VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

three F-box proteins Fbx15, Fbx23 and Fbx29 in the opportunistic pathogenAspergillus fumigatus. Deletion of these genes results in defects in polarizedgrowth during oxidative stress. Further analysis revealed that these genes arerequired for proper growth under amino acid starvation conditions inducedby 3-amino-triazole or 5-methyl-tryptophan, which causes histidine- andtryptophan-starvation by false feedback inhibition. The A. nidulans sconBhomologin A. fumigatus, which is the F-box protein encoding gene involvedin the regulation of cysteine synthesis pathway of A. nidulans, is essentialfor A. fumigatus. We could further show that the gene for the F-box proteinFbx15 is required for virulence of A. fumigatus in a murine model.Functional GFP-tagged versions of Fbx15 and Fbx25 are localized in thenucleus. Future studies aim to identify the potential targets of these F-boxproteins.This work is supported by the Deutsche Forschungsgemeinschaft, DFGResearch Unit 1334.[1] Nahlik K. et al (2010): The COP9 signalosome mediates transcriptional and metabolic response tohormones, oxidative stress protection and cell wall rearrangement during fungal development. Mol.Microb. 78: 962- 979.[2] Braus G. H. et al (2010): Fungal development and the COP9 signalosome. Curr. Opin. Microbiol.13: 1-5.FBP016Identification of regulatory proteins involved in sexualdevelopment in Mucor mucedoM. Park*, J. Wetzel, C. Schimek, J. WöstemeyerInstitute for Microbiology, Friedrich-Schiller-University, Jena, GermanySexual development in mucoralean fungi is regulated by retinoid-like betacarotenederivatives, the trisporoids. Trisporoids serve as pheromones inpartner recognition and possibly as internal transcription regulators.Numerous studies deal with their complex, co-operative synthesis. Some ofthe biosynthesis enzymes have been studied in detail, both, at the enzymaticlevel and at the level of their genes, but their control and especially thetrisporoid signal perception events are essentially unknown.We have now started to search for regulatory proteins involved in sexualdevelopment of Mucor mucedo. Towards this goal, we have made use ofcommercially available antibodies against mammalian retinoid bindingtranscription factors and other regulatory proteins. Using such antibodies,we identified a putative retinoid binding protein, cross-reacting with anantibody against cellular retinaldehyde-binding protein CRALBP, and aputative homeobox protein. We identified and cloned the putative CRALBPgene using PCR and inverse PCR, as well as Southern hybridization. Asanalytical tool, CRALBP was overexpressed in Escherichia coli forinvestigating the role of the putative CRALBP in early sexual development.FBP017Development of a tool for genetic manipulation of thezygomycete Mucor mucedoM. Park*, J. Wetzel, A. Burmester, J. WöstemeyerInstitute for Microbiology, Friedrich-Schiller-University, Jena, GermanyGenetic analysis of Mucor mucedo, a zygomycete model organism forstudying sexual communication and action of the trisporoid pheromonesystem, is hampered by a strong tendency towards autonomous replicationof introduced plasmids instead of stable integration. We are working ondevelopment of reliable tools for genetic manipulation based on interferencewith the DNA double strand break-repair system.In several fungi, targeted gene disruption via homologous recombination isused for analysis of gene function. Although integration of exogenous DNAat homologous sites in the genome occurs easily in Saccharomycescerevisiae, and is at least possible in some filamentous fungi, it is rare inthose fungi where DNA integration occurs predominantly by nonhomologousend joining (NHEJ), leading for DNA integration at ectopicsites in the genome. Direct ligation of DNA strands in NHEJ is mediated bya DNA-dependent protein kinase, a DNA ligase complex, and the Ku70-80heterodimer. Homologoues of Ku70 and Ku80 have been identified in manyorganisms. Recent studies in filamentous fungi have shown increased genetargeting frequencies in Ku-deficient mutants, indicating that Ku-disruptionstrains are efficient recipients for gene targeting. We are searching for a M.mucedo homologoues to the human KU70 and KU80 genes using PCR,inverse PCR, and Southern hybridization.FBP018Do NOX enzymes and GSA alpha subunits participate inidentical signaling pathways in Sordaria macrospora?D. Dirschnabel*, I. Teichert, U. KückDepartment General and Molecular Botany, Ruhr-University, Bochum,GermanyFilamentous fungi in general can undergo sexual and asexual reproduction.Both developmental processes are rather complex and involve regulation ofgene expression, specialization of cell types and intercellularcommunication. In filamentous ascomycetes, asexual development leads tothe production of conidia, nonmotile asexual propagules that are separatedfrom specialized sporogenous cells. In contrast, sexual spores are formed inmulticellular fruiting bodies. The proper development of these fruitingbodies with mature sexual spores is crucial for sexual development ofascomycetes. To explain how this complex process takes place and isregulated is the aim of our work.The ascomycete Sordaria macrospora is an excellent model organism tostudy the complex development from vegetative hyphae to sexual structureslike fruiting bodies. This is due to the fact that no asexual reproduction takesplace and no mating partner is needed. In the last few years, importantplayers involved in sexual development in S. macrospora could be identifiedand characterized, indicating a complex regulatory protein network.Components of this protein network are three G-protein alpha subunits(GSA) which participate in two different signaling pathways leading tosexual development: GSA1 and GSA2 are involved in the formation ofmature fruiting bodies, whereas GSA3 is important for spore germination.Similar results could be obtained for the NAD(P)H oxidases NOXA, NOXBand their regulator NOXR in other fungi. Our current work is now focusedon the connection of NOX and GSA proteins in the regulation of sexualdevelopment.FBP019Will be presented as oral presentation with the ID FBV025!FBP020Proteomic profiling of the short-term response ofAspergillus fumigatus to hypoxic growth conditionsK. Kroll* 1,2 , M. Vödisch 1,2 , M. Roth 3 , A.A. Brakhage 1,2 , O. Kniemeyer 1,21 Department o f Molecular and Applied Microbiology, Hans-Knöll-Institute(HKI), Jena, Germany2 Friedrich-Schiller-University, Jena, Germany3 Bio Pilot Plant, Hans-Knöll-Institute (HKI), Jena, GermanyThe filamentous fungus Aspergillus fumigatus is an opportunistic airbornepathogen causing systemic infections in immunocompromised patients. It isobligate aerobe and requires molecular oxygen for growth. However, duringthe infection process A. fumigatus has to adapt quickly to very low oxygenconcentrations when it grows in inflammatory, necrotic tissue. Recently, itwas shown that hypoxia is involved in virulence of A. fumigatus [1]. In ourlab, the metabolic long-term response of this fungus has recently beenanalyzed by using an oxygen-controlled chemostat. Still little is knownabout the short-term adaptive mechanisms of A. fumigatus to low oxygenconcentrations. To gain more insights, we aimed to investigate theimmediate response of A. fumigatus after oxygen depletion on the proteinlevel by proteomic approaches. A. fumigatus was cultivated as a batchculture in a 3 L bioreactor. After pre-cultivation of A. fumigatus at 21 %(vol/vol) molecular oxygen concentration, the oxygen supply was shifted to0.21 % (vol/vol) and several samples were taken during a 24 hour period ofhypoxia. Cytosolic protein levels were analyzed by 2D-DIGE gelelectrophoresis and differentially regulated proteins were identified byMALDI-TOF/TOF-analysis. Significant changes in the amino acid,carbohydrate and energy metabolism were observed within 24 hours ofhypoxic growth. Glycolytic enzymes and proteins involved in amino acidmetabolism were up-regulated. Furthermore, there was an increasedproduction of proteins involved in respiration, electron transport and ageneral stress response. In contrast, enzymes which catalyze steps in sulfurmetabolism and the biosynthesis of fatty acids were down-regulated.Furthermore, proteins of the pentose phosphate pathway (PPP) and the TCAcycle were down-regulated during the short-term response, as well.Strikingly, we determined also a strong up-regulation of the NO-detoxifyingflavohemoprotein FHp under hypoxic conditions. In summary, hypoxia hasa strong influence on the metabolic regulation of A. fumigatus and thespektrum | Tagungsband 2011