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

linear, branched and cyclic alkanes. Linear hydrocarbons from C3 to C16become hydroxylated at the 2- and 3-position. Branched alkanes werehydroxylated like linear ones, if there is a free hydrogen to abstract. In asecond hydroxylation step the corresponding ketones are formed.Unlike intracellular MMO and P450s, A. aegerita peroxygenase is highlystable and does not need complex cofactors and electron transport proteins(NAD[P]H, flavin reductases). Even in organic solvents such as n-hexane oracetone solution up to 60% nearly no loss in activity can be observed withintwo hours.Beyond that we could show, that the hydroxylation of pentane, hexane,heptane and octane is enantioselctive with an enatiomeric excess between36% for (S)-pentanol and 99.9% for (R)-3-octanol.FBP012Mating type - specific proteins from the zygomyceteMucor mucedoY. Rudigier*, J. WöstemeyerInstitute for Microbiology, Friedrich-Schiller-University, Jena, GermanyDuring their sexual interactions, zygomycetous fungi communicate via anelaborate series of carotene-derived compounds, namely trisporic acid andits biosynthetic progenitors. The number of proteins affected by sexualstimulation, however, seems to be low.In this project, genes that are exclusively expressed in one of the matingtypes during sexual and asexual development and whose proteins are used toinduce sexual structures in the complementary partner are identified andcharacterized.2D-gel electrophoresis is used as a tool to identify different spot-patterns inthe complementary mating types. The following developmental stages areanalysed: substrate mycelia (no zygophores, no asexual structures), trisporicacid induced mycelia, harvested 18h after induction and uninduced andmycelia as controls. All stages are investigated in the (+)-type and the (-)-type as well as in (+)-types cultivated together. In addition, cell wall proteinsof (+)-, (-)- and (+)-cultures grown in liquid medium have been isolated andinvestigated on 2D-gels.Spots that were expressed in only one of the mating types were chosen andanalysed by MALDI-TOF. For the resulting sequences degenerated primerswere constructed. Fragments resulting from PCR were cloned into a vectorand multiplied in E.coli. Sequencing the plasmid inserts was used to confirmwhether the right insert was ligated into the vector.To confirm whether the chosen genes are transcribed differentially, NorthernBlots will be performed and antibodies against the proteins will be made inorder to analyze expression levels during growth and differentiation. At thepresent state of the project, the low amount of differentially expressedproteins confirms previous data found in the zygomycete Absidia glauca [1,2].[1] Teepe, H. et al: FEBS - Letters 234, 100-106.[2] Vetter, M. et al: Microbiol. Res. 149, 17-22.FBP013Molecular analysis of gene expression in Mucor mucedo:Manipulation by transformation with antisensemorpholino oligonucleotidesJ. John*, J. WöstemeyerInstitute for Microbiology, Friedrich-Schiller-University, Jena, GermanycrgA in Mucor mucedo and other zygomycetes codes for a negatively actingtranscriptional regulator. Thus, negative regulation of crgA gene in Mucormucedo, using morpholino oligonucleotides (MO), has a positive effect onlight-regulated carotenogenesis. This is the first demonstration of using MOto down-regulate a gene in fungus. The crgA gene from Mucor mucedo wascloned and sequenced. The MO were designed using the sequenced crgAgene to suppress expression of the crgA gene, resulting in additionalaccumulation of carotene.This system has been used as a proof-of-principle for the morpholinooligonucleotideapproach in mucoralean fungi. We now concentrate ondown-regulation of the ku70 gene in Mucor mucedo in order to achievestable integrative transformation events in this organism. For establishingthe technique, we make use of the cloned leuA gene that we intend tointroduce into a leuA mutant. Gene and mutant are available for Mucorcircinelloides. A silent mutation with a unique XbaI restriction site wascreated in the leucine gene of plasmid pEUKA4. The KU 70 MO wasdesigned and used for transformation of protoplasts of the Mucorcircinelloides leucine auxotroph together with the above mutated plasmid.This approach allows easy screening of transformants in minimal mediumwithout leucine. Transformation can further be confirmed by XbaI digestionof the transformant DNA followed by Southern blot analysis.[1] Navarro, E. et al (2000): S.Eur J Biochem. 2000 Feb; 267(3):800-7.FBP014Molecular analysis of horizontal gene transfer betweentwo mucoralean fungi: Parasitella parasitica and AbsidiaglaucaA. Burmester, S. Karimi*, J. Wetzel, J. WöstemeyerInstitute for Microbiology, Friedrich-Schiller-University, Jena, GermanyParasitella parasitica, a facultative parasite of zygomycetous fungi,transfers nuclei and thus DNA to its host Absidia glauca. Interspecificrecombinants (para-recombinants) contain both, parasite and host DNAinformation [1]. The mechanism of this transfer, as well as the stability andfate of the transferred DNA is not clear. In this respect we used an adenineauxotrophic P. parasitica strain for infection of a methionine auxotrophic A.glauca host strain to analyze prototrophic recombinants and the direction ofthe gene transfer.The mutant strains were characterized at the molecular level. The myceliumof the P. parasitica ade- mutant shows an orange-red color similar to ade1or ade2 mutants of baker’s yeast. DNA sequence analysis of the ade2 geneencoding the phosphoribosyl-aminoimidazole carboxylase of the P.parasitica mutant strain shows the interruption of the open reading frame bya stop codon as the result of a single point mutation.Feeding experiments with different methionine precursors of the A. glaucamutant reveal growth on cystathionine or homocysteine but no growth withhomoserine complementation. The cystathionine-gamma-synthase (CGS) orthe homoserine-acetyl-transferase (HAT) may thus be blocked in function.The genes were cloned and sequenced. The mutant A. glauca HAT geneshows an insertion of a new DNA element which interrupts the open readingframe. Two putative HAT genes of P. parasitica were cloned andsequenced.For four successive sporulation cycles of para-recombinants, the percentagesof complemented, prototrophic phenotypes were determined. In most casesthe para-recombinants show an Absidia morphology.Hybridization analysis of the para-recombinant DNA with the A. glaucaHAT gene probe shows no homologous recombination at the HAT genelocus. No hybridization signals could be detected with the two P. parasiticaHAT probes, indicating a copy number far below single copy or thecomplementation by an unknown third HAT gene. To analyze thecomplementation ability of the isolated HAT genes, transformationexperiments were started with the A. glauca mutant strain using vectorscontaining the A. glauca wild type HAT gene as well with the P. parasitellagenes. Prototrophic transformants with the A. glauca HAT gene wereisolated.[1] Wöstemeyer et al (2002): in Horizontal gene transfer (eds. Syvanen, Kado) 21; pp. 241-247.FBP015Interplay between protein degradation, oxidative stressresponse, polar growth and virulence in the pathogenAspergillus fumigatusB. Jöhnk* 1 , Ö. Bayram 1 , T. Heinekamp 2 , A.A. Brakhage 2 , G.H. Braus 11 Department of Molecular Microbiology & Genetics, Georg-August-University, Göttingen, Germany2 Department of Molecular and Applied Microbiology, Hans-Knöll-Institute(HKI), Jena, GermanyThe ability of eukaryotic cells to rapidly adapt to environmental changes ismainly achieved by a tightly controlled protein turnover. A conservedmechanism for controlled protein degradation is the ubiquitin proteasomesystem. Target proteins are attached to ubiquitin within the ubiquitin-proteinligase (E3) and therefore marked for degradation via the 26S-proteasome.The largest group of E3-enzymes is the SCF Cullin1 Ring ligases (CRL),which are multisubunit enzymes. The F-box subunit functions as a substrateadaptor and therefore is responsible for the substrate specificity of the E3enzyme. The exchange of F-box proteins requires the COP9 signalosomeCSN. Defects in CSN result in increased oxidative stress, impairment ofpolar growth and development, and a misregulated secondary metabolism inthe mold Aspergillus nidulans [1, 2]. We have analyzed the genes for thespektrum | Tagungsband 2011