TY - CONF A1 - Erdmann, Eileen A1 - Kunze, R. A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - Tools for Reverse and Forward Genetics in Knufia petricola N2 - Microcolonial black fungi are inhabitants of exposed natural and man-made surfaces in all climate zones. Genetic studies are hampered by slow growth, lacking sexual cycles and difficulties to transform these fungi. Recently, we implemented CRISPR/Cas9-based genetic tools enabling efficient targeted mutations of the rock inhabitant Knufia petricola (Eurotiomycetes/Chaetothyriales) for studying stress responses, the unusual types of cell division, mineral weathering and symbiotic interactions with phototrophic microbes (Voigt, Knabe et al. 2020, Sci Rep; Erdmann et al. 2022, Front Fungal Biol). To further explore the genome of this fungus as representative of the polyphyletic group of black fungi and to assign functions to yet unknown genes, a forward genetics approach is aimed. We chose the two-component Activator/ Dissociation (Ac/Ds) transposon system from maize for generating a collection of insertional mutants by in-vivo mutagenesis. For the optimal use of this genetic tool, an inducible promoter for the expression of the Ac transposase and by this the regulatable transposition of the resistance cassette-containing Ds transposon is requested. However, endogenous promoters for nitrate assimilation and galactose catabolism that are often used in fungi for regulatable gene expression are not inducible by their substrates in K. petricola suggesting that the regulatory networks for nutrient acquisition differ significantly in the oligotrophic black fungi. Therefore, the metabolism-independent Tet-on system was adopted and validated using a Ptet::gfp construct. No GFP fluorescence was detected in absence of the inducer doxycycline (DOX) while fluorescence intensities increased with increasing inducer concentrations. Consequently, the Tet-on system is suitable for regulatable expression of endogenous and foreign genes in K. petricola. T2 - VAAM Fachgruppentagung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - Rock-inhabiting fungus KW - Genetic tools KW - Controllable gene expression PY - 2022 AN - OPUS4-55678 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Erdmann, Eileen A1 - Kunze, R. A1 - Gorbushina, Anna A1 - Schumacher, Julia 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 - Erdmann, Eileen A1 - Kunze, R. A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - A random mutagenesis approach to elucidate the biology of extremotolerant black fungi N2 - Microcolonial black fungi ubiquitously inhabit sun-exposed natural and man-made surfaces of our planet. To promote genetic studies, which are hindered by slow growth, lack of sexual cycles and transformation difficulties, CRISPR/Cas9-based genetic tools were implemented (Erdmann et al. 2022, Front Fungal Biol). Now efficient targeted mutagenesis of the rock inhabitant Knufia petricola (Eurotiomycetes/Chaetothyriales) - as a representative of the polyphyletic group of black fungi - enables the elucidation of extremotolerance, oligotrophism, unusual types of cell division, mineral weathering and symbiotic interactions. Still more progress on assigning functions to yet unknown genes can be expected if a forward genetics approach is available. We chose the two-component Activator/ Dissociation (Ac/Ds) transposon system from maize for generating a collection of insertional mutants by in-vivo mutagenesis of K. petricola. For the optimal use of this genetic tool, an inducible promoter for the expression of the Ac transposase (AcTPase) and by this the regulatable transposition of the resistance cassette-containing Ds transposon is desired. However, endogenous promoters for nitrate assimilation and galactose catabolism - often used in fungi for regulatable gene expression - are not inducible by their substrates in K. petricola suggesting that the regulatory networks for nutrient acquisition differ significantly in oligotrophic fungi. Therefore, the metabolism-independent Tet-on system was combined with the AcTPase coding sequence and subsequently transformed into Ds-carrying K. petricola strains. In total, four auxotrophic Ac|Ds starter strains containing the Ds transposon at different position of ade2 or ura3 were generated. The cultivation of these strains with doxycycline for induction of TET::Ac and subsequent selection of cells on ADE/URA-lacking media resulted in prototrophic colonies (revertants) for some but not all Ac|Ds strains. Currently, the transposition events in the obtained revertants are studied to validate the procedure. First amplicon sequencing of excision sites revealed footprint patterns, proving the transposon jumped. T2 - 16th European Conference on Fungal Genetics CY - Innsbruck, Austria DA - 05.03.2023 KW - Rock-inhabiting fungi KW - Foward genetics KW - In-vivo mutagenesis PY - 2023 AN - OPUS4-57144 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Dittrich, Maria A1 - Paulo, Carlos A1 - Knabe, Nicole A1 - Sturm, Heinz A1 - Zaitsev, Vladimir A1 - Gorbushina, Anna T1 - Microscopic Raman study of fungal pigment using the genetically amenable rock inhabitant Knufia petricola as a model organism N2 - Fungal pigments such as melanin and carotenoids are distinctive markers of animal and plant pathogenic fungi as well as their environmental relatives. These complex pigments play important roles in pathogenicity and stress tolerance while also being useful as biomarkers. Accordingly, it is important to be able to identify in situ the pigments in black fungi, a group of clinical and environmental importance. In this study, wild-type and genetically modified strains of Knufia petricola A95 and wild fungal cells attached to ancient rock were investigated for their spectroscopic and microscopic Raman features and morphological appearance. Knockout mutants of melanin synthesis genes pks1 (polyketide synthase), sdh1 (scytalone dehydratase), and both pks1 and the carotenoid synthesis gene phd1 (phytoene desaturase) were studied We applied two different Raman microscopes using two lasers, with 633 nm and 488 nm wavelengths. We analyzed and compared Raman spectra between the measured reference substances and the mutant and wild-type strains. In the wild strain WT:A95, the peaks close to melanin peals were found at 1353 cm−1 and 1611 cm−1. There are no characteristic melanin peaks at 1580–1600 cm−1 and around 1350 cm−1 at the spectrum of the Δpks1/Δphd1 mutant and the Δsdh1 mutant. The Δpks1 mutant spectrum has the peaks at the beta-carotene v2 C-C in-plane stretch at 1155 cm−1 and v3 C-CH3 deformation at 1005 cm−1. The peaks of carotenoids and melanin were found in all mutants and the wild strain, except the Δpks1/Δphd1 mutant. Raman spectra allow for discrimination between the various pigments. Hence, interactions between natural fungal melanin, as well as other protective pigments, and complex environmental matrices can be characterized on a range of spatial and temporal scales. KW - Raman Spectroscopy KW - Instrumentation KW - Analytical Chemistry KW - Knufia petricola KW - Confocal microscopy KW - Atomic and Molecular Physics and Optics PY - 2023 DO - https://doi.org/10.1016/j.saa.2023.123250 SN - 1386-1425 VL - 303 SP - 1 EP - 11 PB - Elsevier BV AN - OPUS4-58792 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl A1 - Gorbushina, Anna A1 - Gerrits, Ruben T1 - Modelling biofilm formation and material colonisation for Knufia petricola N2 - Rock-inhabiting black fungi are adapted to survive extreme stresses such as radiation and lack of water and are thus able to colonise arid material surfaces like solar panels and marble monuments. Black fungi can typically grow as one of two cell types: depending on the environmental conditions, they are able to form yeast-like cells or hyphal filaments. Knufia petricola is one member of the rock-inhabiting black fungi which functions as a model reference species. As there is no mathematical model for biofilm formation of RIF, we are going to develop a model for K. petricola which describes fungal growth on the level of individual cells adn biofilm formation. T2 - Mini conference at University of Birmingham CY - Birmingham, UK DA - 05.10.2022 KW - Knufia petricola KW - Rock-inhabiting black fungi KW - individual-based modelling PY - 2022 AN - OPUS4-56697 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - 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 - JOUR A1 - de Vallée, A. A1 - Bally, P. A1 - Bruel, C. A1 - Chandat, L. A1 - Choquer, M. A1 - Dieryckx, C. A1 - Dupuy, J.W. A1 - Kaiser, S. A1 - Latorse, M.-P. A1 - Loisel, E. A1 - Mey, G. A1 - Morgant, G. A1 - Rascle, C. A1 - Schumacher, Julia A1 - Simon, A. A1 - Souibgui, E. A1 - Viaud, M. A1 - Villalba, F. A1 - Poussereau, N. T1 - A similar secretome disturbance as a hallmark of non-pathogenic Botrytis cinerea ATMT-mutants? N2 - The gray mold fungus Botrytis cinerea is a necrotrophic pathogen able to infect hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to decipher its infectious strategy, a library of 2,144 mutants was generated by random insertional mutagenesis using Agrobacterium tumefaciensmediated transformation (ATMT). Twelve mutants exhibiting total loss of virulence toward different host plants were chosen for detailed analyses. Their molecular characterization revealed a single T-DNA insertion in different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably deficiencies in the secretion of proteases and hemicellulases, were observed and validated by biochemical tests. They were also a hallmark of the remaining eight non-pathogenic strains, suggesting the importance of these secreted Proteins in the infection process. In the twelve non-pathogenic mutants, the Differentiation of infection cushions was also impaired, suggesting a link between the Penetration structures and the secretion of proteins involved in the virulence of the pathogen. KW - Fungus KW - Plant pathogen KW - Virulence PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-502710 DO - https://doi.org/10.3389/fmicb.2019.02829 SN - 1664-302X VL - 10 SP - Article 2829 PB - Frontiers Media CY - Lausanne AN - OPUS4-50271 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Davis, Hannah A1 - Meconcelli, Stefania A1 - Radek, R. A1 - McMahon, Dino Peter T1 - Termites shape their collective behavioural response based on stage of infection N2 - Social insects employ a range of behaviours to protect their colonies against disease, but little is known about how such collective behaviours are orchestrated. This is especially true for the social Blattodea (termites). We developed an experimental approach that allowed us to explore how the social response to disease is co-ordinated by multistep host-pathogen interactions. We infected the eastern subterranean termite Reticulitermes flavipes with the entomopathogenic fungus Metarhizium anisopliae, and then, at different stages of infection, reintroduced them to healthy nestmates and recorded behavioural responses. As expected, termites groomed pathogen-exposed individuals significantly more than controls; however, grooming was significantly elevated after fungal germination than before, demonstrating the importance of fungal status to hygienic behaviour. Significantly, we found that cannibalism became prevalent only after exposed termites became visibly ill, highlighting the importance of host condition as a cue for social hygienic behaviour. Our study reveals the presence of a coordinated social response to disease that depends on stage of infection. Specifically, we show how the host may play a key role in triggering its own sacrifice. Sacrificial self-flagging has been observed in other social insects: our results demonstrate that termites have independently evolved to both recognize and destructively respond to sickness. KW - Social KW - Immunity KW - Cannibalism KW - Entomopathogen PY - 2018 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-463239 DO - https://doi.org/10.1038/s41598-018-32721-7 SN - 2045-2322 VL - 8 SP - 14433, 1 EP - 10 PB - Nature CY - London AN - OPUS4-46323 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Cunliffe, A. J. A1 - Askew, P. D. A1 - Stephan, Ina A1 - Iredale, G. A1 - Cosemans, P. A1 - Simmons, L. M. A1 - Verran, J. A1 - Redfern, J. T1 - How do we determine the efficacy of an antibacterial surface? A review of standardised antibacterial material testing methods N2 - Materials that confer antimicrobial activity, be that by innate property, leaching of biocides or design features (e.g., non-adhesive materials) continue to gain popularity to combat the increasing and varied threats from microorganisms, e.g., replacing inert surfaces in hospitals with copper. To understand how efficacious these materials are at controlling microorganisms, data is usually collected via a standardised test method. However, standardised test methods vary, and often the characteristics and methodological choices can make it difficult to infer that any perceived antimicrobial activity demonstrated in the laboratory can be confidently assumed to an end-use setting. This review provides a critical analysis of standardised methodology used in academia and industry, and demonstrates how many key methodological choices (e.g., temperature, humidity/moisture, airflow, surface topography) may impact efficacy assessment, highlighting the need to carefully consider intended antimicrobial end-use of any product. KW - Antimicrobial materials KW - Antimicrobial testing KW - ISO 22196 KW - Antimicrobial surfaces KW - Antibacterial coatings PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-532138 DO - https://doi.org/10.3390/antibiotics10091069 SN - 2079-6382 VL - 10 IS - 9 SP - 1 EP - 14 PB - MDPI CY - Basel AN - OPUS4-53213 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Cook, Jake Thomas A1 - Tonon, Chiara T1 - Assessment of concrete bioreceptivity in algal biofilm green façade systems N2 - Algal biofilm façades are an alternative to traditional green façades which can help to improve biodiversity and air quality within cities. They present a low maintenance approach in which subaerial algae are grown directly on concrete substrates. The intrinsic bioreceptivity of the substrate is a critical factor in successful facade colonisation. Existing research has identified several environmental and material properties which influence concrete bioreceptivity, however a consensus has yet to be made on which properties are most influential and how the interaction between properties may promote algal biofilm growth under specific conditions. T2 - International Conference on Bio-Based Building Materials CY - Vienna, Austria DA - 21.06.2023 KW - Concrete KW - Façade KW - Bio-receptive KW - Extracellular polymeric substances KW - Fractional factorial PY - 2023 UR - https://www.rilem.net/agenda/5th-international-conference-on-bio-based-building-materials-1501 AN - OPUS4-58976 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -