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 - Frelih, Milena T1 - Material-Microbiome Interactions N2 - Estimations show that the anthropogenic mass, the technosphere, already exceeds the biomass on Earth. Understanding the technosphere-environment interactions and their impacts is key to safe and healthy ecosystems and human societies. At BAM, we aim at promoting awareness for microorganisms living and evolving in contact with human-made materials and technical systems. We are creating datasets of biofilms on plastic and other anthropogenic materials and in the context of important technical challenges (e.g. microbially influenced corrosion or biocide-induced antimicrobial resistance). This enables us to identify and examine key organisms in the investigated systems that potentially harbor new, also industrially relevant, species and traits. Further, we explore material effects on aquatic microbial communities, aiming at the development and establishment of innovative and environmentally relevant methodologies. By using bioinformatic and molecular tools and interdisciplinary research, we enhance the understanding of material-microbiome interactions for a safe and sustainable future. T2 - NFDI4Microbiota Annual Conference CY - Cologne, Germany DA - 30.09.2025 KW - Microbiome KW - Microplastics KW - Black fungi KW - Nanoparticles PY - 2025 AN - OPUS4-64637 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl T1 - How Single Cells and Biofilms of the Extremotolerant Black Fungus Knufia petricola Respond to Oligotrophic Environments N2 - Melanised black fungi, such as Knufia petricola, are renowned for their constitutive melanin production, extremotolerance, and ability to thrive in hostile, oligotrophic natural and built environments from Antarctic outcrops to marble monuments and solar panels. These traits make them compelling models for astrobiological research, offering insights into survival strategies under conditions analogous to extraterrestrial habitats. Here, we quantified the responses of K. petricola A95 to carbon and nitrogen supply, varying sources (C: glucose vs. sucrose; N: ammonium vs. nitrate) and concentrations (C:N ratios of 0.6 – 600), including nutrient-free controls. Biofilm radial extension and thickness, morphology, biomass, produced CO2, and carbon-use efficiency (CUE) were analysed across controlled C:N ratios after 28 days. we combined macroscale assays of biofilm formation on agar with microscale tracking of single-cell development into microcolonies in a microfluidic device. This dual-scale approach allows the study of the nutritional requirements and growth strategies of K. petricola under chronic oligotrophy and provides quantitative inputs for individual-based simulations using iDynoMiCS 2.0. Macroscale analysis identified a C:N ratio of 60 as optimal, resulting in the highest biomass and colony thickness and the highest cumulative CO2 release. Depletion or low concentrations of C or N reduced biomass and increased peripheral filamentation and higher substrate penetration. Notably, nitrate as the N source produced biofilms with more peripheral filaments and caused more wrinkles on the surface of biofilm, whereas ammonium yielded compact, smooth biofilms with little to no filamentation. The CUE ranged from 0.4 to 0.6, being lowest under nutrient limitation. Microscale observations of single cells revealed that increasing the C:N ratio from 0.6 to 60 shortened cell cycles and promoted branching, no branching formed without C and N. Thus, a denser colony at optimal conditions and a rather extended morphology under C or N limitations were observed. T2 - Berlin Early-career Space Research (BESR) conference 2025 CY - Berlin, Germany DA - 06.10.2025 KW - Black fungi KW - Oligotrophs KW - Rock-inhabiting fungi PY - 2025 AN - OPUS4-65034 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - 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 - Schumacher, Julia T1 - CRISPR-Cas9 in der Materialforschung - Den gesteinsbesiedelnden Pilzen auf der Spur T1 - CRISPR-Cas9 in materials research - On the trail of rock-inhabiting fungi N2 - Das mikroskopische Leben auf exponierten Oberflächen ist genügsam und kooperativ. Gesteinsbesiedelnde schwarze Pilze, Grünalgen und Cyanobakterien unterstützen einander in der Eroberung von Felsen, Mauern, Denkmälern, Dächern, Fassaden und Sonnenkollektoren. Bedeutend sind die schwarzen Pilze als Gesteinszerstörer und Biofilmbildner. Ihre massiven Zellwände und ihr langsames Wachstum machen sie stresstolerant und fordern zugleich die experimentelle Forschung heraus. In der Materialforschung können Biofilme erwünscht oder unerwünscht sein. Biofilme auf Fassaden können das Innenstadtklima positiv beeinflussen, während sie auf einem Marmordenkmal unwillkommen sind. Ohne tieferes Verständnis der angepassten Mikroben ist weder ihre Bekämpfung noch ihre gezielte Förderung auf Materialien möglich. Hier treffen sich Genetik und Materialforschung: Die CRISPR-Cas9-Technologie ermöglicht es, die Genome der Pilze für funktionale Analysen zu editieren, um die Mechanismen der Materialbesiedlung und Materialschädigung zu entschlüsseln. N2 - Rock-inhabiting black fungi are adapted to the harsh life on rocks in deserts and release minerals from the rocks. The same adaptations enable these fungi to colonize man-made surfaces such as monuments, building facades and solar systems. Black fungi are often associated with phototrophic microorganisms. The slow growth and the melanized cell walls, which protect the fungi from extreme environmental infuences, render molecular biological and genetic engineering methods diffcult, which is why little is known about the biology of these fungi. Knufa petricola was selected to understand the processes of material colonization and damage with the help of adapted methods such as CRISPR-Cas9-mediated genome editing. KW - Knufia petricola KW - Schwarze Pilze KW - Bioflme KW - Genomeditierung KW - Pigmente KW - Multiplexing KW - Resistenzkassette KW - Transformanten KW - Knufia petricola KW - Black fungi KW - Biofilms KW - Pigments KW - Genome editing KW - Multiplexing KW - Resistance cassette KW - Transformants PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-620500 DO - https://doi.org/10.11576/biuz-7595 SN - 0045-205X SN - 1521-415X N1 - Volltext (PDF) in deutsch und englisch - Full text (PDF) in German and English VL - 54 SP - 41 EP - 50 PB - Verband Biologie, Biowissenschaften und Biomedizin in Deutschland (VBiO) CY - München AN - OPUS4-62050 LA - mul 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 - Gerrits, Ruben T1 - Genetic engineering of melanised fungi to study the role of melanin in mineral weathering and metal corrosion N2 - Melanins are organic, dark pigments produced by many organisms. Fungi either fix these pigments in their cell wall or secrete them into their environment to protect themselves against an array of physicochemical stresses (e.g., UV irradiation, desiccation, ...). However, melanins can also adsorb metals like iron and therefore might have a role in the fungal capacity to deteriorate iron-containing minerals and metals. To study this, we focus on the fungi Knufia petricola, a rock-inhabiting fungus with melanised cell walls which deteriorates minerals, and Amorphotheca resinae, a soil-borne fungus able to secrete melanin, degrade alkane-containing fuels, and corrode metals. The broader significance of this work lies in the mitigation of climate change via the negative emission technology enhanced rock weathering for the former to the mitigation of corrosion of biofuel infrastructure for the latter. Genes of K. petricola involved in melanin synthesis and iron uptake were deleted and down- and up- regulated to generate strains with a different melanin content and ability to take up iron. These strains and the wild type (WT) were exposed to a growth solution containing the iron-magnesium silicate olivine of which dissolution rates were obtained by measuring aqueous Mg concentrations using ICP-OES. Thus, the specific mineral deterioration mechanisms of melanised fungi were identified. Abiotically, these experiments showed that iron oxidation at the surface inhibits olivine dissolution. K. petricola was able to enhance dissolution when this abiotic inhibition is strongest (at pH 6) but prevented dissolution when this inhibition is weakest (at pH 4). The fungus therefore dissolves olivine by interacting with the oxidised iron at the olivine surface. Its iron uptake pathways do not seem to be involved as mutants deficient in iron uptake dissolved olivine at the same rate as the WT. The higher dissolution rates of a mutant which secretes a melanin precursor and the lower rates of a melanin-deficient mutant, however, indicate that the iron-adsorbing and -reducing capacities of melanin play a key role. This hypothesis is further supported by carbon steel corrosion experiments with six strains of A. resinae isolated from anthropogenic and natural environments. Their varying ability to secrete melanin correlated with their corrosion rates. We now plan to develop CRISPR/Cas9-mediated genome editing techniques for A. resinae to figure out whether this correlation is also a causation. In summary, our data show that the iron-binding capacity of melanins enables fungi to deteriorate iron-containing substrates at a higher rate. T2 - IBBS19 CY - Berlin, Germany DA - 09.09.2024 KW - Black fungi KW - Weathering KW - Corrosion PY - 2024 AN - OPUS4-61171 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl T1 - From microscale to macroscale: how environmental conditions impact the growth of the black fungus Knufia petricola N2 - Environmental, black fungi are known for their oligotrophic lifestyle and melanin production and ubiquitous colonisers of natural and anthropogenic subaerial surfaces like marble monuments, washing machine soap dispensers and solar panels. Even though their growth is generally unappreciated, prevention or inhibition thereof is still to be accomplished. We have chosen the black fungus Knufia petricola as a model species to study these organisms as it is genetically amenable, able to colonise and deteriorate minerals and produces extracellular polymeric substances allowing attachment. Our goal is simple: the quantitative description of its growth on various environmental conditions to create a mathematical model which could allow the development of a mitigation strategy. The conditions to be tested are the quantity and type of carbon and nitrogen sources, the pH and the presence of neighbouring colonies. The mathematical model is in development using the iDynoMiCS 2.0 platform. Our first results showed that the growth rate is negatively correlated to the colony forming units (i.e. the presence of a neighbouring colony). Although interesting with respect to mitigation, we therefore have chosen to track the growth of single colonies. On a macro-scale, the highest extension rates of colonies were at pH 5, 0.01M NO3, 0.01M NH4, 0.1M glucose, and 0.1M sucrose. Interesting are the similar rates in the range of 0.01M to 0.1M for carbon, and that CO2 production on similar conditions correlates well with these results. On a microscale, we have followed the growth of single cells using a custom-made microfluidic flow cell. Overall, first results show that medium containing more carbon not necessarily affected the growth rate but rather the quantity of branches produced by a single cell. This rapidly results in a more compact microcolony versus the more extended colonies on C-deficient media. This more profound knowledge on the growth of black fungi will allow to dissect their role in material deterioration and colonisation. T2 - International Biodeterioration & Biodegradation Society (IBBS) CY - Berlin, Germany DA - 09.09.2024 KW - Black fungi KW - Rock-inhabiting fungi KW - Fungal biofilm KW - Individual-based modelling PY - 2024 AN - OPUS4-62141 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben T1 - Quantifying and modelling growth of a subaerial black fungus N2 - Environmental, black fungi are known for their oligotrophic lifestyle and melanin production and are ubiquitous colonisers of natural and anthropogenic subaerial surfaces like marble monuments, washing machine soap dispensers and solar panels. Even though their growth is generally unappreciated, prevention or inhibition thereof is still to be accomplished. We have chosen the genetically amenable, black fungus Knufia petricola as a model species to study these organisms. Our goal is simple: the quantitative description of its growth on various environmental conditions to create a mathematical model using the iDynoMiCS 2.0 platform which could allow the development of a mitigation strategy. The conditions to be tested are the quantity and type of carbon and nitrogen sources, the pH and the presence of neighbouring colonies. First results showed that colonies grown on agar have the highest extension rates at pH 5, 0.01M NO3 or NH4, and 0.1M glucose or sucrose. Interestingly, extension rates but also CO2 production was unaffected by an increase in the concentration of the C-source from 0.01M to 0.1M. On a microscale, we observed that medium containing more C resulted in a higher quantity of branches produced by a single cell and not in a higher single cell growth rate. This rapidly results in a more compact microcolony versus the more extended colonies on C-deficient media. We believe that such deeper knowledge on the growth of black fungi will help mitigate their material colonisation and deterioration. T2 - IMC12 CY - Maastricht, Netherlands DA - 09.08.2024 KW - Black fungi KW - Modelling KW - Carbon and nitrogen limitation PY - 2024 AN - OPUS4-61170 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl T1 - From microscale to macroscale: environmental conditions and growth patterns of the black fungus Knufia petricola N2 - Environmental, black fungi are known for their oligotrophic lifestyle and melanin production and ubiquitous colonisers of natural and anthropogenic subaerial surfaces like marble monuments, washing machine soap dispensers and solar panels. Even though their growth is generally unappreciated, prevention or inhibition thereof is still to be accomplished. We have chosen the black fungus Knufia petricola as a model species to study these organisms as it is genetically amenable, able to colonise and deteriorate minerals and produces extracellular polymeric substances allowing attachment. Our goal is simple: the quantitative description of its growth on various environmental conditions to create a mathematical model which could allow the development of a mitigation strategy. The conditions to be tested are the quantity and type of carbon and nitrogen sources, the pH and the presence of neighbouring colonies. The mathematical model is in development using the iDynoMiCS 2.0 platform. Our first results showed that the growth rate is negatively correlated to the colony forming units (i.e. the presence of a neighbouring colony). Although interesting with respect to mitigation, we therefore have chosen to track the growth of single colonies. On a macro-scale, the highest extension rates of colonies were at pH 5, 0.01M NO3, 0.01M NH4, 0.1M glucose, and 0.1M sucrose. Interesting are the similar rates in the range of 0.01M to 0.1M for carbon, and that CO2 production on similar conditions correlates well with these results. On a microscale, we have followed the growth of single cells using a custom-made microfluidic flow cell. Overall, first results show that medium containing more carbon not necessarily affected the growth rate but rather the quantity of branches produced by a single cell. This rapidly results in a more compact microcolony versus the more extended colonies on C-deficient media. This more profound knowledge on the growth of black fungi will allow to dissect their role in material deterioration and colonisation. T2 - CurvoBio 2024 CY - Warsaw, Poland DA - 28.08.2024 KW - Black fungi KW - Rock-inhabiting fungi KW - Fungal biofilm KW - Individual-based modelling KW - Biocorrosion PY - 2024 AN - OPUS4-62270 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 - How does light affect rock-inhabiting fungi? 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 with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses, 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). Less apparent are other light-dependent processes such as light-driven DNA repair by photolyases (photoreactivation) or ion pumping by microbial opsins. Fungi that can share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess many proteins for absorbing UV/blue, green, red and far-red light, produce the black 1,8 dihydroxynaphthalene (DHN) melanin and orange-red carotenoids, and may live in multispecies biofilms. 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 light (UV-B) tolerance of K. petricola. T2 - 32nd Fungal Genetics Conference CY - Pacific Grove, CA, USA DA - 12.03.2024 KW - Knufia petricola KW - Black fungi KW - Light-induced stress PY - 2024 AN - OPUS4-59732 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Erdmann, Eileen T1 - In-vivo mutagenesis of the rock inhabitant Knufia petricola by a customized Ac/Ds transposon system N2 - Microcolonial black fungi ubiquitously inhabit sun-exposed natural and man-made surfaces of our planet. To promote genetic studies, CRISPR/Cas9-based genome editing was implemented in the rock-inhabiting fungus Knufia petricola (Eurotiomycetes/ Chaetothyriales). Now efficient targeted mutagenesis of K. petricola - 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. Even more progress on assigning functions to yet unknown genes can be achieved by a forward genetics approach. We chose the two-component Activator/Dissociation (Ac/Ds) transposon system from maize for generating K. petricola insertional mutants by in-vivo mutagenesis. For the optimal use of this genetic tool, an inducible promoter i.e, from the metabolism-independent Tet-on system, was combined with the AcTPase-coding sequence enabling the regulatable transposition of the resistance cassette-containing Ds transposon. In total, six auxotrophic Ac/Ds starter strains containing the Ds transposon at different position of ade2, ura3 or ppt1 were generated. The cultivation of these strains with doxycycline for induction of TET::Ac and subsequent selection of cells on ADE/URA/LYS-lacking media resulted in prototrophic colonies (revertants) for most Ac/Ds strains. Amplicon sequencing of excision sites revealed characteristic footprint patterns, proving that the transposon jumped. For identifying unknown Ds re-insertions sites, the thermal asymmetric interlaced (TAIL)-PCR was successfully implemented. First identified Ds re-insertion sites suggest that the distribution pattern may depend on the excision site. Currently, transposition frequencies and genome-wide distribution of re-insertion sites are studied in different Ac/Ds starter strains to identify the best candidate for generating saturated mutant libraries. This transposon mutagenesis strategy is also interesting for studying other black fungi, because once the Ac and Ds components are integrated in the genome, the fungus ‘only’ needs to be cultivated for generating insertional mutants. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Black fungi KW - Genetics KW - Transposon KW - Mutagenesis PY - 2023 AN - OPUS4-58427 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 - 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 - 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 - Selbmann, L. A1 - Benkő, Z. A1 - Coleine, C. A1 - de Hoog, S. A1 - Donati, C. A1 - Druzhinina, I. A1 - Emri, T. A1 - Ettinger, C. L. A1 - Gladfelter, A. S. A1 - Gorbushina, Anna A1 - Grigoriev, I. V. A1 - Grube, M. A1 - Gunde-Cimerman, N. A1 - Karányi, Z. A. A1 - Kocsis, B. A1 - Kubressoian, T. A1 - Miklós, I. A1 - Miskei, M. A1 - Muggia, L. A1 - Northen, T. A1 - Novak-Babič, M. A1 - Pennacchio, C. A1 - Pfliegler, W. P. A1 - Pòcsi, I. A1 - Prigione, V. A1 - Riquelme, M. A1 - Segata, N. A1 - Schumacher, Julia A1 - Shelest, E. A1 - Sterflinger, K. A1 - Tesei, D. A1 - U’Ren, J. M. A1 - Varese, G. C. A1 - Vázquez-Campos, X. A1 - Vicente, V. A. A1 - Souza, E. M. A1 - Zalar, P. A1 - Walker, A. K. A1 - Stajich, J. E. T1 - Shed Light in the DaRk LineagES of the Fungal Tree of Life—STRES N2 - The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the “Shed light in The daRk lineagES of the fungal tree of life” (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments. KW - Adaptation KW - Black fungi KW - Dothideomycetes KW - Eurotiomycetes KW - Extremophiles KW - Genomics KW - Metabolomics KW - Secondary metabolites KW - Stress conditions KW - Transcriptomics PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-519328 DO - https://doi.org/10.3390/life10120362 VL - 10 IS - 12 SP - 362 PB - MDPI CY - Basel AN - OPUS4-51932 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -