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The Forty Fifth Annual Meeting of the Arizona-Nevada Branch of the American Society for Microbiology

Contributors

2006

ASM

Waksman Foundation for Microbiology Lecturers

VWR International

McGraw Hill

EM Scientific

Mettler-Toledo

Becton Dickinson

Promega

Department of Biological Sciences University of Nevada Las Vegas

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Program

The Forty Fifth Annual Meeting of the Arizona-Nevada Branch of the American Society for Microbiology

Saturday February 25, 2006

University of Nevada Las Vegas

Las Vegas, NV 7:30-8:15 Registration: Foyer Bigelow Physics Building (BPB), UNLV Refreshments: Foyer BPB Hang posters: Foyer BPB Load talks onto computer: 102 BPB 8:15-8:30 Opening remarks: Ron Yasbin, Dean College of Sciences, UNLV

President Elect Dr. Brian Hedlund (102 BPB) 8:30-9:30 Keynote address: Chatterbugs: Cell-cell communication and its

consequences among the bacteria. Dr. Clay Fuqua, Indiana University, Department of Biology, Bloomington, IN. Sponsored by the ASM Foundation for Microbiology Lecturers Program.

9:30-10:00 Guest presentation: Stable isotope analysis in DNA: a powerful

new tool in microbial ecology. Dr. Egbert Schwarz, Department of Biological Sciences, Northern Arizona University.

10:00-10:20 Refreshment break: Foyer BPB 10:20-12:00 Oral presentations: 102 BPB

OP1 A Comparison of Ammonia-Oxidizing Communities in Northern Arizona Soils. K. L Adair1* and E. Schwartz1 1Northern Arizona University, Flagstaff, AZ. (graduate student)

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OP2 Microbial Community Characterization: Who Is Decomposing Leaf Litter in Fossil Creek, Arizona? B. L. Harrop*, R. M. Rieder, J. C. Marks, M. E. Watwood. Northern Arizona University, Flagstaff, AZ. (graduate student)

OP3 Assessing Three Field Sites for Bioremediation Potential Using

Enzyme Activity Dependent Probes. S. R. Clingenpeel*1,2, M. H. Lee3, and M. E. Watwood2. 1Idaho State University, Pocatello, ID; 2Northern Arizona University, Flagstaff, AZ; 3Northwind Environmental, Idaho Falls, ID. (graduate student)

OP4 Life in Great Basin Hot Springs. J. B. Navarro* and B. P. Hedlund.

University of Nevada Las Vegas, Las Vegas, NV. (undergraduate student)

OP5 Molecular genetics of cyanobacterial sunscreens. T. Do1*, V.

Stout1, and F. Garcia-Pichel1. 1Arizona State University, Tempe, AZ. (undergraduate student)

12:00-2:00 Lunch and poster session

Lunch and business meeting (for faculty): 102 BPB Lunch for students: Foyer BPB Poster session: Foyer BPB Vendor tables: Foyer BPB

(VWR, EM Scientific, Mettler-Toledo, Becton-Dickinson, Promega) 2:00-2:30 Guest presentation: Microbial Life in the Deepest Reaches of

Earth’s Terrestrial Biosphere. D.P. Moser1*, T.C. Onstott2, T.M. Gihring3; F.J. Brockman4; J.K. Fredrickson4. 1Desert Research Institute, Las Vegas, NV; 2Princeton University, Princeton, NJ; 3Florida State University, Tallahassee, FL; 4Pacific Northwest National Lab, Richland, WA.

2:30-3:30 Oral presentations: 102 BPB

OP6 Analysis of a toxin regulatory pathway in Pseudomonas tolaasii. C. Dobbin*. Arizona State University, Tempe, AZ. (graduate student)

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OP7 Virulence for chickens of Clostridium perfringens isolated from poultry and other sources. K. Cooper*, M.K. Keel, B. Stewart, H. Trinh, B.H. Jost, and J.G. Songer. Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ. (graduate student)

OP8 Pili production during biofilm growth in Campylobacter jejuni.

J.R. Theoret1*, R.J. Reeser1, S.J. Billington1, and L.A. Joens1 Dept. of Veterinary Science & Microbiology, University of Arizona, Tucson, AZ. (graduate student)

3:30-3:45 Refreshment break: Foyer BPB 3:45-4:45 Oral presentations: 102 BPB

OP9 Selective Role of YfgL in Escherichia coli Outer Membrane Protein Biogenesis. E. S. Charlson*, J. N. Werner, E. R. Coon, and R. Misra. Arizona State University, Tempe, AZ. (undergraduate student)

OP10 Importance of AcrB Hairpin 1 in a Functional Interaction with

TolC. S. Al-Khatib, F. Husain, and R. Misra. School of Life Sciences, Arizona State University, Tempe, AZ. (undergraduate student)

OP11 Importance of TolC periplasmic turns in protein-protein

interaction. T. Celaya Kolb*, S. Pecora, R. Treuer, R. Misra. School of Life Sciences, Arizona State University, Tempe, AZ. (research specialist)

5:00 Announcement of student presentation awards: 102 BPB 6:00 Dinner at Bahama Breeze. Courtesy of VWR and Department of

Biological Sciences, UNLV.

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Guest Presentations Stable isotope analysis of DNA; a powerful new technology for microbial ecology Egbert Schwartz1. 1Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86011-5640 Stable isotopes in biomolecules can indicate metabolic functions of microorganisms in the environment. My research group has used two different analyses of isotope composition of DNA extracted from soil to study microbial growth and Nitrogen cycling. First we have developed an experimental approach to measure the natural abundance of 15N and 13C in DNA from soil, which shows there are significant differences in δ15N. We suggest δ15N-DNA is increased with higher nitrogen mineralization rates. In a second analysis, we used 18O-water to label DNA, which was separated from unlabeled DNA on a cesium chloride gradient. Three separate bands of DNA formed in the ultra centrifuge tubes during this analysis. We propose that the middle band contains DNA with one labeled strand while both strands of the DNA are labeled in the bottom band. Alternatively, the bottom band is generated by predators whose DNA obtains label from both 18O-water and 18O labeled prey, while the middle band is generated by microorganisms that assimilate unlabeled food. Stable isotope probing with 18O-water allows identification of newly grown as well as dormant cells and avoids fertilization affects since water's main role is that of a solvent and is never limiting as a substrate. Microbial Life in the Deepest Reaches of Earth’s Terrestrial Biosphere D.P. Moser1, T.C. Onstott2, T.M. Gihring3; F.J. Brockman4; J.K. Fredrickson4 1Desert Research Institute, Las Vegas, NV; 2Princeton University, Princeton, NJ; 3Florida State University, Tallahassee, FL; 4Pacific Northwest National Lab, Richland, WA Numerous studies have revealed the presence of microbial communities occupying oceanic and terrestrial deep subsurface Settings. Owing to its enormous volume, this habitat may host the majority of Earth’s prokaryotes, yet surprisingly little is known concerning the diversity, distribution, and energetic foundations of microbial populations at great depth. This talk will summarize the findings of a large, international project run out of Princeton University, which has explored deep subsurface microbial ecology utilizing the ultradeep mines of South Africa. This work ultimately revealed surprisingly simple archaeal and bacterial communities composed mostly of novel lineages, some at the predicted phylum-level. In this system, evidence for microbial life and activity can be extended to at least 4 - 5 km depth. The possibility that natural radioactivity provides the energetic foundation for such isolated ecosystems, totally independent of photosphere inputs will be discussed.

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Oral Presentations OP1 A Comparison of Ammonia-Oxidizing Communities in Northern Arizona Soils. Karen L Adair1 and Egbert Schwartz1. 1Northern Arizona University, Flagstaff, AZ. (graduate) Nitrification, the oxidation of ammonia to nitrite and nitrate by microorganisms, is an important component of the nitrogen cycle in terrestrial ecosystems. Autotrophic ammonia-oxidizing bacteria are thought to be responsible for much of the nitrification that occurs in soils; however recent studies have demonstrated the ubiquity of ammonia-oxidizing archaea in marine systems. The goal of this research is to characterize the ammonia-oxidizing communities in soils from five northern Arizona ecosystems (great basin, grassland, pinyon-juniper, ponderosa pine and mixed conifer) and two seasons (post-snowmelt and monsoon). Nitrification potentials were significantly lower following the monsoon season than post-snowmelt at all sites, except for the ponderosa pine. When averaged across all sites, abundance of ammonia-oxidizing bacterial amoA (the gene coding for the active site of ammonia monooxygenase) was more than 2,000 times greater in the monsoon season than following the spring snowmelt. Abundance of ammonia-oxidizing archaeal amoA differed between ecosystems. OP2 Microbial Community Characterization: Who Is Decomposing Leaf Litter in Fossil Creek, Arizona? B. L. Harrop, R. M. Rieder, J. C. Marks, M. E. Watwood. Northern Arizona University, Flagstaff, AZ. (graduate) Preliminary studies of Fossil Creek suggested greater primary production and decomposition rates in the travertine pools reach than in free-flowing reaches below the hydropower dam, providing more energy to support the food web in the travertine pools reach. In association with a leaf litter decomposition study, fungal and bacterial communities were characterized and compared between two leaf species and two study sites along the travertine gradient. Using submerged litter bags, decomposition rates were determined for alder and sycamore leaves. Ash free dry mass lost was calculated, and the negative exponential decay model (Nt = N0e-kt) was used to determine instantaneous rate of loss (k). Decomposition rates ranged from 0.0035 to 0.2033 day-1 and varied significantly between leaf species (P<0.0001) and between travertine pools and free-flowing study sites (P<0.0001). Differing rates were predicted to correspond with distinct microbial communities. Terminal restriction fragment length polymorphism analysis was used to generate community profiles for each treatment based on 16S rDNA for bacteria or ITS regions for fungi. The total number of fragments ranged from 28 to 45 for bacteria and 7 to 21 for fungi. Community profiles were compared using PCA analyses and cluster analyses. Results suggest that fungal community composition varies primarily with study site location, while bacterial community composition varies both with study site location and with leaf species. Future research is planned to determine if dam decommissioning and stream recovery corresponds with alterations in microbial community structure and function; the current work will provide baseline data for those studies.

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OP3 Assessing Three Field Sites for Bioremediation Potential Using Enzyme Activity Dependent Probes. S. R. Clingenpeel1,2, M. H. Lee3, and M. E. Watwood2. 1Idaho State University, Pocatello, ID; 2Northern Arizona University, Flagstaff, AZ; 3Northwind Environmental, Idaho Falls, ID. (graduate)

Trichloroethylene (TCE) is a common groundwater contaminant in the United States. Many bacteria, including those with toluene oxygenases, are capable of cometabolically degrading TCE. Our group has developed a suite of enzyme-activity probes that target the toluene 2,3-dioxygenase, toluene 2-monooxygenase, and xylene monooxygenase pathways, and produce a quantifiable, fluorescent signal when the enzyme of interest is active.

Groundwater was collected from inside and outside the contamination plumes at three sites: Test Area North (TAN) site of the Idaho National Laboratory, Test Area V (TA-V) site of the Sandia National Laboratories, and the Arizona Department of Environmental Quality Park-Euclid (PE) site. Toluene oxygenase activity, based on enzyme-activity probe response, was detected in all of the samples analyzed from both the TA-V and PE sites and in most samples at the TAN site. All three sites had some activity from each of the toluene oxygenase pathways. Toluene oxygenase genes were successfully PCR amplified in most samples, and the amplified sequences corresponded to the specific pathways expected based on enzyme-activity probe response. Toluene degrading isolates were cultured from several wells at the TAN and PE sites. At all three sites, active TCE degrading oxygenases were detected both inside and outside the contamination plumes, suggesting natural attenuation processes may be occurring.

This study applies integrated molecular and functional approaches to three distinct field sites, and a clear demonstration of toluene oxygenase activity at all three sites. These data support consideration of in situ bioremediation or monitored natural attenuation as remedial options at these sites. OP4 Life in Great Basin Hot Springs. J. B. Navarro and B. P. Hedlund. University of Nevada Las Vegas, Las Vegas, NV. (undergraduate) Thermophilic or “heat-loving” life is diverse, yet many of the organisms that live in hot environments are poorly understood and have never been cultivated or studied in the laboratory. This study targets three hot springs in Northern Nevada: Great Boiling Spring (GBS, 80°C), Sandy’s Spring West (SSW, 84°C), and Sandy’s Spring East (SSE, 75°C). A large cultivation-independent census has been completed at each spring using five primer sets targeting the 16S rRNA gene for Bacteria and Archaea, the results of which show that these Great Basin hot springs host moderately diverse microbial communies dominated by organisms with an unknown role in the environment. Known primary producers make up only a small fraction of the GBS and SSW/E communities, raising the question of which microbes are fixing carbon in the spring. A sediment core terminal Restriction Fragment Length Polymorphism (tRFLP) analysis reveals some vertical stratification of microbes at GBS, suggesting that certain microbes depend on electron donors or acceptors that become depleted in the sediment. Inorganic electron acceptors and donors were measured from hot spring water and will be used to calculate which redox reactions are predicted to yield the most energy for microorganisms living in the spring. Subsequent field experiments will test our thermodynamic hypotheses by measuring electron acceptor flux in situ.

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OP5 Molecular genetics of cyanobacterial sunscreens. T. Do1*, V. Stout1, and F. Garcia-Pichel1. 1Arizona State University, Tempe, AZ. (undergraduate) Some cyanobacteria synthesize sunscreen pigments that serve as passive protective screens against UV radiation-induced damage. The indole alkaloid scytonemin is the most common and widespread among them and provides efficient protection in the UV-A range. Previous research on scytonemin has focused on its distribution, ecology, role, physiology, and biochemistry but the molecular genetics has not been explored. To determine the genetic elements responsible for the biosynthesis and regulation of scytonemin, a scytonemin-deficient mutant was obtained in Nostoc punctiforme ATCC 29133 using transposon mutagenesis. We characterized the mutant’s phenotype and have identified the genomic region interrupted by the transposon in the completely sequenced genome of Nostoc 29133. At least six potential genes in an operon-like arrangement appear to be involved in the biosynthesis of scytonemin. Genomic comparisons indicate that these genes do not currently have any function associated with them. Future work will focus on elucidating their genetic role and mode of regulation by directed mutagenesis. A comparative analysis in other scytonemin-producing organisms will be undertaken to search for analogous sequences in order to conduct a phylogenetic study that may tell us more about the evolution of UV adaptations. OP6 Analysis of a toxin regulatory pathway in Pseudomonas tolaasii. C. Dobbin*. Arizona State University, Tempe, AZ. (graduate) Brown blotch disease in the commercial mushroom, Agaricus bisporus, is the most serious disease of mushrooms and results in economic losses for growers. The disease is caused by Pseudomonas tolaasii, which can be found in two forms, the pathogenic wild-type and the non-pathogenic variant, which cannot produce the toxin, tolaasin, and other virulence factors. One such variant was found to have a spontaneous mutation within the gacS gene, which appears to encode a sensor kinase regulator. Using transposon and allele exchange mutagenesis, we have initiated studies of the GacS regulon to characterize the regulatory network responsible for the expression of tolaasin biosynthesis genes of P. tolaasii. Although it is known that the hybrid sensor kinase regulator, GacS, is required for toxin production, its cognate response regulator has yet to be identified. Our lab has isolated the putative response regulator in P. tolaasii which shows 94% amino acid identity to the GacA response regulator protein of P. fluorescens. We have also generated a library of approximately 10,000 transposon mutants which were screened for variant phenotypes. Of those mutants, 464 were tolaasin negative, 60 had the variant phenotype, although none of the sequenced mutants had mutations in genes of the predicted GacS regulon. However, one of the tolaasin null mutants was found to have an ABC-type transporter interrupted. We believe that the toxin is secreted via this type I secretion system. We currently are characterizing the GacS regulon and identifying other regulators of tolaasin.

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OP7 Virulence for chickens of Clostridium perfringens isolated from poultry and other sources. K. Cooper*, M.K. Keel, B. Stewart, H. Trinh, B.H. Jost, and J.G. Songer. Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ. (graduate) Clostridium perfringens type A is a common cause of necrotic enteritis, which costs the poultry industry $2 billion annually. Little is known of mechanisms of pathogenesis. We investigated the virulence for poultry of isolates from a variety of enteric sources. Newly-hatched Cornish x Rock male chicks were fed a low protein diet for one week, a high protein diet for a second weeks, and then challenged with log-phase cultures mixed 1.25:1 with high protein feed. Strain JGS 4143 [genotype A, beta2 (CPB2) positive, from a field case of necrotic enteritis] produced gross lesions compatible with necrotic enteritis in 83.3% of inoculated birds, but strains JGS 1714 (enterotoxigenic genotype A, human food poisoning), JGS 1936 (genotype A, bovine neonatal enteritis), JGS 4142 (genotype A, CPB2+, bovine jejunal hemorrhage syndrome), JGS 1473 (genotype A, CPB2+, chicken normal flora), JGS 1070 (genotype C, CPB2+, porcine hemorrhagic enteritis), and JGS 1882 (genotype A, CPB2+, porcine neonatal enteritis) failed to do so. Further work is needed to clarify the virulence effects of in vitro passage, but our results suggest that some strains of type A may be specifically equipped for infection of chickens. OP8 Pili production during biofilm growth in Campylobacter jejuni. J.R. Theoret1*, R.J. Reeser1, S.J. Billington1, and L.A. Joens1 Dept. of Veterinary Science & Microbiology, University of Arizona, Tucson, AZ. . (graduate) Campylobacterosis is a leading cause of gastroenteritis both in the US and world wide, resulting in an estimated 2.4 million cases annually. Although poultry is considered the major source of infection, cattle, sheep, and swine have also been reported as sources. Given the high number of infections and large host range, much emphasis has been placed on determining the pathogenesis of campylobacter infections. In this study we report the discovery of a novel pilus expressed by Campylobacter jejuni under biofilm conditions. Crude preparations of the pilus were prepared from C. jejuni by differential centrifugation in ethanolamine, followed by ammonium sulfate precipitation. Through the use of proteomics, the amino acid sequence was determined, ultimately leading to the identification of the gene encoding the major pilin subunit. The gene, which shares homology with the major pilin subunit of fine tangled pili produced by Haemophilus ducreyi, has been mutated by homologous recombination using a suicide vector with selection by chloramphenicol. This resulted in the apiliate mutant NCTC 11168::∆pil as demonstrated by scanning electron microscopy (SEM). Initial studies of the mutant have demonstrated a decreased ability to form biofilms when compared to the NCTC 11168 type strain. This data represents a new facet of campylobacter pathogenesis that should greatly aid our understanding and prevention of this disease.

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OP9 Selective Role of YfgL in Escherichia coli Outer Membrane Protein Biogenesis. E. S. Charlson*, J. N. Werner, E. R. Coon, and R. Misra. Arizona State University, Tempe, AZ. (undergraduate)

In Escherichia coli, YfgL together with NlpB, YfiO, and YaeT form a protein complex thought to facilitate the insertion of proteins into the outer membrane. Of the three lipoproteins - NlpB, YfgL, and YfiO - YfiO, like YaeT, is essential for cell growth. This study examines the role of YfgL in outer membrane biogenesis. The absence of YfgL interferes with the biogenesis of a number of outer membrane proteins, including OmpA, OmpF, and LamB, but not that of TolC. In contrast, biogenesis of all model outer membrane proteins, including TolC, is affected in a strain depleted of YaeT (Werner, J., and R. Misra. 2005. Mol. Microbiol. 57:1450-1459). Thus, despite being a part of the same protein complex, YfgL and YaeT play distinct roles in the biogenesis of outer membrane proteins. Biogenesis of OmpA, OmpF, and LamB, but not that of TolC, is also affected in a mutant background expressing defective lipopolysaccharide, a lipid component exclusively localized in the outer membrane. Mutants lacking yfgL do not show reduced lipopolysaccharide levels or mislocalization, but rather elevated lipopolysaccharide levels in the outer membrane. Thus, an effect of yfgL deletion on lipopolysaccharide-dependent outer membrane proteins is unlikely due to a defect in lipopolysaccharide biogenesis. A selective decrease in the lipopolysaccharide-dependent outer membrane protein levels together with an increase in the lipopolysaccharide level in the yfgL deleted strain suggest that YfgL may play a role in coupling certain outer membrane proteins with lipopolysaccharide to facilitate their insertion into the outer membrane.

OP10 Importance of AcrB Hairpin 1 in a Functional Interaction with TolC. Sofian Al-Khatib, Fasahath Husain, and Rajeev Misra. School of Life Sciences, Arizona State University, Tempe, AZ. (graduate)

The TolC protein is an outer membrane protein in Escherichia coli that interacts with AcrA and AcrB proteins to efflux various antimicrobials from the cell. AcrB is the energy driven pump in the inner membrane and AcrA which is primarily located in the periplasm, is believed to seal the AcrB-TolC complex. Homologues of these three proteins are found in most gram-negative organisms, and are known to confer a very high degree of resistance to a wide variety of antimicrobials. Cross-linking studies have shown that the lower end of TolC and the top end of AcrB, both of which are exposed in the periplasm, interact with each other. The focus of this study is to evaluate the functional interactions of TolC and AcrB. We focused on the periplasmic hairpin-1 of the AcrB, including the residues 252 to 258 out of 1059 residues of AcrB. Altering all the residues in this hairpin disabled the efflux complex, causing hypersensitivity to antibiotics. We selected for antibiotic resistant revertants and identified second-site suppressor mutations in tolC. According to the crystal structure TolC forms a conduit through which the antimicrobials have to pass. Normally, the periplasmic aperture of TolC’s conduit remains in the closed state. All the suppressor mutations in tolC resulted in the open state TolC protein. Based on our results, we suggest that TolC’s periplasmic aperture has to open during antimicrobial efflux and AcrB hairpin-1 is involved in opening of TolC’s aperture.

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OP11 Importance of TolC periplasmic turns in protein-protein interaction. Teresa Celaya Kolb*, Sara Pecora, Robin Treuer, Rajeev Misra. School of Life Sciences, Arizona State University, Tempe, AZ (research specialist) Two components of the AcrAB-TolC efflux pump involved in resistance to toxic compounds have been crystallized. The crystal structure of TolC shows trimeric protein with extra cellular loops and periplasmic turns. The TolC external loops have been involved with host–parasite interactions. Each TolC monomer contains two turns connecting the alpha helices at the bottom of channel. Mutations in turn 2, which folds inward, indicate its involvement in opening and closing the TolC channel. Turn 1 folds outward and its specific role has not been determined. This turn may either play a structural role contributing to the proper helix organization or, due to its position at the periplasmic entrance, turn 1 may assist in the interactions with the AcrAB proteins, allowing the TolC AcrAB complex to function properly when assembled together. We mutagenized the turn 1 to determine the behavior of TolC and thus, turn 1 function. The TolC mutant antibiotic sensitivity pattern suggests that turn 1 is not involved in opening and closing of the TolC aperture. Reversion analysis reveled six mutations in AcrA protein, all of them mapping different domains of AcrA. These findings suggest no domain specificity for the suppression interaction but maybe conformational changes in AcrA structure that compensates for the lack of proper interaction of TolC turn 1 mutant with AcrA and/or AcrB. Poster Presentations PP1 Genomic Instability in Yeast and Cancer. Marlene Begay1*, Ted Weinert2 and Alison Adams1. 1Northern Arizona University and 2Arizona Cancer Center, UA. (undergraduate) We are using yeast in this study as a model organism to study genomic instability and cancer. Our long-term goal is to understand how environmental exposure and genetic factors affect genomic instability. During cancer development, translocations result when a segment of DNA breaks off and attaches to a different chromosome. Often, the resulting chromosome is unstable, leading to further breakage and attachment at a new location. Genomic instability may result in altered gene products or changes in gene expression, which may affect cell growth and consequently lead to cancer. To further understand this process, which occurs at high frequency in cancer cells, we are using yeast to learn about genes required to stabilize the genome. RAD9 is a yeast checkpoint gene. Mutations in RAD9 lead to increased levels of genomic instability. In order to study the role of the RAD9 protein in genomic instability, we are using recombinant DNA technology to make a yeast strain carrying a mutation in RAD9. This strain will then be used in genetic crosses to analyze the role of this, and other genes, in genomic instability.

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PP2 Identification of genes that, when over-expressed, increase genomic instability in yeast. Patricia Chan1*, Ted Weinert2 and Alison Adams1. 1Northern Arizona University and 2Arizona Cancer Center, UA. (graduate) Cancer results from uncontrollable cell division, which occurs as a result of an accumulation of mutations. One of the mechanisms leading to such mutations is ‘genomic instability’, which occurs when specific regions of the chromosome break. The resulting chromosome fragments then recombine with novel regions of the genome to form translocations. These translocations lead to altered gene products and/or gene expression. We are using yeast as a model organism to study this process. To detect genomic instability in yeast, we are using a ‘colony sectoring’ assay, which allows us to detect particular breakage and recombination events. We are using this assay to identify genes that normally have a role in stabilizing the genome of yeast. To this end, we are looking for genes that, when over-expressed, increase the rate of genomic instability. Thus, we are screening a library of high-copy-number plasmids, containing ~10kB inserts of yeast genomic DNA, to look for those that cause an increase in colony sectoring (indicative of an increase in genomic instability). In a pilot study, we have screened 240 plasmids, and have identified one that increases colony sectoring. We are currently screening an additional ~2,500 plasmids, so ensure that ~98% of all yeast genes are tested for an effect on genomic instability when over-expressed. PP3 Assessment of Bioremediation Potential in Contaminated Groundwater in Tucson, AZ. D. M. Conley1*, K. S. Shamrell1, S. R. Clingenpeel1,2, and M. E. Watwood1. 1Northern Arizona University, Flagstaff, AZ; 2Idaho State University, Pocatello, ID. (undergraduate)

Trichloroethylene (TCE), a common groundwater contaminant, is degraded by many bacteria, including those with toluene oxygenases. To identify these oxygenases enzyme activity probes, developed by our lab, were used that target the toluene 2,3-dioxygenase, toluene 2-monooxygenase, and xylene monooxygenase pathways, and yield a quantifiable, fluorescent signal if the specific enzyme is active.

The Park-Euclid site has shallow and deep aquifers and is contaminated with chlorinated ethenes, including TCE, and diesel fuel. Most of the contamination is in the shallow aquifer and is migrating into the deeper aquifer which provides drinking water to Tucson, AZ.

Groundwater was collected from inside and outside the contamination zone in both the shallow and deep aquifers during two consecutive semiannual samplings. Toluene oxygenase activity, based on enzyme probe response, was detected in all samples analyzed. Highly contaminated samples had the highest levels of enzymatic activity, with up to 75% of total cells expressing an active oxygenase; and multiple pathways were usually active. Samples from areas with low or no contamination typically had 5-25% of total cells expressing active toluene oxygenases with fewer active pathways. These results were consistent over the two samplings. Toluene oxygenase genes were successfully PCR amplified in most samples, and the amplified sequences corresponded to the pathways expected based on enzyme probe response.

This study provides clear evidence of toluene oxygenase activity inside and outside the contamination zones at the Park-Euclid site. These data support consideration of in situ bioremediation and monitored natural attenuation as remedial options at this site.

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PP4 Assessing Genomic instability using Brewer’s Yeast Saccharomyces cerevisiae. Danita Davis1*, Ted Weinert2 and Alison Adams1. 1Northern Arizona University and 2Arizona Cancer Center, UA. (undergraduate) The impact of cancer on American Indians and Alaskan natives of all ages was shown to be the third leading causing of death for these individuals and the second leading cause of death for those individuals over 45 years of age (IHS Pub No. 97-615-23). However, understanding the genetic mechanisms and pathways that result in an increase in cancer is still very poorly understood. Cancer is caused by mutations, some of which lead to genomic instability. The purpose of this study is to analyze genomic instability in brewer’s yeast, Saccharomyces cerevisiae. One particular instability event has been described previously (Admire et al., 2006) and will be analyzed further using a strain that we are generating in this study. In particular, the pathway of instability is believed to occur via an intra-chromosomal recombination event, involving the left and right arms of chromosome VII. We are testing whether this intra-chromosomal event is essential for instability, by generating a modified version of chromosome VII in which one of the regions involved in the recombination event has been deleted. PP5 Survival of Shigella flexneri strains in distilled water. Julius M. de Leon*, Kylie Siek, Eduardo Robleto, and Helen Wing. Department of Biological Sciences, University of Nevada, Las Vegas. (undergraduate) Shigella flexneri causes severe dysentery, and it is prevalent in developing countries that are prone to flooding. I propose Shigella strains carrying the large virulence plasmid survive for longer periods in water than Shigella strains which have lost the virulence plasmid. Unfavorable environments, such as water can induce a dormant state in Shigella increasing its survival. Induction of the dormant state may be attributed to genes encoded on the virulence plasmid. Microcosms were set up for both strains, and monitored for culturability by a plate count assays on TSB and Congo-Red plates. My results show that in microcosms incubated at 30 oC the avirulent strain retained the culturability state longer than the wild type. However, in the microcosms incubated at 37 oC these results were reversed; wild type strain retained culturability longer than the avirulent strain. BacLight is a mixture of dyes that can distinguish between live and dead bacterial cells. These dyes can be used to monitor the viability of strains in water. My data indicates SYTO 9 and propidium iodide dyes label the appropriate cells. In conclusion, my data shows that the virulent strain retains culturability better at a temperature of 37 oC, possibly due to virulent genes being turned on, and the avirulent strain retains culturability better in water at 30 oC. These studies will improve our knowledge of Shigella’s survival in water enabling us to determine the possible health risks associated with Shigella in water.

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PP6 Cultivation-intensive study of a Mojave recharge playa during a wet cycle. Andrea Flores1*, Jason B. Navarro1, Duane P. Moser1, 2, and Brian P. Hedlund1

1Department of Biological Sciences, University of Nevada, Las Vegas, NV; 2Desert Research Institute, Las Vegas, NV. (undergraduate) Rainfall over much of the U.S. Great Basin is insufficient to connect hydrologic drainages and thus solutes accumulate in terminal basins, the low-points of which are often occupied by ephemeral playa lakes. In this study, we examined the geochemical and corresponding microbial community development over one hydration/desiccation cycle of a large recharge playa, Silver Lake, CA. To accomplish this, we employed approaches for culture-dependent and -independent microbial community analysis in combination with measurements of solute chemistry. Over the course of the study, total dissolved solids increased by 10 fold and the pH increased from 8.4 to 9.2. Extremely high concentrations of culturable heterotrophs (>1 x 109 cells/ml) were detected, particularly in the later time points, which correlated with the near-complete depletion of soluble nitrate. 16S rRNA gene sequencing and phylogenetic analysis of isolates revealed a wide diversity of Firmicutes (43 isolates), Actinobacteria (42 isolates), Bacteroidetes (33 isolates), α-, β-, and γ-Proteobacteria (48, 15, and 15 isolates, respectively), and halophilic Euryarchaeota (10 isolates). Several novel lineages were detected and the population density for a number of groups appeared to respond to changing lake conditions. PP7 Evolution of microbial commensals of desert mammals. Tim Foley* and Brian P. Hedlund. Department of Biological Sciences, University of Nevada, Las Vegas, NV. (undergraduate) Mammals are host to an incredibly diverse population of prokaryotic organisms. These complex prokaryotic communities consist of hundreds, if not thousands of microbial species. Despite the potential importance of these microbial commensals, the evolutionary and natural history relationships between mammals and their normal flora remain relatively unexplored. This study examines the evolution of oral Streptococcus spp. in desert mammals focusing on four alternative evolutionary models: the cospeciation model, the biogeography model, the host ecology model, and the free dispersal or null hypothesis model. Bacteria have been identified by PCR amplifying and sequencing 16S rRNA genes. Subsequently, these sequences were used to establish phylogenetic trees to compare them to known organisms. To date, we have isolated 32 strains of Streptococcus from 8 different species of desert rodents that were sampled from two different geographical regions. The strains belong to two novel monophyletic groups, designated Desert Mammal Group I, II, and III. Thus far, it appears that certain Streptococcus phylotypes are particular to certain hosts. Streptococcus in Ammospermophilus spp. form a tight phylogenetic group, including a Nevada clade and an Arizona clade, consistent with the biogeography hypothesis.

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PP8 Stationary phase mutagenesis in bacillus subtilis: the role of CodY. Dalia Hawwass*, Christine Pybus, Eduardo Robleto and Ronald E. Yasbin. Department of Biological Sciences, University of Nevada, Las Vegas. (undergraduate) Bacillus subtilis is a model of development and differentiation, showing such processes as sporulation and competence (uptake of foreign DNA). We previously demonstrated that non-growing B. subtilis cells undergo mutagenesis when subjected to a non-lethal selective pressure (starvation for an amino acid). Several gene products play a role in this process, including developmental regulators ComA and ComK, mismatch repair protein MutS, error-prone DNA polymerase YqjH, transcription repair coupled factor Mfd and elongation factor GreA. Our goal is to elucidate the genetic pathway(s) responsible for regulating this important process. This project examines the effect of global regulator CodY on stationary phase mutagenesis. Stationary phase mutagenesis is an adaptive and genetically programmed response to stress, and may play a role in the development of pathogenicity and the formation of cancer. PP9 Phenotypic differences of pseudomonas fluorescens mutants in two types of soil. Chris Fayeghi1, Katrina Cabradilla1*, Mark Silby2 and Eduardo Robleto1. 1Department of Biological Sciences, University of Nevada, Las Vegas. 2Department of Molecular Biology and Microbiology, Sackler School of Medicine, Tufts University. (undergraduate) Pseudomonas fluorescens is a soil denizen used as a model to identify microbial traits that are important for adaptation to fluctuating environments. Three P. fluoresecens strains carrying insertional disruptions in genes required for the establishment of populations in soils from the North East (Silby and Levy, 2004) were examined for their ability to colonize arid soils, from the Mojave Desert. The disrupted genes are involved in carbon metabolism, two component regulation or yet to be identified functions. In sterile soils with higher water and organic matter content, strains iiv2, iiv3 and iiv7 roughly attained population levels equivalent to 1/10 of those attained by the wild type. Interestingly, inoculation of the same strains in live soils subject to dessication and lower organic matter content showed no significant differences compared to the wild type. Our results suggest that Pseudomonas uses a set of genes to adapt to arid soil environments different from those required for moist and high organic matter conditions.

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PP10 Insights into Arcanobacterium pyogenes pathogenesis from a draft genome sequence. M.S. John*, B.H. Jost, M.V. Pier, J. Yan and S.J. Billington University of Arizona, Tucson, AZ. (graduate) Arcanobacterium pyogenes is a commensal and opportunistic pathogen in important commercial livestock and causes several diseases such as mastitis, liver and lung abscessation, and pneumonia. However, little is known about how A. pyogenes causes disease. Genome sequencing of A. pyogenes strain BBR1 was undertaken to identify novel virulence determinants and mechanisms of survival in the host. A draft genome sequence was generated using pyrosequencing to 20x coverage, revealing a genome that is approximately 2.2 Mb in length and contains 2187 open reading frames. The gene encoding the major virulence factor, a cholesterol dependent cytolysin, pyolysin, was shown to be encoded on a genomic islet. This islet is not present in the related organism Arcanobacterium haemolyticum suggesting that acquisition of this sequence may have been a major event in the separation of these species. Extracellular proteolytic activity is thought to play a role in protection from host defenses and promote tissue damage. Genes encoding three proteases, EspA, EspB and ColA, were identified in the genome sequence. Adhesion plays a major role in both the commensal and pathogenic lifestyles of A. pyogenes. In addition to known adhesion factors such as neuraminidases and a collagen binding protein, CbpA, four fimbrial operons and a putative laminin binding protein were also identified in the genome sequence and may account for the wide variety of hosts that A. pyogenes can colonize. The draft sequence will provide the basis for a targeted approach to the investigation of A. pyogenes disease pathogenesis. PP11 Surface Distribution of Shigella Protease IcsP. Lisa Locaynia* and Helen Wing. Department of Biological Sciences, University of Nevada Las Vegas (undergraduate) Shigella is an intracellular pathogen that invades the colonic epithelium of a human host causing approximately 1 million deaths and 163 million cases of dysentery per year. Shigella virulence involves three key steps: penetration of target cells, multiplication within cells, and spread between cells. Shigella surface proteins IcsP and IcsA are important virulence factors. The protease IcsP cleaves its substrate IcsA from the bacterial surface, therefore negatively regulating actin tail formation. Previous studies were not able to identify the location of IcsP on the surface of wild type Shigella strains. It is possible that this was due to steric hindrance by bacterial LPS or low expression of IcsP. A rough LPS mutant and determination of optimal IcsP expression will address these issues and allow for continuation of these experiments. The aim of this study is to determine the bacterial surface location of IcsP. Our approach uses indirect immunofluorescence. Results show that IcsP is expressed over the entire bacterial surface in a rough LPS mutant. Induction studies will indicate if this is the true surface location of the protease or a result of the fluidity of a truncated LPS. This work will provide further understanding of the location of the protease, its proximity to the substrate, and therefore Shigella virulence.

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PP12 The Genome Sequence of Arcanobacterium haemolyticum: Identification of Novel Virulence Determinant. E.A. Lucas*, S.J. Billington and B.H. Jost University of Arizona, Tucson, AZ. (graduate) Arcanobacterium haemolyticum is a Gram positive, non-motile rod that causes pharyngitis and skin infections in humans. In addition, this organism can invade into deeper tissues resulting in more severe infections, such as meningitis, pneumonia and osteomyelitis. Despite this, very little is known about the pathogenesis of A. haemolyticum infections. To date, only one putative virulence factor has been cloned, a phospholipase D. A draft genome of A. haemolyticum ATCC 9345 was determined by pyrosequencing to >20X coverage, resulting in 75 contigs. Outward firing primers were designed to the ends of each contig and PCR amplification was performed with pools of primers. PCR products were cloned into pGEM-T Easy and subjected to nucleotide sequencing in a an effort to close the genome. The A. haemolyticum ATCC 9345 genome is ~1.95Mb with a 53.1% G+C content and contains no plasmids. Automated annotation identified ~1870 open reading frames, one of which, encoded a novel cholesterol-dependent cytolysin (CDC), arcanolysin (ALN). ALN contains a PEST-like sequence, which is not usually found in other CDCs, and may be important for the function of this toxin. Other interesting findings included identification of genes encoding a collagen-binding protein, a neuraminidase, a cell wall-anchored DNase and three fimbrial gene operons, all of which may play a role in disease pathogenesis. Determination of the draft genome of A. haemolyticum allows a more targeted approach of studies aimed at better understanding the pathogenesis of infections caused by this organism. PP13 Analysis of the biosynthesis of a bacterial toxin that acts on mushrooms. J.R. Lytle1*, C.L. Dobbin1, V. Stout1, Arizona State University1. (Undergraduate) Brown blotch disease in the commercial mushroom, Agaricus bisporus, is the most serious disease of mushrooms and results in huge economic losses for growers each year. The disease is caused by Pseudomonas tolaasii, which can be found in two forms: the wild-type, which is pathogenic; and the variant, which is non-pathogenic due to loss of production of the toxin tolaasin. A current model for the regulation of tolaasin production proposes that within the cell’s inner membrane the protein, GacS, which receives an unknown environmental signal becomes phosphorylated and then activates GacA by transferring its phosphate to GacA. GacA then activates transcription of the tolaasin biosynthesis genes. To find other regulators involved in tolaasin biosynthesis, we are creating a reporter gene to fuse to one of the genes involved in tolaasin biosynthesis. We have selected the gene, sypB, to create the reporter fusion mutation. Studies in our lab have shown that the sypB gene may be involved in tolaasin synthesis. When the tolaasin biosynthesis genes are activated they produce a peptide synthetase, which then makes the toxin. The sypB gene encodes a peptide synthetase. This gene will be fused to the lacZ reporter. Following the creation of this reporter strain, transposon mutagenesis will be used to initiate studies of the gacS regulon.

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PP14 Comparative analysis of two bacterial proteases that belong to the Omptin family. Tim Struve*, Noam Moas*, Kylie Siek and Helen J. Wing. Department of Biological Sciences, University of Nevada, Las Vegas, NV (*-these authors contributed equally to this work)(undergraduate) Omptins are a family of proteases, found associated with the outer membrane of a number of gram negative bacteria. Strikingly, most omptins proteases are found associated with bacterial pathogens or symbionts, suggesting these proteases may function at the bacterial:host interface. In Salmonella, the omptin protease PgtE has been shown to help defend Salmonella from the effects of one of our body’s defense mechanisms: cationic antimicrobial peptides (CAMPs). In Shigella, a similar omptin protease has been shown to remove the actin-tail-formation protein IcsA from the bacterial surface (IcsA is a key virulence determinant in Shigella spp.). PgtE and IcsP are over 38% identical at the amino acid level. Furthermore, the active site residues of PgtE and IcsP are 100% conserved. The overall aim of this study is to determine whether PgtE and IcsP can functionally substitute for each other. These studies will broaden our knowledge of the omptin family and may allow us to ascribe additional functions to these proteases. Initially we will address two questions: 1. Does IcsP protect Salmonella from cationic antimicrobial peptides? 2. Does PgtE cleave IcsA from the Shigella outer membrane? Our preliminary studies are described here. PP15 Genomic Instability in Yeast and Cancer: Does over-expression of the RRM3 gene of yeast cause an increase in genomic instability? Jason Tidwell1*, Ted Weinert2 and Alison Adams1. 1Northern Arizona University and 2Arizona Cancer Center, UA (undergraduate) The long-term goal of this study is to understand how genetic factors influence the risk of genomic instability in normal and cancer cells. Normal cells can become cancerous after chromosome breakages occur and lead to rearrangements (e.g., translocations). Such rearrangements may be unstable, and lead to further rearrangements. Such changes often result in mutations and/or alterations in gene expression that in turn, can affect growth control and consequently lead to cancer. We are using yeast as a model organism to study genomic instability. We are looking for genes that, when over-expressed, increase genomic instability. We are starting with genes that, on the basis of their functions and mutant phenotypes, may normally have a role in stabilizing the genome when expressed at normal levels. In this study, we are testing whether the yeast RRM3, which encodes a DNA helicase, causes increased levels of genomic instability when over-expressed in yeast.

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PP16 Bacteriophage and transposon association of erm(X) in Arcanobacterium pyogenes. B.C. Vona*, B.H. Jost and S.J. Billington. University of Arizona, Tucson AZ. (Undergraduate) Arcanobacterium pyogenes is a commensal and opportunistic pathogen of economically important food animals. Consequently, it is exposed to antibiotics such as the macrolide tylosin, fed to cattle and pigs for growth promotion and disease prophylaxis. Nearly, 25% of A. pyogenes isolates are resistant to tylosin, with erm(X) being the primary resistance gene. The erm(X) gene was previously characterized on a plasmid pAP2 from strain 98-4277-2. However, dotblot analysis indicated that this plasmid was not present in other erm(X) containing A. pyogenes strains. In this study the erm(X) genes of two additional strains, B167 and 52785-99, were cloned and the gene regions sequenced. In B167, a fragment containing erm(X) and truncated plasmid genes appeared to have integrated into a prophage, Apy01. PCR experiments indicated that Apy01 was present in five of nine erm(X) containing strains, and in each case erm(X) was associated with Apy01. The Apy01 genes appeared to be intact and thus Apy01 may be a functional phage capable of transferring erm(X) through transduction. In 52785-99, erm(X) was flanked by identical insertion sequences forming a composite transposon. This erm(X) transposon appeared to have inserted into, and partially deleted, the DNA methylase gene from a second prophage, Apy02. erm(X) was linked by PCR to the flanking insertion sequence in the three erm(X) strains that didn’t carry either Apy01 or pAP2. However, Apy02 was present in eight of nine erm(X) strains, including strains carrying Apy01, and was not always associated with the erm(X) transposon. PP17 Life after death - peptidoglycan accumulation in the Antarctic cryptoendolithic microbial communities results in high amino acid D/L ratios and possibly slows down carbon cycling. Henry J. Sun1*, Jianchun, Bao1, Gene D. McDonald2, Christopher P. McKay3, E. Imre Friedmann3, 4. 1*Division of Earth and Ecosystem Sciences, Desert Research Institute, 755 E. Flamingo Rd, Las Vegas, NV, 2Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, TX , 3NASA Ames Research Center, MS 245-3, Moffett Field, CA 4Department of Microbiology, University of Washington, Seattle, WA. (PI) Unusually high concentrations of D-amino acids accumulate in the cryptoendolithic microbial communities living in sandstone rocks in the Antarctic desert (McMurdo Dry Valleys). Geochemical racemization (aging) of L-amino acids is ruled out as an explanation because the communities are relatively young (<900 y) and because racemization in the cold Antarctic environment is slow. The only feasible explanation for our results is that remnants of bacterial cell walls (peptidoglycans), which contain D-amino acids (i.e. D-alanine, D-glutamic acid, D-aspartic acid, D-serine, D-leucine), accumulate. The recycling of these compounds may represent a bottleneck in the carbon cycle and would explain why the communities carry a high 14C age despite the fact that they are “open” systems.