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Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic 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) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. 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 the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). 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. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi.
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.
How the rock-inhabiting fungus K. petricola A95 enhances olivine dissolution through attachment
(2020)
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.
Due to their ability to penetrate, deteriorate and discolour stone surfaces, rock-inhabiting black fungi represent a remarkable issue for cultural heritage conservation. Black microcolonial fungi (MCF) can also adapt to different environmental conditions, by converting from yeast-like morphology to a peculiar meristematic development with swollen cells (torulose hyphae, TH), to extremely thin structures (filamentous hyphae, FH). Furthermore, black MCF produce protective pigments: melanin, dark pigment particularly evident on light stone surfaces, and carotenoids. Black fungi produce melanin in critical, oligotrophic conditions as well as constitutively. Melanin function is mostly related to stress resistance and the ability of fungi to generate appressorial turgor to actively penetrate plant cells in pathogenic species. An involvement of melanins in stone surface penetration has been suggested, but not experimentally proved. In this work, we tested the role of hyphal melanisation in penetration mechanisms on the model black fungus Knufia petricola A95 in lab conditions. The wild-type and three mutants with introduced targeted mutations of polyketide-synthases (melanin production) and/or phytoene dehydrogenase (carotenoid synthesis) were inoculated on artificial carbonate pellets (pressed Carrara marble powder) of different porosity. After 5, 10, 17 and 27 weeks, hyphal penetration depth and spread were quantified on periodic acid Schiff-stained cross-sections of the pellets, collecting measurements separately for TH and FH. Droplet assay of the mutants on different media were conducted to determine the role of nutrients in the development of different fungal morphologies. In our in vitro study, the hyphal penetration depth, never exceeding 200 μm, was proven to be consistent with observed penetration patterns on stone heritage carbonate substrates. Pellet porosity affected penetration patterns of TH, which developed in voids of the more porous pellets, instead than actively opening new passageways. Oppositely, the thin diameter of FH allowed their penetration independently of substrate porosity. Instead, the long-hypothesized crucial role of melanin in black MCF hyphal penetration should be rejected. TH were developed within the pellets also by melanin deficient strains, and melanized strains showed an endolithic component of non-melanized TH. FH were non-melanized for all the strains, but deeply penetrated all pellet types, with higher penetration depth probably related to their potential exploratory (nutrient-seeking) role, while TH may be more related to a resistance to surface stress factors. In the melanin deficient strains, the absence of melanin caused an increased penetration rate of FH, hypothetically related to an earlier necessity to search for organic nutrients.
Interface between the atmosphere and mineral substrates is the oldest terrestrial habitat. Morphologically simple microbial biofilms were the first settlers on these inhospitable surfaces at times when the Earth was inhabited only by microorganisms and the solid substrates represented only by natural rock surfaces i.e. lithosphere. Miniature, self-sufficient microbial ecosystems continue to develop on subaerial (i.e. air-exposed) solid surfaces at all altitudes and latitudes where direct contact with the atmosphere and solar radiation occurs – on rocks, mountains, buildings, monuments, solar panels. All these sub-aerial biofilms develop under fluctuating and hostile conditions – and thus frequently harbour stress-tolerant black fungi inherently able to cope with the stresses of bright sunlight and constantly changing atmospheric conditions. Black fungi – a polyphyletic group of Ascomycetes– accumulate the dark pigment DHN melanin, diverse carotenoids and mycosporines in their cells and thus successfully colonise sunlight-flooded habitats from phyllosphere to rock surfaces. Various chemical and physical extremes and fluctuating environments belong to the challenges effectively mastered by black fungi. In our laboratory we isolate novel black fungi from man-made habitats like building materials and solar panels. Using Knufia petricola A95 as a model we conduct experiments to clarify interactions of black fungi with inorganic substrates. We use available mutants to determine the functional consequences of changes in the outer cell wall envelopes – from excreted EPS to layers of protective pigments. A genetic toolbox to manipulate this Chaetothyriales representative is in further development. Our long-term goal is to understand the fundamental mechanisms how black fungi are able (i) to adhere to dry atmosphere-exposed surfaces, (ii) to survive multiple stresses and (iii) to change the underlying substrates including rocks.
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish subaerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that exhibits all the characteristics of microcolonial black fungi such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis (Nai et al. 2013, Fungal Genet Biol). We developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis. The differences between the wild-type fungus and its melanin-deficient mutants were tested in geomicrobiological experiments and measured are now discussed in detail, with special accent on the possible effects of the mutation on EPS and other exuded substances.
The story of how black fungi survive harsh conditions on sun-exposed desert rocks and material surfaces is fascinating. In the presentation examples of how knowledge of these organisms can be of practical value (e.g., in biodeterioration studies, such as on stone markers in cemeteries) would be given. Among other stories, the example of how roof tiles amended with a titanium oxide layers as a biocide actually selected for the black fungi will be told. A hypothesis about how these organisms would likely be found on solar panels and some early BAM work in that area will be presented. This then brings the connection to a study ripe for investigation in North Carolina. The talk presents the development of a study aiming at deciphering the influence of microbial biofilm formation on the energy conversion efficiency of solar photovoltaic panels or modules at two facilities (one facility under the impact of high intensity of animal agriculture and high deposition of ammonia from atmosphere and the other with low animal agriculture and lower atmospheric ammonia deposition) in North Carolina. The main hypothesis of the study is that microbial biofilm formation on solar photovoltaic panels will lead to significant decreases in energy conversion efficiency of solar photovoltaic modules and biofilm formation will also be accelerated by high ammonia concentration in the ambient atmosphere and high nitrogen deposition.
Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. But 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 fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black [dihydroxynaphthalene (DHN) melanin-containing] fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers ofPRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for fungi that receive nutrients through cooperation with phototrophs. Genetic tools for manipulating K. petricola exist and will be used to test this idea.
In a controlled growth experiment we found that the cyanobacterium Nostoc punctiforme has a bulk cell 26Mg/24Mg ratio (expressed as δ26Mg) that is −0.27‰ lower than the growth solution at a pH of ca. 5.9. This contrasts with a recently published δ26Mg value that was 0.65‰ higher than growth solution for the black fungus Knufia petricola at similar laboratory conditions, interpreted to reflect loss of 24Mg during cell growth. By a mass balance model constrained by δ26Mg in chlorophyll extract we inferred the δ26 Mg value of the main Mg compartments in a cyanobacteria cell: free cytosolic Mg (−2.64‰), chlorophyll (1.85‰), and the nonchlorophyll-bonded Mg compartments like ATP and ribosomes (−0.64‰). The lower δ26Mg found in Nostoc punctiforme would thus result from the absence of significant Mg efflux during cell growth in combination with either (a) discrimination against 26Mg during uptake by desolvation of Mg or transport across protein channels or (b) discrimination against 24Mg in the membrane transporter during efflux. The model predicts the preferential incorporation of 26Mg in cells and plant organs low in Mg and the absence of isotope fractionation in those high in Mg, corroborated by a compilation of Mg isotope ratios from fungi, bacteria, and higher plants.
In the modern world there is an increased understanding that design and performance monitoring of materials have to be tested in connection to chemical, physical and (micro)biological challenges. A systematic study on how biofilms interact with materials and what could be done to engineer biofilms and/or materials in order to maximize the resistance of the material (surface) or the resistance the biofilm-modified material (bulk) is in strong need. In the Department “Materials and the Environment” of the BAM new experimental platform is being developed. With the help of different type of device for high throughput and microbiologically-controlled environment simulation we establish a new approach to clarify the mechanisms of biofilm/material interactions. Despite the focus on fundamental research, the main results of this project proposal will be transferable into material technology and construction chemistry and will influence the development of standardization in this topic. As the interactions of biofilms and materials have implications for most constructions as well as climate change, the results of the research generates additional value.