Untersuchung von Signalkomponenten der sexuellen Entwicklung bei dem Basidiomyceten Schizophyllum commune

In dieser Arbeit wurde gezeigt, dass Ras1 und Gap1 des Homobasidiomyceten S. commune an der sexuellen Entwicklung des Pilzes beteiligt sind. Konstitutiv aktive Ras1- und Dgap1-Mutanten zeigten ein verändertes Verhalten im polaren Wachstum des Pilzmyzels auf Festmedien. Während die Hyphen der Wildtyp-Stämme ihre geradlinige Wachstums-richtung beibehielten, veränderten die Hyphen der Mutanten-Stämme ihre Wachstums-richtung und erschienen kurvig. Bei konstitutiv aktiven Ras1-Stämmen konnte man zusätzlich die Ausbildung vermehrter Seitenzweige beobachten. Kompatible konstitutiv aktive Ras1-Mutanten waren nicht in der Lage, ein Dikaryon auszubilden, d.h. sich sexuell zu vermehren. Kreuzungen konstitutiv aktiver Ras1-Mutanten mit kompatiblen Wildtypstämmen führte zur Ausbildung von Fruchtkörpern und Sporen, die aber eine verringerte Keimungsrate gegenüber dem Wildtyp zeigten. Homozygote Dgap1/Dgap1- Kreuzungen waren in der Lage Fruchtkörper auszubilden, die aber einen Defekt in der Ausbildung des Hymeniums mit fehlender Sporenbildung aufwiesen. Untersuchungen der cAMP-abhängigen Proteinkinase A hat gezeigt, dass konstitutiv aktive Ras1- und Dgap1- Mutanten eine deutliche Zunahme ihrer PKA-Aktivität aufwiesen. Dies deutet auf eine Ras1/cAMP vermittelte Signaltransduktionskaskade in S. commune hin

Analysis of Clock-Regulated Genes in Neurospora Reveals Widespread Posttranscriptional Control of Metabolic Potential

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level

Genome Sequence of the Model Mushroom Schizophyllum Commune

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species\u27s unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals

Analysis of clock-regulated genes in Neurospora reveals widespread posttranscriptional control of metabolic potential

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.Keywords: Clock-controlled genes, Circadian, Transcription, Neurospora, RNA-Se

The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering

The roles extracellular polymeric substances (EPS) play in mineral attachment and weathering were studied using genetically modified biofilms of the rock-inhabiting fungus Knufia petricola strain A95. Mutants deficient in melanin and/or carotenoid synthesis were grown as air-exposed biofilms. Extracted EPS were quantified and characterised using a combination of analytical techniques. The absence of melanin affected the quantity and composition of the produced EPS: mutants no longer able to form melanin synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, the melanin-producing strains attached more strongly to the mineral olivine and dissolved it at a higher rate. We hypothesise that the pullulan-related linkages, with their known adhesion functionality, enable fungal attachment and weathering. The released phenolic intermediates of melanin synthesis in the Δsdh1 mutant might play a role similar to Fe-chelating siderophores, driving olivine dissolution even further. These data demonstrate the need for careful compositional and quantitative analyses of biofilm-created microenvironments

Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions

We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 μg of vector DNA and 106 protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure

Maximal ectoine yield for different ATP loads (ATP demands external to the network) calculated using Flux Balance analysis (FBA).

<p>The black solid line shows the values when the full network is operational. Additional lines show yields when only the indicated subset of anaplerotic pathways and Na transporters are active (see text for details).</p