The 55th annual asm az/nv branch meeting

Interdisciplinary Science and Technology Building 4 (ISTB 4), Arizona State University

Saturday April 16th, 2016

Abstracts

Oral Presentations

O1

Effects of Acute Alcohol Toxicity on Burkholderia thailandensis E264 – Macrophage Interaction

Victor Jimenez1, Ryan Moreno1, Emily Kaufman*, Heidie O’Neill*, Erik Settles*, Paul Keim*, Fernando P. Monroy1

1Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86011 *Microbial Genetics and Genomics (MGGen), Northern Arizona University, Flagstaff, AZ, 86011

Alcohol consumption has diverse and well-documented effects on the human immune system and the ability to defend against infective agents. One example is melioidosis, a disease caused by Burkholderia pseudomallei that is of public health importance in Southeast Asia and Northern Australia. While B. pseudomallei infections can occur in healthy humans, the incidence is increasingly associated with excess alcohol consumption. Although alcohol consumption has been considered as a risk factor for the development of melioidosis, no experimental studies have investigated the outcomes of ethanol on Burkholderia spp. infection. Therefore, we propose the use of non-pathogenic B. thailandensis E264 as a useful BSL-1 model system to study the effects of alcohol on bacteria and alveolar macrophage interactions in vitro. Alveolar macrophages incubated in ethanol (0.08%) for 3 h prior to infection showed significantly lower bacterial uptake at 2 and 8 h. Activated (IFN-γ), AMs pre-incubated in ethanol and exposed to B. thailandensis released lower nitric oxide (NO) concentrations compared to non-alcoholic controls and alcoholic recovery treatment. As a result B. thailandensis survival and replication increased ~2.5 fold compared to controls. The presence of ethanol (1%) also increased bacterial survival within AM cells. Ethanol significantly decreased bacterial motility compared to non-alcoholic controls. Increased biofilm formation was observed with CFU’s and qPCR at 3 and 6 h when bacteria were pre-incubated in (0.08%) ethanol. The proinflammatory cytokine TNF-α was significantly decreased 24 h post alcohol exposure in vitro. These results provide insights into alcohol consumption, a culturally prevalent risk factor, as a predisposing factor for melioidosis.

O2

Identification and Characterization of a Novel Function of a Transcriptional Regulator Essential for Virulence in Shigella flexneri

Michael A. Picker, Juan C. Duhart, Joy A. Immak, and Helen J. Wing

School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154-4004 Shigella flexneri, a bacterial pathogen that causes bacillary dysentery in humans, harbors a large virulence plasmid encoding many virulence genes that are transcriptionally repressed by H-NS. VirB is a transcriptional regulator essential to Shigella virulence and functions to counteract H-NS-mediated repression. Details of this regulatory interplay have not been fully elucidated. Previous studies have focused on comparisons between VirB and its closest homolog, the plasmid partitioning protein ParB. Similar to ParB, we have recently shown that VirB can facilitate changes in DNA supercoiling. My overall goal is to identify if these changes in supercoiling are involved in VirB-dependent regulation. As a first step toward this goal, I aim to characterize the requirements of VirB-dependent changes in supercoiling. I show that VirB-dependent changes in supercoiling require an inverted repeat that is also essential for VirB-dependent regulation. Next I show that VirB-dependent transcription is not driving changes in DNA supercoiling. Together these data suggest that the VirB:DNA interaction is crucial for the observed changes in supercoiling. Importantly, the VirB protein is not sufficient to cause a change in DNA supercoiling in vitro, suggesting the involvement of another factor. Overall my work here provides important information that aid in defining the role that VirB-dependent changes in supercoiling play in overcoming H-NS-mediated transcriptional repression, a process essential to Shigella virulence.

O3

Low-Toxin Producing Clostridium difficile Isolates Elicit Severe Disease

Farhan Anwar1, Michael Mallozzi1, Neil Ampel3, VK Viswanathan1,2, and Gayatri Vedantam1,2,3

1 School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson AZ 85721

2 BIO5 Institute, University of Arizona, Tucson AZ 85721

3 Southern Arizona VA Healthcare System, Tucson AZ

Clostridium difficile (CD) is a leading cause of healthcare-associated infections. The toxins TcdA and TcdB are the primary virulence determinants, but there is increasing appreciation for non-toxin factors impacting CD virulence. During routine surveillance of Tucson-area hospitals, we discovered the presence of low-toxin CD strains that were PCR-positive for the toxin genes but enzyme-immunoassay (EIA)-negative for toxin. Despite the low-toxin phenotype, these strains were potently cytotoxic. They also belonged to multiple molecular types (ribotypes), including

the outbreak-associated type RT027. Remarkably, all low-toxin RT027 isolates we tested were lethal in the Golden Syrian hamster model of CD infection, with disease kinetics comparable to those of a reference (and high-toxin producing) RT027 strain. These results suggested that non-toxin virulence factors might play a significant role in CD pathogenesis. To begin elucidating the nature of these factors, we performed fully-quantitative, comparative proteomic analyses of multiple low-toxin CD isolates, which revealed (among others) that oxidative stress-tolerance proteins were significantly dysregulated in these strains. Collectively, our data underscore the limitations of the EIA for diagnostic purposes, highlight the significance of low-toxin CD isolates in the clinical context, and draw attention to the contribution(s) of non-toxin virulence factors in CD pathogenesis.

O4

Ascending Vaginal Microbiota Impacts Human Endometrial Epithelial Barrier Function and Integrity.

Paweł Łaniewski, Geoffrey Hire, Adriana Gomez, Melissa Herbst-Kralovetz

Department of Basic Medical Sciences, University of Arizona, College of Medicine-Phoenix, Phoenix, AZ, 85004

Bacterial vaginosis (BV) is the most common vaginal infection in the Unites States and is characterized by a shift in the vaginal microbial milieu from a Lactobacillus predominant flora to a diverse collection of anaerobes (e.g. Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae). This dysbiotic microbiome associated with BV may cause damaging local inflammation and disrupt the epithelial barrier function and integrity. Here we test the hypothesis that BV-associated bacteria that ascend into the upper reproductive tract can cause the endometrial epithelial barrier breach. To analyze the epithelial barrier breach, we utilized semi-permeable cell culture inserts and 3-D bioreactor-derived endometrial models. Differentiated human endometrial cells (HEC-1a) were infected with G. vaginalis (BV-associated) or Lactobacillus crispatus (associated with vaginal health) strains. Barrier integrity was determined using transepithelial electric resistance (TEER) and paracellular flux (PF) of FITC-labeled dextrans. Cell viability was determined by Trypan blue exclusion. Scanning electron microscopy (SEM) was utilized to visualize host-microbe interactions on the surface of 3-D aggregates. Following G. vaginalis infection, decreased TEER and increased PF measurements were observed as a result of the endometrial cell barrier disruption. These inversely proportional changes were not observed after colonization with commensal L. crispatus. G. vaginalis infection had cytotoxic effect on endometrial cells in a dose-dependent fashion. In conclusion, G. vaginalis infection caused endometrial epithelial barrier breach, which coupled with local inflammation, impact overall reproductive health at this site.

O5

The Impact of Autophagy on Neisseria gonorrhoeae Infection

Won Jong Kim 1,2, Nathan Weyand3, Magdalene So1,2

1BIO5 Institute and 2Department of Immunobiology, University of Arizona, Tucson, AZ, USA 3Department of Biological Sciences, Ohio University, Athens, OH, USA

N. gonorrhoeae (Ngo) attaches to epithelial cells for prolonged periods without causing damage. Ngo invades cells early, yet few viable intracellular bacteria are recovered until 4-5 hours post-infection. We observed lysosome inhibitors increase the viable intracellular CFU by 6-10-fold as early as 1 and 2 hours post-infection. As autophagy is a lysosome-dependent catabolic process that is crucial for defense against intracellular pathogens as well as for maintaining cellular homeostasis, we determined whether autophagy affects intracellular survival of Ngo. Using RNAi, chemical inhibitors, immunoblots, and immunofluorescence microscopy, we demonstrate that Ngo induces autophagy through the Type 1 membrane protein CD46-cyt1 and its interacting partner, GOPC. This autophagic response kills early invaders.

We propose a model to reconcile our autophagy findings with the ability of Ngo to survive inside cells at later stages of infection. Atkinson has shown that Ngo induces shedding of CD46; we have shown that Ngo remodels lysosomes via its secreted IgA protease (IgAP). Swanson and Song have shown that Ngo activates Epidermal Growth Factor Receptor (EGFR), a process that increases the number of viable intracellular CFU. We propose that (1) Ngo-induced shedding of CD46 reduces the intracellular pool of CD46, diminishing the ability of the infected cells to initiate autophagy; (2) IgAP remodeling of lysosomes inhibits the fusion of lysosomes to autophagosome and/or prevents degradation of the contents of the autophagolysosome; and (3) Ngo-induced phosphorylation of EGFR decelerates autophagy by sequestering a key autophagic component, Beclin-1.

O6

Heterotrophic Bacteria from Three Contrasting Peatlands of the Amazon Basin

Vanessa Hendrix, Kenneth Barker, Karen Barker, Fatema Kermani, Jessica Spring, Mahoro Uwingiyhana, Reena Ygot, Analissa Sarno, Hinsby Cadillo-Quiroz

School of Life Sciences, Arizona State University, Arizona, 85287

The Amazon Basin is recognized to be a thriving biological hotspot supporting a vast array of life of both plant and animal species. While much is understood regarding the diversity and profusion of macro species living in this tropical climate, little is known about the microorganisms inhabiting this ecosystem. The role of heterotrophic soil microorganism has been found to have an extensive impact on important ecological cycles, including decomposition and the carbon cycle. For this reason, it is essential to understand the identity and the potential

functional role of these microorganisms have in tropical peatland soils. We amassed a collection of heterotrophic soil bacterial isolates from three locations within an Amazon peatland. After performing dilutions on the soil samples, dilutions were plated in duplicate onto two types of media (CAT and R2G). Colonies were counted and isolated onto individual plates for purification through restreaking. The purified isolates were prepared for storage at -80 degrees Celsius. A unique method for expeditious DNA extraction was developed to allow for rapid preparation of the over 600 frozen isolates for 16S sequencing. Initial sequencing results of 100 fast growing isolates indicate the dominant presence of Pseudomonas dinitricans (30%), Cupriavidus sp (22%, Bulkholderiaceae), Novosphingobium (15%, Sphingomonadaceae), and new taxa within the Pseudoclavibacter sp (2%, Microbacteriacea), and novel Chitinophagaceae strains. Further identifications are underway. Our early results indicate that the soils of the Amazon seem to support an important variety of heterotrophic bacteria whose role in organic carbon decomposition can be experimentally evaluated with recovered isolates. Future evaluations of isolated diversity will reveal the ecophysiological role of known and novel heterotrophs in Amazon peatland soils.

O7 Marine Synechococcus Aggregation

Wei Deng, Bianca Cruz, and Susanne Neuer

School of Life Sciences, Arizona State University, Tempe, AZ

Cyanobacteria are considered to play an important role in the oceanic biological carbon pump, especially in oligotrophic regions. But as single cells are too small to sink, their carbon export has to be mediated by aggregate formation and/or consumption by zooplankton that produce sinking fecal pellets. Here, we report results on the aggregation of the ubiquitous marine pico-cyanobacterium Synechococcus as a model organism. We first investigated the mechanism behind such aggregation by studying the role of transparent exopolymer particles (TEP), and the effects of nutrient (nitrogen or phosphorus) limitation on the TEP production and aggregate formation of these pico-cyanobacteria. We further studied the aggregation and subsequent settling in roller tanks and investigated the effects of the clay kaolinite in increasing concentrations. Our results show that despite the lowered growth rates, Synechococcus in nutrient limited cultures had larger cell-normalized TEP production, formed a greater volume of aggregates, and resulted in higher settling velocities compared to results from replete cultures. In addition, we found that despite their small size and lack of natural ballasting minerals, Synechococcus cells could still form aggregates and sink at measureable velocities in seawater. Clay minerals increased the number and reduced the size of aggregates, and their ballasting effects increased the sinking velocity and carbon export potential of aggregates. These results contribute to our understanding of the role of marine Synechococcus in the ecology and biogeochemistry of oligotrophic oceans.

O8

Culturing Coccidioides: Optimizing In Vitro Culture Media to Reflect Nutrient Availability In Vivo

H.L. Mead1, 2, B.M. Barker1, 2

1Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 2Pathogen Genomics Division, Translation Genomics Research Institute-North, Flagstaff, AZ

INTRODUCTION: The pathogenic soil dwelling fungi, Coccidioides immitis and C. posadasii cause an estimated 150,000 cases of coccidioidomycosis, also known as valley fever, in the United States each year1. The current media for culturing spherules is not nutritionally representative of a mammalian respiratory system or conducive to in vitro immunology experiments. Supplemented RPMI media was used to produce the spherule form in a previous study1.

METHODS: Using the supplemented media and an attenuated biosafety level two (BSL2) Coccidioides, preliminary trials were conducted to determine which factors, such as cell density, temperature, time, CO2 and O2 concentration, would affect spherule development. Potential conditions were identified, reproduced in triplicate, and compared to determine the most successful method of growth.

RESULTS: The BSL2 strain cultured in the supplemented RPMI media successfully grew spherules at several conditions similar to the host environment. Spherule development between the two media types was relativity equivalent, and the highest percentage of parasitic morphology occurred at 15 % CO2.

DISCUSSION: The supplemented RPMI media is an excellent candidate for culturing Coccidioides spherules and can be applied to future research in the BSL3 laboratory. The components of the media provide the organism with nutrients that are similar to those found in a host respiratory system at conditions which are favorable for in vitro immunology experiments.

1 CDC. Final 2011 Reports of Nationally Notifiable Infectious Diseases. MMWR. 2012;61:624-37 1 Petkus AF, et al. Pure spherules of Coccidioides immitis in continuous culture. JCM. 1985;22:165-167.

O9

E3L Inhibits Vaccina Induced Necroptosis

Heather Harrington 1,2, Samantha Cotsmire1, Corwin Bernard1, Jeffery Langland1, Bertram Jacobs1,2

1 Arizona State University, Biodesign Institute, Tempe, AZ 85287, USA

2 Arizona State University, School of Life Sciences, MCB Graduate Program, Tempe, AZ 85287, USA

Vaccinia virus (VACV) is the current vaccine for smallpox. VACV encodes an innate immune evasion gene, E3L, encoding for the E3 protein that contains a ZNA binding domain (Z-NA BD) in the N terminus and a dsRNA binding domain in the C-terminus. E3 is responsible for inhibiting the host IFN pathway and this phenotype in cell culture has been mapped primarily to the dsRNA binding domain of E3 in the carboxy-terminus. The Z-NA BD of E3 has been shown to be dispensable for replication and IFNR in most human cells in culture; however, it is required for pathogenicity in mice mode. Despite little evidence for a role of the N-terminus in cells in culture, the N-terminus is highly conserved within poxviruses, which indicates it may contribute an essential function. Recently, we have demonstrated that the N-terminus is necessary to inhibit virally induced necroptosis. Inhibition of necroptosis in VACV infected L929 cells is specific to the regions of the N-terminal that correspond to Z-NA binding and is dependent on both RIP3 and DAI. This represents novel insight into the elusive function of the Z-NA binding domain of the N-terminus.

O10

Coronavirus Viroporin Envelope (E) Protein Transmembrane Domain Role in Virus Assembly

Bereket Estifanos, Jonathan Carrillo, Henry H. Hogue, Sasha M. Daskalova, Michael Goryll, Brenda G. Hogue

Arizona State University, Tempe, AZ USA

Coronaviruses (CoV) assemble at intracellular membranes of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). Virus budding into the ERGIC lumen is driven primarily by interactions between the membrane (M) and nucleocapsid (N) proteins. The E protein is expressed well in infected cells, but very little is assembled into virions. Deletion of the E gene results in variable results when deleted from CoVs, ranging from no virus production to very crippled viruses. The role of E in virus assembly is still not fully understood. E proteins are viroporins that exhibit a hydrophobic nature and a molecular mass of 10-12 kDa. Oligomers of E form ion channels in planar lipid bilayers that have cation selectivity. To address the importance of MHV E ion channel activity in virus assembly, we introduced specific point mutations (Q15A, Q15E, Q15N, Q15R, Q15T and V26F) in the mouse hepatitis coronavirus (MHV) E

transmembrane (TMD) domain. Mutations were selected based on a NMR model of SARS-CoV E TMD and residues predicted to play a role in ion selectivity and/or channel structure. Ion channel activity of each mutant E protein was measured. All of the proteins exhibited ion channel activity except for the protein harboring the Q15R substitution. The mutations were introduced by reverse genetics into the MHV genome to directly examine the impact of the substitutions on virus production. All recovered viruses exhibited lower virus yields and smaller plaque phenotypes. Viruses with only the introduced substitutions and no secondary changes were recovered in all cases, except for the Q15R mutant. After 5 passages a heterogeneous virus population had replaced the parental Q15R virus. Plaque isolates from passage 5 contained the Q15R substitution, but also harbored additional changes. Our results indicate that ion channel activity is not absolutely required for MHV production and provides further insight about its role in virus assembly.

O11

IL-36γ limits herpes simplex virus replication in human vaginal epithelial cells

Jameson Gardner, Sean Winkle and Melissa Herbst-Kralovetz

Department of Basic Medical Sciences, University of Arizona College of Medicine - Phoenix, Phoenix, Arizona, 85004

Herpes Simplex Virus (HSV) is one of the most common sexually transmitted infections (STI) affecting approximately 20% of all women. HSV infections of the female reproductive tract (FRT) are persistent, lifelong infections that increase risk of acquiring subsequent STI, including HIV. The IL-36 cytokines are members of the IL-1 superfamily, and have been associated with chronic inflammatory diseases at mucosal sites. Expression of these cytokines is regulated in part by the Th17 cytokines. We have recently shown that IL-36γ is expressed in human vaginal tissue and is upregulated in response to microbial products and HSV-2 infection. We hypothesize that IL-36γ is a key immunoregulatory cytokine in the FRT, that functions to limit HSV replication in the vaginal epithelium. We conducted a timecourse analysis to measure expression of IL-36γ in human vaginal epithelial cells (VEC) following HSV infection and found that IL-36γ expression peaks at 4 hours post infection. Pre-treatment of three dimensional organotypic VEC with recombinant IL-36γ 24 hours and 2 hours before HSV-2 challenge significantly increased IL-36γ expression and significantly decreased HSV-2 replication in a dose-dependent manner. Pre-treatment with rIL-36γ 2 hours before HSV-2 challenge significantly decreased HSV replication at the same magnitude as pretreatment for 24 hours. Co-delivery of IL-17 and IL-22 produced a synergistic effect that significantly induced IL-36γ expression and significantly reduced HSV replication in 3-D VEC. Our recent HSV findings suggest that IL-36γ could be an important regulator of the mucosal immune response to HSV-2 in the FRT.

O12

Optimizing Combination Oncolytic Viral and Immunotherapy Treatment Strategies with a Predictive Model

Ilyssa Summer1, Angela Peace2 and John Nagy3

1Simon A. Levin Mathematical, Computational and Modeling Sciences Center, Arizona State

University, Tempe, AZ, 85287 2Department of Mathematics & Statistics, Texas Tech University, Lubbock, TX, 79409

3 School of Mathematical & Statistical Sciences, Arizona State University, Tempe, AZ, 85287 Combination therapy has shown to improve success for cancer treatment. Oncolytic virotherapy is cancer treatment that uses engineered viruses to specifically infect and kill cancer cells, without harming healthy cells. Immunotherapy boosts the body's natural defenses towards cancer. This combination is explored through a deterministic system of nonlinear differential equations, constructed to match experimental data for murine melanoma, with the underlying goal to seek optimal treatment regimens; for both frequency and dose quantity. The model was first used to estimate parameters from preclinical experimental data. The values for the experiments optimal treatment were then used in various permutations for treatment regimens of combination oncolytic virotherapy and dendritic vaccinations. Observations from the produced data lead to in silico exploration of immune-viral interactions. Results suggest under optimal settings, combination treatment works better than monotherapy of either type. The most optimal result indicates administering treatment over a longer period of time, with fractioned doses, while reducing the total dendritic vaccination quantity, and maintaining the maximum virotherapy used in the experimental work.

O13

Microbial Community and Antibiotic Resistance in Biological Wastewater Treatment

Majid Neyestani, Daniel Gerrity

Department of Civil and Environmental Engineering and construction, University of Nevada, Las Vegas, Nevada, 89119

Wastewater treatment plants play an important role to protect human and environmental health from pollution in wastewater, however, they are also considered as significant reservoirs for antibacterial resistance. The presence of antibiotics in wastewater can form a selective pressure, which increases the concentration of antibiotic resistant bacteria by inhibiting antibiotic-susceptible bacteria and also increases the chance of mutation and horizontal gene transfer. In this research, the main goal was to provide a better understanding of the effect of different variables such as operational conditions and influent wastewater quality on the microbial community and also the occurrence, proliferation, and mitigation of antibiotic resistance of activated sludge systems. In this study, four laboratory-scale sequencing batch reactors (SBRs) with different solids retention times (SRTs) were developed to mimic biological

treatment process. Samples were collected from the SBRs for 16s rDNA sequencing and isolation of Staphylococcus/Streptococcus to identify bacterial species and detect antibiotic resistant bacteria in SBRs, respectively. Antibiotic resistant bacteria were determined in the presence of different antibiotics, including ampicillin, tetracycline, sulfamethoxazole / trimethoprim, and vancomycin. The results showed that multiple bacterial species like Enterococcus gallinarum and Salmonella enterica were not detected in primary effluent but were increasingly abundant at longer SRTs. Data obtained from this study revealed that SRT can change the microbial community and may select for antibiotic resistance in biological wastewater treatment systems.

O14

Small Multidrug Resistance Transporters Enhance Furfural Tolerance

Kurgan, G., Rodriguez, Y., Panyon, L., Pacheco, E., Nieves, L., and Wang, X.

School of Life Sciences, Arizona State University, Tempe, AZ, 85287

Efficient biological conversion of lignocellulose into valuable products is hindered by the presence of furfural, a dehydration product of pentose sugars produced during chemical pretreatments. To develop a cost-effective conversion, robust biocatalysts are needed that can tolerate toxic inhibitors while maintaining optimal metabolic conversion. A library of multidrug efflux transporters were screened for furfural tolerance in an ethanologenic Escherichia coli. Expression of small multidrug resistance (SMR) transporters enhanced furfural tolerance, suggesting a novel function of SMR transporters as a furfural efflux pump. Furthermore, chromosomal integration of SMR transporter genes such as mdtJI under a furfural inducible promoter created a furfural-responsive detoxification mechanism with an increased furfural tolerance. This work ascribes a new function to the SMR transporters and provides a platform for further enhancing efflux of furfural using directed evolution.

O15

Characterization of the importance of bacterial motility in counter-gradient isolations using discrete-element modeling and physical experimentation

Jaime Lopez1, Steffen Buessecker2, Hinsby Cadillo-Quiroz2 and Bruce Rittmann1

1Swette Center for Environmental Biotechnology, Tempe, AZ, 85281 2Arizona State University School of Life Sciences, Tempe, AZ, 85281

The counter-gradient isolation method is based on creating opposing substrate gradients that allow microorganisms of interest to self-segregate into visible bands. The common understanding of this method is that it is highly dependent on bacterial motility. However, this

dependence has not been rigorously evaluated. Here, the role of motility in the dynamics of this isolation method is examined via modeling and physical experiments. A reaction-diffusion (i.e. without motility) non-steady-state model of the isolation method was created. Counter-gradient isolations using pure Methylosinus trichosporium as inoculum were performed in conditions that limited motility. Data from these approaches were compared with data from a study characterizing M. trichosporium in a similar gradient system without motility inhibition.

The results suggest that counter-gradient isolation is better conceptualized as a reaction-diffusion-based isolation that is enhanced by motility, rather than as a primarily motility-based isolation. It was shown that with limited or no motility, the band of microorganisms still appears but is spatially extended, often to the point of being invisible to the human eye. The spatial extension is negatively correlated with initial cell population size and cell growth rate. Both the model and the experiments demonstrated that successful counter-gradient isolation is possible with severely limited motility under certain conditions. By comparing the data to the aforementioned study, it can be seen that motility drastically reduces the band spatial expansion. Therefore, motility allows counter-gradient isolation to generate visible bands from microorganisms that grow slowly or are not present in large numbers.

O16

pH Shifts in the Anode Potential Response from Thermincola ferriacetica Suggest the Presence of a Rate Limiting Proton-Coupled Electron Transfer Protein

Bradley G. Lusk1, Prathap Parameswaran2, Sudeep C. Popat1, Bruce E. Rittmann1, Cesar I. Torres1,3

1 Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, Arizona 85287−5701, United States of America

2 Department of Civil Engineering, Kansas State University, 2123 Fiedler Hall, Manhattan, Kansas 66502, United States of America 3 School for Engineering of Matter, Transport and

Energy, Arizona State University, 501 E Tyler Mall, Tempe, Arizona 85287, United States of America

Background: Thermincola ferriacetica, a thermophilic, Gram-positive, anode respiring bacterium (ARB) was grown in biofilms in microbial electrochemical cells (MXCs) to investigate its external electron-transport (EET) limitations. Electrochemical studies, including low scan cyclic voltammetry (LSCV), are often used to elucidate the rate limiting step of electron transport in ARB biofilms. Previously reported CV analysis of T. ferriacetica biofilms indicated a sigmoidal Nernst-Monod response in electrical current (j) to changes in anode potential (V). This response suggests that a single proton (H+) coupled electron (n = 1) transport reaction is responsible for the rate-limiting step in T. ferriacetica metabolism. The specific protein responsible for this response is thought to be a c-type cytochrome. Although T. ferriacetica has been shown to contain 35 c-type cytochromes, the one(s) responsible for EET has yet to be identified.

Methods: To determine the effect of pH on Eka, biofilms were grown at 50 mM bicarbonate buffer and 25 mM acetate as the electron donor. After achieving a steady j, pH was altered by the addition of HCl or NaOH. Then, LSCV was performed to determine the effect of pH on Eka. To assess the effect of bicarbonate buffer on Eka, biofilms were grown with 10, 25, 50, and 100 mM bicarbonate with 25 mM acetate as the electron donor. After achieving a steady j, LSCVs were performed at 1.0 mV s-1 and 10 mV s-1. This was repeated at 10, 25, 50, or 100 mM bicarbonate by either starting at 10 mM and increasing to 100 mM or by starting at 100 mM and decreasing to 10 mM. Results: T. ferriacetica’s response under certain growth conditions is composed of at least two separate n = 1 Nernst-Monod relationships; suggesting the presence of more than one pathway for anode respiration. Altering bulk pH reveals that biofilms in neutral to high pH (6.9-8.3) show a very broad redox peak while biofilms in low pH (5.2) reveal multiple redox peaks. Altering bicarbonate buffer concentration shows a similar trend, with lower bicarbonate leading to the presence of multiple redox peaks; consistent with pH gradients developing inside the T. ferriacetica biofilm. Conclusions: T. ferriacetica contains more than one H+ coupled EET pathway and EET pathways within T. ferriacetica are sensitive to changes in bulk pH.

Poster Presentations

P1

Electrochemical Techniques Reveal that Ammonia Stress Stimulates High Respiration Rates in Bacterial Anode Biofilm: What Does Not Kill Them Makes Them Stronger

Mohamed Mahmoud, Prathap Parameswaran, César I. Torres, and Bruce E. Rittmann

Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State

University, P.O. Box 875701, Tempe, AZ 85287–5701, USA

The unique ability of metal-reducing bacteria, including anode-respiring bacteria (ARB), to respire electrons from organic matter into solid electron acceptors is important in global biogeochemical cycles, and in microbial electrochemical cells (MXCs)) for generating renewable energy. ARB normally harvest only a small fraction of the free energy in the electron donor, since the anode potential often is only a few tenths of a volt higher than the donor potential. Thus, the main goal of MXCs is to achieve high organic matter oxidation rates with fast rate of electron transfer into the anode surface, and a high ratio of respiration rate to substrate-consumption rate. Here, we showed that relatively high total ammonium-nitrogen (TAN) concentration imposed a significant stress on the ARB biofilm. When induced with relatively high ammonium concentration (i.e., 2.2 g TAN-N/L), the anode biofilm boosted the electron fluxes toward current generation, a likely result of an energetic penalty, and consequently lowered biomass yield. As the biofilm anode grew with high TAN concentration, the ARB grew slower with a longer lag phase compared to anode biofilm grown with media having the normal ammonia concentration required to support the growth (i.e., 0.2 g TAN-N/L). The doubling times and electrons used for biomass synthesis (fs°) for the anode biofilm were 9.0 h and 0.09 e– eq biomass/e– eq acetate, and 28.6 h and 0.04 e– eq biomass/e– eq acetate in the presence of 0.2 and 2.2 g TAN–N/L, respectively. However, further increase in influent TAN concentration (i.e., to 3 and then to 4.4 g/L) completely inhibited ARB. Upon shifting the MXC’s feed into control TAN (i.e., 0.2 g TAN-N/L), the ARB had the capability to recover their initial activity, suggesting that high TAN concentrations cause a reversible deactivation of electron donor consumption; rather than damaging the biofilm. In summary, our results suggest that ARB biofilms have the ability to adapt to most of the environmental niches, mainly due to their physiological resilience that enables them to thrive by changing their energy gain per mol of substrate consumed under given environmental conditions.

Keywords: Renewable energy; Biofuels; Microbial electrochemical cells; Anode-respiring bacteria; Microbial stress response; total ammonium-nitrogen.

P2

Ecotin in the Extracellular Matrix Protects P. aeruginosa Biofilms from Proteolytic Attack

Boo Shan Tseng1, Courtney R. Reichhardt2, Gennifer Merrihew3, Michael J. MacCoss3 and Matthew R. Parsek2

1School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154

2Department of Microbiology, University of Washington School of Medicine, Seattle, WA, 98195 3Department of Genome Sciences, University of Washington, Seattle, WA, 98195

Bacterial biofilms cause chronic infections that are difficult to eradicate because of their higher tolerance against antimicrobials. This increased tolerance is in part due to the extracellular matrix, which encases the bacteria and holds the biofilm together. Although proteins have long been known to be an important part of the biofilm matrix, few matrix proteins have been identified and little is known about their functions. We hypothesize that the matrix provides more than just structural support by selectively retaining biochemically active proteins that aid in the protection of the biofilm. To identify proteins in the biofilm matrix of P. aeruginosa, we performed a screen for proteins enriched in the biofilm matrix relative to the total biofilm proteome. Here we describe one candidate matrix protein: ecotin (PA2755), a serine protease inhibitor. This protein is of interest because of its ability to inhibit neutrophil elastase, an enzyme produced by the host immune system. Ecotin was approximately 4-fold over-represented in the matrix proteome relative to the total biofilm proteome. Supporting the localization of ecotin in the matrix, we show that ecotin coprecipitated with the biofilm exopolysaccharide Psl. Our work shows not only that ecotin protected planktonic P. aeruginosa from neutrophil elastase in vitro, but also that ecotin inhibited neutrophil elastase while bound to a Psl matrix. These results suggest that ecotin in the matrix may protect biofilms from proteolytic attack.

P3

Testing the Pathogenicity of Close Phylogenetic Relatives of Burkholderia pseudomallei in a Laboratory Mouse Model

Lindsay Sidak-Loftis1, Victor Jimenez, Courtney Seavey, Erik Settles, Isaiah Self, Rachel Meyer1, Heather Centner1, Anthony Baker2, Vanessa Theobald, Mark Mayo2, Bart J. Currie2,

Paul Keim, Joseph D. Busch1, and David M. Wagner1

1Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86001

2Menzies School of Health Research, Casuarina, NT, Australia 3Tasmanian Institute of Agriculture, UTAS Sandy Bay Campus, Private Bag 54, Hobart

Tasmania, 7001

Burkholderia pseudomallei is a gram-negative bacterium that causes melioidosis, a severe disease prevalent in Southeast Asia and Northern Australia. Close phylogenetic relatives of B. pseudomallei, or “near neighbors”, also occur in these regions but data on their pathogenicity is limited. Near neighbors are important because they are often cultured during field surveys for B.

pseudomallei in endemic and non-endemic regions. In this study we tested the ability of seven near neighbor species to cause illness or mortality in laboratory mice (BALB/c). Past studies of B. thailandensis, the closest relative of B. pseudomallei, have suggested that this near neighbor is not highly pathogenic. Therefore, we hypothesized that other recently isolated near neighbors would not be highly pathogenic but might have the ability to cause illness in mice. We tested seven near neighbor species by challenging mice with a range of infectious doses (104, 105, or 106 bacterial cells per mouse). Only two species (B. thailandensis and B. vietnamiensis) caused noticeable illness. The most severe illness was caused by two environmental isolates of B. vietnamiensis, one from Florida and one from Australia, both of which led to visible sickness, a strong antibody response, and even death in one mouse. These findings suggest that B. vietnamiensis may be a highly immunogenic species, even though it is not considered a dangerous pathogen. The results of this experiment provide new information on the pathogenic potential of near neighbor species and can be used as a BSL2 infection model for comparison to B. pseudomallei.

P4

Sequence-Specific Detection of Different Strains of LCMV in a Single Sample Using Tentacle Probes

Lina S. Franco1, Susan A. Holechek2,3, Michael Caplan4, Joseph N. Blattman2

1School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287 2School of Life Sciences, Arizona State University, Tempe, AZ, 85287

3Simon A. Levin Mathematical, Computational and Modeling Sciences Center Arizona State University, Tempe, AZ 85287

4School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, 85271

Viral infections often result in quasi-species of virus strains that can have dramatic impacts on disease outcomes or experimental results. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity in order to design appropriate treatment, and can be useful for many experimental systems. We have developed a novel molecular method for simple and accurate detection of RNA virus genetic variants using Tentacle probes coupled with quantitative PCR. Tentacle probes are modified molecular beacons that are less expensive and dramatically improve false positive rates in routine qPCR. To validate this approach we have designed two tentacle probes for two strains of Lymphocytic Choriomeningitis Virus (LCMV): the parental Armstrong strain and the more virulent clone-13 strain, which differ by only 3 nucleotide substitutions. Using this approach we were able to distinguish these two strains of LCMV. Furthermore, in virus mixing experiments we were able to detect down to 5% of clone-13 diluted in Armstrong. Thus, we have developed a fast, cost-effective approach for differentiating between closely related viruses.

P5

Ecological Niche Modeling of Tick-Borne Relapsing Fever in the Western US

Kylie M Sage1, Mike B Teglas2, Nathan C Nieto1

1Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86001 2Department of Agriculture, Nutrition, and Veterinary Science, University of Nevada, Reno, NV,

89557

Tick-borne relapsing fever (TBRF) is a zoonotic, vector-borne disease transmitted by Ornithodoros hermsi in the western United States (US). The disease is caused by a spirochete bacterium, Borrelia hermsii, which is exclusively transmitted through the bite of an O. hermsi tick. The pathogen is maintained in small rodent reservoir hosts such as chipmunks, ground squirrels, and deer mice. In order for these ticks to be active and produce viable eggs, a narrow set of environmental parameters must be met, namely temperature and precipitation. Maximum Entropy Species Distribution Modeling (Maxent) was used in this study to predict the population distribution of Ornithodoros hermsi and B. hermsii through time and space based on current climatic trends and future projected climate changes. From this modeling process, we found that the projected current distribution of TBRF aligns with the known endemic foci for the disease. Further, future-cast models predict a decrease in overall suitable habitat for the tick vector, but the density of the tick populations at higher elevations will increase. These data could be used for targeting surveillance efforts in areas of high risk, increasing the efficiency and accuracy of a public health investigation or their control efforts.

P6

Towards a Model Organism for Phototrophic Biofilm Formation: Axenic Biofilms of Synechocystis PCC 6803 Require Cell Surface Structures and Occur Under Nutrient

Limitation

Rey Allen1, Willem Vermaas1, and Roy Curtiss III2

1Swette Center for Environmental Biotechnology, Arizona State University, Tempe, AZ, 85281 2Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, 85281

3Department of Infectious Disease and Pathology, University of Florida, Gainesville, FL, 32611

Phototrophic biofilms are key to nutrient cycling in natural and engineered environments. The literature on mixed-species phototrophic biofilms is extensive, but there are few studies describing biofilm formation by a single (axenic) species of phototrophic microbe. The cyanobacterium Synechocystis PCC 6803 is a model organism for the study of oxygenic photosynthesis, and a promising candidate for biofuel production. We hypothesize that phototrophic bacteria such as Synechocystis are able to form axenic biofilms, and use cell surface structures such as pili to attach and adhere to surfaces, similar to other biofilm-forming heterotrophic bacteria. Using microscopy and the crystal violet biofilm assay, we found that

axenic wild-type (WT) Synechocystis forms biofilms of cells and extra-cellular material when shifted to nutrient-depleted medium. WT Synechocystis does not form biofilms in nutrient-replete medium (BG11). Unlike WT Synechocystis, mutants lacking genes required for synthesis of cell surface structures such as type IV pili and the S-layer do not form biofilms under nutrient limitation. We conclude that pili and the S-layer are necessary but not sufficient for biofilm formation by WT Synechocystis: additional factor(s) must be induced, such as through nutrient limitation, in order for pili and S-layer to facilitate biofilm formation. We will use mass spectrometry and RNA sequence of induced and uninduced WT cultures to further develop this model. These studies have applications towards customizing adhesive properties of Synechocystis biofuel feedstock strains, including biomass harvesting by induced aggregation, and engineering non-biofouling strains by deletion of genes for pili or S-layer.

P7

Activity of Synbody 14 Against Methicillin-Resistance Staphylococcus aureus Cell Membrane

Carlyn Harris

Barrett, The Honors College, Arizona State University

Background: Internationally, antibiotic resistance has emerged as a major public health issue and resistant strains of bacteria are responsible for hundreds of thousands of deaths per year. Synthetic antibodies (“Synbodies”) offer promise in the fight of superbugs such as Methicillin-resistant Staphylococcus aureus by attacking the bacterial cell membrane—a largely under-researched drug target. ASU’s Synbody-14 is made up of two peptide arms (similar to arms on an antibody)—“147 Peptide” and “Lytic Peptide”. Lytic peptide exhibits membrane lysis activity while peptide 147 exhibits membrane-binding activity. Objectives: To further characterize the mechanism of action of Synbody-14 against MRSA’s gram-positive bacterial cell membrane. Previous experiments have shown that both Synbody-14 and Ly Peptide inhibit growth of MRSA while peptide 147 on its own does not. In this experiment, we investigate the effect of Synbody-14 and its peptides on MRSA’s cell membrane potential. Methods: Bacterial cells are loaded with a cationic dye that accumulates in the cytoplasmic membrane. Cells are treated with Synbody-14, Peptide 147 and Lytic peptide at different concentrations in a 96-well plate and monitored for fluorescence over a 20 minute period in a MicroTek Plate reader. If fluorescence in the medium increases, the membrane potential is disrupted. Results: Syn-14 and Peptide 147 each cause the medium to exhibit a rapid increase in fluorescence to near 600 RFU’s each while the medium treated with only Lytic peptide exhibited about 300 RFUs. Conclusion: Synbody-14 alone as well as the two peptides that compose it cause membrane depolarization of Methicillin-resistant Staphylococcus aureus. Though Ly peptide inhibits MRSA growth, it causes relatively low disruption in membrane potential. However, Peptide 147, which

does not inhibit bacterial cell growth alone, causes a large and rapid depolarization of the bacterial membrane potential.

P8

Characterization of the Protist Hindgut Community in Heterotermes aureus

Garcia MD*, Jasso-Selles D*, Merrell TL*, Peterson KD*, and Gile GH

School of Life Sciences, Arizona State University, Tempe, Arizona, 85281 * These authors contributed equally.

Heterotermes aureus (Rhinotermitidae) is the most common subterranean termite in the dry and hot Sonoran Desert and the most destructive urban termite pest in Arizona. As generalist feeders, they are often found in and burrowed around deadwood. Wood eating termites like H. aureus have gut dwelling symbiotic protists to help them break down their lignocellulose diet. This obligate symbiotic relationship is ancient, dating back to the common ancestor of termite and the wood roach, Cryptocercus. Each termite and Cryptocercus species (except for termites in the family Termitidae) has a characteristic assemblage of protist symbionts. We collected H. aureus from an empty lot in Gilbert, Arizona and characterized the hindgut protist community with morphological and molecular methods. The hindgut eukaryotic microbiome of H. aureus was previously reported to consist unnamed species of Holomastigotoides, Spirotrichonympha, and Pseudotrichonympha and our light microscopical observations agree with these classifications. We isolated single cells of each protist with micropipettes, amplified and sequenced the small subunit ribosomal RNA gene (SSU rDNA) and performed phylogenetic analyses. Each protist branched as expected: Pseudotrichonympha within the Trichonymphidae, and Holomastigotoides and Spirotrichonympha in the Spirotrichonymphidae. Based on their distinct host species and differences in their SSU rDNA sequences, we suspect the H. aureus protists should be considered new species.

P9

Coronavirus Viroporin Envelope (E) Protein Transmembrane Domain Role in Virus Assembly

Bereket Estifanos, Jonathan Carrillo, Henry H. Hogue, Sasha M. Daskalova, Michael Goryll, Brenda G. Hogue

Arizona State University, Tempe, AZ USA

Coronaviruses (CoV) assemble at intracellular membranes of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). Virus budding into the ERGIC lumen is driven primarily by interactions between the membrane (M) and nucleocapsid (N) proteins. The E protein is expressed well in infected cells, but very little is assembled into virions. Deletion of the E gene results in variable results when deleted from CoVs, ranging from no virus production to very

crippled viruses. The role of E in virus assembly is still not fully understood. E proteins are viroporins that exhibit a hydrophobic nature and a molecular mass of 10-12 kDa. Oligomers of E form ion channels in planar lipid bilayers that have cation selectivity. To address the importance of MHV E ion channel activity in virus assembly, we introduced specific point mutations (Q15A, Q15E, Q15N, Q15R, Q15T and V26F) in the mouse hepatitis coronavirus (MHV) E transmembrane (TMD) domain. Mutations were selected based on a NMR model of SARS-CoV E TMD and residues predicted to play a role in ion selectivity and/or channel structure. Ion channel activity of each mutant E protein was measured. All of the proteins exhibited ion channel activity except for the protein harboring the Q15R substitution. The mutations were introduced by reverse genetics into the MHV genome to directly examine the impact of the substitutions on virus production. All recovered viruses exhibited lower virus yields and smaller plaque phenotypes. Viruses with only the introduced substitutions and no secondary changes were recovered in all cases, except for the Q15R mutant. After 5 passages a heterogeneous virus population had replaced the parental Q15R virus. Plaque isolates from passage 5 contained the Q15R substitution, but also harbored additional changes. Our results indicate that ion channel activity is not absolutely required for MHV production and provides further insight about its role in virus assembly.

P10

Restoration in Arid Lands: Can we produce Biocrusts in Greenhouse Facilities?

S. Velasco Ayuso, A.M. Giraldo Silva, C.J. Nelson and F. Garcia-Pichel

1School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA

Biological soil crusts are topsoil communities comprise of cyanobacteria, algae, lichens, and mosses that grow intimately associated with soil particles in dry-lands around the world. Biocrusts have central ecological roles in desert areas as sources of carbon and nutrients, and efficiently retain water and prevent soil erosion, which improves soil structure and promotes soil fertility. However, human activities, such as cattle grazing, hiking or military training, are rapidly striking biocrusts, leading to a loss of the services that they provide. Using soil and inoculum from four different sites located in one cold desert (UT) and in one hot desert (NM), we performed a fractional factorial experiment involving six factors (water, light, nutrients, calcium carbonate, trace metals and type of inoculum) to screen their effects on the growth of biocrusts. After four months, we measured the concentration of chlorophyll a, and we discovered that water, light and nutrients were the most important factors controlling the growth of biocrusts in all sites. In the experimental units involving these three factors we measured a similar concentration of chlorophyll a (or even higher) to this found in the field locations. Amplification of the 16S rRNA gene revealed a microbial community composition in the biocrusts grown that corresponds to initial measurements made on each field location. Based on our clear success in obtaining biocrust biomass from natural communities in greenhouse facilities, we are paving the road to propose a protocol to produce a high quality-nursed inoculum aiming to assist restoration of arid and semiarid ecosystems.

P11

Recovering Arid Lands: Cyanobacteria Conditioning for Biological Soil Crust Restoration

Giraldo Silva, A.1, Nelson, C.1, Barger, N.2 and Garcia-Pichel, F.1

1School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA 2Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309,

USA

Biological soil crust (biocrusts) communities provide important ecosystems services to arid lands regarding soil fertility and stability. Filamentous cyanobacteria, such as Microcoleus vaginatus and Microcoleus steenstrupii, are considered biocrusts pioneers and assist soil surface stabilization. Soil stabilization promotes the successional establishment of other biocrusts organisms, including other cyanobacteria. Human activities, ranging from cattle grazing to military training, have resulted in the significant deterioration of biocrusts surface cover of soils. Aiming at developing an effective restoration strategy, we established a “biocrust-nursery” that serves as an inoculum supply for restoration. We isolated and grew large quantities of the main biocrusts cyanobacteria from a variety of soil types in cold and warm deserts of military lands in southwestern US. Traditional scale-up methods from the algae industry gave good outcomes when growing some of the target cyanobacteria (Nostoc sp., Tolypothrix sp. and Scytonema sp.). Yet, when growing Microcoleus spp., biomass yields were very low. We developed a method that gives excellent biomass outcomes. By implementing this new approach, we were able to obtain exponential and fast growth of the biocruts pioneers M. vaginatus and M. steenstrupii. Our inoculum formulation is based on pedigreed laboratory cultures that match the cyanobacterial relative abundance of the original sites, and additionally, have been conditioned to dry-wet cycles and increasing light exposure, with the goal of increasing field adaptation and survival rates. Ultimately, this nursery approach should help us to increase field recovery rates.

P12

Engineering A Co-Culture System for Enhanced Lignocellulose-Derived Sugar Mixture Utilization

Andrew Flores1, David Nielsen1 and Xuan Wang2

1School for Engineering of Matter, Transport, and Energy- Chemical Engineering Program, Arizona State University, Tempe, Arizona, 85287

2The School of Life Sciences, Arizona State University, Tempe, Arizona, 85287

Cellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical processes. The United States has vast amounts of land suitable to grow energy crops. Lignocellulosic-derived sugars derived from these crops can be converted by microorganisms into high-value products. Microbial consortia, or heterogeneous communities,

are a naturally occurring phenomenon found throughout nature. Heterogeneous communities often form symbiotic relationships to complete complex tasks, such as assisting in food digestion or metabolic cooperation. Inspired by natural systems, we are engineering synthetic microbial communities to overcome the limitations encountered in traditional metabolic engineering of a single microbial species. A major limitation that arises in engineering monocultures for lignocellulose conversion is the low-efficiency at sugar mixture utilization. We demonstrate that this limitation can be overcome by engineering a microbial co-culture system composed of different ‘specialists’ to metabolize only one specific sugar. Here, we report the design and optimization of an Escherichia coli co-culture that successfully co-utilizes 50 g/L of the sugar mixture glucose and xylose in a batch fermentation. Our overall research goal is to engineer and explore the behaviors and performances of co-cultures for efficient conversion of lignocellulose-derived sugars into high-value products.

P13

The Impact of Autophagy on Neisseria gonorrhoeae Infection

Won Jong Kim 1,2, Nathan Weyand3, Magdalene So1,2

1BIO5 Institute and 2Department of Immunobiology, University of Arizona, Tucson, AZ, USA 3Department of Biological Sciences, Ohio University, Athens, OH, USA

N. gonorrhoeae (Ngo) attaches to epithelial cells for prolonged periods without causing damage. Ngo invades cells early, yet few viable intracellular bacteria are recovered until 4-5 hours post-infection. We observed lysosome inhibitors increase the viable intracellular CFU by 6-10-fold as early as 1 and 2 hours post-infection. As autophagy is a lysosome-dependent catabolic process that is crucial for defense against intracellular pathogens as well as for maintaining cellular homeostasis, we determined whether autophagy affects intracellular survival of Ngo. Using RNAi, chemical inhibitors, immunoblots, and immunofluorescence microscopy, we demonstrate that Ngo induces autophagy through the Type 1 membrane protein CD46-cyt1 and its interacting partner, GOPC. This autophagic response kills early invaders.

We propose a model to reconcile our autophagy findings with the ability of Ngo to survive inside cells at later stages of infection. Atkinson has shown that Ngo induces shedding of CD46; we have shown that Ngo remodels lysosomes via its secreted IgA protease (IgAP). Swanson and Song have shown that Ngo activates Epidermal Growth Factor Receptor (EGFR), a process that increases the number of viable intracellular CFU. We propose that (1) Ngo-induced shedding of CD46 reduces the intracellular pool of CD46, diminishing the ability of the infected cells to initiate autophagy; (2) IgAP remodeling of lysosomes inhibits the fusion of lysosomes to autophagosome and/or prevents degradation of the contents of the autophagolysosome; and (3) Ngo-induced phosphorylation of EGFR decelerates autophagy by sequestering a key autophagic component, Beclin-1.

P14

HIV-1 Virus-like Particles as Stimulators of Innate Immunity

William Martelly1, 2, Lydia Meador1, 2, and Bertram Jacobs1, 2

1School of life Sciences, Arizona State University, Tempe, AZ, 85281 2Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Tempe, AZ, 85281

Development of a safe but effective vaccine against HIV-1 is an attractive strategy for the prevention of new infections needed for pandemic control. Numerous vaccine trials have indicated that viral vector and subunit based prime-boost vaccines have protective capacities against HIV-1 infection though low efficacy. HIV virus-like particles (VLPs) produced in N. benthamiana plants have consequentially natural adjuvancy due to their method of production involving a gram-negative bacteria. To assess this immunogenic potential, we seek to characterize the VLPs’ interaction with and activation of the innate immune system. We treated multiple human and mouse cell lines in vitro with VLPs to look for different pathway activation including: toll-like receptors (TLR), MAPK, SAP/JNK, IRF3, and NFkB. We have found these particles activate via TLR and p38 pathways, potentially leading to the stimulation of antiviral genes; a beneficial property if used as a booster to a vaccine.

P15

Co-occurrence and Timing of Fungal Endophyte Infection of Aphid Galls

Julia B. Hull1, Ron J. Deckert1, Thomas G. Whitham1,2, and Catherine A. Gehring1,2

1 Department of Biological Sciences, Northern Arizona University, Flagstaff AZ 86011 2Merriam-Powell Center for Ecological Research, Flagstaff AZ 86011

Fungal endophytes inhabit healthy plant tissues. Many endophytes have a negative effect on plant enemies, such as herbivores. An important herbivore of cottonwoods (Populus spp.) are gall-forming aphids (Pemphigus spp.). Galls are abnormal growths of plant tissue induced by the aphids, where the aphids asexually reproduce and are in intimate contact with fungal endophytes. Because aphids may seek to avoid areas of high endophyte infection, we tested the hypothesis that gall tissue will have lower rates of fungal endophyte infections. A follow-up study tested the hypothesis that endophyte infections increase as the gall tissue ages. In the first study, Pemphigus betae galls and ungalled leaves were collected from narrowleaf cottonwood trees in June 2013. The second study examined immature and mature galls from 10 Fremont cottonwood trees (P. fremontii). Plant tissues were surface sterilized and cultured for four weeks, during which time the plates were monitored for contamination. Two patterns emerged: 1) gall tissue had 3-fold greater fungal endophyte infection than surrounding leaf tissue and 2) gall tissue endophyte infection was only present in mature galls. These results show that while gall tissues housed a significantly greater number of fungal endophytes, the infections occur late in the lifecycle of the gall structure. However, neither of these studies examined the endophyte communities of unsuccessful galls. Future studies will test the

hypotheses that unsuccessful galls house different communities of endophytes than successful galls and that the timing of endophyte infection will influence gall success rates.

P16

Small Multidrug Resistance Transporters Enhance Furfural Tolerance

Kurgan, G., Rodriguez, Y., Panyon, L., Pacheco, E., Nieves, L. and Wang, X.

School of Life Sciences, Arizona State University, Tempe, AZ, 85287

Efficient biological conversion of lignocellulose into valuable products is hindered by the presence of furfural, a dehydration product of pentose sugars produced during chemical pretreatments. To develop a cost-effective conversion, robust biocatalysts are needed that can tolerate toxic inhibitors while maintaining optimal metabolic conversion. A library of multidrug efflux transporters were screened for furfural tolerance in an ethanologenic Escherichia coli. Expression of small multidrug resistance (SMR) transporters enhanced furfural tolerance, suggesting a novel function of SMR transporters as a furfural efflux pump. Furthermore, chromosomal integration of SMR transporter genes such as mdtJI under a furfural inducible promoter created a furfural-responsive detoxification mechanism with an increased furfural tolerance. This work ascribes a new function to the SMR transporters and provides a platform for further enhancing efflux of furfural using directed evolution.

P17

Development of the K-state Promotes Mutagenesis, Independent of DNA-uptake, in Stressed Bacillus subtilis cells.

Amanda A. Kidman1, Holly A. Martin1, Carmen Vallin1, John Creech1 and Eduardo A. Robleto1

1School of Life Science, University of Nevada, Las Vegas, NV, 89145

Mutagenesis is central to the evolutionary process. We currently view evolution as a gradual process affecting all cells within a population. However, I aim to study an underappreciated part of the evolutionary process, mutations generated during stationary phase (caused by nutritional stress or growth arrest) within a subpopulation. Stationary phase cultures of Bacillus subtilis) develop subpopulations that exhibit different survival strategies including competence, secondary metabolite production, biofilm formation, cannibalism and endospore. The development of competence permits cells to uptake exogenous DNA and incorporate it into their genome. During competence, new alleles can be acquired and recombine into the hosts genome leading to genetic diversity. Published results from my research group have shown that i) defects in genetic factors that control competence (ComK and ComA) result in decreases in mutagenesis in non-growing cells; and ii) the observed decrease is independent of recombination. We speculate that some other mechanism, activated during the K-state,

regulated by the transcriptional activator ComK, in which more than just competence genes are activated, is responsible for most of the mutations seen during stationary phase. My project seeks to bring together these separate observations into a coherent understanding of how competence or the K-state leads to increases in mutagenesis. Here we test the hypothesis that the population of cells that develops competence experiences increased levels of mutagenesis during stationary phase.

P18

How Neisseria gonorrhoeae Avoids Killing by the Complement Cascade Morgan Brown1, Nathan Weyand PhD2 and Magdalene So PhD3

1Microbiology, University of Arizona, Tucson, Arizona, 85715 2Biological Sciences, Ohio University, Athens, Ohio, 45701

3Immunobiology, University of Arizona, Tucson, Arizona, 85715

The complement system (C’) is an integral component of the innate immune response. It is composed of a large number of plasma proteins that work as a cascade to produce a series of inflammatory responses against a pathogen. C’ is regulated by several related proteins such as CD46, a Type I transmembrane glycoprotein that binds to C3b/C4b C’ components and inhibits activation, preventing damage to the host cell by unchecked C’. CD46 also interacts with Neisseria gonorrhoeae (Ngo) at various levels. During infection of epithelial cells, Ngo recruits host CD46 proteins to the bacterial attachment site, causes CD46 phosphorylation, and promotes CD46 shedding by host cells. We hypothesize that CD46 clustering at the site of infection protects Ngo from being killed by C’. To test this hypothesis we followed two approaches: serum bactericidal assays to determine Ngo survival after treatment with Normal Human Serum, and CD46 downregulation in an MCF7 cell line using specific shRNAs. We found that these data support the hypothesis that CD46 clustering protects Ngo from C’ killing, but future experiments are necessary to examine the roles of both CD46 and the related complement protein CD55 in Ngo survival of C’.

P19

Attenuation of the Type IV pilus Retraction Motor Affects the Social and Infection Behavior of Neisseria gonorrhoeae

Alyson M. Hockenberry, Danielle M. Hutchens, Al Agellon and Magdalene So

Department of Immunobiology and the BIO5 Institute, University of Arizona, Tucson, Arizona, USA

Retraction of the Type IV pilus (Tfp) mediates DNA uptake, motility, and social behavior in a wide variety of prokaryotes. The cytosolic ATPase PilT powers retraction of the Neisseria gonorrhoeae (Ngo) Tfp; mutants deleted of pilT are nontransformable, nonmotile, and unable to

organize into microcolonies. Tfp retraction facilitates invasion of epithelial cells and alters transcriptional and signaling programs in these cells. Studies imply the speed and force of single pilus retraction events are quite heterogeneous and sensitive to environmental stimuli such as oxygen. To date, studies on the role of Tfp retraction in Ngo biology have used non-retractile pilT deletion strains; it is unclear whether altering the rate of ATP hydrolysis of PilT may influence Tfp retraction-dependent phenotypes.

We constructed a mutant in Ngo (pilTL201C) that expresses a PilT motor that displays half-maximal ATPase activity. pilTL201C is motile and competent, implying Tfp retraction occurs. However, it aggregates into abnormally shaped microcolonies in vitro and is defective in epithelial cell invasion. Ngo invasion is known to involve Epidermal Growth Factor Receptor (EGFR) activation and our data show the DpilT and pilTL201C strains are unable to activate EGFR by multiple mechanisms. Addition of exogenous EGFR ligand to DpilT- and pilTL201C-infected cultures partially restored the invasion defect of these mutants. Thus, the invasion defect of these mutants lies in their inability to activate EGFR.

Our findings show PilT enzymatic activity strongly influences Ngo social behavior and epithelial cell infection, but not DNA uptake or crawl speed. They imply that Tfp retraction dynamics, and environmental factors that influence retraction events, are determinants of these two Ngo behaviors. The pilTL201C mutant will be useful for establishing the link between Tfp retraction force/speed and Ngo social interactions.

P20

Investigation of Lactate Export in Escherichia coli

Schneider, A.; Kurgan, G.*; Kurgan, L.; Rodriguez, Y.; Xiao, J. and Wang, X.

School of Life Sciences, Arizona State University, Tempe, AZ, 85287 Microbial fermentation is a cost-effective method used worldwide for enhancing food nutritional value and creating valuable products. In fermentative metabolism, glycolysis is generally the main source of ATP generation and many fermentative organisms convert pyruvate into lactate in order to maintain redox balance. Although metabolic mechanisms behind this phenomena are well described, the determinants of export of this key metabolic end product from bacterial cells remains uncharacterized. Here we use a lactate producing Escherichia coli to investigate genes involved in lactate export. Using reverse genetics, eight candidate genes were evaluated for a role in lactate export. Knockouts of well-documented lactate permeases LldP and GlcA only caused minimal perturbations for lactate export and production, whereas a ProP knockout showed large deficiencies. The finding that LldP and GlcA cause minimal deficiencies demonstrates the current knowledge gap in lactate export, and in organic acid export in general. Ongoing investigations plan to further characterize this network of lactate exporters and provide more knowledge on this fundamental metabolic process.

P21

Developing Breath-Based Diagnostics to Detect P. aeruginosa Exoproducts in CF Lung Infections

Darrin Anderson, Charity Bhebhe, Nathan Dacasin and Heather D. Bean

School of Life Sciences, Arizona State University, Tempe, AZ 85287

Cystic fibrosis (CF) is a genetic disease affecting approximately 30,000 persons in the U.S. (70,000 worldwide). The genetic defect causes the body to produce thick, sticky mucus that clogs the airways of the lungs and also impairs the lungs’ ability to clear out inhaled microorganisms, which leads to life-threatening lung infections. The leading cause of CF lung infections is Pseudomonas aeruginosa. Although P. aeruginosa is a ubiquitous bacterium, its opportunistic nature makes it a particularly dangerous pathogen to those with compromised immune systems, including those afflicted with CF. In addition, due to its ability to adapt to the lung environment through phenotypic and genotypic changes, P. aeruginosa can cause infections that last for years to decades. Early P. aeruginosa treatment is essential for long-term patient outcomes; once the infection is established, strains of the bacteria that are mucoid (slimy), quorum-sensing deficient, non-motile and antibiotic resistant are established, and these traits are correlated to lung function declines. The goal of this project is to phenotype and genotype P. aeruginosa CF lung isolates for exoproduct production. In particular, we are focused on P. aeruginosa proteases, surfactants, exopolysaccharides, quorum sensing molecules, and siderophores. The quantities of these exoproducts will be correlated to the isolates’ metabolomes to discover unique volatile biomarkers of P. aeruginosa phenotypes, leading to new, improved medical devices for early diagnosis and characterization of chronic lung infections.

P22

Evaluating Phage Effect on Heterotrophic Decomposition in Amazon Peatland Soils

Jessica Spring1, Hinsby Cadillo-Quiroz1, and Analissa Sarno2

1School of Life Sciences, Arizona State University, Tempe, AZ, 85287

Little is known about the diversity and role of bacteriophages in carbon (C) rich ecosystems such as peatlands in tropical and temperate regions. To better understand how bacteriophages influence organic C cycling to final products like CO2 and CH4, phage communities and phage like particles were isolated or manipulated from freshwater sources from Amazon peatlands. Here we present initial findings on bacteriophages and their effects on heterotrophic bacteria, which in turn affects C cycling and decomposition to CH4 in peatlands. To assess diversity, phages were enriched from water samples from Amazon peatlands by filtering to remove organic matter and bacteria, and iron flocculation to bind the phages and allow for precipitation onto a filter. Phage community enrichments were screened using an OD absorbance and cross-

streaking method, against 50 heterotrophic bacterial isolates obtained from the same Amazon peatlands to identify phage hosts. Once a host was found, the phage was isolated. Total phage community numbers were assessed using fluorescent microscopy. The efficiency of iron flocculation was determined using fluorescent microscopy counts of phage pre and post extraction method. The effect of iron flocculation on infectivity was determined by counting plaques pre and post extraction method. Selected phages will be sequenced for accurate identification. Our results provide the groundwork for further characterizing the role that bacteriophage play in C cycling and greenhouse gas production from Amazon peatland soils.

P23

Salmonella Typhi Peyer’s Patches Targeting Strategies Tested in an M cell Culture Model

Amanda Gonzales, Shyra Wilde, Andrea Segerstrom, Nyga Brown and Kenneth L. Roland

Biodesign Institute, Arizona State University, Tempe, AZ 85287

Live Salmonella vaccines show great potential as vaccines because of their ability to elicit a strong mucosal IgA response. Live Salmonella Typhimurium-based recombinant vaccines are highly immunogenic and protective in mice. S. Typhimurium preferentially invades the M cells of Peyer’s patches (PPs), stimulating a strong pro-inflammatory immune response. By invading PPs, S. Typhimurium is encounter the host mucosal immune system resulting in production of pro-inflammatory cytokines, which recruit and activate neutrophils, monocytes and dendritic cells. Translating these successes to S. Typhi-based vaccines for humans has been problematic because S. Typhi uses a number of strategies to evade the host immune system. For example, in S. Typhi infections, the bacteria tend to invade intestinal epithelial cells, bypassing the PPs.

Differences in intestinal trafficking observed for S. Typhi and S. Typhimurium is linked to their fimbrial profiles. The S. Typhi-specific Stg fimbriae promote adhesion to the intestinal epithelial cell surface. We deleted the stg fimbrial operon to reduce epithelial cell targeting.

The stg mutants and wild type parental strains were evaluated in a tissue culture model in which M-like cells are derived from Caco-2 epithelial cells co-cultured with the human lymphoblastoid B cell line, RajiB. Like human M cells, the M-like cells in this model exhibit reduced binding to Ulex europaeus Agglutinin I lectin and show upregulation of surface protein galectin-9 compared to Caco-2 cells. In this study we have characterized the M-like cells produced in this co-culture model and tested the model’s usefulness in identifying S. Typhi PPs targeting mechanisms.

P24

Characterization of a Synergistic Network of Malate Exporters in Escherichia coli

Kurgan, L. *, Kurgan, G. *, Rodriguez, Y., Nieves, L. and Wang, X. School of Life Sciences, Arizona State University, Tempe, AZ, 85287

Besides being a key metabolite in the citric acid cycle, L-malate is an important specialty chemical used in the food, pharmaceutical, and polymer industries. It is also a desirable renewable chemical with the potential for mass production from biomass. The export of organic acids represents a potential bottleneck for bioproduction, but malate export has not been characterized in bacterial hosts. In this work, we use a reverse genetics approach to characterize a malate export system and explore whether this export system can enhance production in an L-malate biocatalyst. Ten candidate genes were evaluated for a role in malate export in an L-malate producing Escherichia coli. One transporter of the dicarboxylate uptake family (Dcu) and two transporters of the dicarboxylate anion: sodium symporter (DASS) family together appear to play a primary role in malate export. Combinatorial evaluation of the transporters demonstrates that TtdT and DcuA play a crucial role in this synergy, and removal of these genes results in elevated intracellular L-malate concentrations.

P25

Developing Breath-Based Diagnostics to Detect P. aeruginosa Motility and Antibiotic Resistance in CF Lung Infections

Jonathan Kiermayr1, Amritha Venguideshe2, Lea Witzel2 and Heather D. Bean2

1School of Molecular Sciences, Arizona State University, Tempe, AZ 85287 2School of Life Sciences, Arizona State University, Tempe, AZ 85287

The primary cause of lung function decline and eventual death for persons with cystic fibrosis (CF) is pulmonary damage from chronic bacterial lung infections, with Pseudomonas aeruginosa responsible for half of all CF infections, and establishing chronic lung infections in 75% of adults with CF.1 During chronic infection, P. aeruginosa acquire phenotypes, such as antibiotic resistance, loss of motility, and mucoidy, that are significantly correlated to lung function decline.2,3 However, accurately diagnosing these phenotypes in the clinical microbiology laboratory is incredibly challenging due to rapid phenotypic switching by the bacteria once they are cultured outside of the lung environment.4 The long-term vision of the Bean Lab is to identify volatile biomarkers to detect these clinically-important bacterial phenotypes directly from the patients’ breath, making it possible to detect the pathogens and their phenotypes in situ. The goal of this project is to phenotype and genotype P. aeruginosa CF lung isolates for motility and antibiotic resistance. In particular, we are focused on P. aeruginosa swimming, swarming, and twitching motilities, and resistance to cephalosporin, fluroquinolone, carbapenem, aminoglycoside, monobactam, and polymyxin antibiotics. These traits will be correlated to the isolates’ volatile metabolomes to identify

biomarkers of these P. aeruginosa phenotypes, which will be developed into breath-based diagnostics for early, non-invasive detection and characterization of chronic lung infections.

P26

IBT-Resistance and Murine Cell Tropism are Unlinked Traits in Monkeypox Virus.

Brian Patrick Johnson, Bertram Lewis Jacobs

Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ, 85281

Monkeypox virus (MPXV) is a member of the orthopoxvirus genus, which also includes variola virus, the causative agent of smallpox, and vaccinia virus (VACV), used as the smallpox vaccine. Despite their close relation, MPXV has several distinguishing features from VACV. The MPXV homolog of the VACV virulence gene E3L has an n-terminal deletion of 37 amino acids. Introducing this truncation into VACV E3L significantly reduces viral replication in some murine cell lines; MPXV replicates efficiently in these same cells. Additionally, we have found that MPXV is naturally resistant to the antiviral drug isatin-β-thiosemicarbazone (IBT) compared to the WR strain of VACV.

Because E3L is known to inhibit dsRNA-mediated host immune responses and IBT is known to increase viral synthesis of dsRNA, we hypothesized that IBT-resistance and rescue of murine cell tropism in MPXV are linked traits. To test this, permissive cells were co-infected with MPXV and VACV E3Ldel37N NeoGFP. Virus was then harvested and grown in murine cells permissive to MPXV but not VACV E3Ldel37N, and green plaques were picked to select for hybrid virus. IBT resistance and growth kinetics for the hybrid were then measured.

We have isolated a hybrid virus with murine tropism but IBT sensitivity, suggesting that that these traits are unlinked. We have performed next-gen sequencing on this hybrid, and are analyzing the results in order to determine which portions of the hybrid genome derive from MPXV and which from VACV E3Ldel37N as a means of identifying the gene or genes responsible for murine tropism.

P27

Influence of the Mycobacterial PrrAB Two-Component System on Metabolism and Lipid Biosynthesis

Jason D. Maarsingh1,2 and Shelley E. Haydel1,2

1School of Life Sciences, Arizona State University, Tempe, Arizona 85287,United States 3The Biodesign Institute, Arizona State University, Tempe, Arizona 85287,United States

Mycobacterium tuberculosis is estimated to infect a third of the world’s population and was responsible for 1.5 million deaths in 2014. Only HIV kills more people from a single infectious agent. Upon inhalation, M. tuberculosis is phagocytosed by alveolar macrophages. If not

cleared, the bacillus may persist for decades until the host experiences an immunocompromised state, upon which bacterial replication ensues with subsequent tissue destruction.

Two-component systems (TCS) are signal transduction pathways that recognize and allow bacteria to adapt and survive in diverse environmental conditions. TCS are ubiquitous in bacteria yet absent in humans, making them attractive therapeutic targets. Upon phagocytosis, multiple TCS act to program M. tuberculosis responsiveness, thus contributing to survival in this inhospitable intracellular environment. The prrAB TCS is essential in M. tuberculosis and is induced during the early stages of intracellular infection, suggesting a role for adaptation and survival within the macrophage.

Nonpathogenic Mycobacterium smegmatis shares numerous genetic homologues with M. tuberculosis, some of which are virulence factors in the latter species. We generated a prrAB knockout in M. smegmatis mc2155 and introduced M. tuberculosis prrA isoforms into mc2155::ΔprrAB. Differential lipid expression upon nitrogen-limitation was evident in mc2155::ΔprrAB harboring M. tuberculosis PrrA phosphomimetic D58E and PrrA phosphoablative D58A constructs. Preliminary RNA-Seq data reveal differential gene expression in pathways for short-chain carbon metabolism. The mc2155::ΔprrAB strain exhibited growth defects in media containing glycerol, acetate, or propionate as carbon sources, each of which are available substrates within the phagosome. Ongoing studies are focused on determining how PrrAB regulates essential metabolic pathways and influences mycobacterial pathogenesis.

P28

pH Shifts in the Anode Potential Response from Thermincola ferriacetica Suggest the Presence of a Rate Limiting Proton-Coupled Electron Transfer Protein

Bradley G. Lusk1, Prathap Parameswaran,2 Sudeep C. Popat,1 Bruce E. Rittmann,1 Cesar I. Torres1,3

1 Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, Arizona 85287−5701, United States of America

2 Department of Civil Engineering, Kansas State University, 2123 Fiedler Hall, Manhattan, Kansas 66502, United States of America 3 School for Engineering of Matter, Transport and

Energy, Arizona State University, 501 E Tyler Mall, Tempe, Arizona 85287, United States of America

Background: Thermincola ferriacetica, a thermophilic, Gram-positive, anode respiring bacterium (ARB) was grown in biofilms in microbial electrochemical cells (MXCs) to investigate its external electron-transport (EET) limitations. Electrochemical studies, including low scan cyclic voltammetry (LSCV), are often used to elucidate the rate-limiting step of electron transport in ARB biofilms. Previously reported CV analysis of T. ferriacetica biofilms indicated a sigmoidal

Nernst-Monod response in electrical current (j) to changes in anode potential (V). This response suggests that a single proton (H+) coupled electron (n = 1) transport reaction is responsible for the rate-limiting step in T. ferriacetica metabolism. The specific protein responsible for this response is thought to be a c-type cytochrome. Although T. ferriacetica has been shown to contain 35 c-type cytochromes, the one(s) responsible for EET has yet to be identified. Methods: To determine the effect of pH on Eka, biofilms were grown at 50 mM bicarbonate buffer and 25 mM acetate as the electron donor. After achieving a steady j, pH was altered by the addition of HCl or NaOH. Then, LSCV was performed to determine the effect of pH on Eka. To assess the effect of bicarbonate buffer on Eka, biofilms were grown with 10, 25, 50, and 100 mM bicarbonate with 25 mM acetate as the electron donor. After achieving a steady j, LSCVs were performed at 1.0 mV s-1 and 10 mV s-1. This was repeated at 10, 25, 50, or 100 mM bicarbonate by either starting at 10 mM and increasing to 100 mM or by starting at 100 mM and decreasing to 10 mM. Results: T. ferriacetica’s response under certain growth conditions is composed of at least two separate n = 1 Nernst-Monod relationships; suggesting the presence of more than one pathway for anode respiration. Altering bulk pH reveals that biofilms in neutral to high pH (6.9-8.3) show a very broad redox peak while biofilms in low pH (5.2) reveal multiple redox peaks. Altering bicarbonate buffer concentration shows a similar trend, with lower bicarbonate leading to the presence of multiple redox peaks; consistent with pH gradients developing inside the T. ferriacetica biofilm. Conclusions: T. ferriacetica contains more than one H+ coupled EET pathway and EET pathways within T. ferriacetica are sensitive to changes in bulk pH.

P29

Characterization of spore germination profiles and coat and protein composition of epidemic and non-epidemic strains of Clostridium difficile

Kevin Lewis1, Michael Mallozzi1,2, V.K. Viswanathan1, and Gayatri Vedantam1,2,3,4

1Dept. of Veterinary Science and Microbiology, 2Southern Arizona VA Healthcare System, 3BIO5Research Institute, and 4Dept. of Immunobiology, University of Arizona, Tucson, AZ

Clostridium difficile (CD) is a spore-forming bacterium, which causes a diarrheic disease that is precipitated upon treatment with antibiotics. Many of the problems associated with the transmissibility and persistence of CD are likely mediated by the spore, which acts as the infectious particle for this otherwise fastidious anaerobic bacterium. Many characteristics of the spore such as its ability to become aerosolized, adhere to biotic and abiotic surfaces, and resist decontamination, facilitate the spread of CDI in hospitals. Spores remain metabolically dormant until ingested. After passing through the low pH of the stomach, spores encounter bile acids, which are specific germination-triggering biomolecules for CD, and germinate in the small bowel initiating vegetative cell outgrowth and the infection.

We hypothesize that endospore structure, resistance, germination, and outgrowth properties play an important role in determining which strains are epidemic-associated (EPI) or not epidemic-associated (non-EPI).

In this study, we assessed spore protein composition, ultrastructure, and germination rates, and evaluated their differences between epidemic and non-epidemic strains of CD. Using an assay which quantitatively measured the rate of spore germination, we identified a broad range of strain-specific germination profiles. Thin-section and scanning electron microscopy revealed that the spores’ ultrastructure differed on a strain-to-strain basis, but shared notable elements. Further, spore protein composition was determined by mass spectrometry and was further analyzed to identify a proteome of spore-coat-associated proteins. Two proteins which were either absent or differentially abundant in the non-EPI vs. EPI strains were identified, and selected for mutagenesis and/or over-expression to examine their role in spore germination and ultrastructure.

P30

Investigation of Virus Structure with X-ray Free-Electron Laser (XFEL) Methods

Robert M. Lawrence1,2,3, Brenda G. Hogue1,2,3,4

1Biodesign Institute, 2Center for Infectious Diseases and Vaccinology, 3Center for Applied Structural Discovery, 4School of Life Sciences, Arizona State University, Tempe, AZ, 85287

As with proteins, whole virus particles can be crystallized and probed with X-ray light to produce 3-dimensional structure models from measured diffraction. A new generation of linear X-ray free-electron laser (XFEL) light sources are capable of producing unprecedented levels of flux that can be focused to submicron dimensions. XFEL methods have recently been applied successfully to the study of crystallized proteins. In principle, larger biological samples such as viruses can be used to diffract X-rays as single particles, thus obviating the prerequisite of crystallization for x-ray studies. As a model for investigating the application of XFEL techniques to virology, we have produced microcrystals of the Sindbis virus and measured diffraction. Ongoing efforts with other viruses are being pursued in order to develop the single particle XFEL approach as well. Preparation of the sample involves the use of Dynamic Light Scattering and Electron Microscopy as analytical methods, as well as inactivation of the virus particle for biosafety purposes. Ultimately we anticipate that XFEL methods will enable time-resolved structural investigations of whole viruses, as has been recently accomplished with proteins.

P31

Fungal Twig Endophytes of Pinus edulis (Pinyon Pine) Respond to Host Plant Genetics

Ron J. Deckert1 and Catherine A. Gehring1,2

1 Department of Biological Sciences, Northern Arizona University, Flagstaff AZ 86011 2Merriam-Powell Center for Ecological Research, Flagstaff AZ 86011

Fungal endophytes are ubiquitous and symptomless endosymbionts of plants. Host specificity varies among endophytes but little is known about the response of endophytes to genotypic differences within a host species. In pinyon pine (Pinus edulis), microbial symbiont communities such as ectomycorrhizal fungi, have been demonstrated to vary with the genetics of the host. In the Sunset Crater area, two phenotypes of P. edulis represent different genotypes that are differentially susceptible to a phytophagous insect (Dioryctria albovittella, the pinyon tip moth) and drought, the herbivore susceptlble genotype being more drought resistant. We conducted preliminary testing of the hypothesis that endophyte morphotypes isolated from genetically distinct P. edulis will also vary according to host genetics. In order to test the hypothesis, 20 three-year old needle fascicles were collected from each of four branches representing the four cardinal directions on an herbivore susceptible and an herbivore resistant pine for a total of 160 needles and 80 fascicle bases (anatomically, short shoots or twigs). Samples were surface-sterilized and plated on PDA and scored after three weeks for emerging fungal hyphae and scored for infection frequency and morphotype frequency. Several patterns emerged: 1) both host genotypes exhibited very low levels of leaf infection (<1%). 2) Twig endophytes were isolated significantly more often on susceptible trees (37.5%) than on resistant trees (23%). 3) Susceptible trees harbored morphotypes that were not present in resistant trees. Future studies will characterize twig endophyte communities of the pine genotypes using ITS and LSU sequence data.

P32

Evaluating the Drivers of Chemodenitrification in Tropical Peat Soil

Kaitlyn Tylor1, Steffen Buessecker1, Hinsby Cadillo-Quiroz1

1School of Life Sciences, Arizona State University, Tempe, AZ, 85281

Nitrous oxide (N2O) is a major contributor to the greenhouse effect and to ozone depletion, and remains in the atmosphere for 114 years. The infrared radiative forcing of one N2O molecule is 206 times that of one CO2 molecule. In soils, nitrogen reduction is performed by biotic and abiotic processes, including microbial denitrification, microbial nitrifier denitrification and chemical denitrification. Chemical denitrification, or chemodenitrification, is the abiotic step-wise reduction of nitrate (NO3

-), nitrite (NO2-), or nitric oxide (NO) to N2O in anoxic environments, with

high turnover rates particularly in acidic soils. Chemodenitrification was identified in various environments, but the mechanism is still not understood. In this study, the factors influencing

abiotic reduction of NO2- to N2O in acidic tropical peat soil are examined. These factors include

pH, organic matter content, and dissolved ferrous iron. Anoxic peat soil from sites located in the Peruvian Amazon was used for incubations. Our results show that peat soil (pH 4.5) reduces nitrite at a maximum rate of 150 µM per day. Soil adjusted to pH 2.6 reduced 200 µM NO2

- at a 1.3-fold increased rate compared with soil adjusted to pH 6.9. In addition, soil containing ferrous iron additions of 0 µM, 100 µM, and 500 µM reduced NO2

- by 0 µM, 50 µM, and 100 µM, respectively, during a two-hour time period. How and to what extent the pH and the concentrations of organic matter and ferrous iron affect the kinetic rate of chemodenitrification will lend insight into the N2O production potential of natural tropical peatlands.

P33

Characterization of the DNA Binding Site for a Transcriptional Regulator Essential for Virulence of the Human Pathogen: Shigella

Joy A. Immak1, Natasha Weatherspoon-Griffin1, Michael Picker1, and Helen J. Wing1

1School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV, 89154-4004

Shigella species are intracellular bacterial pathogens that are the causative agents of shigellosis in humans. Due to the low infectious dose and simple mode of transmission through contaminated water, foods, or direct contact, Shigella infections continue to persist across the world. The virulence of Shigella species is attributed to its large virulence plasmid whose genes are up regulated by the transcriptional regulator, VirB. Regulatory properties of VirB are dependent upon it binding to DNA in a sequence-specific manner. However, the DNA sequence required for VirB-binding has been greatly debated and varies between two virulence gene promoters studied thus far: icsB and icsP. In both virulence gene promoters, the VirB binding site is dependent upon the presence of specific DNA sequences organized as an inverted repeat with a single nucleotide in between. The half sites of the inverted repeats have been named: Box 1 and 2. For the icsB VirB-binding site, Box 1 is much less important for VirB binding, whereas Box 2 is essential. To contrast, icsP requires both half sites. I aim to more thoroughly characterize the DNA sequences required for VirB binding by analyzing eleven additional VirB-dependent genes, and their associated VirB-binding sites. Thus far, this study has identified highly similar Box 1 and 2 sequences in the regulatory regions of the following genes across all Shigella species: icsP, ipgD, icsB, ospD1, virB, spa15, phoN2, and virA. This suggests that VirB binding and subsequent regulation is dependent on the presence of both half sites: Box 1 and 2.

P34

Optimization of Measles Viral Antigen Expression for Serologic Assays

Zuena Mushtaq1, Karen S. Anderson1,2

1Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, Tempe, AZ;

2School of Life Sciences, College of Liberal Arts and Sciences, Arizona State University, Tempe, AZ

Background: Measles (MV) is a contagious, vaccine-preventable disease that continues to be the leading cause of death in children below the age of 5. We have found that expression of MV antigens as C-terminal GST fusion proteins was insufficient to detect IgG immunity in healthy donors. This study aimed to improve protein expression of MV antigens for rapid point of care measles serologic screening. Methods: MV genes were subcloned into the pJFT7_nGST vector to generate N-terminal GST fusion proteins. MV antigens were expressed using IVTT with human cell lysate. Expression of GST-tagged proteins was measured. Relative light units (RLUs) as luminescence was measured. Antibodies to MV antigens were measured in 12 serum samples from healthy subjects. Results: Protein expression of three measles antigens, N, F, and H, was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001). Average RLU values for F and H were 2.37 x 109 (p<0.001) and 5.46 x 107 (p=0.001), respectively. All proteins expressed at least 50% greater than vector control. There was over an 8-fold improvement in expression levels using N-terminal GST fusion proteins. 11/12 serum samples had IgG to N protein (Ag:GST ratio>1.5), and no samples had detectable IgG to H. Conclusion: These data indicate that the in vitro expression of measles proteins N and F were markedly improved using N-terminal GST fusion proteins. A large scale screening study is planned.

P35

Ontogeny of the Hypopharyngeal and Salivary Glands in Honey Bees

Rachna Nath, Alan Rawls and Juergen Gadau

School of Life Sciences, Arizona State University, Tempe, AZ

Exocrine glands perform crucial functions in insect societies and several exocrine glands like the hypopharyngeal gland in honey bees show phenotypic plasticity linked to age, caste and task. Honey bees have three exocrine glands in their head, the salivary (head part), hypopharyngeal and mandibular glands. Structural complexity of these exocrine glands in honey bee (Apis mellifera) has been defined by Noirot and Quennedey (1974) and also by

Johan Billen (1986). Post embryonic development of these gland has also been studied by Emmert(1968) and the adult glands by Kratky(1931),Simpson(1962),Snodgrass(1956), Cruz Landim(1957 to 2010). But despite their importance, little is known about the gene regulatory network underlying the ontogeny and phenotypic plasticity in honeybees. Here we characterized the anatomical changes of these glands during metamorphosis (transition from larval to adult stage). The larval salivary gland is almost completely reorganized during metamorphosis whereas the other two glands have no progenitor in larvae and develop de novo.

The goals of this study is to 1. describe in detail when, which structural and histological changes occur in glandular development during metamorphosis and 2. determine the gene regulatory network/s underlying gland development and phenotypic plasticity in adults, i.e. to link differential gene expression with the different caste and task phenotypes. Here, we present a detailed description and histology of the ontogeny of the salivary and hypopharyngeal glands in Apis mellifera using paraffin embedding with a hematoxylin and eosin stain.

P36

Manipulation Experiments on N2O Reduction in Amazon Peatland Soils

Zacary Zamora, Steffen Buessecker, Hinsby Cadillo-Quiroz

School of Life Sciences, Arizona State University, Tempe, Arizona, 85281

A yet uncovered diversity of microbes are capable of using nitrous oxide (N2O) to generate energy. The redox couple N2O/N2 is very powerful, with potential of 1.35V as compared to that of NO3- /NO2- 0.43V. Novel peatlands of the Amazon show high potential to reduce N2O under anoxic conditions thus increasing the importance of the understanding of these communities. In order to access its role in the peat ecosystem, microbial N2O reduction was quantified and followed in incubations under inhibiting conditions.

Nitrous oxide reductase (Nos) is the periplasmic enzyme that catalyzes the reduction of N2O to N2. Acetylene (C2H2) prevents the reduction of N2O to N2 by inhibiting Nos. Carbon Monoxide (CO) prevents the degradation of C2H2 by blocking all Nitrogenase based reactions (nitrogenase reduces acetylene). Acetylene Prepared slurries of tropical peat soil were manipulated with 200 µM nitrite (precursor of N2O), CO and C2H2. All C2H2 treatments were effective for up to 6 days before being degraded by microbes. CO had no statistically significant effects. N2O production observed in C2H2 treatments averaged 126.4(±2.67) ppm and 123.4(±5.68) ppm across days where C2H2 was viable and 14.5(±20.5) ppm in non-inhibited controls. Results indicated that there is high microbial reduction potential for nitrous oxide and that more studies observing N2O reduction model in acidic soils (pH: 6.02).

P37

In Vitro and In Vivo Binding of a Regulator Essential for Shigella Virulence

Natasha Weatherspoon-Griffin, Monika M. Karney, Joy A. Immak, Eliese Potocek, and Helen J. Wing

School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV. 89154-4004

VirB is a transcriptional regulator that is essential for the virulence of the human pathogen, Shigella flexneri. Using the icsP promoter (PicsP) as a model virulence promoter, our laboratory has previously shown that its regulation requires both VirB and an inverted repeat located more than 1 kb upstream of its transcription start site. Although the regulation of PicsP by VirB has never been shown to be direct or indirect, it has been speculated that VirB binds to the inverted repeat to exert its regulatory function. Here, we hypothesize that VirB directly binds to the inverted repeat required for the VirB-dependent regulation of the PicsP. We evaluated the binding of VirB to the inverted repeat using a novel in vivo binding tool as well as the in vitro DNaseI footprinting and electromobility shift assays. In addition, we investigated the requirement of each half-site of the inverted repeat also using the in vivo binding tool. Our data demonstrate that VirB binds to the inverted repeat both in vivo and in vitro. Conversely, when the inverted repeat is mutated, we observe either a loss or reduction of VirB binding in vivo and in vitro suggesting that VirB specifically interacts with the wild type inverted repeat. Moreover, we demonstrate that each half-site of the inverted repeat is required for the binding of VirB which refutes claims that only a single site is required for VirB to bind. Cumulatively, this data is the first to demonstrate a direct and specific interaction between VirB and the inverted repeat required for VirB-dependent regulation of the icsP promoter.

P38

Neuropathogenesis of LCMV in Clone-13 Carriers

Britney M. Tillis1,2, Susan A. Holechek1,2,3, Jason Newbern1, and Joseph N. Blattman1,2

1School of Life Sciences, Arizona State University, Tempe, AZ, 85287 2Center for Infectious Diseases, Biodesign Institute, Arizona State University, Tempe, AZ, 85287

3Simon A. Levin Mathematical Computational Biology and Modeling Sciences Center, Arizona State University, Tempe, AZ, 85287

Lymphocytic choriomeningitis virus (LCMV) is an arenavirus that infects about 10% of the rodent population in the wild. Rodents can be infected as adults or congenitally infected. In adult-infected immune response there is an anti-viral immune response while in congenitally infected carriers lack of response. When mice are infected with LCMV they will suffer from persistent infection without having an immune response. These persistently infected mice are typically called carriers or super-spreaders and are different from infected mice since they show no antibody response while having high titers of virus in their blood and body fluids. We have seen LCMV in the brain and nervous system, which suggests neuropathogenesis is happening.

Carrier infected mice are runted and neurological and physiological defects have been observed. We will be looking into the neurological effects of the virus at different gestational stages during vertical transmission from mother to fetus. This will happen by looking at both the histology of the brain as well as doing immunohistochemistry on the brain tissues. At different stages of gestation, the brain will be dissected from mice to see if there are any differences of the brain region sizes and immunological markers in the brain from persistently infected mice and mock-infected.

P39

The Exercise Microbiome Project, Assessing the Effect of Physical Activity on the Human Gut Microbiome

Arron Shiffer1, Anthony Santos2, Dierdra Bycura2, Kyle Winfree3, Talima Pearson1, J. Gregory Caporaso1

1Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86001 2Department of Health Sciences, Northern Arizona University, Flagstaff, AZ, 86001

3Department of Informatics and Computing, Northern Arizona University, Flagstaff, AZ, 86001

The aim of this project is to track changes in the composition of the human gut microbiome that happen in conjunction with prescribed changes in physical activity. We are interested in determining whether increase in human physical activity can cause variation in the human gut microbiome. We will Recruit 30-35 healthy but self-described sedentary college-age subjects, who will be put on a cardiorespiratory exercise regimen, and we will study the relationship between changes in physical activity and microbiome composition. These data will help in understanding how changes in weight and fitness correlate with changes in the microbiome. Additionally, these data may be informative for developing microbiome-based treatments for weight gain or loss (for example, by identifying organisms that should be included in probiotics).

The temporal variability, the rates of variability, and drivers for these temporal changes of the human gut microbiome are largely unknown. The current study is designed to explore possible drivers of change and study the temporal variation of the human gut microbiome.

The entire project will last 14 weeks with an 8-week exercise intervention, and twice weekly we will obtain fecal samples to track changes in the gut microbiome. There will be baseline periods at the beginning and end to compare the microbiome changes in the exercise intervention. We will track subject’s heart rate and physical activity via FitbitTM physical tracking devices. We will perform a pre/post VO2 max treadmill, skin fold, and BMI testing. All microbiome data will be analyzed with the QIIME analysis pipeline.

P40

Flagellin Glycosylation Influences Flagellar Assembly, Autoaggregation, and Toxin Production in Clostridium difficile

Aaron R. Brussels1, Andrew E. Clark1, John S. Costanza1, Bryan P. Roxas1, VK Viswanathan1,2,3, and Gayatri Vedantam1,2,3,4

1School of Animal and Comparative Biomedical Sciences, 2Department of Immunobiology, 3BIO5 Institute, University of Arizona, Tucson, AZ, 4Southern Arizona VA Healthcare

System, Tucson, AZ

Clostridium difficile (CD) has emerged as the most prevalent healthcare-associated infection in the United States, responsible for an estimated annual cost of $4 billion. Disease manifests as a fulminant diarrhea and is mediated through the action of two glucosylating toxins, TcdA and TcdB, which target cellular GTPases of intestinal epithelial cells leading to apoptosis. Events leading to CD host colonization remain largely undefined. CD flagella are critical for motility and play a pivotal role in bacterial-host interactions. Whole-genome sequencing has revealed two clade-specific glycosylation islands within the CD flagellar biosynthesis locus that are responsible for the post-translational modification of the flagellin protein (FliC). Mutagenesis of genes within these islands demonstrates that FliC glycosylation impacts motility, flagellin export, secreted toxin levels. The contribution of the CD flagellar glycosylation system toward CD biology and disease pathogenesis remains unclear and warrants further study.

Our lab has previously demonstrated that recent outbreak strains of CD downregulate flagellar genes when compared to historical strains, providing a mechanism for dampening of the host immune response, potentially aiding colonization. We hypothesize that flagellar glycosylation in CD functions in a similar manner, allowing CD to avoid immune response recognition and activation, leading to facilitated colonization and establishment of C. difficile infection.

P41

Creating an Optimal Multiple Sequence Alignment Workflow for Phylogenetic Diversity Analyses

William Mercurio, Arron Shiffer, Evan Bolyen, Jai Ram Rideout, John Chase, and J. Gregory Caporaso

Department of Biology, Northern Arizona University, Flagstaff, AZ, 86001

Microbiome surveys regularly make use of phylogenetic diversity metrics to examine community similarity. This requires the generation of a phylogenetic tree, which in turn requires the generation of a multiple sequence alignment.

We present the results of a benchmark experiment to compare iterative and progressive multiple sequence alignment tools (Muscle, MAFFT) to a reference based multiple sequence aligner (PyNAST), and a structure based multiple sequence aligner (SSU-ALIGN), to identify an

approach that can be used as a replacement in QIIME’s phylogenetic tree building workflow, which is known to be suboptimal. We additionally compare dynamic entropy-based filtering and conservation based filtering to the traditional static Lane mask-based filtering. We test the hypothesis that multiple sequence alignment followed by dynamic positional alignment filtering will yield phylogenetic diversity results that are as good or better than QIIME’s default alignment and filtering workflow.

Our analyses were performed on three real-world microbiome data sets. We first compared phylogenetic trees to determine how similar the resulting trees were to each other using scikit-bio’s implementation of the Robinson-Foulds metric, and then compared the alpha and beta diversities of the communities. Preliminary results showed that SSU-ALIGN derived trees maximize the expected signals in real-world data, and may therefore be suitable as the default workflow in QIIME. Additional analyses on simulated communities, where the actual composition is known in advance, are currently in progress and will complement the analyses based on real data.

P42

Detecting Melioidosis: Development of MagPix Assays to Limit Disease

Rachel K. Meyer1, Carina M. Hall1, Jason Sahl1, Vanessa Theobald2, Mark Mayo2, Erin Price2, Joseph D. Busch1, Bart J. Currie2, Paul Keim1, David M. Wagner1

1 Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011 2 Menzies School of Health Research, Casuarina, NT, Australia

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative soil bacterium endemic to Southeast Asia and Northern Australia. Rapid and accurate identification of B. pseudomallei in clinical or soil samples has the potential to improve treatment options or assist in the identification of B. pseudomallei should it be used as a biological weapon. B. pseudomallei has many close relatives so we also need to include many near-neighbor strains in our panel. Due to frequent recombination within the B. pseudomallei genome, a wide variety of strains must be considered. We have developed molecular tools to allow for the detection and differentiation of B. pseudomallei and near neighbor species using the Luminex MagPix platform. The MagPix uses colored magnetic microspheres to identify each assay, allowing for the simultaneous screenings of multiple Burkholderia species targets per sample. Using four multiplex PCRs with a total of 50 targets, we examined the sensitivity of these assays by testing the limit of detection. We also screened 192 diverse samples within the Burkholderia genera to confirm the specificity of the 50 targets. We investigated the performance of each of the markers in their multiplexes by determining how sensitive and specific each target is. Overall, these tools will have the power to identify B. pseudomallei in both a clinical setting and in a malicious act of bio-warfare.

P43

Creating an Artificial Antigen Presenting Cell System for HPV16 Proteins

Bianca Varda1,2, Sri Krishna Sundaresan1,3, Karen S. Anderson1,2

1Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, Tempe, AZ;

2School of Life Sciences, College of Liberal Arts and Sciences, Arizona State University, Tempe, AZ;

3School of Biological and Health Systems Engineering Arizona State University, Tempe, AZ

Background: High risk types of human papillomavirus (HPV) are known to cause cancer, including cervical (99%) and oropharyngeal cancer (70%). HPV type 16 is the most common subtype. Three antigens that are critical for integration or tumor progression are E2, E6 and E7. In this study, we developed a systematic approach to identify naturally-processed HPV16-derived HLA class I epitopes for immunotherapy development. Methods: K562 cells, which lack HLA expression, were transduced with each HPV16 antigen using lentivirus and supertransfected with HLA-A2 by nucleofection. Stable cell lines expressing each antigen were selected for and maintained throughout the investigation. In order to establish a Gateway-compatible vector for robust transient gene expression, a Gateway recombination expression cloning cassette was inserted into the commercial Lonza pMAX GFP backbone, which has been experimentally shown to display high transfection expression efficiency. GFP was cloned into the vector and plain K562 cells were transfected with the plasmid by nucleofection. Results: Expression of K562-A2 was tested at various time points by Western blot analysis and A2 expression was confirmed. Protein expression was shown for the transduced K562 E7 by Western blot analysis. High transfection efficiency of the pMAX_GFP_Dest vector (up to 97% GFP+ cells) was obtained 48 hours post transfection, comparable to the commercial GFP-plasmid. Conclusion: We have established a rapid system for target viral antigen co-expression with single HLA molecules for analysis of antigen presentation. Using HPV as a model system, our goal is to identify specific antigenic peptide sequences to develop immunotherapeutic treatments for HPV-associated cancers. P44

Recognition of Two New Cryptic Species of Wilmottia (Oscillatoriales, Cyanobacteria) Based on 16S rRNA and ITS Molecular Analyses

Náthali Maria Machado de Lima1*, Mariéllen Dornelles Martins1, Luís Henrique Zanini Branco1

1Department of Botany and Zoology, Sao Paulo State University, Sao Jose do Rio Preto, SP 15054-000

In 2011, Strunecký et al. studied the species Phormidium murrayi West & West and similar morphotypes, demonstrating their genetic distinction from other Phormidium species despite

their morphological similarity and erecting the new genus Wilmottia Strunecký, Elster & Komárek (type species Willmottia murrayi. During a work about the diversity of the sub-family Phormidioideae in Brazil, 10 strains morphologically similar to Phormidium were studied by morphological, molecular (using 16S rRNA and ITS genetic markers) and ecological criteria. The strains did not present significant differences in morphology, however, the molecular analysis to the 16S RNAr separated them in six clades positioned inside the Wilmottia cluster with high supported values in ML, NJ and IB analysis (87%, 99% and 1, respectively). Though, the relation among the clades was not clear, considering the high similarity between some sequences. The ITS showed informative to the region D1-D1’ that converged with 16 rRNA data, however, showed subspecific level differentiation to Box-B and V3 structures, not solving the specific distinction, what was just possible after ecological considerations. We could conclude that Wilmittia is a real genus composed of different species and the definition of species based on 16S rRNA and ITS until need more studies.

References: Strunecky, O., Elster, J., Komarek, J. Taxonomic revision of the freshwater cyanobacterium “Phormidium” murrayi = Wilmottia murrayi. Fottea, v. 11, n. 1, p. 57-71, 2011.

P45 Stationary Phase Mutagenesis in Bacillus subtilis Mediated by Induction of comK

Tatiana S. Ermi, Carmen Vallin, Amanda Kidman and Eduardo A. Robleto

Department of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154 The gram-positive bacterium Bacillus subtilis has the notable ability of differentiating into distinct subpopulations when it encounters environmental stress. Under conditions of stress the ability to generate genetic diversity becomes important. Of interest to my project is the competence subpopulation. Competence propagates genetic diversity and mediates the acquisition of new alleles through uptake and recombination of exogenous DNA. In B. subtilis, it is believed that the competence subpopulation also undergoes stationary phase mutagenesis (SPM) or mutations arising in non-growing conditions. Previous work in our lab has shown that when ComK, a master regulator of competence, was knocked out, SPM levels were effected indicating that genes under its control could be important for SPM. My project is looking at two genes under ComK control, maf and comGA. By utilizing an inducible comK strain, I will place cells under non-dividing conditions and measure mutations.

Results from these studies will provide evidence for how the competent subpopulation creates genetic diversity in response to stress, in this case through SPM. Ultimately, these experiments will increase our understanding of how mutations and evolution occur in all domains of life.

P46 Immunological Characterization of Virus-Like Particles Produced Both in Plants and by

Live Vaccinia Virus Vectors as a Vaccine Candidate for HIV

Lydia R. Meador1,2,3, Will Martelly2, Tsafrir S. Mor2,3, and Bertram L. Jacobs2,3

1Ira A. Fulton Schools of Engineering, Arizona State University, Tempe, AZ 85287 2School of Life Sciences, Arizona State University, Tempe, AZ 85287

3Center for Infectious Diseases and Vaccinology at The Biodesign Institute, Arizona State University, Tempe, AZ, 85287

While antiretroviral therapy has greatly slowed progression to AIDS, the majority of infected people live in poor or impoverished countries without access to treatment making the lack of a preventative vaccine candidate more apparent than ever. The RV144 clinical trial produced the most promising results to-date using a non-replicating canarypox viral vector and boosting with proteins, but the modest 31% efficacy left room for improvement. Our project builds upon this system via a novel combination of a replicating but highly attenuated strain of Vaccinia virus and plant-produced HIV virus-like particles (VLPs), making a cost-effective, scalable vaccine production platform. VLP production in plants involves a gram-negative bacterium and a viral-based expression vector. We sought to determine whether the method of production can stimulate the innate immune system. VLP activation of toll-like receptors (TLRs) was assayed both in vitro and in vivo using cell lines and MyD88-/- and TICAM-/- mice. VLPs stimulate a TLR-dependent activation of macrophages resulting in production of cytokines correlated with a Th2 immune response. IgG isotyping confirms the Th2 bias induced by VLPs and antibody production in mice is dependent upon TLR stimulation. These results indicate that production of HIV VLPs in plants is uniquely placed to activate innate immunity, which do factor in immunogenicity of this vaccine candidate, making plants an attractive system for future VLP-based vaccines for many infectious diseases.

P47 Analysis of the Acyl Carrier Protein in the Cyanobacterium Synechocystis sp. PCC 6803

Sharon Wu, Daniel Brune and Wim Vermaas

School of Life Sciences, Arizona State University, Tempe, AZ, 85287

Acyl Carrier Protein (ACP) is a small, acidic protein that plays an essential role in fatty acid synthesis by elongating fatty acid chains. ACP was extracted from a strain of Synechocystis sp. PCC 6803 where ACP is overexpressed, where acyl-ACP synthetase has been deleted, and that contains a thioesterase enabling production of free fatty acids (laurate). The purpose of this study is to determine whether an acyl (fatty acid) group is typically bound to ACP as acyl-ACP is an inhibitor of early steps in fatty acid biosynthesis. Using ammonium sulfate precipitation to isolate a crude protein fraction containing ACP, immunoblot analysis was performed to determine relative amounts of free and acyl-ACP in the cell. The nature of fatty acids attached to ACP was determined by creating butylamide derivatives that were analyzed using GC/MS.

Immunoblot analysis showed a 1:1 ratio of acylated ACP to free ACP in the cell. From GC/MS data it was determined that palmitic acid was the predominate component of acyl groups attached to ACP. The results indicate that the significant presence of acyl-ACP is likely to be a limitation in fatty acid biosynthesis, and that it is necessary for ACP to be overexpressed and/or to have the palmitic acid cleaved off from acyl-ACP in order to synthesize optimal amounts of lauric acid to be used for cyanobacterial biofuel production.

P48 Clostridium difficile Infection Surveillance in a Tertiary Medical Center Reveals

Significant Strain Variation

Asad Mansoor1, Kareem Shehab2, Farhan Anwar1, V.K. Viswanathan1 and Gayatri Vedantam1

1School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, 85721

2College of Medicine, University of Arizona, Tucson, AZ, 85724

Clostridium difficile infection (CDI) is typically precipitated by antibiotic-mediated clearance of gut microbiota. While community-associated CDIs have been reported, few studies have parsed between hospital-acquired and hospital-precipitated CDI. Currently, there are no clear molecular ‘signatures’ that distinguish hospital-acquired from community-associated strains. Objective: To monitor Clostridium difficile (CD) frequency, and compare isolates from patients, as well as those recovered from the hospital environment. Methods: CD prevalence was monitored in a 487-bed hospital during two 3-month periods in Years 2011 and 2015. In 2015, only Intensive Care Units (ICUs) were monitored, and both patients and hospital surfaces targeted. For patient specimens, CD was cultured from stool, and characterized via ribotype analysis. For abiotic surfaces, wards and common areas were swabbed, and cultured for CD. Unique isolates were also assessed for toxin production. Results: A total of 53 (in 2015) and 21 (in 2011) unique CD isolates respectively, were obtained from patients, and belonged to a wide range of ribotypes (>30). However, the 2015 study revealed an increase in the “outbreak-associated” RT027 ribotype. CD isolates – even those of identical ribotype - expressed a wide range of toxin levels, suggesting that they were not clonal. Of 150 ICU surface swabs, CD was isolated from two localized areas, and was of RT027. Conclusions: The distribution of CD ribotypes in patient isolates suggests that patients may acquire CD in the community, but that CDI itself is likely hospital-precipitated. Prospective monitoring of all patients could therefore be effective in defining the burden of community-acquired CDI.

P49

Modulating T Cell Homing and Differentiation via Culturing with Vitamin A Analogues

Jonathan P. Burbige1,2, Kavita Manhas1,2, Joseph N. Blattman1,2

1Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University, Tempe, AZ, 85281

2School of Life Sciences, Arizona State University, Tempe, AZ, 85281

Vitamins A and D are known to serve important roles in modulating the T cell homing and responses, particularly to skin and mucosal sites. Our previous work has shown that the T cell response is dependent on the form of vitamin A used. Administration of exogenous ATRA during vaccination improved mucosal responses during viral challenge. Additionally, transfection with a RALDH2 containing plasmid concurrent with vaccination also resulted in an improved mucosal response. However, ATRA administered at the time of vaccination increased the amount of central memory T cells (TCM) detected in the spleen, which wasn’t seen with the RALDH2 treatment. However, ATRA is relatively unsuitable for use as an adjuvant in vaccines due to teratogenic activity, incompatibility with recombinant vaccines, and general instability when exposed to light and heat. Rexinoids, however, are synthetic vitamin A analogues that are more stable, being non-reactive to light and withstanding a wider range of temperatures, and are potentially more compatible to be used as adjuvants during vaccination. We began by testing whether rexinoids are able to induce similar changes in phenotypic expression in mucosal homing. T-cells were harvested from healthy p14 mice and plated in RPMI with IL-2 and gp33-41 as well as varying forms of rexinoids. Initial results showed that some of the rexinoids induced greater amounts of CCR9 expression, indicating that they can induce similar increases to mucosal responses as transfection with ATRA and a plasmid bearing RALDH2. Following this, the next step will be to repeat the experiment with the same experimental conditions but to instead measure the degree to which the rexinoids are able to alter the differentiation of T cells into TCM and TEM as well as tests to determine how altering the concentration of rexinoid used may change the phenotype of the T cell.

P50 Exploring the Role of the Mycobacterium tuberculosis Putative Toxin-Antitoxin Rv3642c-

Fic During Macrophage Infection.

Ryan A. LaMarca1, D. Mitchell Magee2, and Shelley E. Haydel1,3

1School of Life Sciences, Arizona State University, Tempe, Arizona 85287,United States

2The Biodesign Institute Center for Personalized Diagnostics, Arizona State University, Tempe, Arizona 85287,United States

3The Biodesign Institute Center for Infectious Disease and Vaccinology, Arizona State University, Tempe, Arizona 85287,United States

Fic proteins, found throughout nature, perform post-translational modifications (PTM) with the majority of characterized Fic proteins catalyzing AMPylation, the transfer of an AMP to a target

protein. Some bacterial Fic proteins with AMPylation activity function as secreted virulence toxins that target host GTPases, while other Fic proteins inhibit bacterial growth by regulating DNA replication. Furthermore, the activity of bacterial Fic proteins can be regulated by a small α-inhibitor protein, allowing the two proteins to function as a toxin-antitoxin (TA) module. Mycobacterium tuberculosis (Mtb) Fic can hydrolyze ATP in vitro, demonstrating that the recombinant Fic protein has catalytic activity. To determine if Fic has PTM activity, we examined Fic expression in HeLa cells and observed that Fic, but not catalytically inactive Fic H144A, caused cell rounding. We also found that Mtb Rv3642c, a putative α-inhibitor protein, forms a complex with Fic when both proteins are co-expressed in E. coli, implying possible TA module interactions. To investigate recombinant Mtb Fic catalytic activity, we are currently performing in vitro AMPylation assays with the Rac1 GTPase and HeLa and Mtb protein cell lysates. Additionally, we generated Mtb fic, Rv3642c, and Rv3642c-fic allelic exchange mutants and are determining the ability of these mutants to infect and survive in THP-1 human macrophage-like cells. With these ongoing studies, we aim to identify Mtb Fic targets and delineate the role of the Rv3642c-Fic complex during Mtb macrophage infection.

P51

Growth Phase Affects MALDI-TOF Mass Spectra of Chlorella vulgaris Cultures

Duane Barbano1, Anthony Gutierrez2,3 and Todd R. Sandrin2

1School of Life Sciences, Arizona State University, Tempe, AZ, 85281 2School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, 85069

3Biosciences Department, Phoenix College, Phoenix, AZ, 85013

Current methods for monitoring microalgae growth over time are limited in terms of accuracy, time requirements, and cost. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a viable alternative for monitoring microalgae growth. Growth of Chlorella vulgaris UTEX 395 was measured using optical density at 750 nm followed by MALDI-TOF mass spectrometry analysis every 24 hours. The overarching objective of this work was to determine if MALDI-TOF MS is sensitive enough to detect changes in a microalgae culture based on culture age. Rapid mass spectrometry based analysis of Chlorella vulgaris UTEX 395 was facilitated through the use of a common protein extraction method. Resulting spectra contained between 21 and 73 peaks within the m/z 2,000 to 20,000 range. MALDI-TOF MS yielded unique mass spectra for C. vulgaris depending on the age of the culture. For example, spectra from the late stationary phase of growth were different from other growth phases with many growth phase-specific peaks in the low mass range. Spectra from earlier growth phases shared peaks, such as m/z 2043, 2638, and 2659. Interestingly, spectra from late stationary phase cultures did not share these peaks and exhibited unique peaks at m/z 2033, 2862, and 3018. This report of growth phase-specific MALDI-TOF spectra of microalgae suggests that: 1) the performance of MS-based approaches to microalgae characterization may require standardization and 2) MALDI-TOF may be useful for the rapid characterization of microalgae growth phases and monitoring of culture health.

P52 Genes Within a Clostridium difficile Capsule Biosynthetic Locus Influence Bacterial

Shape, Polysaccharide Production and Virulence

Michele Chu1, Michael Mallozzi1, Bryan P. Roxas1, Lisa Bertolo5, Mario Monteiro5, Al Agellon1, V.K. Viswanathan1,2,3, Gayatri Vedantam1,2,3,4

1School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, 85721

2Department of Immunobiology, University of Arizona, Tucson, AZ, 85721 3BIO5 Institute for Collaborative Research, University of Arizona, Tucson, AZ, 85721

4Southern Arizona VA Healthcare System, Tucson, AZ, 85723 5Department of Chemistry, University of Guelph, Guelph, Ontario, N1G 2W1 Canada

Clostridium difficile (CD) has been designated as an urgent threat by the Centers for Disease Control. Our studies have revealed key roles for non-toxin virulence factors in CD infections (CDI). We previously reported on the immunogenic potential of PSII, a cell wall glycopolymer (CWG). In this work, we explored the contribution of a conserved CWG biosynthetic locus to PSII production, bacterial morphology, and CD virulence. Attempts at disruption of various genes (5 tested, including the predicted CWG flippase, mviN) failed to recover mutants, suggesting that these genes are essential for CD survival. asRNA-mediated mviN downregulation retarded CD growth, reduced capsule production, and decreased PSII deposition. A genetic disruption was successful, however, for lcpB, a gene with a putative role in CWG cell-surface attachment. The lcpB mutant was defective in growth and PSII deposition, and displayed a distinct helical and elongated morphology, suggestive of a cell septation defect. The lcpB mutant also had a hyper-sporulation phenotype, but no difference in toxin production compared to the parent strain. Further, the lcpB mutant displayed heightened virulence in the hamster model of infection (100% lethality by day 4 post-infection, compared to day 7 for the parent strain). Overall, genes in the CD CWG biosynthetic cluster are essential for survival and, likely, for production/assembly of key antigenic surface polysaccharides, such as PSII. Since CWGs fundamentally affects key aspects CD pathogenesis, we are currently exploiting their biology to engineer next-generation anti-CDI biotherapeutics and vaccine candidates.

P53

Interactions between the HIV-1 Protein gp41 and Calmodulin and its Effect on Cytotoxicity When Expressed in Nicotiana benthamiana

Naji-Talakar, Siavosh1; Meador, Lydia1,2; and Mor, Tsafrir S.1,2

1School of Life Science, Arizona State University, Tempe, Arizona, 85281 2The Biodesign Institute at Arizona State University, Tempe, Arizona, 85281

HIV-1 virions display two surface proteins, gp120 and gp41, with key antibody targets. Gp120 protein is very immunogenic but highly mutable and used by HIV as a decoy. The gp41 protein is also immunogenic but is more highly conserved in specific regions. Using a modified version

of gp41, deconstructed gp41 (dgp41), we may expose the membrane proximal external region (MPER). The MPER is a target for broad neutralizing antibodies (bNAbs) such as 2F5, 4E10, 10E8. These antibodies are very valuable in the immune system, however, they may take years to develop in an infected patient. Gp41 is highly toxic in plant and mammalian expression systems. Recent data suggests this could be due to ER stress through a calmodulin (CaM) dependent Ca2+ signaling pathway. Gp41 binds CaM at its C-terminus. In this project, we will clone a point mutation, A835W, into the dgp41 gene. This mutation has been shown to disrupt CaM interactions with gp41 and reduces cytotoxicity in mammalian cell culture. If this mutation succeeds in preventing CaM to bind to dgp41, then we may produce the dgp41 protein with reduced toxicity in the plant system. We can then proceed to make transgenic plants, which can successfully express the protein and further the progress of achieving a HIV vaccine.

P54 Reevaluation of DNA Sequences Required for Transcriptional Regulation of the Virulence

Gene, icsP, in Shigella flexneri

Alexander D. Karabachev, Makensie E. Millar, Krystle L. Pew and Helen J. Wing

School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154-4004

The VirB protein is a key regulator of virulence promoters in the human bacterial pathogen Shigella flexneri, the causative agent of bacillary dysentery. VirB works through transcriptional antisilencing via an unknown mechanism to alleviate H-NS mediated repression. Our work at the icsP promoter has shown that VirB-dependent regulation occurs from a remotely located inverted repeat over 1 kb upstream of the transcription start site and must function in cis. While a 7 to 8 nucleotide-binding site for VirB has been proposed, the VirB binding site at the icsP promoter requires 15 nucleotides, a 14 nucleotide inverted repeat separated by 1 nucleotide. Here, I show that VirB-dependent regulation of the icsP promoter requires four guanines directly upstream of the essential inverted repeat. Further analysis of this site demonstrates that the four guanines that lie on only one side of the inverted repeat are also required for binding. Since the four guanines do not appear in any previously proposed VirB binding sites, my finding challenges our current understanding of the DNA sequences required for VirB binding and regulation. An in silico analysis of other VirB-dependent virulence promoters in S. flexneri show that in many cases, guanines upstream from the inverted repeat are conserved. A firmer understanding of the DNA sequences required for VirB-dependent regulation helps elucidate a key aspect of transcriptional regulation in this group of enteric bacteria.

P55 Plant-Derived Monoclonal Antibodies as Efficacious and Safe Therapeutics to Treat

Dengue Virus Infection

Adrian Esqueda, Jonathan Hurtado, Matthew Dent, Huafang Lai, Qiang “Shawn” Chen

Center for Infectious Disease and Vaccinology, The Biodesign Institute at Arizona State University

School of Life Sciences, Arizona State University

Dengue Virus (DENV) is the most common mosquito-transmitted virus in the world, with ~50 to 100 million infections per year. DENV infection causes a spectrum of disease ranging from Dengue Fever (DF) to the life-threatening Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS). One of the major impediments towards developing vaccines and antibody-based therapeutics for DENV is the risk of Antibody-Dependent Enhancement (ADE), which may render vaccinated or anti-DENV antibody treated subjects more susceptible to DENV infection. ADE occurs because pre-existing sub-neutralizing concentrations of antibodies, including therapeutic monoclonal antibodies (MAbs), and the infecting DENV form complexes that bind to Fc-γ receptor (FcγR)-bearing cells, resulting in increased virus uptake and subsequent viral replication. The binding of human IgG to FcγR is highly sensitive to the presence and the nature of N-linked glycosylation at position N297 in its CH2 domain. However, the underlying mechanism of how individual glycoforms affect MAb binding to FcγRs and their role in modulating effector functions and ADE remains unclear. This study aims to define how Fc glycosylation of MAbs affects ADE by taking advantage of the unique ability of plants in producing proteins with homogenous glycans. Our study demonstrated that specific glycoforms of an anti-DENV MAb that neutralizes all four DENV serotypes diminishes the risk of ADE, thus, gaining therapeutic activity in vivo. In addition to generating safer therapeutic reagents for DENV, this study will enhance our understanding of how glycan variation on IgG MAbs modulates effector function, which could potentially lead to the development of MAbs against other diseases by manipulating the selection of particular glycoforms best suited for efficacy and safety.

P56 Subcellular Localization of VirB in Shigella flexneri

Jillian N. Socea1, Grant R. Bowman2, and Helen J. Wing1

1School of Life Sciences, University of Nevada-Las Vegas, Las Vegas, NV 89154 2Department of Molecule Biology, University of Wyoming, Laramie, WY 82071

VirB is an essential transcriptional regulator of genes located on the virulence plasmid of the bacterial pathogen Shigella flexneri, which causes dysentery in humans. VirB is unique in the sense that it is not related to any known transcriptional regulators. Its closest homolog is a

plasmid-partitioning protein known as ParB. Importantly though, VirB and ParB do not share any functional overlap. During plasmid partitioning, the ParB protein is seen to form discrete foci at the cell poles. This subcellular localization is essential for the plasmid partitioning function of ParB. The goal of my project is to investigate whether VirB has a subcellular localization and if so, to determine what role this plays in the transcription regulation of Shigella virulence genes. I hypothesize that VirB will form multiple discrete foci in each cell and that these foci will be indicative of VirB binding to specific DNA sites on the Shigella virulence plasmid. In order to test this hypothesis, I have constructed two plasmids containing virB fused to a multi-superfolder GFP (msfGFP) at either the 5’ or 3’ end of the virB gene. To determine if the msfGFP-tagged VirB proteins are capable of regulating gene expression at the same level as untagged VirB, b-galactosidase assays using a PicsP-lacZ transcriptional reporter plasmid will be used. To determine the concentration of inducer required to express these tagged proteins at physiologically relevant levels, Western blots will be used. Finally, the GFP-tagged VirB constructs will be visualized in cells using both phase contrast and fluorescence microscopy and subcellular localization phenotypes will be quantified. In combination, this work will provide a better understanding of the function of this important virulence factor of Shigella pathogenesis.

P57

Quantifying the Effect of Anti-viral Vaccination by Memory CD8+ T Cells

Charles K. Miller1, Susan A. Holechek1,2,3, Joseph N. Blattman1,2

1School of Life Sciences, College of Liberal Arts and Sciences, Arizona State University, Tempe, AZ, 85281

2Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ, 85281

3Simon A. Levin Mathematical Computational Biology and Modeling Sciences Center, Arizona State University, Tempe, AZ, 85287

Memory CD8+ T-cells can persist in the absence of antigen, primed for immediate activation and proliferation if later exposed to the same antigen. These cytotoxic lymphocytes provide long-term immunity following an acute infection. Studies have observed that intermediate levels of general T cell transfer prior to infection may cause an inappropriate response resulting in increased pathology rather than prevention. Therefore, our study focused on a memory CD8 T-cell therapy using lymphocytic choriomeningitis virus (LCMV) specific splenocytes, which activate and proliferate at an accelerated pace compared to that of naive T-cells. LCMV is a natural murine pathogen, which also poses a zoonotic infection threat to humans, and the effect of immune cell vaccination therapies for LCMV is not fully understood. We observed the effect of multiple memory CD8 T cell dosage levels on overall disease and memory CD8+ T-cell response to the virus. Infection by exposure to a carrier was shown to have a reduced impact on mice receiving higher doses of memory T cells prior to infection compared to mice receiving less or no memory cells. Higher presence of activated memory cells were shown to correlate with less disease-related weight loss and accelerated recovery times. Survival rate was not shown to

be affected by dosage level, suggesting that memory T cells are not directly responsible for pathology observed in other studies.

P58

Role of the Secreted Protein EspZ in the Virulence of Enteropathogenic Escherichia coli

Shylaja Ramamurthy1, Jennier Lising Roxas1, John Scott Wilbur1, Gresa Sylejmani1, Gayatri Vedantam1,2,3, V.K. Viswanathan,1,2,

1School of animal and comparative biomedical sciences, Tucson, AZ 85721 2Department of Immunobiology, Tucson, AZ 85721.

3The Bio5 institute for collaborative research, University of Arizona, Tucson, AZ-85721.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea, particularly in developing countries. EPEC belongs to the attaching and effacing (A/E) family of pathogens. All A/E pathogens harbor a type III secretion system (T3SS) that delivers virulence proteins directly into host epithelial cells. These proteins mediate diverse structural and functional alterations that likely facilitate pathogenesis. We recently demonstrated that EspZ, a secreted protein unique to A/E pathogens, is a critical virulence factor and that mutant strains lacking espZ are impaired for pathogenesis in both mouse and rabbit models of infection. EspZ prevents premature death of cultured intestinal epithelial cells by inhibiting intrinsic apoptosis. We hypothesized that EspZ promotes cell survival by engaging host proteins. Yeast two-hybrid studies identified the mitochondrial fission protein, hFis1, as a putative EspZ interactor. Co-immunoprecipitation studies confirmed EspZ-hFis1 interaction, and hFis1 was shown to be re-distributed in infected cells. These observations are consistent with the established role of hFis1 in intestinal cell survival pathways. The goal of my studies is to validate hFis1-EspZ interactions in epithelial cell cyto-protection and, eventually, to establish the significance of this pathway in EPEC virulence.

P59 Establishing a Mouse Model for Congenital LCMV Infection

Kristen N. Morrow1, Susan A. Holechek1,2 and Joseph N. Blattman1,2

1School of Life Sciences, Arizona State University, Tempe, AZ 85281

2Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85281

3Simon A. Levin Mathematical Computational Biology and Modeling Sciences Center, Arizona State University, Tempe, AZ, 85287

Pathogens such as lymphocytic choriomeningitis virus (LCMV) cause spontaneous abortions in mice when exposure occurs during early pregnancy; however, exposure at later stages of pregnancy and after birth causes a chronic, lifelong infection associated with T cell tolerance. Although tolerated, the virus is responsible for major neurological effects that impair the

behavior of these mice. To study the evolution and neuropathogenesis of the virus, we needed mice chronically infected with LCMV. To establish congenital infection, newborn C57BL/6J mice were intra-cerebrally (i.c.) injected with 1 x 103 PFU LCMV Armstrong. Infanticide and abandonment following administration of the virus resulted in 87% mortality after two weeks, and surviving mice were seronegative for LCMV. Although known to be less efficient at producing congenital carriers, intraperitoneal (i.p.) injection has reduced mortality, so we attempted to establish carriers in this way. While mortality among the pups was greatly reduced in the two weeks following injection, no mice were seropositive for the virus. A higher concentration of LCMV may be necessary to induce this state through i.p. injection, and a lower concentration of LCMV may decrease the mortality following i.c. injection and result in the generation of carriers. Additionally, injection of pregnant mice may be necessary to establish the carrier state in offspring.

P60

Clostridium difficile Flagellar Components Influence Toxin Synthesis, Pathogenicity and Activation of Innate Immune Responses

Andrew E. Clark1, Bryan A.P. Roxas1, VK Viswanathan,1,2,3 and Gayatri Vedantam1,2,3,4

1School of Animal & Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, 85721

2Department of Immunobiology, University of Arizona, Tucson, AZ, 85721 3BIO5 Institute for Collaborative Research, University of Arizona, Tucson, AZ, 85721

4Sourthern Arizona VA Healthcare System, Tucson, AZ, 85723

Clostridium difficile (CD) causes antibiotic-associated diarrhea (CDI), and outbreak-associated C. difficile strains (OA-CD) have worldwide prevalence. OA-CD strains are associated with increased morbidity, mortality, disease severity and incidence of relapse; however, the biological basis for the elevated virulence of these strains remains undefined. In this study, a comparative proteomics approach identified differentially abundant proteins in 11 OA-CD strains when compared to a genetically-related, non-OA-CD ancestor. Numerous OA-CD specific dysregulated proteins were identified, including components of the flagellar system. Isogenic flagellin (fliC) and capping protein (fliD) mutations were constructed in both OA-CD and ancestral strain backgrounds. Both genes were found to be necessary for motility, and both mutants secreted elevated levels of toxins. Both mutants were non-motile and did not assemble flagella, and fliD mutants hypersecrete FliC. Loss of FliC and FliD also led to altered levels of attachment to human intestinal epithelial cells (Caco2) in vitro, changes in cell surface properties, and differential disease and pathology in the hamster model of acute CDI. Flagellar mutants did not exhibit colonization differences in a wild type (C57B6) mouse model, but impacted both pathology and disease progression in a TLR5-deficient transgenic mouse. These mutants also differentially induced inflammatory markers in Caco2 cells, suggesting that CD fliC and fliD mutants influence CDI through non-redundant mechanisms. CD flagellar mutants are currently being further assessed for their ability to activate the innate immune response, and to

interrogate the mechanistic relationship between the synthesis of flagellar components and toxin secretion.

P61

Combination Therapy for Pediatric Metastatic Osteosarcoma

Joseph N. Blattman1,2, Nicole Appel1,2, Rebecca McCall Cook1,2 and Nicole Labban1,2

1School of Life Sciences, Arizona State University, Tempe, AZ 85281 2Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State

University, Tempe, AZ 85281

Osteosarcoma is one of the most common bone cancers in children and adolescents, with 85% of metastatic osteosarcoma cases resulting in pulmonary metastases. The survival rate for this disease remains at 20%, and is raised only to about 35% when treating with aggressive resection of pulmonary metastases. The remarkably low survival rate of metastatic osteosarcoma calls for the development of novel therapies: our past studies have shown that using the combinatorial blockade therapy utilizing the mAbs, PD-L1 and CTLA-4, led to complete control of metastatic disease in 60% of Balb/c mice. Combinatorial blockade therapy evaded the issue of mAb PD-L1 resistant tumors, which was previously seen when mAb PD-L1 was used alone. Other studies have shown that irradiation therapy, a current standard of care in cancer treatment, paired with a PD-L1 blockade therapy amplified tumor sensitization to the PD-L1 mAbs by increasing PD-L1 expression on tumor cells. We wanted to see if pairing the combinational immunotherapy with radiation would increase survival in mice with metastatic osteosarcoma. To determine if the treatments worked together synergistically, the PD-L1 & CTLA-4 blockade therapy was administered with irradiation therapy in Balb/c mice using the K7M2 model of metastatic osteosarcoma. Although further data is needed, the combination of therapies slightly increased average lifespan while also appearing to alleviate symptoms of disease in the mice.

P62

Mutational Activation of Drug Efflux Pump Genes in Escherichia coli

Hyun Jae Cho, Rajeev Misra

School of Life Sciences, Arizona State University, Tempe, AZ, 85287

Multi-drug resistance (MDR) among human bacterial pathogens has been steadily rising at an alarming rate. A means by which bacteria achieve MDR is by expelling drugs though the membrane-bound efflux system. In Escherichia coli, one of the major and constitutively expressed drug efflux systems is composed of TolC, AcrA, and AcrB proteins. In addition to AcrAB, TolC—the outer membrane component—can interact with seven other efflux pumps to

confer a MDR phenotype. However, unlike AcrAB, the other TolC-dependent MDR pumps are either weakly or not expressed. This study was undertaken to investigate how the dormant MDR pumps can be activated in the absence of AcrAB.

Initially drug resistant mutants were isolated from an E. coli strain expressing a defective AcrAB efflux pump. Strikingly, in all mutants the resistance was determined to be caused by the activation of the AcrEF pump, which has the substrate specificity very similar to AcrAB. Consequently, in our second attempt drug resistant mutants were sought using a strain simultaneously lacking AcrAB and AcrEF. All resistant mutants analyzed were found to be TolC dependent. We then employed a knockout library of the non-essential E. coli genes, and determined that efflux pumps that are responsible for the drug resistance phenotypes as in MdtEF, MdtABC, and MacAB efflux pumps.

As the efflux pumps were determined, HFR conjugation was introduced as a mapping strategy to locate the mutation that activated the efflux pumps. Among mdtABC dependent mutants (ΔacrAB ΔacrEF mdtABC), HFR conjugation and subsequent knockout strains introduction revealed that the mutations in baeSR two component regulatory system activated transcription of mdtABC efflux pump. To confirm the locations of mutations, primers were built to amplify baeS gene. As a result, four different mutations within baeS were found.

Together, our analyses demonstrate the potential for bacteria to mutationally activate the dormant drug efflux pumps when the constitutively expressed AcrAB pump is either disabled by an inhibitor or a mutation.

P63

Inhibition of VACV E3 N-Terminal Deletion Mutants can be PKR-Independent

Samantha Cotsmire1,2,3, Heather Harrington1,2, Shizuka Barclay1,2, Bertram Jacobs1,2

1Arizona State University Center for Infectious Diseases and Vaccinology 2ASU School of Life Sciences

3ASU School of Biological and Health Systems Engineering

Pox viruses such as Vaccinia virus (VACV) and Monkeypox virus (MPXV) produce RNA transcripts that extend beyond the transcribed gene which can form double-stranded RNA (dsRNA). Because the innate immune system can easily recognize dsRNA, poxviruses produce E3 to mask dsRNA. It contains a dsRNA binding domain in its C-terminus; the N-terminus of E3 contains a domain that binds to Z-form nucleic acids. Both must be present for pathogenicity in murine models, but the mechanism of the N-terminus is unclear. When we compared a vaccinia virus with a wild-type E3 protein (VACV wt) to one with an E3 N-terminal truncation of 37 amino acids (VACV E3Δ37N), phenotypic differences appeared in several cell lines. In HeLas and certain MEFs, dsRNA recognition pathways like PKR become activated during VACV E3Δ37N infections; unlike VACV wt. Additionally, VACV E3Δ37N cannot grow in the murine cell line JC whereas VACV wt can. The MPXV homologue, F3, has an N-terminal truncation of 37 amino

acids. However, MPXV does not activate PKR in HeLas or MEFs and replicates in JCs. Additionally, our investigation determined that MPXV produces less dsRNA than VACV. We made VACV E3Δ37N more similar to MPXV by selecting mutants that produce less dsRNA. These mutants no longer activated PKR in HeLa cells or MEFs restoring the wild-type phenotype. However, these mutants were still unable to grow in the JC cells. We have explored the differences between these cell lines to determine that JCs inhibit virus growth without being completely dependent on PKR.

P64

Genotyping Yersinia pestis from North America Using Amplicon Sequencing

Heather Centner1, Carina M. Hall1, Jason Sahl1, Jim Schupp2, Paul Keim1 and David M. Wagner1

1Center for Microbial Genetics and Genomics, Northern Arizona University, 1298 S Knoles Drive, Flagstaff, AZ 86011, USA

2Translational Genomics Research Institute, Flagstaff, AZ, USA Yersinia pestis, the causative agent of plague, is a gram-negative bacterium responsible for the deaths of millions of people worldwide. Transferred via fleas on ship rats, plague came to North America from Hong Kong at the turn of the twentieth century. Since then, plague has established itself in native prairie dog and ground squirrel populations in the western half of the United States. As Y. pestis has spread throughout the U.S., it has developed mutations called single nucleotide polymorphisms (SNPs). These SNPs can be used to create a unique DNA fingerprint which can then be used to determine where plague was introduced into the U.S. and how it has spread to other areas of the country. In order to better understand this movement of plague, we whole genome sequenced a subset of Y. pestis DNAs from North America. The sequence data we obtained allowed us to identify 385 novel SNPs. In an effort to identify the SNP states in 820 other un-sequenced samples, we utilized a technique called amplicon sequencing. This cutting edge approach offers distinct advantages over more traditional SNP typing techniques. We were able to optimize the amplicon sequencing system to process a remarkably large subset of Y. pestis DNAs with unparalleled efficiency and cost-effectiveness. The data we will generate using the amplicon sequencing approach will allow us to create a more comprehensive Y. pestis phylogeny in a timely and cost-effective manner.

P65

MAGPIX: a New Technological Tool for Diagnostics and New Vaccines Development for Infectious Diseases

Isaiah K. Self, Erik Settles, Dawn N. Birdsell, David M. Wagner, and Paul Keim

Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USA

Microbial pathogens of diverse origin (bacteria, fungi, viruses) have the ability to compromise human health by causing infectious diseases of varying magnitudes. Developing diagnostic tools for disease confirmation and vaccines for disease prevention are important strategies for combatting infectious diseases. Many different traditional methods (i.e. ELISAs) are currently used to provide disease diagnoses and aid in vaccine development, but these methods are labor intensive and time-consuming. However, recent advancements in technology have allowed for the development of a new tool (MAGPIX) that holds promise to increased efficiency of diagnoses and in identifying biological components important for vaccine development as compared to traditional methods. The MAGPIX technology allows for the screening of single samples for multiple microbial infections or targets simultaneously within the same reaction. This design allows the MAGPIX platform to serve as a powerful potential diagnostic tool for identifying diseases from a broad range of diverse microbes. The purpose of our project was to validate the diagnostic capabilities of the new MAGPIX platform. In this study, we developed validation tools, identified optimal protocol conditions tested on a specific bacterial microbe (using Burkholderia pseudomallei as a test model), and demonstrated the enhanced performance capabilities of MAGPIX compared to traditional methods, such as the ELISA. Our study demonstrates that the MAGPIX technology is powerful and effective for rapid diagnostic confirmation and an informative tool for vaccine development.

P66 Characterization of TolC-AcrB Interactions in Efflux Pumps in Escherichia coli

Megan McFeely1, Rajeev Misra2

1Barrett, the Honors College, Arizona State University, Tempe, AZ, 85281 2School of Life Sciences, Arizona State University, Tempe, AZ, 85281

The spread of antibiotic resistant bacteria is currently a pressing global health concern, especially considering the prevalence of multi-drug resistance. Efflux pumps, bacterial machinery involved in various active transport functions, are capable of non-specific removal of antibiotics from the periplasmic space, frequently conferring multi-drug resistance. Many aspects of efflux machinery’s structure, functions, and inter-protein interactions are still not fully understood; further characterization of these components of efflux will provide a strong foundation for combating this resistance mechanism. In this project, we further characterize the channel protein TolC as a part of the AcrAB-TolC protein complex in Escherichia coli by first

determining the specificity of compensatory mutations in TolC as a response to defects in AcrA and AcrB, and then identifying TolC residues that might influence TolC aperture dynamics or stability when altered. Specificity of compensatory mutations was determined using an array of TolC mutants generated from defects in either AcrA or AcrB transformed to possess a different defect in AcrB; these new mutants were then analyzed with efflux and antibiotic susceptibility assays. A vancomycin sensitive mutant – a phenotype that has been associated with constitutively open yet unengaged TolC channels – was then used to generate vancomycin-resistant revertants which were evaluated with DNA sequencing, protein quantification, and efflux assays to identify residues important for TolC aperture dynamics and stability.

P67

Development of Antibody Reactive Markers to Rapidly Diagnose Patients Infected with Burkholderia pseudomallei

Lauren Pristo1, Karen Hernandez1, Jinhee Yi1, Christopher Allender1, Heidie Hornstra O’Neill1, Joseph D. Busch1, Erik Settles1, Francis Simpanya1, Mitch Keener1, Erin Price2, Mark Mayo2,

Bart J. Currie2, and Paul Keim1

1The Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USA

2 Global and Tropical Health Division, Menzies School Health Research and Infectious Diseases, Royal Darwin Hospital, Darwin, NT, Australia

Melioidosis is a lethal infectious disease caused by the bacterial pathogen Burkholderia pseudomallei. This soil-dwelling bacterium is endemic to Southeast Asia and Northern Australia. Individuals that acquire melioidosis face a 10-40% mortality rate. The disease is difficult to diagnose rapidly, especially at early stages of infection – when treatment is most needed – and currently there is no approved vaccine for it. Subsequently, there is a demand to develop rapid and dependable molecular assays based on blood antibodies that will facilitate rapid diagnosis and proper antibiotic treatment. To this end, we used clinical samples from 50 Australian melioidosis patients to characterize each person’s antibody response to the specific proteins in a B. pseudomallei culture grown from that person. Using immunogenic assays, we determined that patient antibody profiles were highly variable, with anywhere from 6 to 154 proteins raising an antibody response in each patient. Of these proteins, 17 have been selected as potential diagnostic markers because they reacted in up to 98% of the patients. Melioidosis patient samples collected at varying times after hospital admittance will be screened with these 17 protein markers to determine how soon in the infection melioidosis patients can be diagnosed. In future studies we will verify the specificity of these diagnostic markers by comparing blood samples from confirmed cases of melioidosis against patients that were hospitalized for a different reason. Our eventual goal is to confirm a set of candidate bacterial proteins to be used as diagnostic markers and in a potential vaccine against B. pseudomallei.

P68

Characterization of Functionally Relevant Residues Associated with the Drug Binding and Translocation Pathway of Escherichia coli Multi-drug Efflux Pump Protein AcrB

Mellecha Blake, Rajeev Misra

School of Life Sciences, Arizona State University, Tempe, AZ, 85287

Multidrug resistance bacteria are a major concern to global health. Their abundance seems to increase faster than the development of drugs that eliminate them. One of the major mechanisms MDR bacteria utilize is drug efflux. In E.coli, AcrAB-TolC proteins constitute the major chromosomally-encoded drug efflux system. AcrB, an asymmetric trimeric membrane protein of the Resistance-Nodulation-Division superfamily, is well-known for its promiscuity. It has the ability to efflux a broad spectrum of substrates alongside compounds such as dyes, detergent, bile salts and metabolites. The anatomy and mechanism of the drug binding and translocation pathway of AcrB is still largely unknown. However, work in our lab has identified several novel AcrB residues that were shown to be functionally relevant in drug binding and translocation pathway using a positive selection strategy. These residues — Y49S, V127A, V127G, D153E, G288C, F453C, and L486W — were identified as suppressors of an alteration, F610A, that confers a drug hypersensitivity phenotype. In this study we expanded our work to determine the function of these novel residues in AcrB activity. Using site-directed mutagenesis (SDM), a real-time efflux assay, and a classical minimum inhibitory concentration (MIC) assay, we were able to characterize their properties.

Three approaches were used for the characterization of these residues. The first approach focused on side chain specificity. Our results showed that certain suppressor sites prefer a particular side chain property, such as size, so as to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors, such as phe-arg-β-naphthlyamide (PAβN). Our results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the inhibitors. Our final approach was to determine whether suppressors mapping in the periplasmic (G288C) and trans-membrane (e.g. L486W) domains act by influencing the same or different pathways. Our results show that some combinations, e.g. D153E and L486W, demonstrate synergism (different pathway) while others, e.g. G288C and L486W show independence (same pathway).

To conclude, our approaches have given a deeper understanding of the mechanism by which novel suppressor residues of AcrB may overcome the functional defect of the drug binding domain alteration, F610A. Future studies could elucidate the anatomy and mechanism of this pathway for more effective drug developments.

P69

T-Cell Inducing Vaccines Offer Promising Potential and Significant Challenges

Megan McAfee, Joshua Carmen, Joseph Blattman

Center for Infectious Disease and Vaccinology, Arizona State University, Tempe, AZ 85287

T-cell responses are an extremely important part of the adaptive immune response. Antibody responses are sufficient to control some diseases, however, the importance of long lasting cellular immunity continues to gain greater recognition as research has grown, particularly in the last ten years. Unfortunately, cellular immunity is a double edged sword, equally capable of protecting the host from a virulent pathogen and creating massive disease in response to a mild infection. Pathology in infection is quite common and many diseases are deadly because of the host response to the pathogen rather than the effects of the infection itself. This is true for the Arenavirus family, which includes viruses which cause hemorrhagic fevers, often leading to death. This is directly contributable to the host’s production of cytokines in response to the infection. Using lymphocytic choriomeningitis virus (LCMV), the prototypic arenavirus, our lab previously demonstrated that CD8 T cells generated in response to infection or vaccination can induce either protection or pathology based largely on the number of T cells responding to the

pathogen. In short, small numbers of T cells result in T cell exhaustion and viral persistence, but low pathology and host survival. Large numbers of T cells result in viral clearance, low pathology and host survival. Intermediate numbers of T cells, however, result in high pathology and host mortality. Further, though the figure shows these results as memory CD8 T cells, we have shown the same results utilizing naive cells specific for LCMV. This shows that CD8 specificity has very little to do with the level of immunopathology. Moving forward from this point, we will begin to study the interaction of CD8 T cells with CD4 T cells during infection. The primary question is whether an active population of effector CD4 T cells will reduce

the immunopathology seen previously using only CD8 T cells. To accomplish this, we will utilize adoptive transfer of both CD4 and CD8 T cells specific for a known LCMV epitope from a transgenic mouse. By adding small, intermediate and large numbers of CD4 T cells along with varying numbers of CD8 T cells, we will determine what effect, if any, T helper cells have on cytotoxic T lymphocyte induced pathology during active infection.

P70

Regulation of Mycobacterium tuberculosis Cytochrome bd by the Essential PrrAB Two-Component System

Michelle D. Stephens1,2, Jason D. Maarsingh1, and Shelley E. Haydel1,2,3

1School of Life Sciences, 2Barrett, The Honors College, and 3 The Biodesign Institute Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287

In 2014, Mycobacterium tuberculosis (Mtb) caused 1.5 million deaths and latently infected another 2 billion people globally. The success of Mtb as a pathogen is dependent on its ability to adapt to the intracellular environment of macrophages and persist within the human host. To survive within the human host, Mtb utilizes two-component systems (TCSs) to sense environmental cues and respond by altering gene expression. The PrrAB TCS was initially identified during early stages of Mtb intracellular replication in human macrophages and was subsequently deemed essential for Mtb viability. This TCS consists of PrrB, a membrane-bound histidine kinase, and PrrA, a transcriptional regulator with a receptor domain and DNA-binding domain. Upon phosphotransfer, PrrA binds to target DNA sequences to modulate gene expression. We have generated phosphomimetic (D58E) and phosphoablative (D58A) forms of PrrA to mimic these phosphorylation states. In 2014, diarylthiazole (DAT) was identified as an anti-tuberculosis compound, which targets PrrB, thus confirming our hypothesis that the PrrAB TCS serves as a bona fide drug target. Mtb PrrA D58E overexpression repressed cydA, one of the genes encoding the cytochrome bd terminal oxidase, which is induced during hypoxia and chronic Mtb infection. Conversely, DAT-mediated PrrB inhibition induced cydA expression. Electrophoretic mobility shift assays demonstrated that PrrA specifically binds to the cydA promoter. These data reveal that the PrrAB TCS represses the non-proton-pumping cytochrome bd terminal oxidase, an enzyme that enables Mtb to transition from acute to chronic infection of mouse lungs and tolerate nitrosative and oxidative stresses.

P71

Factors that Modulate Production of Tryptophan and Serotonin by Gut Bacteria

Emily Yee1, Zehra Esra Illhan1, Rosa Krajmalnik-Brown

Swette Center for Environmental Biotechnology, Arizona State University, Tempe, AZ, 85287

Microorganisms can produce metabolites in the gut including short chain fatty acids, vitamins, and amino acids. Certain metabolites produced in the gut can affect the brain through changes in neurotransmitter concentrations, which is known as the brain-gut-axis. Serotonin, a neurotransmitter, is associated with mood, appetite, and sleep. Up to 80 or 90% of serotonin synthesis is located in the gut, by human enterochromaffin cells. Bacteria known to biosynthesize tryptophan, precursor to serotonin, include Escherichia coli, Enterococcus and Streptococcus. Tryptophan is synthesized by bacteria with the enzyme tryptophan synthase

and requires Vitamin B6, or Pyridoxal. We hypothesize that gut isolates from surgical weight loss patients can enhance tryptophan production, which relies on vitamin B6 availability. We isolated Escherichia and Klebsiella from human gastric bypass patient stool samples. Bacterial cultures were enriched with yeast and tested for tryptophan production using GC-FID. Bacterial cultures were also enriched with serine and indole, substrates necessary for tryptophan biosynthesis. Between the Escherichia and Klebsiella isolates, Klebsiella produced more tryptophan while under the serine and indole condition. We also tested a gradient of vitamin B6 on tryptophan production using Klebsiella cultures.

P72 Heterotrophic Bacteria from Three Contrasting Peatlands of the Amazon Basin

Kenneth Barker, Vanessa Hendrix, Karen Barker, Fatema Kermani, Jessica Spring, Mahoro Uwingiyhana, Reena Ygot, Analissa Sarno, Hinsby Cadillo-Quiroz

School of Life Sciences, Arizona State University, Arizona, 85287 The Amazon Basin is recognized to be a thriving biological hotspot supporting a vast array of life of both plant and animal species. While much is understood regarding the diversity and profusion of macro species living in this tropical climate, little is known about the microorganisms inhabiting this ecosystem. The role of heterotrophic soil microorganism has been found to have an extensive impact on important ecological cycles, including decomposition and the carbon cycle. For this reason, it is essential to understand the identity and the potential functional role these microorganisms have in tropical peatland soils. We amassed a collection of heterotrophic soil bacterial isolates from three locations within Amazon peatlands. After performing dilutions on the soil samples, the dilutions were plated in duplicate onto two types of media (CAT and R2G). Colonies were counted and isolated onto individual plates for purification through restreaking. The purified isolates were prepared for storage at -80 degrees Celsius. A unique method for expeditious DNA extraction was developed to allow for rapid preparation of the over 600 frozen isolates for 16S sequencing. Initial sequencing results of 100 fast growing isolates indicate the dominant presence of Pseudomonas dinitricans (30%), Cupriavidus sp (22%, Bulkholderiaceae), Novosphingobium (15%, Sphingomonadaceae), and new taxa within the Pseudoclavibacter sp (2%, Microbacteriacea), and novel Chitinophagaceae strains. Further identifications are underway. Our early results indicate that the soils of the Amazon seem to support an important variety of heterotrophic bacteria whose role in organic carbon decomposition can be experimentally evaluated with recovered isolates. Future evaluations of isolated diversity will reveal the ecophysiological role of known and novel heterotrophs in Amazon peatland soils.