abstract band final ncb asm meeting - south dakota …€¦ · providing us with the opportunity to...

College of Agriculture and Biological SciencesDepartment of Biology and MicrobiologySDS 228, Box 2104A, South Dakota State UniversityBrookings, SD 57007-2142From: Volker BrözelPhone: 605 688 6141Fax: 605 688 6677Email: [email protected]

South Dakota

State University

Welcome Microbiologists! Welcome to Brookings, South Dakota for the 73rd annual meeting of the North Central Branch of the American Society for Microbiology. The organizing committee is proud to host the meeting on the campus of South Dakota State University, home to the state’s premier microbiology program. We offer undergraduate majors in Microbiology and Biotechnology, and MS and PhD programs in Microbiology. The Department of Biology and Microbiology is honored to provide a forum for students to present results of their own research. We are pleased to host speakers presenting on recent advances in microbiology. These include recent findings in two-component signal transduction, presented by SDSU distinguished alumnus Dr. Jim Hoch of the Scripps Institute, as well as new information on virulence factors of E. coli, presented by Dr. Philip Hardwidge, and on the biology and evolution of Pantoea, presented by Fanus Venter from the University of Pretoria. The committee is grateful to ASM for providing us with the opportunity to invite ASM Distinguished Lecturer Dr. Jeffrey Wilusz of Colorado State University to this meeting. Professional and career development of students of microbiology has been the historical emphasis of the North Central Branch. Oral and poster presentations will be evaluated by audiences using clickers, and prizes will be awarded in undergraduate and graduate categories. On Saturday, students will have the opportunity to engage on career considerations with our panel of invited speakers. This meeting would not be possible without the support of sustaining members, sponsors and exhibitors and the American Society for Microbiology. Please spend some time interacting with our exhibitors. On Friday night we invite you to join us in the Old Fire Hall, 310 4th Street for social hour and the conference dinner. Participation is included in your registration fee. The organizing committee hopes that you will enjoy the meeting and your stay in Brookings. Sincerely Volker S. Brözel, President North Central Branch, American Society for Microbiology

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PROGRAM

1

Friday, October 11, 2013

07:30 – 08:25 REGISTRATION – Volstorff

08:30 – 08:40 WELCOME – Volstorff

Dr. Barry Dunn Dean, College of Agriculture and Biological Sciences Dr. Volker Brözel President, ASM North Central Branch

08:40 – 09:30 KEYNOTE SPEAKER – Volstorff

Dr. Jeffrey Wilusz Colorado State University ASM Distinguished Lecturer

How viruses escape the wrath of the cellular RNA decay machinery during infection

09:40 – 10:40 SESSION 1 – Environmental Microbiology – Campanile Room

09:40 – 10:00 Laura White South Dakota State University

Effect of isoflavone rhizodeposits of Glycine max on rhizoplane bacterial diversity

10:00 – 10:20 Xiurong Wang South Dakota State University

Growth responses and phosphate uptake of soybean as affected by arbuscular mycorrhizal fungi

10:20 – 10:40 Jerry Mensah South Dakota State University

Differences in fungal phosphate metabolism and the impact on plant growth benefit in the arbuscular mycorrhizal symbiosis

09:40 – 10:40 SESSION 3 – Microbial Biotechnology – Volstorff

09:40 – 10:00 Maureen B. Quin University of Minnesota

Mushroom hunting using bioinformatics: sesquiterpenoid discovery in basidiomycota

10:00 – 10:20 Charles Halfmann South Dakota State University

Engineering cyanobacteria for the photosynthetic production of long chain hydrocarbons

10:20 – 10:40 Richard Kramer St. Cloud State University

Expression and characterization of lytic Staphylococcus aureus phage P68 holin proteins in Escherichia coli

10:40 – 11:00 BREAK – Volstorff

11:00 – 12:00 SESSION 2.1 – Medical and Veterinary Microbiology – Volstorff

11:00 – 11:20 Mahmoud Darweesh South Dakota State University

Characterization of the cytopathic BVDV strains isolated from 13 mucosal disease cases arising in one cattle herd

11:20 – 11:40 Meredith Irsfeld North Dakota State University

Phenylethylamine as a potential biofilm prevention substrate

11:40 – 12:00 Megan Constans North Dakota State University

Computational analysis of the basis for vaccine escape by a porcine circovirus strain 2B (PCV2B) virus isolate

11:00 – 12:00 SESSION 4.1 – Microbial Cell Biology – Campanile Room

11:00 – 11:20 Yeyan Qui South Dakota State University

Proteomic study on akinetes, heterocysts and vegetative cells in Anabaena cylindrica

11:20 – 11:40 Kangming Chen South Dakota State University

Identification of a site‐2 metalloprotease required for cold acclimation in cyanobacteria

11:40 – 12:00 Xinyi Xu South Dakota State University

ALR4853, an O2‐sensitive aspartate aminotransferase is specifically required for N2‐based growth in Anabaena sp. PCC 7120

PROGRAM

2

Friday, October 11, 2013

12:00 – 01:00 LUNCH

01:00 – 02:30 POSTER PRESENTATIONS – Volstorff



Dr. Philip R. Hardwidge Kansas State University

E. coli virulence factors and the innate immune system


Dr. James A. Hoch The Scripps Institute

Understanding two‐component signal transduction

04:50 Adjourn

06:00 Dinner – The Old Firehouse – Downtown Brookings

PROGRAM

3

Saturday, October 12, 2013

08:00 – 08:50 BUSINESS MEETING – Campanile Room

08:00 – 09:00 Career Meeting – Volstorff All students are encouraged to meet the invited speakers and to discuss career opportunities in Microbiology


Dr. Stephanus N. Venter University of Pretoria

Genomes: Exploration of the biology and evolution of Genus Pantoea


10:20 – 11:20 SESSION 2.2 – Medical and Veterinary Microbiology – Volstorff

10:20 – 10:40 Milton Thomas South Dakota State University

Differential expression of viral pattern recognition receptors in porcine airway and intestinal epithelial cells in response to influenza infection

10:40 – 11:00 Anuradha Vegi North Dakota State University

Diagnostic tool for detecting swine torque teno virus 1 (TTV1)

11:00 – 11:20 Runxia Liu South Dakota State University

Genetic and biological characterization of a novel influenza virus in US cattle and swine

10:20 – 11:20 SESSION 4.2 – Microbial Cell Biology – Campanile Room

10:20 – 10:40 Adam Edwinson North Dakota State University

Host glycoproteins as triggers of Cryptosporidium development

10:40 – 11:00 Joseph Madison University of Wisconsin

Can old genes give rise to new genes? A test of the gene duplication hypothesis with HisB of Escherichia coli

11:00 – 11:20 Michael Schultz University of Wisconsin

Evolution of serine phosphate phosphatase activity in the HisB gene of Escherichia coli

11:30 – 12:00 AWARDS – Volstorff

LIST OF ORAL AND POSTER PRESENTATIONS

4

KEYNOTE SPEAKERS Friday, October 11, 2013 and Saturday, October 12, 2013 Volstorff

Hardwidge, Philip Keynote E. coli virulence factors and the innate immune system

Hoch, James Keynote Understanding two‐component signal transduction Wilusz, Jeffrey Keynote How viruses escape the wrath of the cellular RNA decay

machinery during infection Venter, Stephanus Keynote Genomes: Exploration of the biology and evolution of Genus

Pantoea

SESSION 1 – ENVIRONMENTAL MICROBIOLOGY – ORAL PRESENTATIONS

Friday, October 11, 2013 09:40 – 10:40 Campanile Room

White, Laura Session 1 OP 1 Effect of isoflavone rhizodeposits of Glycine max on rhizoplane bacterial diversity

Wang, Xiurong Session 1 OP 2 Growth responses and phosphate uptake of soybean as affected by arbuscular mycorrhizal fungi

Mensah, Jerry A. Session 1 OP 3 Differences in fungal phosphate metabolism and the impact on plant growth benefit in the arbuscular mycorrhizal symbiosis

SESSION 1 – ENVIRONMENTAL MICROBIOLOGY – POSTER PRESENTATIONSFriday, October 11, 2013 01:00 – 02:30 Volstorff

Werner, Sarah Session 1 PP 1 Isolation of anaerobes from the intestinal tract of Arion fasciatus, an aquatic slug

Anower, M. Rokebul Session 1 PP 2 Mechanism of salinity tolerance in alfalfa (Medicago sativa L.) root physiology

Kanchupati, Praveena Session 1 PP 3 Study of expression patterns of MiZ1‐like genes in maize (Zea mays L.)

NandaKafle, Gitanjali Session 1 PP 4 Population dynamics of Escherichia coli at an experimental MOB‐grazing site

Mensah, Jerry A. Session 1 PP 5 Fungal nutrient allocation in common mycelia networks is regulated by the carbon source strength of individual host plants

Johnson, Tylor J. Session 1 PP 6 Increasing tolerance of cyanobacteria to long‐chain chemicals via directed evolution

SESSION 2.1 – MEDICAL AND VETERINARY MICROBIOLOGY – ORAL PRESENTATIONSFriday, October 11, 2013 11:00 – 12:00 Volstorff

Darweesh, Mahmoud Session 2 OP 1 Characterization of the cytopathic BVDV strains isolated from 13 mucosal disease cases arising in one cattle herd

Irsfeld, Meredith Session 2 OP 2 Phenylethylamine as a potential biofilm prevention substrate

Constans, Megan Session 2 OP 3 Computational analysis of the basis for vaccine escape by a porcine circovirus strain 2B (PCB2B) virus isolate

SESSION 2.2 – MEDICAL AND VETERINARY MICROBIOLOGY – ORAL PRESENTATIONSSaturday, October 12, 2013 10:20 – 11:20 Volstorff

Thomas, Milton Session 2 OP 4 Differential expression of viral pattern recognition receptors in porcine airway and intestinal epithelial cells in response to influenza infection

Vegi, Anuradha Session 2 OP 5 Diagnostic tool for detecting swine torque teno virus 1 (TTV1)

Liu, Runxia Session 2 OP 6 Genetic and biological characterization of a novel influenza virus in US cattle and swine


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SESSION 2 – MEDICAL AND VETERINARY MICROBIOLOGY – POSTER PRESENTATIONSFriday, October 11, 2013 01:00 – 02:30 Volstorff

Edquist, Mitchell Session 2 PP 1 Optimization of the inhibition of Listeria monocytogenes by a carnobacteria bacteriocin

Eklund, Bridget Session 2 PP 2 Development and optimization of amoeba and insect virulence assays for the high‐throughput analysis of Stenotrophomonas maltophilia virulence determinants

Domfeh, Yayra E. Session 2 PP 3 Does stress exposure in the hospital environment impact subsequent virulence in Stenotrophomonas maltophila?

Leisen, Erin Session 2 PP 4 Prevalence of MRSA colonization of patients and staff in a regional hospital in Cuenca, Ecuador

McCormick, Kara Session 2 PP 5 Construction and immunogenicity evaluation of recombinant influenza A viruses containining chimeric HA genes from genetically divergent influenza H1N1 viruses

Park, Kaci Session 2 PP 6 Effect of bovine herpesvirus 1 and bovine viral diarrhea virus (BVDV) on bovine monocyte‐derived dendritic cells

Wang, Xiuqing Session 2 PP 7 Virus‐like particles generated from expressing the membrane (M) and nucleocapsid (N) proteins of porcine reproductive and respiratory syndrome virus (PRRSV)

Martin, Ben Session 2 PP 8 Development and validation of insect virulence assays for the high‐throughput analysis of Francisella spp and Burkholderia spp virulence determinants: yet another way to get rid of cockroaches!

Xiang, Xiaoxiao Session 2 PP 9 M165A mutation in the matrix 1 (M1) protein of influenza A virus blocks virus replication by disrupting an early postentry step

Ran, Zhiguang Session 2 PP 10 Triple‐serine‐motif (S224S225S226) of the M1 protein is essential to influenza A virus replication

SESSION 3 – MICROBIAL BIOTECHNOLOGY – ORAL PRESENTATIONSFriday, October 11, 2013 09:40 – 10:40 Volstorff

Quin, Maureen B. Session 3 OP 1 Mushroom hunting using bioinformatics: sesquiterpenoid discovery in basidiomycota

Halfmann, Charles Session 3 OP 2 Engineering cyanobacteria for the photosynthetic production of long chain hydrocarbons

Kramer, Richard Session 3 OP 3 Expression and characterization of lytic Staphylococcus aureus phage P68 holin proteins in Escherichia coli

SESSION 3 – MICROBIAL BIOTECHNOLOGY – POSTER PRESENTATIONSFriday, October 11, 2013 01:00 – 02:30 Volstorff

West, Thomas P. Session 3 PP 1 Glycerol‐based citric acid production by an analogue‐resistant Candida guilliermondii mutant

Peterson, Jessica L. Session 3 PP 2 Pullulan production by a deoxyglucose‐resistant fungal mutant on dilute acid hydrolyzed prairie cordgrass

Tyagi, Deepti Session 3 PP 3 Influence of pre‐harvest environmental factors on stress resistance in E. coli and Salmonella on lettuce

Croat, Jason R. Session 3 PP 4 Metabolism of antinutritional components in Brassica carinata to produce high protein feed

Karki, Bishnu Session 3 PP 5 Optimization of extrusion processing conditions on enzymatic hydrolysis and fermentation of Distiller´s low oil wet cake

Murthy, Naveen Kumar Srinivasa

Session 3 PP 6 Examination of Bacillus biological control agents for Itu D and Ipa 14genes using polymerase chain reaction

Gu, Liping Session 3 PP 7 Direct photosynthetic conversion of CO2 and H2O into perfume linalool by engineered cyanobacteria

Zhu, Huilan Session 3 PP 8 Genetic transformation of Anabaena cylindrica ATCC 29414

Quin, Maureen B. Session 3 PP 9 Biosynthesis and designer microbes


6

SESSION 4.1 – MICROBIAL CELL BIOLOGY – ORAL PRESENTATIONS

Friday, October 11, 2013 11:00 – 12:00 Campanile Room

Qiu, Yeyan Session 4 OP 1 Proteomic study on akinetes, heterocysts and vegetative cells in Anabaena cylindrica

Chen, Kangming Session 4 OP 2 Identification of a site‐2 metalloprotease required for cold acclimation in cyanobacteria

Xu, Xinyi Session 4 OP 3 ALR4853, an O2‐sensitive aspartate aminotransferase, is specifically required for N2‐based growth in Anabaena sp. PCC 7120

SESSION 4.2 – MICROBIAL CELL BIOLOGY – ORAL PRESENTATIONS

Saturday, October 12, 2013 10:20 – 11:20 Campanile Room

Edwinson, Adam Session 4 OP 4 Host glycoproteins as triggers of Cryptosporidium development

Madison, Joseph Session 4 OP 5 Can old genes give rise to new genes? A test of the gene duplication hypothesis with HisB of Escherichia coli

Schultz, Michael Session 4 OP 6 Evolution of serine phosphate phosphatase activity in the HisBgene of Escherichia coli

SESSION 4.1 – MICROBIAL CELL BIOLOGY – POSTER PRESENTATIONSSaturday, October 12, 2013 01:00 – 02:30 Volstorff

Lucas, Andrea L. Session 4 PP 1 Examination of trans‐golgi snare syntaxin 10 at the chlamydial inclusion

Hernandez, Kristina Session 4 PP 2 Cell shape determination in Helicobacter pyroli: characterizing the roles of CSD1 and CSD2

Zhu, Huilan Session 4 PP 3 A 9‐BP GC hairpin sequence is sufficient for transcriptional repression

Zhang, Hanmo Session 4 PP 4 Role of DSRNA‐dependent protein kinase R (PKR) and eukaryotic translation initiation factor 2‐Alpha (EIF2A) in porcine reproductive and respiratory syndrome virus (PRRSV) replication

KEYNOTE SPEAKERS

7

Keynote 1

HOW VIRUSES ESCAPE THE WRATH OF THE CELLULAR RNA DECAY MACHINERY

DURING INFECTION.

Jeff Wilusz

Colorado State University, Department of Microbiology, Immunology & Pathology,

Microbiology Bldg, Rm B321, 1682 Campus Delivery, Fort Collins, CO 80523‐1682

As our appreciation increases for the pervasive nature of transcription in the cell, so too has our

appreciation for the major role of RNA decay/stability in regulating both the quantity and the

quality of gene expression. As soon as viral RNAs appear in the cell, they must be prepared to

combat or avoid cellular RNA decay pathways. This talk will describe several ways that we

have uncovered that RNA viruses use to deal with the general host RNA decay machinery that

is active in the cell immediately upon viral infection – turning what at first appears to be very

hostile territory for a foreign transcript into a sort of ‘promised land’ for viral gene expression.

In addition, these strategies used by RNA viruses result in the dysregulation of cellular mRNA

stability and have a significant impact on cellular gene expression. This limits the ability of the

infected cell to effectively respond to the virus and likely has a significant impact on viral

replication and pathogenesis. Finally, viral RNA stability represents an attractive but largely

unexplored avenue for the development of broad‐spectrum antiviral therapeutics.

KEYNOTE SPEAKERS

8

Keynote 2

E. COLI VIRULENCE FACTORS AND THE INNATE IMMUNE SYSTEM

Philip R. Hardwidge1 1 Kansas State University, Department of Diagnostic Medicine and Pathobiology, Manhattan,

KS, USA

Enteric bacterial pathogens cause diarrheal disease outbreaks, thus constituting enormous

health burdens. The molecular mechanisms of how these pathogens inhibit innate immune

responses to colonize their host are under intense investigation. The Shiga toxin‐producing E.

coli (STEC) use a type III secretion system (T3SS) to inject virulence proteins (effectors) into host

cells. While T3SS effectors clearly play important roles in bacterial virulence, the mechanisms by

which they subvert host functions to promote pathogen survival are incompletely

characterized.

Through studies of the mechanism of the NleB effector, an interaction between the mammalian

glycolysis enzyme GAPDH, and an innate immunity scaffolding protein, TRAF2, was

identified. TRAF2 regulates the pro‐inflammatory NF‐kB pathway. Maximal TRAF2

polyubiquitination and NF‐B activation requires the TRAF2‐GAPDH interaction. NleB

functions as a â‐D‐N‐acetylglucosamine (GlcNAc) transferase that modifies GAPDH to inhibit

its function in innate immunity. Protein O‐GlcNAcylation regulates many cellular processes

such as cell division and metabolism, but relatively little was known about the role of O‐

GlcNAc in intestinal immunity. Eliminating NleB O‐GlcNAcylation activity attenuated

Citrobacter rodentium colonization in a mouse infection model, confirming its significance to

bacterial virulence.

KEYNOTE SPEAKERS

9

Keynote 3

UNDERSTANDING TWO‐COMPONENT SIGNAL TRANSDUCTION

James A. Hoch

Department of Molecular and Experimental Medicine, The Scripps Research Institute MEM116

10550 North Torrey Pines Road, La Jolla CA 92037, Phone: 858 784 7905

Two‐component signal transduction systems consisting of Sensor Kinases and Response

Regulators along with accessory regulatory proteins are embedded in control systems for a

wide variety of cellular processes in bacteria, fungi and plants. There are innumerable signal

inputs that regulate these systems and a variety of outputs but their major role is to control gene

transcription. This presentation will focus on the fundamental mechanisms by which these

regulatory switches work, including transduction mechanisms and protein recognition,

regardless of function or cellular source.

KEYNOTE SPEAKERS

10

Keynote 4

GENOMES: EXPLORATION OF THE BIOLOGY AND EVOLUTION OF THE

GENUS PANTOEA

Stephanus N. Venter

Department of Microbiology and Plant Pathology; Forestry and Agricultural Biotechnology

Institute; Genomics Research Institute, University of Pretoria, Private Bag x 20, Hatfield

Pretoria, South Africa, 0028.

Genome sequencing and comparisons are currently one of the leading drivers in biological

research. With the development and advances in high throughput sequencing platforms and

the reduction in associated costs, sequencing and assembly of genomes have come within reach

of most laboratories. The genome sequence of an organism not only serves as the blue print of

all the genes present but also provides the opportunity to study the genetic diversity,

pathogenicity and population dynamics within certain species, as well as the broader evolution

of species and genera. Based on the availability of 8 genomes, comparative genomic studies are

currently undertaken to explore the pathogenicity of the emerging plant pathogen, Pantoea

ananatis. This is an attempt to understand how this organism can cause disease in a diverse

range of plants such as onions, maize and Eucalyptus. Various factors, including the Type 6

secretion system, have been identified and studied.The genomes of at least 26 isolates

representing 12 of the described species of the genus Pantoea are available and several were

used to validate genomic approaches for the delineation of the genus and species within this

genus. This data is also used for phylogenomic studies to understand the evolution of the genus

and its associated traits.

SESSION 1 – ORAL PRESENTATIONS

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Session 1 – OP 1

EFFECT OF ISOFLAVONE RHIZODEPOSITS OF GLYCINE MAX ON

RHIZOPLANE BACTERIAL DIVERSITY

Laura White1, Senthil Subramanian1,2, Volker S. Brözel1 1Department of Biology and Microbiology; 2Plant Science Department, South Dakota State

University, Brookings, SD, USA

In soil, the largest bacterial diversity is located nearby plant roots mainly due to “rhizodeposits”

(compounds released from plant roots that act as a carbon source or as signaling molecules to

soil bacteria). Plant family‐ or species‐specific compounds (e.g. isoflavonoids in soybeans) are

particularly interesting due to their signaling roles. Isoflavonoids help soybeans defend against

pathogens and regulate nod genes in Rhizobium. We aimed to determine if and how isoflavones

affect the composition of root surface bacterial communities. Composite soybean (Glycine max)

plants with either normal or reduced isoflavonoid levels (achieved using RNAi) were grown in

soil pooled from multiple soybean fields for 1 and 3 weeks. Loose soil, tightly adhered soil, and

rhizoplane soil samples were obtained by consecutive sonication steps, DNA was isolated, and

16S rRNA gene amplicons were examined for bacterial diversity by pyrosequencing or

denaturing gradient gel electrophoresis (DGGE). Resulting DNA sequences from

pyrosequencing were analyzed using MOTHUR software and examined for differences among

operational taxonomic units. DGGE analysis noted differences among root surface preparations

as well as between growing times and samples from plants with normal and reduced isoflavone

levels, which suggests isoflavonoids influence soybean root surface bacteria. Initial MOTHUR

analyses further clarified those bacterial communities that preferentially colonize soybean roots.


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Session 1 – OP 2

GROWTH RESPONSES AND PHOSPHATE UPTAKE OF SOYBEAN AS

AFFECTED BY ARBUSCULAR MYCORRHIZAL FUNGI

Xiurong Wang1,2, Heike Bücking1 1 South Dakota State University, Biology and Microbiology, Brookings, SD, USA; 2 South China

Agricultural University, Root Biology Center, Guangzhou, Guangdong, China

Soybean (Glycine max (L.) Merr.) is one of the most important leguminous crops worldwide and

plays a key role for food security. Phosphate (P) is an essential macronutrient for growth and

development, and a higher P availability can improve the productivity of soybean. Arbuscular

mycorrhizal (AM) fungi are important soil fungi that can have a significant impact on the P

nutrition of their host plant. However, so far very little is known about the growth responses of

soybean to different AM fungi. In the present study, we first evaluated the growth benefits of

two soybean varieties with different P efficiency after colonization with three closely‐related

AM fungal species, Glomus interadices, Glomus aggregatum and Glomus custos. The results showed

that an inoculation with Glomus intraradices led to high colonization rates, and caused a large

growth promotion in both soybean varieties under low‐P conditions, but that even a low

colonization with Glomus aggregatum caused growth depressions in soybean. Application of P

decreased the plant growth responses to AM colonization. Under P starvation, the P‐inefficient

soybean variety obtained more benefits from AM fungi possibly due to the smaller root

diameter. We also used 33P to quantify the contribution of Glomus intraradices and Glomus

aggregatum to P uptake by soybean grown in compartmented pots. The results showed that both

fungi independent on their growth benefits were able to deliver P to their host plant. Further

studies are planned to examine the P transport regulation in these two soybean cultivars.


13

Session 1 – OP 3

DIFFERENCES IN FUNGAL PHOSPHATE METABOLISM AND THE IMPACT ON

PLANT GROWTH BENEFIT IN THE ARBUSCULAR MYCORRHIZAL SYMBIOSIS

Jerry A. Mensah1, Alexander M. Koch2, Pedro Antunes3, Miranda Hart2, E. Toby

Kiers4, Heike Bücking1 1South Dakota State University, Biology and Microbiology Department, Brookings, SD 57006,

USA; 2University of British Columbia Okanagan, Department of Biology, Kelowna, British

Columbia, V1V 1V7, Canada; 3Algoma University, Department of Biology, Sault Ste. Marie,

Ontario, P6A2G4, Canada; 4Vrije Universiteit; Institute of Ecological Science; Amsterdam,

Netherlands.

The application of arbuscular mycorrhizal (AM) fungi in sustainable agriculture is still hindered

by our limited understanding of the fungal nutrient metabolism and its effect on plant growth

benefit. The goal of these studies was to examine the effect of the fungal phosphate metabolism

on plant growth benefit. We tested the nutritional benefits of Medicago sativa plants after

colonization with 31 different AM fungi isolates from 10 fungal species and analyzed the P and

N contents in root and shoots, the P pool distribution, and examined whether differences in

nutritional benefits can be related to the P metabolism of the fungal symbiont. There were high

inter‐ and intra‐specific differences in the plant growth benefit of different AM associations.

Plant growth benefit was strongly correlated to the increase in P and N uptake by the AM

fungus. In particular, the positive impact of the high performance isolates on N nutrition set

these fungal isolates apart from medium performance isolates that only increased the P

nutrition of the host. Low and high performance isolates differed in their phosphate handling

and here particularly in their polyphosphate metabolism. In mycorrhizal roots that were

colonized with low performance isolates, a higher percentage of P was found in form of

polyphosphates, and thereby in a fungal specific P pool that is unavailable for the host. The

results will be discussed with regard to different fungal strategies in P and N handling that

determines plant growth benefit and nutrition of the host.

SESSION 1 – POSTER PRESENTATIONS

14

Session 1 – PP 1

ISOLATION OF ANAEROBES FROM THE INTESTINAL TRACT OF ARION FASCIATUS,

AN AQUATIC SLUG

Sarah Werner, Bonnie Bratina

University of Wisconsin‐La Crosse, Microbiology, La Crosse, WI, USA

Arion fasciatus is an invasive slug species, which typically lives exclusively in terrestrial

environments. Some populations, however, have been found in Wisconsin and Minnesota that

live in streams. When fecal smears from these slugs were viewed microscopically, many

unusually shaped microbes were observed. The goal of my research project was to isolate strict

anaerobic or novel bacteria from the intestinal tract of Arion fasciatus. Several methods were

used to better capture the bacterial diversity found in the slug guts: dissecting the slugs at

various times after being removed from the streams, using several different growth media and

gelling agents, and testing a variety of carbon and energy sources and growth factors. It was

found that waiting at least 24 hours to dissect the slug after removing it from the stream

decreased colony diversity and fungal growth. Changing the gelling agent did affect colony

morphology diversity, but unexpectedly the gelling agents also had a differential effect with the

delayed dissection protocol. A number of unusual carbon and energy sources and growth

factors were tested in various combinations. Some combinations resulted in heavy growth,

whereas others resulted in very little growth. Colony morphology and Gram stains were used

to determine probable unique isolates for further identification.


15

Session 1 – PP 2

MECHANISM OF SALINITY TOLERANCE IN ALFALFA (MEDICAGO SATIVA L.)

ROOT PHYSIOLOGY.

M. Rokebul Anower1, Ivan Mott2, Michael Peel2, Yajun Wu1, #

1 Biology & Microbiology Department, South Dakota State University, Brookings, SD‐57006,

USA

2 USDA, Forage & Range Research Lab, Utah State University, Logan, UT 84322‐6300, USA

Alfalfa (Medicago sativa L.) is an important forage legume crop worldwide that is relatively

susceptible to soil salinity. Improved cultivars with high biomass production on saline soil will

profit many land rangers. This study reports biomass production of three experimental alfalfa

half‐sib families, HS‐A, HS‐B and HS‐C, developed to survive in high saline conditions in

greenhouse. Six‐week‐old seedlings were subjected to salinity treatment by dipping roots in

NaCl‐nutrient solution starting at an electrical conductivity (EC) of 3.0 dS m‐1. The treatment

was continued for 4 weeks with an increment of 3.0 dS m‐1 per week in the salt solution,

reaching 12.0 dS m‐1 in the 4th week. HS‐A and HS‐B showed 47% and 63%, respectively, greater

shoot dry biomass production under salinity treatment compared to their parental lines, while

HS‐C showed no difference in dry weight from its parental lines. Root dry weight, however,

was greater in all the selection lines, showing 30%, 47% and 50% increase for HS‐A, HS‐B and

HS‐C respectively compared to their parental lines. Root/shoot dry weight ratio was not

affected in parental lines but increased in all the selection lines. Salinity treatment resulted in

browning of the roots regardless genetic backgrounds. The selection lines however showed

larger and more branched roots systems compared to their parental lines after salt treatment.

These results suggested that root growth in selection lines is less sensitive to salinity stress and

an increase in root/shoot ratio may play an important role for overall salinity tolerance in plants.


16

Session 1 – PP 3

STUDY OF EXPRESSION PATTERNS OF MIZ1‐LIKE GENES IN MAIZE

(ZEA MAYS L.)

Praveena Kanchupati, Eric Oines, Andrew Nelson, Viet Tran, Donald Auger

and Yajun Wu

Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57006

Roots respond to the moisture gradient, through a phenomenon called hydrotropism.

However, very little is known about the molecular mechanism underlying hydrotropism. The

first gene involved in hydrotropism was cloned from an Arabidopsis mutant named mizu‐

kussei1 (miz1) that lacks hydrotropism. Gene miz1 encodes a novel protein and is only found in

terrestrial plants. Thus, hydrotropism involving miz1 represents a unique molecular pathway in

higher plants. A recent study showed that overexpression of miz1 in Arabidopsis enhanced the

hydrotropic response in roots. The results lead to our hypothesis that an enhancement of

hydrotropism in roots of major crops may result in a better water acquisition and an improved

performance under drought stress. The B73 maize (Zea mays L.) genome contains 15 miz1‐like

genes. To identify miz1 orthologs in maize, we studied the expression patterns of these genes.

Our preliminary expression analysis revealed that seven genes were expressed at relatively high

levels in primary roots. Three of the miz1‐like genes were expressed at greater levels in the first

2 mm of the root compared to other regions of the root and other tissues of the maize seedlings;

four were expressed at higher level across the whole root tip in both apical and basal regions

compared to other tissues of the maize seedlings. Currently we are studying expression patterns

of miz1‐like genes in various tissues of mature maize plants, and examining the effect of plant

hormones and various abiotic stresses, especially drought stress on the expression of the miz1‐

like genes.


17

Session 1 – PP 4

POPULATION DYNAMICS OF ESCHERICHIA COLI AT AN EXPERIMENTAL

MOB‐GRAZING SITE

Gitanjali NandaKafle1*, Madhav Nepal1, Stephanus N. Venter2 and Volker S. Brözel1,2

South Dakota State University, Brookings, SD1,

University of Pretoria, Pretoria, South Africa2

Commensal and pathogenic strains of Escherichia coli are commonly associated with the

gastrointestinal tract of warm blooded animals, their primary habitat. However, E. coli strains

also spend a considerable part of their life in secondary environments such as soil, sediments

and water. The ability of E. coli to survive in different environments may contribute to their

genetic diversity. The aim of this work was to examine the population density and diversity of

E. coli in cattle pasture under mob and rotational grazing. Soil cores were taken before cattle

were introduced for the season. After stocking, soil cores, run‐off water and manure samples

were collected over four weeks from two mob‐grazed and two rotational grazed sites in Volga,

SD during July 2013. Run‐off was generated using a Cornell Infiltrometer. E. coli were

enumerated by filtering 1 and 10ml samples through 0.45 μm membrane filter, and incubating

on Membrane Lactose Glucuronide Agar. Green colonies were selected, purified and their uidA

gene amplified by PCR and sequenced. Sequences (368) were aligned using ClustalW and

trimmed in Se‐Al. A Maximum‐ Likelihood tree was constructed using MEGA5.2. E. coli counts

were significantly higher in samples from mob‐grazed plots than rotational ones, despite

identical stocking density. Isolates grouped into three distinct clusters. All four samples types

occurred across all clusters, but one large subcluster of cluster 2 contained no manure isolates.

This subcluster contained a larger proportion of isolates taken before cattle entered the pastures.

This subcluster likely comprises E. coli not associated with cattle, but endemic to the pasture

environment.


18

Session 1 – PP 5

FUNGAL NUTRIENT ALLOCATION IN COMMON MYCELIA NETWORKS IS

REGULATED BY THE CARBON SOURCE STRENGTH OF INDIVIDUAL HOST

PLANTS

Jerry A. Mensah1*, Carl R. Fellbaum1*, Adam J. Cloos1, Gary E. Strahan2, Philip E.

Pfeffer2, E. Toby Kiers3, Heike Bücking1 1South Dakota State University, Biology and Microbiology, Brookings, SD, USA; 2United States

Department of Agriculture, Agriculture Research Service, Eastern Regional Research Center,

Wyndmoor, PA, USA; 3Vrije Universiteit, Institute of Ecological Science, Amsterdam,

Netherlands

*Both authors contributed equally

The common mycorrhizal networks (CMN) of arbuscular mycorrhizal (AM) fungi provide

multiple host plants with nutrients, but the mechanisms by which the nutrient transport to

individual host plants within one CMN is controlled, are currently unknown. We followed, by

radioactive and stable isotope labeling, the transport of phosphate (P) and nitrogen (N) by

CMNs to Medicago plants, that differed in their photosynthetic capability and correlated the

nutrient transport to the expression of plant P and ammonium transporters in the mycorrhizal

interface. AM fungi discriminated between host plants that shared a CMN and both AM fungi

preferentially allocated nutrients to plants that were able to provide more benefit. Fungal P

transport was correlated to the expression of MtPt4 in mycorrhizal roots. When the CMN had

access to N, the putative ammonium transporter 1723.m00046 was induced, suggesting that this

transporter plays a role in the N transport across the mycorrhizal interface. Plants compete with

other plants for limited resources and the carbon source strength plays a role in the fungal

nutrient allocation within CMNs. However, AM fungi also transfer nutrients to low quality

hosts to ensure a continuous carbon supply for their obligate biotrophic life style.


19

Session 1 – PP 6

INCREASING TOLERANCE OF CYANOBACTERIA TO LONG‐CHAIN

CHEMICALS VIA DIRECTED EVOLUTION

Tylor J. Johnson1, Charles Halfmann1, Ruanbao Zhou1, William R. Gibbons1

1South Dakota State University, Biology and Microbiology, Brookings, SD, USA

Three wild‐type strains of filamentous, nitrogen‐fixing cyanobacteria were grown in the

presence of various concentrations of long‐chain chemicals to establish the tolerance level for

each strain. These long‐chain chemicals are volatile and can prove difficult to maintain in

culture fluid without evaporation. Therefore, cultures of cyanobacteria were grown in sealed

test tubes that contained progressively higher levels of each chemical. The growth medium was

BG‐11, which was supplemented with 0.5 g/L NaHCO3 to provide a carbon source. Tubes were

incubated for 72 h at 30oC under constant illumination. Tubes were constantly inverted at 20

rpm to maximize mixing of the insoluble chemical in water, and to prevent the filamentous

cyanobacteria from fouling tube surfaces. After incubation the cultures were plated on solid BG‐

11 medium to recover isolates that could tolerate higher concentrations of long‐chain chemicals.

These isolates were then inoculated into tubes containing even higher farnesene concentrations.

A. variablis consistently had the highest chemical tolerances while N. punctiforme consistently

had the lowest level of chemical tolerance. For example, in farnesene A. variabilis can tolerate

0.43 g/L for 72 h while N. punctiforme can tolerate 0.21 g/L. This acclimation methodology will be

continued to establish the maximal chemical tolerance. These methods will be used to increase

tolerance of cyanobacteria strains that are being engineered to produce long‐chain chemicals

from carbon dioxide.


20

Session 2.1 – OP 1

CHARACTERIZATION OF THE CYTOPATHIC BVDV STRAINS ISOLATED

FROM 13 MUCOSAL DISEASE CASES ARISING IN ONE CATTLE HERD

Mahmoud Darweesh1*, Mrigendra Rajput1, Lyle Braun1, Julia Ridpath2, John

Neil2, Alan Young1 and Christopher Chase1

1South Dakota State University, Department of Veterinary and Biomedical Sciences,

Brookings, SD, 57006, USA; 2 Unit, National Animal Disease Center, Ruminant Diseases and

Immunology Research Agricultural Research Service, United States Department of Agriculture,

Ames, IA, 50010, USA

Bovine viral diarrhea viruses (BVDV) are positive single stranded RNA viruses belonging to

Pestivirus genus of the Flaviviridae family. Noncytopathic (ncp) BVDV may establish lifelong

persistent infections in calves. Cytopathic (cp) BVDV strains arise from noncytopathic strains

via mutations, the most common of which is an insertion of RNA derived from either host or a

duplication of viral sequences into the region of the genome coding for the NS2/3 protein.

Superinfection of a persistently infected animal with a cytopathic virus can give rise to mucosal

disease, a condition that is invariably fatal. A herd of 136 bred first calf heifers was assembled

by a cattle buyer from several different sources. These heifers gave birth to 36 PI animals of

which 13 succumbed to mucosal disease. In this study, we characterized the noncytopathic and

cytopathic viruses isolated from these 13 animals. All viruses belonged to the BVDV2a genotype

and were highly similar. All the cytopathic viruses contained an insertion in the NS2/3 coding

region consisting of the sequences derived from the transcript encoding a DnaJ protein.

Comparison of the NS2/3 regions of the 13 cytopathic isolates revealed sequence identities

ranging from 96% ‐ 98% in the dnaJ insertion and 99% in the viral sequences with common

borders for the insertion. This suggested that one animal likely developed a cytopathic virus

that then progressively spread to the other 12 animals. This is the first characterization of the

evolution of a cytopathic bovine viral diarrhea virus under natural conditions.


21


PHENYLETHYLAMINE AS A POTENTIAL BIOFILM PREVENTION SUBSTRATE

Meredith Irsfeld, Birgit Pruess, Shelley M. Horne

North Dakota State University, Veterinary and Microbiological Sciences, Fargo, ND, USA

Previous research in our lab has found PEA to be the most effective of 95 carbon and 95

nitrogen sources screened for their effect on Escherichia coli O157:H7 growth, bacterial counts,

and biofilm amounts on treated pieces of beef (Lynnes et al., 2013). It may exert this function by

playing a role in the signal transduction pathway of the FlhD/FlhC complex which controls

flagellar formation (Stevenson et al., 2013).

A range of concentrations of PEA (0 to 100mg/mL) has then been applied to E. coli K‐12 strains

to see the effects on biofilm amounts, growth, bacterial counts, and flhD expression. Biofilm

amounts were reduced by 50% at 2mg/mL. Growth and bacterial counts didn’t show a decrease

in the amount of bacteria until around 10mg/mL of PEA; this leads us to believe that PEA has

an effect on initial biofilm formation. To focus on early biofilm, we looked at flhD expression,

which has been shown to be vital in the reversible stage of biofilm formation. This is reflected

by our results of flhD expression, which showed no expression of flhD after 0.25mg/mL of PEA.

Altogether, these results support the hypothesis that PEA plays a role in the initial attachment

required for the formation of biofilms. Another important conclusion is that PEA seems to be

controlling bacterial phenotypes, without applying selective pressure which would induce

resistance. This leads us to believe PEA would be a good candidate as a potential biofilm

prevention substrate.

The research was funded by 1R15AI089403 from the NIH/NIAID.

Lynnes T, Horne SM & Prüß BM (2013) ß‐phenylethylamine as a novel nutrient treatment to

reduce bacterial contamination due to Escherichia coli O157:H7 on beef meat. Meat Sci 96: 165‐

171.

Stevenson LG, Szostek BA, Clemmer KM & Rather PN (2013) Expression of the DisA amino acid

decarboxylase from Proteus mirabilis inhibits motility and class 2 flagellar gene expression in

Escherichia coli. Res Microbiol 164: 31‐37.


22


COMPUTATIONAL ANALYSIS OF THE BASIS FOR VACCINE ESCAPE BY A

PORCINE CIRCOVIRUS STRAIN 2B (PCV2B) VIRUS ISOLATE.

Constans Megan1, $, Chelladurai. J. J1,$, Semmadali. M1,2, and Ramamoorthy. S1*. 1Department of Veterinary and Microbiological Sciences, North Dakota State University, Dakota

State University, Fargo, ND.

2School of Biosecurity, Biotechnical and Laboratory Sciences, College of Veterinary Medicine,

Animal resources and Biosecurity, Makerere University. P.O BOX 7062, Kampala, Uganda.

$Equal contributing author

Porcine circovirus strain 2 is responsible for post‐weaning multi‐systemic wasting disease

(PMWS), an economically important swine disease. Current vaccines against PCV2 were

designed using the PCV2a genotype. In recent years, the predominant genotype has shifted

from PCV2a to the PCV2b in the U.S. Moreover, recent studies have documented the emergence

of new recombinant viral strains. These findings suggest that current vaccines may be inducing

selection pressure and driving viral evolution. In this study, a PCV2b infectious clone was

created using DNA from a natural case of PMWS in a vaccinated herd. The infectious clone was

sequenced and differing epitopes between PCV2a and PCV2b were identified using in‐silico

analysis. Predicted B and T cell epitopes were identified in all proteins except ORF4 where only

one B cell epitope was detected. Using the NetMHCspan server, a majority of predicted high

affinity MHC‐I epitopes binding to Swine SLA1‐0401, SLA2‐0401, and SLA3‐0401 were

conserved. Variations between epitopes occurred within ORF1 at positions 67, 274 and 275,

within ORF2 at positions 48, 58, 145, 147 and 178 and within ORF3 at position 95. The MHC‐II

epitopes predicted using the PROPRED server were conserved with variations occurring within

ORF1 at aa 274, ORF2 at aa 184 and within ORF3 at aa 37. Using the linear B cell epitope tools

provided by the Immune Epitope Database, variations were detected in ORF1, four in ORF2

and another in ORF3. Epitope variations of B and T cell epitopes were concentrated within

ORF2; providing a likely basis for vaccine escape.


23


DIFFERENTIAL EXPRESSION OF VIRAL PATTERN RECOGNITION RECEPTORS

IN PORCINE AIRWAY AND INTESTINAL EPITHELIAL CELLS IN RESPONSE TO

INFLUENZA INFECTION

Milton Thomas1, Zhiguang Ran2, Laihua Zhu2, Ben Hause3, Mahesh Khatri4, David

Francis2, Feng Li1, 2, and Radhey S. Kaushik1, 2

1Department of Biology and Microbiology, 2Department of Veterinary and Biomedical Sciences,

South Dakota State University, Brookings, SD, USA; 3Newport Laboratories, Worthington, MN,

USA; 4Food Animal Health Research Program, Ohio Agricultural Research and Development

Center, The Ohio State University, Wooster, Ohio, USAThe epithelial cells of swine respiratory tract are highly susceptible while digestive tract

epithelia are considered more resistant to influenza infection. The MK1‐OSU cells, a clonally

derived porcine cell line from tracheal and bronchial epithelial cells, express both α‐2, 3 and α‐2,

6‐linked sialic acid receptors and are infected by influenza A, B, and C viruses. The SD‐PJEC

cell line, a sub‐clone of porcine intestinal epithelial IPEC‐J2 cells, supports growth of influenza

A strains but resists influenza B infection. The objective of this study was to investigate the Toll‐

like receptors (TLRs) and RIG‐1 like receptors (RLRs) expressions in both cell lines in response

to Influenza viruses. The MK1‐OSU cells were infected at 0.01 MOI with B/Florida/4/2006,

A/swine/Iowa/0855/2007(H3N2), A/swine/Minnesota/2073/2008(H1N1) whereas SD‐PJEC cells

were infected with Influenza A strains only. The protein expressions of TLRs 2‐9, RIG1, and

MDA5 were quantified using flow cytometry. At 24h post infection, TLR 7, RIG1, and MDA5

proteins decreased (P<0.05) for Influenza A strain infected MK1‐OSU cells compared with

uninfected control cells. TLR7 expression in SD‐PJEC cells infected with H1N1 strain decreased

compared with control. RIG1 expression for infected SD‐PJEC cells was similar to control cells.

Also, MDA5 expression increased, but not significantly (P<0.1), in infected SD‐PJEC cells.

However, there was no change in expressions of these proteins at 6h post infection in both cell

lines. The decrease in expression of anti‐viral TLR7, RIG1 and MDA‐5 in influenza infected

respiratory epithelium suggests the down regulation of TLRs and RLRs‐mediated immunity by

influenza virus.


24


DIAGNOSTIC TOOL FOR DETECTING SWINE TORQUE TENO VIRUS 1 (TTV1)

Anuradha Vegi1, Sheela Ramamoorthy1

1 North Dakota State University, Veterinary & Microbiological Sciences, Fargo, ND, USA

Swine Torque Teno viruses (TTSuVs) which are related to human TTVs of family Anelloviridae

are non‐enveloped, circular, negative sense single stranded DNA viruses and are widely

distributed in swine populations. TTSuV’s are highly associated with the porcine respiratory

disease complex (PRDC) causative agents. However, their role in pathogenesis is not yet

understood. Hence, there is need for diagnostic tools to detect swine TTVs. Two main swine

TTSuV groups – TTSuV1 and TTSuV2 exist. The protein encoded TTSuV1 open reading frame 1

is considered to be the major capsid protein. To develop an immunofluorescence assay (IFA) to

detect antibodies to TTSuV1, DNA from a TTV1 positive swine liver sample was used as

template to amplify the TTV1 ORF1, 2 and 3 which were 1.9 kb, 219 bp and 684 bp respectively.

The PCR fragments were cloned into a commercial mammalian expression vector. The plasmids

were then transfected into the pig kidney cells (PK‐15N) cells using lipofectamine. The

subcellular visualization of TTV1 ORF 1, 2, and 3 expression plasmids in PK‐15N via

immunofluorescence assay was achieved using monoclonal antibodies to C terminal epitope

tags. The TTV1 ORF1 was localized in nuclei, ORF2 was located in cytoplasm and ORF3

localized to both the nucleus and cytoplasm of PK‐15 N cells. Hence, TTSuv proteins were

successfully expressed in PK‐15N cells as a first step in optimization of a TTSuV specific IFA.


25


GENETIC AND BIOLOGICAL CHARACTERIZATION OF A NOVEL INFLUENZA

VIRUS IN US CATTLE AND SWINE

Runxia Liu1,2, Emily Colin2,3, Ben Hause3, and Feng Li1.2 1 South Dakota State University, Biology and Microbiology, Brookings, SD, USA; 2 South Dakota

State University, Veterinary and Biomedical Sciences, Brookings, SD, USA;

3 Newport Laboratories, Worthington, Minnesota 56187

Recently, a virus with 50% overall homology to human influenza C viruses (ICVs) was isolated

from a pig in Oklahoma (C/swine/Oklahoma/1334/2011 (C/OK). To further characterize this

novel virus, we employed deep‐RNA sequencing approach to analyze viral and host

transcriptomes in infected swine testicle (ST) cells at 0, 18, and 36 h post infection. High‐

resolution viral transcriptome, further confirmed by traditional RT‐PCR and Sanger sequencing

revealed a novel splicing mechanism to express the M1 protein. Also a previously unrecognized

transcript spliced from PB1 segment was also observed. Analysis of host transcriptome

landscape in response to C/OK virus infection demonstrated a relatively weaker interferon

response compared to those reported for influenza A virus. Virus replication study showed that

swine C/OK virus differed from human ICV in that it is not restricted at 37 °C for growth and

has a much broader cellular tropism. Significantly, this novel virus has been recently isolated

from US cattle population. Cross‐species transmission highlights the potential of this novel

pathogen in transmission to and causing disease in humans. Taken together, these results

suggest that this new group of viruses is genetically and antigenically distinct from human ICV

and warrant classification probably as a new genus of influenza.


26

Session 2 – PP 1

OPTIMIZATION OF THE INHIBITION OF LISTERIA MONOCYTOGENES BY A

CARNOBACTERIA BACTERIOCIN

Mitchell Edquist, Bonnie Bratina

University of Wisconsin‐La Crosse, Microbiology, La Crosse, WI, USA

Bacteriocins are proteinaceous toxins that are produced by non‐pathogenic bacteria that inhibit

the growth of similar strains of bacteria. Bacteriocins have been referred to by some as narrow‐

spectrum antibiotics. The antimicrobial activity has the potential to be useful to the food and

medical industries. We have 24 strains of Carnobacterium that were isolated from Antarctic

oligotrophic lakes. These strains have been subjected to numerous tests to determine which

strains were most effective at inhibiting Listeria monocytogenes, the causative agent of listeriosis

(a disease with a 20 to 30% mortality rate). Carnobacteria from this extreme and unusual

environment may be able to produce effective carnocins, bacteriocins produced by

carnobacteria, which are different from those previously characterized. One of the best

carnobacteria strains was selected for further testing, and experiments are currently underway

to optimize production of carnocin from that strain. Parameters being tested include variables

such as: incubation time, media selection, addition of salts and phosphates to the media.

Preliminary results indicate that more carnocin is produced with incubation times up to 96

hours. By changing this and other parameters to optimize carnocin production, we hope to

achieve the goal of the project, which is to obtain high enough production for the strain to be of

practical use in the food or medical industries.


27

Session 2 – PP 2

DEVELOPMENT AND OPTIMIZATION OF AMOEBA AND INSECT VIRULENCE

ASSAYS FOR THE HIGH‐THROUGHPUT ANALYSIS OF STENOTROPHOMONAS

MALTOPHILIA VIRULENCE DETERMINANTS

Bridget Eklund1, Ben Martin2, Elliott Welker3, and Nathan Fisher1,2,3

1North Dakota State University, Biotechnology Program, Fargo, ND, USA; 2 North Dakota State

University, Veterinary & Microbiological Sciences, Fargo, ND, USA; 3North Dakota State

University, Genomics & Bioinformatics Program, Fargo, ND, USA

Stenotrophomonas maltophilia is an emerging, multi‐drug‐resistant (MDR) pathogen that

frequently colonizes ventilator tubes and indwelling medical devices where it forms biofilms.

Initial colonization can lead to severe, life‐threatening infection. Currently, the incidence and

prevalence of S. maltophilia infections are increasing, especially in immune‐compromised, cystic

fibrosis (CF), chronic obstructive pulmonary disease (COPD), and cancer patients. Clinical

treatment is challenging because of S. maltophilia’s natural resistance to most antibiotics.

Moreover, in vitro sensitivity testing often leads to ineffective treatment due to in vivo up‐

regulation of drug efflux systems. We have initiated preliminary studies aimed at identifying

key virulence determinants in S. maltophilia as part of a long‐term effort to develop novel

treatment options that circumvent the S. maltophilia efflux systems by targeting secreted factors

or by pharmacologically bolstering the otherwise inadequate host immune response. Here, we

present our work in developing effective, high‐throughput assays that will facilitate a genome‐

scale analysis of S. maltophilia virulence determinants. A panel of 20 S. maltophilia isolates, from

both clinical and environmental origin, was tested for the ability to (a) resist predation by the

amoeba Dictyostelium discoideum and (b) cause toxicity when injected into the hemoceol of

juvenile Blaptica dubia (orange‐spotted cockroach) and Galleria mellonella (greater wax moth).

The former is used to assay bacteria‐phagocyte interactions while the latter is used to assay

bacteria‐innate immune interactions. Details regarding experimental set up and phenotypic

differences between isolates will be discussed, as will the applicability of both assays for high‐

throughput analysis of S. maltophilia mutant libraries currently under construction.


28

Session 2 – PP 3

DOES STRESS EXPOSURE IN THE HOSPITAL ENVIRONMENT IMPACT

SUBSEQUENT VIRULENCE IN STENOTROPHOMONAS MALTOPHILA?

Yayra E. Domfeh, Julie Sherwood, Teresa M. Bergholz and Nathan A. Fisher

North Dakota State University, Department of Veterinary and Microbiological Sciences,

Fargo, ND

Stenotrophomonas maltophilia is an environmental, opportunistic, gram‐negative bacterium that is

emerging as an important nosocomial pathogen worldwide. Typically, S. maltophilia affects

patients suffering from co‐morbid illnesses, who had been hospitalized over a long period of

time. The bacteria can be acquired in the hospital environment and from health professionals. S.

maltophilia infections pose a significant challenge to immunocompromised individuals such as

cystic fibrosis patients. Our research goal is to determine if stresses encountered in the hospital

environment can impact subsequent virulence of the pathogen. It is likely that S. maltophilia

encounters osmotic stress in the non‐host environment such as on surgical equipment or

medical devices, and we wanted to test if exposure to osmotic stress influences resistance to

cationic antimicrobial peptides, which are part of the innate immune response. To accomplish

this, five strains of S. maltophilia were exposed to salt and nisin in HEPES buffer at 37°C. The

strains were exposed to 2.5% NaCl for two hours and exhibited an average log decrease of 1.3 ±

0.14 cfu/ml while strains exposed to 15mg/ml nisin for two hours exhibited an average log

decrease of 2.1 ± 1.08 cfu/ml. This has implications for assessing the gram negative cell envelope

stress response systems in S. maltophilia upon exposure to salt and cationic antimicrobials. The

results obtained from this study are an early step to understanding how non‐host

environmental stress can impact virulence of this nosocomial pathogen.


29

Session 2 – PP 4

PREVALENCE OF MRSA COLONIZATION OF PATIENTS AND STAFF IN A

REGIONAL HOSPITAL IN CUENCA, ECUADOR

Erin Leisen, Annie Szmanda, and Daniel Herman

University of Wisconsin – Eau Claire, Biology, Eau Claire, WI, USA

Methicillin‐resistant Staphylococcus aureus (MRSA) is an antibiotic‐resistant strain of the

bacterium Staphylococcus aureus that is responsible for many hospital‐acquired infections world‐

wide. MRSA commonly colonizes individuals on the mucous membranes of the anterial nares,

as well as the skin of the axilla and groin and can be spread between individuals via direct

contact. Very little information is currently available on the prevalence of MRSA colonization

of patients and staff in Ecuadorian hospitals. During the summer of 2012, nasal swabs were

collected from 550 volunteers in a regional public hospital in Cuenca, Ecuador to determine the

prevalence of MRSA colonization within the hospital. The nasal swabs were inoculated onto

mannitol salt agar supplemented with 4 mg/ml oxacillin to select for potential MRSA. Mannitol

fermenting colonies were further examined using Gram stain and catalase tests. Colonies

consisting of catalase positive Gram positive cocci were tested further using a latex

agglutination test to detect bacteria producing coagulase and/or protein A. Isolates that were

oxacillin‐resistant, mannitol fermenting, catalase positive, Gram positive cocci with a positive

latex agglutination reaction were presumptively identified as MRSA. Thirty presumptive

MRSA isolates were identified from the 550 volunteers that were sampled resulting in a

prevalence of 5.5% for MRSA colonization. These results indicate that MRSA is present within

the Ecuadorian hospital examined and the potential for hospital‐acquired infections exists.


30

Session 2 – PP 5

CONSTRUCTION AND IMMUNOGENICITY EVALUATION OF RECOMBINANT

INFLUENZA A VIRUSES CONTAINING CHIMERIC HA GENES FROM

GENETICALLY DIVERGENT INFLUENZA H1N1 VIRUSES

1Kara McCormick, 2Longchao Zhu, 2Zhiyong Jiang, 2Steven R. Lawson, 2Robert

Langenhorst, 2Russell Ransburgh, 1Colin Brunick, 1Victor Huber, 2Ying Fang 1Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of

South Dakota, Vermillion, SD 57069. 2Department of Veterinary and Biomedical

Sciences, South Dakota State University, Brookings, SD 57007.

Influenza A virus causes highly contagious respiratory diseases in a variety of

mammalian hosts, including humans and pigs. To develop a vaccine that can be

broadly effective against genetically divergent strains of viruses, in this study, we

employed molecular breeding (DNA shuffling) technology to create a panel of chimeric

HA genes. Each chimeric HA gene contains genetic elements from four parental viruses

that represent major phylogenetic clusters of influenza A H1N1 viruses. Eight shuffled

HA constructs were initially evaluated in mice by DNA immunization. The result

showed that six HA constructs, HA‐111, HA‐113, HA‐123, HA‐116, HA‐107 and HA‐

129, induced broad antibody response against either four or five of the genetically

distinct parental viruses. These chimeric HAs were subsequently cloned into the

backbone of A/Puerto Rico/8/34 and A/swine/Texas/4199‐2/98 infectious clones, and

HA‐129 recovered viable recombinant viruses using reverse genetics, designated as

PR8‐129 and TX98‐129. The PR8‐129‐infected mice developed HI antibody titer greater

than 1:80 to parental viruses. The broad protective immunity induced by HA‐129 was

further assessed in a pig challenge model. Pigs were immunized with live or inactivated

TX98‐129 virus. The result showed all of the immunized pigs developed HI antibody

titer higher than 1:40 to four parental viruses at 28 days post immunization, with

significant difference between the inactivated TX98‐129 immunized group and non‐

immunized group of pigs. This study established a platform for creating novel HA

genes of influenza viruses using molecular breeding approach, which will have

important implications in future development of broadly protective influenza virus

vaccines.


31

Session 2 – PP 6

EFFECT OF BOVINE HERPESVIRUS 1 AND BOVINE VIRAL DIARRHEA

VIRUS (BVDV) ON BOVINE MONOCYTE‐DERIVED DENDRITIC CELLS

Kaci Park* Mrigendra Rajput, Lyle J Braun and C.C.L. Chase1

1Department of Veterinary and Biomedical Sciences, SDSU, Brookings, SD, 57007, USA

Corresponding author email: [email protected]

Dendritic cells (DC) are antigen presenting cells that initiate naive T‐cell activation and regulate

both innate and acquired immunity. Viral infection may adversely affect the immune system by

hindering these functions. In this study we evaluated the effects of BVDV, an RNA virus, and

BHV1, a DNA virus, on DC viability and on cell surface marker expression.

Monocytes were differentiated into bovine monocyte‐derived dendritic cells (MDDC) using

bovine recombinant IL‐4 and GMCSF. Four strains of BVDV were used, including the severe

acute non‐cytopathic (ncp) BVDV2a‐1373, a mild acute strain ncp BVDV2a‐28508‐5, and a virus

pair, cytopathic (cp) BVDV1b‐TGAC and ncp BVDV1b‐TGAN. The Cooper strain of BHV1 was

used in the study. Results revealed none of the BVDV strains used affected MDDC viability up

to 72 hr p.i., while the BHV1 killed around 18% of MDDCs by 72 hr p.i. The cp BVDV1b‐TGAC

up regulated MHCI and MHCII expression on MDDCs as early as 1 hr p.i. The other three ncp

BVDV strains reduced MHCI and MHCII expression in MDDC p.i. The cp BVDV1b‐TGAC up

regulated the CD86 expression at 48 hr and 72 hr p.i,. However, none of the ncp BVDV used

had any effect on CD86 expression. The Cooper strain of BHV1 down regulated MHCI and

MHCII expression with course of infection, but up regulated CD86 expression at 96 hr p.i.

Further studies are needed to observe how different strains of BHV1 modify MDDCs, along

with effect of viral infection of MDDCs on T cell activation and cytokine production.


32

Session 2 – PP 7

VIRUS‐LIKE PARTICLES GENERATED FROM EXPRESSING THE MEMBRANE (M) AND

NUCLEOCAPSID (N) PROTEINS OF PORCINE REPRODUCTIVE AND RESPIRATORY

SYNDROME VIRUS (PRRSV)

Xiuqing Wang, Hanmo Zhang, April Malsam, Martha Reed

Department of Biology and Microbiology,

South Dakota State University, Brookings, SD 57007

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive

failure in pregnant sows and acute respiratory disease in young pigs. It is a leading infectious

agent of swine respiratory complex, which has significant negative economic impact on the

swine industry. Commercial markets currently offer both live attenuated and killed vaccines;

however, increasing controversy exists about their efficacy providing complete protection.

Therefore, a pressing need exists for a safe and effective vaccine to control and prevent this

devastating disease. Virus‐like particles (VLPs) possess many desirable features of a potent

vaccine candidate and have similar structure and antigenic properties as their native infectious

virus particles, but they are not pathogenic since they do not contain any viral genome.

Heterologous protection can be easily achieved by immunization of animals with multivalent

VLPs generated by using the exact genetic match of the circulating virus strains. More

importantly, VLPs have been clinically proven to be highly immunogenic and protective against

virus infections. In this study, we investigated the role of different viral structural proteins of

PRRSV in the formation of VLPs. We have cloned several structural proteins of PRRSV into a

mammalian expression vector and assessed their ability in the formation of VLPs. We have sub‐

cloned some of the genes into a transfer vector for generating recombinant baculoviruses.

Future studies will be focused on the characterization of VLPs generated from recombinant

baculovirus and the evaluation of the immunogenicity and protection efficacy of the VLPs

against PRRSV.


33

Session 2 – PP 8

DEVELOPMENT AND VALIDATION OF INSECT VIRULENCE ASSAYS FOR THE HIGH‐

THROUGHPUT ANALYSIS OF FRANCISELLA SPP AND BURKHOLDERIA SPP

VIRULENCE DETERMINANTS: YET ANOTHER WAY TO GET RID OF COCKROACHES!

Ben Martin1 and Nathan Fisher1

1North Dakota State University, Veterinary and Microbiological Sciences, Fargo, ND, USA

Invading pathogens first interact with the innate immune system of the host, which presents

significant barriers to survival and growth of microorganisms, including physical sequestration

of the pathogen, nutrient limitation, enzymatic degradation of pathogen cell walls and

membranes, actions of antimicrobial peptides, and engulfment and subsequent destruction by

phagocytes. Characterizing these interactions between the pathogen and host is vital to the

ultimate goals of disease prevention and treatment. Saturating transposon mutagenesis has

recently been achieved in Francisella tularensis subspecies novicida and Burkholderia thailandensis

and several groups have begun to use readily available mutant libraries as a genetic basis for

the evaluation of interactions between these bacteria and the host. Unfortunately, small

mammal host model systems do not offer adequate throughput for individual interrogation of

mutant strains. Various attempts have been made to analyze mutant strains pooled together,

but these have thus far met with limited success and an efficient mechanism by which to

individually test a large number (thousands) of strains is preferred. Fortunately, this level of

throughput is possible with insect virulence assays designed for specific analysis of the innate

immune system. Since the innate immune systems of insects are strikingly robust and highly

similar to those of mammals, their use as a tool to study this interaction has been described for a

number of important human pathogens and a large body of research has described extensive

regulatory, structural, and mechanistic similarities between the innate immune systems of

insects and mammals. Larvae of the greater wax moth, Galleria mellonella, are a common host

choice primarily because they thrive at 37 degrees Celsius, are large enough for easy handling

and inoculation, and are commercially available at a very low cost. Here we describe the

development and validation of two assays for determining relative resistance to innate

immune‐mediated killing of Francisella spp and Burkholderia spp isolates. The first assay is

based on the popular alternative host G. mellonella, while the second is based on infection of

larvae of the orange‐spotted cockroach, Blaptica dubia. The new assay based on B. dubia solves

significant logistical difficulties associated with the care and maintenance of G. mellonella larvae

and is also well suited for use by undergraduate researchers. The applicability of both assays

for discovery of novel virulence determinants via high‐throughput mutant testing is also

discussed.


34

Session 2 – PP 9

M165A MUTATION IN THE MATRIX 1 (M1) PROTEIN OF INFLUENZA A VIRUS BLOCKS VIRUS REPLICATION BY DISRUPTING AN EARLY POSTENTRY STEP

Xiaoxiao Xiang1,2, Zhiguang Ran1,2, Zhao Wang1,2, and Feng Li1.2

1South Dakota State University, Biology and Microbiology, Brookings, SD, USA; 2 South Dakota State University, Veterinary and Biomedical Sciences, Brookings, SD, USA;

The influenza A virus matrix protein M1 is a multifunctional protein that plays numerous roles in viral replication including assembly, viral RNA replication, and early post-entrance to the host cell. It forms a protein shell just beneath the lipid bilayer membrane and forms the connection structurally between the viral transmembrane envelope proteins and the soluble vRNP complex. The M1 protein is a highly conserved 252-amino acid protein that contains two globular domains, an N-terminal domain (residues 1-164) and a C-terminal domain (residues 165-252). It has long been hypothesized that the proteolysis-sensitive junction region between the domains of M1 is important for virus replication. To address this hypothesis, we generated a panel of mutations targeting a four-residue segment (residues 163-166, RQMV) spanning the junction site between N- and C-domain of M1 protein and characterized them for viral replication and other functional properties such as particle production and RNA polymerase activity. We found that all mutations, regardless of whether they were conservative or non-conservative in nature, resulted in a replication-competent virus except for the M165A mutation. A detailed characterization of the M165A mutant demonstrated that a defect in early postentry event is likely responsible for the replication-incompetent phenotype observed in the M165 mutant virus. Thus, our data provides evidence that the junction region, particularly residue 165 of M1, is essential for influenza A virus replication. The data also indicates that the M1 protein plays a role in early postentry step.


35

Session 2 – PP 10

TRIPLE‐SERINE‐MOTIF (S224S225S226) OF THE M1 PROTEIN IS ESSENTIAL TO

INFLUENZA A VIRUS REPLICATION

Zhiguang Ran1,2, Qiji Deng1,2, Dan Wang1.2, Zhao Wang1.2, Milton Thomas1, Ben

Hause2,3, Radhey S. Kaushik1.2, Weidong Zhou4, and Feng Li1.2 1South Dakota State University, Biology and Microbiology, Brookings, SD, USA; 2South

Dakota State University, Veterinary and Biomedical Sciences, Brookings, SD, USA; 3Newport Laboratories, Worthington, Minnesota 56187

4George Mason University, Virginia

Previous studies have demonstrated that influenza A M1 protein is a phosphorylated protein. Here using the immunoprecipitation assay coupled with the Mass spectrometry (MS) approach, we observed that M1 S195 and S224 were phosphorylated. Knockout of phosphorylation at M1 S224 by substitution of serine for alanine reduced influenza A WSN33 virus replication by approximately 10-fold. Interestingly, same substitution at M1 S195 had no detectable effect on virus replication. Replacement of S225 or S226 with alanine also resulted in an attenuation similar to that observed for S224A mutation. Furthermore, double or triple mutations centering S224S225S226 all resulted in a significantly attenuated phenotype (10-50 fold less infectious) but levels of attenuation seemed not to reflect an additive effect. It is intriguing to notice that a phosphomimetic mutation at M1 S224 (S224D) still retained the attenuated phenotype, indicating that the phosphorylation mimics does not reproduce the effect of S224 phosphorylation on the structural and functional properties of the M1 protein in support of influenza A virus replication. Out studies identify a functionally significant motif that is required for efficient influenza A virus replication.


36

Session 3 – OP 1

MUSHROOM HUNTING USING BIOINFORMATICS: SESQUITERPENOID

DISCOVERY IN BASIDIOMYCOTA

Maureen B. Quin, Christopher M. Flynn, Grayson T. Wawrzyn,

Claudia Schmidt‐Dannert

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota,

Saint Paul, MN, USA

Basidiomycota are known to produce a large number of secondary metabolites, in particular

sesquiterpenes, one of the largest and most diverse classes of natural products. Many

sesquiterpenes have antifungal, antimicrobial and cytotoxic properties, leading to their

adaption and isolation for pharmaceutical purposes. Recently, we reported the genome

sequencing, mining, discovery and characterization of a suite of sesquiterpene synthases and

their associated biosynthetic enzymes in Omphalotus olearius. Subsequent phylogenetic analyses

of all putative sesquiterpene synthases in all genome sequenced Basidiomycota indicated a

conservation of both sequence and initial cyclization mechanisms. Using this information we

developed a predictive framework to facilitate the genome mining and discovery of

sesquiterpene synthases in Basidiomycota. The accuracy of our predictive framework was

confirmed in the selective identification of several novel sesquiterpene synthases that follow a

1,6‐, 1,10‐, and 1,11‐cyclization mechanism in the crust fungus Stereum hirsutum. Our predictive

framework is a robust and reliable roadmap for the discovery of specific sesquiterpene

synthases from Basidiomycota. Our work represents an important step forward in natural

product discovery, and lays a path for the discovery and bioengineering of enzymes that

produce pharmaceutically relevant compounds.


37

Session 3 – OP 2

ENGINEERING CYANOBACTERIA FOR THE PHOTOSYNTHETIC

PRODUCTION OF LONG CHAIN HYDROCARBONS

Charles Halfmann, Liping Gu, and Ruanbao Zhou

South Dakota State University, Biology and Microbiology, Brookings, SD, USA

Terpenes are a large class of organic molecules with important applications in pharmaceuticals,

cosmetics, and biofuels. Current strategies for harvesting terpenes from plant biomass require

arable land, impose high energy demands due to industrial processing, and release CO2 as a

harmful waste byproduct. Here, we focus on a direct photosynthesis‐to‐biofuel approach in

terpene generation by metabolically engineering the filamentous cyanobacterium Anabaena 7120

as cellular machinery to over‐produce and excrete farnesene (C15H24) and limonene (C10H16) into

a photo‐bioreactor using CO2, H2O, and light. To maximize terpene yield, a farnesene synthase

gene (faS) and a limonene synthase gene (limS) were individually spliced into separate synthetic

MEP operons and expressed in Anabaena 7120 for the microbial production of their respective

products. Our study reveals that expression of faS and limS in Anabaena 7120 allows for

photosynthetic production of farnesene and limonene, respectively, which is excreted across the

cell membrane and volatilized into the culture headspace, allowing for quick separation of the

target compound from the culture biomass. We envision this platform of using CO2 as a

sustainable feedstock to be applicable for the production of a wide range of commodity

chemicals and drop‐in‐fuels.


38

Session 3 – OP 3

EXPRESSION AND CHARACTERIZATION OF LYTIC STAPHYLOCOCCUS

AUREUS PHAGE P68 HOLIN PROTEINS IN ESCHERICHIA COLI

Kramer Richard

St. Cloud State University, Cell and Molecular Biology, St. Cloud, MN, USA

Bacteriophages require a host cell to complete the replication phase of their life cycle. To

release the new virions into the environment the host cell is often lysed. P68, a bacteriophage of

Staphylococcus aureus codes for a pair of holin proteins that integrate into the host cell membrane

causing disruption and generating cell lysis. In this study we aim to examine if expression of a

holin from S. aureus in Escherichia coli will cause lysis. The holin gene known as hol15 was codon

optimized for expression in E. coli. This optimized gene was then subcloned into the pET‐28a(+)

inducible vector backbone. If lysis is induced, the event is to be characterized through serial

dilution plating and optical density readings. Determination of the time and efficiency of this

process will help to identify if this has potential use in the production of biomolecules for

S. aureus, by E. coli.


39

Session 3 – PP 1

GLYCEROL‐BASED CITRIC ACID PRODUCTION BY AN ANALOGUE‐

RESISTANT CANDIDA GUILLIERMONDII MUTANT

Thomas P. West


The ability of a Candida guilliermondii mutant resistant to the analogue fluorocitrate to utilize

glycerol for citric acid production was investigated. The specialty chemical citric acid is

commercially valuable as an acidulant and a food additive. In this study, the yeast C.

guilliermondii ATCC 9058 was used because of its ability to convert glycerol into citric acid. The

mutant was isolated by selecting a colony able to grow on a medium containing yeast nitrogen

base, ammonium sulfate, raffinose and 200 mg/l fluorocitrate after 96‐120 hours at 30oC. The

ability of the mutant strain to produce citric acid and biomass from a medium containing 6%

glycerol was compared to its parent strain following growth for 120 hours at 30oC. To determine

citric acid content, a coupled enzyme assay was used where the reaction was monitored at 340

nanometers. After each culture was centrifuged to pellet the cells, the supernatant was

spectrophotometrically assayed for citric acid production. The collected cells were used to

compare biomass production by the parent and mutant strains. The results showed that the

mutant strain produced a 1.6‐fold higher citric acid level from glycerol relative to the parent

strain. The biomass level produced by the mutant strain from glycerol was slightly higher

compared to the parent strain. It was concluded that the fluorocitrate resistant mutant strain of

C. guilliermondii was capable of increased glycerol‐based citric acid production. This mutant

strain could be of value in the production of citric acid from crude glycerol from soy biodiesel.


40

Session 3 – PP 2

PULLULAN PRODUCTION BY A DEOXYGLUCOSE‐RESISTANT FUNGAL

MUTANT ON DILUTE ACID HYDROLYZED PRAIRIE CORDGRASS

Jessica L. Peterson, Thomas P. West


The extracellular polysaccharide pullulan is a biopolymer elaborated by the fungus

Aureobasidium pullulans. The gum has a variety of applications based upon its low viscosity and

water solubility. Prairie cordgrass is a plant that is composed of approximately 30% cellulose.

Degradation of its cellulose using dilute acid hydrolysis to glucose provides a substrate for

fungal pullulan production. In this study, a previously isolated deoxyglucose‐resistant mutant

of A. pullulans was used to determine how effectively pullulan could be produced on the acid

hydrolyzed cordgrass. The prairie cordgrass was treated with 0.05%‐0.30% sulfuric acid in an

autoclave for 30 minutes at 121oC under 17 pounds/square inch of pressure. After the

hydrolysates were filtered, the filtrates were utilized in a 0.5% phosphate buffered medium (pH

6.0) containing 0.1% yeast extract, 0.1% sodium chloride and 0.02% magnesium sulfate. Pullulan

production by the mutant strain grown on the hydrolysate‐containing medium was analyzed

following growth for 168 hours at 30oC. Pullulan was measured gravimetrically following

alcohol precipitation of the polysaccharide from the culture medium. The highest pullulan

concentration was produced by the mutant strain grown on the 0.25% sulfuric acid hydrolysate‐

containing medium. After 168 h of growth, the mutant strain produced more than a 2‐fold

higher pullulan level on the medium containing the 0.25% acid hydrolysate compared to the

0.05% acid hydrolysate. In conclusion, a dilute acid‐treated plant biomass can be used to

support pullulan production by a deoxyglucose‐resistant mutant with the dilute acid

concentration selected being important to observe maximum polysaccharide elaboration.


41

Session 3 – PP 3

INFLUENCE OF PRE‐HARVEST ENVIRONMENTAL FACTORS ON STRESS

RESISTANCE IN E.COLI AND SALMONELLA ON LETTUCE

Deepti Tyagi, Julie Sherwood, Katherine Sanders, Teresa M. Bergholz

North Dakota State University, Department of Veterinary and Microbiological Sciences, Fargo,

North Dakota, USA

Produce‐associated foodborne outbreaks have been increasing over the last few decades. The

potential exists for contamination of produce with enteric pathogens to occur in the pre‐harvest

environment. Fresh produce is often minimally processed and consumed raw, thereby

increasing consumer’s risk of exposure to severe gastrointestinal illnesses. Little is known about

the physiological state of these pathogens in the pre‐harvest field and how that state affects

subsequent pathogen survival, stress resistance and virulence. The goal of this research is to

determine the physiological state of two enteric pathogens: Enterohemorrhagic E. coli (EHEC)

and Salmonella on pre‐harvest lettuce. Changes in gene expression of four strains of EHEC and

four strains of Salmonella will be studied over different lettuce cultivation conditions. In order to

assess changes in gene expression over time, quantifying survival of these pathogens on lettuce

is necessary. Strains were inoculated on lettuce plants in the greenhouse. Five days post‐

inoculation, the average log reduction in two Salmonella Typhimurium strains was found to be

1.2 ± 0.2 and 0.8 ± 0.1 log cfu/g of lettuce, respectively. These data demonstrate that enteric

pathogens are capable of surviving on pre‐harvest lettuce, with only minor changes in cell

numbers. Based on this survival data, we will utilize transcriptional profiling to understand the

ability of these pathogens to survive in a non‐host environment. Further, we propose that stress

induced by environmental changes could affect their ability to survive post‐harvest

decontamination treatments or gastric acidity in the food chain.


42

Session 3 – PP 4

METABOLISM OF ANTINUTRITIONAL COMPONENTS IN BRASSICA CARINATA TO

PRODUCE HIGH PROTEIN FEED

Jason R. Croat1 and William R. Gibbons1

1Biology & Microbiology Department, South Dakota State University, Brookings, SD 57007, USA

Carinata or Ethiopian mustard (Brassica carinata) is being developed as a source of alternative oil

for biodiesel/jet fuel and as an alternative feed. A limitation with the feed usage of Carinata

oilseed meal is the presence of glucosinolates (GSL). GSL and the enzyme myrosinase are

compartmentally stored separately in the plant. Upon disruption of plant tissues, myrosinase

cleaves the glucose molecule from GSL and releases toxic compounds such as nitriles,

thiocyanates, and isothiocyanates to defend the plant. For this reason feed inclusion rates of

GSL‐containing crops are limited to 10% of the diet for ruminants and poultry, but only 2% for

swine. The objective of this research is to develop a microbial process to metabolize GSL and

the resulting breakdown products into non‐toxic components so that higher levels can be

included in livestock rations. An additional objective is to boost the protein levels and

digestibility of the converted meals via the microbial treatment. Several metabolically diverse

fungal cultures are being evaluated. Aurobasidium pullulans has been grown in submerged

culture on both cold pressed and hexane extracted carinata meal. A. pullulans, Trichoderma reesei,

and Fusarium venenatum, have also been grown on hexane extracted carinata meal at a 50%

moisture level. Assays for protein, GSL, and hydrolysis products are currently underway.


43

Session 3 – PP 5

OPTIMIZATION OF EXTRUSION PROCESSING CONDITIONS ON ENZYMATIC

HYDROLYSIS AND FERMENTATION OF DISTILLER’S LOW OIL WET CAKE

Bishnu Karki1,2, Kasiviswanathan Muthukumarappan1, William Gibbons2

1Agricultural and Biosystems Engineering Department; 2Biology and Microbiology Department;

South Dakota State University, Brookings, SD, 57007

Distiller’s low oil wet cake (DLOWC) is one of the major co‐products of the dry grind ethanol

facilities. Although it is a highly potential feedstocks of cellulosic ethanol, due to the high

moisture content of product (~70%, db), very limited studies have explored this material as an

additional feedstock of ethanol industry. In this study, we aim to explore the effect of single‐

screw extrusion (SCE) pretreatment of DLOWC on enzymatic hydrolysis and fermentation

yield. SCE pretreatment will be carried out using a central composite design at 5 level of

moisture content (25 to 72%, db) and 5 level of temperature (80‐180°C). The central point will be

48.5% moisture content and 130°C temperature, replicated for five times. The screw speed of 50

rpm will be used. Enzymatic saccharification and fermentation will then be conducted on the

pretreated extrudates for 72 hrs using enzymes Cellic Ctech2 and Htech2. Initial solid loadings

of 5% will be used. For the simultaneous saccharification and fermentation, saccharomyces

cerevisiae will be used. Sugar and ethanol released from these sets of experiments will then be

used to identify the optimal extrusion conditions for the pretreatment of DLOWC.


44

Session 3 – PP 6

EXAMINATION OF BACILLUS BIOLOGICAL CONTROL AGENTS FOR Itu D and

Ipa 14 GENES USING POLYMERASE CHAIN REACTION

Naveen Kumar Srinivasa Murthy1, Bruce Bleakley1, and Kay Ruden2

1South Dakota State University, Biology and Microbiology, Brookings, SD, USA 2South Dakota State University, Plant Science Department, Brookings, SD, USA

Corresponding email – [email protected]

Abstract – Fusarium Head Blight (FHB), caused by Fusarium graminearium is an economically

important disease, primarily observed in wheat, barley and some corn varieties. Strains of

Bacillus species can act as biological control agents (BCAs) to control FHB, and are typically

applied onto wheat heads at anthesis to protect against the disease. After spraying Bacillus

strains onto wheat heads, the wheat heads should be examined over time for presence of the

bacteria. We are attempting to monitor presence of Bacillus BCAs in wheat plots inoculated

with these BCAs. As a first step in the project, it was necessary to optimize the DNA extraction

and PCR protocols. DNA was extracted from Bacillus amyloliquefaciens strains 1BA, 1BC, 1BE,

1D3 and 1BAC, then subjected to PCR to examine them for presence of Itu D (1203bp) and Ipa 14

(675bp) genes. Further, Bacillus strains 1BA and 1D3 were sprayed onto previously

uninoculated, blank wheat heads, which were then air‐dried and subjected to processing for

recovery of Bacillus. Upon recovery of Bacillus from wheat heads, DNA was extracted and

subjected to PCR. All five strains of Bacillus amyloliquefaciens (1BA, 1BC, 1BE, 1D3 and 1BAC)

possessed Itu D and Ipa 14 gene fragments. Bacillus strains 1BA and 1D3 were successfully

recovered from wheat heads. DNA extracted from the recovered BCA’s was shown to have Itu

D and Ipa 14 gene fragments. Currently, DNA extraction from endospores is being optimized.

Upon optimization, the wheat heads will be examined for the presence of Bacillus strains (both

vegetative cells and endospores).


45

Session 3 – PP 7

DIRECT PHOTOSYNTHETIC CONVERSION OF CO2 AND H2O INTO PERFUME

LINALOOL BY ENGINEERED CYANOBACTERIA

Liping Gu, Xianling Xiang, Chuck Halfmann, William Gibbons, Ruanbao Zhou


Biorefineries typically release one third of the carbohydrate carbon as CO2 during fermentation,

as well as significant amounts of low grade heat. Ideally, a photosynthetic organism could be

engineered to convert these unused resources into high value chemicals. The current microalgae

production model suffers from technical challenges including harvest cells, extract oils, and

then convert them into a final product. We circumvented these challenges by engineering

cyanobacteria as a cellular factory to directly produce and secrete linalool using sunlight and

CO2 as the feedstock. Linalool was selected as our model product because of its wide

commercial applications, such as use in perfumery, potential anti‐cancer drugs as well as drop‐

in biofuels. Linalool is naturally produced from the MEP pathway where the universal

isoprenoid intermediate geranyl diphosphate (GPP) is converted to linalool by linalool

synthase.

Cyanobacteria have native metabolic pathways to photosynthetically convert CO2 and H2O to a

variety of reduced carbon compounds including geranyl diphosphate (GPP), the precursor for

making linalool. However, cyanobacteria lack the enzyme for converting GPP into linalool. In

this research, the linalool synthase gene from a linalool‐emitting plant was fused to Anabaena

promoters and subcloned into an Anabaena shuttle vector for transformation of Anabaena. The

transgenic Anabaena has been confirmed to produce and secrete linalool. In summary, our

engineered cyanobacteria can convert CO2 (released from corn‐based ethanol plants) directly

into high‐value perfume linalool, driven by solar energy and waste biorefinery heat.


46

Session 3 – PP 8

GENETIC TRANSFORMATION OF ANABAENA CYLINDRICA ATCC 29414

Huilan Zhu1, Peter Wolk2, Michael Hildreth1, Ruanbao Zhou1

1South Dakota State University, Biology and Microbiology, Brookings, SD, USA

2Michigan State University, MSU‐DOE Plant Research Laboratory, East Lansing, USA

Unlike Anabaena sp. PCC 7120, whose vegetative cells can differentiate only into N2‐fixing

heterocysts, vegetative cells of A. cylindrica can differentiate also into another distinct type of

cell, the spore‐like akinete. Akinetes develop within the same filament, and normally adjacent to

heterocysts. We report the first successful transformation of A. cylindrica. Further development

of transformation of A. cylindrica should permit genetic analysis of akinetes and of their

juxtaposition to heterocysts.

The gene acaK is a homolog of the previously identified akinete marker gene, avaK (Zhou &

Wolk, 2002. J. Bacteriol. 184: 2529‐32). acaK and its upstream region was PCR‐amplified from A.

cylindrica genomic DNA. The 1.5‐kb PCR product was fused to a promoter‐less gfp gene in

pAM1956, a derivative of Nostoc plasmid pDU1.The resulting construct, pZR963, was

transferred into A. cylindrica ATCC 29414 by conjugation. Transformants were selected on AAN

medium supplemented with 25 μg kanamycin ml‐1. Five colonies observed in a Petri dish were

confirmed as exconjugants by colony PCR. In these transformants, akinetes formed earlier, and

in greater abundance, than in the wild‐type strain in medium AA/8 with and without fixed

nitrogen. GFP‐based fluorescence from PacaK–gfp originated primarily in mature akinetes or

developing akinetes. To further study the role of AcaK in akinete formation, we are trying to

inactivate acaK in A. cylindrica and have made a Pnir‐acaK construction (Pnir, a nitrate‐inducible

promoter) in a pDU1‐based plasmid and are transferring it to A. cylindrica to determine whether

nitrate can induce akinete formation.


47

Session 3 – PP 9

BIOSYNTHESIS AND DESIGNER MICROBES

Maureen B. Quin & Claudia Schmidt‐Dannert

Schmidt‐Dannert Laboratory

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Saint

Paul, MN, USA

Plants, fungi and microbes produce a diverse array of chemical compounds, many of which

cannot be produced using chemically synthetic methods. We can, however, utilize the existing

metabolic machineries of plants, fungi and microorganisms to biologically synthesize valuable

compounds in recombinant microbial hosts. By combining various enzyme functions we can

create new metabolic reaction pathways, allowing us to custom synthesize valuable

compounds. Both in vitro directed evolution and computational rational design strategies can be

used to alter and optimize the catalytic function of enzymes in these assembled pathways1.

Together, these strategies result in the creation of tailor designed microbes for biosynthetic

purposes.

Currently, we investigate and engineer the biosynthesis of diverse isoprenoid‐derived

compounds using enzymes isolated from fungi, to produce chemicals with medicinal and

industrial properties2,3. We are also designing and building versatile protein nanobioreactors

within microbial hosts for the strategic targeting of metabolic pathways for optimal multi‐step

biosynthesis4,5. Overall, we aim to engineer tailor‐designed biocatalysts for the efficient

production of compounds which cannot be produced by chemical means.

1 Quin, M.B. and C. Schmidt-Dannert, ACS Catal, 2011. 1(9): p. 1017-1021. 2 Quin, M.B., C.M. Flynn, et al., ChemBioChem, 2013. In press. 3 Wawrzyn, G.T., M.B. Quin, et al., Chem Biol, 2012. 19(6): p. 772-83. 4 Choudhary, S., M.B. Quin, et al., PLoS One, 2012. 7(3): p. e33342. 5 Held, M., M.B. Quin, et al., J Mol Microbiol Biotechnol, 2013. 23(4-5): p. 308-20.

Session 4 – ORAL PRESENTATIONS

48


PROTEOMIC STUDY ON AKINETES, HETEROCYSTS AND VEGETATIVE CELLS IN

ANABEANA CYLINDRICA

Yeyan Qiu, Liping Gu and Ruanbao Zhou

Department of Biology and Microbiology, South Dakota state University, SD, USA The vegetative cells of Anabaena cylindrica can differentiate into both heterocysts and akinetes in medium AA/8 with and without fixed nitrogen. The heterocyst is accepted to be the site for nitrogen fixation in heterocystous cyanobacteria. The akinete is a spore-like cell capable of germination and of withstanding certain environmental extremes such as cold, desiccation, and phosphate limitation. The first akinete-specific protein AvaK from Anabaena variabilis was previously reported (Zhou & Wolk, 2002. J. Bacteriol.184: 2529-32), but no proteomic study of akinetes has been presented. Anabaena cylindrica was selected for proteomic analysis of akinetes because its akinetes are large and easily isolated. The akinetes and heterocysts were harvested by passing the A. cylindrica culture grown in AA/8 medium through a High Pressure Homogenizer at 4500 psi twice to completely break the vegetative cells, and then purified by CsCl density gradient centrifugation, respectively (Wolk & Simon, 1969. Planta 86:92-97). The total proteins extracted from the purified akinetes, heterocysts and vegetative cells were subjected to SDS-PAGE, in-gel tryptic digestion and LC-MS/MS for protein identification. LC-MS/MS identified a total of 634 proteins from the purified akinetes, a total of 924 proteins from the purified heterocysts, and a total of 1241 proteins from vegetative cells, including 107 proteins found exclusively in akinetes while 417 proteins including nif gene products are heterocyst specific. Since we, in collaboration with C. Peter Wolk, recently succeeded in genetic transformation of A. cylindrica, the identification of an akinete-specific proteome should enable us to pursue a genetic study of akinete formation.


49


IDENTIFICATION OF A SITE‐2 METALLOPROTEASE REQUIRED FOR COLD

ACCLIMATION IN CYANOBACTERIA

Kangming Chen1, Ruanbao Zhou1 1 South Dakota State University, Biology and Microbiology, Brookings, SD

In response to environmental changes, cells must transduce signals across their membranes to

elicit transcriptional responses. One mechanism by which information is transduced across the

membrane involves regulated intramembrane proteolytic activation of membrane‐tethered

transcription factors or membrane‐bound signaling proteins. Site‐2 metalloprotesae (S2P) is a

member in the family of intramembrane‐cleaving proteases (I‐CLip). Like other I‐CLips, this

S2P with catalytic‐site motifs embedded within transmembrane segments cleave the

transmembrane helix of a protein substrate inside the lipid bilayer, a hydrophobic environment

that is distinct from the aqueous environment required for conventional proteases. Thus, I‐Clips

can accomplish many important membrane‐involved tasks beyond the capability of their

soluble cousins.

In this study, Ava_4785 was identified to be a Site‐2 metalloprotease required for cold

acclimation in Anabaena variabilis ATCC 29413. Two ava_4785 knock‐out mutants showed a

phenotype that is incapable of survival at 4C after cold acclimation treatment from 30C to 4C,

while same treated wild‐type strain can still survive. Furthermore, the ava_4785 promoter

activity was monitored by a transcriptionally fused promoter‐less green fluorescence protein

(GFP). Strong GFP fluorescence signals were observed under fluorescent microscope after 1 h

shift from 30C to 4C indicating that expression of ava_4785 is indeed regulated by cold signal.

A complementation experiment for the two mutants is being performed to ascertain that

ava_4785 is responsible for the phenotype of the mutant. Nevertheless, we conclude, for the first

time, that an intramembrane metalloprotease Ava_4785 may be required for cold acclimation in

Anabaena variabilis.


50


ALR4853, AN O2‐SENSITIVE ASPARTATE AMINOTRANSFERASE, IS

SPECIFICALLY REQUIRED FOR N2‐BASED GROWTH IN Anabaena sp. PCC 7120

Xinyi Xu, Liping Gu, Ping He, Ruanbao Zhou


Anabaena sp. PCC 7120 is a filamentous heterocyst‐forming cyanobacterium. Lacking of fixed

nitrogen in the growth media triggers differentiation of N2‐fixing cells called heterocysts.

Heterocysts lack the oxygen‐producing photosystem II; respire rapidly; and have an additional

envelope that impedes the entry of oxygen, providing a micro‐oxic environment for oxygen

sensitive enzymes such as nitrogenase.

Thirty‐five proteins were identified in the growth medium by tandem MS when Anabaena sp.

PCC 7120 grown in the dinitrogen condition. To understand the functions of those putatively

secreted proteins, we performed a systematic study by simultaneously inactivating each target

gene and monitoring its promoter activity. A putative aspartate aminotransferase (AST) gene

alr4853 was inactivated. Two independently isolated alr4853 mutants showed growth deficiency

on N2‐media. The alr4853 mutants can still form heterocyst after 24 hour nitrogen step‐down

and its promoter is constitutively active as indicated by GFP reporter. The recombinant alr4853

protein overexpressed in E. coli was purified and tested for AST activity in vitro. Our data

showed that Alr4853 has AST activity and the AST activity increases at least 50% in a micro‐oxic

environment, suggesting that Alr4853 is an oxygen sensitive AST. Since there are totally five

putative AST genes in Anabaena PCC 7120 genome, we are currently investigating the biological

functions of these putative AST genes. In conclusion, we identified that alr4853 is a fox gene (fix

dinitrogen in an oxygen‐containing milieu) encoding an aspartate aminotransferase that

functions in both aerobic and anaerobic condition in vitro but favors the latter.


51


HOST GLYCOPROTEINS AS TRIGGERS OF CRYPTOSPORIDIUM DEVELOPMENT

Adam Edwinson, John McEvoy

North Dakota State University, Veterinary and Microbiological Sciences, Fargo, ND, USA

The obligate intracellular parasite Cryptosporidium causes the diarrheal disease

cryptosporidiosis, which has been linked to stunted growth of toddlers and infants, and

mortality in malnourished and immunocompromised individuals. Upon infection the

Cryptosporidium life cycle begins with the release of linear motile sporozoites which attach to,

and infect, the host intestinal epithelium forming a rounded non‐motile trophozoite. The

parasite then transitions from this feeding stage to an asexually replicating meront. Although

the life cycle has been well characterized little is known regarding the mechanisms underlying

the distinct biological changes seen in Cryptosporidium during infections. Our research aims to

determine what triggers sporozoite invasion, but also examines factors that promote parasite

development. Our approach is novel in that we have developed a method of examining

potential activators of Cryptosporidium development in a host cell free system. Using this model

we have established glycoproteins, a major constituent of the intestinal matrix, are released

from host cells and induce motile sporozoites to round independent of host cell attachment.

This morphological change to a rounded non‐motile trophozoite‐like structure indicates host

glycoproteins modulate progression of the Cryptosporidium life cycle. We have also found

varying species of Cryptosporidium respond differently to host secretions, and that glycans can

initiate the asexual replication in the absence of host cells. Understanding the role glycoproteins

have in promoting Cryptosporidium development will help improve our understanding of the

overall invasion process, leading to novel anti‐cryptosporidial drug therapies which will aid in

disease prevention.


52


CAN OLD GENES GIVE RISE TO NEW GENES? A TEST OF THE GENE

DUPLICATION HYPOTHESIS WITH HISB OF ESCHERICHIA COLI

Joseph Madison*, Michael Schultz, and Ralph Seelke

University of Wisconsin‐Superior, Natural Sciences, Superior, WI, USA

We have been testing the ability of a gene that is performing two functions to evolve into two

separate genes. E. coli strain RS301 has hisB (required for histidine biosynthesis) cloned into a

high copy number, IPTG‐inducible vector, pCA24N; it also has chromosomal deletions for both

serB and hisB. Upon IPTG induction, the hisB gene is able to substitute for serB, required for

serine biosynthesis. The hisB gene codes for histindinol phosphate phosphatase (HPP), but it

also has weak serine phosphate phosphatase (SPP) activity, the activity of the serB gene

product. Two populations of RS301 have been evolving for over 1000 generations through

serial transfer in minimal medium lacking both serine and histidine; both grow much better

than RS301 in this medium. If two genes were produced, some plasmids would specialize in

producing histidine (similar to the ancestral gene), while others would specialize in producing

serine, allowing good growth in the absence of serine but not histidine. This was tested by

transforming (at a low plasmid:cell ratio) the ancestral host with plasmids from the 500 and

1000 generation populations, and screening for transformants that that grew poorly in the

absence of histidine but not serine. All transformants tested (~400) showed the phenotype of

good growth in the absence of histidine when supplied with serine. Thus, selection has

apparently resulted in a multifunctional hisB gene, rather than two genes. Evidence for

additional changes in these populations (loss of IPTG sensitivity to hisB induction and lowered

copy number) will also be presented.


53


EVOLUTION OF SERINE PHOSPHATE PHOSPHATASE ACTIVITY IN THE HISB

GENE OF ESCHERICHIA COLI

Michael Schultz*, Joseph Madison, and Ralph Seelke

University of Wisconsin‐Superior, Natural Sciences, Superior, WI, USA

We have been examining the evolutionary paths taken by an E. coli strain, RS301, in which hisB

is found on a multicopy plasmid, while both serB and hisB are deleted from the chromosome.

The hisB gene product (HBGP) is required to serve as both a serine phosphate phosphatase

(SPP) and in its original role as a histindinol phosphate phosphatase. Two populations of RS301

(A150&B150) have evolved in a medium lacking serine and histidine for 1,000 generations, and

grow 1.7‐2X faster than RS301 in this medium. The objective of this project was to isolate HBGP

from the ancestral and evolved populations and determine the SPP activity in each population.

Isolation of HBGP was carried out by sonication of stationary phase cultures and separation of

the his‐tagged HBGP from the supernatant using a nickel agarose column. SPP activity was

determined using a phosphate assay in which serine phosphate was the enzyme substrate and

in which the production of phosphate was measured spectrophotometrically. As expected the

SPP activity in the evolved strains was much greater than in the ancestral strain, and far greater

than expected from the growth rate increase. The SPP activity (nmoles phosphate/hr/ug HBGP)

from A150 was approximately 52 times greater than that of RS301, which had barely detectable

SPP activity. B150 had approximately a 10‐fold higher SPP activity. Further work will be

needed to clarify the basis for this increase, and the unexpected lack of a linear relationship

between the growth rate increase and the SPP activity increase.

Session 4 – POSTER PRESENTATIONS

54

Session 4 – PP 1

EXAMINATION OF TRANS‐GOLGI SNARE SYNTAXIN 10 AT THE CHLAMYDIAL

INCLUSION

Andrea L. Lucas, Emily Kabeiseman, and Elizabeth R. Moore

University of South Dakota, Basic Biomedical Sciences, Vermillion, SD, USA

Chlamydia trachomatis, an obligate intracellular pathogen, takes advantage of host vesicular

transport pathways to maintain an infection. Upon infection the elementary body (EB), which is

the infectious but metabolically inactive form, inhabits a vacuole termed inclusion where it

differentiates into the reticulate body (RB), the non‐infectious metabolically active form.

Following endocytosis the inclusion localizes to the Golgi region to acquire Golgi‐derived

sphingolipids. Previous studies demonstrated that the inclusion preferentially intercepts

basolaterally‐targeted, sphingomyelin‐containing vesicles. In order to identify which host

trafficking pathways interact with the inclusion, we investigated several trans‐Golgi associated

soluble N‐ethylmaleimide–sensitive factor attachment proteins (SNAREs). We demonstrated

that trans‐Golgi SNARE syntaxin 10 colocalizes with the chlamydial inclusion; where direct

contacts between syntaxin 10 and Golgi structural proteins remained localized at the inclusion

despite Golgi structures being chemically disrupted. We hypothesize that syntaxin 10’s

localization with the inclusion tethers Golgi subunits to the inclusion membrane and play a role

in host derived lipid acquisition. To test this hypothesis we examined the role of syntaxin 10 in

chlamydial lipid acquisition and development of infectious progeny. When inclusions are

grown in syntaxin 10 knock down cells a fluorescent lipid analog, NBD‐sphingomyelin is

trafficked to and retained in inclusions significantly more than inclusions grown in control cells.

However, there is a decrease of infectious progeny when inclusions are grown syntaxin 10

knock down cells, indicating a there is a balance between chlamydia lipid acquisition and

optimal chlamydial infectivity. Defining the relationship between syntaxin 10 and C. trachomatis

will increase our understanding of SNARE‐mediated Golgi‐inclusion interactions.


55

Session 4 – PP 2

CELL SHAPE DETERMINATION IN HELICOBACTER PYLORI: CHARACTERIZING THE

ROLES OF CSD1 AND CSD2

Kristina Hernandez, Ellie Hofer, Julie Bonham, Timna Wyckoff

University of Minnesota, Morris, Division of Science and Mathematics, Morris, MN, USA

Helicobacter pylori is a bacterium that colonizes 50% of the world’s human population. H. pylori

infection has been linked to stomach ulcers and gastric cancer. The helical shape of the

bacterium allows it to colonize the gastric mucosa, thus avoiding the acidity of the

stomach. Understanding how H. pylori generates shape could lead to the development of

species‐specific therapy. Cell Shape Determining (CSD) proteins, of which 8 are currently

known, modify the peptidoglycan wall in ways that create curve and twist. Genetic evidence

suggests that Csd1 and Csd2 are zinc‐containing endopeptidases that work to create twist

(Sycuro et al. 2010. Cell 141, 822‐833). However, Csd2 is missing two conserved active site

residues. In addition, Csd1 is unstable in the absence of Csd2 (personal observation, T.

Wyckoff). Therefore, we hypothesize that Csd2 may be a scaffold protein that stabilizes and

directs Csd1 hydrolysis of the peptidoglycan wall. In this study, we confirmed the importance

of two putative active site residues in Csd1 through site‐directed mutation and shape

analysis. Work is underway to probe the importance of the analogous residues in Csd2.


56

Session 4 – PP 3

A 9‐BP GC HAIRPIN SEQUENCE IS SUFFICIENT FOR TRANSCRIPTIONAL

REPRESSION

Huilan Zhu, Ping He, Liping Gu and Ruanbao Zhou

Department of Biology and Microbiology, South Dakota State University, Brookings, SD57007

The impact of a specific sequence and its context on gene expression has been well documented

in prokaryotes. The formation of the intrinsic hairpin‐dependent terminator and/or

antiterminator plays important roles in transcriptional regulation. But little is known about the

minimum hairpin sequence required for transcriptional attenuation. Here, we report that a GC‐

rich segment inhibits GFP expression at transcription level using an elegant in vivo system to

study transcriptional termination. A GC‐rich segment insertion immediately upstream of the

start codon of GFP gene abolished GFP expression as readily visualized by fluorescence

microscope and confirmed by Western blot. Real‐time PCR showed that this high GC segment

inhibited GFP expression at transcriptional level. Mutations were introduced to the original 15‐

bp GC‐rich fragment by site directed mutagenesis to finally delimit a 9‐bp G/C segment as a

minimum hairpin sequence required for transcriptional attenuation in E. coli and yeast.


57

Session 4 – PP 4

ROLE OF DSRNA‐DEPENDENT PROTEIN KINASE R (PKR) AND EUKARYOTIC

TRANSLATION INITIATION FACTOR 2‐ALPHA (EIF2Α) IN PORCINE REPRODUCTIVE

AND RESPIRATORY SYNDROME VIRUS (PRRSV) REPLICATION

Hanmo Zhang, Alex M. Abel, Xiuqing Wang

Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007

Protein kinase R (PKR) is involved in apoptotic cell death and anti‐viral activities in

response to many virus infections. Several recent studies, however, suggest the pro‐viral

properties of PKR, which may or may not be dependent on the catalytic activity of PKR. To

reveal the role of PKR in the replication of porcine reproductive and respiratory syndrome

(PRRSV), we first examined the kinetics of PKR activation during PRRSV infection. Results

showed that PRRSV transiently activates PKR during 12 and 24 h post infection. Surprisingly,

eIF‐2α was only significantly phosphorylated compared to mock‐infected cells at late time

points of infection. A reduced viral protein synthesis and virus titer were detected in cells

transfected with PKR silencing RNA prior to PRRSV infection, indicating the role of PKR in

facilitating virus replication. This is further confirmed by the reduced virus titer in cells treated

with a PKR specific inhibitor, 2‐aminopurine (2‐AP). Overall, PKR plays a pro‐viral role in

PRRSV replication, which does not seem to be mediated through eIF‐2α phosphorylation.

Author Index

58

Author Name Session presentation Page

Abel, Alex M. Session 4 PP 4 57

Anower, M. Rokebul Session 1 PP 2 15

Antunes, Pedro Session 1 OP 3 13

Auger, Donald Session 1 PP 3 16

Bergholz, Teresa M. Session 2 PP 3, Session 3 PP 3 28, 41

Bleakley, Bruce Session 3 PP 6 44

Bonham, Julie Session 4 PP 2 55

Bratina, Bonnie Session 1 PP 1, Session 2 PP 1 14, 26

Braun, Lyle Session 2 OP 1, Session 2 PP 6 20, 31

Brözel, Volker Session 1 OP 1, Session 1 PP 4 11, 17

Brunick, Colin Session 2 PP 5 30

Bücking, Heike Session 1 OP 2, Session 1 OP 3, Session 1 PP 5 12, 13, 18

Chase, Christopher Session 2 OP 1, Session 2 PP 6 20, 31

Chelladurai, J. J. Session 2 OP 3 22

Chen, Kangming Session 4 OP 2 49

Cloos, Adam J. Session 1 PP 5 18

Colin, Emily Session 2 OP 6 25

Constans, Megan Session 2 OP 3 22

Croat, Jason R. Session 3 PP 4 42

Darweesh, Mahmoud Session 2 OP 1 20

Deng, Qiji Session 2 PP 10 35

Domfeh, Yayra E. Session 2 PP 3 28

Edquist, Mitchell Session 2 PP 1 26

Edwinson, Adam Session 4 OP 4 51

Eklund, Bridget Session 2 PP 2 27

Fang, Ying Session 2 PP 5 30

Fellbaum, Carl R. Session 1 PP 5 18

Fisher, Nathan Session 2 PP 2, Session 2 PP 3, Session 2 PP 8 27, 28, 33

Flynn, Christopher M. Session 3 OP 1 36

Francis, David Session 2 OP 4 23

Gibbons, William R. Session 1 PP 6, Session 3 PP 4, Session 3 PP 5, Session 3 PP 7

19, 42, 43, 45

Gu, Liping Session 3 OP 2, Session 3 PP 7, Session 4 OP 1, Session 4 OP 3, Session 4 PP 3

37, 45, 48, 50, 56

Halfmann, Charles Session 1 PP 6, Session 3 OP 2, Session 3 PP 7 19, 37, 45

Hardwidge, Philip R. Keynote 8

Hart, Miranda Session 1 OP 3 13

Hause, Ben Session 2 OP 4, Session 2 OP 6, Session 2 PP 10 23, 25, 35

He, Ping Session 4 OP 3, Session 4 PP 3 50, 56

Herman, Daniel Session 2 PP 4 29

Hernandez, Kristina Session 4 PP 2 55

Hildreth, Michael Session 3 PP 8 46

Hoch, James A. Keynote 9

Author Index

59


Hofer, Ellie Session 4 PP 2 55

Horne, Shelley M. Session 2 OP 2 21

Huber, Victor Session 2 PP 5 30

Irsfeld, Meredith Session 2 OP 2 21

Jiang, Zhiyong Session 2 PP 5 30

Johnson, Tylor Session 1 PP 6 19

Kabeiseman, Emily Session 4 PP 1 54

Kanchupati, Praveena Session 1 PP 3 16

Karki, Bishnu Session 3 PP 5 43

Kaushik, Radhey S. Session 2 OP 4, Session 2 PP 10 23, 35

Khatri, Mahesh Session 2 OP 4 23

Kiers, E. Toby Session 1 OP 3, Session 1 PP 5 13, 18

Koch, Alexander M. Session 1 OP 3 13

Kramer, Richard Session 3 OP 3 38

Langenhorst, Robert Session 2 PP 5 30

Lawson, Steven R. Session 2 PP 5 30

Leisen, Erin Session 2 PP 4 29

Li, Feng Session 2 OP 4, Session 2 OP 6, Session 2 PP 9, Session 2 PP 10

23, 25, 34, 35

Liu, Runxia Session 2 OP 6 25

Lucas, Andrea L. Session 4 PP 1 54

Madison, Joseph Session 4 OP 5, Session 4 OP 6 52, 53

Malsam, April Session 2 PP 7 32

Martin, Ben Session 2 PP 2, Session 2 PP 8 27, 33

McCormick, Kara Session 2 PP 5 30

McEvoy, John Session 4 OP 4 51

Mensah, Jerry A. Session 1 OP 3, Session 1 PP 5 13, 18

Moore, Elizabeth R. Session 4 PP 1 54

Mott, Ivan Session 1 PP 2 15

Murthy, Naveen Kumar Srinivasa

Session 3 PP 6 44

Muthukumarappan, Kasiviswanathan

Session 3 PP 5 43

NandaKafle, Gitanjali Session 1 PP 4 17

Neil, John Session 2 OP 1 20

Nelson, Andrew Session 1 PP 3 16

Nepal, Madhav Session 1 PP 4 17

Oines, Eric Session 1 PP 3 16

Park, Kaci Session 2 PP 6 31

Peel, Michael Session 1 PP 2 15

Peterson, Jessica L. Session 3 PP 2 40

Pfeffer, Philip E. Session 1 PP 5 18

Pruess, Birgit Session 2 OP 2 21

Author Index

60


Qiu, Yeyan Session 4 OP 1 48

Quin, Maureen B. Session 3 OP 1, Session 3 PP 9 36, 47

Rajput, Mrigendra Session 2 OP 1, Session 2 PP 6 20, 31

Ramamoorthy, Sheela Session 2 OP 3, Session 2 OP 5 22, 24

Ran, Zhiguang Session 2 OP 4, Session 2 PP 9, Session 2 PP 10 23, 34, 35

Ransburgh, Russell Session 2 PP 5 30

Reed, Martha Session 2 PP 7 32

Ridpath, Julia Session 2 OP 1 20

Ruden, Kay Session 3 PP 6 44

Sanders, Katherine Session 3 PP 3 41

Schmidt‐Dannert, Claudia Session 3 OP 1, Session 3 PP 9 36, 47

Schultz, Michael Session 4 OP 5, Session 4 OP 6 52, 53

Seelke, Ralph Session 4 OP 5, Session 4 OP 6 52, 53

Semmadali, M. Session 2 OP 3 22

Sherwood, Julie Session 2 PP 3, Session 3 PP 3 28, 41

Strahan, Gary E. Session 1 PP 5 18

Subramanian, Senthil Session 1 OP 1 11

Szmanda, Annie Session 2 PP 4 29

Thomas, Milton Session 2 OP 4, Session 2 PP 10 23, 35

Tran, Viet Session 1 PP 3 16

Tyagi, Deepti Session 3 PP 3 41

Vegi, Anuradha Session 2 OP 5 24

Venter, Stephanus N. Keynote, Session 1 PP 4 10, 17

Wang, Dan Session 2 PP 10 35

Wang, Xiuqing Session 2 PP 7, Session 4 PP 4 32, 57

Wang, Xiurong Session 1 OP 2 12

Wang, Zhao Session 2 PP 9, Session 2 PP 10 34, 35

Wawrzyn, Grayson T. Session 3 OP 1 36

Welker, Elliott Session 2 PP 2 27

Werner, Sarah Session 1 PP 1 14

West, Thomas P. Session 3 PP 1, Session 3 PP 2 39, 40

White, Laura Session 1 OP 1 11

Wilusz, Jeffrey Keynote 7

Wolk, Peter Session 3 PP 8 46

Wu, Yajun Session 1 PP 2, Session 1 PP 3 15, 16

Wyckoff, Timna Session 4 PP 2 55

Xiang, Xianling Session 3 PP 7 45

Xiang, Xiaoxiao Session 2 PP 9 34

Xu, Xinyi Session 4 OP 3 50

Young, Alan Session 2 OP 1 20

Zhang, Hanmo Session 2 PP 7, Session 4 PP 4 32, 57

Author Index

61


Zhou, Ruanbao Session 1 PP 6, Session 3 OP 2, Session 3 PP 7, Session 3 PP 8, Session 4 OP 1, Session 4 OP 2, Session 4 OP 3, Session 4 PP 3

19, 37, 45, 46, 48, 49, 50, 56

Zhou, Weidong Session 2 PP 10 35

Zhu, Huilan Session 3 PP 8, Session 4 PP 3 46, 56

Zhu, Laihua Session 2 OP 4 23

Zhu, Longchao Session 2 PP 5 30

Addresses of Attendees

62

Name Address

Anover, Mohammed

South Dakota State University, College of Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 605‐651‐4714

Bleakley, Bruce South Dakota State University, College of Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 605‐688‐5498

Bonham, Julie University of Minnesota Morris, 600 East 4th Street, Morris, MN 56267 Email: [email protected], Phone: 651‐500‐1382

Bratina, Bonnie University of Wisconsin ‐ La Crosse, Microbiology, 3029 Cowley Hall, La Crosse, WI 54601Email: [email protected], Phone: 608‐785‐6994

Brözel, Volker South Dakota State University, College of Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected]

Bücking, Heike South Dakota State University, College of Agriculture and Biological Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Carlson, Diane North Dakota State University, Veterinary and Microbiological Sciences, 1523 Centennial Boulevard, Fargo, ND 58102 Email: [email protected], Phone: 734‐646‐0588

Chaussee, Michael University of South Dakota, Basic Biomedical Sciences, 414 East Clarke Street, Vermillion, SD 57069 Email: [email protected], Phone: 605‐677‐6681

Chen, Kangming South Dakota State University, College of Agriculture and Biological Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007Email: [email protected], Phone: 605‐688‐6968

Constans, Megan North Dakota State University, Veterinary and Microbiological Sciences, NDSU Department 7690, P. O. Box 6050, Fargo, ND 58108 Email: [email protected],Phone: 701‐231‐8504

Croat, Jason South Dakota State University, College of Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 507‐370‐3979

Damodaran, Suresh

South Dakota State University, Department of Plant Science, Box 2207A, Brookings, SD 57007 Email: [email protected], Phone: 605‐651‐2830

Domfeh, Yayra North Dakota State University, Agriculture, Food Systems and Natural Resources, Veterinary and Microbiological Sciences, NDSU VMS P. O. 6050, Department 7690, Fargo, ND 58108 Email: [email protected], Phone: 701‐231‐7977

Edquist, Mitchell University of Wisconsin‐La Crosse, 327 North 15th Street, La Crosse, WI 54601 Email: [email protected]

Edwinson, Adam North Dakota State University, Agriculture, Food Systems, and Natural Resources, Veterinary and Microbiological Science, 1523 Centennial Boulevard, Fargo, ND 58102 Email: [email protected], Phone: 612‐202‐5324

Effertz, Karl North Dakota State University, 1523 Centennial Boulevard, Fargo, ND 58102 Email: [email protected]

Eibensteiner, Janice

South Dakota State University, College of Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2104D, Brookings, SD 57007 Email: [email protected], Phone: 507‐360‐9592

Eklund, Bridget North Dakota State University, Veterinary and Microbiological Sciences, 1523 Centennial Boulevard, Fargo, ND 58102 Email: [email protected]; Phone: 7346460588


63

Name Address

Gibson, Susan South Dakota State University, College of Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 605‐688‐4805

Greenfield, Tony Southwest MN State University, Biology, 1501 State Street, Marshall, MN 56258 Email: [email protected], Phone: 507‐537‐7291

Halfmann, Charles South Dakota State University, College of Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Hartwidge, Philip Kansas State University, 1600 Denison Avenue, Manhattan, KS 66506 Email: [email protected], Phone: 785‐532‐2506

Herman, Daniel University of Wisconsin ‐ Eau Claire, Biology, 105 Garfield Avenue, Eau Claire, WI 54701Email: [email protected], Phone: 715‐836‐5386

Hernandez, Kristina

University of Minnesota‐Morris, Biology, 600 East 4th Street, Morris, MN 56267 Email: [email protected]

Hoch, Jim Department of Molecular and Experimental Medicine, The Scripps Research Institute MEM116, 10550 North Torrey Pines Road, La Jolla CA 92037 Email: [email protected], Phone: 858‐784‐7905

Hofer, Ellie University of Minnesota Morris, Division of Math and Science, 600 East 4th Street, Morris, MN 56267 Email: [email protected], Phone: 605‐362‐9346

Huber, Victor University of South Dakota, BBS, 414 East Clark Street, Vermillion, SD 57069 Email: [email protected], Phone: 605‐677‐5163

Irsfeld, Meredith North Dakota State University, Veterinary and Microbiological Sciences, 1523 Centennial Boulevard, Fargo, ND 58102 Email: [email protected], Phone: 701‐566‐4826

Johnson, Tylor South Dakota State University, Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 605‐688‐6141

Kafle, Arjun South Dakota State University, Agricultural and Biological and Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Kamrowski, Nina South Dakota State University, Agricultural and Biological and Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Kanchupati, Praveena

South Dakota State University, Agricultural and Biological and Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 409‐291‐1605

Karki, Bishnu South Dakota State University, Agricultural and Biological Sciences, Department of Biology and Microbiology, Box 2120, Brookings, SD 57007 Email: [email protected], Phone: 701‐566‐1433

Katwal, Pratik South Dakota State University, Agriculture and Biological Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 208‐240‐5393

Kramer, Richard St. Cloud State University, Biology, 720 4th Avenue South, St. Cloud, MN 56301 Email: [email protected], Phone: 218‐760‐8330

Leisen, Erin University of Wisconsin ‐ Eau Claire, 105 Garfield Avenue, Eau Claire, WI 54701 Email: [email protected], Phone: 507‐876‐2492

Li, Feng South Dakota State University, Agriculture and Biological Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]


64

Name Address

Lin, Tao South Dakota State University, Art and Sciences, Chemistry and Biochemistry, SAV 306, Brookings, SD 57007 Email: lintao‐[email protected], Phone: 605‐592‐0586

Liu, Runxia South Dakota State University, Agriculture and Biological Sciences, Department of Biology and Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 605‐592‐0716

Lucas, Andrea University of South Dakota, Basic Biomedical Sciences, 414 East Clark Street, Vermillion, SD 57069 Email: [email protected]

Madison, Joseph University of Wisconsin‐Superior, Natural Sciences, 2000 Catlin Avenue, Superior, WI 54880 Email: [email protected], Phone: 715‐394‐8322

Mahdi, Osama North Dakota State University, Veterinary and Microbiological Sciences, 1523 Centennial Boulevard, Fargo, ND 58102 Email: [email protected], Phone: 734‐646‐0588

Martin, Ben North Dakota State University, Veterinary and Microbiological Sciences, 1523 Centennial Boulevard, Fargo, ND 58102 Email: [email protected], Phone: 734‐646‐0588

McCormick, Kara University of South Dakota, Basic Biomedical Sciences, 414 East Clark Street, Vermillion, SD 57069 Email: [email protected], Phone: 605‐421‐1161

Mensah, Jerry South Dakota State University, Agriculture and Biological Sciences, Department of Biology and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 6056956149

Mikel, Cassandra University of Wisconsin ‐ La Crosse, 1725 State Street, La Crosse, WI 54601 Email: [email protected]

Moore, Elizabeth University of South Dakota, SSOM, BBS, 414 East Clark Street, Vermillion, SD 57069Email: [email protected], Phone: 605‐677‐5164

NandaKafle, Gitanjali

South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 512‐809‐0284

Nelsen, April South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Okino, Cintia Embrapa Swine and Poultry, 4100 West Shirley Place, Sioux Falls, SD 57106 Email: [email protected]

Ouellette, Scot University of South Dakota, Basic Biomedical Sciences, 414 East Clark Street, Vermillion, SD 57069 Email: [email protected]

Park, Kaci South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected]

Peterson, Jessica South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 605‐688‐5470

Priyankar, samanta North Dakota State University, Veterinary and Microbiological Sciences, 1523 Centennial Boulevard, Fargo, ND 58102 Email: [email protected], Phone: 701‐306‐2870

Qiu, Yeyan South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 605‐592‐0606


65

Name Address

Quast, Megan South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 605‐593‐3070

Quin, Maureen University of Minnesota, BMBB/BTI, 1479 Gortner Avenue, Saint Paul, MN 55108 Email: [email protected]

Reed, Martha South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Ruden, Kay South Dakota State University, Department of Plant Science, Box 2207A, Brookings, SD 57007 Email: [email protected]

Schmidt‐Dannert, Claudia

University of Minnesota, Department of Biochemistry, 1479 Gortner Avenue, St. Paul, MN 55108 Email: [email protected]

Schultz, Michael University of Wisconsin‐Superior, Department of Natural Sciences, 2000 Catlin Avenue, Superior, WI 54880 Email: [email protected], Phone: 715‐394‐8322

Seelke, Ralph University of Wisconsin‐Superior, Department of Natural Sciences, 2000 Catlin Avenue, Superior, WI 54880 Email: [email protected], 715‐394‐8320

Sreenivasan, Chithra

South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected]

Srinivasa Murthy, Naveen Kumar

South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 617‐800‐5981

Subramanian, Sen South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected]

Szmanda, Annie University of Wisconsin ‐ Eau Claire, 422C Chancellors Hall, 820 University Drive, Eau Claire, WI 54701 Email: [email protected], Phone: 715‐551‐8866

Thomas, Milton South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Thorstad, Mason South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 701‐388‐0578

Tyagi, Deepti North Dakota State University, Agriculture, Food Systems and Natural Resources, VMS, P. O. Box 6050, Department 7690, Fargo, ND 58108 Email: [email protected], Phone: 701‐231‐7977

Vegi, Anuradha North Dakota State University, Veterinary and Microbiological Sciences, NDSU Department 7690, P. O. Box 6050, Fargo, ND 58108 Email: [email protected], Phone: 402‐310‐9611

Venter, Fanus University of Pretoria, Department of Microbiology and Plant Pathology, Private Bag X20, Hatfield, 0028, South Africa

Email: [email protected] Wang, Xiurong South Dakota State University, Agriculture and Biological Sciences, Department of Biology

and Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]


66

Name Address

Wang, Xiuqing South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 605‐688‐5502

Wang, Zhao South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Werner, Sarah University of Wisconsin ‐ La Crosse, UW ‐ La Crosse, Microbiology, 3335 East Avenue South, # 220; La Crosse, WI 54601 Email: [email protected], Phone: (831) 869‐8747

West, Thomas South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected], Phone: 605‐688‐5469

White, Laura South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2104A, Brookings, SD 57007 Email: [email protected]

Wilusz, Jeff Colorado State University, Department of Microbiology, Immunology and Pathology, Room B321, 1682 Campus Delivery, Fort Collins, CO 80523‐1682 Email: [email protected], Phone: 970 491 0652

Wyckoff, Timna University of Minnesota‐Morris, Science and Math, 600 East 4th Street, Morris, MN 56267Email: [email protected], Phone: 3205896352

Xu, Xinyi South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Zhang, Hanmo South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected], Phone: 605‐592‐6642

Zhou, Ruanbao South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

Zhu, Huilan South Dakota State University, Agriculture and Biological Sciences, Department of Biologyand Microbiology, Box 2140D, Brookings, SD 57007 Email: [email protected]

abstract band final ncb asm meeting - south dakota …€¦ · providing us with the opportunity to...

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