Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS34. Functional Analysis of a Novel Diaminopimelate Decarboxylase from the Oomycete Saprolegnia parasitica. Lingxiao ge 1 , Josie Hug 1 , Stan Oome 2 , PaulMorris 1 . 1) Biological Sci, Bowling Green State Univ, Bowling Green, OH; 2) Plant-Microbe Interactions, Utrecht University, The Netherlands.In bacteria and plants, the lysine precursor L, L-diaminopimelate (DAP) is first converted to meso-diaminopimelate by an epimerase. Then meso-DAP isconverted to lysine by a DAP decarboxylase. Comparative analysis of seven sequenced oomycete genomes, revealed that only Saprolegnia parasiticacontains a predicted epimerase. Sequence homology in all of the predicted DAP decarboxylases in oomycetes is strongly conserved, suggesting that theseproteins have similar biochemical activity. The oomycete DAP gene appears to have been acquired by horizontal transfer from Archaea sp. Notably, theseparticular Archaea sp. have all the genes needed to synthesize lysine, except for epimerase. Thus we postulated that the oomycete DAP might be a novelenzyme capable of converting L, L-DAP directly to lysine. To test this hypothesis, we codon-optimized the DAP gene from S. parasitica and expressed it inan E. coli DAP mutant. Complementation assays of the mutant expressing the S parasitica gene in lysine-minus media indicate that the gene functions as aDAP decarboxylase. To determine the substrate specificities of the S. parasitica DAP gene, we have developed an HPLC method to separate the D, L, andmeso isomers of chemically synthesized DAP. Authentic L, L-DAP has also been purified from the culture filtrates of an E coli epimerase mutant. Functionalassays of the affinity-purified protein will enable us to characterize the substrate specificities of the oomycete enzyme. If the S. parasitica DAP enzyme canutilize L, L-DAP as a substrate, then the retention of epimerase in this genome may indicate that meso-DAP is incorporated into the cell wall of this groupof organisms.35. Living on Air?: Ustilago maydis cells grow without being provided nitrogen in their growth media. Michael H Perlin, Michael Cooper. Dept Biol andProgram on Disease Evolution, Univ Louisville, Louisville, KY, USA.Nitrogen is an essential nutrient for all living creatures. Ammonium is one of the most efficiently used and thus preferred, sources of nitrogen. As withother dimorphic fungi, yeast-like cells of Ustilago maydis, the fungal pathogen of maize, switch to filamentous growth when starved fornitrogen/ammonium. U. maydis carries two genes, ump1 and ump2, encoding ammonium transporters that facilitate both uptake of ammonium and thefilamentous response to its absence. While no obvious phenotype is observed when ump1 is deleted, cells without ump2 are unable to filament inresponse to low ammonium, although they can still grow. Surprisingly, ump1ump2 double mutants can also grow on low ammonium. More amazing still,both wild type and mutant cells continue to grow, even after strenuous efforts were made to remove all nitrogen sources from their growth media. Toinvestigate these unusual observations further, we grew wild type and mutant cells in the absence or presence of added nitrogen, as ammonium orsupplied as 15 N gas. Septum bottles with rich, low ammonium and no ammonium media were inoculated with rinsed overnight wild type and mutant cells,injected with +0.1% 15 N 2 and were then incubated for seven days. The resulting biomass was sampled for microscopic examination, collected by filtration,dried and loaded into tin sample capsules for d 15 N analysis by the Stable Isotope Research Unit at Oregon State University. The wild type cells under rich,minimal and no ammonium conditions had mean d 15 N ratios of 0.7, 10.8 and 45.2, respectively, while the mutant cells had mean d 15 N ratios of 3.29, 49.5and 134.8, respectively, for these growth conditions. This indicated significant incorporation of the 15 N tracer from the injected gas into the cellularbiomass. We are currently investigating additional candidate genes that may play a role in this novel capability by a fungus.36. Saprotrophic metabolism of the White-Nose Syndrome fungus Geomyces destructans in bat hibernacula. Hannah Reynolds 1 , Tom Ingersoll 2 , HazelBarton 1 . 1) Department of Biology University of Akron Akron, OH 44325; 2) National Institute for Mathematical and Biological Synthesis (NIMBioS)University of Tennessee Knoxville, TN 37996.Geomyces destructans (Myxotrichaceae, Leotiomycetes), an emerging epizootic disease of hibernating bats in North America has arisen from apredominately saprotrophic genus. We have isolated multiple, non-infectious Geomyces species from cave surfaces and healthy bats for physiological andgenetic comparison with G. destructans to better understand its disease ecology. In particular, we are interested in 1) whether G. destructans retainssaprotrophic ability, acting as a facultative rather than an obligate pathogen and 2) identifying the microhabitats that support Geomyces and presumablyG. destructans growth. Identifying an environmental niche for G. destructans would aid in understanding future disease ecology. Comparative genomicsindicates the presence of multiple enzymes involved in saprotrophic metabolism, including endoglucanases, b-glucosidases and chitinases, while in vitrosaprotrophic assays demonstrate similar cellulase and lipase functions in both pathogenic and non-pathogenic Geomyces. To understand the nativemicrobial habitats that might inhibit or promote G. destructans growth we used molecular phylogenetic analyses of environmental fungal ITS sequences toexamine both the overall fungal diversity and the diversity of Geomyces in multiple cave microhabitats. Knowledge of the specific habitat of G. destructanswill allow us to determine the likelihood for saprophytic growth within caves and estimate the role that subsidies can play in disease ecology. Indeed,disease modeling indicates that an environmental subsidy for the growth of G. destructans increases the likelihood of bat host extinction events.37. Cellulose acting enzymes of the white-rot fungus Dichomitus squalens: expression of the genes and characterization of the enzymes. JohannaRytioja, Aila Mettälä, Kristiina Hildén, Annele Hatakka, Miia Mäkelä. Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.Plant biomass is a diverse raw material that has great potential to be exploited e.g. in second generation biorefinery applications. In order to overcomethe economic and technological thresholds in biomass utilization, novel cellulose attacking enzymes and optimal enzyme mixtures are needed. Thesynergistic effect of cellulose hydrolyzing enzymes, namely endoglucanases (E.C. 3.2.1.4), cellobiohydrolases (CBH, E.C. 3.2.1.91) and b-glucosidases (E.C.3.2.1.21), during cellulose degradation is a well-defined phenomenon, which has also been reported for cellulose oxidizing enzymes. The fungal producedoxidative enzymes related to cellulose degradation include cellobiose dehydrogenases (CDH, E.C. 1.1.99.18) and the proteins of glycoside hydrolase (GH)family 61 (www.cazy.org).Basidiomycetous white-rot fungi are able to efficiently degrade all the wood polymers, i.e. cellulose, hemicelluloses and lignin. Their lignin-modifyingoxidoreductases (peroxidases and laccases) are rather well characterized, whereas their cellulose acting enzymes (CAZymes) have so far gained lessattention. In the large screening of hydrolytic enzymes of basidiomycetous fungi from the Fungal Biotechnology Culture Collection (FBCC, University ofHelsinki), the white-rot fungus Dichomitus squalens was found to produce high cellulolytic activity and appeared as a promising source of novel CAZymes.In this work, the expression of selected hydrolytic and oxidative CAZyme encoding genes (cdh, four cbhs, five putative gh61s) was followed withquantitative real-time RT-PCR during the growth of D. squalens on solid Norway spruce (Picea abies) wood and in semi-solid microcrystalline cellulose(Avicel) -peptone liquid medium. The enzymatic activities of cellulases and xylanase as well as lignin-modifying oxidoreductases were measured from thesemi-solid cultures. In addition, CBHI and CDH enzymes of D. squalens were purified and characterized.130