NW ASM Branch Conference 2010

Abstracts

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Endophytes for Sustainable Bioenergy Crop Growth
Authors Title Abstract Format
SHARON LAFFERTY DOTY1, Gang Xin2, Regina Redman1, Zareen Khan1, Soo-Hyung Kim1, and Gregory Ettl1 1School of Forest Resources, College of the Environment, University of Washington, Seattle, USA 2 Department of Civil and Environmental Engineering, University of Washington, Seattle, USA. sldoty@u.washington.edu Endophytes for Sustainable Bioenergy Crop Growth The interior of plants provides habitat for a wide range of bacteria and fungi, termed endophytes, that benefit the plant host in multiple ways including phytohormone production, increasing nutrient acquisition, stress tolerance, pathogen resistance, and aiding in phytoremediation of environmental pollutants. Nitrogen fixed biologically by plant-symbiotic bacteria is ecologically friendly and has been effectively exploited for important leguminous crop species, but associations of other N-fixing (diazotrophic) bacteria with non-legume crops, especially non-tropical crops, have been less studied. A variety of microorganisms able to grow under nitrogen-limitation were isolated from the stems of native poplar (Populus trichocarpa) and willow (Salix sitchensis). Cloning of nitrogenase sequences from the isolates, positive results from the acetylene reduction assay, and incorporation of 15N from labeled dinitrogen gas support our hypothesis that some of these endophytes can fix nitrogen for the host plant. Inoculation of these poplar and willow endophytes into commercially important plants including turf grass, rice, and corn significantly increased plant growth in the absence of fertilizer. Considering the enormous potential impact of the use of nitrogen-fixing endophytes on global food and bioenergy crop production, awareness of research in this area is limited. There is a strong need for the development of sustainable agricultural practices that use resources more efficiently, maintain environmental health, and yet increase the food and bioenergy biomass supply. With more study of the multiple benefits of diazotrophic endophytes on plants, we can better understand the role of endophytes in natural systems as well as utilize that information for a new revolution in agriculture that is better for the environment. Oral
Marion Brodhagen
Department of Biology, Western Washington University, Bellingham, WA
Host-pathogen crosstalk: Aspergillus, aflatoxin, and oxylipins Plants and microbes engage in symbioses ranging from mutualistic to pathogenic, and the exchange of chemical signals between the organisms underpins and defines these interactions. Among these signals are oxylipins (oxygenated polyunsaturated fatty acids), which act as hormones in eukaryotic organisms. This talk will describe the molecular mechanism for oxylipin crosstalk between seeds and Aspergillus, a necrotrophic fungal pathogen that contaminates its substrate with potent mycotoxins. Oral

Joseph Mougous
University of Washington
Seattle, WA

Bacterial cell-targeting type VI secretion systems Bacteria possess complex multi-component secretion machines that allow highly efficacious delivery of proteins to specifically targeted cells. The type VI secretion system (T6SS) is the most recently described of the large bacterial protein export machines. This pathway, which can be readily identified in the genomes of hundreds of sequenced bacteria, is encoded by a core group of approximately 15 conserved genes and a variable number of accessory elements. Through studies of the secretion system in Vibrio cholerae, and later in Aeromones hydrophila, it was ascertained that a subset of T6SSs are able to deliver bacterial effectors to the cytoplasm of eukaryotic cells. Our laboratory recently reported the surprising finding that certain T6SSs efficiently target other bacteria. Studying Pseudomonas aeruginosa, we identified a small family of T6S substrates; among these we found Tse2 (type VI secretion exported 2), a previously undescribed toxin. Remarkably, we found that T6S specifically targets this toxin to other bacterial cells, and that its delivery is exquisitely dependent on cell-cell contact. Now focusing our efforts on the Burkholderia, we have since expanded upon this initial discovery. Using B. thailandensis as a surrogate for B. pseudomallei, we showed that a single T6SS can target multiple bacterial species. Moreover, we demonstrated that T6SSs within the same organism have distinct roles in host versus bacterial cell interactions. Oral

Lakshmi Rajagopal
University of Washington
Seattle, WA

 

Eukaryotic like signaling mediates S. aureus virulence and antibiotic resistance S. aureus are Gram positive bacteria that cause a wide array of invasive infections in humans. Increasing incidence of S. aureus infections due to methicillin resistant strains has been observed in hospital and community settings. S. aureus encodes signaling enzymes commonly found in eukaryotes; these are a serine/threonine kinase and a serine threonine phosphatase. In both methicillin sensitive and resistant S. aureus, these signaling enzymes regulate alpha toxin expression and virulence. However, these enzymes affect resistance to certain β-lactam antibiotics only in methicillin resistant S. aureus. Further characterization of their role in S. aureus virulence and antibiotic resistance will be presented. Oral

Thomas E Besser, DVM PhD.
Food and Waterborne Disease Research Program
Veterinary Microbiology and Pathology
Washington State University College of Veterinary Medicine
Pullman, WA

Enough to Eat vs Safe to Eat: the Economics, Politics and Science of Escherichia coli O157:H7 With the world's most abundant and inexpensive food supply, produced by the lowest percentage of the country's workforce in the world, do United States residents have good reason to worry about the safety of their food? A seemingly constant stream of food-borne disease outbreaks publicized in the media would suggest the answer is yes. In this talk, I will highlight some of the scientific, policy and economic issues relevant to the prevention and control (or lack thereof) of one specific agent of food-borne illness, Escherichia coli O157:H7. Almost twenty years after the 'Jack in the Box' outbreak, why hasn't this problem gone away? Oral

Wesley C. Van Voorhis MD, Ph.D.
Professor and Head
Allergy and Infectious Diseases Division
Department of Medicine
University of Washington
Seattle, WA 98195-6423

Calcium Dependent Protein Kinases as a Drug Target for Malaria, Toxoplasomosis, and Cryptosporidiosis: An Achilles Heel New therapeutics are needed for infections caused by Cryptosporidium parvum, Toxoplasma gondii, and Plasmodium falciparum. Calcium dependent protein kinase 1 (CDPK1) of T. gondii is thought to be critical for the invasion process of T. gondii and thus is a drug target for toxoplasmosis. By inference, the homologous CDPK of C. parvum and P. falciparum are likely to be critical for infection by cryptosporidium and malaria. We have solved the crystal structures of TgCDPK1 and CpCDPK1 and have shown the active sites are susceptible to "bumped" kinase inhibitors (BKI) that do not inhibit mammalian kinases. This differential sensitivity is due to the absence of a bulky gatekeeper sidechain in the ATP binding site of both TgCDPK1 and CpCDPK1 that is present in mammalian protein kinases. Thus, these BKI offer tremendous selectivity for inhibition of TgCDPK1 & CpCDPK1 vs. human kinases. Furthermore, these BKI compounds show good activity against small gatekeeper CDPK's of P. falciparum. BKI compounds have shown minimal toxicity in mice when administered in the course of other work. Our preliminary results show that multiple BKI compounds based on a known scaffold can inhibit TgCDPK1, P. falciparum CDPK1 & 4, & CpCDPK1 and also inhibit T. gondii and C. parvum cell invasion at low nanomolar concentrations. Expression of a mutant TgCDPK1 with a Met gatekeeper in T. gondii cells leads to resistance to the BKI effect, demonstrating the BKI inhibits cell entry via CDPK1. Oral

Gita Bangera
Bellevue College
Bellevue, WA

Sequencing the genome of Pseudomonas fluorescens L5.1-96: A way to engage undergraduate students in science P. fluorescens L5.1-96 is a plant growth-promoting rhizosphere bacterium that colonizes wheat roots efficiently and is an effective biocontrol agent of a wide range of soilborne fungal plant pathogens. Sequencing the genome of this strain will help to identify genes important in the colonization and persistence of populations of the strain on wheat roots. ComGen is an innovative project supported by CCLI grant from NSF (#0717470) at Bellevue College aimed at providing Community College students with Authentic Research Experiences early in their academic careers.. Community College students isolate plasmids from a genomic library, perform quality control of DNA and perform DNA sequencing reactions and sequence analysis. Early sequencing results have identified genes in strain L5.1-96 that are not present in two previously sequenced strains of fluorescent Pseudomonas spp., Pf-5 and Pf01. Our assessment has found that this has a huge impact on the students in terms of their ability to conduct independent research and their confidence in lab work and in visualizing themselves as scientists. Oral
Derek Wood
Department of Biology
Seattle Pacific University
Department of Microbiology
University of Washington
Seattle, WA
The Genome Education National Initiative: Supporting classroom based research in genomics The Genomics Education National Initiative is sponsored by a distributed consortium of genomics researchers dedicated to the incorporation of original research into high school, community college and undergraduate classrooms and laboratories. The sponsors develop hypothesis driven original research questions and genetic resources to facilitate these projects in the classroom setting. The disseminated nature of the program is coordinated through a website and database that tracks users and project data, provides access to level appropriate protocols, lists reagents, strains and materials and integrates assessment modules. This presentation will describe several of the experimental modules currently under investigation by the group including the finishing of the Agrobacterium rhizogenes A4 genome. This genome was shotgun sequenced and initially assembled at the Monsanto finishing center using 454 reads resulting in 140 contigs. A single enzyme optical map was generated and scaffolding to the closely related A. radiobacter K84 genome was performed to associate 95 of these contigs to known replicons. Using these tools undergraduate researchers in the classroom have closed 44 of the 62 gaps in chromosome I. Oral
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Joe J. Harrison, Yasuhiko Irie, Henrik Almblad, Julie M. Silverman and Matthew R. Parsek Evolution of attenuated virulence in infectious bacterial biofilms Rugose small colony variants (RSCVs) of Pseudomonas aeruginosa are frequently isolated from chronic infections as well as from laboratory biofilm culture. An analysis of these isolates has revealed adaptations that might impair invasiveness of infected tissues. Previous work has established that activating mutations in the wsp pathway cause the RSCV phenotype by increasing cyclic-di-GMP, a key intracellular signal regulating biofilm formation. However, it is also known that mutations independent of the wsp system can cause the RSCV phenotype. By analyzing diversity amongst P. aeruginosa grown in drip-flow biofilm reactors, we show that RSCVs numerically dominate the biofilm population and that mutations in the wsp pathway account for <10% of these variants. Transposon mutagenesis was then used to identify putative chromosomal loci linked to the rugose phenotype. This approach identified five additional classes of genetic elements linked to this phenotype, namely: 1) digu anylate cyclases and phosphodiesterases, 2) genes in the regulator of secondary metabolism (rsm) pathway, 3) genes of the flagellum biosynthesis pathway, 4) the periplasmic disulfide isomerase dsbA, and 5) the ribonuclease orn. Whole genome sequencing of rugose isolates from drip-flow reactors identified that many of these mutations were selected during biofilm growth, but most mutations occurred in genes encoding the flagellum. Subsequently, unmarked mutations were reconstructed in the wild type strain and biochemical analysis revealed that every RSCV class had elevated intracellular cyclic-di-GMP pools when compared to the wild type. Suppressor mutagenesis identified that this was dependent on the diguanylate cyclases siaD and sadC. RSCVs thus overproduced several cyclic-di-GMP regulated extracellular factors important for biofilm formation, including the polysaccharides Pel and Psl. Altogether, these data suggest that mutations activating cyclic-di-GMP signalling pat hways are under positive selection during biofilm growth. Since activation of cyclic-di-GMP signalling pathways is correlated with suppression of several known virulence determinants, evolution of genetic variants with attenuated virulence may be a natural part of biofilm growth for this human bacterial pathogen Poster
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