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Black fungi belong to different classes of Ascomycota but evolved similar morpho-physiological adaptations such as yeast-like growth and constitutive melanin formation to colonize extreme competition-free environments. They are ubiquitously found on air-exposed surfaces, from ancient marble monuments to modern solar panels. The rock inhabitant Knufia petricola was chosen to become a model for these extremotolerant black fungi. Plasmid-based and ribonucleoprotein-based CRISPR/Cas9 techniques were introduced to precisely introduce one to multiple double strand breaks into the DNA to modify, replace or add sequences to the genome either using the available selection marker systems (hygR, natR, genR, baR, suR) or by marker-free approaches. Multiplexing is very efficient, allowing for four or more simultaneous genome editing events. The newly generated cloning vectors containing the Tet on construct for doxycycline-controlled gene expression, and the validated sites in the K. petricola genome for color-selectable (pks1, phs1, ade2) or neutral insertion (igr1 to 5) of expression constructs complete the reverse genetics toolbox. One or multiple endogenous or heterologous genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides. We thus can express genes from synthetic clusters in a non-pigmented background (Δpks1/Δphs1). The fact that K. petricola only produces few secondary metabolites (DHN melanin, carotenoids, mycosporines and a siderophore) and plant cell-wall degrading enzymes but is capable to take the burden of acetyl-CoA-consuming metabolism and protein secretion renders K. petricola a promising host for the expression of heterologous genes encoding high-end secondary metabolites and enzymes.
Any surface in the environment acts as hotspot for microbial attachment and activity. These biofilms represent the interface between humans and the environment.
While in the past biofilms were often seen as disturbance, we now start to understand the enormous potential of beneficial biofilms. They can be used in a broad range of applications and are sources for new microorganisms and traits. After all, biofilms represent a great example for a collaborative lifestyle.
Since nowadays people spend most of their time indoors, a healthy environment is essential. Volatile organic compounds (VOCs) emitted from furniture and building materials are reported to cause health complaints. Therefore, the usage of low emitting materials will improve the indoor air quality. Quantitative VOC emission testing is usually conducted in emission test chambers under specified controlled conditions as described in DIN 16000-9 and DIN EN 16516.
For reasons of quality control/quality assurance (QC/QA) and for a better comparability of test results from different laboratories, suitable emission reference materials (ERM) are needed. Here, it is important to have a homogenous material with known emission rates over a specific time. Different approaches can be found in literature, inter alia polymer films loaded with the target compound to be released again, or a lacquer material to which a VOC mixture is added. After curing of the lacquer, the material can be loaded into a test chamber. Drawback of those approaches are their relatively fast decreasing emission profiles. For QC/QA purposes according to the test standards, VOC sources with constant emission profiles are desirable.
The EU-funded research project MetrIAQ “Metrology for the determination of emissions of dangerous substances from building materials into indoor air” is working on a multi-component ERM with an envisaged instability of ≤ 10 % in the emission rate over at least 14 days.
Within a doctoral thesis porous materials are impregnated with VOCs. Supercritical CO2 is used as solvent. Thus, the impregnated material does not contain any solvent that may show a measurable amount of emission in the emission test chamber. Furthermore, CO2 has the benefits to have a good availability and low costs. For the selection of porous materials several properties like the pore size, the surface, and the interaction with the components in the atmosphere need to be considered. The impregnation method is optimised while the different porous materials are tested. For the selection of porous materials the pores need to be large enough for the VOC molecules, further influence of the pore size is tested.
Sunlight is an almost unavoidable environmental cue and plays a fundamental role in the biology of pro- and eukaryotic organisms. 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 organisms to accurately sense and respond to changes in light. The benefits of light are obvious for green organisms such as cyanobacteria, algae and plants which use light as an energy source (photosynthesis). Less apparent are other light-dependent processes such as light-driven DNA repair by photolyases (photoreactivation) or ion pumping by microbial opsins. Fungi that can share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess many proteins for absorbing UV/blue, green, red and far-red light, produce the black 1,8 dihydroxynaphthalene (DHN) melanin and orange-red carotenoids, and may live in multispecies biofilms. Here, we are addressing the question to which extent constitutive pigment formation (melanin and carotenoids) and responses mediated by the stress-activated mitogen-activated protein (MAP) kinase contribute to the observed light (UV-B) tolerance of K. petricola.
Bacterial adhesion on surfaces of medical, water and food applications may lead to infections, water or food spoilage and human illness. In comparison to traditional static and macro flow chamber assays for biofilm formation studies, microfluidic chips allow in situ monitoring of biofilm formation under various flow regimes, have better environment control and smaller sample requirements.
In this work, a novel microfluidic platform is developed to investigate biofilm adhesion under precisely controlled bacteria concentration, temperature, and flow conditions. This platform central unit is a single-inlet microfluidic flow cell with a 5 mm wide chamber designed and tested to achieve ultra-homogenous flow in the central area of chamber. Within this area, defined microstructures are integrated that will disturb the homogeneity of the flow, thus changing bacterial adhesion pattern.
Here we present the monitoring of bacterial biofilm formation in a microfluidic chip equipped with a microstructure known as micro-trap. This feature is based on a 3D bacteria trap designed by Di Giacomo et al. and successfully used to sequester motile bacteria.
At first, fluorescent particles similar in size to Escherichia coli (E. coli) are used to simulate bacteria flow inside the flow cell and at the micro-trap. The turbulences induced by the trap are analyzed by imaging and particle tracking velocimetry (PTV). Secondly, the model strain E. coli TG1, ideal and well described for biofilm studies, is used to analyze biofilm formation in the micro-trap. Therefore, a stable fluorescent strain E. coli TG1-MRE-Tn7-141 is constructed by using Tn7 transposon mutagenesis according to the method described by Schlechter et al. Sequestering of E. coli cells within the micro-trap was followed using epifluorescence microscopy.
The novel microfluidic platform shows great potential for assessment of bacterial adhesion under various flow regimes. The performance of structural feature with respect to the generation of turbulences that promote or reduce bacterial adhesion can be systematically examined.
The combination of flow analysis and fluorescent strain injection into the microfluidic chip shows that the micro-trap is useful for capturing bacteria at defined positions and to study how flow conditions, especially micro-turbulences, can affect biofilm formation. It represents a powerful and versatile tool for studying the relation between topography and bacteria adhesion.
Sunlight is an almost unavoidable environmental cue and plays a fundamental role in the biology of pro- and eukaryotic organisms. To cope with sunlight-associated stresses e.g., high temperatures, UV radiation, accumulation of reactive oxygen species, desiccation, and osmotic stress, it is important for organisms to accurately sense and respond to changes in light. The benefits of light are obvious for green organisms such as cyanobacteria, algae and plants which use light as an energy source (photosynthesis). Fungi that can share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Examples are the plant pathogen Botrytis cinerea, the gray mold fungus, and the rock inhabitant Knufia petricola, a microcolonial black fungus which forms multispecies biofilms with bacteria and algae.
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.
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.
Melanins are organic pigments produced by most fungi. These organisms either fix these pigments in their cell wall or secrete them into their extracellular environment to protect themselves against an array of physicochemical stresses (e.g., UV irradiation, desiccation, ...). Melanin can adsorb metals like Fe. How this affects fungal uptake of Fe and deterioration of Fe-containing minerals and metals is however less known. To study this, we use the model fungi Knufia petricola A95, a rock-inhabiting fungus known to deteriorate minerals and have melanised cell walls, and Amorphotheca resinae, able to contaminate fuel tanks, secrete melanin and corrode metals. In K. petricola, we have deleted genes involved in melanin production and Fe uptake using CRISPR/Cas. Through comparison of the geochemical signatures of these gene deletion mutants with those of the wild type (WT), we explore the specific mineral/metal deterioration mechanisms of melanised fungi.
Fe isotope signatures of the biomass of melanin- and Fe uptake-deficient mutants of K. petricola revealed that Fe adsorbed either directly onto melanin or after being reduced by Fe reductases. Importantly, once adsorbed to melanin, Fe could not be mobilised and taken up into the cell: both the WT and its melanin-deficient mutant, previously grown at Fe replete conditions, showed similar growth at Fe deficient conditions.
Olivine dissolution experiments revealed that Fe oxidation inhibits dissolution. K. petricola was able to enhance dissolution when this inhibition is strongest (at pH 6) and prevented dissolution when this inhibition is weakest (at pH 4). The fungus therefore dissolves olivine by interacting with the oxidised Fe at the olivine surface. However, Fe uptake did not seem to be involved: mutants deficient in various Fe uptake mechanism dissolved olivine at the same rate as the WT. This indicates that Fe adsorption onto melanin might play a key role. This is also shown by K. petricola’s ability to enhance olivine dissolution even further if secreting a melanin precursor and A. resinae’s corrosion of carbon steel whilst secreting melanin. Combined, our results imply that the Fe adsorbed to melanin cannot be taken up but enables fungi to deteriorate Fe-containing substrates at a higher rate.
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.