TY - JOUR A1 - Catanzaro, Ilaria A1 - Gerrits, Ruben A1 - Feldmann, Ines A1 - Gorbushina, Anna A. 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 SP - 1 EP - 19 PB - Wiley AN - OPUS4-60659 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Catanzaro, Ilaria A1 - Nitsche, Sarah A1 - Gorbushina, Anna A1 - Schumacher, Julia A1 - Onofri, S. T1 - Generation of Cryomyces antarcticus mutants to explore the importance of DHN melanin for survival N2 - Cryomyces antarcticus, a cryptoendolithic melanized fungus endemic to Antarctica (phylum Ascomycota, class Dothideomycetes incertae sedis), has demonstrated high capability to survive extreme environmental conditions like those found in space (e.g., ionizing radiation, vacuum, microgravity), thus fueling fundamental astrobiological questions like “the search for life beyond Earth”. Its extraordinary resilience has been attributed to the presence of thick, highly melanized cell walls, which may contain both DHN and DOPA melanins. To better understand the contribution of DHN melanin to the overall resilience of C. antarcticus, we decided to generate melanin-deficient mutants by genetic engineering. For this, the melanin-PKS (polyketide synthase)- encoding ortholog was identified in the C. antarcticus CBS 116301 genome and used to design primers for re-sequencing of the capks1 locus in the strain CCFEE 515. Based on the genetic toolkit developed for the black fungus Knufia petricola, we designed a strategy for mutating capks1. Protoplasts of C. antarcticus CCFEE 515 were generated and co-transformed with a circular AMA-containing plasmid for expression and in-vivo assembly of Cas9 and two capks1-specific sgRNAs and a PCR-generated donor DNA i.e., a hygromycin resistance cassette flanked by ~75-bp-long sequences homologous to the capks1 locus. Transformation of C. antarcticus is challenging because of its very slow growth and uncertain gene copy number. It took six months from obtaining enough biomass for cell wall lysis to transferring the putatively resistant transformants for genotyping, but we eventually managed to generate three independent non-melanized (whitish) Δcapks1 mutants! We are now awaiting first complemented strains as ultimate genetic transformation control. They are considered to have a wild-type-like pigmentation due to the reintroduction of the wild-type capks1 into the Δcapks1 mutant. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Black fungus KW - Genetics KW - UV tolerance KW - Melanin PY - 2023 AN - OPUS4-58458 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - 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 - JOUR A1 - C., Ruibal A1 - L., Selbmann A1 - Serap, Avci A1 - Martin-Sanchez, Pedro Maria A1 - Gorbushina, Anna ED - Gorbushina, Anna T1 - Roof-Inhabiting Cousins of Rock-Inhabiting Fungi: Novel Melanized Microcolonial Fungal Species from Photocatalytically Reactive Subaerial Surfaces N2 - Subaerial biofilms (SAB) are an important factor in weathering, biofouling, and biodeterioration of bare rocks, building materials, and solar panel surfaces. The realm of SAB is continually widened by modern materials, and the settlers on these exposed solid surfaces always include melanized, stress-tolerant microcolonial ascomycetes. After their first discovery on desert rock surfaces, these melanized chaetothyrialean and dothidealean ascomycetes have been found on Mediterranean monuments after biocidal treatments, Antarctic rocks and solar panels. New man-made modifications of surfaces (e.g., treatment with biocides or photocatalytically active layers) accommodate the exceptional stress-tolerance of microcolonial fungi and thus further select for this well-protected ecological group. Melanized fungal strains were isolated from a microbial community that developed on highly photocatalytic roof tiles after a long-term environmental exposure in a maritime-influenced region in northwestern Germany. Four of the isolated strains are described here as a novel species, Constantinomyces oldenburgensis, based on multilocus ITS, LSU, RPB2 gene phylogeny. Their closest relative is a still-unnamed rock-inhabiting strain TRN431, here described as C. patonensis. Both species cluster in Capnodiales, among typical melanized microcolonial rock fungi from different stress habitats, including Antarctica. These novel strains flourish in hostile conditions of highly oxidizing material surfaces, and shall be used in reference procedures in material testing. KW - Microcolonial fungi KW - Multilocus phylogeny KW - Photocatalytic surfaces KW - Subaerial biofilms KW - Stress tolerance KW - Constantinomyces PY - 2018 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-455182 DO - https://doi.org/10.3390/life8030030 VL - 8 IS - 3 SP - 30 EP - 44 PB - MDPI CY - Basel, Schweiz AN - OPUS4-45518 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Bucek, A. A1 - Sobotnik, J. A1 - He, Shulin A1 - Shi, M. A1 - McMahon, Dino Peter A1 - Holmes, E.C. A1 - Roisin, Y. A1 - Lo, N. A1 - Bourguignon, T. T1 - Evolution of Termite Symbiosis Informed by Transcriptome-Based Phylogenies N2 - Termitidae comprises 80% of all termite species that play dominant decomposer roles in Tropical cosystems. Two major events during Termite evolution were the loss of cellulolytic gut protozoans in the ancestor of Termitidae and the subsequent gain in the termitid subfamily Macrotermitinae of fungal symbionts cultivated externally in ‘‘combs’’ constructed within the nest. How these symbiotic transitions occurred remains unresolved. Phylogenetic analyses of mitochondrial data previously suggested that Macrotermitinae is the earliest branching termitid lineage, followed soon after by Sphaerotermitinae, which cultivates bacterial symbionts on combs inside its nests. This has led to the hypothesis that comb building was an important evolutionary step in the loss of gut protozoa in ancestral termitids. We sequenced genomes and transcriptomes of 55 termite species and reconstructed phylogenetic trees from up to 4,065 orthologous genes of 68 species. We found strong support for a novel sister-group relationship between the bacterial comb-building Sphaerotermitinae and fungus comb-building Macrotermitinae. This key finding indicates that comb building is a derived trait within Termitidae and that the creation of a comb-like ‘‘external rumen’’ involving bacteria or fungi may not have driven the loss of protozoa from ancestral termitids, as previously hypothesized. Instead, associations with gut prokaryotic symbionts, combined with dietary shifts from wood to other plant-based substrates, may have played a more important role in this symbiotic transition. Our phylogenetic tree provides a platform for future studies of comparative termite evolution and the evolution of symbiosis in this taxon. KW - Molecular clock KW - Fungiculture KW - Gut symbionts KW - Insect evolution KW - Isoptera PY - 2019 DO - https://doi.org/10.1016/j.cub.2019.08.076 VL - 29 IS - 21 SP - 3728 EP - 3734.e4 PB - Elsevier Ltd. AN - OPUS4-49647 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Bucar, K. A1 - Zitnik, M. A1 - Stabile, L. A1 - Ozan, J. A1 - Seeger, Stefan T1 - Performance of a Sharp GP2Y low-cost aerosol PM sensor N2 - Simple particulate matter sensors are gaining popularity due to their low price, easy handling and good temporal resolution. In this presentation, we report on the performance of a Sharp optical PM sensor GP2Y1010AU0F, which costs less than 15 €. The sensor is built around an infrared emitting diode (ILED) and a phototransistor detecting the light scattered from the aerosol particle. An electronic circuit shapes the detected light in a pulsed signal. The manufacturer advises sampling the output signal 280 microseconds after the ILED pulse. The measured output voltage is an indicator of dust concentration. We have built two identical simple PM monitoring devices using Raspberry Pi 3 computer interfacing the PM sensor with Microchip’s MCP3002 ADC via SPI. The ADC is capable of more than 100 ksamples/s at 10-bit resolution. The Rpi3 was pulsing the sensor at 10Hz, digitizing and saving the data and sending the results wirelessly. Sensor’s output pulse shape was sampled with 10 microsecond time steps and saved, thus making offline analysis possible. A time jitter of output pulses can be observed and suggests a peak fitting as a better approach to the signal readout compared to the single sampling at a fixed time after pulse triggering We compared both methods. T2 - European Aerosol Coference EAC 2019 CY - Gothenburg, Sweden DA - 25.08.2019 KW - Aerosol KW - Low cost aerosol PM sensor KW - PM PY - 2019 AN - OPUS4-49581 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Brusamarello-Santos, L. C. C. A1 - Alberton, D. A1 - Valdameri, G. A1 - Camilios-Neto, D. A1 - Covre, R. A1 - Lopes, K. d. P. A1 - Zibetti Tadra-Sfeir, M. A1 - Faoro, H. A1 - Adele Monteiro, R. A1 - Barbosa-Silva, A. A1 - Broughton, William John A1 - Oliveira Pedrosa, F. A1 - Wassem, R. A1 - de Souza, E.M. T1 - Modulation of defence and iron homeostasis genes in rice roots by the diazotrophic endophyte Herbaspirillum seropedicae N2 - Rice is staple food of nearly half the world’s population. Rice yields must therefore increase to feed ever larger populations. By colonising rice and other plants, Herbaspirillum spp. stimulate plant growthand productivity. However the molecular factors involved are largely unknown. To further explore this interaction, the transcription profiles of Nipponbare rice roots inoculated with Herbaspirillum seropedicae were determined by RNA-seq. Mapping the 104 million reads against the Oryza sativa cv. Nipponbare genome produced 65 million unique mapped reads that represented 13,840 transcripts each with at least two-times coverage. About 7.4% (1,014) genes were differentially regulated and of these 255 changed expression levels more than two times. Several of the repressed genes encoded proteins related to plant defence (e.g. a putative probenazole inducible protein), plant disease resistance as well as enzymes involved in flavonoid and isoprenoid synthesis. Genes related to the synthesis and efflux of phytosiderophores (PS) and transport of PS-iron complexes were induced by the bacteria. These data suggest that the bacterium represses the rice defence system while concomitantly activating iron uptake. Transcripts of H. seropedicae were also detected amongst which transcripts of genes involved in nitrogen fixation, cell motility and cell wall synthesis were the most expressed. KW - Herbaspirillum seropedicae KW - Pathogen KW - Rice KW - qPCR KW - Genome PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-490122 DO - https://doi.org/10.1038/s41598-019-45866-w SN - 2045-2322 VL - 9 SP - 10573-1 EP - 10573-15 PB - Nature CY - London AN - OPUS4-49012 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 - 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 - THES A1 - Breitenbach, Romy T1 - Biochemische und physiologische Charakterisierung der extrazellulären Matrix eines Modellbiofilms N2 - An der Grenzschicht zwischen einer festen Oberfläche und der umgebenden Luft wachsende subaerische Biofilme (SAB) zeichnen sich durch eine erhöhte Toleranz gegenüber extremen Umweltbedingungen und dem Eintrag von Bioziden aus. Dieser Schutz vor äußeren Umwelteinflüssen wird vornehmlich durch den Beitrag von extrazellulären polymeren Substanzen (EPS) und Pigmenten, wie Melanin und Carotinoiden, vieler unterschiedlicher Organismen gewährleistet. Deren Synthese wird wiederum durch intrazelluläre Botenstoffe reguliert. Das Cyanobakterium Nostoc punctiforme und der mikrokoloniale Pilz Knufia petricola, als Partner eines etablierten SAB-Modells und Vertreter zweier typischer Organismengruppen in SAB, wurden genutzt, um Botenstoffe und Pigmente genetisch zu manipulieren und die Biofilmmatrix dieser Mutanten strukturell zu untersuchen. In dieser Arbeit konnten erstmals die EPS beider Organismen extrahiert und die Struktur der extrazellulären Polysaccharide beschrieben werden. Daneben wurden die extrazellulären Polysaccharide von K. petricola Wildtyp mit denen verschiedener Pigmentmutanten verglichen. Das Fehlen des Schutzpigmentes Melanin führte zu einer ausgeprägteren extrazellulären Matrix in den Biofilmen. Gleichzeitig änderte sich die Struktur der extrazellulären Polysaccharide. Während der Wildtyp zu ~80% ein α-Glucan und zu ~20% ein α/β-Galaktomannan sekretierte, war der Anteil des Galaktomannans bei den Melaninmutanten erhöht. Das Ausschalten der Carotinoid-Synthese hatte jedoch keinen Einfluss auf die Beschaffenheit der extrazellulären Polysaccharide. Ein deutlich komplexeres extrazelluläres Polysaccharid aus acht verschiedenen Monosaccharid-Einheiten bildete N. punctiforme. Durch die Überproduktion des bakteriellen sekundären Botenstoffes c-di-GMP konnte zudem ein grundlegender Einfluss auf die Reaktion des Cyanobakteriums gegenüber externen Signalen und die damit verbundene Zelldifferenzierung gezeigt werden. Ein artifiziell erhöhtes c-di-GMP-Level in den Zellen führte zur Ausbildung eines sessilen Lebensstils durch Hemmung der Differenzierung motiler Hormogonien und vermehrte Produktion von EPS. Die Struktur der extrazellulären Polysaccharide wurde dadurch nicht verändert. Neben der strukturellen Analyse konnten die Veränderungen der extrazellulären Matrix beider Organismen zusätzlich durch mikroskopische Methoden visualisiert werden. Beide Organismen steuern komplexe Polymere, deren Produktion maßgeblich mit intrazellulären Faktoren verknüpft ist, zu der extrazellulären Matrix des Modellbiofilms bei. KW - Biofilm KW - EPS KW - Extrazelluläre Polysaccharide PY - 2018 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-471747 SP - 1 EP - 198 CY - Berlin AN - OPUS4-47174 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -