TY - CONF A1 - Catanzaro, Ilaria A1 - Nitsche, Sarah A1 - Gorbushina, Anna A1 - Schumacher, Julia A1 - Onofri, S. 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 - Erdmann, Eileen A1 - Nitsche, Sarah A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - Tools for Knufia petricola: new techniques for CRISPR/Cas9-based genome editing N2 - Black microcolonial fungi represent a group of ascomycetes with similar adaptations for existing in natural and anthropogenically created extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) in the multilayered cell walls. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a representative for developing methods for genetic manipulation, simulation of mineral weathering and study of symbiotic interactions. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and three additional resistance selection markers. The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter, while the black-white screening due to the concurrent elimination of pks1 (melanin) and phs1 (carotenoids) was used to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. In addition, two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series (Schumacher, 2012) and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for use in other fungal systems as well. T2 - 31st Fungal Genetics Conference CY - USA, CA, Pacific Grove DA - 15.03.2022 KW - Microcolonial fungi KW - Genetic engineering KW - Fluorescent proteins PY - 2022 AN - OPUS4-54586 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nitsche, Sarah A1 - Gerrits, Ruben A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - The role of the stress-activated MAP kinase SAK1 in Knufia petricola N2 - Mitogen-activated protein (MAP) kinases are highly conserved in animals, plants, and fungi and represent fundamental parts of signaling networks in eukaryotic cells. Black DHN (1,8-dihydroxynaphthalene) melanin and orange carotenoids are produced by many fungi in specific cell types/under certain conditions for protecting cells from abiotic and/or biotic stresses. Microcolonial black fungi constitutively produce DHN melanin, contain the conserved carotenogenic gene cluster, exhibit slow yeast-like growth and survive in extreme environments. Thus, the question arises to which extent pigment formation and responses mediated by the stress-activated MAP kinase module contribute to the observed extremotolerance. We address this question in the rock inhabitant Knufia petricola, the only representative of the extremotolerant black fungi that is genetically amendable. Here, the mutations of pks1, phs1 and both genes result in melanin-free (pink), carotenoid-free (black) and pigment-free (white) strains, respectively (Voigt Knabe et al. 2020, Sci Rep). The gene encoding the stress-activated MAP kinase was deleted in the wild-type and different pigment-deficient backgrounds. In addition, strains were generated that express a GFP-SAK1 fusion protein from the sak1 locus to follow the cytosolic/nuclear shuttling of SAK1 upon stress. Growth of the obtained single, double and triple deletion mutants was tested by dropping cell suspensions on solid media supplemented with different stress-inducing agents. The Δsak1 mutants show slightly reduced growth rates even under non-stress conditions and are hypersensitive to different stress conditions: reduced growth is observed on media inducing, for instance, osmotic, oxidative, membrane, and pH stress, and upon incubation at 30 °C (heat stress). Melanin-free Δsak1 mutants are more sensitive than black Δsak1 mutants to some but not all stress conditions, suggesting that melanin and the SAK1 pathway have complementary roles in protecting K. petricola from stress. T2 - VAAM Fachgruppentragung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - Fungus KW - Extremotolerance KW - Signal transduction PY - 2022 AN - OPUS4-55677 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - Iron uptake by the rock-inhabiting fungus Knufia petricola N2 - Iron is arguably the most essential metal in living organisms. For rock-inhabiting fungi its acquisition might be unconventional as they (1) tend to inhabit iron-deficient, oxygen-rich surfaces like marble monuments and solar panels and (2) produce the black, iron-adsorbing pigment melanin. We used a range of analytical methods, ongoing mineral dissolution experiments and gene deletion mutants of the model rock-inhabiting fungus Knufia petricola to figure out the mechanisms and substrate deteriorating effects of iron uptake by these organisms. To study both siderophore-mediated and reductive iron assimilation (RIA), genes like sidC, encoding a putative siderophore synthetase and ftr1 and fet3 encoding the subunits of an iron permease-oxidase were deleted. At iron deficient conditions, growth of the wild type (WT) and ΔsidC mutant was similar, whereas growth of the Δftr1-fet3 mutant and the double mutant ΔsidC/Δftr1-fet3 was diminished and absent, respectively. We were not able to detect the siderophore of K. petricola and the WT and mutants were not able to grow at low concentrations of strong iron chelators. However, in a cross-feeding experiment, an overexpression strain of sidC allowed more growth of ΔsidC/Δftr1-fet3 on iron deficient medium than the WT, whereas the ΔsidC mutant could not do so at all. Compared to the WT, the sidC overexpression strain also withstood oxidative stress better and had a shorter lag time and higher growth rate. Combined, these results indicate that K. petricola relies more on RIA than siderophore-mediated uptake as it likely excretes low quantities of a primarily intracellular siderophore. Interestingly, Δftr1-fet3 had a higher iron content than the WT at iron deficient conditions. This difference disappeared upon deletion of melanin synthesis (Δpks1 vs. Δpks1/Δftr1-fet3): melanin-bound iron can likely not be used without RIA. K. petricola’s chelation incapacity implies a habitat free of competition for iron while offering us a mitigation strategy. T2 - ECFG16 CY - Innsbruck, Austria DA - 05.03.2023 KW - Siderophore KW - Melanin KW - Reductive iron assimilation PY - 2023 AN - OPUS4-57148 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl A1 - Gerrits, Ruben A1 - Schumacher, Julia A1 - Kreft, J.-U. A1 - Gorbushina, Anna T1 - Fungal biofilms on materials: describing and modelling growth of the black fungus Knufia petricola N2 - Fungi that grow as biofilms are associated with clinical settings as well as various cases of material fouling and material damage. Black fungi as biofilm formers have been rarely studied so far. Their conspicuous dark pigmentation, EPS production, adhesion capabilities and adaptations to stresses allow black fungi to develop biofilms on materials under harsh conditions. For example, rock-inhabiting black fungi withstand sun irradiation and dehydration and are therefore ubiquitous on arid surfaces like solar panels and marble monuments. To understand and control their ability to colonise and deteriorate materials, one should assess and model black fungi’s growth patterns. But so far, no mathematical model has been developed to describe their growth. Knufia petricola A95, representing rock-inhabiting fungi from Chaetothyriales, is genetically amenable and can serve as a model for biofilm studies in black fungi. The primary objective of this project is to develop a growth model for K. petricola A95 which will enable to define and predict material colonisation of black fungi. Dedicated experimental work with K. petricola will allow the quantitative assessment of the impact of environmental conditions (e.g. pH, nutrients, etc.) on the growth behaviour at the biofilm and single cells level. Data which will be used to validate and develop an individual-based model (based on the iDynoMICS modelling platform) that explains how fungal biofilms form, colonise materials, and cause deterioration. Thus far, research has been conducted on the impact of different concentrations and sources of major elements (e.g. C, N, …), as well as trace elements (e.g. Cu, Mg, …), on the colony shape and biomass of Knufia petricola A95 biofilms. To study the behaviour of single cells, the length of the cell cycle in different growth media has been determined via the combined use of microfluidic devices and confocal microscopy. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Biofilm KW - Rock-inhabiting fungus KW - Mathematical modelling PY - 2023 AN - OPUS4-58438 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Heeger, Felix A1 - Whitfield, Daniel A1 - Knabe, Nicole A1 - Nai, Corrado A1 - Schumacher, Julia A1 - Broughton, William A1 - Cuomo, C. A1 - de Souza, E. A1 - Lespinet, O. A1 - Mazzoni, C. A1 - Monaghan, M. A1 - Gorbushina, Anna T1 - Exploring the genome of the black yeast Knufia petricola N2 - Black yeasts form a polyphyletic group of Ascomycota that colonize bare surfaces like rocks, facades of buildings, and solar panels. Their protective adaptations enable an adequate response to fluctuating and diverse temperature, water and UV radiation stresses. Together with bacteria and algae they form sub-aerial biofilms (SAB) this way discoloring and weathering the surfaces they grow on. Strain A95 of Knufia petricola (Eurotiomycetes, Chaetothyriales) displays both typical yeast-like cell growth and constitutive dihydroxynaphthalene (DHN) melanogenesis. Along with the cyanobacterium Nostoc punctiforme as photobiont, it is already used in a model system for studying SAB formation and bio-weathering. Applying the recently developed tools for the generation of deletion mutants will allow to define gene functions and to identify genes critical for abiotic and biotic interactions. We present a chromosome-level genome assembly and annotation for K. petricola A95. The genome was assembled with MaSuRCA using a hybrid assembly approach of Illumina MiSeq and PacBio SMRT sequencing data. The resulting assembly consists of 17 contigs including the complete mitochondrial genome and five complete chromosomes. It shows indication of repeat-induced point mutations (RIP). Supported by RNA sequencing data from eight different growth conditions, 10,994 genes were predicted with the BRAKER2 pipeline. Functional annotation of genes was obtained from general functional annotation databases and the fungal specific database FungiPath. Comparative analyses are in progress to identify genes specific to black yeasts, that may facilitate the survival on exposed surfaces. In sum, the genome sequence of K. petricola is a valuable resource to gain insight into the protein inventory and functional pathways of extremotolerant and symbiosis-capable fungi. T2 - Fungal Genetics Conference 2019 CY - Asilomar, CA, USA DA - 12.03.2019 KW - Knufia petricola KW - Black fungus KW - Genome sequence PY - 2019 AN - OPUS4-49636 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Brandhorst, Antonia A1 - Voigt, Oliver A1 - Nitsche, Sarah A1 - Erdmann, Eileen A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - DHN melanin – synthesis, regulation, and functions in Knufia petricola 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, microcolonial black fungi exhibit 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 regulation and relevance of 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 deletion mutants are studied for specifying the functions of DHN melanin in K. petricola. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubliana, Slovenia DA - 19.09.2023 KW - Pigment KW - Fungus KW - Heterologous expression PY - 2023 AN - OPUS4-58430 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Brandhorst, Antonia A1 - Erdmann, Eileen A1 - Nitsche, Sarah A1 - Voigt, Oliver A1 - Gorbushina, Anna T1 - DHN melanin synthesis in the rock inhabitant Knufia petricola N2 - DHN (1,8-dihydroxynaphthalene) melanin is produced by different Ascomycetes via slightly differing biosynthetic routes. The polyketide synthases (PKS) release the heptaketide YWA1, the hexaketide AT4HN or the pentaketide T4HN. The first two products are deacetylated by ‘yellowish-green’ hydrolases to T4HN, and T4HN is further converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases (MCOs). The involved genes are tightly clustered, partially clustered or widely distributed in the genomes of DHN melanin-producing fungi. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g. in melanized reproduction, survival and/or infection structures. In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeast) dwelling in hostile habitats such as bare rock surfaces in hot and cold deserts, exhibits constitutive melanogenesis. Here, we report on the identification and functional characterization of the DHN melanogenic genes of Knufia petricola as a representative of the Chaetothyriales, the sister order of the Eurotiales. Orthologs for all melanogenic genes were identified in the genome of K. petricola A95, including one gene encoding the polyketide synthase (KpPKS1), two genes encoding ‘yellowish-green’ hydrolases (KpYGH1,2), two genes encoding THN reductases (KpTHR1,2) and one gene encoding a scytalone dehydratase (KpSDH1). Ten genes encoding MCOs were identified, all MCOs are predicted to be secreted. The genes are not clustered in the genome but are highly expressed. Gene functions are studied by generation of single, double, and multiple deletion mutants in K. petricola and by heterologous expression in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. T2 - VAAM Fachgruppentagung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - DHN melanin KW - Genetics KW - Biodegradation PY - 2022 AN - OPUS4-55676 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Brandhorst, Antonia A1 - Erdmann, Eileen A1 - Nitsche, Sarah A1 - Voigt, Oliver A1 - Gorbushina, Anna T1 - DHN melanin synthesis in the rock inhabitant Knufia petricola N2 - DHN (1,8-dihydroxynaphthalene) melanin is produced by Ascomycetes via slightly differing synthetic routes. Polyketide synthases release YWA1, AT4HN or T4HN. YWA1 and AT4HN are deacetylated by ‘yellowish-green’ hydrolases, and T4HN is converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases. The melanogenic genes are tightly, partially or not clustered in the genomes, and are often regulated in a spatial and/or temporal fashion. By contrast, microcolonial fungi/black yeasts – a polyphyletic group of Ascomycetes dwelling in hostile habitats such as bare rock surfaces – feature constitutive DHN melanogenesis. Here, we report on the DHN melanogenic genes of Knufia petricola (Eurotiomycetes/Chaetothyriales). T2 - 16th European Conference on Fungal Genetics CY - Innsbruck, Austria DA - 05.03.2023 KW - DHN melanin KW - Fungus KW - Biosynthesis PY - 2023 AN - OPUS4-57143 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Abdallah, Khaled A1 - Knabe, Nicole A1 - Breitenbach, Romy A1 - Dementyeva, Polina A1 - Voigt, Oliver A1 - Gerrits, Ruben A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - Developing a genetic toolbox for Knufia petricola A95: a model for stress-tolerant and symbiose-competent rock-inhabiting fungi N2 - Black microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in hostile desert environments. In these niches, MCF have evolved mineral-weathering and symbiotic capabilities as well as mechanisms to cope with multiple stresses such as solar irradiation, temperature extremes and low water activity. Due to their stress tolerance these ascomycetes are prominent in modern terrestrial ecosystems – like man-made material surfaces from roof to solar panels. MCF interactive capabilities support their facultative symbiotic relationships with cyanobacteria and ensure their rock-weathering geochemical activity. Using the rock-inhabiting fungus K. petricola A95 (Chaetothyriales), we developed transformation protocols and deleted genes responsible for production of the protective pigments melanins and carotenoids. To confirm that the mutant phenotypes were not due to hidden mutations, melanin synthesis was restored by complementing the mutants with the respective wild type genes. Strains of K. petricola carrying gene variants for fluorescent proteins EGFP and DsRed are available. We successfully labelled the cytoplasm, nuclei, peroxisomes and mitochondria. Targeted and ectopic integrations result in stable transformants suitable for further phenotypical characterization. As K. petricola is a non-pathogenic fungus with all characteristic features of MCF, including meristematic growth, melanized cell-walls, extracellular polymeric substances and extensive pigment production, our results will shed light on protective role of pigments during cell wall maturation and oxidative stress defence in rock-inhabiting MCF. Genes involved in environmental sensing or substrate and phototroph interactions are currently targeted. With the help of a mutant collection and fluorescently labelled K. petricola we will be able to investigate interactions of MCF with environmental stressors, mineral substrates, soil matrices and phototrophic symbionts. T2 - Fungal Genetics Conference 2019 CY - Asilomar, CA, USA DA - 12.03.2019 KW - Knufia petricola KW - Genetics KW - Melanin PY - 2019 AN - OPUS4-49635 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -