TY - CONF A1 - Schumacher, Julia T1 - Light-dependent development in Botrytis cinerea N2 - Sunlight is an important environmental factor is almost all ecosystems by being a source of energy, information, and stress. All organisms must protect themselves from the harmful effects of light such as UV radiation, ROS accumulation, heat, and desiccation. Finally, light qualities and quantities can be used for decision making, timing and as guide for directed growth when they are sensed and transduced into intracellular signals. Botrytis cinerea and other plant pathogens infecting the sun-exposed parts of the plant must cope with the high light conditions the host plant seeks. Further they experience an altered light spectrum (‘green gap’) when they colonize shaded parts of the plant; it is depleted for blue and red light that is absorbed by the plant chlorophyll and enriched for green and far-red light that is reflected or transmitted by the plant tissue. As these ambient light conditions trigger the shade avoidance response in the plant, the pathogens may trigger their own ‘shading response’ such as the upregulation of virulence determinants and inoculum production. B. cinerea maintains a highly sophisticated light signaling machinery that senses different light qualities to trigger a variety of responses, that are protection, morphogenesis, positive and negative tropisms, and entrainment. These characteristics render B. cinerea a valuable model to enlighten the role of light in parasitic fungus-plant interactions and beyond. The vegetative mycelium – the core of all infection and developmental programs – is not visibly pigmented and thus considered to be sensitive to biotic and abiotic stresses. However, the vegetative hyphae have a very limited half-life and are usually restricted to the invasive growth phase in which they are protected from light by the plant tissue. Fast colonization of host tissues and by this proper nutrient acquisition enables the rapid formation of long-lasting reproduction structures (melanized conidiophores with conidia, sclerotia) on the surfaces of rotted plant tissues. Depending on the light and temperature conditions, conidiation or sclerotial development is initiated. Taken together, B. cinerea uses light-regulated signaling networks to avoid light whenever possible; for example, by minimizing the half-life of sensitive cells that are hiding in plant tissues and by scheduling critical steps such as conidiogenesis, conidial germination and penetration of plant tissues for the night. T2 - BotrySclero2022 CY - Avignon, France DA - 13.06.2022 KW - Fungus KW - Light KW - Stress KW - Melanin PY - 2022 AN - OPUS4-55248 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Fungi in extreme habitats: Lessons from the microcolonial black fungus Knufia petricola N2 - Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. But to cope with sunlight-associated stresses [e.g. high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses] it is important for fungi to accurately sense and respond to changes in light. The genomes of black [dihydroxynaphthalene (DHN) melanin-containing] fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers of PRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for fungi that receive nutrients through cooperation with phototrophs. T2 - Gordon Research Conference "Cellular and Molecular Fungal Biology" CY - Holderness, NH, USA DA - 26.06.2022 KW - Light KW - Stress KW - Photoperception KW - Pigments PY - 2022 AN - OPUS4-55249 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CHAP A1 - Schumacher, Julia ED - Scott, B. ED - Mesarich, C. T1 - Role of light in the life cycle of Botrytis cinerea T2 - Plant Relationships. The Mycota N2 - The fungus Botrytis cinerea (Botryotinia fuckeliana) infects more than 500 plant species and causes a wide range of symptoms: soft rots, accompanied by collapse and water-soaking of tissues followed by the appearance of gray masses of conidia on leaves and soft fruits (gray mold), and spots that may turn brown to full-scale soft rotting on flower petals (Botrytis blight). In general, B. cinerea is responsible for severe economic losses that are either due to the damage of growing plants in the field or the rot of harvested fruits, flowers, and vegetables during storage under cold and humid conditions. B. cinerea has adapted to the plant host and its environment by evolving strategies to use plant tissues for proliferation in terms of a necrotrophic lifestyle, and to survive biotic stresses (host responses) as well as abiotic factors of the host’s environment such as sunlight and concomitant stresses. B. cinerea maintains a complex regulatory network of light-sensitive proteins and signal transduction pathways to use light for coordinating stress responses, virulence, and reproduction. Different light-controlled reproduction cycles enable B. cinerea to live in moderate climate zones by infecting and propagating in summer and resting in winter when green host tissues are unavailable. KW - Gray mold fungus KW - Plant pathogen KW - Light KW - Photoreceptors KW - Development PY - 2023 DO - https://doi.org/10.1007/978-3-031-16503-0_14 VL - 5 SP - 329 EP - 346 PB - Springer, Cham AN - OPUS4-56724 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben T1 - Fungal biofilms are supported by EPS and protective pigments in their substrate attachment and desiccation tolerance N2 - Rock-inhabiting fungi are known to colonise air-exposed substrates like minerals, photovoltaic panels building facades and monuments, withstanding the various stresses these extreme habitats are known for. Here we show how both colonisation and stress resistance are linked to the properties of the fungal cell surface. By deleting genes involved in the synthesis of melanin and carotenoid pigments of the model rock-inhabiting fungus Knufia petricola via CRISPR-Cas and comparing the behaviour of the gene-deletion mutants with the wild type (WT), we studied the role of these genes in mineral colonisation and stress sensitivity. The extracellular polymeric substances (EPS) of biofilms of the WT and mutants were extracted, quantified and chemically characterised. We observed that the absence of melanin affected the quantity and composition of the produced EPS: melanin-deficient mutants synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, in mineral dissolution experiments, these mutants showed a lower ability to colonise the mineral olivine. We hypothesise that not melanin, but the pullulan-related linkages enabled the melanin-producing strains to attach more strongly to the mineral. Moreover, ICP-OES analysis of the aqueous mineral-derived solutes showed that biofilms of the K. petricola mutants which could attach were able to dissolve the olivine faster than those that could not. The same mutants were also characterised by their sensitivity to desiccation stress: only the mutant deficient in both melanin and carotenoid synthesis was more sensitive to desiccation compared to the WT, indicating that a combination of both pigments is critical to withstand desiccation. Overall, these results show the critical role of the cell surface in the specific capacities of rock-inhabiting fungi. T2 - Biofilms 10 CY - Leipzig, Germany DA - 09.05.2022 KW - Bio-weathering KW - Desiccation KW - Fungal biofilms PY - 2022 AN - OPUS4-56675 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Breitenbach, Romy A1 - Erdmann, Eileen A1 - Gerrits, Ruben A1 - Heeger, Felix A1 - Nitsche, Sarah A1 - Tonon, Chiara A1 - Voigt, Oliver A1 - Gorbushina, Anna T1 - Knufia petricola – a model for exploring the biology of black rock-inhabiting fungi N2 - Black fungi also called black yeasts, rock-inhabiting fungi or microcolonial fungi are a group of Ascomycetes [Eurotiomycetes, Arthoniomycetes and Dothideomycetes] that exhibit high stress tolerance, yeast‑like or meristematic growth, and constitutive 1,8-dihydroxynaphthalene (DHN) melanin formation. They dominate a range of hostile natural and man‑made environments – from desert rocks and salterns to dishwashers, roofs, and solar panels. Due to their slow growth and the lack of sexual cycles and genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We consider the rock inhabitant K. petricola [Eurotiomycetes, Chaetothyriales] a suitable model for studying the phenotypic characteristics of black fungi. With K. petricola the regulation of pigment synthesis, general stress responses and the unusual modes of cell division can be dissected by advanced reverse and forward genetics approaches. The genome of K. petricola strain A95 was sequenced using a combination of short high quality Illumina reads and long PacBio reads. The final assembly consists of twelve contigs: five complete chromosomes and six contigs with one telomer each. Gene annotation supported by transcriptomics and proteomics data was manually curated. Recently, we developed a set of genetic tools to manipulate the genome for analyzing gene functions and studying the cell biology. This set includes CRISPR/Cas9-based genome editing and live-cell imaging using genetically encoded fluorescent proteins, as well as protocols for -omics approaches and for simulation of mineral weathering in the laboratory. Mutants defective in DHN melanogenesis, carotenogenesis or both processes are currently studied to elucidate the role of these protective pigments in tolerance of natural and man-made stresses, weathering of olivine, penetration of marble, and adhesion to surfaces. Further, the established protocols and knowledge gained from K. petricola form a starting point for making other extremotolerant black fungi accessible to genetic manipulation. T2 - 31st Fungal Genetics Conference CY - USA, CA, Pacific Grove DA - 15.03.2022 KW - Black fungi KW - Melanin KW - Genome editing PY - 2022 AN - OPUS4-54585 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Erdmann, Eileen A1 - Nitsche, Sarah A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - Tools for Knufia petricola: new techniques for CRISPR/Cas9-based genome editing N2 - Black microcolonial fungi represent a group of ascomycetes with similar adaptations for existing in natural and anthropogenically created extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) in the multilayered cell walls. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a representative for developing methods for genetic manipulation, simulation of mineral weathering and study of symbiotic interactions. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and three additional resistance selection markers. The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter, while the black-white screening due to the concurrent elimination of pks1 (melanin) and phs1 (carotenoids) was used to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. In addition, two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series (Schumacher, 2012) and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for use in other fungal systems as well. T2 - 31st Fungal Genetics Conference CY - USA, CA, Pacific Grove DA - 15.03.2022 KW - Microcolonial fungi KW - Genetic engineering KW - Fluorescent proteins PY - 2022 AN - OPUS4-54586 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - von Laar, C. A1 - Baar, C. A1 - Plarre, Rüdiger A1 - McMahon, Dino Peter T1 - Genetic relationships of local infestations by Anobium punctatum, Xestobium rufovillosum and their associated predator Korynetes caeruleus from buildings in North-Eastern Germany T2 - Proceedings IRG Annual Meeting 2021 N2 - Wood-destroying pests such as Anobium punctatum and Xestobium rufovillosum cause damage to art and cultural objects as well as to buildings. Monitoring population dynamics of pest species as well as of their naturally occurring counterparts are an essential part in the development of biological control measures as alternatives to conventional wood protection. Therefore, both the dispersal and homogeneity of pest and beneficial insect populations across multiple sites and buildings were investigated in the present study using DNA barcoding. Specifically, beetles of Anobium punctatum (de Geer 1774) (Coleoptera, Ptinidae), Xestobium rufovillosum (de Geer, 1974) (Coloeptera, Ptinidae), and Korynetes caeruleus (de Geer 1775) (Coleoptera, Cleridae) were collected from buildings at four different sites