4 Material und Umwelt
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Traceable and reliable chemical element analysis of aerosols by X-ray spectrometry was investigated using aerosol samples from field campaigns which have been measured in the GIXRF-beamline at BESSY.
The reference-free XRF approach allows for a traceable analysis of the mass deposition.
Traceable quantification by means of XRF can be transfered to benchtop instrumentation used in the laboratory Chemical and dimensional analysis of deposited aerosol allows for a comprehensive analysis of aerosols, e.g. for toxicity assessment and determination of the source The folowing elements could be identified and quantified in the field samples: Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Y, W, Pb.
Quantitative chemical analysis of airborne particulate matter (PM) is vital for the understanding of health effects in indoor and outdoor environments, as well as for enforcing air quality regulations. Typically, airborne particles are sampled over long time periods on filters, followed by lab-based analysis, e.g., with inductively coupled plasma mass spectrometry (ICP-MS). Within the EURAMET EMPIR AEROMET project, cascade impactor aerosol sampling was combined for the first time with on-site total reflection X-ray fluorescence (TXRF) spectroscopy to develop a tool for quantifying particle element compositions within short time intervals and even on-site. This makes variations of aerosol chemistry observable with time resolution of only a few hours and with good size resolution in the PM10 range. A proof of principles of this methodological approach and the comparison to standard methods within the scope of a field campaign will be presented. Secondly, aerosol sampling and TXRF analysis seems suitable for the quantification of elements in indoor aerosols as well and may provide an important enhancement of existing methods for the analysis of organic species in aerosols (such as sampling and TD-GC/MS). As an example, the TXRF analysis of particles emitted from laser printers under controlled conditions in an environmental test chamber will be presented.
Recent studies have shown that host-microbiota interactions can lead to dramatic changes in host phenotype especially behaviour. We investigate the causal drivers of microbe-associated shifts in host phenotype by examining how feeding behaviour is modulated by a bacterial infection in an omnivorous cockroach: Blatta orientalis. We conducted food-choice experiments after challenging hosts with the common entomopathogenic soil bacterium Pseudomonas entomophila to understand the impact of bacterial pathogens on host macronutrient preference. We find that immune challenge by this bacterium drives a sharp decline in carbohydrate intake and results in a relative increase in the ratio of protein to carbohydrate consumed. Additionally, infected cockroaches reduce their overall nutrient intake. We show for the first time that cockroach feeding behaviour is dynamically modulated by a pathogen. In contrast to studies on Spodoptera moths, this modulation does not impact any of the immune parameters we measured: it does not affect the abundance of immune related proteins in the hemolymph, hemolymph antimicrobial activity, or survival. This leads to the possibility that as long-lived omnivorous species B. orientalis may be better adapted to unpredictable variation in food availability and quality. An illness-induced anorexia-like response which is thought to assist hosts in limiting nutritional resources available to pathogens is therefore the most likely cause of the observed phenotype. Reduction of carbohydrate intake by sick individuals would be consistent with such an explanation.
In another cockroach species Blatella germanica we also investigate whether in addition to pathogens, host microbiota especially gut commensals are causally responsible for shifts in host behavioural phenotype and immune competence. To address this, we conduct food-choice and immunechallenge experiments in germ-free cockroaches and naïve cockroaches.
Human-made systems, also called “build environment” or “technosphere”, sustain human comfort as well as our industrial activities. These systems have become particularly widespread since the Industrial Revolution, i.e., since the 17th century. At the same time, these technical systems – buildings, monuments, energy production, transformation and transmission, water purification and supply systems - serve as new habitats for living organisms.
Life is ubiquitously present on our planet since a very long time: the Earth is 4.54 billion years old and microbial communities have played a key role on our planet for 3.7 billion years. Once human-made system appeared, microorganisms became an integral part of all types of technosphere infrastructure as well.
Here we will illustrate biosphere-technosphere interactions using a specific example of the black fungi and their impact on the efficiency of solar (photovoltaic) panels. This expanding renewable infrastructure for electricity generation is growing on all continents - and create a specific, arid habitat for stress-tolerant black fungi.
