvania.
Dr. Goulian received his Ph.D. in Theoretical High Energy Physics in 1990. At the Center for Physics and Biology at Rockefeller, University, he spent most of his time working on problems
in membrane biophysics and cell biology, primarily in the
laboratory of Sandy Simon. In the midst of this work, he became fascinated with cell signaling in bacteria. He therefore
started learning bacterial genetics and exploring twocomponent signaling in E. coli in the background. After five
years at Rockefeller, he came to the University of
Pennsylvania. His lab has continued to focus on twocomponent signaling in E. coli. Perturbing, Imaging, Modeling, and Evolving Two-Component Signaling Systems in E. coli
Mark Goulian, Department of Biology, University of Pennsylvania
All cells sense and respond to physical and chemical cues in their environment. They do this through
elaborate signal transduction networks of interacting proteins that detect, interpret, and carry out
responses to specific input signals. In bacteria, one of the major modes of signal transduction is
mediated by two-component systems. These regulatory circuits, which in their simplest forms are
characterized by two proteins - a sensor kinase and a response regulator, have been uncovered in
remarkable numbers within individual organisms and across different bacterial species. They play a
central role in regulating basic aspects of microbial physiology and mediate responses to diverse environmental signals. I will describe some of the recent work from my lab in which we have explored
aspects of the basic design of these systems in E. coli. I will specifically focus on the phosphorylation cycle, feedback, and cell-to-cell variability in these circuits.
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Dr. Goulian’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 Eanas Aboobakar
The phospholipid-binding protein Cts1 may function downstream of
calcineurin during high temperature stress response in Cryptococcus
neoformans
2 Jamila Broadway
Contact-regulated gene A (crgA) Modulates Virulence Determinants in
Neisseria gonorrhoeae
3 Denene Blackwood
Rapid Quantitative PCR for Measuring Recreational Water Quality
4 Mary Carmichael
Peptide-based probe capture of Mn oxides and associated bacteria in
various environments including deep-sea samples near Loihi Seamount, Hawaii, and Carter Salt Peter Cave, Tennessee
5 Rebecca Cooper
Role of potABC genes on polyamine transport and biofilm formation
in Vibrio cholerae
6 Carter Dillow
Detection of ammonia oxidizing archaea from the roots of Eastern
Hemlock (Tsuga canadensis) from Great Smoky Mountains National
Park
7 Floyd Inman, III
The Creation of a Plasmid Vector for the Genomic Integration of the
Gene for Green Fluorescent Protein (GFP) into Salmonella enteritidis.
8 Ben Jeuck
Identifying bacteria producing antibacterial agents from soil samples
of Albright Grove, an old growth forest in Great Smoky Mountains
National Park
9 Adam Kulp
Outer membrane vesicles relieve stress of misfolded proteins
10 Shannon Landvater
CREATION OF A DSRA CLASS II MUTANT IN HAEMOPHILUS DUCREYI
STRAINS HMC112 AND DMC111
11 Hatajai Lassiter
Lytic Transglycosylases Affect Carbohydrate and Pyrimidine Metabolism in Neisseria gonorrhoeae
12 Charles Li
Investigation of the sex locus and construction of a mutant library of
Mucor circinelloides, a human pathogenic zygomycete
13 Johnathan Locklear
Research on the use of bioluminescent organisms Pyrocystis fusiformis and transformed E. coli MM294Lux+ as tools to study the effects of shear during hypergravity and microgravity experiments.
14 David Martinson
Exploring a Link Between Hfq and bhuA Expression in B. abortus
15 Evan Menscher
Metal responsive regulation of the manganese transport gene mntH
in Brucella abortus 2308
16 MD MOTALEB
Role of cyclic-di-GMP in Borrelia burgdorferi motility and virulence
17 Bidong Nguyen
Genetic Analysis of Chlamydia trachomatis
18 Shakir Ratani
Identification of a Copper Resistance Determinant in Listeria monocytogenes Using a mariner-Based Transposition System
19 Sadondria Richardson
The Effect of Gravitational Change on Growth Rate of Non-typeable
Haemophilus influenza 86-028 NP
20 Camile Semighini
Apoptotic-like cell death in the human pathogen Cryptocococcus neoformans
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21 Stephanie Skipper
Lytic Transglycosylases Affect Cell Wall and Pilus Biosynthesis Gene
Expression in Neisseria gonorrhoeae
22 Jeffrey Spontak
Arginine Catabolic Mobile Element (ACME) and Polyamine Resistance
in USA-300 Staphylococcus aureus
23 Antonia Tetteh
A global analysis of transcriptional profiling of E. coli under toxic
and beneficial conditions of selenium
24 Oscar Tirado-Acevedo
Biological Catalysts for Conversion of Gas to Ethanol and Methane
25 Ruchi Yadav
Exploring selenium toxicity to inhibit genetically modified PC3 cancer cells with over-expression of bacterial or human selenocysteine
synthase gene
26 Shuqing Zhao
Genetics of chromosomally mediated intermediate resistance to
expanded-spectrum cephalosporins in Neisseria gonorrhoeae
27 Sobhan Nandi
Identification of novel mtrR and penB mutations in N. gonorrhoeae
isolates from New Caledonia with increased resistance to penicillin.
Abstracts (posters)
1. The phospholipid-binding protein Cts1 may function downstream of calcineurin during high temperature stress response in Cryptococcus neoformans
Eanas Aboobakar, Lukasz Kozubowski, and Joseph Heitman
Department of Molecular Genetics and Microbiology, Duke University Medical Center
Adaptation to environmental stress within the host is critical for the virulence of the human fungal
pathogen Cryptococcus neoformans. Calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, regulates many physiological processes necessary for C. neoformans virulence, including morphogenesis, cell wall biogenesis, ion homeostasis, and stress response. The phospholipidbinding protein Cts1 has been previously identified as a multicopy suppressor of a calcineurin cna1 mutant in C. neoformans. Similar to the calcineurin mutant, the cts1 strain is not viable at 37°C and exhibits cell wall defects. In addition, the cts1 cna1 double mutant strain is inviable at 24°C, suggesting
that Cts1 and calcineurin function in parallel or in branched pathways. Here, we further characterize
the function of Cts1 and the links between Cts1 and the calcineurin signaling pathway. Microscopic
analysis of Cts1 tagged to GFP at the N-terminus has revealed that Cts1 localizes at punctate structures
in the cytoplasm and colocalizes with the endosomal marker FM4-64. GFP-Cts1 was detected by Western blot analysis, which revealed slower electrophoretic mobility of GFP-Cts1 in cells grown at 37°C as
compared to cells grown at 24°C and a shift to even higher molecular weight in the presence of FK506,
an inhibitor of calcineurin. In-vitro treatment with CIP phosphatase resulted in faster electrophoretic
mobility of GFP-Cts1, suggesting that Cts1 is phosphorylated during high temperature stress and that
the phosphorylation state of Cts1 may be dependent on calcineurin.
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2. Contact-regulated gene A (crgA) Modulates Virulence Determinants in Neisseria gonorrhoeae
Jamila Broadway and Daniel Williams, Ph.D.
Department of Biology, North Carolina Central University
Contact-regulated gene A (crgA) is a member of the Lys-R family of transcriptional regulators that are
critical for the adherence of Neisseria meningtidis to host epithelial cells. Adherence has been shown
to be mediated through negative regulation of crgA, pilE, pilC1, and sia. However, recent findings suggest that CrgA does not control the expression of pilE, pilC1, and sia and is not responsible for the
down-regulation of pili during intimate cell contact. We hypothesize that CrgA may have a similar role
in regulating virulence genes in Neisseria gonorrhoeae that are involved in establishing infection. Our
long term goal of the proposed research is to define the mechanisms by which CrgA regulates virulence
determinants in N. gonorrhoeae. Results generated from real-time PCR show that CrgA activates pilE
expression. We were also interested in whether negative regulation of the lipooligosaccharide biosynthesis genes, sia affected levels of susceptibility to antibiotics. Therefore, using minimum inhibitory
concentration (MIC) assays, we determined that a mutant crgA strain had a 2-fold increased susceptibility to ampicillin, vancomycin, and oxacillin.
3. Rapid Quantitative PCR for Measuring Recreational Water Quality
Denene Blackwod, Presentor, Rachel T Noble, and Reagan Converse
Institute of Marine Sciences, University of North Carolina at Chapel Hill
Current recreational water quality management is inaccurate due to outdated science and monitoring
methods, unnecessarily putting beachgoers at risk for being exposed to pathogens of waterborne illnesses that cause gastroenteritis, dysentery, hepatitis, respiratory ailments and other serious health
problems. At present it takes 18 to 72 h for EPA-approved culture-based methods to yield results;
meaning that the beach remains open when it is polluted and closed after the threat has already
passed. In addition, there are great inaccuracies in specificity and agreement between traditional
based culture methods. There is a tremendous need for the implementation of rapid methods for
measurement of fecal indicators, such as the use of quantitative PCR, which can yield results the same
day as samples are taken. The implementation of these methods would translate into better beach
management strategies and prevent swimming related illnesses and lost tourism dollars. The EPA BEACH
Act of 2000 and the proposed Beach Protection Act (2008) requires the institution of new rapid methods
and updated standards for water quality indicators based on new epidemiological studies by 2012.
Our lab has developed several rapid quantitative PCR methods for measurement of the fecal indicator
bacteria, Enterococcus, E. coli and Bacteroides. The pitfalls and advantages of the use of these rapid
methods compared to traditional methods will be discussed and data from a recent epidemiological
study will be presented. The study includes two different beaches in southern CA. Comparisons of results for each beach (n= >450 in each case) show that method relationships are highly variable depending upon sites of sample collection, year sampled, and extent of contamination. For example, rapid
Enterococcus QPCR during summer 2007 at Doheny State Beach showed a strong relationship to EPA
1600 (r=0.85, n=112), but during summer 2008 this relationship was significantly weaker. This presentation will provide information to help explain the methodological and endpoint based differences and
similarities between rapid QPCR-based methods and culture-based methods, and improve our abilities
to consider the ramifications of widespread implementation of rapid QPCR-based methods for recreational water quality monitoring.
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4. Peptide-based probe capture of Mn oxides and associated bacteria in various environments including
deep-sea samples near Loihi Seamount, Hawaii, and Carter Salt Peter Cave, Tennessee
Carmichael, MJ, Presentor, BM Tebo, & SL Brauer
Department of Biology, Appalachian State University
Cave microorganisms are actively involved in creating organic matter from CO2, in rock weathering
processes, and in forming ferromanganous crusts and biofilms on rock surfaces. Phylogenetic analyses
alone are insufficient to determine what organisms are involved in microbially induced manganese
oxidation; thus, we are attempting to develop a molecular method that employs a peptide probe specific for biogenic Mn oxides. 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 and Carter Salt Peter
Cave. 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 at Loihi Seamount found that 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 determined 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 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. The
procedure was repeated at Carter Salt Peter Cave and preliminary analysis indicated a modest enrichment of Pseudomonads. Each sample type requires at least some optimization, so we plan to repeat
these experiments with higher stringency to verify the results.
5. Role of potABC genes on polyamine transport and biofilm formation in Vibrio cholerae
Rebecca Cooper & Ece Karatan
Department of Biology, Appalachian State University
Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera disease, which is
characterized by the colonization of the gastrointestinal tract by V. cholerae, with symptoms including
severe diarrheal disease, dehydration, and decreased blood pressure. The bacterium is also a natural
inhabitant of aquatic environments, including freshwater, estuarine, and marine environments and has
the capacity to form biofilms. One of the environmental signals that are known to influence biofilm
formation in V. cholerae is the presence or absence of polyamines, specifically norspermidine and
spermidine. Polyamines are small aliphatic hydrocarbon molecules with quaternary nitrogen groups
that possess a net positive charge at physiological pH. Polyamines are ubiquitous among all organisms,
and are required for cell growth. Polyamines are produced within the cell, but exogenous polyamines
can also be imported in. In E. coli, polyamine transport, specifically spermidine, is facilitated by the
ATP-binding cassette (ABC) transporter encoded by the PotABCD operon. In V. cholerae, an ABC transporter responsible for the preferential uptake of spermidine known as the PotABCD2D1 operon has
been identified. Our lab has previously shown that potD1 functions in spermidine uptake, while potD2
does not. In addition, the deletion of potD1 results in an increase in biofilm formation while the deletion of potD2 does not. Our current research efforts focus on the characterization of the genes that
comprise the remainder of the PotABCD2D1 operon, specifically potA, potB, and potC. To assess the
function of PotA, PotB, and PotC proteins, we have started constructing in-frame deletions in the potA,
potB, potC genes. Preliminary results show that the potC strain shows a phenotype similar to the
potD2 strain, but different from the potD1 strain. That is, the potD2 strain does not show an increase
in biofilm formation. This result suggests that PotC may not be required for spermidine uptake. We
propose the hypothesis PotC and PotD2 work together with PotA to import a yet uncharacterized
polyamine whereas PotB and PotD1 work together with PotA to import spermidine. Deletion experiments and subsequent biofilm polyamine content analyses for potA, potB and potC strains will be performed to test this hypothesis.
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6. Detection of ammonia oxidizing archaea from the roots of Eastern Hemlock (Tsuga canadensis) from
Great Smoky Mountains National Park
Carter Dillow & Sean O'Connell
Biology Department, Western Carolina University
Eastern Hemlock (Tsuga canadensis) is an ecologically important species under attack by the Hemlock
Wooly Adelgid (Adelges tsugae). Understanding the microbial communities in hemlock rhizospheres
may be key to saving this species. Of particular interest in rhizospheres is the cycling of elements essential for life, such as nitrogen. We sampled soil attached to hemlock roots from three sites in Great
Smoky Mountains National Park; an old growth forest (Albright Grove), a forest formerly clear cut (Purchase Knob), and a forest that had suffered massive chestnut death (Cataloochee). DNA was extracted
from triplicate soil samples and then PCR performed using bacterial ammonia oxidizing subunit A (AOB
amoA) and archaeal ammonia oxidizing subunit A gene primers (AOA amoA) with the 5’-primer labeled
with 6-FAM dye. Following PCR, the products were digested with a combination of two restriction enzymes and the terminal restriction fragment length polymorphism (T-RFLP) products were run in a capillary DNA sequencer. PCR products were generated for AOA amoA from all samples taken, but for only
two of nine AOB amoA reactions. The AOA amoA T-RFLP electroferrograms for Albright Grove had an
average of 47.7 ± 9.3 peaks and 82 total peaks, Cataloochee had an average of 57.3 ± 1.2 peaks and
105 total peaks, and Purchase Knob had an average 50.7 ± 11.7 peaks and 103 total peaks. This overall pattern of diversity for archaeal populations reflects general bacterial diversity patterns for these
same sites, i.e., diversity is higher in the disturbed forests. Principal components analysis (PCA) of the
T-RFLP data showed common microbial distribution patterns in Albright Grove and Purchase Knob while
Cataloochee was very different. The role that Archaea plays in the nitrogen cycle is of great interest
in the microbial ecology world and this study documents for the first time that that ammonia oxidizing
species exist in Eastern Hemlock and may be more important than Bacteria. This is of particular interest since this tree is rapidly dying in its native habitat and may require knowledge of existing microflora for its survival.
7. The Creation of a Plasmid Vector for the Genomic Integration of the Gene for Green Fluorescent Protein (GFP) into Salmonella enteritidis.
Floyd L. Inman III, Heather Walters and Len Holmes
Department of Chemistry and Physics, UNC at Pembroke
The aim of this work is to integrate the gene for Green Fluorescent Protein (GFP) into the chromosomal
DNA of Salmonella enteritidis. Our approach is to apply standard cloning techniques for the insertion of
the PCR amplified GFP gene which is restricted from the Clontech™ plasmid (pGFP®) into an electrophoretically purified, enzymatically digested chromosomal fragment containing a selected non-coding
region of the Salmonella enteritidis genome. The resulting recombinant DNA fragment will then be
ligated to form a non-replicating 3.3 kb plasmid that is then amplified and inserted into Salmonella
enteritidis for GFP integration using electroporation.
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8. Identifying bacteria producing antibacterial agents from soil samples of Albright Grove, an old
growth forest in Great Smoky Mountains National Park
Ben Jeuck, Sean O'Connell
Biology Department, Western Carolina University
Prokaryotic biodiversity is high in soil and especially in old growth forests such as in Great Smoky Mountains National Park (GSMNP). Bacterial species, previously discovered and uncultured, are capable of
providing the biotechnology and drug-development fields with countless new biological agents, of
which antibacterial compounds are one. The purpose of our experiment was to culture bacteria that
exhibited antibiotic characteristics and to identify these species. Soil samples were collected from
forest soil near Albright Grove Trail. Six soil samples were taken from below different tree species,
pooled, and diluted and spread-plated on dilute nutrient broth (DNB) agar, R2A, and Luria broth (LB).
The colonies that appeared to display antibiotic characteristics and the colonies affected were
streaked for isolation. Only R2A produced such colonies. The isolates were competed against each
other to verify the presence of an antibiotic agent. Those that continued to exhibit antibiotic characteristics (production or sensitivity) were subjected to partial 16S rDNA sequencing. The aggressor species were also competed against Escherichia coli and Bacillus cereus to determine the sensitivity of
these common lab cultures. The four presumptive antibiotic producing strains belonged to phyla including Firmicutes (Clostridia) and Actinobacteria, while the sensitive strains were aligned with Firmicutes (Bacillus), Betaproteobacteria, and one that is unknown at this time. Tests of the antibiotic
producers against E. coli and B. cereus showed an effect from two strains against these species. Bacteria can find competitive advantages in their production of bioactive compounds. Most species of bacteria have not been cultured, yet they represent a wealth of novel biochemicals, some of which can be
used in developing new drugs, antibiotics, and other products. This research yielded species that were
selected on low-nutrient media and may represent undiscovered bacteria with unusual chemical products. More work is planned to identify the compounds produced by these bacteria and to look for even
more extreme growers that have not yet been brought into culture.
9. Outer membrane vesicles relieve stress of misfolded proteins
Adam Kulp and Meta Kuehn
Duke University
Gram negative bacteria have many ways of dealing with misfolded proteins in that accumulate from
various environmental stressors. Several envelope stress response pathways have been described in
Escherichia coli, including the SigmaE, Bae and Cpx pathways. The SigmaE pathway in particular responds to misfolded outer membrane proteins in the periplasm by recognizing an exposed three amino
acid sequence at the C-terminus of the denatured protein. The SigmaE pathway upregulates periplasmic chaperones and proteases, and has been shown to increase the production of outer membrane
vesicles (OMVs). OMVs are 20-200 nm structures whose surfaces are composed of outer membrane lipids and proteins while the interior contains periplasmic proteins. Misfolded proteins can be expelled
from bacteria via these OMVs, thus an increase in vesiculation can serve as an envelope stress response
pathway. While vesiculation and the SigmaE pathway are clearly linked, there is evidence that vesiculation can be induced by misfolded proteins independently. The data presented here provides evidence
that E. coli uses vesiculation as a vital independent envelope stress response pathway, and that strains
deficient in vesiculation are more susceptible to the harmful effects of misfolded envelope proteins.
We hope that further understanding this response will yield insights into the survival of Gram negative
bacteria in general, as well as help elucidate the mechanism behind vesicle formation. This is important because vesicles can be used to disseminate toxins such as the heat labile enterotoxin of Enterotoxic E. coli.
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10. CREATION OF A DSRA CLASS II MUTANT IN HAEMOPHILUS DUCREYI STRAINS HMC112 AND DMC111
Shannon W. Landvator, and C. Dinitra White
Department of Biology, North Carolina Agricultural and Technical State University
Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease
that increases the risk of HIV transmission. Chancroid ulcers are characterized by soft, painful ulcers
that bleed readily and may provide a suitable environment for the acquisition, maintenance, and
spread of HIV. The well studied type strain H. ducreyi 35000HP was previously sequenced and annotated by Robert Munson and colleagues. Compared to 35000HP, several clinical isolates possessed variations in the amino acid composition and/or protein expression of virulence-associated outer membrane
proteins and lipooligosaccharide. Therefore, 35000HP-like strains were designated class I (CI), and
strain variants were designated class II (CII). We have previously been unable to generate an isogenic
mutant in the H. ducreyi CII strains using traditional methods. Therefore, the purpose of this study was
to construct a non-plasmid, CII dsrAII-deletion cassette by employing a novel technique consisting of
overlapping extension polymerase chain reaction (oePCR). We designed PCR primers that were specific
to the upstream region of the dsrAII gene (this product included 500 base pairs of upstream flanking
DNA and approximately 100 base pairs of the beginning of the gene). In addition, we designed PCR
primers that were specific to the downstream region of the dsrAII gene (this product included 500 base
pairs of downstream flanking DNA and approximately 100 base pairs of the end of the gene). Finally, we
constructed primers that would amplify a chloramphenical acetyltransferase cassette (CAT) to be used
in development of the deletion cassette. Whole genomic DNA from class II strain HMC112 was used as a
template in the dsrAII PCR reactions, while the CAT cassette was amplified using pRY112 plasmid DNA.
Thus far, we have successfully amplified the upstream and downstream PCR products from HMC112 in
addition to the approximately 950 bp CAT DNA product. Studies are currently underway to complete
construction of the dsrAII-deletion using oePCR.
11. Lytic Transglycosylases Affect Carbohydrate and Pyrimidine Metabolism in Neisseria gonorrhoeae
Hatajai Lassiter and Daniel Williams, Ph.D
Department of Biology, North Carolina Central University
Neisseria gonorrhoeae (gonococcus) is the etiologic agent of the sexually-transmitted disease gonorrhea. Peptidoglycan (PG) is a critical cell wall component that is recycled and released at high frequency via lytic transglycosylases (LT) during cell division. LTs cleave the β-1,4-glycosidic bond linking
N-acetylmuramic acid (NAM) and N-acetylglucosmine (NAG) glycan residues of PG. Although several LTs
are encoded in the gonococcal genome, ltgA and ltgD are responsible for a significant percentage
(~90%) of PG recycling and release. PG release has been also implicated in the pathogenesis of pelvic
inflammatory disease (PID) and disseminated gonococcal infection (DGI). We hypothesize that lytic
transglycosylase A and D modulate glucosamine-fructose-6-phosphate aminotransferase (glmS) and uracil phosphoribosyltransferase (upp) expression during normal growth and/or exposure to drugs targeting
the cell wall. Mutant LT strains showed no growth abnormalities compared to wild-type, but conventional and real-time PCR did show negative modulation of glmS and upp expression during normal
growth. It is plausible that increased glmS and upp expression in mutant LT strains aids in maintaining
the cell wall during normal growth. We also hypothesize that disruption of PG recycling and release
will increase gonococcal susceptibility to host innate immune responses (e.g. antimicrobial peptides)
and antimicrobials. Results from minimum inhibitory concentration (MIC) assays indicate that mutant
LT strains have increased susceptibility to drugs inhibiting cell wall synthesis; therefore, lytic transglycosylases may be potential targets of antibiotic therapy.
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12. Investigation of the sex locus and construction of a mutant library of Mucor circinelloides, a human
pathogenic zygomycete
Charles H. Li, Santiago R. Torres-Martinez, Soo Chan Lee, Joseph Heitman
Department of Molecular Genetics and Microbiology, Duke University
Mucor circinelloides is a zygomycete fungus that is an opportunistic pathogen that can infect immunosuppressed and transplant patients, resulting in life-threatening mucormycosis. M. circinelloides is
heterothallic, forming a specialized sexual structure, the zygospore, when two mating type strains are
co-cultured. The sex locus, which governs and orchestrates sexual reproduction, of a related zygomycete Phycomyces blakesleeanus was characterized to consist 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. Genome searches and sequence analysis were
used to analyze the sex locus of the subspecies. Additionally, Agrobacterium tumefaciens induced mutagenesis was performed in an attempt to identify genes related to virulence. The pyrG gene, encoding
OMP decarboxylase, is often used as a marker in M. circinelloides, in which a mutation in pyrG results
in an auxotrophic requirement for uracil/uridine and confers resistance to 5-FOA. Random mutagenesis
was conducted in a pyrG mutant strain to construct a mutant library and mutants were screened for
virulence using a heterologous host model with the larvae of the wax moth, Galleria mellonella. We
found that 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. Multi-locus sequence typing
(MLST) was used to construct a phylogeny of the Mucor subspecies. Our findings suggest that the subspecies are closely related but their HMG-domains are highly diverged. Searches for virulence genes
are ongoing. These findings may further unravel the evolution of the sex locus and sex determination in
basal fungi.
13. Research on the use of bioluminescent organisms Pyrocystis fusiformis and transformed E. coli
MM294Lux+ as tools to study the effects of shear during hypergravity and microgravity experiments.
Johnathan Locklear, Dr. Len Holmes, Sarah Brown
Department of Chemistry, University of North Carolina at Pembroke
Fluid dynamics has direct effects on basic environmental parameters of suspension cultures of microorganisms, both within the cell and in the bulk fluid. Batch suspension cultures of the bioluminescent
microorganisms E. coli MM294Lux+ and the marine dinoflagellate Pyrocystis fusiformis may provide
useful models to observe and measure fluid dynamic effects on cell physiology. It is the purpose of this
research to investigate the utility of such an approach. The effects on bioluminescent response will be
recorded via a luminometer. Cell population data, pH and oxygen saturation under experimental conditions will also be collected.
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14. Exploring a Link Between Hfq and bhuA Expression in B. abortus
David A. Martinson, Jennifer M. Gaines, Eric S. Anderson, James T. Paulley, R. Martin Roop II
Department of Microbiology, East Carolina University
Brucella abortus is a small, gram-negative, facultative intracellular zoonotic pathogen. B. abortus infections can lead to abortion and infertility in cattle and undulant fever, a chronic, debilitating zoonotic disease in humans. Infection is established when bacteria enter the host though inhalation or ingestion where they penetrate the mucosal epithelium and are transported to the regional lymph nodes. B.
abortus enters host macrophages and nonprofessional phagocytes, such as trophoblasts in pregnant cattle, where they survives and multiply in the endoplasmic reticulum. One challenge the bacteria must
overcome to survive in this niche is the lack of free iron within the intracellular compartment of the
phagosome. A major biological role of the macrophage is to recycle iron containing heme molecules in
red blood cells. As a result, heme is in a constant rate of flux within the macrophage. B. abortus overcomes the low iron conditions by taking advantage of the heme flux rate within the macrophage by internalizing heme molecules through a heme uptake system. The Ton-B dependent outer membrane protein BhuA transports heme though the outer membrane into the periplasmic space where another set of
proteins transports heme into the cytoplasm. Many bacterial outer membrane genes have been shown
to be regulated at the posttranscriptional level through the action of small noncoding RNAs (sRNAs) and
the RNA chaperone Hfq. Here, we have begun to explore the regulation of the B. abortus bhuA gene at
the post-transcriptional level. Transcriptional fusion experiments indicate high levels of bhuA transcriptional activity under low iron conditions, however low levels of BhuA were detected by Western blot,
suggesting there is a mechanism for inhibiting translation of bhuA mRNA. In an hfq deletion B. abortus
strain, bhuA translational fusion activity levels were greatly increased in iron depleted conditions as
compared to wild type. In B. abortus, the data suggests there is a mechanism of post-transcriptional
regulation in which Hfq is required for normal expression of bhuA.
15. Metal responsive regulation of the manganese transport gene mntH in Brucella abortus 2308
Evan A. Menscher, Eric S. Anderson, & R.M. Roop II
Department of Microbiology, East Carolina University
The Brucella spp. are Gram-negative bacteria that reside in host macrophages and cause a debilitating
febrile illness in humans. The capacity of the Brucella spp. to survive and replicate in the phagosomal
compartment of host macrophages is critical for their virulence. Within this intracellular environment,
the brucellae face the limited availability of several important micronutrients including divalent cations. Work in our laboratory has shown that the H+-dependent manganese transporter MntH serves as
the sole high-affinity manganese transporter in B. abortus 2308, and an isogenic mntH mutant derived
from this strain exhibits extreme attenuation in both cultured murine macrophages and experimentally
infected mice. The nucleotide sequences upstream of mntH in B. abortus 2308 contain motifs that resemble predicted binding sites for the manganese-responsive transcriptional repressor Mur. Gel shift
assays with Mur performed in the presence of 100 µM MnCl2 show direct binding to the mntH promoter,
but not to known iron responsive genes, indicating a strong preference for Mn and not Fe. Transcriptional fusions were constructed with the mntH promoter fused to lacZ and β-galactosidase activity was
measured in the presence and absence of exogenous MnCl2. Mur represses the expression of mntH in a
Mn dose-dependent manner in B. abortus 2308, but this Mn-responsive repression of mntH expression is
not observed in an isogenic mur mutant. Studies are currently ongoing to identify the specific nucleotides to which Mur binds in the region upstream of the mntH promoter and evaluate the relative importance of Mur-mediated Mn-responsive repression of mntH expression in the virulence of B. abortus 2308
in experimental hosts.
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16. Role of cyclic-di-GMP in Borrelia burgdorferi motility and virulence
Syed Z. Sultan1, Josh Pitzer1, Michael Miller2, Michael Betteken1 and Md A. Motaleb1
1Department
of Microbiology and Immunology, School of Medicine, East Carolina University, Greenville,
NC 27834 2Department of Biochemistry, West Virginia University, Morgantown, WV 26506
Lyme disease is caused by the spirochete Borrelia burgdorferi and transmitted to humans by Ixodes
(deer) ticks. The disease is the most prevalent vector borne disease in the United States and Europe.
Lyme disease has various clinical manifestations including erythema migrans rash, neurological symptoms, Lyme arthritis, and carditis. Deaths have also been attributed to Lyme disease. A small percentage of patients who develop Lyme arthritis do not respond to repeated courses of oral or intravenous
antibiotics. No vaccine is currently available. Understanding factors important in causing Lyme disease
is fundamental for developing effective prevention/treatment regimes. Two-component signaling systems are required for gene regulation governing bacterial virulence, adaptation, osmoregulation and
sporulation. Only three two-component signaling systems have so far been reported in B. burgdorferi.
These include hpk1-rrp1, hpk2-rrp2, and a chemotaxis system. The hpk1-rrp1 two-component system
of B. burgdorferi appears to be responsible for regulating the level of cyclic-dimeric-guanosine monophosphate (cdG). In many pathogenic bacteria, cdG is involved in diverse physiological functions including motility, virulence and biofilm formation. However, a role of cdG in B. burgdorferi pathogenesis has
not been reported. Genome sequence and published reports indicate that B. burgdorferi contains a
complete set of cdG-synthesizing and -hydrolyzing enzymes (Fig 1). Rrp1 (gene locus number BB0419)
encodes a GGDEF active site domain containing diguanylate cyclase (DGC) that is active when phosphorylated. Rrp1 is also reported to be involved in regulating the expression of many genes, although its
role in vivo has not been reported. BB0363 and BB0374 are putative cdG-specific phosphodiesterases
containing the highly conserved domains EAL and HD-GYP, respectively, which are essential for cdG
phosphodiesterase activity. Our results demonstrate for the first time that a putative cdG phosphodiesterase, BB0363 has a critical role in B. burgdorferi motility and virulence. The BB0363 mutant cells
have an altered motility that rendered the cells non-infectious in mice.
17. Genetic Analysis of Chlamydia trachomatis
Bidong D Nguyen, Raphael H. Valdivia
Department of Molecular Genetics and Microbiology, Duke University
The lack of a system to enable genetic manipulation of Chlamydia species has limited our understanding of how these medically important pathogens cause disease. To address this limitation, we devised a
methodology based on deep sequencing technologies to perform genetic analysis in Chlamydia. We
generated a collection of EMS-induced mutants that produce small plaques in Vero cells. A small
plaque (spq) phenotype may indicate defects in growth, infectivity, invasion, establishment/
maintenance of the chlamydial parasitophorous vacuole (inclusion) and developmental transitions. Six
spq mutants were characterized by electron microscopy and immunohistochemistry. We found that
mutants display abnormal cellular morphology, as well as defects in the localization of inclusion markers. Interestingly, one subset of mutants displays a hyper-accumulation of glycogen in the lumen of the
inclusion, a phenotype that has not been previously reported. EMS-induced single nucleotide polymorphisms (SNP) in these mutants will be identified by whole genome sequencing using a Solexa/Illumina
format. PCR and traditional Sanger-based sequencing will be used to confirm SNPs in mutants. To link
phenotypes with genotypes, we will generate chlamydial recombinants by co-infection of mutant and
wild type strains and the inheritance of SNPs will be assessed by PCR. This strategy represents our first
forays into developing a system for genetic analysis in Chlamydiae and other bacteria that may not be
amenable to genetic manipulation.
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NC ASM 2009
18. Identification of a Copper Resistance Determinant in Listeria monocytogenes Using a mariner-Based
Transposition System
Shakir S. Ratani, Vikrant Dutta, Reha O. Azizoglu, & Sophia Kathariou
Department Food Science, Bioprocessing, and Nutrition Science, North Carolina State University
The factors underlying the environmental persistence of Listeria monocytogenes have not been completely understood. One of the intriguing aspects of this persistence is the heavy metal resistance to
toxic metals such as cadmium, arsenic, and copper. Genetic determinants for cadmium resistance have
been identified, and resistance to cadmium and arsenic has been extensively used for strain typing.
However genes encoding copper resistance remain to be identified. In this work, using a mariner-based
transposition system, we have identified a possible determinant for copper resistance in the 10403S
strain of L. monocytogenes. Transposition vector pMC38 was transferred into L. monocytogenes strain
10403S, and transformants were screened for enhanced susceptibility to copper on TSA-YE plates with
10 µM CuSO4.5H20. The insertion of the transposon was localized on Lmon1_020100001620 annotated as
Uracil-DNA glycosylase downstream of a multidrug resistance protein. The insertion of the transposon
was confirmed using PCR. In future studies we intend to perform the functional analysis of this mutant
and confirm our results using genetic complementation.
19. The Effect of Gravitational Change on Growth Rate of Non-typeable Haemophilus influenza 86-028
NP
Sadondria Richardson,C. Dinitra White, & Gregory Goins
Department of Biology, North Carolina Agricultural & Technical State University
Haemophilus influenza, an opportunistic human pathogen which has evolved to thrive in the mucosal
tissues, specifically the nasopharynx and respiratory tract. Non-typeable H. influenza (NTHi) infection
may result in development of Chronic Obstructive Pulmonary Disease or other upper respiratory disorders. Immunocompromised individuals are especially susceptible to infection with NTHi. The National
Aeronautics and Space Administration are exploring the possibility of long-term colonization and a future manned mission to Mars. However; exposure to microgravity results in decreased immune function
in humans. Although little is known about the effect of microgravity on human microflora or known
pathogens, studies have shown that Salmonella enterica, a food-borne pathogen, grown in a microgravity environment expressed additional factors which lead to higher mortality rates in a mouse model of
infection. To date, few studies of other human pathogens have been conducted. Therefore, we examined the effect of simulated micro-gravity on NTHi 86-028 NP growth rate and biofilm formation. To
simulate microgravity, liquid cultures were grown at 1 RPM in an Erlenmeyer flask fixed at a 90° angle
(horizontal). However, controls were grown at 1 RPM in a flask placed upright (vertical) to maintain
normal gravity conditions. Samples were collected from the flasks every hour for 12 hours and optical
density (OD) determined at 600nm using a spectrophotometer. To determine the growth rate of bacteria in static cultures, NTHi 86-028 NP were grown in 96-well tissue culture plates and the OD at 490nm
was determined. Finally, Variations in biofilm formation was detected in microtiter plates by allowing
the bacteria to attach to the wells, staining the biofilm with crystal violet, and measuring the OD at
570 nm. Preliminary data indicate that there was no statistical difference in growth rates of NTHi 86028 NP subjected to simulated microgravity as compared to those grown under normal gravitational
conditions. However, bacteria grown under simulated microgravity conditions produced significantly
less biofilm than those grown under normal conditions.
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NC ASM 2009
20. Apoptotic-like cell death in the human pathogen Cryptocococcus neoformans
Camile P. Semighini, Yen-Ping Hsueh and Joseph Heitman
Department of Molecular Genetics & Microbiology, Duke University Medical Center
Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular
organisms. Apoptotic-like cell death (ALCD) has been described in several fungi including the opportunistic human pathogen C. neoformans, which undergoes ALCD in response to oxidative stress. Conversely, capsular polysaccharides from C. neoformans are known to induce apoptosis in host immune
cells, contributing to its virulence. Our goals are to characterize the apoptotic-signaling cascade in C.
neoformans as well as its unique features when compared to the host machinery, which can be potentially targeted as a novel antifungal strategy. In order to functionally analyze candidate apoptotic proteins in C. neoformans, we identified two caspase-like genes (MCA1 and MCA2) and a putative
apoptosis-inducing factor (AIF1) homolog, and the three genes were deleted in the serotype A background. Phenotypic characterization of aif1, mca1, mca2 and mca1 mca2 mutant strains indicate that
they are resistant to hydrogen peroxide and that they show a delay in hydrogen peroxide-induced
ALCD. The mutant strains also exhibit mating defects suggesting apoptosis may occur during sexual development. Apoptosis was also shown to be important for population fitness, however virulence analysis
using the Galleria mellonella larvae fungal pathogenicity model and an inhalation murine model of
cryptococcosis showed that the apoptotic mutant strains have no significant difference in virulence
when compared to the wild-type strain H99.
21. Lytic Transglycosylases Affect Cell Wall and Pilus Biosynthesis Gene Expression in Neisseria gonorrhoeae
Stephanie Skipper and Daniel Williams Ph.D.
Department of Biology, North Carolina Central University
Neisseria gonorrhoeae (gonococcus) is the causative agent of the sexually-transmitted disease (STD)
gonorrhea. Gonococcal infections are acquired by sexual contact and usually attach via the Type IV Pilus System (Tfp) to the mucosal membranes of the urethra in males and the endocervix and urethra in
females. Lytic transglycosylases (LT) are a special class of virulence determinants responsible for the
recycling of PG monomers and release of cytotoxic PG fragments that cause lysis of cervical epithelial
cells. It is conceivable that LTs may indirectly affect Tfp assembly and biogenesis during remodeling of
the cell wall; therefore, we hypothesize that lytic transglycosylases, LtgA and AtlA, modulate expression of penicillin binding protein-A1 (ponA1) and pilin biosynthesis gene-Q (pilQ) during cell division.
Real-time PCR results showed negative modulation of ponA and pilQ expression in a mutant LT strains.
We also determined that a mutant LT strains have an increased susceptibility to drugs interfering with
cell wall synthesis.
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NC ASM 2009
22. Arginine Catabolic Mobile Element (ACME) and Polyamine Resistance in USA-300 Staphylococcus aureus
Jeffrey S. Spontak, Anthony R. Richardson
Department of Microbiology and Immunology, University of North Carolina
Staphylococcus aureus is a major human pathogen causing skin and soft tissue infections, pneumonia,
endocarditis and septicemia. The multidrug resistance associated with clinical staphylococcal isolates
complicates treatment of these infections especially those caused by methicillin resistant S. aureus
(MRSA). In the last decade, a hyper virulent MRSA clone known as USA-300 has emerged and is now responsible for most community and hospital acquired infections. Here we show that S. aureus is almost
universally susceptible to small polycationic aliphatic hydrocarbons known as polyamines. These compounds contain two ore more primary amino groups that net a positive charge at physiological pH.
Polyamines are ubiquitous in nature and are said to be produced by all living organisms. Curiously, of
the fourteen sequenced Staphylococcus aureus genomes, none harbor recognizable polyamine biosynthetic genes. In contrast to all other tested S. aureus isolates, USA-300 displayed complete resistance
to all polyamines. USA-300 contains a newly acquired genetic island known as the Arginine Catabolic
Mobile Element (ACME). We found that USA-300 ACME is sensitive to polyamines in the same manner as
other non-ACME containing S. aureus. Located within the ACME cassette, is an annotated Nacetyltransferase that displays homology to Spermidine/Spermine N-acetyltransferase (speG). USA-300
speG gives rise to polyamine sensitivity and pspeG confers polyamine resistance in non-ACME containing
S. aureus. Thus, ACME-encoded speG is necessary and sufficient for the polyamine resistance associated with USA-300 S. aureus. Given the copious amounts of polyamines produced by the host at sites
of inflammation, this newly acquired polyamine-resistance may provide a selective advantage to USA300 explaining the remarkable success of this clone.
23. A global analysis of transcriptional profiling of E. coli under toxic and beneficial conditions of selenium
Antonia Y Tetteh, Chiu-Yueh Hung and Jiahua Xie
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise,
North Carolina Central University, Durham, NC 27707, USA
Selenium (Se) is an essential micronutrient for mammals and bacteria. It is a component of a novel
amino acid (AA) selenocysteine (commonly referred to as the 21st AA), which is required to produce the
antioxidant selenoproteins. However, Se becomes toxic when its concentration is elevated. Se pollution
caused by both natural and anthropogenic sources is a big problem in the USA. In nature, some soils in
the western United States (about 160 Kmiles2) formed from shale rocks contain high concentrations of
Se. Understanding the mechanisms of Se metabolism and detoxification is very important. Unlike
mammals, bacteria can tolerate relatively high selenium environment and some species have been directly used to clean up Se contamination. Some genes and their roles in responding Se treatments have
been studied individually, but the genome-wide transcript changes under different Se conditions have
not been reported. The objectives of this research are to: a. study the transcriptional profiling of E.
coli under toxic and beneficial Se concentrations, b. identify some key genes related to Se accumulation and detoxification for phytoremediation. Our primary study showed that the beneficial concentration of sodium selenite on E. coli K12 strain is around 10 µM with the increase of cell number approximately 20% while the 50% inhibitory concentration is 1mM. Based on these data, we are using microarray technique to analyze the transcriptional profiling under stimulatory (10 µM) and inhibitory (1mM)
concentrations. qRT-PCR will be used to validate the differentially expressed genes. This work may allow us to screen some key genes related to selenium accumulation and detoxification, which can be
transferred into plants for phytoremediation. Acknowledgments: supported by a USDA grant (200935318-05032) and a NC Biotechnology Research Grant (2007-BRG-1223).
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NC ASM 2009
24. Biological Catalysts for Conversion of Gas to Ethanol and Methane
Oscar Tirado-Acevedo1, Mari S. Chinn2, and Amy M. Grunden1
1Department
of Microbiology, NCSU, 2Department of Biological and Agricultural Engineering, NCSU
There is a need to find new and improved ways to obtain alternative fuels that do not compete with
food sources. Any carbonaceous material can be converted to syngas (a mixture of CO, CO2, N2, and
H2) by means of gasification. Some acetogenic bacteria are able to ferment syngas resulting in alcohol,
organic acids and hydrogen as fermentation byproducts. Conversion of syngas to ethanol using microbial strains achieves high yields and is more environmentally friendly than traditional chemical catalysis. Clostridium ljungdahlii ferments syngas to ethanol resulting in the conversion of the CO2 and CO
present in the syngas to ethanol and acetic acid. ATP synthesis resulting from acetate production
skews the final liquid products to higher acetate production compared to ethanol. Therefore, development of a microbial fermentation system that can support the conversion of acetic acid to other fuel
components, such as methane, would increase the effective fuel yield of the gasification-fermentation
process. Methanosarcina species can convert acetate to methane. These aceticlastic methanogens
could be used in the production of biofuels from the acetate generated by the fermentation of syngas
by C. ljungdahlii. In addition, it is desirable to find appropriate fermentation conditions that will shift
product ratios towards ethanol. There have been previous studies reporting that certain acetogens can
produce higher amounts of ethanol compared to acetate when oxygen was added to the cultures. For
the two-step fuel production, C. ljungdahlii was grown anaerobically on liquid media and syngas was
used in the headspace. Spent media was collected and use to grow Methanosarcina barkeri and ethanol, acetate and methane concentrations were quantified. For the oxygen exposure experiments, different concentrations of oxygen were added to the culture headspace and ethanol and acetate production was quantified. In the present study we demonstrate the use of microbial catalysts for the orchestrated production of ethanol and methane and show that the addition of oxygen to the acetogen culture improves ethanol production as well as ethanol to acetate ratios.
25. Exploring selenium toxicity to inhibit genetically modified PC3 cancer cells with over-expression of
bacterial or human selenocysteine synthase gene
Ruchi Yadav, Tomilowo Abijo and Jiahua Xie
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise,
North Carolina Central University, Durham, NC 27707, USA
Selenium (Se) is an essential nutrient for mammals and other organisms, but it becomes toxic when its
concentration is elevated. The window between beneficial and deleterious effects is very narrow. Selenocysteine is a novel amino acid, which is formed by the replacement of sulfur in cysteine with Se. This
amino acid, commonly referred to the 21st amino acid, is required for the production of various antioxidant selenoproteins. However, overproduction of selenocysteines in cells can result in non-specific
incorporation into proteins, cause protein degradation, and subsequently induce apoptosis. Our goal
in this research is to explore selenium toxicity to inhibit the proliferation of cancer cells through the
induction of apoptosis. The overexpression of selenocysteine synthase gene, a rate limited gene in
selenocysteine biosynthesis pathway, may increase the production of selenocysteine. Both bacterial
(SelA) and human (SecS) selenocysteine synthase genes were amplified and cloned separately into the
expression vector. Currently, we are over-expressing them in PC3 (Prostate Carcinoma) cancer cells.
Thereafter, we will analyze the sensitivities between transformed and wild type PC3 cells under different Se treatments and determine potential mechanism(s) of cell death caused by Se treatments. Our
final goal is to combine the chemopreventive properties of Se with gene therapy to find a long lasting
cure for cancer.
Acknowledgments: supported by a USDA grant (2009-35318-05032) and a NC Biotechnology Research Grant (2007-BRG-1223).
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NC ASM 2009
26. Genetics of chromosomally mediated intermediate resistance to expanded-spectrum cephalosporins
in Neisseria gonorrhoeae
Shuqing Zhao1, Margaret Duncan1, Joshua Tomberg1, Christopher Davies2, Magnus Unemo3, and Robert
A. Nicholas1
1Department
of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 2Department
of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, and 3National Reference Laboratory for Pathogenic Neisseria, Department of Clinical Microbiology,
Örebro University Hospital, Örebro, Sweden
The recent emergence of intermediate resistance to expanded-spectrum cephalosporins in Neisseria
gonorrhoeae sounds a warning bell for the future effectiveness of these antibiotics in treating gonococcal infections. The hallmark of intermediate cephalosporin-resistant (cephI) strains is the presence of
mosaic penA alleles encoding altered forms of penicillin-binding protein 2 (PBP 2) with 55-60 amino
acid differences compared to PBP 2 from penicillin-susceptible strains. It remains unclear, however,
what role other chromosomal determinants play in increasing resistance to ceftriaxone and cefixime.
To address this question, we undertook a systematic study to define the role of penicillin-resistance
determinants in cephI resistance. The mosaic penA allele from one of these cephI strains (penA35) confers increased resistance to expanded-spectrum cephalosporins, but other determinants such as mtrR
(increased expression of the MtrC-MtrD-MtrE efflux pump) and penB (mutations in the major outer
membrane porin) are also needed to increase to the level of cephI clinical isolates. The majority of
cefixime resistance is conferred by the penA35 allele, with less contribution from other determinants,
whereas ceftriaxone resistance is nearly equally dependent on both. The gene(s) required to reach donor levels of cephI resistance appear to be the same as those that confer high-level penicillin resistance in CMRNG strains, but the molecular identities of these genes remain unknown. Our data also
suggest that cephI resistant strains arose from a single transformation event in which penA alleles from
commensal Neisseria species recombined into a penicillin-resistant strain harbouring mtrR, penB,
ponA1 (mutation in PBP 1), and the unknown gene(s) involved in high-level penicillin resistance.
27. Identification of novel mtrR and penB mutations in N. gonorrhoeae isolates from New Caledonia
with increased resistance to penicillin.
Sobhan Nandi1, Margaret Duncan1, Shuqing Zhao1, Frédérique Vernel-Pauillac2, Cyrille Goarant2 and
Robert A. Nicholas1
1University
of North Carolina at Chapel Hill, Department of Pharmacology, Chapel Hill, NC 27599-7365,
USA and 2Institut Pasteur de Nouvelle-Calédonie, Laboratoire de Recherche en Bactériologie, Nouméa
Cedex, New Caledonia
mtrR and penB are two important determinants in chromosomally mediated penicillin and tetracycline
resistance. The mtrR determinant, which causes overexpression of the MtrC-MtrD-MtrE efflux pump, is
due to mutations either in the promoter or coding region of mtrR, while the penB determinant encodes
mutations in the major outer membrane PorB1b porin. The most common mutation in mtrR is deletion
of a single nucleotide in a 13-bp inverted repeat in the mtrR promoter (-A), which ablates transcription
of mtrR and increases transcription of mtrCDE. The two mutations in penB most commonly associated
with penicillin resistance are the double mutation G101D/A102D and the single mutation G101K, which
reside on the constriction loop within the porin channel. Identification of additional mutations in these
two determinants is critical for comprehensive epidemiological monitoring of antibiotic resistance
trends in N. gonorrhoeae. In a genotyping assay to track the existence of known resistance alleles in
gonococcal isolates in New Caledonia, we identified strains with increased penicillin resistance (Minimum Inhibitory Concentration or MIC=0.13-0.16 µg/ml) but lacking the common mutations usually associated with chromosomally mediated penicillin-resistant strains1. We show that these strains contain
novel resistant alleles of mtrR and penB, which together with penA confer reduced susceptibility to
penicillin.
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NC ASM 2009
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 Poston was a longtime employee of Duke University who contributed much
to the NC Branch and she was held in high esteem both by her colleagues 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 Medicine
Structure-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 University. Prior to this position, Dr. Melton was a member of NC State University'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 Research Training Award from ASM. This award honors an individual who
has made significant contributions toward fostering the research training of underrepresented minorities in microbiology.
Last years winner:
Nathan Rigel, University of North Carolina at Chapel Hill
Characterization of the SecA2 specialized secretion pathway of mycobacteria.
The Best Poster award is open to anyone presenting a poster at the NC
ASM meeting.
Last years winner:
Alice Lee, NC State University
Recombinant Expression of Superoxide Reductase from Pyrococcus furiosus in Arabidopsis thaliana Enhances Heat and Drought Tolerance.
The Paul Phibbs Award is awarded for the best presentation by an undergraduate student at NC ASM Branch meetings.
Last years winner:
Brittany Baffer & Amanda Tarnowicz, University of North Carolina at
Chapel Hill
Isolation of Gene(s) Responsible for Binding of Agrobacterium tumefaciens to Plant Host Cells.
A check for $100 will be given for each of these awards at the conclusion of the meeting.
Page 27
LNAME
Aboobakar
Allen
Anderson
Atkinson
Baber
Ball
BECK
Bennett
Betteken
Blackman
Blackwood
Blanton
Blenman
Brauer
Braunstein
Broadway
Brown
Brown
Brown
Byrd
Carmichael
Caswell
Clement
Coleman
Cooper
Crotts
Davis
Dillow
Doyle
Doyle
Duncan
Dunn
Ernest
FNAME
Eanas
Sherrice
Eric
Tim
Caitlin
Sarah
RACHEL
Channell
Michael
Rob
Denene
Jody
Anthony
Suzanna
Miriam
Jamila
James
Sherri
Forina
Matthew
Mary
Clayton
Camille
Jim
Rebecca
Caren
Amanda
Carter
Ashlie
Ashlie
Margaret
J.D.
Rachel
Turkey
Turkey
Turkey
Turkey
Veggie wrap
Grad student (no preference)
Vendor/Exhibitor
Roast Beef
Technician
Roast Beef
Undergraduate Roast Beef
Undergraduate (no preference)
Faculty
Turkey
Faculty
Veggie wrap
Grad student Turkey
Faculty
Roast Beef
Undergraduate Turkey
Undergraduate Turkey
Grad student Turkey
Grad student Roast Beef
Postdoc
Turkey
Undergraduate Turkey
Faculty
Turkey
Grad student Veggie wrap
Undergraduate Turkey
(unspecified) Veggie wrap
Grad student Veggie wrap
Position
LUNCH
Undergraduate Veggie wrap
Faculty
Roast Beef
Postdoc
Roast Beef
Vendor/Exhibitor
Roast Beef
Undergraduate Roast Beef
Undergraduate Turkey
Undergraduate Turkey
Undergraduate Turkey
Undergraduate
doylean@guilford.edu
doylean@guilford.edu
Undergraduate
margaret_duncan@med.unc.edu Grad student
jd.dunn@duke.edu
Postdoc
Grad student
raernest@gmail.com
mib0405@ecu.edu
rblackman@bioexpress.com
adb@med.unc.edu
blantonjw@guilford.edu
yomela1@yahoo.com
brauersl@appstate.edu
braunste@med.unc.edu
jbroadwa@eagles.nccu.edu
james_brown@ncsu.edu
brownsg@guilford.edu
flo-brown@hotmail.com
mabyrd@wfubmc.edu
carmichaelmj@appstate.edu
caswellc@ecu.edu
clementcm@guilford.edu
colemanj@ecu.edu
cooperre@appstate.edu
carebear804@hotmail.com
davisaj@guilford.edu
ctdillow1@catamount.wcu.edu
East Carolina University
ISC BioExpress
University of North Carolina at Chapel Hill
Guilford College
Guilford College
Appalachian State University
University of North Carolina Chapel Hill
North Carolina Central University
NC State University
Guilford College
Guilford College
Wake Forest University School of Medicine
Appalachian State University
East Carolina University
Guilford College
Brody School of Medicine
Appalachian State University
Guilford College
Guilford College
Western Carolina University
Guilford College
Guilford College
UNC CH School of Medicine
Duke University Medical Center
NCSU
Email
eanas.aboobakar@duke.edu
svallen@uncfsu.edu
andersone@ecu.edu
tim.atkinson@carolina.com
baber@email.unc.edu
brockingtonsl@guilford.edu
beckra@guilford.edu
bennettc@guilford.edu
INSTITUTION
Duke University
Fayetteville State Univ.
ECU School of Medicine
Association of Southeastern Biologists
UNC-Chapel Hill
Guilford College
GUILFORD COLLEGE
Guilford College
Poster
Poster
Short talk
Poster
Short talk
Poster
Poster
Short talk
PRESENT
Poster
Gerry
Janetta
toby
Johnathan
Shannon
Hatajai
Katherine
Sunhee
Melanie
Lisa
Kim
Charles
Johnathan
Sunny
Heather
Elizabeth
Rose
Lukasz
Adam
Scott
Marian
Ine
David
Taryn
Garner
Giddings
Goulian
Haungs
Inman, III
Janney
Jeuck
Amber
Morgan
Mark
Amanda
Floyd
Molly
Ben
Guilford College
UNC
ECU Brody School of Medicine
Guilford College
The University of North Carolina at Chapel
Guilford College
UNC Pembroke
Guilford
Western Carolina University
Association of Southeastern Biologists
Jewell
Johnson-ThompsonRetired (NIEHS), Prof Emerita UDC
Jorgensen
Duke University
Keaton
Guilford College
Kern
Guilford College
Guilford College
Key
Killion
Guilford
King
UNC-Chapel Hill
Kozubowski
Duke University Medical Center
Kulp
Duke University
NC Central University
Ladapo
Landvater
NC A&T State Univ
Lassiter
North Carolina Central University
Lawrence
Guilford College
Lee
Duke Unviersity
Lee-Brown
Guilford College
Lee-Phillips
Guilford College
Lewis
Guilford College
Li
Duke University Medical Center
Locklear
UNC-Pembroke
Long
Guilford College
NC State University
Luginbuhl
Magee
Guilford College
A-B Tech
Mapes
Frazier
Fuller
Gaines
Shanna
James
Jennifer
Undergraduate Turkey
Postdoc
Roast Beef
Postdoc
Turkey
Undergraduate Turkey
Hill
easecres@email.unc.edu
Faculty
Turkey
goulian@sas.upenn.edu
Faculty
Turkey
haungsam@guilford.edu
Undergraduate Turkey
fli001@bravemail.uncp.edu
Undergraduate Turkey
janneymj@guilford.edu
Undergraduate Roast Beef
bnjeuck1@catamount.wcu.edu Undergraduate Roast Beef
a2zconvention@yahoo.com
Vendor/Exhibitor
Roast Beef
mjohnsonthompson@aol.com
Faculty
Turkey
ij2@duke.edu
Grad student Veggie
Undergraduate Turkey
keatondl@guilford.edu
Undergraduate Roast Beef
tarynkern@earthlink.net
secretmakeup@yahoo.com
Undergraduate Roast Beef
killionea@guilford.edu
Undergraduate Turkey
Undergraduate Veggie wrap
rmking@email.unc.edu
lukasz.kozubowski@duke.edu
Postdoc
Veggie wrap
ajk26@duke.edu
Grad student Veggie wrap
ladapo@nccu.edu
Faculty
Roast Beef
Grad student Turkey
swlandva@ncat.edu
hlassit2@nccu.edu
Grad student (no preference)
lawrencekc@guilford.edu
Undergraduate Turkey
sunhee.lee@duke.edu
Faculty
Veggie wrap
Faculty
Turkey
mleebro@guilford.edu
leephillipsld@guilford.edu
Undergraduate Turkey
lewiskl@guilford.edu
Undergraduate Roast Beef
charles.li@duke.edu
Undergraduate Turkey
Undergraduate Turkey
jrl026@bravemail.uncp.edu
longsg@guilford.edu
(unspecified) Veggie wrap
gerry_luginbuhl@ncsu.edu
Faculty
Veggie wrap
mageej@guilford.edu
Undergraduate Turkey
Faculty
Roast Beef
tmapes@abtech.edu
frazierse@guilford.edu
jrfuller@med.unc.edu
gainesj@ecu.edu
Poster
Poster
Poster
Poster
Poster
Short talk
Poster
Poster
Plenary
Short talk
Short talk
Miles
Miller
MOTALEB
Mudrak
Murray
Nandi
Neumann
Nguyen
Nicholas
Normann
Nyoromoi
O'Connell
Pawlak
Pendley
Penland
Perkowski
Pitzer
Pitzer
Ponnusamy
Ratani
Rice
Richardson
Russell
Mischa
Jennifer
MD
Ben
Nichol
Sobhan
Kurt
Bidong
Robert
Grace
Okot
Sean
Joel
Marilyn
Kelsey
Ellen
Josh
Josh
Loganathan
Shakir
Yasmin
Sadondria
Deneca
Alina
Saez
Hector Alex Saka
Malotky
Martin
Martin
Martinson
Mastrovito
Matthysse
McElvania TeKippe
Menscher
Metera
Michelle
Shyniece
Sheri
David
Michelle
Ann
Erin
Evan
Kimberly
Guilford College
Duke
saezam@guilford.edu
alex.saka@duke.edu
(unspecified)
Postdoc
Technician
pitzerj@ecu.edu
pitzerj@ecu.edu
Technician
loganathan_ponnusamy@ncsu.edu
(unspecified)
ssratani@ncsu.edu
Undergraduate
Undergraduate
sdrichar@ncat.edu
Grad student
russellda@guilford.edu
Undergraduate
East Carolina University
East Carolina University
NCSU
North Carolina State University
Guilford College
NC A&T SU
Guilford
Roast Beef
Roast Beef
Roast Beef
Roast Beef
Turkey
Turkey
Turkey
Turkey
Turkey
Faculty
Roast Beef
Undergraduate Turkey
Undergraduate Turkey
Grad student Roast Beef
Undergraduate Turkey
Faculty
Roast Beef
Grad student Veggie wrap
Grad student Turkey
Faculty
Roast Beef
Undergraduate Roast Beef
Faculty
Turkey
Faculty
Turkey
Grad student Roast Beef
Grad student Turkey
Grad student (no preference)
Vendor/Exhibitor
Turkey
Grad student Turkey
Faculty
Roast Beef
Undergraduate Roast Beef
Faculty
(no preference)
Faculty
Turkey
Faculty
Roast Beef
Vendor/Exhibitor
Roast Beef
Undergraduate Turkey
Grad student Turkey
kneumann@nikon.net
bdn@duke.edu
nicholas@med.unc.edu
normanngl@gmail.com
onyormoi@NCCU.EDU
soconnell@email.wcu.edu
jjpawlak@ncsu.edu
mpendley@cccti.edu
penlandks@gmail.com
foot@email.unc.edu
smarti26@mail.nccu.edu
martinsl@guilford.edu
dam0224d1@ecu.edu
mastrovitomc@guilford.edu
ann_matthysse@unc.edu
emac@med.unc.edu
Eam1007@ecu.edu
kametera@waketech.edu
mmiles@guilford.edu
jen_miller@ncsu.edu
motalebm@ecu.edu
bm36@duke.edu
nichol.murray@gmail.com
Guilford College
NC Central University
Guilford College
East Carolina University
Guilford
University of North Carolina at Chapel Hill
UNC-Chapel Hill
East Carolina University
Wake Technical Community College
Guilford College
North Carolina State University
EAST CAROLINA UNIVERSITY
Duke University
NCA&T University
University of North Carolina at Chapel Hill
Nikon Instruments Inc.
Duke University
University of North Carolina at Chapel Hill
Guilford
NC Central University
Western Carolina University
NC State University
Association of Southeastern Biologists
Guilford College
University of North Carolina, Chapel Hill
Short talk
Poster
Poster
Poster
Poster
Poster
Short talk
Short talk
Short talk
Poster
Poster
Salisbury
Scholle
Semighini
Simpkins
Simpkins
Skipper
Spontak
Stephenson
Strickland
Stubblefield
Su
Sullivan
Sultan
Swanson
Swords
Taylor
Tayyarah
Tetteh
Tirado-Acevedo
Van Arsdale
Ward
Weerakoon
Weimer
Wells
Wilks
Williams
Williams
Wolf
Wolfinger
Yadav
Zarilla
Zhao
Russell
Frank
Camile
Mary
Mary
Stephanie
Jeffrey
Partick
Danielle
Paul
Hsun-Cheng
Tabb
Syed
Shauna
Ed
Latesha
Rana
Antonia
Oscar
Julie
Raji
Dilan
Kristin
Henry
Melissa
Alexandra
Daniel
Joe
Liz
Ruchi
Kathy
Shuqing
Guilford College
University of North Carolina Chapel Hill
Wake Forest University School of Medicine
Guilford College
Guilford College
Guilford College
NC Central University
Peace College
Meredith College
NC Central University
Durham Tech Comm College
University of North Carolina at Chapel Hill
East Carolina University
UNC Chapel Hill
Wake Forest University Health Sciences
NCA&T University
Guilford College
NC Central University
NCSU
Guilford College
UNC School of Medicine
UNC School of Medicine
North Carolina Central University
University of North Carolina
Guilford College
Guilford College
Guilford College
UNC-Chapel Hill
UNC, Chapel Hill
Fisher Scientific
NCSU
Duke University Medical Center
zarillak@durhamtech.edu
wilksmk@guilford.edu
williamsam2@guilford.edu
dwilliams@nccu.edu
jwolf@peace.edu
wolfingere@meredith.edu
dilanw@hotmail.com
kdew@wfubmc.edu
otirado@ncsu.edu
buttrflyja3030@yahoo.com
wswords@wfubmc.edu
lstaylor@ncat.com
tayyarahr@guilford.edu
sultans@ecu.edu
stricklanddl@guilford.edu
stubblefieldsp@guilford.edu
seansu@email.unc.edu
jontabb@email.unc.edu
simpkinsmj@yahoo.com
simpkinsmj@yahoo.com
stephanie_shawnta@yahoo.com
jeffrey_spontak@med.unc.edu
Undergraduate
(unspecified)
Grad student
Undergraduate
Undergraduate
Undergraduate
Faculty
Faculty
Faculty
Grad student
Faculty
Grad student
Postdoc
Grad student
Faculty
Grad student
Undergraduate
Grad student
Grad student
(unspecified)
Professional
Professional
Grad student
Technician
Undergraduate
Undergraduate
Undergraduate
Postdoc
Grad student
Short talk
Poster
Poster
Poster
Veggie wrap
Turkey
Turkey/Chicken Short talk
Turkey
Turkey
Roast Beef
Roast Beef
Turkey/Chicken
Roast Beef
(no preference) Poster
Turkey
(no preference) Poster
Turkey
Turkey
Veggie wrap
Turkey
Roast Beef
(no preference) Poster
Roast Beef
Poster
Roast Beef
Veggie wrap
Veggie wrap
Turkey
Roast Beef
Turkey
Turkey
Turkey
Veggie wrap
Veggie wrap
russell.salisbury@thermofisher.com
Vendor/Exhibitor
Turkey
frank_scholle@ncsu.edu
Faculty
Roast Beef
camile.semighini@duke.edu
Postdoc
Turkey
NC ASM 2009
Meeting Organization Committee / NC ASM Officers
Daniel Williams
President-elect
NC Central University
Kathy Zarilla
President
Durham Tech Community College
Melanie Lee-Brown
Past-president
Guilford College
Jim Brown
Secretary
NC State University
Geraldine Luginbuhl
Treasurer
NC State University
Sherrice Allen
Councilor
Fayetteville State University
Wrennie Edwards
Alternate Councilor
Novartis/UCFCC
Joe Wolf
Historian
Peace College
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