in Mecklenburg-Western Pomerania, North-Eastern Germany. DNA analysis was performed using mitochondrial cytochrome c oxidase subunit I (COI). For A. punctatum, low base pair variability was found in the gene segment studied (4-5 SNPs) within one building (Greven) and between four spatially separated sites. Conversely, in X. rufovillosum, the sequences from two sites studied were homogeneous within a site but differed between locations by nine base pair positions (SNPs). The main result of this study is that the pests A. punctatum and X. rufovillosum showed a higher variability in the investigated gene segment than the natural counterpart K. caeruleus. T2 - IRG52 Webinar on Wood Protection CY - Online meeting DA - 01.11.21 KW - Anobium punctatum KW - Xestobium rufovillosum KW - Korynetes caeruleus KW - DNA barcode PY - 2021 SN - 2000-8953 VL - 21 SP - 1 EP - 11 PB - The International Research Group on Wood Protection CY - Stockholm, Sweden AN - OPUS4-54197 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Abdallah, Khaled A1 - Stock, S. C. A1 - Heeger, Felix A1 - Koester, M. A1 - Nájera, F. A1 - Merino, C. A1 - Spielvogel, S. A1 - Gorbushina, Anna A1 - Kuzyakov, Y. A1 - Dippold, M. A. T1 - Nitrogen Gain and Loss Along an Ecosystem Sequence: From Semi-desert to Rainforest JF - Frontiers in Soil Science N2 - Plants and microorganisms, besides the climate, drive nitrogen (N) cycling in ecosystems. Our objective was to investigate N losses and N acquisition strategies along a unique ecosystem-sequence (ecosequence) ranging from arid shrubland through Mediterranean woodland to temperate rainforest. These ecosystems differ in mean annual precipitation, mean annual temperate, and vegetation cover, but developed on similar granitoid soil parent material, were addressed using a combination of molecular biology and soil biogeochemical tools. Soil N and carbon (C) contents, δ15N signatures, activities of N acquiring extracellular enzymes as well as the abundance of soil bacteria and fungi, and diazotrophs in bulk topsoil and rhizosphere were determined. Relative fungal abundance in the rhizosphere was higher under woodland and forest than under shrubland. This indicates toward plants' higher C investment into fungi in the Mediterranean and temperate rainforest sites than in the arid site. Fungi are likely to decompose lignified forest litter for efficient recycling of litter-derived N and further nutrients. Rhizosphere—a hotspot for the N fixation—was enriched in diazotrophs (factor 8 to 16 in comparison to bulk topsoil) emphasizing the general importance of root/microbe association in N cycle. These results show that the temperate rainforest is an N acquiring ecosystem, whereas N in the arid shrubland is strongly recycled. Simultaneously, the strongest 15N enrichment with decreasing N content with depth was detected in the Mediterranean woodland, indicating that N mineralization and loss is highest (and likely the fastest) in the woodland across the continental transect. Higher relative aminopeptidase activities in the woodland than in the forest enabled a fast N mineralization. Relative aminopeptidase activities were highest in the arid shrubland. The highest absolute chitinase activities were observed in the forest. This likely demonstrates that (a) plants and microorganisms in the arid shrubland invest largely into mobilization and reutilization of organically bound N by exoenzymes, and (b) that the ecosystem N nutrition shifts from a peptide-based N in the arid shrubland to a peptide- and chitin-based N nutrition in the temperate rainforest, where the high N demand is complemented by intensive N fixation in the rhizosphere. KW - Nitrogen KW - Rhizosphere KW - Microbial abundance KW - Natural abundance of 15N KW - Nitrogen fixation KW - Nitrogen uptake PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-543708 DO - https://doi.org/10.3389/fsoil.2022.817641 SN - 2673-8619 VL - 2 SP - 1 EP - 14 PB - Frontiers Media CY - Lausanne AN - OPUS4-54370 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - Schwarze Pilze – Wüstenbesiedler finden neue Lebensräume JF - BIOspektrum N2 - Schwarze mikrokoloniale Pilze besiedeln zunehmend von Menschen geschaffene Habitate, wie schadstoffbelastete Böden, Statuen, Gebäudefassaden und Dächer. Sie verfärben und zersetzen die Oberflächen von anfälligen Materialien oder reduzieren die Lichtausbeute von Solaranlagen. Die Biologie dieser Pilze und ihre Relevanz für die Materialforschung stehen im Fokus unserer Studien an der Bundesanstalt für Materialforschung und -prüfung (BAM). Mit einer Kollektion schwarzer Pilze, die von Solaranlagen isoliert wurden, bringen wir klima- und materialrelevante Biodiversität in den Stammbaum des Lebens. Die Überlebensstrategien dieser Organismen versuchen wir mit molekularbiologischen und genetischen Untersuchungsansätzen zu entschlüsseln. KW - Pilze KW - Genetik KW - Diversität PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-541037 DO - https://doi.org/10.1007/s12268-021-1646-9 VL - 27 IS - 6 SP - 665 EP - 666 PB - Springer AN - OPUS4-54103 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Photoperception in plant- and rock-associated black fungi N2 - Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Sunlight-associated stresses are however multiple: high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses. Ascomycota dominating light-flooded habitats accurately sense and respond to changes in light using it as a cue to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships. Two species from two light-flooded habitats – phyllosphere and sun-exposed solid surfaces – were analysed for their photoreceptor distribution. In both habitats phototroph-associated and black [dihydroxynaphthalene (DHN) melanin-containing] fungi are prevalent. This diversity was sampled with the plant-associated fungus Botrytis cinerea (Leotiomycetes), while Knufia petricola (Eurotiomycetes) was included as a typical biofilm-former on sun-exposed solid surfaces e.g. rocks, building facades, roofs, and solar panels. The analysis has shown that genomes of black fungi contain more photoreceptors than animal pathogens and saprophytes such as Aspergillus nidulans and Neurospora crassa1,2. B. cinerea that causes the grey mould disease by infecting the above-ground parts of more than 200 dicots has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts1. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers of photoreceptors along with the same set of protective metabolites i.e. melanin, carotenoids and mycosporines2. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for sun-stressed fungi that receive nutrients through cooperation with phototrophs. CRISPR/Cas9-based genetic tools for manipulating K. petricola were established3 and are currently used for elucidating the functions of the different photoreceptors in the biology of rock-inhabiting fungi. This work was supported by the grant SCHU 2833/4-1 from the German Research Foundation (DFG) and internal funds of the BAM. T2 - 19th Congress of the European Society for Photobiology CY - Online meeting DA - 30.08.2021 KW - Fungi KW - Light KW - Extreme environments PY - 2021 AN - OPUS4-53191 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Brandhorst, Antonia A1 - Erdmann, Eileen A1 - Nitsche, Sarah A1 - Voigt, Oliver A1 - Gorbushina, Anna T1 - DHN melanin synthesis in the rock inhabitant Knufia petricola N2 - DHN (1,8-dihydroxynaphthalene) melanin is produced by different Ascomycetes via slightly differing biosynthetic routes. The polyketide synthases (PKS) release the heptaketide YWA1, the hexaketide AT4HN or the pentaketide T4HN. The first two products are deacetylated by ‘yellowish-green’ hydrolases to T4HN, and T4HN is further converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases (MCOs). The involved genes are tightly clustered, partially clustered or widely distributed in the genomes of DHN melanin-producing fungi. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g. in melanized reproduction, survival and/or infection structures. In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeast) dwelling in hostile habitats such as bare rock surfaces in hot and cold deserts, exhibits constitutive melanogenesis. Here, we report on the identification and functional characterization of the DHN melanogenic genes of Knufia petricola as a representative of the Chaetothyriales, the sister order of the Eurotiales. Orthologs for all melanogenic genes were identified in the genome of K. petricola A95, including one gene encoding the polyketide synthase (KpPKS1), two genes encoding ‘yellowish-green’ hydrolases (KpYGH1,2), two genes encoding THN reductases (KpTHR1,2) and one gene encoding a scytalone dehydratase (KpSDH1). Ten genes encoding MCOs were identified, all MCOs are predicted to be secreted. The genes are not clustered in the genome but are highly expressed. Gene functions are studied by generation of single, double, and multiple deletion mutants in K. petricola and by heterologous expression in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. T2 - VAAM Fachgruppentagung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - DHN melanin KW - Genetics KW - Biodegradation PY - 2022 AN - OPUS4-55676 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nitsche, Sarah A1 - Gerrits, Ruben A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - The role of the stress-activated MAP kinase SAK1 in Knufia petricola N2 - Mitogen-activated protein (MAP) kinases are highly conserved in animals, plants, and fungi and represent fundamental parts of signaling networks in eukaryotic cells. Black DHN (1,8-dihydroxynaphthalene) melanin and orange carotenoids are produced by many fungi in specific cell types/under certain conditions for protecting cells from abiotic and/or biotic stresses. Microcolonial black fungi constitutively produce DHN melanin, contain the conserved carotenogenic gene cluster, exhibit slow yeast-like growth and survive in extreme environments. Thus, the question arises to which extent pigment formation and responses mediated by the stress-activated MAP kinase module contribute to the observed extremotolerance. We address this question in the rock inhabitant Knufia petricola, the only representative of the extremotolerant black fungi that is genetically amendable. Here, the mutations of pks1, phs1 and both genes result in melanin-free (pink), carotenoid-free (black) and pigment-free (white) strains, respectively (Voigt Knabe et al. 2020, Sci Rep). The gene encoding the stress-activated MAP kinase was deleted in the wild-type and different pigment-deficient backgrounds. In addition, strains were generated that express a GFP-SAK1 fusion protein from the sak1 locus to follow the cytosolic/nuclear shuttling of SAK1 upon stress. Growth of the obtained single, double and triple deletion mutants was tested by dropping cell suspensions on solid media supplemented with different stress-inducing agents. The Δsak1 mutants show slightly reduced growth rates even under non-stress conditions and are hypersensitive to different stress conditions: reduced growth is observed on media inducing, for instance, osmotic, oxidative, membrane, and pH stress, and upon incubation at 30 °C (heat stress). Melanin-free Δsak1 mutants are more sensitive than black Δsak1 mutants to some but not all stress conditions, suggesting that melanin and the SAK1 pathway have complementary roles in protecting K. petricola from stress. T2 - VAAM Fachgruppentragung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - Fungus KW - Extremotolerance KW - Signal transduction PY - 2022 AN - OPUS4-55677 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Erdmann, Eileen A1 - Kunze, R. A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - Tools for Reverse and Forward Genetics in Knufia petricola N2 - Microcolonial black fungi are inhabitants of exposed natural and man-made surfaces in all climate zones. Genetic studies are hampered by slow growth, lacking sexual cycles and difficulties to transform these fungi. Recently, we implemented CRISPR/Cas9-based genetic tools enabling efficient targeted mutations of the rock inhabitant Knufia petricola (Eurotiomycetes/Chaetothyriales) for studying stress responses, the unusual types of cell division, mineral weathering and symbiotic interactions with phototrophic microbes (Voigt, Knabe et al. 2020, Sci Rep; Erdmann et al. 2022, Front Fungal Biol). To further explore the genome of this fungus as representative of the polyphyletic group of black fungi and to assign functions to yet unknown genes, a forward genetics approach is aimed. We chose the two-component Activator/ Dissociation (Ac/Ds) transposon system from maize for generating a collection of insertional mutants by in-vivo mutagenesis. For the optimal use of this genetic tool, an inducible promoter for the expression of the Ac transposase and by this the regulatable transposition of the resistance cassette-containing Ds transposon is requested. However, endogenous promoters for nitrate assimilation and galactose catabolism that are often used in fungi for regulatable gene expression are not inducible by their substrates in K. petricola suggesting that the regulatory networks for nutrient acquisition differ significantly in the oligotrophic black fungi. Therefore, the metabolism-independent Tet-on system was adopted and validated using a Ptet::gfp construct. No GFP fluorescence was detected in absence of the inducer doxycycline (DOX) while fluorescence intensities increased with increasing inducer concentrations. Consequently, the Tet-on system is suitable for regulatable expression of endogenous and foreign genes in K. petricola. T2 - VAAM Fachgruppentagung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - Rock-inhabiting fungus KW - Genetic tools KW - Controllable gene expression PY - 2022 AN - OPUS4-55678 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl A1 - Gorbushina, Anna A1 - Gerrits, Ruben T1 - Modelling biofilm formation and material colonisation for Knufia petricola N2 - Rock-inhabiting black fungi are adapted to survive extreme stresses such as radiation and lack of water and are thus able to colonise arid material surfaces like solar panels and marble monuments. Black fungi can typically grow as one of two cell types: depending on the environmental conditions, they are able to form yeast-like cells or hyphal filaments. Knufia petricola is one member of the rock-inhabiting black fungi which functions as a model reference species. As there is no mathematical model for biofilm formation of RIF, we are going to develop a model for K. petricola which describes fungal growth on the level of individual cells adn biofilm formation. T2 - Mini conference at University of Birmingham CY - Birmingham, UK DA - 05.10.2022 KW - Knufia petricola KW - Rock-inhabiting black fungi KW - individual-based modelling PY - 2022 AN - OPUS4-56697 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - Iron uptake by the rock-inhabiting fungus Knufia petricola N2 - Iron is arguably the most essential metal in living organisms. For rock-inhabiting fungi its acquisition might be unconventional as they (1) tend to inhabit iron-deficient, oxygen-rich surfaces like marble monuments and solar panels and (2) produce the black, iron-adsorbing pigment melanin. We used a range of analytical methods, ongoing mineral dissolution experiments and gene deletion mutants of the model rock-inhabiting fungus Knufia petricola to figure out the mechanisms and substrate deteriorating effects of iron uptake by these organisms. To study both siderophore-mediated and reductive iron assimilation (RIA), genes like sidC, encoding a putative siderophore synthetase and ftr1 and fet3 encoding the subunits of an iron permease-oxidase were deleted. At iron deficient conditions, growth of the wild type (WT) and ΔsidC mutant was similar, whereas growth of the Δftr1-fet3 mutant and the double mutant ΔsidC/Δftr1-fet3 was diminished and absent, respectively. We were not able to detect the siderophore of K. petricola and the WT and mutants were not able to grow at low concentrations of strong iron chelators. However, in a cross-feeding experiment, an overexpression strain of sidC allowed more growth of ΔsidC/Δftr1-fet3 on iron deficient medium than the WT, whereas the ΔsidC mutant could not do so at all. Compared to the WT, the sidC overexpression strain also withstood oxidative stress better and had a shorter lag time and higher growth rate. Combined, these results indicate that K. petricola relies more on RIA than siderophore-mediated uptake as it likely excretes low quantities of a primarily intracellular siderophore. Interestingly, Δftr1-fet3 had a higher iron content than the WT at iron deficient conditions. This difference disappeared upon deletion of melanin synthesis (Δpks1 vs. Δpks1/Δftr1-fet3): melanin-bound iron can likely not be used without RIA. K. petricola’s chelation incapacity implies a habitat free of competition for iron while offering us a mitigation strategy. T2 - ECFG16 CY - Innsbruck, Austria DA - 05.03.2023 KW - Siderophore KW - Melanin KW - Reductive iron assimilation PY - 2023 AN - OPUS4-57148 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Sieksmeyer, Thorben A1 - McMahon, Dino Peter A1 - He, Shulin T1 - Beyond nutrition: host-microbiota interactions drive shifts in the behavioural phenotypes of cockroaches N2 - Recent studies have shown that host-microbiota interactions can lead to dramatic changes in host phenotype especially behaviour. We investigate the causal drivers of microbe-associated shifts in host phenotype by examining how feeding behaviour is modulated by a bacterial infection in an omnivorous cockroach: Blatta orientalis. We conducted food-choice experiments after challenging hosts with the common entomopathogenic soil bacterium Pseudomonas entomophila to understand the impact of bacterial pathogens on host macronutrient preference. We find that immune challenge by this bacterium drives a sharp decline in carbohydrate intake and results in a relative increase in the ratio of protein to carbohydrate consumed. Additionally, infected cockroaches reduce their overall nutrient intake. We show for the first time that cockroach feeding behaviour is dynamically modulated by a pathogen. In contrast to studies on Spodoptera moths, this modulation does not impact any of the immune parameters we measured: it does not affect the abundance of immune related proteins in the hemolymph, hemolymph antimicrobial activity, or survival. This leads to the possibility that as long-lived omnivorous species B. orientalis may be better adapted to unpredictable variation in food availability and quality. An illness-induced anorexia-like response which is thought to assist hosts in limiting nutritional resources available to pathogens is therefore the most likely cause of the observed phenotype. Reduction of carbohydrate intake by sick individuals would be consistent with such an explanation. In another cockroach species Blatella germanica we also investigate whether in addition to pathogens, host microbiota especially gut commensals are causally responsible for shifts in host behavioural phenotype and immune competence. To address this, we conduct food-choice and immunechallenge experiments in germ-free cockroaches and naïve cockroaches. T2 - XI EUROPEAN CONGRESS OF ENTOMOLOGY CY - Neapel, Italy DA - 02.07.2018 KW - Bacterial infection KW - Feeding behaviour KW - Gut microbiota KW - Illness-induced anorexia KW - Proteomics PY - 2018 AN - OPUS4-45414 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - C., Ruibal A1 - L., Selbmann A1 - Serap, Avci A1 - Martin-Sanchez, Pedro Maria A1 - Gorbushina, Anna ED - Gorbushina, Anna T1 - Roof-Inhabiting Cousins of Rock-Inhabiting Fungi: Novel Melanized Microcolonial Fungal Species from Photocatalytically Reactive Subaerial Surfaces JF - Life N2 - Subaerial biofilms (SAB) are an important factor in weathering, biofouling, and biodeterioration of bare rocks, building materials, and solar panel surfaces. The realm of SAB is continually widened by modern materials, and the settlers on these exposed solid surfaces always include melanized, stress-tolerant microcolonial ascomycetes. After their first discovery on desert rock surfaces, these melanized chaetothyrialean and dothidealean ascomycetes have been found on Mediterranean monuments after biocidal treatments, Antarctic rocks and solar panels. New man-made modifications of surfaces (e.g., treatment with biocides or photocatalytically active layers) accommodate the exceptional stress-tolerance of microcolonial fungi and thus further select for this well-protected ecological group. Melanized fungal strains were isolated from a microbial community that developed on highly photocatalytic roof tiles after a long-term environmental exposure in a maritime-influenced region in northwestern Germany. Four of the isolated strains are described here as a novel species, Constantinomyces oldenburgensis, based on multilocus ITS, LSU, RPB2 gene phylogeny. Their closest relative is a still-unnamed rock-inhabiting strain TRN431, here described as C. patonensis. Both species cluster in Capnodiales, among typical melanized microcolonial rock fungi from different stress habitats, including Antarctica. These novel strains flourish in hostile conditions of highly oxidizing material surfaces, and shall be used in reference procedures in material testing. KW - Microcolonial fungi KW - Multilocus phylogeny KW - Photocatalytic surfaces KW - Subaerial biofilms KW - Stress tolerance KW - Constantinomyces PY - 2018 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-455182 DO - https://doi.org/10.3390/life8030030 VL - 8 IS - 3 SP - 30 EP - 44 PB - MDPI CY - Basel, Schweiz AN - OPUS4-45518 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - How light determines the life of the plant pathogen Botrytis cinerea N2 - The rotation of the Earth causes a day/night cycle that is characterized by changing light and temperature conditions. Fungi actively sense the environmental light conditions to induce protective mechanisms against the light-associated stresses and to regulate development. Since fungi adapted to habitats with different light regimes, the complexities of their ‘visual’ systems and photoresponses may vary significantly. Fungi that are associated with plants experience a special light regime because the host seeks optimum light conditions for photosynthesis – and the fungus must cope with them. Moreover, fungi living under the canopy are confronted with an altered spectrum enriched for green and far-red light. Plants sense light to coordinate growth and morphogenesis: a low red:far-red ratio indicates the presence of competitors (shading by other plants) and triggers the shade avoidance response which is accompanied by increased susceptibility to nectrophic pathogens. Botrytis cinerea, the causal agent of gray mold diseases on many plant species in moderate climate zones (high humidity, temperatures around 20°C, seasons), exhibits striking photoresponses accompanied by a broad action spectrum and a high number of photoreceptors. Light is the most important factor controlling morphogenesis: it induces conidiation for disease spreading (summer cycle) and represses sclerotial development for survival and/or sexual recombination (winter cycle). In the past years, we identified several cellular components involved in photoperception and regulation of photomorphogenesis and virulence indicating that the same signaling pathways are of relevance for both processes, propagation and infection. Notably, we recognized the role of the phytochromes (red/far-red light sensors) for coordinated responses to light and elevated temperatures (photo-/thermomorphogenesis) – similar to the function of Arabidopsis PhyB. BcPHY2-like thermosensors are likely restricted to the Leotiomycetes, which include several plant pathogens of the moderate climate zones. T2 - Fungal Genetics Conference 2019 CY - Asilomar, CA, USA DA - 12.03.2019 KW - fungus KW - light KW - plant pathogen KW - signaling KW - virulence PY - 2019 AN - OPUS4-48128 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Photoperception in black fungi that share niches with green organisms N2 - Sunlight, the major source of energy, drives life but in excess it provokes UV-induced DNA damage, accumulation of reactive oxygen species, desiccation, osmotic stresses and so on. Biological strategies to survive excess light include protection, avoidance and active utilisation. Phototrophic organisms including cyanobacteria, green algae and plants use all three strategies. Fungi occupying phototrophic niches may profit from the surplus photosynthetic products which also obliges them to cope with the same light-induced stresses. One way of handling these stresses would be to use similar signalling pathways to sense the presence and to interact with their phototrophic partners. Obviously, the levels of adaptation and responses to light will depend on the environment as well as fungal genotype and phylogenetic position. Black fungi – a polyphyletic group – accumulate the dark pigment DHN melanin in their cell walls and often occupy light-flooded habitats from phyllosphere to rock surfaces. Here we compare two sequenced melanised fungi of different lifestyles in their response to light. The Leotiomycete Botrytis cinerea is an aggressive pathogen that primarily infects the above-ground parts of plants. It possesses large numbers of photoreceptors that respond to a broad spectrum of light. As a consequence, light controls morphogenesis in which it induces conidiation for disease spreading and represses sclerotial development for survival and/or sexual recombination. Cellular components involved in photo-perception and regulation of morphogenesis, stress responses and virulence have been identified and appear to regulate propagation, survival and infection. These include phytochromes, a group of photoreceptors which are particularly enriched in the Leotiomycetes and that mediate coordinated responses to light and elevated temperatures. Assuming that photo-regulation may be equally important for fungi that live in mutualistic relationships with phototrophs either by forming composite organisms or biofilms, we investigate the role of light in the rock-inhabiting Eurotiomycete Knufia petricola. Like other black yeasts, K. petricola grows slowly, does not form specialised reproductive structures and constitutively produces DHN melanin as well as carotenoids. Combining K. petricola with the cyanobacterium Nostoc punctiforme, we developed a model system for studying biofilm formation and bio-weathering. A genetic toolbox to manipulate this model system is being developed. K. petricola strain A95 possesses ten putative photoreceptors, more than found in filamentous Eurotiomycetes suggesting that light plays an important role for abiotic and biotic interactions in extremo-tolerant and symbiosis-capable fungi. T2 - International Symposium on Fungal Stress (ISFUS) CY - Sao Jose dos Campos, Brazil DA - 19.05.2019 KW - Botrytis KW - carotenoids KW - Knufia KW - melanin KW - Nostoc PY - 2019 AN - OPUS4-48130 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Torge, Manfred A1 - Seneschal-Merz, Karine A1 - Günther, Tobias A1 - Wilsch, Gerd T1 - Determination of boron in 19th and 20th century paint layers using LIBS N2 - 19th and 20th centuries glass paint layers consist of a colour body and a colourless lead silicate flux, in which borax or boric acid was added as further component to improve the paint ability and to reduce the firing temperature for multiple layers of paint. Model glasses were used in laboratory tests to investigate the stability of glass paints with additions of boron oxide. To determine boron in paint layers, a LIBS-system with pulsed NdYAG-laser was used. T2 - Technart 2019 CY - Brugge, Belgien DA - 07.05.2019 KW - Stained glass windows KW - Glass paints KW - LIBS PY - 2019 AN - OPUS4-48229 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna T1 - Life on the rocks and other hard surfaces N2 - After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish subaerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that exhibits all the characteristics of microcolonial black fungi such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis (Nai et al. 2013, Fungal Genet Biol). We developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis. The differences between the wild-type fungus and its melanin-deficient mutants were tested in geomicrobiological experiments and measured are now discussed in detail, with special accent on the possible effects of the mutation on EPS and other exuded substances. T2 - Departmental colloquium of the Tuscia University CY - Viterbo, Italy DA - 14.02.2020 KW - Ascomycetes KW - Fungal biofilms KW - Material surface colonisation KW - Genetics KW - Genomics KW - Extremophilic fungi PY - 2020 AN - OPUS4-50643 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Many-sided DHN melanin – spotlight on its function in microcolonial black fungi N2 - Dihydroxynaphthalene (DHN) melanin is produced by different Ascomycetes via slightly differing biosynthetic routes. The polyketide synthases (PKS) release the heptaketide YWA1, the hexaketide AT4HN or the pentaketide T4HN. The first two products are deacetylated by ‘yellowish-green’ hydrolases to T4HN, and T4HN is further converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g. in melanized reproduction, survival and/or infection structures. Remarkable is the regulation of the DHN melanogenesis in the foliar plant pathogen Botrytis cinerea: it involves two differently expressed PKSs providing the precursor in conidia and sclerotia, respectively (Schumacher 2016, Mol Microbiol). In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeast) dwelling in hostile habitats such as bare rock surfaces in hot and cold deserts, exhibits constitutive melanogenesis. Here, DHN melanin builds a protective layer around all vegetative cells thus contributing to the survival of diverse environmental stresses even without specialized reproduction structures. As part of our continuing research on microcolonial rock-inhabiting fungi, we chose the genetically amenable Knufia petricola strain A95 (Nai et al. 2013, Fungal Genet Biol; Noack-Schönmann et al. 2014, AMB Express) for detailed studies. DHN-deficient mutants generated by targeted mutation of biosynthetic genes were studied with regard to the architecture of the cell wall and the EPS (extracellular polymeric substances) matrix, attachment to and weathering of olivine, as well as the tolerance to abiotic and biotic stresses. We will discuss the critical role of the outer cell surface (DHN melanin and EPS) in adhesion to the substrate and subsequent damage of the colonized surface. T2 - European Conference on Fungal Genetics (ECFG15) CY - Rome, Italy DA - 17.02.2020 KW - fungus KW - melanin KW - pigmentation PY - 2020 AN - OPUS4-50591 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Genetic manipulation of the microcolonial black fungus Knufia petricola N2 - Microcolonial black fungi, a polyphyletic group of ascomycetes, exhibit constitutive melanin formation, yeast-like growth and high stress tolerances. They dominate – often together with bacteria and algae in sub-aerial biofilms – a range of hostile environments including natural and man-made ones, from salterns to dishwashers, roofs and solar panels. Because of lacking genetic tools and the slow growth of most isolates, the genetic bases for these specific properties are largely unknown. The rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales) exhibits all characteristics of microcolonial black fungi and was selected as recipient for genetic engineering to study gene functions and genetic interactions. Different variants of green and red fluorescent proteins were successfully expressed indicating that fluorescence microscopy using genetically encoded fluorescent proteins and fluorescent dyes enables various cell biology approaches. Furthermore, genes of biosynthetic pathways (DHN melanin, carotenoids, uracil, adenine) were successfully mutated by applying traditional gene replacement and plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9 or silenced by RNA interference (RNAi). The availability of this advanced and efficient genetic toolbox and the annotated genome sequence of strain A95 makes K. petricola an excellent model for exploring the secrets of microcolonial black fungi. T2 - Departmental colloquium of the Tuscia University CY - Viterbo, Italy DA - 14.02.2020 KW - black fungus KW - genetics KW - Crispr/Cas9 PY - 2020 AN - OPUS4-50592 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna A1 - Knabe, Nicole A1 - Voigt, Oliver A1 - Heeger, Felix A1 - Schumacher, Julia T1 - A genetic toolbox for exploring the life style of the rock-inhabiting black fungus Knufia petricola N2 - After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish subaerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of microcolonial black fungi such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis (Nai et al. 2013, Fungal Genet Biol). We developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence of the strain A95 is paving the way for studying interactions of K. petricola with environmental stressors, material surfaces, soil matrices and phototrophic symbionts. T2 - European Conference on Fungal Genetics (ECFG15) CY - Rome, Italy DA - 17.02.2020 KW - black fungi KW - genomics KW - genetics PY - 2020 AN - OPUS4-50593 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna T1 - Microbiology / biofilms in material research and testing N2 - In the modern world there is an increased understanding that design and performance monitoring of materials have to be tested in connection to chemical, physical and (micro)biological challenges. A systematic study on how biofilms interact with materials and what could be done to engineer biofilms and/or materials in order to maximize the resistance of the material (surface) or the resistance the biofilm-modified material (bulk) is in strong need. In the Department “Materials and the Environment” of the BAM new experimental platform is being developed. With the help of different type of device for high throughput and microbiologically-controlled environment simulation we establish a new approach to clarify the mechanisms of biofilm/material interactions. Despite the focus on fundamental research, the main results of this project proposal will be transferable into material technology and construction chemistry and will influence the development of standardization in this topic. As the interactions of biofilms and materials have implications for most constructions as well as climate change, the results of the research generates additional value. T2 - Initialgespräch - DFG-Forschungsgruppe "Mikrobiologie/Biofilme" CY - Karlsruhe, Germany DA - 14.11.2019 KW - Biofilm KW - Microbiology KW - Black fungi KW - Solar panel PY - 2019 AN - OPUS4-50199 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna T1 - Life on the rocks - Black fungi from biofilms on material-atmosphere interface N2 - Interface between the atmosphere and mineral substrates is the oldest terrestrial habitat. Morphologically simple microbial biofilms were the first settlers on these inhospitable surfaces at times when the Earth was inhabited only by microorganisms and the solid substrates represented only by natural rock surfaces i.e. lithosphere. Miniature, self-sufficient microbial ecosystems continue to develop on subaerial (i.e. air-exposed) solid surfaces at all altitudes and latitudes where direct contact with the atmosphere and solar radiation occurs – on rocks, mountains, buildings, monuments, solar panels. All these sub-aerial biofilms develop under fluctuating and hostile conditions – and thus frequently harbour stress-tolerant black fungi inherently able to cope with the stresses of bright sunlight and constantly changing atmospheric conditions. Black fungi – a polyphyletic group of Ascomycetes– accumulate the dark pigment DHN melanin, diverse carotenoids and mycosporines in their cells and thus successfully colonise sunlight-flooded habitats from phyllosphere to rock surfaces. Various chemical and physical extremes and fluctuating environments belong to the challenges effectively mastered by black fungi. In our laboratory we isolate novel black fungi from man-made habitats like building materials and solar panels. Using Knufia petricola A95 as a model we conduct experiments to clarify interactions of black fungi with inorganic substrates. We use available mutants to determine the functional consequences of changes in the outer cell wall envelopes – from excreted EPS to layers of protective pigments. A genetic toolbox to manipulate this Chaetothyriales representative is in further development. Our long-term goal is to understand the fundamental mechanisms how black fungi are able (i) to adhere to dry atmosphere-exposed surfaces, (ii) to survive multiple stresses and (iii) to change the underlying substrates including rocks. T2 - International Symposium on Fungal Stress (ISFUS) CY - São José dos Campos, Brazil DA - 19.05.2019 KW - Subaerial biofilm KW - Melanins KW - Carotenoids KW - Knufia KW - Mineral weathering PY - 2019 AN - OPUS4-50200 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna T1 - Life on the rocks and other hard surfaces N2 - The story of how black fungi survive harsh conditions on sun-exposed desert rocks and material surfaces is fascinating. In the presentation examples of how knowledge of these organisms can be of practical value (e.g., in biodeterioration studies, such as on stone markers in cemeteries) would be given. Among other stories, the example of how roof tiles amended with a titanium oxide layers as a biocide actually selected for the black fungi will be told. A hypothesis about how these organisms would likely be found on solar panels and some early BAM work in that area will be presented. This then brings the connection to a study ripe for investigation in North Carolina. The talk presents the development of a study aiming at deciphering the influence of microbial biofilm formation on the energy conversion efficiency of solar photovoltaic panels or modules at two facilities (one facility under the impact of high intensity of animal agriculture and high deposition of ammonia from atmosphere and the other with low animal agriculture and lower atmospheric ammonia deposition) in North Carolina. The main hypothesis of the study is that microbial biofilm formation on solar photovoltaic panels will lead to significant decreases in energy conversion efficiency of solar photovoltaic modules and biofilm formation will also be accelerated by high ammonia concentration in the ambient atmosphere and high nitrogen deposition. T2 - Dr. Michael D. Aitken Symposium CY - Chapel Hill, NC, USA DA - 08.11.2019 KW - Black fungi KW - Solar panel KW - Biofilm PY - 2019 AN - OPUS4-50125 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Gerrits, Ruben T1 - Data of "An experimental study of fungal olivine weathering" N2 - This file contains all the data used for the figures shown in the Dissertation of Ruben Gerrits with the title "An experimental study of fungal olivine weathering". In this study, the weathering-affecting, rock-inhabiting fungus, Knufia petricola A95 and the Fe-bearing olivine (Fe0.2Mg1.8SiO4) were selected to investigate fungi-induced effects on mineral dissolution. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, that produced more extracellular polymeric substances (EPS) than the wild type (WT), enabled comparative studies of the role of melanin and EPS in weathering processes. KW - Olivine KW - Weathering KW - Fungus PY - 2019 DO - https://doi.org/10.26272/opus4-48770 PB - Bundesanstalt für Materialforschung und -prüfung (BAM) CY - Berlin AN - OPUS4-48770 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - THES A1 - Gerrits, Ruben T1 - An experimental study of fungal olivine weathering N2 - Free-living and (ecto)-mycorrhizal fungi enhance rock weathering. In their roles as mineral weathering agents and mutualistic partners of phototrophs, fungi supply primary producers like plants and phototrophic microorganisms with mineral-derived nutrients. The exact mechanisms behind fungus-induced mineral weathering processes are however not well understood. Progress can be achieved here by reproducible experimental simulations of the natural processes, using well-characterised model organisms and minerals. In this study, the weathering-affecting, rock-inhabiting fungus, Knufia petricola A95 and the Fe-bearing olivine (Fe0.2Mg1.8SiO4) were selected to investigate fungi-induced effects on mineral dissolution. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, that produced more extracellular polymeric substances (EPS) than the wild type (WT), enabled comparative studies of the role of melanin and EPS in weathering processes. Three experimental systems, which generate long-term microbiological stability, were developed to study the impact of the WT and ΔKppks on olivine weathering: (1) batch and (2) mixed flow dissolution experiments, and (3) biofilm cultivation experiments. In addition, state-of-the-art analytical techniques were used to monitor changes in the growth medium, as well as of the mineral surface and biofilm-mineral interface. Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis of the Mg, Si and Fe concentrations in the reacted growth medium was used to quantify olivine dissolution. In abiotic controls, Mg and Si dissolved congruently, while Fe precipitated. The measured olivine dissolution rates at pH 6 were two orders of magnitude lower than previously reported, but similar at acidic pH. X-ray photoelectron spectroscopy (XPS) analyses of the olivine surface confirmed the presence of Fe (oxyhydr)oxide precipitates. Transmission electron microscopy (TEM) imaging of an abiotically reacted polished olivine section from the long-term cultivation experiment showed the presence of an amorphous layer enriched in Fe. All these observations indicate that the precipitation of Fe (oxyhydr)oxides on the olivine surface inhibits olivine dissolution. Both tested rock-inhabiting fungal strains affect Fe precipitation as well as olivine dissolution. Evaluation of the WT and ΔKppks revealed that the WT formed less biomass but could take up higher amounts of metals (e.g. Fe) and was more efficient in its attachment to olivine. The WT and ΔKppks enhanced olivine dissolution as demonstrated by higher Mg and Si concentration in the reacted growth medium. They furthermore prevented Fe precipitation by binding Fe and retaining it in solution, thereby allowing olivine dissolution to proceed. The WT cells that were attached to the olivine surface were particularly efficient at inhibiting Fe precipitation. By binding Fe directly at the olivine surface, the WT cells removed the inhibition of olivine dissolution almost completely. TEM analysis of polished olivine sections, colonised by a fungal biofilm for seven months, supported this hypothesis. After long-term fungus-olivine interaction, the Fe-enriched, amorphous layer did not develop, and the olivine surface was stronger etched compared to the abiotic control. To study the effect of mutualism on mineral weathering, K. petricola was grown with the cyanobacterium, Nostoc punctiforme ATCC 29133. Both partners showed an enhanced growth and formed a stratified biofilm which attached more strongly to olivine. Nevertheless, the olivine dissolution rate of the fungus-cyanobacterium consortium was moderate. Rock weathering simulation systems developed here are promising research instruments. The experimental conditions allow for the alteration of the studied mineral surface, while the clear definition of these conditions delivers a stable growth of microorganisms. The latter makes these systems universally applicable, especially in combination with integrative multidisciplinary analytics. Processes underlying environmental and biological effects on rock weathering, metal corrosion, plastic degradation, or the deterioration of any other substrate can be studied reproducibly and over a long period of time. The chemical and biological complexity of these simulation systems mimics natural rock weathering processes. The mineral dissolution rates generated in this study are therefore relevant to natural ecosystems. KW - Olivine KW - Weathering KW - Fungus PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:188-refubium-25125-1 DO - https://doi.org/10.17169/refubium-2880 SP - 1 EP - 178 PB - Freie Universität Berlin CY - Berlin AN - OPUS4-48825 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Photoregulation of Metabolism in Plant- & Rock-associated Black Fungi N2 - Fungi that share light-flooded habitats with phototrophs may profit from excess photosynthetic products. But to cope with sunlight-associated stresses it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The filamentous foliar plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideo- and Eurotiomycetes including Knufia petricola possess equal numbers of PRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and photoregulation are important for fungi that avoid loss of energy and nutrients through cooperation with phototrophs. T2 - European Fungal Secondary Metabolism Symposium CY - Hannover, Germany DA - 30.09.2019 KW - Fungi KW - Secondary metabolism KW - Light regulation KW - Pigments PY - 2019 AN - OPUS4-49638 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - McMahon, Dino Peter A1 - Esparza, M. A1 - Davis, H. A1 - Margy, A. T1 - Infection stage and pathogen life cycle determine collective termite behaviour N2 - Social insects nesting in soil environments are in constant contact with entomopathogens and have evolved disease resistance mechanisms within a colony to prevent the occurrence and spread of infectious diseases. Among these mechanisms: mutual grooming reduces the cuticular load of pathogens, and burial of cadavers and cannibalism can prevent pathogens from replicating within the group. We explored how the rate and type of collective behavioural response is determined by stepwise infection dynamics operating at the level of the individual. Specifically, we infected the eastern subterranean termite Reticulitermes flavipes with different types of infectious particle and infection route of the entomopathogenic fungus Metarhizium anisopliae and recorded behavioural responses of nestmates to individuals at different times during the progression of infections. As expected, termites groomed conidia-exposed individuals significantly more than controls. Interestingly, grooming was significantly elevated after fungal germination than before, suggesting that pathogen growth cues act as strong stimulators of allogrooming. Conidia-exposed termites were cannibalized, but only after they became visibly ill. By contrast, termites did not groom blastospore-injected individuals more than controls at any time-point following infection. Instead, we found that blastospore-injected individuals were continually cannibalized at a low-level following injection with either viable or heat-killed blastospores, with a marked increase in cannibalism after termites injected with viable blastospores became visibly ill and were close to death. Together, these findings point to the importance of host condition as a cue for social hygienic behavior, and that the host itself appears to emit essential sickness cues that act as targets for its own sacrifice. This demonstrates that termites have independently evolved to both identify and destructively respond to sickness. T2 - VI Central European Meeting of the IUSSI 2019 CY - Wien, Austria DA - 19.03.2019 KW - Termite KW - Evolution KW - Social immunity PY - 2019 AN - OPUS4-49643 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - McMahon, Dino Peter T1 - The evolution of termite immunity N2 - The termites are a derived eusocial lineage of otherwise non-social cockroaches. Understanding the proximal and ultimate drivers of this major evolutionary transition represents an important goal in biology. One outstanding question concerns the evolution of termite immunity, which is thought to have undergone broad-sweeping adaptations in order to enhance group-level immune protection. To understand the evolutionary origins of termite immunity, we conducted qualitative and quantitative transcriptome analyses along a gradient of sociality. Firstly, we aimed to identify large-scale genetic shifts in immune traits linked to eusociality by comparing immune gene repertoires in solitary and subsocial cockroaches and across a range of eusocial termite lineages. Secondly, we compared the responses of a solitary cockroach, a subsocial wood-roach and different castes of a lower termite species to a non-specific immune challenge, in order to understand how sociality may have influenced the evolution of immune gene regulation. Firstly, we found that termites have a broadly representative repertoire of canonical immune genes as compared to gregarious cockroaches and subsocial wood-roaches. Secondly, with respect to immune challenge, the solitary cockroach and the subsocial wood-roach displayed a similarly comprehensive induced response, while the termite response was considerably dampened by comparison and strongly influenced by caste; with reproductives displaying a generally higher constitutive level of immune-gene expression compared to sterile castes. In summary we did not find termite eusociality to be associated with significant changes in immune gene diversity, but rather to be linked with significant modifications to the regulation of immunity following the origin of division of labour. T2 - Institute for Evolution and Biodiversity Lecture Series, Universität Münster CY - Münster, Germany DA - 20.02.2019 KW - Immunity KW - Evolution KW - Ecology KW - Termite KW - Molecular PY - 2019 AN - OPUS4-49644 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - McMahon, Dino Peter T1 - G-BOP kick-off meeting proposal ideas Ecology and evolution of termite immunity N2 - Results suggest a reduction in immune gene repertoires in termites and possible complementary expression between termite castes. With comparative genomics we will investigate the evolution of gene families related to immunity, try to understand where reductions and expansions take place and relate these changes to shifts in sociality and ecology. The role of TEs in expansions and contractions of immune gene families will be investigated. For these analyses, we propose to generate high quality, highly contiguous genomes of species from different levels of sociality, covering all major termite families. With comparative transcriptomics we will investigate the expression of immune genes in different castes. Via network analyses we will identify pathways indicated in differential immunity between castes and between species of different sociality levels. We will investigate how these pathways have been rewired along the transitions to higher levels of sociality and how, intra-specifically, they change between castes. T2 - Rundgespräch zur Vorbereitung eines SPP G-BOP - Genomic Basis Of Phenotypic Innovations in Insect Evolution CY - Zoologisches Forschungsmuseum, Bonn, Germany DA - 16.05.2019 KW - Bioinformatics KW - Evolution KW - Termites PY - 2019 AN - OPUS4-49645 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - McMahon, Dino Peter T1 - The evolution of termite immunity N2 - A broad suite of immune adaptations have evolved in social insects which hold close parallels with the immune systems of multicellular individuals. However, comparatively little is known about the evolutionary origins of immunity in social insects. We tackle this by identifying immune genes from 18 cockroach and termite species, spanning a gradient of social lifestyles. Termites have undergone contractions of major immune gene families during the early origin of the group, particularly in antimicrobial effector and receptor proteins, followed by later re-expansions in some lineages. In a comparative gene expression analysis, we find that reproductive individuals of a termite invest more in innate immune regulation than other castes. When colonies encounter immune-challenged nestmates, gene expression responses are weak in reproductives but this pattern is reversed when colony members are immune-challenged individually, with reproductives eliciting a greater response to treatment than other castes. Finally, responses to immune challenge were more comprehensive in both subsocial and solitary cockroaches compared to termites, indicating a reduced overall ability to respond to infection in termites. Our study indicates that the emergence of termite sociality was associated with the evolution of a tapered yet caste-adapted immune system. T2 - 112th Annual Meeting of the German Zoological Society CY - Jena, Germany DA - 10.09.2019 KW - Social KW - E$volution KW - Termite KW - Immunity PY - 2019 AN - OPUS4-49646 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna A1 - Knabe, Nicole A1 - Voigt, Oliver A1 - Heeger, Felix A1 - Schumacher, Julia T1 - A Genetic Toolbox for Exploring the Life Style of the Rock-inhabiting Black Fungus Knufia petricola N2 - After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish sub-aerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of black yeasts such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis. For this environmental strain we developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence is paving the way for studying interactions of K. petricola and other black yeasts with environmental stressors, material surfaces, soil matrices and phototrophic symbionts. T2 - VAAM symposium 'Molecular Biology of Fungi' CY - Göttingen, Germany DA - 19.09.2019 KW - Knufia petricola KW - Rock-inhabiting fungus KW - Genetics KW - Crispr-Cas9 PY - 2019 AN - OPUS4-49634 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Abdallah, Khaled A1 - Knabe, Nicole A1 - Breitenbach, Romy A1 - Dementyeva, Polina A1 - Voigt, Oliver A1 - Gerrits, Ruben A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - Developing a genetic toolbox for Knufia petricola A95: a model for stress-tolerant and symbiose-competent rock-inhabiting fungi N2 - Black microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in hostile desert environments. In these niches, MCF have evolved mineral-weathering and symbiotic capabilities as well as mechanisms to cope with multiple stresses such as solar irradiation, temperature extremes and low water activity. Due to their stress tolerance these ascomycetes are prominent in modern terrestrial ecosystems – like man-made material surfaces from roof to solar panels. MCF interactive capabilities support their facultative symbiotic relationships with cyanobacteria and ensure their rock-weathering geochemical activity. Using the rock-inhabiting fungus K. petricola A95 (Chaetothyriales), we developed transformation protocols and deleted genes responsible for production of the protective pigments melanins and carotenoids. To confirm that the mutant phenotypes were not due to hidden mutations, melanin synthesis was restored by complementing the mutants with the respective wild type genes. Strains of K. petricola carrying gene variants for fluorescent proteins EGFP and DsRed are available. We successfully labelled the cytoplasm, nuclei, peroxisomes and mitochondria. Targeted and ectopic integrations result in stable transformants suitable for further phenotypical characterization. As K. petricola is a non-pathogenic fungus with all characteristic features of MCF, including meristematic growth, melanized cell-walls, extracellular polymeric substances and extensive pigment production, our results will shed light on protective role of pigments during cell wall maturation and oxidative stress defence in rock-inhabiting MCF. Genes involved in environmental sensing or substrate and phototroph interactions are currently targeted. With the help of a mutant collection and fluorescently labelled K. petricola we will be able to investigate interactions of MCF with environmental stressors, mineral substrates, soil matrices and phototrophic symbionts. T2 - Fungal Genetics Conference 2019 CY - Asilomar, CA, USA DA - 12.03.2019 KW - Knufia petricola KW - Genetics KW - Melanin PY - 2019 AN - OPUS4-49635 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Heeger, Felix A1 - Whitfield, Daniel A1 - Knabe, Nicole A1 - Nai, Corrado A1 - Schumacher, Julia A1 - Broughton, William A1 - Cuomo, C. A1 - de Souza, E. A1 - Lespinet, O. A1 - Mazzoni, C. A1 - Monaghan, M. A1 - Gorbushina, Anna T1 - Exploring the genome of the black yeast Knufia petricola N2 - Black yeasts form a polyphyletic group of Ascomycota that colonize bare surfaces like rocks, facades of buildings, and solar panels. Their protective adaptations enable an adequate response to fluctuating and diverse temperature, water and UV radiation stresses. Together with bacteria and algae they form sub-aerial biofilms (SAB) this way discoloring and weathering the surfaces they grow on. Strain A95 of Knufia petricola (Eurotiomycetes, Chaetothyriales) displays both typical yeast-like cell growth and constitutive dihydroxynaphthalene (DHN) melanogenesis. Along with the cyanobacterium Nostoc punctiforme as photobiont, it is already used in a model system for studying SAB formation and bio-weathering. Applying the recently developed tools for the generation of deletion mutants will allow to define gene functions and to identify genes critical for abiotic and biotic interactions. We present a chromosome-level genome assembly and annotation for K. petricola A95. The genome was assembled with MaSuRCA using a hybrid assembly approach of Illumina MiSeq and PacBio SMRT sequencing data. The resulting assembly consists of 17 contigs including the complete mitochondrial genome and five complete chromosomes. It shows indication of repeat-induced point mutations (RIP). Supported by RNA sequencing data from eight different growth conditions, 10,994 genes were predicted with the BRAKER2 pipeline. Functional annotation of genes was obtained from general functional annotation databases and the fungal specific database FungiPath. Comparative analyses are in progress to identify genes specific to black yeasts, that may facilitate the survival on exposed surfaces. In sum, the genome sequence of K. petricola is a valuable resource to gain insight into the protein inventory and functional pathways of extremotolerant and symbiosis-capable fungi. T2 - Fungal Genetics Conference 2019 CY - Asilomar, CA, USA DA - 12.03.2019 KW - Knufia petricola KW - Black fungus KW - Genome sequence PY - 2019 AN - OPUS4-49636 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Photoregulation in Plant- & Rock-associated Black Fungi N2 - Fungi that share light-flooded habitats with phototrophs may profit from excess photosynthetic products. But to cope with sunlight-associated stresses it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The filamentous foliar plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideo- and Eurotiomycetes including Knufia petricola possess equal numbers of PRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and photoregulation are important for fungi that avoid loss of energy and nutrients through cooperation with phototrophs. T2 - Scientific Colloquium at the Department of Genetics, University of Seville CY - Seville, Spain DA - 24.10.2019 KW - Light sensing KW - Knufia petricola KW - Botrytis cinerea PY - 2019 AN - OPUS4-49637 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Rock-EATING FUNGI - Genetic Dive into the Biology of the Microcolonial Black Fungus Knufia petricola N2 - Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast‑like growth and constitutive melanin formation. They dominate a range of hostile natural and man‑made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock‑inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. The state‑of‑the‑art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi. T2 - VAAM-Jahrestagung 2021 CY - Online meeting DA - 18.03.2021 KW - Fungus KW - Genetics KW - Marble PY - 2021 AN - OPUS4-52496 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - McMahon, Dino Peter T1 - Evolution, recombination and virulence of emerging bee viruses N2 - Bee populations have declined significantly in recent years and this is thought to be attributable at least in part to the (re-)emergence of viruses. These viruses are predominantly positive single stranded (+ss) RNA viruses belonging to the Picornavirales. Managed honeybees are often infested with the invasive mite, Varroa destructor, which vectors RNA viruses including Deformed wing virus (DWV, family Iflaviridae): a leading culprit of colony losses. Many bee viruses have been sequenced and structural features are now available for viruses such as DWV. DWV consists of at least 3 distinct genotypes, two of which have been shown to be differentially virulent in honeybees. Molecular studies have demonstrated that DWV has a mean evolutionary rate of 1.35 x 10-3 per site per year. For such viruses – in contrast to their eukaryotic hosts – ecological and evolutionary timescales significantly overlap. This rapid evolution allows RNA viruses to adapt quickly to novel host environments with recombination representing an additional key source of genetic variation. Interestingly, recombination between genotypes of DWV has recently been shown to be a common occurrence in honeybees. A challenge has been to develop bioinformatics tools that can accurately reconstruct viral haplotypes – including recombinants – from heterogenous high-throughput sequence data. The impact of blood-to-blood Varroa destructor transmission on virus evolution represents an important question in bee virus research. Due to the nature of the V. destructor life cycle, predictions can be made about the potential impact of the mite on virus virulence evolution. Specifically, the developing honeybee host pupa should remain alive until close to the completion of metamorphosis to provide sufficient time for successful mite reproduction, including offspring mating. For optimal transmission, any virus found in a mature and mated daughter mite will hold a significant selective advantage over a virus found in an immature or unmated daughter mite – placing a cost on virus virulence that impacts honeybee pupae before mites can mate. On the other side, viruses replicating too slowly and with delayed virulence effects will hold a selective handicap because fewer transmission units will be found in mated mites. I have hypothesized that the evolution of virus virulence shifted following the arrival of V. destructor, with viruses, including recombinants and/or specific viral genotypes being selected for a level of virulence in pupae (and likely also in adults) that maximises R0, which represents the basic reproductive number of the virus in the host population. R0 is defined by the number of subsequent infections caused by a single infection and it must be greater than 1 for an infection to spread in a population. It is enhanced by maximising the number of transmission units passed to uninfected susceptible vectors, and ultimately hosts (Figure 1). Honeybee viruses are also shared with sympatric wild bees and viral prevalence and sequence data indicate frequent virus transmission between managed and wild bee species. In addition to infecting the western honeybee (Apis mellifera), DWV can infect other Asian honeybee species such as Apis ceranae. Outside of honeybees, DWV has been found widely in bumblebees, including solitary bees and wasps and there is evidence that it can actively replicate in several Bombus and solitary bee species. Whether the arrival of the V. destructor mite in A. mellifera has driven viral emergence in non-Apis bees is a target of ongoing research. T2 - 2nd European Virus Bioinformatics Centre (EVBC) conference CY - Utrecht, Netherlands DA - 09.05.2018 KW - Virus KW - Virulence KW - Bees PY - 2018 AN - OPUS4-47155 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - McMahon, Dino Peter T1 - Emerging Viruses in Bees: From Molecules toEmerging bee viruses: from molecules to host and vector ecology Ecology N2 - Technical advances in the study of molecular evolution have crystallized the fundamental insight that many bee pathogens evolve and adapt over timescales that overlap with host ecology. At the same time, the role played by bee host community ecology is increasingly being appreciated in host-parasite interactions. Here, we focus on Deformed wing virus (DWV) and present recent studies exploring the link between virulence, DWV genetic diversity and changes to host ecology - namely the arrival of an invasive ectoparasite, the Varroa destructor mite, which vectors viruses between honeybees. Specifically, we show how V. destructor may have created conditions for the emergence of more virulent strains of DWV in the western honeybee, Apis mellifera. We present a molecule-to-ecology framework to help interpret findings and to guide future hypotheses, emphasizing the role of molecular interactions between viruses and host immunity as drivers of change at the bee population level. T2 - International Union for the Study of Social Insects (IUSSI2018) CY - Guaruja, Brazil DA - 05.08.2018 KW - Disease KW - Virus KW - Emerging KW - Pathogen KW - Bee PY - 2018 AN - OPUS4-47156 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - McMahon, Dino Peter T1 - Exploring RNA viruses in edible insects: a case study using cockroaches, termites N2 - Our understanding of RNA viruses from edible insects is minimal at best, with studies largely focusing on model insect species and those associated with obvious signs of disease. This represents a considerable gap in understanding, given the growing role of insects as a source of food and feed, as well as the more general relevance of insects in agriculture and health. Illness due to entomophagy is rare but well documented, including fatal cases following the consumption of termites. Termites are eaten commonly in tropical Asia, Africa and South America, and are among the insects with the highest recorded fat content. There are many species of termites, with a wide range of diets and habitats centering around the consumption of wood and soil substrates. In this study, we report the results from a survey of more than 30 cockroach and termite transcriptomes, with the aim of understanding the diversity and evolution of RNA viruses as well as other potentially pathogenic organisms that are associated with this relevant but somewhat overlooked group of insects. We discuss our results in the context of the possible zoonotic risk posed by insects, as well as in the context of emerging viral and other disease threats that may face insects being reared at industrial scales. T2 - 4th International INSECTA 2018 Conference CY - Giessen, Germany DA - 05.09.2018 KW - Edible insects KW - Emerging KW - Virus KW - Pathogen KW - Termite KW - Cockroach PY - 2018 AN - OPUS4-47157 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - McMahon, Dino Peter T1 - The evolution of termite immunity N2 - The termites are a derived eusocial lineage of otherwise non-social cockroaches. Understanding the proximal and ultimate drivers of this major evolutionary transition represents an important goal in biology. One outstanding question concerns the evolution of termite immunity, which is thought to have undergone broad-sweeping adaptations in order to enhance group-level immune protection. To understand the evolutionary origins of termite immunity, we conducted qualitative and quantitative transcriptome analyses along a gradient of sociality. Firstly, we aimed to identify large-scale genetic shifts in immune traits linked to eusociality by comparing immune gene repertoires in solitary and subsocial cockroaches and across a range of eusocial termite lineages. Secondly, we compared the responses of a solitary cockroach, a subsocial wood-roach and different castes of a lower termite species to a non-specific immune challenge, in order to understand how sociality may have influenced the evolution of immune gene regulation. Firstly, we found that termites have a broadly representative repertoire of canonical immune genes as compared to gregarious cockroaches and subsocial wood-roaches. Secondly, with respect to immune challenge, the solitary cockroach and the subsocial wood-roach displayed a similarly comprehensive induced response, while the termite response was considerably dampened by comparison and strongly influenced by caste; with reproductives displaying a generally higher constitutive level of immune-gene expression compared to sterile castes. In summary we did not find termite eusociality to be associated with significant changes in immune gene diversity, but rather to be linked with significant modifications to the regulation of immunity following the origin of division of labour. T2 - BeGenDiv Annual Genomics Symposium 2018 CY - Berlin, Germany DA - 02.10.2018 KW - Evolution KW - Immunity KW - Termite PY - 2018 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-471581 AN - OPUS4-47158 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - THES A1 - Breitenbach, Romy T1 - Biochemische und physiologische Charakterisierung der extrazellulären Matrix eines Modellbiofilms N2 - An der Grenzschicht zwischen einer festen Oberfläche und der umgebenden Luft wachsende subaerische Biofilme (SAB) zeichnen sich durch eine erhöhte Toleranz gegenüber extremen Umweltbedingungen und dem Eintrag von Bioziden aus. Dieser Schutz vor äußeren Umwelteinflüssen wird vornehmlich durch den Beitrag von extrazellulären polymeren Substanzen (EPS) und Pigmenten, wie Melanin und Carotinoiden, vieler unterschiedlicher Organismen gewährleistet. Deren Synthese wird wiederum durch intrazelluläre Botenstoffe reguliert. Das Cyanobakterium Nostoc punctiforme und der mikrokoloniale Pilz Knufia petricola, als Partner eines etablierten SAB-Modells und Vertreter zweier typischer Organismengruppen in SAB, wurden genutzt, um Botenstoffe und Pigmente genetisch zu manipulieren und die Biofilmmatrix dieser Mutanten strukturell zu untersuchen. In dieser Arbeit konnten erstmals die EPS beider Organismen extrahiert und die Struktur der extrazellulären Polysaccharide beschrieben werden. Daneben wurden die extrazellulären Polysaccharide von K. petricola Wildtyp mit denen verschiedener Pigmentmutanten verglichen. Das Fehlen des Schutzpigmentes Melanin führte zu einer ausgeprägteren extrazellulären Matrix in den Biofilmen. Gleichzeitig änderte sich die Struktur der extrazellulären Polysaccharide. Während der Wildtyp zu ~80% ein α-Glucan und zu ~20% ein α/β-Galaktomannan sekretierte, war der Anteil des Galaktomannans bei den Melaninmutanten erhöht. Das Ausschalten der Carotinoid-Synthese hatte jedoch keinen Einfluss auf die Beschaffenheit der extrazellulären Polysaccharide. Ein deutlich komplexeres extrazelluläres Polysaccharid aus acht verschiedenen Monosaccharid-Einheiten bildete N. punctiforme. Durch die Überproduktion des bakteriellen sekundären Botenstoffes c-di-GMP konnte zudem ein grundlegender Einfluss auf die Reaktion des Cyanobakteriums gegenüber externen Signalen und die damit verbundene Zelldifferenzierung gezeigt werden. Ein artifiziell erhöhtes c-di-GMP-Level in den Zellen führte zur Ausbildung eines sessilen Lebensstils durch Hemmung der Differenzierung motiler Hormogonien und vermehrte Produktion von EPS. Die Struktur der extrazellulären Polysaccharide wurde dadurch nicht verändert. Neben der strukturellen Analyse konnten die Veränderungen der extrazellulären Matrix beider Organismen zusätzlich durch mikroskopische Methoden visualisiert werden. Beide Organismen steuern komplexe Polymere, deren Produktion maßgeblich mit intrazellulären Faktoren verknüpft ist, zu der extrazellulären Matrix des Modellbiofilms bei. KW - Biofilm KW - EPS KW - Extrazelluläre Polysaccharide PY - 2018 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-471747 SP - 1 EP - 198 CY - Berlin AN - OPUS4-47174 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben A1 - Hennehan, M.J. A1 - Frick, D.A. A1 - von Blanckenburg, F. A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - The role of melanin in fungal mineral weathering and metal corrosion N2 - Melanins are organic pigments produced by most fungi. These organisms either fix these pigments in their cell wall or secrete them into their extracellular environment to protect themselves against an array of physicochemical stresses (e.g., UV irradiation, desiccation, ...). Melanin can adsorb metals like Fe. How this affects fungal uptake of Fe and deterioration of Fe-containing minerals and metals is however less known. To study this, we use the model fungi Knufia petricola A95, a rock-inhabiting fungus known to deteriorate minerals and have melanised cell walls, and Amorphotheca resinae, able to contaminate fuel tanks, secrete melanin and corrode metals. In K. petricola, we have deleted genes involved in melanin production and Fe uptake using CRISPR/Cas. Through comparison of the geochemical signatures of these gene deletion mutants with those of the wild type (WT), we explore the specific mineral/metal deterioration mechanisms of melanised fungi. Fe isotope signatures of the biomass of melanin- and Fe uptake-deficient mutants of K. petricola revealed that Fe adsorbed either directly onto melanin or after being reduced by Fe reductases. Importantly, once adsorbed to melanin, Fe could not be mobilised and taken up into the cell: both the WT and its melanin-deficient mutant, previously grown at Fe replete conditions, showed similar growth at Fe deficient conditions. Olivine dissolution experiments revealed that Fe oxidation inhibits dissolution. K. petricola was able to enhance dissolution when this inhibition is strongest (at pH 6) and prevented dissolution when this inhibition is weakest (at pH 4). The fungus therefore dissolves olivine by interacting with the oxidised Fe at the olivine surface. However, Fe uptake did not seem to be involved: mutants deficient in various Fe uptake mechanism dissolved olivine at the same rate as the WT. This indicates that Fe adsorption onto melanin might play a key role. This is also shown by K. petricola’s ability to enhance olivine dissolution even further if secreting a melanin precursor and A. resinae’s corrosion of carbon steel whilst secreting melanin. Combined, our results imply that the Fe adsorbed to melanin cannot be taken up but enables fungi to deteriorate Fe-containing substrates at a higher rate. T2 - Goldschmidt 2023 Conference CY - Lyon, Frankreich DA - 10.07.2023 KW - MIC KW - Bio-weathering KW - Olivine PY - 2023 AN - OPUS4-58523 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Bell, Jérémy A1 - Climent, Estela A1 - Gotor, Raúl A1 - Tobias, Charlie A1 - Martin-Sanchez, Pedro M. A1 - Rurack, Knut T1 - Dipstick coated with polystyrene-silica core-shell particles for the detection of microbiological fuel contamination N2 - Microbial contamination of fuels by fungi or bacteria poses risks such as corrosion and fuel system fouling, which can lead to critical problems in refineries and distribution systems and has a significant economic impact at every stage of the process. Many factors have been cited as being responsible for microbial growth, like the presence of water in the storage tanks. In fact, only 1 % water in a storage system is sufficient for the growth of microorganisms like bacteria or yeasts, as well as for the development of fungal biomass at the oil/water interface. This work presents a rapid test for the accurate determination of genomic DNA from aqueous fuel extracts. The detection is based on the use of polystyrene-mesoporous silica core-shell particles onto which modified fluorescent molecular beacons are covalently grafted. These beacons contain in the hairpin loop a target sequence highly conserved in all bacteria, corresponding to a fragment of the 16S ribosomal RNA subunit. The designed single-stranded molecular beacon contained fluorescein as an internal indicator and a quencher in its proximity when not hybridized. Upon hybridization in presence of the target sequence, the indicator and the quencher are spatially separated, resulting in fluorescence enhancement. To perform the assay the developed particles were deposited on different glass fibre strips to obtain a portable and sensitive rapid test. The assays showed that the presence of genomic DNA extracts from bacteria down to 50–70 μg L–1 induced a fluorescence response. The optical read-out was adapted for on-site monitoring by fitting a 3D-printed case to a conventional smartphone, taking advantages of the sensitivity of the CMOS detector. Such embedded assembly enabled the detection of genomic DNA in aqueous extracts down to the mg L–1 range and represents an interesting step toward on-site monitoring of fuel contamination. T2 - IMA 2023 CY - Chania, Greece DA - 18.09.2023 KW - Teststreifen KW - Test strip KW - Microbial KW - Mikrobiell KW - Smartphone KW - Particles KW - Partikeln PY - 2023 AN - OPUS4-58526 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna A1 - Schumacher, Julia A1 - Abdallah, Khaled T1 - Black fungi on technosphere surfaces: new niches for roof-inhabiting cousins N2 - Human-made systems, also called “build environment” or “technosphere”, sustain human comfort as well as our industrial activities. These systems have become particularly widespread since the Industrial Revolution, i.e., since the 17th century. At the same time, these technical systems – buildings, monuments, energy production, transformation and transmission, water purification and supply systems - serve as new habitats for living organisms. Life is ubiquitously present on our planet since a very long time: the Earth is 4.54 billion years old and microbial communities have played a key role on our planet for 3.7 billion years. Once human-made system appeared, microorganisms became an integral part of all types of technosphere infrastructure as well. Here we will illustrate biosphere-technosphere interactions using a specific example of the black fungi and their impact on the efficiency of solar (photovoltaic) panels. This expanding renewable infrastructure for electricity generation is growing on all continents - and create a specific, arid habitat for stress-tolerant black fungi. Black fungi were once discovered in hot and cold natural deserts – and now belong to the persistent colonisers of human-made deserts of solar parks. This new niche is evolving an impressive biodiversity. So far more than 60 isolates of black fungi belonging to Arthoniomycetes, Eurotiomycetes and Dothideomycetes were obtained from solar panels in Europe and Americas. Here we will present the analysis of this emerging anthropogenic biodiversity. Opportunities for future research in the field include quantification of the microbial load on technosphere surfaces – along with characterisation of the corresponding microbial diversity. The strategy of precise measurement and characterisation will enable us to reliably determine the beneficial and harmful functions that living microorganisms play in the functioning of energy-generating systems – and technosphere in general. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2923 KW - Biosphere-technosphere interaction KW - Microbial communities KW - Solar parks PY - 2023 AN - OPUS4-58451 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Catanzaro, Ilaria A1 - Nitsche, Sarah A1 - Gorbushina, Anna A1 - Schumacher, Julia A1 - Onofri, S. T1 - Generation of Cryomyces antarcticus mutants to explore the importance of DHN melanin for survival N2 - Cryomyces antarcticus, a cryptoendolithic melanized fungus endemic to Antarctica (phylum Ascomycota, class Dothideomycetes incertae sedis), has demonstrated high capability to survive extreme environmental conditions like those found in space (e.g., ionizing radiation, vacuum, microgravity), thus fueling fundamental astrobiological questions like “the search for life beyond Earth”. Its extraordinary resilience has been attributed to the presence of thick, highly melanized cell walls, which may contain both DHN and DOPA melanins. To better understand the contribution of DHN melanin to the overall resilience of C. antarcticus, we decided to generate melanin-deficient mutants by genetic engineering. For this, the melanin-PKS (polyketide synthase)- encoding ortholog was identified in the C. antarcticus CBS 116301 genome and used to design primers for re-sequencing of the capks1 locus in the strain CCFEE 515. Based on the genetic toolkit developed for the black fungus Knufia petricola, we designed a strategy for mutating capks1. Protoplasts of C. antarcticus CCFEE 515 were generated and co-transformed with a circular AMA-containing plasmid for expression and in-vivo assembly of Cas9 and two capks1-specific sgRNAs and a PCR-generated donor DNA i.e., a hygromycin resistance cassette flanked by ~75-bp-long sequences homologous to the capks1 locus. Transformation of C. antarcticus is challenging because of its very slow growth and uncertain gene copy number. It took six months from obtaining enough biomass for cell wall lysis to transferring the putatively resistant transformants for genotyping, but we eventually managed to generate three independent non-melanized (whitish) Δcapks1 mutants! We are now awaiting first complemented strains as ultimate genetic transformation control. They are considered to have a wild-type-like pigmentation due to the reintroduction of the wild-type capks1 into the Δcapks1 mutant. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Black fungus KW - Genetics KW - UV tolerance KW - Melanin PY - 2023 AN - OPUS4-58458 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl A1 - Gerrits, Ruben A1 - Schumacher, Julia A1 - Kreft, J.-U. A1 - Gorbushina, Anna T1 - Fungal biofilms on materials: describing and modelling growth of the black fungus Knufia petricola N2 - Fungi that grow as biofilms are associated with clinical settings as well as various cases of material fouling and material damage. Black fungi as biofilm formers have been rarely studied so far. Their conspicuous dark pigmentation, EPS production, adhesion capabilities and adaptations to stresses allow black fungi to develop biofilms on materials under harsh conditions. For example, rock-inhabiting black fungi withstand sun irradiation and dehydration and are therefore ubiquitous on arid surfaces like solar panels and marble monuments. To understand and control their ability to colonise and deteriorate materials, one should assess and model black fungi’s growth patterns. But so far, no mathematical model has been developed to describe their growth. Knufia petricola A95, representing rock-inhabiting fungi from Chaetothyriales, is genetically amenable and can serve as a model for biofilm studies in black fungi. The primary objective of this project is to develop a growth model for K. petricola A95 which will enable to define and predict material colonisation of black fungi. Dedicated experimental work with K. petricola will allow the quantitative assessment of the impact of environmental conditions (e.g. pH, nutrients, etc.) on the growth behaviour at the biofilm and single cells level. Data which will be used to validate and develop an individual-based model (based on the iDynoMICS modelling platform) that explains how fungal biofilms form, colonise materials, and cause deterioration. Thus far, research has been conducted on the impact of different concentrations and sources of major elements (e.g. C, N, …), as well as trace elements (e.g. Cu, Mg, …), on the colony shape and biomass of Knufia petricola A95 biofilms. To study the behaviour of single cells, the length of the cell cycle in different growth media has been determined via the combined use of microfluidic devices and confocal microscopy. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Biofilm KW - Rock-inhabiting fungus KW - Mathematical modelling PY - 2023 AN - OPUS4-58438 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Erdmann, Eileen A. A1 - Nitsche, Sarah A1 - Gerrits, Ruben A1 - Heeger, Felix A1 - Gorbushina, Anna T1 - Genetic engineering of black fungi: lessons learned from Knufia petricola N2 - The exponential rise in the number of fungal genomes sequenced by next-generation sequencing techniques makes it necessary to increase efforts to correctly annotate and assign gene functions. There are two possibilities to explore a genome and its gene functions. The hypothesis-based method proves the function of already existing gene/allele candidates by targeted mutagenesis - so called reverse genetics. The basis of forward genetics approaches is the random mutagenesis of the genome, followed by screening of obtained mutants for the phenotype of interest, and identification of the mutated genes in the respective mutants. This strategy is hypothesis-generating, means it is necessary to verify the relationship between the detected mutations and the observed phenotype by targeted mutagenesis of the identified gene. We developed a toolbox for editing the genome of the rock inhabitant Knufia petricola [Eurotiomycetes, Chaetothyriales] that allows the study of the phenotypic characteristics of black fungi such as the regulation of pigment synthesis, general stress responses, oligotrophy, and the unusual modes of cell division by advanced reverse and forward genetics approaches. The toolbox includes the annotated genome sequence of strain A95, efficient strategies for CRISPR/Cas9-based genome editing and live-cell imaging using genetically encoded fluorescent proteins, as well as protocols for -omics approaches and for simulation of mineral weathering. A forward genetics approach using transposon mutagenesis is currently developed for identifying essential genes. The established protocols and knowledge gained from K. petricola form a starting point for making other fungi from extreme environments accessible to genetic manipulation. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Fungus KW - Extremotolerance KW - Genetics KW - Model organism PY - 2023 AN - OPUS4-58424 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -