TY - CONF A1 - Gerrits, Ruben T1 - Genetic engineering of a black rock-inhabiting fungus shows its strengths and weaknesses  N2 - Environmental black fungi are known to inhabit anthropogenic habitats like marble monuments, solar panels, indoor walls and even washing machines. These pests are generally assumed to persist and grow in these environments due to their melanin production and oligotrophic lifestyle, respectively. We aimed to confirm these assumptions and to find methods to mitigate their substrate colonisation and deterioration by looking at their ability to produce melanin and take up iron, the latter being a general weakness of aerobes. This was done by means of selecting one species, Knufia petricola, and applying CRISPR/Cas9-mediated genome editing techniques. This showed that K. petricola produces one siderophore; deletion of its encoding gene showing no strong phenotypic effects. Deletion of the other iron uptake mechanism – reductive iron assimilation (RIA) – did however cause a strong growth deficiency at iron depleted conditions. Both together reveal that K. petricola has a weak siderophore and relies on RIA to acquire iron, reflecting a natural habitat free of competition for iron. This comes at a price: K. petricola but also other black fungi isolated from solar panels are extremely sensitive to mediocre iron chelators, offering us a mitigation strategy. Melanin, although adsorbing and reducing iron, did not have an observable effect on iron uptake. The pigment did however affect the fungal ability to withstand desiccation: pigment-deficient mutants were not able to withstand a prolonged incubation in a desiccator. This is a clear advantage in the colonisation of dry subaerial surfaces. T2 - 12th International Mycological Congress (IMC12) CY - Maastricht, Netherlands DA - 12.08.2024 KW - Iron uptake KW - Black fungi KW - Melanin PY - 2024 AN - OPUS4-62269 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Catanzaro, Ilaria 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 - Schumacher, Julia 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 - 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 - Gerrits, Ruben 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 - JOUR A1 - Gerrits, Ruben A1 - Wirth, R. A1 - Schreiber, A. A1 - Feldmann, Ines A1 - Knabe, Nicole A1 - Schott, J, A1 - Benning, L.G. A1 - Gorbushina, Anna T1 - High-resolution imaging of fungal biofilm-induced olivine weathering N2 - Many microorganisms including free-living and symbiotic fungi weather minerals through the formation of biofilms on their surface. Weathering thus proceeds not only according to the mineral’s chemistry and the environmental conditions but also according to the local biofilm chemistry. These processes can be dissected in experiments with defined environmental settings and by employing genetic tools to modify traits of the fungal biofilm. Biofilms of the rock-inhabiting fungus Knufia petricola strain A95 (wild-type, WT) and its melanin-deficient mutant (ΔKppks) were grown on polished olivine sections in subaerial (air-exposed) and subaquatic (submerged) conditions. After seven months of interaction at pH 6 and 25°C, the fungus-mineral interface and abiotic olivine surface were compared using high resolution transmission electron microscopy (HRTEM). The abiotic, subaquatic olivine section showed a 25 nm thick, continuous amorphous layer, enriched in Fe and depleted in Si compared to the underlying crystalline olivine. This amorphous layer formed either through a coupled interfacial dissolution reprecipitation mechanism or through the adsorption of silicic acid on precipitated ferric hydroxides. Its thickness was likely enhanced by mechanical stresses of polishing. Directly underneath a fungal biofilm (WT and mutant alike), the surface remained mostly crystalline and was strongly etched and weathered, indicating enhanced olivine dissolution. The correlation between enhanced olivine dissolution and the absence of a continuous amorphous layer is a strong indication of the dissolution-inhibiting qualities of the latter. We propose that the fungal biofilm sequesters significant amounts of Fe, preventing formation of the amorphous layer and driving olivine dissolution onwards. The seemingly similar olivine surface underneath both WT and mutant biofilms illustrates the comparably insignificant role of specific biofilm traits in the weathering of olivine once biofilm attachment is imposed. Under subaerial conditions, the absence of water on the abiotic surface prohibited olivine dissolution. This was overcome by the water retention capacities of both the WT and mutant biofilm: the olivine surface underneath subaerial fungal biofilms was as weathered as the corresponding subaquatic olivine surface. Under the studied environmental settings, the effect of fungal biofilms on olivine weathering seems to be universal, independent of the production of melanin, the composition of extracellular polymeric substances (EPS) or air-exposure. KW - Bio-weathering KW - Forsterite KW - Extracellular polymeric substances KW - Melanin KW - Black fungi PY - 2021 DO - https://doi.org/10.1016/j.chemgeo.2020.119902 VL - 559 SP - 119902 PB - Elsevier B.V. AN - OPUS4-51403 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Looking through the eyes of fungi: from photoperception to photoresponses and beyond N2 - Sunlight is an almost unavoidable environmental cue and plays a fundamental role in the biology of pro- and eukaryotic organisms. To cope with sunlight-associated stresses e.g., high temperatures, UV radiation, accumulation of reactive oxygen species, desiccation, and osmotic stress, it is important for organisms to accurately sense and respond to changes in light. The benefits of light are obvious for green organisms such as cyanobacteria, algae and plants which use light as an energy source (photosynthesis). Fungi that can share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Examples are the plant pathogen Botrytis cinerea, the gray mold fungus, and the rock inhabitant Knufia petricola, a microcolonial black fungus which forms multispecies biofilms with bacteria and algae. T2 - 20th Symposium of the Research Training Group on Bioactive Peptides – The colorful tree of life CY - Berlin, Germany DA - 23.01.2024 KW - Black fungi KW - Melanin KW - Stress tolerance PY - 2024 AN - OPUS4-59543 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Biodiversity on Materials: Black Fungi Colonizing Solar Panels N2 - Sun-exposed surfaces are extreme environments characterized by intense UV radiation, temperature fluctuations, desiccation, and nutrient scarcity. These habitats are colonized by extremotolerant microorganisms that often form multispecies biofilms. Among them, black fungi (Ascomycota) stand out for their unique morphophysiological traits: slow growth, small compact colonies, reproduction by budding or meristematic growth, and continuous deposition of 1,8-dihydroxynaphthalene (DHN) melanin on thickened cell walls. This melanin is thought to provide protection against abiotic stress, for both the fungi and their microbial partners. Originally associated with natural substrates such as rocks in hot, cold, or saline deserts, black fungi have recently been detected on human-made surfaces, including solar panels, where they may reduce system efficiency. Despite their simple morphology, black fungi represent a vast and underestimated genetic diversity with representatives in Eurotiomycetes, Dothideomycetes, and Arthoniomycetes. They exhibit diverse lifestyles, including lichen symbioses, opportunistic pathogenicity, and saprobic growth. However, only a few genomes of black fungi have been sequenced to date. Initial analyses indicate haploid or diploid states, occasional genome duplications, and gene loss, reflecting adaptation to extreme conditions. For even fewer species, genetic engineering tools are available. To address this gap, we develop genetic tools for functional studies and compare genome sequences. The STRES Community Science Program, funded by the Joint Genome Institute (JGI), aims to sequence up to 600 black fungal genomes from diverse extreme environments (https://stresblackfungi.org/). Our contribution includes black fungi isolated from solar panels in Germany and the United States – among some several new species. Annotated genomes are now becoming available, enabling comparative analyses to discover novel genes for enzymes and secondary metabolites. These efforts also support strategies to mitigate undesired biofilm formation on man-made surfaces such as facades and solar panels. T2 - Annual Conference of the Association for General and Applied Microbiology (VAAM) 2026 CY - Berlin, Germany DA - 22.03.2026 KW - Biodiversity KW - Fungi KW - Melanin PY - 2026 AN - OPUS4-65750 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Erdmann, Eileen A. T1 - In-vivo Transposon Mutagenesis in the Black Fungus Knufia petricola N2 - Microcolonial black fungi, commonly found on sun-exposed natural and man-made surfaces worldwide, belong to different classes within the Ascomycota but convergently evolved similar morpho-physiological adaptations to colonize extreme low-competitive environments. Genetic studies of these organisms have long been hampered by slow growth, lack of sexual cycles and difficulties in transformation. To overcome these limitations, CRISPR/Cas9-based genome editing was implemented in the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales). This enables efficient generation of deletion mutants and overexpression strains for functional analyses, and thus hypothesis-driven targeted mutagenesis. However, the unique ability of black fungi to colonize oligotrophic extreme environments remains poorly understood, and genes involved are unknown. To address this, a hypothesis-generating tool for functional assessment of new species- and/or trait-specific genes was implemented in K. petricola. Specifically, we adapted the two-component Activator/Dissociation (Ac/Ds) transposon system from maize for generating insertional mutants by in vivo mutagenesis. For controlling the transposition of a Ds transposon carrying a resistance cassette, the inducible and metabolism-independent Tet-on promoter system was combined with the Ac transposase (AcTPase) coding sequence fused to a functional nuclear localization signal. In total, six auxotrophic Ac/Ds starter strains were generated, each harboring the Ds transposon at different positions within ade2, ura3 or ppt1. Induction of TET::AcTPase with doxycycline followed by selection on ADE/URA/LYS-lacking media resulted in prototrophic revertants for most Ac/Ds strains. Sequencing of excision sites revealed characteristic footprints. Mapping of Ds re-insertion sites demonstrated transpositions both within the same chromosome and across different chromosomes, identifying 30 genes as non-essential. Current efforts include scaling up mutant generation, as the generation and sequencing of saturated mutant libraries combined with mutation mapping will enable the systematic identification of essential genes under diverse culture conditions. T2 - Annual Conference of the Association for General and Applied Microbiology (VAAM) 2026 CY - Berlin, Germany DA - 22.03.2026 KW - Genetics KW - Material colonizer KW - Melanin PY - 2026 AN - OPUS4-65749 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -