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 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 - CONF A1 - Fuentes, Elsa A1 - Prieto, Beatriz A1 - Gorbushina, Anna T1 - Mixed model biofilm: interactions between algae, fungi and cyanobacteria and their effects on granite biodeterioration N2 - Granite, despite being one of the most widely used rocks for building material cultural heritage, is also one of the least studied. Previous works have shown that environmental parameters modulated by climate change will lead to changes in the microbial composition of biofilms grown on stone, but also to changes in the production of EPS, in the composition of pigments and, as a consequence, in the biodeterioration processes that this rock undergoes (Fuentes, 2023). Considering that the colonisation process is highly dynamic, small changes in climatic conditions can lead to the formation of different types of biofilms and diverse interactions between species. Knowing the contribution of the different organisms to the deterioration process is important both to anticipate the consequences that climate change will have on the deterioration of the granite, but also when addressing the processes of recolonization after cleaning, to ensure an improvement of the present situation. So, in order to continue my line of research on granite biodeterioration, the aim of this new project is to study the contribution of each type of organism (green algae, cyanobacteria and fungi) to the process of granite deterioration as well as the specific effect on each mineral, in addition to its role in the resilience and response of biofilms to environmental changes. With this main objective, the effect of the type of substrate on the composition of a model mixed biofilm formed by the fungus Knufia petricola, the algae Jaagichlorella sp. and the cyanobacterium Synechocystis sp. will be studied. The results obtained on granite will be compared with those obtained with other lithic types such as marble or limestone. Furthermore, it will be observed if the different ratios between organisms lead to changes in the capacity of these organisms to generate biodeterioration and to buffer climatic changes. Finally, the susceptibility of each mineral that makes up the granite - quartz, feldspars, micas - to biodeterioration mediated by each type of organism will be analysed. To address these questions, on the one hand, the response of the mixed biofilms as a whole will be evaluated by means of confocal microscopy, PAM, as well as the quantification of pigments and EPS, and on the other hand, the effect of these organisms on the deterioration of the substrates, through the use of SEM and TEM, which will allow the assessment of both the changes in porosity and the appearance of deterioration in the surface layers of the rock. This research will deepen the knowledge of the processes of granite deterioration, which have been limitedly studied in relation to microorganisms; but also aims to provide the scientific community with a model mixed biofilm to increase the reproducibility between experiments, one of the main limitations of this field of study. T2 - 19th International Biodeterioration and Biodegradation Symposium CY - Berlin, Germany DA - 09.09.2024 KW - Biofilm KW - Stone KW - Algae KW - Fungi KW - Cyanobacteria KW - Cultural heritage PY - 2024 AN - OPUS4-62272 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Vogel, Christian A1 - Urban, Klaus A1 - Ackerhans, C. A1 - Gorbushina, Anna T1 - Analysis of Carbon and Nitrogen from Atmospheric Sources by Bulk Deposition Sampling at various locations in Germany N2 - Atmospheric deposition of particulate matter is an important indicator of air pollution and a significant factor in material surface fouling. The elemental composition of this nutrient-containing dust depends largely on the exposure region and time as well as on climate. Therefore, in this paper we report an analysis of atmospheric pollutions with a self-made low-cost bulk deposition sampler directed at sampling deposition via air transport and rainfall. We used the device in diverse environments - thus comparing an urban region, an area surrounded by forest and an area mainly dominated by agriculture. The total organic carbon (TOC) and total nitrogen (TN) amounts were selected as indicator parameters and analyzed in a biweekly rhythm for three and a half and two years, respectively. The TOC value responded to particulate matter in the urban area, especially significant were the influences of the New Year's firework in urban and pollen in the rural forest area. In contrast, the TN value was more under the influence of the nitrogen emissions in the agriculture-dominated area. However, the TN value did not correlate with the NOx values in the urban area because the atmospheric nitrogen emissions in the city might originate from various emission sources. Summarizing, the TOC and TN values of the self-made low-cost bulk deposition sampler were in good agreement with environmental events of their immediate surrounding. Moreover, the selected containers and sampling procedures are universally applicable to monitor and analyze organic as well as inorganic parameters (e.g. metal ions) of atmospheric deposition. KW - Passive sampling KW - Biomonitoring KW - Air Analysis PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-609373 DO - https://doi.org/10.1016/j.envadv.2024.100583 SN - 2666-7657 VL - 17 SP - 1 EP - 9 PB - Elsevier Ltd. AN - OPUS4-60937 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - 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 - Schumacher, Julia T1 - The roles of DHN melanin and the stress-activated MAP kinase in the rock inhabitant Knufia petricola N2 - Black fungi/yeasts exhibit high stress tolerance, yeast‑like or meristematic growth, and constitutive 1,8-dihydroxynaphthalene (DHN) melanin formation. Due to their slow growth, robust cell walls and the lack of sexual cycles and genetic tools, the underlying mechanisms of their phenotypic traits have remained largely unexplored. Using recently developed genetic tools, it is now possible to manipulate the genome of the rock-inhabiting model fungus Knufia petricola. Thus, gene functions and the cell biology of black fungi can be studied using CRISPR/Cas9-based genome editing and live-cell imaging with genetically encoded fluorescent proteins. Here, we are addressing the question to which extent constitutive pigment formation (melanin and carotenoids) and responses mediated by the stress-activated mitogen-activated protein (MAP) kinase contribute to the observed extremotolerance of K. petricola. The mutations of pks1, phs1 and both genes result in melanin-free (pink), carotenoid-free (black) and pigment-free (white) strains, respectively. The other putative melanogenic genes were identified in the genome, deleted to confirm their involvement in DHN melanogenesis and co-expressed in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. Sak1 encoding the stress-activated MAP kinase was deleted in the wild-type and different pigment-deficient backgrounds. Growth of the obtained single, double and triple deletion mutants was tested by droplet tests on media supplemented with different stress-inducing agents. The Δsak1 mutants show slightly reduced growth rates even without environmental pressure and are hypersensitive to different stresses: e.g. osmotic, oxidative, membrane, pH and 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 - 16th European Conference on Fungal Genetics CY - Innsbruck, Austria DA - 05.03.2023 KW - Black fungus KW - Extremotolerance KW - Pigments PY - 2023 AN - OPUS4-57147 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Catanzaro, Ilaria T1 - Understanding the role of DHN melanin in Cryomyces antarcticus N2 - Cryomyces antarcticus – a cryptoendolithic black fungus endemic to Antarctica – is taxonomically classified in phylum Ascomycota, class Dothideomycetes incertae sedis. C. antarcticus has shown high capability to survive extreme environmental conditions like those found in space (ionizing radiation, vacuum, microgravity), thus fueling fundamental astrobiological questions like “searching for life beyond Earth” (Onofri et al. 2020, Extremophiles Astrobiol Model). Its extraordinary resilience has been attributed to the presence of thick, highly melanized cell walls, which may contain both DHN and DOPA melanins (Pacelli et al. 2020, Appl Microbiol Biotechnol). To better understand the contribution of DHN melanin to the overall resilience of C. antarcticus, we initially adopted chemicals e.g., tricyclazole to inhibit the DHN melanin synthetic pathway; however, these studies gave inconclusive results. Eventually, we decided to generate melanin-deficient mutants by genetic engineering. Using the genetic toolkit developed for the black fungus Knufia petricola (Voigt et al. 2020, Sci Rep; Erdmann et al. 2022, Front Fungal Biol), we designed a strategy for mutating the key enzyme (polyketide synthase)-encoding gene capks1 by transient delivery of Cas9 and capks1-specific sgRNA from AMA-containing plasmids and PCR-generated donor DNA i.e., resistance cassettes flanked by ~75-bp-long sequences homologous to capks1. For this, the melanin-PKS encoding ortholog was identified in the C. antarcticus CBS 116301 genome (mycocosm.jgi.doe.gov) and used to design primers for re-sequencing of the capks1 locus in the strain CCFEE 515. Transformation of C. antarcticus is challenging because of its very slow growth; we expect that 4-6 months are needed from obtaining enough biomass for cell wall lysis until transferring putatively resistant transformants for genotyping. Important parameters were evaluated: protoplasts can be generated, and they survive the transformation procedure, and suitable concentrations of selective agents have been identified. Nowadays, we are waiting for the first C. antarcticus mutants considered to be deficient in DHN melanogenesis. T2 - 16th European Conference on Fungal Genetics CY - Innsbruck, Austria DA - 05.03.2023 KW - Cryptoendolithic black fungus KW - DHN melanin KW - Astrobiology PY - 2023 AN - OPUS4-57145 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - von Werder, Julia T1 - Biofilme auf Gebäuden - Biorezeptivität statt Biofilme N2 - Der Vortrag erläutert das Konzept der Gebäudebegrünung mit Biofilmen. Beton wird dabei als künstliches Gestein betrachtet, dessen Besiedelbarkeit durch Mikroorganismen gezielt verbessert werden soll. Ein dreistufiges Testsystem – Laborversuche, simulierte Bewitterung und Freilandtests – analysiert, wie physikalische und chemische Substrateigenschaften das Wachstum beeinflussen. Ein definierter Algen‑Pilz‑Modellbiofilm dient zur reproduzierbaren Bewertung der Besiedlung, erfasst über PAM‑Fluorometrie. Die Ergebnisse zeigen, dass Rauheit, Textur und Porosität entscheidend für Anhaftung, Feuchteretention und Biofilmwachstum sind. Zudem wird deutlich, dass die Organismen hohe Stresstoleranz benötigen, um variierende Umweltbedingungen an realen Fassaden zu überstehen. Insgesamt belegt das Projekt das Potenzial biorezeptiver Betone als low‑tech‑Fassadenbegrünung mit ökologischem Mehrwert. T2 - Workshop Ökologische Campusentwicklung: Wasser und Vegetationsmanagement in Helmholtz-Zentren CY - Potsdam, Germany DA - 03.04.2025 KW - Biofilm KW - Fassade KW - Biorezeptivität KW - Low-tech Begrünung KW - Modellbiofilm PY - 2025 AN - OPUS4-65448 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Stohl, Leonie A1 - Tonon, Chiara A1 - Cook, Jake A1 - Gorbushina, Anna A1 - Dehn, Frank A1 - von Werder, Julia A1 - Manninger, Tanja T1 - Understanding bioreceptivity of concrete: material design and characterization N2 - The climate crisis is driving an increasing demand for ecologically oriented concepts. In the building sector, this demand includes not only the use of environmentally friendly materials but also the greening of urban areas. One promising approach is the development of bioreceptive concrete façades, which support the growth of green biofilms directly on their surfaces. These innovative façades are anticipated to deliver benefits comparable to those of macroscopically greened façades, such as enhanced biodiversity and improved air quality, while offering the advantages of being more self-sustaining and stable systems once fully established. However, the development of bioreceptive concrete presents substantial challenges. Due to the interdisciplinarity and novelty of this field, standardized methods for material characterization and bioreceptivity assessment are currently lacking. This study proposes an approach for evaluating surface properties crucial for bioreceptivity, developed on differently structured samples of ultra-high-performance concrete (UHPC). Existing methods and standards from concrete technology are critically reviewed and, where necessary, modified to meet the unique requirements of measuring bioreceptive material properties. Special attention is given to the surface pH value and water retention characteristics, as these are essential for promoting microbial growth and ensuring the long-term stability of green biofilms. The observed surface characteristics vary according to the imprinted surface structures, offering a spectrum of material properties and enabling the evaluation of their impact on bioreceptivity. The findings presented form the foundation for subsequent laboratory weathering experiments, which will be discussed in a complementary publication. KW - Novel methods KW - Bioreceptivity testing PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-655023 DO - https://doi.org/10.1617/s11527-025-02863-y SN - 1359-5997 VL - 58 SP - 1 EP - 18 PB - Springer Science and Business Media LLC AN - OPUS4-65502 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna T1 - From rocks to riches: Knufia petricola as model and expression platform N2 - Black fungi belong to different classes of Ascomycota but evolved similar morpho-physiological adaptations such as yeast-like growth and constitutive melanin formation to colonize extreme competition-free environments. They are ubiquitously found on air-exposed surfaces, from ancient marble monuments to modern solar panels. The rock inhabitant Knufia petricola was chosen to become a model for these extremotolerant black fungi. Plasmid-based and ribonucleoprotein-based CRISPR/Cas9 techniques were introduced to precisely introduce one to multiple double strand breaks into the DNA to modify, replace or add sequences to the genome either using the available selection marker systems (hygR, natR, genR, baR, suR) or by marker-free approaches. Multiplexing is very efficient, allowing for four or more simultaneous genome editing events. The newly generated cloning vectors containing the Tet on construct for doxycycline-controlled gene expression, and the validated sites in the K. petricola genome for color-selectable (pks1, phs1, ade2) or neutral insertion (igr1 to 5) of expression constructs complete the reverse genetics toolbox. One or multiple endogenous or heterologous genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides. We thus can express genes from synthetic clusters in a non-pigmented background (Δpks1/Δphs1). The fact that K. petricola only produces few secondary metabolites (DHN melanin, carotenoids, mycosporines and a siderophore) and plant cell-wall degrading enzymes but is capable to take the burden of acetyl-CoA-consuming metabolism and protein secretion renders K. petricola a promising host for the expression of heterologous genes encoding high-end secondary metabolites and enzymes. T2 - 12th International Mycological Congress (IMC12) CY - Maastricht, Netherlands DA - 11.08.2024 KW - Fungus KW - Genetic engineering KW - Heterologous gene expression KW - Cell factory PY - 2024 AN - OPUS4-60838 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Breitenbach, Romy A1 - Gerrits, Ruben A1 - Dementyeva, Polina A1 - Knabe, Nicole A1 - Schumacher, Julia A1 - Feldmann, Ines A1 - Radnik, Jörg A1 - Ryo, M. A1 - Gorbushina, Anna T1 - Data for "The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering" N2 - Data for the publication "The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering" (https://doi.org/10.1038/s41529-022-00253-1). It includes: - The Summary of the EPS concentration, EPS sugar components and EPS linkages. - The Summary of the XPS analysis of freeze-dried biofilm samples of all strains. - The Summary of the pH, Mg, SI and Fe concentration, biomass and olivine dissolution rate for each time point of all dissolution experiments. KW - Biofilms PY - 2022 DO - https://doi.org/10.26272/opus4-54901 PB - Bundesanstalt für Materialforschung und -prüfung (BAM) CY - Berlin AN - OPUS4-54901 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 - CHAP A1 - Schumacher, Julia ED - Scott, B. ED - Mesarich, C. T1 - Role of light in the life cycle of Botrytis cinerea 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 - Gorbushina, Anna 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 - 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 - Dehkohneh, Abolfazl T1 - Effect of Carbon and Nitrogen on Growth of the Oligotrophic and Extremotolerant Fungus Knufia petricola N2 - Introduction: Rock-inhabiting black fungi are renowned for their constitutive melanin production, extremotolerance and ubiquitous colonization of oligotrophic, natural or anthropogenic subaerial surfaces, including marble monuments and solar panels. Knufia petricola is a model for these fungi as it is genetically amenable and has their typical characteristics. It is capable of colonizing and deteriorating minerals and produces extracellular polymeric substances that facilitate attachment. Objectives: The objective of this study is to understand how such an organism is able to grow in environments depleted of C and/or N. Method: K. petricola growth parameters (colony extension and thickness, biomass, CO2 respiration and carbon-use efficiency (CUE)) were analysed for different C to N ratios (C:N), changing both the C and N concentrations and sources. The study utilized a dual-scale approach, examining biofilm formation on agar (macroscale) and growth of a single cell into a microcolony in a microfluidic device (microscale). This methodology provided insights into the nutritional requirements and growth behaviours of K. petricola across different spatial scales. Findings: Our macroscale analysis indicated optimal growth at the C:N ratio of 60. The morphological analysis demonstrated that when nitrate is utilized as the N source, colonies tend to exhibit filamentous growth at their edges, especially when glucose is available as C source. In contrast, when ammonium is the N source, the colony surface appears smooth (Fig. 1A). Moreover, C or N depletion led to higher substrate penetration, mostly through filaments at the edge of the biofilm. Notably, the lowest carbon concentration yielded reduced biomass C:N ratio and the peak biomass C:N ratio was observed at the medium C:N ratio of 60, suggesting a potential correlation between C:N ratio and optimal growth (Fig. 1B). The CUE was however quite low, ca. 0.4-0.6, being lowest when C and N were limiting. Concurrently, microscale analyses of single cells revealed that increasing the C:N ratio from 0.6 to 60 caused (1) shorter cell cycles and (2) more branching, no branching formed without C and N (Fig. 1C). This led to a denser colony at optimal conditions and a rather extended morphology under C or N limitations. Conclusion: The findings demonstrate that the depletion of either carbon or nitrogen results in enhanced substrate penetration, predominantly through edge filaments. Notably, the optimal growth and peak biomass C:N ratio of 60 suggest both a correlation with and importance of the medium C:N ratio. Specifically, the lowest carbon input led to a reduced biomass C:N ratio, while the peak biomass C:N ratio was achieved at the medium C:N ratio of 60. T2 - FEMS MICRO conference CY - Milano, Italy DA - 14.07.2025 KW - Black fungi KW - Carbon-use efficiency KW - Polyextremotolerant KW - Oligotrophs KW - Rock-inhabiting fungi PY - 2025 AN - OPUS4-65038 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 - 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 - Erdmann, Eileen T1 - A random mutagenesis approach to elucidate the biology of extremotolerant black fungi N2 - Microcolonial black fungi ubiquitously inhabit sun-exposed natural and man-made surfaces of our planet. To promote genetic studies, which are hindered by slow growth, lack of sexual cycles and transformation difficulties, CRISPR/Cas9-based genetic tools were implemented (Erdmann et al. 2022, Front Fungal Biol). Now efficient targeted mutagenesis of the rock inhabitant Knufia petricola (Eurotiomycetes/Chaetothyriales) - as a representative of the polyphyletic group of black fungi - enables the elucidation of extremotolerance, oligotrophism, unusual types of cell division, mineral weathering and symbiotic interactions. Still more progress on assigning functions to yet unknown genes can be expected if a forward genetics approach is available. We chose the two-component Activator/ Dissociation (Ac/Ds) transposon system from maize for generating a collection of insertional mutants by in-vivo mutagenesis of K. petricola. For the optimal use of this genetic tool, an inducible promoter for the expression of the Ac transposase (AcTPase) and by this the regulatable transposition of the resistance cassette-containing Ds transposon is desired. However, endogenous promoters for nitrate assimilation and galactose catabolism - 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 oligotrophic fungi. Therefore, the metabolism-independent Tet-on system was combined with the AcTPase coding sequence and subsequently transformed into Ds-carrying K. petricola strains. In total, four auxotrophic Ac|Ds starter strains containing the Ds transposon at different position of ade2 or ura3 were generated. The cultivation of these strains with doxycycline for induction of TET::Ac and subsequent selection of cells on ADE/URA-lacking media resulted in prototrophic colonies (revertants) for some but not all Ac|Ds strains. Currently, the transposition events in the obtained revertants are studied to validate the procedure. First amplicon sequencing of excision sites revealed footprint patterns, proving the transposon jumped. T2 - 16th European Conference on Fungal Genetics CY - Innsbruck, Austria DA - 05.03.2023 KW - Rock-inhabiting fungi KW - Foward genetics KW - In-vivo mutagenesis PY - 2023 AN - OPUS4-57144 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl 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 - Schumacher, Julia T1 - Versatile DHN melanin – spotlight on its function in microcolonial black fungi N2 - Dihydroxynaphthalene (DHN) melanin is produced by diverse 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 and survival structures of the foliar plant pathogen Botrytis cinerea (Schumacher 2016, Mol Microbiol). In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeasts) 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. For studying the relevance of constitutive DHN melanogenesis for tolerance of abiotic and biotic stresses, adhesion to substrates and subsequent damage of colonized surfaces, the rock-inhabiting fungus Knufia petricola was chosen as gene functions in this fungus can be studied by CRISPR/Cas9-based genome editing. The putative melanogenic genes were identified in the genome of K. petricola, deleted to confirm their involvement in DHN melanogenesis and co-expressed in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. Phenotypes of DHN-deficient mutants are studied. Here, we will discuss the role of the DHN melanin layer on the outer cell wall in tolerating UV irradiation. T2 - Annual conference 2023 of the Association for General and Applied Microbiology CY - Göttingen, Germany DA - 10.09.2023 KW - Pigment KW - UV radiation KW - Tolerance PY - 2023 AN - OPUS4-58425 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -