Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSJacobsen 3 , Thorsten Heinekamp 1,4 , Axel A. Brakhage 1,4 . 1) Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute,Molecular and Applied Microbiology, Jena, Germany; 2) Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute, SystemsBiology / Bioinformatics, Jena, Germany; 3) Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute, MicrobialPathogenicity Mechanisms, Jena, Germany; 4) Institute of Microbiology, Friedrich Schiller University, Jena, Germany.Aspergillus fumigatus is a saprophytic mould normally inhabiting the soil. The fungus also represents a medically important pathogen causing severesystemic infections in immunocompromised patients. To survive in these entirely different habitats, A. fumigatus needs mechanisms to senseenvironmental signals and transduce them intracellularly. One of these signal transduction pathways is the cAMP dependent protein kinase A (PKA)pathway. For A. fumigatus, components of this signaling cascade have been characterized in detail and its significance for virulence is evident.To identify target genes of PKA, we performed microarray analyses using a mutant strain overproducing the PKA catalytic subunit. Following thisapproach, 23 transcription factors potentially regulated by PKA were identified. From these, 15 were deleted and the mutant phenotypes werecharacterized. A gene encoding a C6 finger domain protein that showed highest upregulation of all identified transcription factors is located in a potentialsecondary metabolite gene cluster. Deletion of the transcription factor gene resulted in reduced growth and sporulation of the mutant strain. Thisphenotype was observed even more drastically for a strain lacking the nonribosomal peptide synthetase of the same cluster. Because genes of this clusterwere shown to be transcribed in infected mouse lungs, a virulence study was performed using an embryonated egg infection model. However, thetranscription factor deletion mutant showed no altered virulence compared to the corresponding wild type.To get deeper insights into the function of the secondary metabolite gene cluster, the gene encoding the C6 finger domain protein was overexpressedusing an inducible promoter. Overproduction of the transcription factor resulted in induced transcription of all cluster genes and furthermore in theformation of a brown substance which is currently under investigation.360. Aspergillus nidulans galactofuranose biosynthesis affects antifungal drug sensitivity. Md. Kausar Alam, Susan Kaminskyj. Biology, University ofSaskatchewan, Saskatoon, SK, Canada.The cell wall is essential for fungal survival in natural environments. Many fungal wall carbohydrates are absent from humans, so they are a promisingsource of antifungal drug targets. Galactofuranose (Gal-f) is a sugar that decorates certain carbohydrates and lipids. It comprises about 5% of theAspergillus fumigatus cell wall, and may play a role in systemic aspergillosis. We are studying Aspergillus wall formation in the tractable model system, A.nidulans. Previously we showed single-gene deletions of three sequential A. nidulans Gal-f biosynthesis proteins each caused similar hyphalmorphogenesis defects and 500-fold reduced colony growth and sporulation. Here, we controlled A. nidulans ugeA, ugmA or ugtA using the alcA(p) orniiA(p) promoter. For repression and expression, alcA(p)-regulated strains were grown on complete medium with glucose or threonine, whereas niiA(p)-regulated strains were grown on minimal medium with ammonium or nitrate. Expression was assessed by qPCR and colony phenotype. The alcA(p) andniiA(p) strains produced similar effects: colonies resembling wild type for gene expression, and resembling deletion strains for gene repression. Gal-fimmunolocalization using the L10 monoclonal antibody showed that ugmA deletion and repression phenotypes correlated with loss of hyphal wall Gal-f.None of the gene manipulations affected itraconazole sensitivity, as expected. Deletion of any of ugmA, ugeA, ugtA, their repression by alcA(p) or niiA(p),OR, ugmA overexpression by alcA(p), increased sensitivity to Caspofungin. Strains with alcA(p)-mediated overexpression of ugeA and ugtA had lowercaspofungin sensitivity. Gal-f appears to play an important role in A. nidulans growth and vigor. We are extending these studies to A. fumigatus UgmA andUgtA to determine which amino acids are critical for function and Gal-f generation. Previously, we showed that wild type AfugmA can restore an A.nidulans ugmA deletion strain to wild type phenotype. Our current results show that certain amino acid residues in A. fumigatus UgmA are critical for Gal-fgeneration: constructs with mutated A. fumigatus sequences failed to rescue the AnugmD phenotype.361. WITHDRAWN362. Sulfur metabolism regulatory mutations induce environmental stress response. J. Brzywczy, M. Sienko, R. Natorff, M. Skoneczny, J. Kruszewska, A.Paszewski. Institute of Biochemistry and Biophysics, 02-106 Warszawa, Poland.Mutations in the cysB, sconB and sconC genes affect sulfur metabolism in Aspergillus nidulans in different ways. The cysB mutation blocks synthesis ofcysteine and leads to a shortage of this amino acid while sconB and sconC mutations lead to elevated levels of cysteine and glutathione. We havecompared transcriptomes of these three mutants to the wild type strain finding that expression of 1263 genes is altered at least twofold. Transcripts of908 genes are elevated and 355 genes exhibit decreased levels of transcripts. Despite opposite effect on sulfur metabolism these mutations influenceexpression of overlapping sets of genes. We have assigned categories of Functional Catalogue to up- and down-regulated genes and have identifiedcategories most enriched with differentially regulated genes. Besides genes involved in sulfur metabolism we find that many up-regulated genes arerelated to stress responses. Two component signal transduction system is a category that is fifteen times enriched with genes up-regulated in the sconCmutant and also highly enriched in the cysB and sconB mutants (eight- and tenfold, respectively). Genes encoding heat shock proteins and enzymes ofglutamate degradation pathway are also up-regulated. The glutamate degradation pathway is also known as a GABA shunt which is induced by anoxidative stress. A large group of up-regulated genes is involved in carbohydrate and energy metabolism including genes coding for enzymes of trehaloseand glycerol synthesis. Genes coding for enzymes of alcohol fermentation, which are induced in response to anaerobic stress, are also up-regulated in thesulfur regulatory mutants. Altered expression of carbohydrate metabolism genes is accompanied by changes in sugar accumulation in mutant mycelia andconidia. Among down-regulated genes there are many encoding membrane proteins and enzymes involved in secondary metabolism including penicillinbiosynthesis cluster. Genes coding for lysozyme are down-regulated too. As secondary metabolites often inhibit growth of other organisms, loweredexpression of genes responsible for their synthesis suggests a decreased response to biotic stress in sulfur metabolism mutants.363. RNA 3' tagging - signalling transcript degradation and translational repression. Mark X. Caddick, Meriel G Jones, Daniel Rigden, Igor Y Morozov. DeptBiological Sci, Univ Liverpool, Liverpool, Merseyside, United Kingdom.A large body of work has elucidated the mechanisms associated with mRNA degradation and translational repression - processes which are fundamentalto biological systems. For the majority of eukaryotic transcripts, deadenylation is known to lead to transcript degradation and translational repression.However, the surveillance mechanism which determines the point at which functional transcripts are effectively switched off is poorly defined. Thistransition is generally associated with the mRNA being tagged at the 3' end with a short run of pyrimidine nucleotides. For example, cell cycle-regulateddecapping and degradation of mammalian histone mRNA is triggered by uridylation. In fission yeast and the filamentous fungus, Aspergillus nidulans,polyadenylated transcripts are decapped and degraded in response to 3' tagging. In A. nidulans and Arabidopsis tagging involves the addition of a C/U richelement when the transcript's poly(A) tail is shortened to ~15 nucleotides. Disruption of the Aspergillus nucleotidyltransferases, CutA and CutB, results inloss of tagging, lower rates of degradation and an accumulation of transcripts with short poly(A) tails. Recently we have shown that 3' tagging is also27th Fungal Genetics Conference | 209