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Essential processes necessary for the establishment and maintenance of rock biofilms include photosynthesis, production of extracellular polymeric substances, substrate penetration and nutrient enrichment from the atmosphere. This natural functional diversity is supported by a complex biofilm community consisting of heterotrophic and phototrophic microorganisms. While heterotrophic rock biofilm-formers are dominated by stress-tolerant microcolonial ascomycetes, phototrophs might be represented by diverse algae and cyanobacteria. Reduction of model systems to the genetically tractable minimum, a proven successful experimental strategy in different symbiotic systems, is now applied to rock biofilms. These complex and important natural systems are simulated in the lab using genetic methods coupled with continuous microscopic and analytical (microscopic as well as geochemical and biochemical) observations. Our in vitro bipartite model includes free-living and symbiosis-competent, genetically tractable microorganisms – a rock-inhabiting fungus Knufia petricola A95 and a cyanobacterium Nostoc punctiforme strain ATCC 29133. To accurately reflect the development of a rock biofilm, contacts of these two genetically tractable partners are studied under well-controlled laboratory conditions. This experimental strategy is strongly supported by the knockout mutants of Knufia petricola which have been recently created and will be compared in their action on mineral surfaces. An impact of Knufia petricola strain A95 with or without protective pigments on mineral adhesion and alteration will be presented.
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.