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  • Undergraduate Poster Abstracts
  • Microbiology

    FRI-763 ELUCIDATING THE MECHANISM OF CADAVERINE IN THE NITROSATIVE STRESS RESPONSE OF UROPATHOGENIC ESCHERICHIA COLI

    • Kristen Clemons ;
    • Brittany Fleming ;
    • Matthew Mulvey ;

    FRI-763

    ELUCIDATING THE MECHANISM OF CADAVERINE IN THE NITROSATIVE STRESS RESPONSE OF UROPATHOGENIC ESCHERICHIA COLI

    Kristen Clemons1, Brittany Fleming2, Matthew Mulvey2.

    1Abilene Christian University, Abilene, TX, 2The University of Utah, Salt Lake City, UT.

    During a urinary tract infection, the infectious agent uropathogenic Escherichia coli (UPEC) elicits a number of host inflammatory response pathways, including the increased generation of reactive nitrogen species (RNS). Unlike non-pathogenic K-12 strains, UPEC can respond to and resolve this nitrosative stress. Previous research in our lab has indicated that UPEC adaptation to RNS is linked to the polyamine cadaverine. UPEC strains lacking either the cadA or cadC gene are unable to produce cadaverine; consequently, these mutant strains are unable to grow in the presence of 3mM RNS unless grown in cadaverine-supplemented media. Although implicating cadaverine, this data does not elucidate the mechanism by which cadaverine provides resistance to RNS. To determine this mechanism, the ΔcadA and ΔcadC strains were mutagenized and screened for loss of rescue by exogenous cadaverine. In this project, the genes identified by the screen were mapped via arbitrary PCR. Among the genes mapped were menA and yieN. To confirm the involvement of these genes, splicing overlap extension (SOE) PCR was used to construct menA and yieN SOE PCR products containing a tetR cassette. The menA SOE PCR product has been transformed into the E. coli K12 strain MG1655 and will be moved by phage transduction into UPEC strain UTI89. Upon construction of the UTI89 ΔmenA and ΔyieN strains, these genes’ role in the nitrosative stress response of UPEC will be tested by analyzing the mutant strain growth in the presence of RNS. Furthermore, other genes identified through the arbitrary PCR process will be investigated.

    THU-753 EFFECTS OF VARIABLE SUNLIGHT LEVELS ON GRAPE QUALITY IN BLANC DU BOIS CANOPIES

    • Jacqueline Olvera ;
    • Irvin Romero ;

    THU-753

    EFFECTS OF VARIABLE SUNLIGHT LEVELS ON GRAPE QUALITY IN BLANC DU BOIS CANOPIES

    Jacqueline Olvera, Irvin Romero.

    Scholars Academy, University of Houston-Downtown, Houston, TX.

    It is well-established that light levels in the grapevine canopy can impact the quality of grapes. Texas is currently growing a great deal of the disease-resistant wine grape variety Blanc Du Bois. However, there is no information about the optimal light levels in canopies of this variety. An experiment was conducted in 2 vineyards in Richardson, Texas, in order to determine the best light levels for Blanc Du Bois vineyards. Control vines were compared to 2 leaf removal treatments in a single vineyard row with randomized design and 9 replicates. Photosynthetically active radiation (PAR) levels were taken from each vine, and fruit samples were analyzed for degree Brix, pH, titratable acidity (TA), and pigment levels. Results indicated no significant difference in pH or degree Brix. However, there was a difference in TA levels at one vineyard. Pigment levels appeared to increase with even the moderate leaf removal treatment, and this difference was highly significant at one vineyard (p = 0.017). We plan to run further chemical analyses to determine if the fruit differs in chemical composition. Our results at this point indicate that varying amounts of sunlight do have an effect on some parameters of grape quality in the variety Blanc Du Bois.

    THU-759 MICROBIOTA PROBING AND IDENTIFICATION IN DAPHNIA MAGNA ADULTS

    • Aigbirhemwen Woghiren ;
    • Marilou Sison-Mangus ;

    THU-759

    MICROBIOTA PROBING AND IDENTIFICATION IN DAPHNIA MAGNA ADULTS

    Aigbirhemwen Woghiren, Marilou Sison-Mangus.

    University of California, Santa Cruz, Santa Cruz, CA.

    Daphnia species are commonly used as model systems in environmental genomics, host-parasite interactions, and ecotoxicology studies. A recent study reported that microbiota or the community of bacteria associating with Daphnia greatly influences the host’s fitness and reproductive success. Understanding the contribution of each microbiota member to the evolutionary and ecological adaptations of the daphnid host is therefore essential and needs to be assessed. To address these questions, we isolated and cultured different bacteria from the Daphnia hosts and did a pilot test of probing bacteria by fluorescent in situ hybridization (FISH) using oligonucleotide probes. Bacteria isolates were obtained from 6 adult Daphnia animals and cultured on Luria broth agar. Six bacterial clones were isolated and genotyped by DNA extraction using modified hotshot method, amplification, and sequencing of the bacterial 16s rDNA gene marker. Sequences were identified by basic local alignment search tool (BLAST) and have homologous sequences to Pseudomonas anguilliseptica, Pseudomonas peli, and Pseudomonas gessardii. Furthermore, FISH probing using EUB555 (a universal bacterial probe) and BET647 (beta-proteobacteria probe), were tested on a Pseudomonas (gamma-proteobacteria) isolate. The EUB555 oligonucleotide probe successfully binds to Pseudomonas,while BET647 did not bind to the bacterial DNA, suggesting that the FISH method can be effectively used to distinguish the locations of different microbiota in Daphnia animals. The FISH method will greatly facilitate studies that address questions on the transmission of microbiota in Daphnia. Bacterial isolates will be used in experimentally manipulating Daphnia- bacteria association to answer ecological and evolutionary questions in this crustacean model.

    FRI-762 SEQUENCE-BASED CHARACTERIZATION OF ENTEROCOCCUS PHAGE ΦKKAS003-1

    • Annika Gomez ;
    • Casandra Lyons ;
    • Michael Shiaris ;

    FRI-762

    SEQUENCE-BASED CHARACTERIZATION OF ENTEROCOCCUS PHAGE ΦKKAS003-1

    Annika Gomez1, Casandra Lyons2, Michael Shiaris2.

    1Cornell University, Ithaca, NY, 2University of Massachusetts Boston, Boston, MA.

    Bacteriophages infecting bacteria in the genus Enterococcus are widespread and have been isolated from a variety of sources. Despite their abundance, these phages are not well characterized and only 8 complete genome sequences are available. Using Illumina paired-end next generation DNA sequencing, we characterized a novel Enterococcus phage (ΦKKAS003-1) that was isolated from a sewage treatment plant. Sequencing yielded 128 scaffolds with a total length of 109,365 bp. Quality of the sequence data was too low to assemble a complete genome. The assembly contains 148 putative open reading frames (ORFs). Of these, 120 match sequences in the National Center for Biotechnology Information database, with putative functions identified for 40 of them. Most of the protein-encoding ORFs are structural or involved in capsid assembly or DNA processing. The phage lysed a variety of species in the genus Enterococcus. Phylogenetic analysis showed that ΦKKAS003-1 is a member of the Spounavirinae subfamily of the Myoviridae family. It, therefore, has a double-stranded DNA genome with an isometric capsid, long contractile tail, and is an obligate lytic phage. It is most closely related to Enterococcus phages EFDG1, ΦEF24C, and ECP3. Higher quality sequence data is needed to validate and expand on these results.

    FRI-753 CHARACTERIZING NOVEL SYMBIOSIS MUTATIONS BETWEEN BARREL MEDIC AND SINORHIZOBIUM MELILOTI

    • Hector Trujillo ;
    • Kathrin Wippel ;
    • Sharon Long ;

    FRI-753

    CHARACTERIZING NOVEL SYMBIOSIS MUTATIONS BETWEEN BARREL MEDIC AND SINORHIZOBIUM MELILOTI

    Hector Trujillo, Kathrin Wippel, Sharon Long.

    Stanford University, Stanford, CA.

    Plants require nitrogen for growth and survival. Although nitrogen is freely available in the atmosphere as N2, plants can only use nitrogen as NH3. Nitrogen fixation is the conversion of N2 to NH3, which is catalyzed by soil bacteria known as rhizobia. Certain plants, known as legumes, form an important symbiotic relationship with these rhizobia via the formation of root nodules, where nitrogen fixation takes place. Nodule formation depends on complex chemical interactions between the plant and bacteria that are not fully understood. To investigate the underlying mechanisms of nodule formation, we performed a forward genetics screen where we tried to link Fix- phenotypes (nodules that don’t fix nitrogen) to the underlying genotypes. To isolate Fix- nodules, Medicago truncatula seeds were randomly mutated with high-energy neutron bombardment. Viable seeds were grown and inoculated with their symbiont, Sinorhizobium meliloti, which carried 3 reporter constructs. These reporter constructs each tracked distinct stages of nodule formation and were used to chronologically assess when in the nodulation scheme nitrogen fixation was interrupted. The plants, however, were grown under media that stressed them to the point where the roots died in the process. Thus, we optimized the growth conditions to obtain the most meaningful data. For the future, finding and characterizing these novel Fix- phenotypes will lead to further mechanistic understanding of the general nodulation process.

    THU-772 COMPARING BACTERIAL ATTACHMENT ON THE MARINE DIATOM PSEUDO-NITZSCHIA

    • Sanjin Mehic ;
    • Marilou Sison-Mangus ;

    THU-772

    COMPARING BACTERIAL ATTACHMENT ON THE MARINE DIATOM PSEUDO-NITZSCHIA

    Sanjin Mehic, Marilou Sison-Mangus.

    University of California, Santa Cruz, Santa Cruz, CA.

    Harmful algal blooms are an increasing threat to the Pacific Coast. The year 2015 had one of the spatially largest, and temporally longest, Pseudo-nitzschia blooms in nearly 20 years. Pseudo-nitzschia blooms are often highly toxic and detrimental to wildlife. The neurotoxin produced by the algae can ripple through the entire food web creating a direct risk for seafood consumption and animal conservation. Research has implicated numerous environmental factors in the rise and demise of Pseudo-nitzschia blooms but it is still unclear whether bacterial interactions are mainly responsible for this ecological phenomenon. In addition, previous experiments find differences in Pseudo-nitzschia physiology when co-cultured with specific bacteria, however it remains unknown whether the attachment of bacteria to the host is a contributing factor to the differences in physiology. In this study, we seek to identify direct and indirect interactions between bacteria and Pseudo-nitzschia cells. We employed epifluorescent and scanning electron microscopy to visualize attachment of bacteria to Pseudo-nitzschia. DAPI, and ATTO dyes were used for staining cultures for epifluorescent imaging. Imaging of laboratory grown cultures will be compared to fresh environmental samples and we will discuss the implications of bacterial attachment on phytoplankton biology.

    FRI-754 THE N-TERMINUS OF THE HSV-1 E3 UBIQUITIN LIGASE ICP0 STIMULATES VIRAL REPLICATION AND GENE EXPRESSION IN CELLS EXPOSED TO INTERFERON-fl

    • Jessica van Loben Sels ;
    • Mirna Perusina Lanfranca ;
    • David Davido ;

    FRI-754

    THE N-TERMINUS OF THE HSV-1 E3 UBIQUITIN LIGASE ICP0 STIMULATES VIRAL REPLICATION AND GENE EXPRESSION IN CELLS EXPOSED TO INTERFERON-fl

    Jessica van Loben Sels, Mirna Perusina Lanfranca, David Davido.

    The University of Kansas, Lawrence, KS.

    HSV-1 infects sensory neurons in humans and establishes lifelong latent infections, which can reactivate by stress stimuli. Among the first proteins to be expressed when HSV-1 infects a cell is infected cell protein 0 (ICP0). ICP0 is an E3 ubiquitin ligase that stimulates viral gene expression and enhances viral replication. ICP0 facilitates viral gene expression by impairing the antiviral effects of the cellular factor, interferon (IFN)-β. The mechanisms by which ICP0 impairs IFN-β restriction on HSV-1 replication remain largely unknown. Consequently, the purpose of the present study was to determine which domains in ICP0 contribute to HSV-1 resistance to the antiviral effects of IFN-β. To identify one or more domains, a series of ICP0 truncation mutants was used to perform plaque reduction and gene expression assays in untreated cells and cells pretreated with IFN-β. We determined that the first 388 N-terminal amino acids of ICP0 confer significant resistance of HSV-1 to IFN-β while efficiently stimulating viral gene expression; specifically, amino acids from 312 to 388 are crucial for mediating this resistance. We hypothesize that this N-terminal domain of ICP0 plays a role in counteracting the IFN-β-induced restriction on viral replication through ICP0-host protein interactions. Overall, we conclude that the N-terminal half of ICP0 enables HSV-1 to resist an established IFN-β response with residues from 312 to 388 being required for this function.

    THU-760 INACTIVATED SHIGELLA AS EFFECTIVE VACCINES AND VACCINE VECTORS

    • Ivan Albino Flores ;
    • Manuel Osorio ;
    • Earle Stibitz ;

    THU-760

    INACTIVATED SHIGELLA AS EFFECTIVE VACCINES AND VACCINE VECTORS

    Ivan Albino Flores, Manuel Osorio, Earle Stibitz.

    Laboratory of Enteric and Sexually Transmitted Diseases, Food and Drug Administration, Silver Spring, MD.

    Shigellae and enterotoxigenic Escherichia coli (ETEC) remain major causes of diarrhea among travelers and children in developing countries. Currently, there are no effective vaccines against these enteric pathogens. Previously, we demonstrated the vaccine and vector potential of formalin inactivated Shigellae by demonstrating the strong protective immune responses elicited in the mouse model. In addition, we showed that these inactivated strains can serve as effective vaccine carriers for fimbrial antigens (CFA/I and CS3) of ETEC. However, we have observed that the harsh formalin treatment can negatively affect the immunogenicity of these fimbrial antigens. We, therefore, explored alternative methods of inactivating Shigella. Here, we present our results of examining the vaccine and vector potential of Shigella ghost cells expressing ETEC antigens (CVD1203). Bacterial ghost cells in general have been shown to be immunogenic and possess inherent adjuvant properties. We found that mice immunized orally or intranasally with ghost cells of S. flexneri expressing CFA/I and CS3 induced strong IgG titers to the homologous LPS and to the ETEC antigens. We show that these immune responses are protective as vaccinated animals can be protected 100% from challenge with the live homologous Shigella strain compared to negligible survival in mice given PBS. These studies show that Shigella ghost cells are highly immunogenic and can serve as effective carriers for exogenous antigens such as ETEC fimbrial antigens.

    THU-755 ELUCIDATION OF THE ROLE OF RHOMBOID PROTEINS IN STREPTOMYCES

    • Wilson Nieves ;
    • Naydu Carmona ;
    • Monica Trujillo ;

    THU-755

    ELUCIDATION OF THE ROLE OF RHOMBOID PROTEINS IN STREPTOMYCES

    Wilson Nieves, Naydu Carmona;Monica Trujillo.

    Queensborough Community College, Bayside, NY.

    Streptomycetes are Gram-positive soil bacteria characterized by a complex developmental cycle and by the production of secondary metabolites. These 2 processes involve signaling mechanisms not fully elucidated. Rhomboids are intramembrane proteases with their active site located within the cell membrane. They are found in all branches of life and have a wide range of biological functions. We hypothesize that rhomboids play a role in the signaling cascades of Streptomyces. Using bioinformatics, 5 families of putative rhomboid genes were identified in 30 Streptomyces strains. Transcription assays showed the 4 rhomboid genes from Streptomyces coelicolor (the model organism for the Streptomyces genus) were transcribed. To investigate the role of rhomboids in Streptomyces, we cloned the codon optimized genes SCO3855 and SCO2139, into vectors suitable to complement a Providencia stuartii rhomboid mutant strain (XD37aarA). The P. stuartii rhomboid gene, aarA, has been extensively characterized. The wild-type P. stuartii strains XD37, XD37aarA, XD37.A-pSCO3855, and XD37.A-SCO2139 were compared for their morphology and their capacity to grow in different media. Our results showed that rhomboid gene SCO3855 is capable of recovering the phenotype of the wild-type strain. Rhomboid gene SCO2139 shows unique phenotypic characteristics suggesting it is capable of partial complementation. Our results demonstrate that the 2 rhomboid genes have different functions within the Streptomyces species. Therefore, complementation of XD37aarA with genes SCO3855 and SCO2139 gave us an insight into the role that rhomboids could play in Streptomyces.

    THU-764 EXPLORING THE ENTRY CAPABILITIES OF H5N1 IN THE ABSENCE OF THE SIALIC ACID BINDING REGION IN HEMAGGLUTININ

    • Brenna Dooley ;
    • Emily Rumschlag-Booms ;

    THU-764

    EXPLORING THE ENTRY CAPABILITIES OF H5N1 IN THE ABSENCE OF THE SIALIC ACID BINDING REGION IN HEMAGGLUTININ

    Brenna Dooley, Emily Rumschlag-Booms.

    Northeastern Illinois University, Chicago, IL.

    With high mutation rates and increased pathogenicity, influenza has the potential to cross the species barrier, sparking a global pandemic. H5N1 avian influenza has already jumped the species barrier, and although transfer between humans is not currently viable, elevated mutation rates in the influenza envelope protein, hemagglutinin (HA), may trigger sustainable transmission among humans. The only known mechanism for influenza to attach and enter host cells is via the sialic acid (SA) binding region of HA and host cell SA receptors; however, studies suggest that other entry routes may be possible. Therefore, if H5N1 can mediate entry in the absence of the SA binding region, the virus may be using another mechanism or cofactor to enter the cell. Our research is centered on exploring the entry capabilities of H5N1 by disrupting the SA binding region and measuring viral entry. Using site directed mutagenesis, amino acid residues were substituted, altering the SA binding region of HA. Then, pseudovirus containing the mutated HA was synthesized. After infection, viral entry levels into target cells will be measured using a luciferase assay. If the viruses with the mutated HA and wild-type HA have comparable levels of entry, it suggests that H5N1 may be entering cells via another method or with the help of an unidentified cofactor. Fully understanding H5N1 entry mechanics could help direct antiviral therapeutics and treatments. Future experiments can focus on identifying potential substances, besides sialic acid, that are being used by H5N1 to mediate attachment and entry.

    FRI-772 DETERMINING THE ABILITY OF H5N1 TO MEDIATE ENTRY IN THE ABSENCE OF THE HEMAGGLUTININ SIALIC ACID BINDING POCKET

    • Isabelle Szot ;
    • Emily Rumschlag-Booms ;

    FRI-772

    DETERMINING THE ABILITY OF H5N1 TO MEDIATE ENTRY IN THE ABSENCE OF THE HEMAGGLUTININ SIALIC ACID BINDING POCKET

    Isabelle Szot, Emily Rumschlag-Booms.

    Northeastern Illinois University, Chicago, IL.

    H5N1 avian influenza virus, more commonly known as bird flu, is a highly pathogenic strain of influenza. Since 1997, there have been 650 individuals infected with bird flu with a mortality rate of over 60%. Though direct transmission to humans is possible, sustained human-to-human transmission has not been seen. However, a strengthened interaction between the viral protein, hemagglutinin (HA), and its hot cell receptor, sialic acid (SA), may lead to sustained transmission. This strengthened viral-host cell interaction is predicted to spark a pandemic. It has been proposed that a global pandemic could be sparked by influenza’s use of a co-receptor reinforcing viral attachment and entry. To further explore presence of this co-receptor, this study aimed to disrupt the traditional viral attachment mechanism between HA and SA. To disrupt the interaction of HA-SA, site-directed mutagenesis was used to create alanine substitutions at glycine135, serine136, and tyrosine98 in the sialic acid binding domain of HA. These amino acid residues were chosen due to their stability and to minimize conformational changes. If this interaction is blocked and viral entry is comparable to wild-type, this suggests that continued infection may be due to the presence of an additional co-receptor. Identification of the presence of a co-receptor can then be applied in development of novel therapeutic approaches for treating avian influenza.

    FRI-758 EVALUATION OF CHAGAS' DISEASE IN WILD AND DOMESTIC RESERVOIRS IN EL PASO COUNTY, TEXAS

    • Claudia Manriquez ;
    • Adam Vera ;
    • Eva Iniguez ;
    • Jose Orozco ;
    • Rosa Maldonado ;
    • Douglas Watts ;

    FRI-758

    EVALUATION OF CHAGAS' DISEASE IN WILD AND DOMESTIC RESERVOIRS IN EL PASO COUNTY, TEXAS

    Claudia Manriquez, Adam Vera, Eva Iniguez, Jose Orozco, Rosa Maldonado, Douglas Watts.

    n/a

    American trypanosomiasis, Chagas´ disease (ChD), is caused by the heamoflagellate protozoan parasite Trypanosoma cruzi. This parasitic disease is transmitted through triatominae “kissing bugs,” or less commonly, through blood transfusion from an infected donor. ChD covers a wide variety of mammalian hosts, making dogs and cats the most common in a domestic environment where humans are present. Currently, there are not many studies that could explain the precise role of dogs and cats in the life cycle of T. cruzi and the risk that poses to humans. The objective of this study is to provide a baseline epidemiological assessment of ChD occurrence in stray dogs and cats in El Paso County, Texas. So far, there are no records to indicate ChD is actually endemic in El Paso, Texas. To determine if an animal was infected with T. cruzi, 2 different diagnostic methods were performed using the animal’s blood and sera, which were previously collected. The first assay was a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA), which is commonly used in the diagnosis of ChD in dogs; the second assay was a kinetoplastid DNA (kDNA) polymerase chain reaction (PCR). Samples from 48 dogs and 48 cats have been tested for ChD by kDNA-PCR. Of the canine samples, 19 (39.58%) were positive and 29 (60.41%) resulted negative. Of the feline samples, only 16 (28.07%) were positive for T.cruzi and 41 (71.90%) were negative.

    FRI-750 DIFFERENCE IN THE TRANSLOCATION RATE OF EFFECTOR PROTEINS INTO MUTANT AND WILD-TYPE ARABIDOPSIS LINES

    • Elizabeth Alger ;
    • Mieder Palm-Forster ;
    • Brad Day ;

    FRI-750

    DIFFERENCE IN THE TRANSLOCATION RATE OF EFFECTOR PROTEINS INTO MUTANT AND WILD-TYPE ARABIDOPSIS LINES

    Elizabeth Alger1, Mieder Palm-Forster2, Brad Day2.

    1Division of Science & Environmental Policy, California State University, Monterey Bay, Seaside, CA, 2Michigan State University, East Lansing, MI.

    In order to cause disease in plants, many bacterial plant pathogens use the type III secretion system (T3SS) to inject plants with virulence proteins, termed type-III effectors (T3Es). By developing a deeper understanding of this plant-microbe disease interaction, we can develop plants that can better withstand biotic stressors. This study focused on the interaction between Pseudomonas syringae pv. tomato strain DC3000, and Arabidopsis thaliana. Specifically, we investigated whether there is a significant difference between the translocation of the virulence protein AvrRpt2 into wild-type and mutant Arabidopsis lines. To do so, we are currently working to optimize the calmodulin-dependent adenylate cyclase (Cya) reporter system by collecting Arabidopsis samples at various time points. Based on previous results, we hypothesize that Arabidopsis mutants lacking or with a nonfunctional NDR1 gene, which is required for the activation of disease resistance to AvrRpt2 in Arabidopsis, will have lower rates of AvrRpt2 translocation than the wildtype. The Cya system will allow for better detection of effector protein transduction, which will, in turn, provide new insights into T3SSs. Results have shown no consistent relationship between translocation rates and the mutant and wild-type Arabidopsis lines. We predict this is due to changing factors that may stress the plant and affect translocation rates such as temperature or humidity. Future experiments will be conducted to determine the factors that affect translocation rates of AvrRpt2.

    FRI-773 MICROBIAL DEGRADATION OF ESTROGEN AND TRICLOSAN IN SOIL FROM WASTE WATER EFFLUENT

    • Eduardo Velo Lara ;
    • Deborah Carr ;

    FRI-773

    MICROBIAL DEGRADATION OF ESTROGEN AND TRICLOSAN IN SOIL FROM WASTE WATER EFFLUENT

    Eduardo Velo Lara, Deborah Carr.

    Texas Tech University, Lubbock, TX.

    Pharmaceutical and personal care products (PPCPs) associated with land farming of municipal waste-water effluent may potentially persist in soil and alter soil microbial community processes. Recent experiments with Biolog Ecoplates may demonstrate the ability of microflora to use different carbon sources as a substrate for growth. Soil that had been previously exposed to these chemicals and soil not previously exposed was spiked with estrone and triclosan (a 1:1 mixture of estrone:triclosan), incubated for 90 days, and analyzed for the ability of their microflora to use different carbon sources. Control samples consisting of unspiked, exposed and unexposed soil were included in the analysis. Ecoplate analysis of day-0 and day-90 samples were read every 24 hours for 168 hrs. Estrone and triclosan spiked samples in both unexposed and exposed soil exhibited growth of carboxylic acids, carbohydrates, phenolic compounds, and primarily nitrogen-containing carbon substrates. An overall increase in substrate use activity was observed in day-90 samples. Degradation of estrone and triclosan occurs by microbial metabolic processes in both exposed and unexposed soils.

    FRI-751 FACILITATING ANTIBIOTIC ENTRY INTO BACTERIAL CELLS

    • Bianca Dunn ;
    • Zachary Ruhe ;
    • David Low ;

    FRI-751

    FACILITATING ANTIBIOTIC ENTRY INTO BACTERIAL CELLS

    Bianca Dunn, Zachary Ruhe, David Low.

    University of California, Santa Barbara, Santa Barbara, CA.

    Bacterial antibiotic resistance is a global health problem. Resistant strains of bacteria can cause lethal infections that are refractory to current treatments. Alarmingly, drug-resistant bacteria are emerging faster than we can develop new antibiotics. If we wish to curtail the effects of antibiotic resistance, we must accelerate the development of new antibacterial agents. To be effective, antibiotics must permeate the outer membrane of Gram-negative bacteria and access vital intracellular processes. Our research takes a novel approach to antibiotic development by improving the way these compounds enter bacterial cells. The purpose of the project is to identify peptides that permeate cells, facilitating the entry of antibiotics. In this study, we employed a selection based on sugar transport to isolate peptides that permeate bacterial cells. A subset of these peptides mediate intercellular aggregation, suggesting that permeation is due to interactions with the bacterial cell surface. We will explore the potential therapeutic properties of these peptides by assaying their synergy with current antibiotic treatments.

    THU-763 UNIQUE AZOREDUCTASE ACTIVITY OF THE PSEUDOMONAS AERUGINOSA STRAIN FRD1

    • Marla Ichord ;
    • Gilbert John ;

    THU-763

    UNIQUE AZOREDUCTASE ACTIVITY OF THE PSEUDOMONAS AERUGINOSA STRAIN FRD1

    Marla Ichord, Gilbert John.

    Oklahoma State University, Stillwater, OK.

    Nosocomial respiratory infections in cystic fibrosis patients are associated with Pseudomonas aeruginosa, which contributes to high morbidity, mortality, and antibiotic resistance in immunocompromised patients. One approach to combat this upward trend is to find a unique survival mechanism for P. aeruginosa infections. This study seeks to establish a unique mechanism of bacterial survival for the Pseudomonas aeruginosa strain FRD1, a cystic fibrosis isolate and alginate (mucoid) producing bacterium, which is phenotypically different than the nonmucoid PAO1 strain. The study specifically examines the physiology and molecular functions of strains FRD and PAO1 azoreductase activities. We hypothesize that FRD1 is uniquely different than PAO1 based on its mechanism of survival via azoreductase activity. Other investigators have extensively studied azoreductase in P. aeruginosa PAO1, as 3 proteins (paAzo-1,-2,-3) have been fully characterized. However, no published information is available regarding molecular, biochemical, and physiological parameters for P. aeruginosa FDR1. The results, after testing 10 azo dyes, determined that FRD1 is able to metabolize (OD/time) the azo dye (methyl red), compared to PAO1, suggesting unique azoreductases may be present within the bacterium. Pure enzyme studies further support unique azoreductases based on specific activity (µM dye/mg protein/min) data. Overall, results support the uniqueness of FRD1 and suggest a potential new mechanism for bacterial survival thereby increasing survivability and ultimately pathogenicity of the bacterium.

    FRI-755 INVESTIGATION OF A NOVEL LACTASE-ASSOCIATED GENE IN UNCULTIVATED BACTERIA TM7

    • Cecelia Brown ;
    • Farsheed Ghadiri ;
    • Becky Lee ;
    • Margarita Rangel ;
    • Adam Caldwell ;
    • Cleber Ouverney ;

    FRI-755

    INVESTIGATION OF A NOVEL LACTASE-ASSOCIATED GENE IN UNCULTIVATED BACTERIA TM7

    Cecelia Brown, Farsheed Ghadiri, Becky Lee, Margarita Rangel, Adam Caldwell, Cleber Ouverney.

    San Jose State University, San Jose, CA.

    Despite numerous strides in bacterial cultivation techniques, maintaining uncultivated bacteria cell lines remains a challenge for microbiologists. However, understanding uncultivated bacteria may provide valuable insights into how we can optimize processes such as creation of materials, cleanup of oil spills, and fermentation. In order to characterize uncultivated bacteria, we isolated genes from bacterial samples and expressed the genes to understand what role they may play in the overall metabolism of the organism. In this study, we have identified a lactase gene associated with the uncultivated bacteria phylum TM7 in a fosmid library generated from an environmental microbial community. The gene was cloned into the expression vector pET28a and expressed in E. coli cells. The level of TM7 lactase gene expression was then quantified and compared to a control wild-type bacterium. This is the first time a TM7 lactase enzyme has been characterized.

    THU-752 TARGETING CELLULAR FUNCTIONS IN CYTOMEGALOVIRUS INFECTION AS A THERAPEUTIC STRATEGY FOR ATHEROSCLEROSIS

    • Benni Vargas ;
    • Maite Sabalza ;
    • Deborah Spector ;

    THU-752

    TARGETING CELLULAR FUNCTIONS IN CYTOMEGALOVIRUS INFECTION AS A THERAPEUTIC STRATEGY FOR ATHEROSCLEROSIS

    Benni Vargas1, Maite Sabalza2, Deborah Spector2.

    1University of California, Los Angeles, Los Angeles, CA, 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA.

    Human cytomegalovirus (HCMV) is a significant human pathogen, and recent studies have proposed that HCMV infection plays a role in the pathogenesis of atherosclerosis. New strategies for atherosclerosis treatment targeting HCMV infection are being studied. Statins are used to treat hypercholesterolaemia due to their ability to inhibit the production of cholesterol. In addition, statins have pleiotropic benefits that may include inhibition of HCMV replication. Trehalose, a disaccharide that protects cells from numerous environmental stress factors, is also a promising candidate because it has recently been shown to induce autophagy in certain cells, and induction of autophagy has been shown to inhibit infection by several viruses. We hypothesized that pitavastatin and trehalose would inhibit replication of HCMV in human aortic endothelial cells (HAECs). We studied the effect of pitavastatin and trehalose individually on HAECs infected under static conditions as well as under different patterns of shear stress, mimicking conditions favorable for atherosclerosis. We performed western blot to analyze HCMV viral protein expression in pitavastatin-treated, trehalose-treated, and non-treated HAECs. The results demonstrated that the synthesis of viral proteins was inhibited by both pitavastatin and trehalose treatment. Subsequent analysis will focus on determining viral titers of cell-associated and released virus by performing standard plaque assays in order to see differences in viral particle output between treated and non-treated cells. Insight into the effects of statins and trehalose in viral replication can lead to new therapeutic treatments to help reduce the HCMV risk factor in atherosclerosis.

    FRI-764 IDENTIFICATION AND EXPRESSION OF GENES INVOLVED IN IRON HOMEOSTASIS IN METHYLOBACTERIUM EXTORQUENS AM1

    • Mayra Resnik ;
    • Jennifer Doherty ;
    • Justin Wingett ;
    • Ramen Kanda ;
    • Elizabeth Skovran ;

    FRI-764

    IDENTIFICATION AND EXPRESSION OF GENES INVOLVED IN IRON HOMEOSTASIS IN METHYLOBACTERIUM EXTORQUENS AM1

    Mayra Resnik, Jennifer Doherty, Justin Wingett, Ramen Kanda, Elizabeth Skovran.

    San Jose State University, San Jose, CA.

    Iron is essential for most living organisms. However, too much iron can cause oxidative damage. Cells have evolved to tightly regulate iron uptake in order to maintain homeostasis. Methylobacterium extorquens is a model organism for methylotrophism and is a platform for production of biofuels and biodegradable plastics from methanol. Our results show methanol growth requires higher levels of exogenous iron compared to multi-carbon growth. Microarray data suggested that the methylotrophic carbon assimilation regulator, QscR, may coordinate this difference by modulating expression of iron uptake genes. In addition to QscR, the M. extorquens genome revealed candidates for FecI and FecR, regulators involved in ferric citrate uptake, and Irr, a putative heme uptake regulator. Null mutations in each of these regulators were generated and growth under iron limitation and iron excess was assessed. The qscR and Irr mutant strains grew better than the wild-type strain under iron limiting conditions, consistent with these regulators functioning as repressors of iron uptake. Loss of FecI resulted in growth inhibition under limiting iron conditions consistent with a role for FecI in activation of iron uptake. To assess regulation in vivo, promoter fusion constructs were generated by fusing the promoter regions of genes predicted to be involved in iron regulation to the fluorescent reporter, YFP (yellow fluorescence protein). Preliminary results demonstrated that each promoter tested is differentially regulated by iron. Future work will determine the relationship between the different iron regulators and the genes involved in iron uptake and storage.

    THU-750 ALTERNATE PROTOCOL FOR DETECTING BIOLOGICAL CONTAMINANTS

    • David Berlin ;
    • Erin Lalime ;

    THU-750

    ALTERNATE PROTOCOL FOR DETECTING BIOLOGICAL CONTAMINANTS

    David Berlin1, Erin Lalime2.

    1College of the Sequoias, Visalia, CA, 2Goddard Space Flight Center, NASA, Greenbelt, MD.

    The purpose of this project is to develop a sterile, water-based rapid bioburden test. Contamination engineers used 2 tests to assess the level of biological contamination on hardware: the rapid, 5-minute bioburden test which is a molecular screening for adenosine triphosphate (ATP), a molecule found in all cells on the hardware, and a slower, colony-growth test which is used to give a more accurate representation of the amount of microbes on the hardware. However, the rapid bioburden test has limited application because it leaves a residue that can be detrimental to sensitive hardware. This can cause project delays while waiting for the results from the 3-day colony growth test. We addressed this problem by adapting the commercial, germicide-based ATP system to a sterile, water-based system. The test works by reacting ATP with D-luciferin and luciferase protein to yield light. The light is then detected by a luminometer that outputs a relative light unit (RLU) amount depending on how much ATP is present. To analyze the effectiveness of the new test, we developed a correlation between amounts of ATP and the RLU produced using the germicide-based system. From these experiments, we have generated a consistent relationship between the two in the form of a power curve. From there, we developed a correlation curve between the amount of colonies and the RLU they produced. Initial tests of the new protocol have shown that the water-based system isn't as sensitive as the germicide-based test.

    FRI-771 SYNTHESIS AND CHARACTERIZATION OF MODIFIED BH3 PEPTIDES

    • Hector Nieves-Rosado ;
    • Amy Keating ;

    FRI-771

    SYNTHESIS AND CHARACTERIZATION OF MODIFIED BH3 PEPTIDES

    Hector Nieves-Rosado1, Amy Keating2.

    1University of Puerto Rico, Mayaguez Campus, Mayaguez, PR, 2Massachusetts Institute of Technology, Cambridge, MA.

    A series of small molecules and engineered peptides that inhibit some anti-apoptotic Bcl-2 proteins have been identified; however, they are not effective against cells that overexpress Mcl-1. Hence, the effort to abrogate the cancer cell survival triggered by Mcl-1 is a major focus in experimental therapeutics. One approach uses pro-death BH3 peptides to re-establish mitochondrial sensitivity. However, the application of small peptides to block cancer has been hindered by some characteristic features of natural peptides such as loss of their natural architecture and protease degradation. The goal of this project was to optimize a promising lead peptide inhibitor of Mcl-1 using unnatural elements to provide enhanced peptide stability and affinity. A side-chain, lactam bridge, linking i with i + 4 spaced amino acid pairs is a powerful helix-stabilizing tool for inhibitors that bind their receptors in an a-helical conformation. Therefore, selected lactam bridged BH3 peptides were prepared by solid-phase synthesis. Biophysical analysis of these peptides in solution confirmed helical contents of these peptides. Remarkably, the ability to inhibit Mcl-1 was reduced in lactam-bridged peptides in comparison with i, i + 4 hydrocarbon-stapled analogue. Moreover, new peptides including bulky and helix-inducing nonnatural amino acids that can potentially improve binding to Mcl-1 were synthesized and characterized.

    THU-751 GENERATION OF AN ATTENUATED CRYPTOCOCCUS NEOFORMANS STRAIN FOR THE INDUCTION OF PROTECTIVE IMMUNITY AGAINST CRYPTOCOCCOSIS

    • Madeline Cortez ;
    • Althea Campuzano ;
    • Christopher Mendoza ;
    • Hayley Gutierrez ;
    • Floyd L. Wormley Jr ;

    THU-751

    GENERATION OF AN ATTENUATED CRYPTOCOCCUS NEOFORMANSSTRAIN FOR THE INDUCTION OF PROTECTIVE IMMUNITY AGAINST CRYPTOCOCCOSIS

    Madeline Cortez1, Althea Campuzano2, Christopher Mendoza3, Hayley Gutierrez3, Floyd L. Wormley Jr.2.

    1Honors College, The University of Texas at San Antonio, San Antonio, TX, 2The University of Texas at San Antonio, San Antonio, TX, 3College of Sciences, The University of Texas at San Antonio, San Antonio, TX.

    Cryptococcus neoformans is a fungal pathogen responsible for over 1 million cases of cryptococcal meningitis, predominantly in patients with compromised immune systems. To date, there is no antifungal vaccine available for clinical use. Therefore, our objective is to genetically engineer an attenuated strain of C. neoformans that induces protection against cryptococcal infections. Pulmonary infection in mice with a highly virulent C. neoformans strain, H99, results in the induction of nonprotective immune responses. Alternatively, mice infected with a C. neoformans strain genetically engineered to express a protein that induces pro-inflammatory immune responses, interferon-gamma (IFN-γ), are able to resolve the infection. However, the IFN-γ-producing strain remains virulent in immune-suppressed mice. Consequently, we used a mutant strain of C. neoformans that cannot grow at mammalian host temperatures, 37 °C, as a backbone for insertion of the IFN-γ gene within the gene laccase1 which is necessary for melanin production and virulence. Confirmation of insertion and IFN-γ production will be evaluated using L-DOPA plates to confirm the absence of melanin production, and an enzyme-linked immunosorbent assay (ELISA) will be used to confirm IFN-γ production. Studies will be conducted to evaluate virulence of the attenuated strain in immunocompromised mice and the efficacy of the strain in eliciting protection against a secondary pulmonary challenge with the pathogenic strain of C. neoformans. This study has the potential to generate a novel vaccine candidate that will induce protective immune responses against C. neoformans infections in immune compromised hosts. (Funded by GM007717, 2RO1AI071752, and R21AL100893.)

    THU-758 IDENTIFICATION OF TRANSCRIPTIONAL REGULATORS THAT CONTROL LANTHANIDE-DEPENDENT METHANOL DEHYDROGENASE EXPRESSION IN METHYLOBACTERIUM EXTORQUENS

    • Brandon Almestica ;
    • Elizabeth Skovran ;

    THU-758

    IDENTIFICATION OF TRANSCRIPTIONAL REGULATORS THAT CONTROL LANTHANIDE-DEPENDENT METHANOL DEHYDROGENASE EXPRESSION IN METHYLOBACTERIUM EXTORQUENS

    Brandon Almestica1, Elizabeth Skovran2.

    1Virginia State University, Petersburg, VA, 2San Jose State University, San Jose, CA.

    Lanthanide elements are essential to our modern lifestyle, functioning in items such as hybrid car and smartphone batteries, magnets, and catalytic converters. Until recently, existence of lanthanide-dependent enzymes was believed implausible due to the low solubility of these elements in the environment. This belief has recently been challenged as lanthanides have been shown to be directly required for the activity of XoxF, a widespread but poorly characterized methanol dehydrogenase enzyme used by some bacteria to oxidize methanol for carbon and energy. Using transcriptional reporter fusion assays in Methylobacterium extorquens, we showed that expression of xoxF1 is upregulated by any of the first 4 lanthanide elements and repressed in their absence. As a first step to determine how the xoxF1 gene is transcriptionally regulated, transposon mutagenesis was used in combination with a fluorescent transcriptional reporter fusion. Specifically, the xoxF1 promoter was cloned upstream of Venus, a modified yellow fluorescent protein. Transposon mutants containing this reporter were plated on media containing or lacking lanthanum and imaged for fluorescence. Mutant strains that have increased fluorescence on medium lacking lanthanum are candidates for xoxF1 repressor mutants. Mutant strains that result in decreased fluorescence on medium containing lanthanum are candidates for xoxF1 activator mutants. To identify which genes are disrupted in the putative regulator mutants, the region surrounding the insertions will be amplified, sequenced, and compared to the M. extorquens genome. This work will further our knowledge of how cells sense and respond to lanthanides, an emerging field in biology.

    FRI-752 A BLOODY MESS: INVESTIGATING TYPE VI SECRETION PREVALENCE IN CAMPYLOBACTER ISOLATES FROM HAWAII

    • Hilario Franco ;
    • Daniel Strange II ;
    • Rebecca Kanenaka ;
    • John Berestecky ;

    FRI-752

    A BLOODY MESS: INVESTIGATING TYPE VI SECRETION PREVALENCE IN CAMPYLOBACTER ISOLATES FROM HAWAII

    Hilario Franco1, Daniel Strange II2, Rebecca Kanenaka3, John Berestecky3.

    1University of Hawaii at Manoa, Honolulu, HI, 2College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI, 3Kapiolani Community College, Honolulu, HI.

    Campylobacter jejuni is a major cause of bacterial gastroenteritis world-wide. In about 7% of patients, infections become more severe, involving bloody diarrhea and occasionally progressing to systemic infection. Rarely, Campylobacter infections may also lead to serious neurological, paralytic sequelae such as Guillain-Barrè syndrome. In spite of Campylobacter’s prevalence, little is known about how this organism establishes itself in the gastrointestinal niche or how it manages to adhere to and invade intestinal epithelial cells. A type VI secretion system (T6SS) has recently been identified as a possible virulence factor in this organism. In other T6SS-positive bacterial species, the hcp gene product plays an essential role, both as a secreted effector protein and as a structural component of a needle-like cell-puncturing mechanism effective in bacterial cell-to-cell warfare and in interactions with eukaryotic host cells. We surveyed a library of curated, locally isolated Hawaiian Campylobacter strains to determine the prevalence of the hcp gene by PCR analysis. We have found that only a few of the isolates in our library appear to possess a T6SS. Two Penner reference strains, Penner-2 and Penner-3, appear positive for this complex as well. We speculate that a T6SS might play a role in establishing invasive infections; thus, our ongoing experiments measure the characteristics of T6SS-positive strains in terms of their interactions with mammalian cells in tissue culture and their in vitro growth characteristics.

    FRI-760 EVALUATING RHODIOLA ROSEA FOR CONTROLLING FOOD SPOILAGE AND FOODBORNE ILLNESS

    • Wafa Zeidan ;
    • Christine Case ;

    FRI-760

    EVALUATING RHODIOLA ROSEA FOR CONTROLLING FOOD SPOILAGE AND FOODBORNE ILLNESS

    Wafa Zeidan, Christine Case.

    Skyline College, San Bruno, CA.

    A tremendous amount of the food available for consumption goes to waste annually due to microbial spoilage. Not only does bacterial growth cause food waste, it also leads to millions of cases of foodborne illnesses. Rhodiola rosea is used by Alaskan Native Americans to treat bacterial infections, suggesting antimicrobial properties that might be useful in eliminating or minimizing bacterial growth in food. The goal of this research is to determine whether R. rosea extract inhibits bacterial growth in food. Growth of single strains of Staphylococcus aureus and Escherichia coli were inhibited in nutrient broth containing Rhodiola. The minimum lethal concentration against S. aureus is 0.063 μL/μL. E. coli was inhibited at 0.125 μL/μL. Growth was assessed on foods treated with an aqueous Rhodiola extract in an experimental system. Potato salad and ground beef were inoculated with approximately 105 colony-forming units/cm2 S. aureus and E. coli, respectively, treated with the Rhodiola, and incubated at different temperatures for 24 h. Treatment with Rhodiola killed S. aureus after 2 hours at all temperatures. After 24 hours in treated beef at 22 °C, the E. coli population was 22% less than the control; E. coli was killed at 5 °C. Results suggest that Rhodiola may be useful in preventing the growth of bacteria introduced into food as well as bacteria already present.

    THU-754 CHARACTERIZATION OF THE KEY AUTOANTIGEN, ABERRANTLY GLCOSYLATED IGA1, IN IGA NEPHROPATHY

    • Jocelyne Fadiga ;
    • Jan Novak ;

    THU-754

    CHARACTERIZATION OF THE KEY AUTOANTIGEN, ABERRANTLY GLCOSYLATED IGA1, IN IGA NEPHROPATHY

    Jocelyne Fadiga1, Jan Novak2.

    1University of California, Merced, Merced, CA, 2The University of Alabama at Birmingham, Birmingham, AL.

    IgA nephropathy (IgAN) is a chronic kidney disease with up to 40% of patients progressing to end-stage renal disease (ESRD). IgAN is characterized by IgA1-containing immunodeposits in the mesangium. These immunodeposits, enriched for galactose-deficient IgA1 (Gd-IgA1), likely originate from the circulating immune complexes consisting of Gd-IgA1 bound by antiglycan autoantibodies. Gd-IgA1 is produced by IgA1-producing cells due to abnormal expression of key glycosylation enzymes. Serum levels of Gd-IgA1 are associated with disease progression. Serum IgA1 is predominantly monomeric, with only about 10% in the polymeric form as dimers or higher polymers. To better characterize the autoantigen, we analyzed the O-glycosylation of polymeric and monomeric IgA1 from serum and secreted by IgA1-producing cells. IgA1 in serum and media of immortalized cells derived from healthy controls and patients with IgAN, and the mono/polymeric forms were isolated by affinity and size-exclusion chromatography. The molecular form of IgA1 was assessed by nonreducing SDS-PAGE, and the degree of galactose deficiency was determined by enzyme linked immunosorbent assay (ELISA) with lectin from Helix aspersa (HAA) and mass spectrometry. We tested the separation of polymeric and monomeric IgA1 using several matrices at low pressure vs. standard high-pressure liquid chromatography. We hypothesize that the lectin-reactive serum IgA1 (Gd-IgA1) is predominantly in immune complexes and free polymeric form and that most Gd-IgA1 produced by IgA1-secreting cells is a polymer. In IgAN, polymeric IgA1 forms are more reactive with Gd-IgA1-specific lectin. These findings will need to be validated using a larger cohort of subjects and analyses expanded by mass spectrometry profiling.

    THU-762 EATING UNDERCOOKED CHICKEN WILL MAKE YOU SICK

    • Joanna Fragoso ;
    • Nicholas Negretti ;
    • Christopher Gourley ;
    • Mark Nissen ;
    • Michael Konkel ;

    THU-762

    EATING UNDERCOOKED CHICKEN WILL MAKE YOU SICK

    Joanna Fragoso1, Nicholas Negretti2, Christopher Gourley2, Mark Nissen2, Michael Konkel2.

    1The University of Texas at San Antonio, San Antonio, TX, 2School of Molecular Biosciences, Washington State University, Pullman, WA.

    Campylobacter jejuni is a leading bacterial cause of human foodborne disease. Illness associated with this bacterium is frequently acquired from eating foods cross-contaminated with raw chicken or from eating undercooked chicken. In order to cause disease, this bacterium invades the cells lining the human intestinal tract. The purpose of this study was to determine if C. jejuni behaves differently when cultured in the laboratory versus when cultured with gut intestinal cells. We hypothesized that proteins synthesized by C. jejuni in response to incubation with human intestinal cells will promote bacterial invasion of these cells. We determined the ability of 4 different C. jejuni strains to invade human INT 407 cells over time and in the presence of chloramphenicol. Chloramphenicol is an antibiotic that selectively inhibits C. jejuni protein synthesis. Binding and internalization assays revealed that C. jejuni strains can be divided into 2 groups: highly invasive and poorly invasive strains. Additional work revealed that the number of C. jejuni bacteria internalized significantly increased over time. Moreover, chloramphenicol significantly inhibited the number of bacteria internalized by INT 407 cells for the highly invasive strains. Together, these data demonstrate that C. jejuni adapt to incubation with host gut cells and synthesize proteins that promote host-cell invasion. In summary, these studies will enable us to determine the virulence attributes and conditions that are associated with the ability of C. jejuni to cause diarrhea.

    THU-771 ROLE OF BB0418 IN THE PATHOGENESIS OF BORRELIA BURGDORFERI

    • Antonio Contreras ;
    • Janakiram Seshu ;

    THU-771

    ROLE OF BB0418 IN THE PATHOGENESIS OF BORRELIA BURGDORFERI

    Antonio Contreras, Janakiram Seshu.

    The University of Texas at San Antonio, San Antonio, TX.

    Lyme disease is a common vector-borne disease in the U.S. with more than 300,000 cases reported in 2013. The agent of Lyme disease, Borrelia burgdorferi, is a spirochetal pathogen that is transmitted to humans and other domestic animals via the bite of infected Ixodes scapularis ticks. B. burgdorferi has the ability to metabolize different compounds present in the ticks and vertebrate hosts. Bioinformatic analysis of the borrelial genome revealed that B. burgdorferi has an open reading frame designated BB0418 that may express an outer membrane transport protein specific for the transport/permeability of organic anions, notably C4-dicarboxylates. Previously, our laboratory has shown that supplementation of key nutrients in the media altered the antigenic profile of the agent of Lyme disease, and we have expanded the analysis of a select set of proteins with structural capabilities for localization in the outer membrane of B. burgdorferi. In order to further dissect the role of this protein, we over-expressed BB0418 and generated antisera in mice. A deletion construct to replace bb0418 has been generated, and we are in the process of replacing bb0418 with a streptomycin resistance marker and evaluating the role of bb0418 in the pathogenic mechanisms of the agent of Lyme disease. Analysis of the mutants in the C3H/HeN mouse model of Lyme disease and in the tick-mouse model will help delineate the role of BB0418 in the pathogenesis of B. burgdorferi.

    FRI-759 ELUCIDATING THE VIRAL MECHANISM OF HIF-2α UPREGULATION DURING LATENT KSHV INFECTION

    • Mona Ahmed ;
    • Erica Sanchez ;
    • Hanna Hong ;
    • Michael Lagunoff ;

    FRI-759

    ELUCIDATING THE VIRAL MECHANISM OF HIF-2α UPREGULATION DURING LATENT KSHV INFECTION

    Mona Ahmed, Erica Sanchez, Hanna Hong, Michael Lagunoff.

    University of Washington, Seattle, WA.

    Kaposi’s sarcoma (KS) is a highly angiogenic tumor caused by the Kaposi’s sarcoma-associated herpesvirus (KSHV) which establishes a latent infection in endothelial cells. KS is the most common tumor found in sub-Saharan Africa and in AIDS patients worldwide. KSHV has been shown to alter host-cell metabolism and requires aerobic glycolysis for survival. Hypoxia-inducible factors (HIFs) are a family of transcription factors that respond to low levels of oxygen in the cell. HIFs are involved in inducing the Warburg effect in cancer cells by increasing glycolysis and decreasing mitochondrial activity. HIF-1α and HIF-2α have been found to be upregulated during KSHV infection. HIF-1α is expressed ubiquitously but HIF-2α is specific to certain cell types, including endothelial cells. We hypothesize that HIF-2α is responsible for altering cell metabolism during KSHV infection. To test this hypothesis, we will use a lentiviral approach to overexpress individual latent genes and observe whether HIF-2α is upregulated through RT-PCR and western blot. We will also infect cells with an adenovirus expressing the entire KSHV latency locus and then knock out individual latent genes and observe whether HIF-2α expression is inhibited. Through this approach, we will be able to identify the viral mechanism of HIF-2α upregulation during KSHV infection.

    THU-761 STRUCTURAL IDENTIFICATION AND CHARACTERIZATION OF AN AZOREDUCTASE FOUND IN THE ANAEROBE CLOSTRIDIUM PERFRINGENS

    • Zach Ridge ;
    • Gilbert John ;

    THU-761

    STRUCTURAL IDENTIFICATION AND CHARACTERIZATION OF AN AZOREDUCTASE FOUND IN THE ANAEROBE CLOSTRIDIUM PERFRINGENS

    Zach Ridge, Gilbert John.

    Oklahoma State University, Stillwater, OK.

    Azo dyes are colorants found in food, textiles, pharmaceuticals, and cosmetics that have been found to be environmental pollutants. Azoreductases are enzymes found in several microorganisms that are capable of reducing azo dyes. The reduction of azo dyes can produce carcinogenic compounds, making this an important process to understand. This study focuses on exploring the structural identification and characterization of the azoreductase found in Clostridium perfringens (AzoC), an anaerobic pathogen found in the human intestine. AzoC is unique among other azoreductases due to its specificity for the dye Direct Blue 15. Having a high specificity for Direct Blue 15 is uncommon due to the dye’s large size. Studying AzoC may also reveal some interesting physiological and molecular insights into its function in C. perfringens. In this work, the amino acid methionine was substituted with selenomethionine in order to improve X-ray diffraction results by addressing the phasing problem. Other additives were added to help stabilize AzoC, improving crystallization. To conserve protein, a new technique called acoustic droplet ejection (ADE) was used. Finally, varying pH for multiple buffers above and below the protein’s isoelectric point (PI) was attempted. The outcome of the study will provide the structure of the AzoC protein and allow for further studies investigating the functions and chemical interactions of the enzyme with substrates.

    FRI-757 HIGH INCIDENCE OF EXTENDED SPECTRUM BETA-LACTAMASES IN KLEBSIELLA PNEUMONIAE FROM ENVIRONMENTAL SURFACE WATERS

    • Kyle Kisor ;
    • Luis Mota-Bravo ;
    • Lili Mesak ;

    FRI-757

    HIGH INCIDENCE OF EXTENDED SPECTRUM BETA-LACTAMASES IN KLEBSIELLA PNEUMONIAE FROM ENVIRONMENTAL SURFACE WATERS

    Kyle Kisor, Luis Mota-Bravo, Lili Mesak.

    University of California, Irvine, Irvine, CA.

    Klebsiella pneumoniae is a Gram-negative, opportunistic pathogen causing infection in immunocompromised individuals, frequently in the urinary and respiratory tracts. To treat an infection of this bacterium, beta-lactam antibiotics of extended spectrum are commonly prescribed. Since K. pneumonia is commonly found in the environment, we hypothesize that environmental strains may be an unexplored reservoir of antibiotic resistance genes, particularly extended spectrum beta-lactamases (ESBL). Water samples were collected in Orange County, California from 2011 to 2014. K. pneumonia strains were identified using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Disk susceptibility tests were performed on each isolate and antibiotic resistance was determined according to Clinical Laboratory Standards Institute (CLSI) guidelines. Thirty eight K. pneumoniae isolates were collected and beta-lactam resistance was found in 32% of isolates against cefotaxime with no observed resistance to ceftazidime. When clavulanic acid was combined with 3rd generation cephalosporins, 37% of isolates tested positive for the presence of an extended spectrum beta-lactamase based on a zone of inhibition greater than or equal to 5 millimeters more than cefotaxime or ceftazidime alone. Cefoxitin and cefepime both resulted in 3% resistance. In the presence of ESBLs, resistance to ciprofloxacin, tetracycline, gentamicin, sulfisoxazole, and trimethoprim were observed. Multidrug resistance defined as resistance to 3 or more classes of antibiotics was observed in all isolates which produced ESBLs. Aquatic environments are an important reservoir of antibiotic resistant determinants in ESBL producing K. pneumonia.

    THU-757 OXIDATION OF MANGANESE AS A MECHANISM OF PHYSICAL PROTECTION IN PSEUDOMONAS PUTIDA GB-1

    • Bianca Ruiz ;
    • Hope Johnson ;

    THU-757

    OXIDATION OF MANGANESE AS A MECHANISM OF PHYSICAL PROTECTION IN PSEUDOMONAS PUTIDA GB-1

    Bianca Ruiz, Hope Johnson.

    California State University Fullerton, Fullerton, CA.

    A phylogenetically diverse group of microbes has been found to oxidize manganese (Mn). These microbes produce Mn (III, IV) oxides and are necessary to several of the planet’s biogeochemical cycles. In addition to the role Mn oxides play in biogeochemical cycles, oxides also act as sorbents for various toxic metals. The potential for practical environmental applications of microbial Mn oxidation make it a process that is worth close examination. Although it is clear Mn oxidizing bacteria are important in environmental cycling, we currently do not understand the reason bacteria oxidize Mn. A model microorganism of Mn oxidation is Pseudomonas putida GB-1. When this strain oxidizes Mn, it forms a Mn oxide coat that surrounds the cell. Identifying the purpose of Mn oxidation by P. putida GB-1 is the subject of our research. To explore possible reasons for microbial Mn oxidation, we are conducting competition experiments between P. putida GB-1 and Pseudomonas aeruginosa PA01 in either the presence or absence of Mn. P. aeruginosa PA01 has a Type VI secretion system which lyses neighboring cells. This provides P. aeruginosa PA01 with a fitness advantage when in competition with other microbes for space or nutrient availability. We wish to determine if oxidation of Mn and production of a Mn oxide coat affords P. putida GB-1 protection from the P. aeruginosa PAO1 bacteriolytic secretion system. Our preliminary results suggest that this is probably the case.

    FRI-756 COMPARING DEEP CAVE BACTERIA WITH MARS-SIMULATION-SURVIVING BACTERIA: IMPLICATIONS FOR DETECTING LIFE ON EXTRATERRESTRIAL BODIES

    • Krystal Charley ;
    • Diana E. Northup ;

    FRI-756

    COMPARING DEEP CAVE BACTERIA WITH MARS-SIMULATION-SURVIVING BACTERIA: IMPLICATIONS FOR DETECTING LIFE ON EXTRATERRESTRIAL BODIES

    Krystal Charley, Diana E. Northup.

    The University of New Mexico, Albuquerque, NM.

    Caves on Mars are a likely place to host extant or fossil microbial life because the microbes would be sheltered from harsh surface environments. A good analog for Mars may be Lechuguilla Cave in southeastern New Mexico, because the microorganisms exist in an aphotic, oligotrophic environment without flowing water. Previous studies have not directly compared cultures from Mars analog sites to cultures that have survived under simulated Martian conditions. Bacterial cultures from a prior NASA-funded project that tested the ability of cave and surface bacteria to survive simulated Martian conditions were analyzed. We hypothesize that cultured bacteria from Mars simulation experiments will be similar to those found in deep caves and would share metabolic strategies. Culture plates were inoculated on site with ferromanganese deposits obtained from approximately 300 m below the surface in Lechuguilla Cave. Using an established protocol to subculture the samples, we obtained 30 putatively pure subcultures; those that could not be purified were cloned for sequencing. These subcultures were characterized morphologically and compared to the Mars-survivor cultures. We are currently working to identify the bacteria using cloned full-length 16S rRNA gene sequences (a bacterial fingerprint), which provides taxonomic information and hints as to metabolic strategies employed. This culture-based approach will assess whether caves in New Mexico are good analogs for Martian caves and improve our chances of recognizing life on other planets.

    THU-756 EVOLVING A NEW FUNCTION: THE EBG CASE

    • Amber Eliza Trujillo ;
    • Ulfar Bergthorsson ;

    THU-756

    EVOLVING A NEW FUNCTION: THE EBG CASE

    Amber Eliza Trujillo, Ulfar Bergthorsson.

    The University of New Mexico, Albuquerque, NM.

    How do genes evolve novel functions? It has previously been shown that the ebg (evolved beta-galactosidase) locus in Escherichia coli can evolve a lactase activity in strains where the native lactase gene, lacZ, has been deleted. The natural substrate of the EBG protein is still unknown. We will test if gene amplification plays a role in the evolution of lactase activity of the ebg gene. The specific hypothesis that will be tested is whether selection for multiple copies of ebg on lactose medium facilitates the evolution of lactase activity of the gene. Duplications of the ebg gene will be constructed in lacZ deletion strains using lambda red technology. The rate of lactase evolution will be compared in strains that have 1 or 2 copies of ebg. If there is natural selection for increased gene copy-number of the wild-type ebg gene because of a preexisting insignificant lactase activity, strains with 2 copies of ebg are expected to evolve lactase activity at greater than 2 times the rate relative to the single copy strains. In preliminary experiments done using Berry Hall’s original lacZ deletion strains, early signs of adaptive mutability arose during the first generations of selection on minimal media. Small colonies of E. coli appeared to grow on glycerol-lactose plates, indicating that these colonies have promiscuous lactase activity. Sequencing and further tests will be performed on these lactose-using colonies to verify duplications of ebg, which, if present, will provide support for the amplification model.