TY - JOUR A1 - Catanzaro, Ilaria A1 - Gerrits, Ruben A1 - Feldmann, Ines A1 - Gorbushina, Anna A1 - Onofri, Silvano A1 - Schumacher, Julia T1 - Deletion of the polyketide synthase‐encoding gene pks1 prevents melanization in the extremophilic fungus Cryomyces antarcticus N2 - Cryomyces antarcticus, a melanized cryptoendolithic fungus endemic to Antarctica, can tolerate environmental conditions as severe as those in space. Particularly, its ability to withstand ionizing radiation has been attributed to the presence of thick and highly melanized cell walls, which—according to a previous investigation—may contain both 1,8‐dihydroxynaphthalene (DHN) and L‐3,4 dihydroxyphenylalanine (L‐DOPA) melanin. The genes putatively involved in the synthesis of DHN melanin were identified in the genome of C. antarcticus. Most important is capks1 encoding a non‐reducing polyketide synthase (PKS) and being the ortholog of the functionally characterized kppks1 from the rock‐inhabiting fungus Knufia petricola. The co‐expression of CaPKS1 or KpPKS1 with a 4′‐phosphopantetheinyl transferase in Saccharomyces cerevisiae resulted in the formation of a yellowish pigment, suggesting that CaPKS1 is the enzyme providing the precursor for DHN melanin. To dissect the composition and function of the melanin layer in the outer cell wall of C. antarcticus, non‐melanized mutants were generated by CRISPR/Cas9‐mediated genome editing. Notwithstanding its slow growth (up to months), three independent non‐melanized Δcapks1 mutants were obtained. The mutants exhibited growth similar to the wild type and a light pinkish pigmentation, which is presumably due to carotenoids. Interestingly, visible light had an adverse effect on growth of both melanized wild‐type and non‐melanized Δcapks1 strains. Further evidence that light can pass the melanized cell walls derives from a mutant expressing a H2B‐GFP fusion protein, which can be detected by fluorescence microscopy. In conclusion, the study reports on the first genetic manipulation of C. antarcticus, resulting in non‐melanized mutants and demonstrating that the melanin is rather of the DHN type. These mutants will allow to elucidate the relevance of melanization for surviving extreme conditions found in the natural habitat as well as in space. KW - Astrobiology KW - Black fungi KW - CRISPR/Cas9 KW - DHN melanin KW - Cryptoendolithism PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-606599 DO - https://doi.org/10.1002/iub.2895 SN - 1521-6551 VL - 76 IS - 12 SP - 1072 EP - 1090 PB - Wiley AN - OPUS4-60659 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Mallikarjun, Jhenkhar A1 - Gorbushina, Anna A1 - Kuzyakov, Yakov A1 - Koester, Moritz A1 - Castro, Rodrigo A1 - Yudina, Anna A1 - Abdallah, Khaled A1 - Matus, Francisco J. A1 - Dippold, Michaela A. T1 - Post-fire recovery of temperate and mediterranean ecosystems: An interplay between fire severity, soil nutrients, and vegetation from early-stage to decadal-scale dynamics N2 - Wildfires strongly alter soil properties, which in turn affect ecosystem recovery over extended periods, though long-term impacts are less certain. This study investigated a 14-year post-fire chronosequence in Chile’s mediterranean and temperate humid forests, revealing ecosystem-specific soil properties and nutrient recovery mechanisms. By analysing sites at successional stages, the chronosequence approach assessed temporal changes and ecosystem recovery, revealing long-term wildfire effects on soil dynamics and nutrients recovery. Wildfires raised soil bulk density to 0.9 g cm−3 in humid temperate and 1.2 g cm−3 in mediterranean ecosystems. Mediterranean soils experienced greater compaction from organic matter loss, soil aggregate destruction, ash-clogged pores, and topsoil erosion. Soil texture shifts were ecosystem-dependent: mediterranean soils increased 10–12 % in clay and silt through ash redistribution and aggregation, while temperate soils saw sand content rise by 0.74 % and 0.32 % yearly at 0–5 and 5–10 cm depths from thermal disaggregation and erosion. Ground vegetation recovers quickly, but physical soil properties like bulk density require over 14 years to return to pre-fire conditions. In humid temperate forests, ash input initially increased soil pH (4.8 to 5.8), reducing acidity, mitigating aluminium toxicity, while increasing nutrient availability. Base cation stocks increased in mediterranean woodlands (e.g., Ca: up to 0.41 Mg ha−1 y−1) due to ash retention, lower leaching, and ash infiltration into subsoil. Nutrient stocks in humid forests recovered slowly (Ca: 0.087–0.13 Mg ha−1 y−1) due to rainfall-driven leaching and low subsoil reserves. Carbon and N losses were restricted to the litter horizon in temperate forests, recovering via fire-resistant tree inputs, whereas mediterranean soils suffered severe C and N depletion from vegetation loss, erosion, and low N fixation. Fire effects and recovery are ecosystem-specific, shaped by landscape, geology, hydrology, and vegetation resilience. Understanding how fire regimes affect soil and nutrient recovery is vital for improving projections in fire-prone regions. KW - Historical fire & Wildfire KW - Nutrients Ecosystem recovery & Vegetation succession KW - Chronosequence approach KW - Losses of nutrients & Nutrients stocks KW - Wildfire PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-642918 DO - https://doi.org/10.1016/j.catena.2025.109431 SN - 0341-8162 VL - 260 SP - 1 EP - 13 PB - Elsevier B.V. AN - OPUS4-64291 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Erdmann, Eileen A1 - Nitsche, Sarah A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - Genetic Engineering of the Rock Inhabitant Knufia petricola Provides Insight Into the Biology of Extremotolerant Black Fungi N2 - Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic 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) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. 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 the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). 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. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi. KW - Microcolonial fungi KW - DHN melanin KW - Cloning vectors KW - Genetics PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-546192 DO - https://doi.org/10.3389/ffunb.2022.862429 SN - 2673-6128 VL - 3 PB - Frontiers Media CY - Lausanne AN - OPUS4-54619 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 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 - Gerrits, Ruben A1 - Pokharel, R. A1 - Breitenbach, Romy A1 - Radnik, Jörg A1 - Feldmann, Ines A1 - Schuessler, J.A. A1 - von Blanckenburg, F. A1 - Gorbushina, Anna A1 - Schott, J. T1 - How the rock-inhabiting fungus K. petricola A95 enhances olivine dissolution through attachment N2 - Free-living and mycorrhizal fungi are able to enhance the weathering of rock and other solid substrates. Deciphering the exact mechanisms of these natural processes requires their experimental simulation. Moreover, by performing these simulations with genetically amenable rock-weathering fungi, one can knock-out certain fungal traits and consequently identify their weathering-relevant function. Here, the effect of the rock-inhabiting fungus, Knufia petricola A95, on the dissolution kinetics of an Fe-bearing olivine (Mg1.86Fe0.19SiO4) is investigated at 25 °C and pH 6 using reproducible batch and mixed flow experiments. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, which produces more extracellular polymeric substances (EPS) than the wild type (WT), enables the comparative study of the role of melanin and EPS in olivine dissolution. In abiotic dissolution experiments, the olivine dissolution rate decreased considerably over time at pH 6 but not at pH 3.5. This inhibition of abiotic olivine dissolution at pH 6 was most likely caused by the in-situ oxidation of ferrous Fe and/or the precipitation of ferric hydroxides at the olivine surface. In corresponding biotic experiments at pH 6, both the wild type K. petricola and its melanin-deficient mutant ΔKppks solubilised and bound significant amounts of Fe released by olivine dissolution. Fe oxidation and precipitation were thus prevented and olivine dissolution proceeded faster than in the abiotic experiments. By sequestering Fe directly at the olivine surface, the attached wild type K. petricola cells were particularly efficient at preventing the oxidation of Fe at the mineral surface: the slowdown of olivine dissolution almost completely disappeared. The attachment capacity of these wild type cells is most likely mediated by wild type-specific EPS. Our presented experimental systems allow the oxidation of mineral-released Fe and include a rock-inhabiting fungus, thus simulating chemical, physical and biological conditions that set dissolution rates in a way that is relevant to natural ecosystems. KW - Black fungi KW - Bio-weathering KW - Forsterite KW - Knock-out mutant KW - Extracellular polymeric substances KW - Melanin Adhesion PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-509332 DO - https://doi.org/10.1016/j.gca.2020.05.010 VL - 282 SP - 76 EP - 97 PB - Elsevier Ltd. AN - OPUS4-50933 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Voigt, Oliver A1 - Knabe, Nicole A1 - Nitsche, Sarah A1 - Erdmann, Eileen A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - An advanced genetic toolkit for exploring the biology of the rock‑inhabiting 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. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple genes with or without resistance cassettes were developed. This 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. KW - Subaerial biofilms KW - Biodeterioration KW - Fluorescent microscopy KW - CRISPR/Cas9 KW - RNA interference PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-518601 DO - https://doi.org/10.1038/s41598-020-79120-5 VL - 10 IS - 1 SP - 22021 PB - Springer Nature AN - OPUS4-51860 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Plarre, Rüdiger A1 - Zocca, Andrea A1 - Spitzer, Andrea A1 - Benemann, Sigrid A1 - Gorbushina, Anna A1 - Li, Y. A1 - Waske, Anja A1 - Funk, Alexander A1 - Wilgig, Janka A1 - Günster, Jens T1 - Searching for biological feedstock material: 3D printing of wood particles from house borer and drywood termite frass N2 - Frass (fine powdery refuse or fragile perforated wood produced by the activity of boring insects) of larvae of the European house borer (EHB) and of drywood termites was tested as a natural and novel feedstock for 3D-printing of wood-based materials. Small particles produced by the drywood termite Incisitermes marginipennis and the EHB Hylotrupes bajulus during feeding in construction timber, were used. Frass is a powdery material of particularly consistent quality that is essentially biologically processed wood mixed with debris of wood and faeces. The filigree-like particles flow easily permitting the build-up of woodbased structures in a layer wise fashion using the Binder Jetting printing process. The Quality of powders produced by different insect species was compared along with the processing steps and properties of the printed parts. Drywood termite frass with a Hausner Ratio HR = 1.1 with ρBulk = 0.67 g/cm3 and ρTap = 0.74 g/cm3 was perfectly suited to deposition of uniformly packed layers in 3D printing. We suggest that a variety of naturally available feedstocks could be used in environmentally responsible approaches to scientific material sciences/additive manufacturing. KW - 3D printing KW - X-ray tomographic KW - SEM micrography KW - Drywood termite PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-521517 DO - https://doi.org/10.1371/journal.pone.0246511 VL - 16 IS - 2 SP - e0246511 AN - OPUS4-52151 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR 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 - The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering N2 - The roles extracellular polymeric substances (EPS) play in mineral attachment and weathering were studied using genetically modified biofilms of the rock-inhabiting fungus Knufia petricola strain A95. Mutants deficient in melanin and/or carotenoid synthesis were grown as air-exposed biofilms. Extracted EPS were quantified and characterised using a combination of analytical techniques. The absence of melanin affected the quantity and composition of the produced EPS: mutants no longer able to form melanin synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, the melanin-producing strains attached more strongly to the mineral olivine and dissolved it at a higher rate. We hypothesise that the pullulan-related linkages, with their known adhesion functionality, enable fungal attachment and weathering. The released phenolic intermediates of melanin synthesis in the Δsdh1 mutant might play a role similar to Fe-chelating siderophores, driving olivine dissolution even further. These data demonstrate the need for careful compositional and quantitative analyses of biofilm-created microenvironments. KW - Biofilms PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-549025 DO - https://doi.org/10.1038/s41529-022-00253-1 SN - 2397-2106 VL - 6 SP - 1 EP - 11 PB - Springer Nature CY - London AN - OPUS4-54902 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Alder-Rangel, A. A1 - Idnurm, A. A1 - Brand, A. A1 - Brown, A. A1 - Gorbushina, Anna A1 - Kelliher, C. A1 - Campos, C. A1 - Levin, D. A1 - Bell-Pedersen, D. A1 - Dadachova, E. A1 - Bauer, F. A1 - Gadd, G. A1 - Braus, G. A1 - Braga, G. A1 - Brancini, G. A1 - Walker, G. A1 - Druzhinina, I. A1 - Pocsi, I. A1 - Dijksterhuis, J. A1 - Aguirre, J. A1 - Hallsworth, J. A1 - Schumacher, Julia A1 - Ho Wong, K. A1 - Selbmann, L. A1 - Corrochano, L. A1 - Kupiec, M. A1 - Momany, M. A1 - Molin, M. A1 - Requena, N. A1 - Yarden, O. A1 - Cordero, R. A1 - Fischer, R. A1 - Pascon, R. A1 - Mancinelli, R. A1 - Emri, T. A1 - Basso, T. A1 - Rangel, D. T1 - The Third International Symposium on Fungal Stress - ISFUS N2 - Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in Sao Jose dos Campos, Sao Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology. T2 - International Symposium on Fungal Stress (ISFUS) CY - Sao Jose dos Campos, Brazil DA - 20.05.2019 KW - Agricultural mycology KW - Fungal stress mechanisms and responses KW - Industrial mycology KW - Medical mycology PY - 2020 DO - https://doi.org/10.1016/j.funbio.2020.02.007 VL - 124 IS - 5 SP - 235 EP - 252 PB - Elsevier Ltd. AN - OPUS4-50953 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 -