Black fungi were once discovered in hot and cold natural deserts – and now belong to the persistent colonisers of human-made deserts of solar parks. This new niche is evolving an impressive biodiversity. So far more than 60 isolates of black fungi belonging to Arthoniomycetes, Eurotiomycetes and Dothideomycetes were obtained from solar panels in Europe and Americas. Here we will present the analysis of this emerging anthropogenic biodiversity.
Opportunities for future research in the field include quantification of the microbial load on technosphere surfaces – along with characterisation of the corresponding microbial diversity. The strategy of precise measurement and characterisation will enable us to reliably determine the beneficial and harmful functions that living microorganisms play in the functioning of energy-generating systems – and technosphere in general.
Microbial contamination of fuels by fungi or bacteria poses risks such as corrosion and fuel system fouling, which can lead to critical problems in refineries and distribution systems and has a significant economic impact at every stage of the process. Many factors have been cited as being responsible for microbial growth, like the presence of water in the storage tanks. In fact, only 1 % water in a storage system is sufficient for the growth of microorganisms like bacteria or yeasts, as well as for the development of fungal biomass at the oil/water interface.
This work presents a rapid test for the accurate determination of genomic DNA from aqueous fuel extracts. The detection is based on the use of polystyrene-mesoporous silica core-shell particles onto which modified fluorescent molecular beacons are covalently grafted. These beacons contain in the hairpin loop a target sequence highly conserved in all bacteria, corresponding to a fragment of the 16S ribosomal RNA subunit. The designed single-stranded molecular beacon contained fluorescein as an internal indicator and a quencher in its proximity when not hybridized. Upon hybridization in presence of the target sequence, the indicator and the quencher are spatially separated, resulting in fluorescence enhancement. To perform the assay the developed particles were deposited on different glass fibre strips to obtain a portable and sensitive rapid test. The assays showed that the presence of genomic DNA extracts from bacteria down to 50–70 μg L–1 induced a fluorescence response. The optical read-out was adapted for on-site monitoring by fitting a 3D-printed case to a conventional smartphone, taking advantages of the sensitivity of the CMOS detector. Such embedded assembly enabled the detection of genomic DNA in aqueous extracts down to the mg L–1 range and represents an interesting step toward on-site monitoring of fuel contamination.
Technical advances in the study of molecular evolution have crystallized the fundamental insight that many bee pathogens evolve and adapt over timescales that overlap with host ecology. At the same time, the role played by bee host community ecology is increasingly being appreciated in host-parasite interactions. Here, we focus on Deformed wing virus (DWV) and present recent studies exploring the link between virulence, DWV genetic diversity and changes to host ecology - namely the arrival of an invasive ectoparasite, the Varroa destructor mite, which vectors viruses between honeybees. Specifically, we show how V. destructor may have created conditions for the emergence of more virulent strains of DWV in the western honeybee, Apis mellifera. We present a molecule-to-ecology framework to help interpret findings and to guide future hypotheses, emphasizing the role of molecular interactions between viruses and host immunity as drivers of change at the bee population level.
Environmental sustainability and –stability of Materials concerning the Migration of pollutants
(2018)
MaUS is an acronym for ”Material und Umweltsimulationen“. Plastics are in the focus of environmental politics due to their long-term behaviour and therefore to their persistence. Not only that they appear as visible contaminants in the sea and on the beach, but their unknown behaviour concerning their additives as well as the related transformation products are anxious. Therefore, we wish to establish a certified reference method to provide a method for testing plastics.
Aim of this project is the development of fast motion standard reference methods for testing plastics regarding to their environmental compatibility. To establish these testing methods, we use polystyrene (PS) and polypropylene (PP) with environmental relevant brominated flame retardants, known for their persistent bioaccumulative and toxic (PBT) properties. In case of PS the material contains 1 wt% of 1,2,5,6,9,10-hexabromocyclododecan (HBCD) and in case of PP 0.1 wt% bromodiphenylether (BDE-209), which is known as a substance of very high concern (SVHC). Furthermore, we use polycarbonate (PC), which is still used as material in baby flasks and releases Bisphenol A (BPA), an estrogenic active substance.
As an additional material PTFE is used for its importance as a source for two ubiquitous environmental substances (PFOS and PFOA), whose toxicological effects are still incompletely known.
The focus in this current work is set on the transfer of potential pollutants out of applied materials mentioned above into environmental compartments like water or soil. Here an accelerated aging concept should be developed to shortened time consuming natural processes. For these resulting simulations we use a programmable weathering chamber with dry and wet periods and with high and low temperatures. These programmes run for several weeks and according to a defined sampling schedule we take water samples, run a clean-up procedure by SPE (Molecular imprinted polymers (MiPs) resp. polymer-based cartridges (Waters Oasis HLB)) and analyse them by HPLC-UV resp. LC-MS/MS. Of most interest in case of flame retardants are photocatalytic transformation products. Therefore, we conduct a non-target-screening resp. a suspected target-screening by LC-MS/MS and HRMS.
Bee populations have declined significantly in recent years and this is thought to be attributable at least in part to the (re-)emergence of viruses. These viruses are predominantly positive single stranded (+ss) RNA viruses belonging to the Picornavirales. Managed honeybees are often infested with the invasive mite, Varroa destructor, which vectors RNA viruses including Deformed wing virus (DWV, family Iflaviridae): a leading culprit of colony losses. Many bee viruses have been sequenced and structural features are now available for viruses such as DWV. DWV consists of at least 3 distinct genotypes, two of which have been shown to be differentially virulent in honeybees. Molecular studies have demonstrated that DWV has a mean evolutionary rate of 1.35 x 10-3 per site per year. For such viruses – in contrast to their eukaryotic hosts – ecological and evolutionary timescales significantly overlap. This rapid evolution allows RNA viruses to adapt quickly to novel host environments with recombination representing an additional key source of genetic variation. Interestingly, recombination between genotypes of DWV has recently been shown to be a common occurrence in honeybees. A challenge has been to develop bioinformatics tools that can accurately reconstruct viral haplotypes – including recombinants – from heterogenous high-throughput sequence data.
The impact of blood-to-blood Varroa destructor transmission on virus evolution represents an important question in bee virus research. Due to the nature of the V. destructor life cycle, predictions can be made about the potential impact of the mite on virus virulence evolution. Specifically, the developing honeybee host pupa should remain alive until close to the completion of metamorphosis to provide sufficient time for successful mite reproduction, including offspring mating. For optimal transmission, any virus found in a mature and mated daughter mite will hold a significant selective advantage over a virus found in an immature or unmated daughter mite – placing a cost on virus virulence that impacts honeybee pupae before mites can mate. On the other side, viruses replicating too slowly and with delayed virulence effects will hold a selective handicap because fewer transmission units will be found in mated mites. I have hypothesized that the evolution of virus virulence shifted following the arrival of V. destructor, with viruses, including recombinants and/or specific viral genotypes being selected for a level of virulence in pupae (and likely also in adults) that maximises R0, which represents the basic reproductive number of the virus in the host population. R0 is defined by the number of subsequent infections caused by a single infection and it must be greater than 1 for an infection to spread in a population. It is enhanced by maximising the number of transmission units passed to uninfected susceptible vectors, and ultimately hosts (Figure 1).
Honeybee viruses are also shared with sympatric wild bees and viral prevalence and sequence data indicate frequent virus transmission between managed and wild bee species. In addition to infecting the western honeybee (Apis mellifera), DWV can infect other Asian honeybee species such as Apis ceranae. Outside of honeybees, DWV has been found widely in bumblebees, including solitary bees and wasps and there is evidence that it can actively replicate in several Bombus and solitary bee species. Whether the arrival of the V. destructor mite in A. mellifera has driven viral emergence in non-Apis bees is a target of ongoing research.
Our understanding of RNA viruses from edible insects is minimal at best, with studies largely focusing on model insect species and those associated with obvious signs of disease. This represents a considerable gap in understanding, given the growing role of insects as a source of food and feed, as well as the more general relevance of insects in agriculture and health. Illness due to entomophagy is rare but well documented, including fatal cases following the consumption of termites. Termites are eaten commonly in tropical Asia, Africa and South America, and are among the insects with the highest recorded fat content. There are many species of termites, with a wide range of diets and habitats centering around the consumption of wood and soil substrates. In this study, we report the results from a survey of more than 30 cockroach and termite transcriptomes, with the aim of understanding the diversity and evolution of RNA viruses as well as other potentially pathogenic organisms that are associated with this relevant but somewhat overlooked group of insects. We discuss our results in the context of the possible zoonotic risk posed by insects, as well as in the context of emerging viral and other disease threats that may face insects being reared at industrial scales.
Rock-inhabiting fungi are known to colonise air-exposed substrates like minerals, photovoltaic panels building facades and monuments, withstanding the various stresses these extreme habitats are known for. Here we show how both colonisation and stress resistance are linked to the properties of the fungal cell surface. By deleting genes involved in the synthesis of melanin and carotenoid pigments of the model rock-inhabiting fungus Knufia petricola via CRISPR-Cas and comparing the behaviour of the gene-deletion mutants with the wild type (WT), we studied the role of these genes in mineral colonisation and stress sensitivity. The extracellular polymeric substances (EPS) of biofilms of the WT and mutants were extracted, quantified and chemically characterised. We observed that the absence of melanin affected the quantity and composition of the produced EPS: melanin-deficient mutants synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, in mineral dissolution experiments, these mutants showed a lower ability to colonise the mineral olivine. We hypothesise that not melanin, but the pullulan-related linkages enabled the melanin-producing strains to attach more strongly to the mineral. Moreover, ICP-OES analysis of the aqueous mineral-derived solutes showed that biofilms of the K. petricola mutants which could attach were able to dissolve the olivine faster than those that could not. The same mutants were also characterised by their sensitivity to desiccation stress: only the mutant deficient in both melanin and carotenoid synthesis was more sensitive to desiccation compared to the WT, indicating that a combination of both pigments is critical to withstand desiccation. Overall, these results show the critical role of the cell surface in the specific capacities of rock-inhabiting fungi.
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. 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 of PRs 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.
Results suggest a reduction in immune gene repertoires in termites and possible complementary expression between termite castes. With comparative genomics we will investigate the evolution of gene families related to immunity, try to understand where reductions and expansions take place and relate these changes to shifts in sociality and ecology. The role of TEs in expansions and contractions of immune gene families will be investigated. For these analyses, we propose to generate high quality, highly contiguous genomes of species from different levels of sociality, covering all major termite families. With comparative transcriptomics we will investigate the expression of immune genes in different castes. Via network analyses we will identify pathways indicated in differential immunity between castes and between species of different sociality levels. We will investigate how these pathways have been rewired along the transitions to higher levels of sociality and how, intra-specifically, they change between castes.
Generation of Cryomyces antarcticus mutants to explore the importance of DHN melanin for survival
(2023)
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.
The exponential rise in the number of fungal genomes sequenced by next-generation sequencing techniques makes it necessary to increase efforts to correctly annotate and assign gene functions. There are two possibilities to explore a genome and its gene functions. The hypothesis-based method proves the function of already existing gene/allele candidates by targeted mutagenesis - so called reverse genetics. The basis of forward genetics approaches is the random mutagenesis of the genome, followed by screening of obtained mutants for the phenotype of interest, and identification of the mutated genes in the respective mutants. This strategy is hypothesis-generating, means it is necessary to verify the relationship between the detected mutations and the observed phenotype by targeted mutagenesis of the identified gene. We developed a toolbox for editing the genome of the rock inhabitant Knufia petricola [Eurotiomycetes, Chaetothyriales] that allows the study of the phenotypic characteristics of black fungi such as the regulation of pigment synthesis, general stress responses, oligotrophy, and the unusual modes of cell division by advanced reverse and forward genetics approaches. The toolbox includes the annotated genome sequence of strain A95, efficient strategies for CRISPR/Cas9-based genome editing and live-cell imaging using genetically encoded fluorescent proteins, as well as protocols for -omics approaches and for simulation of mineral weathering. A forward genetics approach using transposon mutagenesis is currently developed for identifying essential genes. The established protocols and knowledge gained from K. petricola form a starting point for making other fungi from extreme environments accessible to genetic manipulation.
Microcolonial black fungi, a polyphyletic group of ascomycetes, exhibit constitutive melanin formation, yeast-like growth and high stress tolerances. They dominate – often together with bacteria and algae in sub-aerial biofilms – a range of hostile environments including natural and man-made ones, from salterns to dishwashers, roofs and solar panels. Because of lacking genetic tools and the slow growth of most isolates, the genetic bases for these specific properties are largely unknown. The rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales) exhibits all characteristics of microcolonial black fungi and was selected as recipient for genetic engineering to study gene functions and genetic interactions. Different variants of green and red fluorescent proteins were successfully expressed indicating that fluorescence microscopy using genetically encoded fluorescent proteins and fluorescent dyes enables various cell biology approaches. Furthermore, genes of biosynthetic pathways (DHN melanin, carotenoids, uracil, adenine) were successfully mutated by applying traditional gene replacement and plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9 or silenced by RNA interference (RNAi). The availability of this advanced and efficient genetic toolbox and the annotated genome sequence of strain A95 makes K. petricola an excellent model for exploring the secrets of microcolonial black fungi.
In-vivo mutagenesis of the rock inhabitant Knufia petricola by a customized Ac/Ds transposon system
(2023)
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.
Social insects nesting in soil environments are in constant contact with entomopathogens and have evolved disease resistance mechanisms within a colony to prevent the occurrence and spread of infectious diseases. Among these mechanisms: mutual grooming reduces the cuticular load of pathogens, and burial of cadavers and cannibalism can prevent pathogens from replicating within the group. We explored how the rate and type of collective behavioural response is determined by stepwise infection dynamics operating at the level of the individual. Specifically, we infected the eastern subterranean termite Reticulitermes flavipes with different types of infectious particle and infection route of the entomopathogenic fungus Metarhizium anisopliae and recorded behavioural responses of nestmates to individuals at different times during the progression of infections. As expected, termites groomed conidia-exposed individuals significantly more than controls. Interestingly, grooming was significantly elevated after fungal germination than before, suggesting that pathogen growth cues act as strong stimulators of allogrooming. Conidia-exposed termites were cannibalized, but only after they became visibly ill. By contrast, termites did not groom blastospore-injected individuals more than controls at any time-point following infection. Instead, we found that blastospore-injected individuals were continually cannibalized at a low-level following injection with either viable or heat-killed blastospores, with a marked increase in cannibalism after termites injected with viable blastospores became visibly ill and were close to death. Together, these findings point to the importance of host condition as a cue for social hygienic behavior, and that the host itself appears to emit essential sickness cues that act as targets for its own sacrifice. This demonstrates that termites have independently evolved to both identify and destructively respond to sickness.
Black fungi also called black yeasts, rock-inhabiting fungi or microcolonial fungi are a group of Ascomycetes [Eurotiomycetes, Arthoniomycetes and Dothideomycetes] that exhibit high stress tolerance, yeast‑like or meristematic growth, and constitutive 1,8-dihydroxynaphthalene (DHN) melanin formation. They dominate a range of hostile natural and man‑made environments – from desert rocks and salterns to dishwashers, roofs, and solar panels. Due to their slow growth and the lack of sexual cycles and genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We consider the rock inhabitant K. petricola [Eurotiomycetes, Chaetothyriales] a suitable model for studying the phenotypic characteristics of black fungi. With K. petricola the regulation of pigment synthesis, general stress responses and the unusual modes of cell division can be dissected by advanced reverse and forward genetics approaches. The genome of K. petricola strain A95 was sequenced using a combination of short high quality Illumina reads and long PacBio reads. The final assembly consists of twelve contigs: five complete chromosomes and six contigs with one telomer each. Gene annotation supported by transcriptomics and proteomics data was manually curated. Recently, we developed a set of genetic tools to manipulate the genome for analyzing gene functions and studying the cell biology. This set includes CRISPR/Cas9-based genome editing and live-cell imaging using genetically encoded fluorescent proteins, as well as protocols for -omics approaches and for simulation of mineral weathering in the laboratory. Mutants defective in DHN melanogenesis, carotenogenesis or both processes are currently studied to elucidate the role of these protective pigments in tolerance of natural and man-made stresses, weathering of olivine, penetration of marble, and adhesion to surfaces. Further, the established protocols and knowledge gained from K. petricola form a starting point for making other extremotolerant black fungi accessible to genetic manipulation.
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.