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

FBP035Activation of a silent secondary metabolite gene cluster inAspergillus fumigatus by co-cultivation with StreptomycesrapamycinicusC. König* 1,2 , K. Scherlach 3 , V. Schroeckh 1 , H.-W. Nützmann 1,2 ,C. Hertweck 3,2 , A.A. Brakhage 1,21 Department of Molecular and Applied Microbiology, Hans-Knöll,-Institute(HKI), Jena, Germany2 Friedrich-Schiller-University, Jena, Germany3 Biomolecular Chemistry, Hans-Knöll-Institute (HKI), Jena, GermanyAspergillus fumigatus is the most important air-borne human fungalpathogen. Its genome exhibits far more gene clusters predicted to encodesecondary metabolites than compounds known. Because these unidentifiedmetabolites could have interesting biological activity and could also serve asdrug candidates, it is crucial to activate these often silent gene clusters.Recently, we were able to mimic physiological conditions under which oneof these gene clusters is very likely active [1]. During these investigationswe discovered the principle that silent gene clusters in the filamentousfungus Aspergillus nidulans are activated by a distinct bacterium, i.e.,Streptomyces rapamycinicus, which resulted in the formation of orsellinicand lecanoric acid. As reported here, interestingly, this streptomycete has thepotential to induce silent gene clusters in other fungi. As an example, wediscuss data obtained by co-culturing the human pathogen A. fumigatus withthe same Streptomyces rapamycinicus that leads to induction of silent geneclusters and the production of novel metabolites.[1] Schroeckh et al (2009): Intimate bacterial-fungal interaction triggers biosynthesis of archetypalpolyketides in Aspergillus nidulans. PNAS. 106(34): 14558-14563.FBP036Identification of conidia-associated surface proteins inthe human pathogenic fungus Aspergillus fumigatusV. Pähtz* 1,2 , O. Kniemeyer 1,2 , A.A. Brakhage 1,21 Department of Molecular and Applied Microbiology, Hans-Knöll-Institute(HKI), Jena, Germany2 Institute of Microbiology, Friedrich-Schiller-University, Jena, GermanyThe saprophytic fungus Aspergillus fumigatus is one of the most importanthuman pathogenic fungi that causes severe invasive lung infections inimmunocompromised patients. The asexual reproduction of A. fumigatusleads to the formation of conidia which are released into the atmosphere.Based on their small size of 2 to 3 μm in diameter, they are inhaled byhumans and can reach the lung alveoli. Hence, conidia are the fungal entitywhich have the initial contact with the host’s immune system. Besides cellwall polysaccharides the conidial surface proteins are the first molecularstructures which are recognised by the host’s immune system. Tocharacterise the composition of the Aspergillus fumigatus conidial surfaceproteome, we released surface proteins, especiallyglycosylphosphatidylinositol-anchored proteins (GPI) by HF-pyridineextraction and subsequent LC-MS/MS analysis. We identified 210 differentproteins, of which 50 showed a signal peptide for secretion and 9 proteins aGPI anchor attachment signal. The most abundant surface proteins ofconidia of the WT strain ATCC 46645 represented the hydrophobin proteinRodA and a hypothetical protein. To elucidate the role of the conidialmelanin layer on the composition of the conidial surface proteome we alsoinvestigated spores of the pksP mutant (Jahn et al., 1997), which produceswhite, melanin-free conidia and which is drastically reduced in virulence.Using spectral counts for peptide quantification we detected four GPIanchoredproteins that were missing in HF-pyridine extracts of the pksPmutant: an extracellular matrix protein, an antigenic cell wallgalactomannoprotein, the glutaminase GtaA and the 1,3-βglucanosyltransferaseGel1. The HF-pyridine extract of the mutant straincontained an increased amount of cytoplasmic proteins, e. g. ribosomalproteins, which might indicate a higher metabolic activity and therefore areduction in dormancy.[1] Jahn, B. et al (1997): Isolation and characterization of a pigmentless-conidium mutant ofAspergillus fumigatus with altered conidial surface and reduced virulence. Infect Immun 65: 5110-5117.FBP037Chitin deacetylase from Podospora anserina with twochitin binding domainsJ. Hossbach*, B. MoerschbacherInstitute of Plant Biology and Biotechnology (IBBP), AK Moerschbacher,Münster, GermanyChitin deacetylases (CDAs) convert the biopolymer chitin into chitosan.CDAs can e.g. be found in plant pathogenic fungi which have been shown tochange their cell wall chitin into chitosan upon penetrating the host tissue.Interestingly, some species, e.g. Fusarium graminearum and Podosporaanserina, harbor genes which consist of the catalytic deacetylase domainand also one or more chitin binding domains (CBDs).The latter are known tohelp chitinases to act on insoluble chitin polymers. In assuming a similarfunction in CDAs, we decided to heterologously express such a gene topurify the corresponding protein and to characterize its enzymatic properties.So far, the activity of an enzyme containing the CDA domain and also oneor more CBDs is not described. Chemically produced chitosans possess onlyrandom patterns of acetylation (PAs), but enzymatically deacetylatedchitosan may have non-random PAs We want to analyze the activity andspecificity of these enzymes and their catalysis products, because the CBDmay influence the mode of action and therefore also the biological activitiesof the produced chitosans.One putative P. anserina CDA is predicted to contain a CBD and CDAdomain and is very similar to the group of known CDAs, mostly related tothe Colletotrichum lindemuthianum CDA in the catalytic domain. Domainidentification by hidden Markov models of the PFAM database shows oneN-terminal and one C-terminal CBD. Because the two chitin bindingdomains are different in sequence and length they may have differentsubstrate affinities and/or specificities. We want to analyze the function ofthe different domains by synthesis of the full length protein and thetruncated protein lacking one or both chitin binding domains followed byanalysis of substrate activity and specificity. The CDA gene is synthesizedand optimized for expression in Hansenula polymorpha.FBP038Pyranose-2-oxidase production by the white rot fungusPycnoporus cinnabarinus: characterization of the enzymeand a putative geneJ. Nüske*, R. HerzogInstitute of Microbiology, Friedrich-Schiller-University, Jena, GermanyPeroxidases and laccase are involved in lignocellulose degradation by director indirect (via mediators) action. Peroxidases depend on the provision ofperoxides as a co-substrate. Therefore lignin degrading fungi needmechanisms for the formation peroxides. Besides glyoxal-oxidase, arylalcohol oxidase and glucose 1-oxidase Pyranose 2-oxidase (POx, pyranose:oxygen 2-oxidoreductase EC 1.1.3.10) is a possible candidate for thisfunction. The presence of POx is relatively widespread among wooddegradingbasidiomycetes but the enzyme has only been isolated from alimited number of fungal species and only a few genes and c-DNAsequences are known.In the presence of molecular oxygen the enzyme catalyse the oxidation ofseveral aldopyranoses at carbon-2 and sometimes but in lesser extent atcarbon-3. Besides oxygen the reduction of some different quinones has alsobeen shown. Therefore three possible functions of the enzyme are proposed:1. formation of H 2O 2 for ligninolytic peroxidases 2. reduction of quinonesinstead of oxygen and 3. involvement in the biosynthesis of cortalcerone anantibiotic of fungal origin.Pycnoporus cinnabarinus has not jet been shown to produce this enzyme.For this organism we could show the formation of manganese peroxidase asthe only known ligninolytic peroxidase, therefore a H 2O 2 generating enzymesystem should be present.POx production is correlated with idiophasic growth and seems not to beextracellularly. A correlation of H 2O 2 in the culture supernatant with POxactivity in the cells is not clear indicating that this enzyme should not be theonly H 2O 2 generating activity under the conditions tested.POx has been isolated, purified to apparent homogeneity and characterizedbiochemically. Besides D-glucose other pyranoses (e.g. L-sorbose, D-xylose, cellobiose) can be oxidized. The protein is a homotetramer with amolecular mass of about 244 kDa containing flavin.Using PCR with degenerated primers leading to partial sequences followedby a genome walking protocol with gene specific primers two open readingframes could be detected cloned and sequenced. A protein model (POX1)derived from one of the gene sequences (POX1) using AUGUSTUSspektrum | Tagungsband 2011