Understanding Fungi in Glacial and Hypersaline Environments.

Hypersaline waters and glacial ice are inhospitable environments that have low water activity and high concentrations of osmolytes. They are inhabited by diverse microbial communities, of which extremotolerant and extremophilic fungi are essential components. Some fungi are specialized in only one of these two environments and can thrive in conditions that are lethal to most other life-forms. Others are generalists, highly adaptable species that occur in both environments and tolerate a wide range of extremes. Both groups efficiently balance cellular osmotic pressure and ion concentration, stabilize cell membranes, remodel cell walls, and neutralize intracellular oxidative stress. Some species use unusual reproductive strategies. Further investigation of these adaptations with new methods and carefully designed experiments under ecologically relevant conditions will help predict the role of fungi in hypersaline and glacial environments affected by climate change, decipher their stress resistance mechanisms and exploit their biotechnological potential.

Experimental evolution of extremotolerant and extremophilic fungi under osmotic stress.

Experimental evolution was carried out to investigate the adaptive responses of extremotolerant fungi to a stressful environment. For 12 cultivation cycles, the halotolerant black yeasts Aureobasidium pullulans and Aureobasidium subglaciale were grown at high NaCl or glycerol concentrations, and the halophilic basidiomycete Wallemia ichthyophaga was grown close to its lower NaCl growth limit. All evolved Aureobasidium spp. accelerated their growth at low water activity. Whole genomes of the evolved strains were sequenced. No aneuploidies were detected in any of the genomes, contrary to previous studies on experimental evolution at high salinity with other species. However, several hundred single-nucleotide polymorphisms were identified compared with the genomes of the progenitor strains. Two functional groups of genes were overrepresented among the genes presumably affected by single-nucleotide polymorphisms: voltage-gated potassium channels in A. pullulans at high NaCl concentration, and hydrophobins in W. ichthyophaga at low NaCl concentration. Both groups of genes were previously associated with adaptation to high salinity. Finally, most evolved Aureobasidium spp. strains were found to have increased intracellular and decreased extracellular glycerol concentrations at high salinity, suggesting that the strains have optimised their management of glycerol, their most important compatible solute. Experimental evolution therefore not only confirmed the role of potassium transport, glycerol management, and cell wall in survival at low water activity, but also demonstrated that fungi from extreme environments can further improve their growth rates under constant extreme conditions in a relatively short time and without large scale genomic rearrangements.

Black yeasts in hypersaline conditions.

Extremotolerant and extremophilic fungi are an important part of microbial communities that thrive in extreme environments. Among them, the black yeasts are particularly adaptable. They use their melanized cell walls and versatile morphology, as well as a complex set of molecular adaptations, to survive in conditions that are lethal to most other species. In contrast to extremophilic bacteria and archaea, these fungi are typically extremotolerant rather than extremophilic and exhibit an unusually wide ecological amplitude. Some extremely halotolerant black yeasts can grow in near-saturated NaCl solutions, but can also grow on normal mycological media. They adapt to the low water activity caused by high salt concentrations by sensing their environment, balancing osmotic pressure by accumulating compatible solutes, removing toxic salt ions from the cell using membrane transporters, altering membrane composition and remodelling the highly melanized cell wall. As protection against extreme conditions, halotolerant black yeasts also develop different morphologies, from yeast-like to meristematic. Genomic studies of black yeasts have revealed a variety of reproductive strategies, from clonality to intense recombination and the formation of stable hybrids. Although a comprehensive understanding of the ecological role and molecular adaptations of halotolerant black yeasts remains elusive and the application of many experimental methods is challenging due to their slow growth and recalcitrant cell walls, much progress has been made in deciphering their halotolerance. Advances in molecular tools and genomics are once again accelerating the research of black yeasts, promising further insights into their survival strategies and the molecular basis of their adaptations. KEY POINTS: • Black yeasts show remarkable adaptability to environmental stress • Black yeasts are part of microbial communities in hypersaline environments • Halotolerant black yeasts utilise various molecular and morphological adaptations.

A method for targeting a specified segment of DNA to a bacterial microorganelle.

Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.

Identification of novel genes involved in neutral lipid storage by quantitative trait loci analysis of Saccharomyces cerevisiae.

BACKGROUND: The accumulation of intracellular fat depots is a polygenic trait. Therefore, the extent of lipid storage in the individuals of a species covers a broad range and is determined by many genetic factors. Quantitative trait loci analysis can be used to identify those genetic differences between two strains of the same species that are responsible for the differences in a given phenotype. We used this method and complementary approaches to identify genes in the yeast Saccharomyces cerevisiae that are involved in neutral lipid storage. RESULTS: We selected two yeast strains, the laboratory strain BY4741 and the wine yeast AWRI1631, with a more than two-fold difference in neutral lipid content. After crossing, sporulation and germination, we used fluorescence activated cell sorting to isolate a subpopulation of cells with the highest neutral lipid content from the pool of segregants. Whole genome sequencing of this subpopulation and of the unsorted pool of segregants implicated several loci that are involved in lipid accumulation. Three of the identified genes, PIG1, PHO23 and RML2, were investigated in more detail. Deletions of these genes and the exchange of the alleles between the two parental strains confirmed that the encoded proteins contribute to neutral lipid storage in S. cerevisiae and that PIG1, PHO23 and RML2 are the major causative genes. Backcrossing of one of the segregants with the parental strains for seven generations revealed additional regions in the genomes of both strains with potential causative genes for the high lipid accumulation phenotype. CONCLUSIONS: We identified several genes that contribute to the phenotype of lipid accumulation in an allele-specific manner. Surprisingly, no allelic variations of genes with known functions in lipid metabolism were found, indicating that the level of storage lipid accumulation is determined by many cellular processes that are not directly related to lipid metabolism.

Greenland and Svalbard glaciers host unknown basidiomycetes: the yeast Camptobasidium arcticum sp. nov. and the dimorphic Psychromyces glacialis gen. and sp. nov.

Sampling campaigns in Greenland and Svalbard were executed to explore fungal diversity in cold habitats. Three very abundant groups of strains were discovered, consisting either of recently described or of yet-undescribed psychrophilic and oligotrophic yeasts and dimorphic fungi, accounting for around 50 % of the total cultivable diversity of basidiomycetes in our studies. The occurrence of these taxa has also been demonstrated by culture-independent methods. Based on phylogenetic analyses of ribosomal gene cluster sequences (D1/D2 domains of 28S (LSU), 18S (SSU), ITS with 5.8S rDNA) and sequences of protein-coding genes for elongation factor one alpha (TEF), cytochrome b (CYTB) and two subunits of the RNA polymerase II (RPB1 and RPB2) obtained from pure cultures, the isolated taxa presented in this study belong to Basidiomycota, subphylum Pucciniomycotina, class Microbotryomycetes, family Camptobasidiaceae. The dataset of the sequences supported the recognition of three species: Camptobasidium gelus, Camptobasidium arcticum sp. nov. (ex-type strain EXF-12713) and Psychromyces glacialis gen. and sp. nov. (ex-type strain EXF-13111). Camptobasidium gelus was found in the Svalbard and Greenland samples, while representatives of the here proposed new species, C. arcticum, were found only in the Greenland Ice Sheet. Psychromyces gen. nov. was erected for the dimorphic/filamentous isolates found in Svalbard and Greenland glacial environments. The taxon, for which the invalid name 'Rhodotorula svalbardensis' has been used, belongs to this genus. Based on ribosomal genes, Camptobasidium arcticum and Psychromyces glacialis are related, phylogenetically most closely related to the genera Glaciozyma and Cryolevonia. Seven genes phylogeny restricted to taxa with available sequences, supported the placement of Psychromyces to Camptobasidiaceae.

Microorganisms populating the water-related indoor biome.

Modernisation of our households created novel opportunities for microbial growth and thus changed the array of microorganisms we come in contact with. While many studies have investigated microorganisms in the air and dust, tap water, another major input of microbial propagules, has received far less attention. The quality of drinking water in developed world is strictly regulated to prevent immediate danger to human health. However, fungi, algae, protists and bacteria of less immediate concern are usually not screened for. These organisms can thus use water as a vector of transmission into the households, especially if they are resistant to various water treatment procedures. Good tolerance of unfavourable abiotic conditions is also important for survival once microbes enter the household. Limitation of water availability, high or low temperatures, application of antimicrobial chemicals and other measures are taken to prevent indoor microbial overgrowth. These conditions, together with a large number of novel chemicals in our homes, shape the diversity and abundance of indoor microbiota through constant selection of the most resilient species, resulting in a substantial overlap in diversity of indoor and natural extreme environments. At least in fungi, extremotolerance has been linked to human pathogenicity, explaining why many species found in novel indoor habitats (such as dishwasher) are notable opportunistic pathogens. As a result, microorganisms that often enter our households with water and are then enriched in novel indoor habitats might have a hitherto underestimated impact on the well-being of the increasingly indoor-bound human population. KEY POINTS: Domestic environment harbours a large diversity of microorganisms. Microbiota of water-related indoor habitats mainly originates from tap water. Bathrooms, kitchens and household appliances select for polyextremotolerant species. Many household-related microorganisms are human opportunistic pathogens.

Across the tree of life, radiation resistance is governed by antioxidant Mn2+, gauged by paramagnetic resonance.

Despite concerted functional genomic efforts to understand the complex phenotype of ionizing radiation (IR) resistance, a genome sequence cannot predict whether a cell is IR-resistant or not. Instead, we report that absorption-display electron paramagnetic resonance (EPR) spectroscopy of nonirradiated cells is highly diagnostic of IR survival and repair efficiency of DNA double-strand breaks (DSBs) caused by exposure to gamma radiation across archaea, bacteria, and eukaryotes, including fungi and human cells. IR-resistant cells, which are efficient at DSB repair, contain a high cellular content of manganous ions (Mn2+) in high-symmetry (H) antioxidant complexes with small metabolites (e.g., orthophosphate, peptides), which exhibit narrow EPR signals (small zero-field splitting). In contrast, Mn2+ ions in IR-sensitive cells, which are inefficient at DSB repair, exist largely as low-symmetry (L) complexes with substantially broadened spectra seen with enzymes and strongly chelating ligands. The fraction of cellular Mn2+ present as H-complexes (H-Mn2+), as measured by EPR of live, nonirradiated Mn-replete cells, is now the strongest known gauge of biological IR resistance between and within organisms representing all three domains of life: Antioxidant H-Mn2+ complexes, not antioxidant enzymes (e.g., Mn superoxide dismutase), govern IR survival. As the pool of intracellular metabolites needed to form H-Mn2+ complexes depends on the nutritional status of the cell, we conclude that IR resistance is predominantly a metabolic phenomenon. In a cross-kingdom analysis, the vast differences in taxonomic classification, genome size, and radioresistance between cell types studied here support that IR resistance is not controlled by the repertoire of DNA repair and antioxidant enzymes.

Fifty Aureobasidium pullulans genomes reveal a recombining polyextremotolerant generalist.

The black yeast Aureobasidium pullulans is a textbook example of a generalistic and ubiquitous fungus thriving in a wide variety of environments. To investigate whether A. pullulans is a true generalist, or alternatively, whether part of its versatility can be attributed to intraspecific specialization masked by cryptic diversification undetectable by traditional phylogenetic analyses, we sequenced and analysed the genomes of 50 strains of A. pullulans from different habitats and geographic locations. No population structure was observed in the sequenced strains. Decay of linkage disequilibrium over shorter physical distances (<100 bp) than in many sexually reproducing fungi indicates a high level of recombination in the species. A homothallic mating locus was found in all of the sequenced genomes. Aureobasidium pullulans appears to have a homogeneous population genetics structure, which is best explained by good dispersal and high levels of recombination. This means that A. pullulans is a true generalist that can inhabit different habitats without substantial specialization to any of these habitats at the genomic level. Furthermore, in the future, the high level of A. pullulans recombination can be exploited for the identification of genomic loci that are involved in the many biotechnologically useful traits of this black yeast.

Genome mining for peptidases in heat-tolerant and mesophilic fungi and putative adaptations for thermostability.

BACKGROUND: Peptidases (EC 3.4) consist of a large group of hydrolytic enzymes that catalyze the hydrolysis of proteins accounting for approximately 65% of the total worldwide enzyme production. Peptidases from thermophilic fungi have adaptations to high temperature that makes them adequate for biotechnological application. In the present study, we profiled the genomes of heat-tolerant fungi and phylogenetically related mesophilic species for genes encoding for peptidases and their putative adaptations for thermostability. RESULTS: We generated an extensive catalogue of these enzymes ranging from 241 to 820 peptidase genes in the genomes of 23 fungi. Thermophilic species presented the smallest number of peptidases encoding genes in relation to mesophilic species, and the peptidases families with a greater number of genes were the most affected. We observed differences in peptidases in thermophilic species in comparison to mesophilic counterparts, at (i) the genome level: a great reduction in the number of peptidases encoding genes that harbored a higher number of copies; (ii) in the primary protein structure: shifts in proportion of single or groups of amino acids; and (iii) in the three-dimensional structure: reduction in the number of internal cavities. Similar results were reported for extremely thermophilic proteins, but here we show for the first time that several changes also occurred on the moderate thermophilic enzymes of fungi. In regards to the amino acids composition, peptidases from thermophilic species in relation to the mesophilic ones, contained a larger proportion of Ala, Glu, Gly, Pro, Arg and Val residues and a lower number of Cys, His, Ile, Lys, Met, Asn, Gln, Ser, Thr and Trp residues (P < 0.05). Moreover, we observed an increase in the proportion of hydrophobic and charged amino acids and a decrease in polar amino acids. CONCLUSIONS: Although thermophilic fungi present less genes encoding for peptidases, these have adaptations that could play a role in thermal resistance from genome to protein structure level.

Genomic evidence for intraspecific hybridization in a clonal and extremely halotolerant yeast.

BACKGROUND: The black yeast Hortaea werneckii (Dothideomycetes, Ascomycota) is one of the most extremely halotolerant fungi, capable of growth at NaCl concentrations close to saturation. Although dothideomycetous fungi are typically haploid, the reference H. werneckii strain has a diploid genome consisting of two subgenomes with a high level of heterozygosity. RESULTS: In order to explain the origin of the H. werneckii diploid genome we here report the genome sequencing of eleven strains isolated from different habitats and geographic locations. Comparison of nine diploid and two haploid strains showed that the reference genome was likely formed by hybridization between two haploids and not by endoreduplication as suggested previously. Results also support additional hybridization events in the evolutionary history of investigated strains, however exchange of genetic material in the species otherwise appears to be rare. Possible links between such unusual reproduction and the extremotolerance of H. werneckii remain to be investigated. CONCLUSIONS: H. werneckii appears to be able to form persistent haploid as well as diploid strains, is capable of occasional hybridization between relatively heterozygous haploids, but is otherwise limited to clonal reproduction. The reported data and the first identification of haploid H. werneckii strains establish this species as a good model for studying the effects of ploidy and hybridization in an extremotolerant system unperturbed by frequent genetic recombination.

Transport Systems in Halophilic Fungi.

Fungi that tolerate very high environmental NaCl concentrations are good model systems to study mechanisms that enable them to endure osmotic and salinity stress. The whole genome sequences of six such fungal species have been analysed: Hortaea werneckii, Wallemia ichthyophaga and four Aureobasidium spp.: A. pullulans, A. subglaciale, A. melanogenum and A. namibiae. These fungi show different levels of halotolerance, with the presence of numerous membrane transport systems uncovered here that are believed to maintain physiological intracellular concentrations of alkali metal cations. Despite some differences, the intracellular cation contents of H. werneckii, A. pullulans and W. ichthyophaga remain low even under extreme extracellular salinities, which suggests that these species have efficient cation transport systems. We speculate that cation transporters prevent intracellular accumulation of Na(+), and thus avoid the toxic effects that such Na(+) accumulation would have, while also maintaining the high K(+)/Na(+) ratio that is required for the full functioning of the cell - another crucial task in high-Na(+) environments. This chapter primarily summarises the cation transport systems of these selected fungi, and it also describes other membrane transporters that might be involved in their mechanisms of halotolerance.

Genome sequencing of four Aureobasidium pullulans varieties: biotechnological potential, stress tolerance, and description of new species.

BACKGROUND: Aureobasidium pullulans is a black-yeast-like fungus used for production of the polysaccharide pullulan and the antimycotic aureobasidin A, and as a biocontrol agent in agriculture. It can cause opportunistic human infections, and it inhabits various extreme environments. To promote the understanding of these traits, we performed de-novo genome sequencing of the four varieties of A. pullulans. RESULTS: The 25.43-29.62 Mb genomes of these four varieties of A. pullulans encode between 10266 and 11866 predicted proteins. Their genomes encode most of the enzyme families involved in degradation of plant material and many sugar transporters, and they have genes possibly associated with degradation of plastic and aromatic compounds. Proteins believed to be involved in the synthesis of pullulan and siderophores, but not of aureobasidin A, are predicted. Putative stress-tolerance genes include several aquaporins and aquaglyceroporins, large numbers of alkali-metal cation transporters, genes for the synthesis of compatible solutes and melanin, all of the components of the high-osmolarity glycerol pathway, and bacteriorhodopsin-like proteins. All of these genomes contain a homothallic mating-type locus. CONCLUSIONS: The differences between these four varieties of A. pullulans are large enough to justify their redefinition as separate species: A. pullulans, A. melanogenum, A. subglaciale and A. namibiae. The redundancy observed in several gene families can be linked to the nutritional versatility of these species and their particular stress tolerance. The availability of the genome sequences of the four Aureobasidium species should improve their biotechnological exploitation and promote our understanding of their stress-tolerance mechanisms, diverse lifestyles, and pathogenic potential.

Degradation Potential of Xerophilic and Xerotolerant Fungi Contaminating Historic Canvas Paintings.

Fungi are important contaminants of historic canvas paintings worldwide. They can grow on both sides of the canvas and decompose various components of the paintings. They excrete pigments and acids that change the visual appearance of the paintings and weaken their structure, leading to flaking and cracking. With the aim of recognizing the most dangerous fungal species to the integrity and stability of paintings, we studied 55 recently isolated and identified strains from historic paintings or depositories, including 46 species from 16 genera. The fungi were categorized as xero/halotolerant or xero/halophilic based on their preference for solutes (glycerol or NaCl) that lower the water activity (aw) of the medium. Accordingly, the aw value of all further test media had to be adjusted to allow the growth of xero/halophilic species. The isolates were tested for growth at 15, 24 °C and 37 °C. The biodeterioration potential of the fungi was evaluated by screening their acidification properties, their ability to excrete pigments and their enzymatic activities, which were selected based on the available nutrients in paintings on canvas. A DNase test was performed to determine whether the selected fungi could utilize DNA of dead microbial cells that may be covering surfaces of the painting. The sequestration of Fe, which is made available through the production of siderophores, was also tested. The ability to degrade aromatic and aliphatic substrates was investigated to consider the potential degradation of synthetic restoration materials. Xerotolerant and moderately xerophilic species showed a broader spectrum of enzymatic activities than obligate xerophilic species: urease, ß-glucosidase, and esterase predominated, while obligate xerophiles mostly exhibited ß-glucosidase, DNase, and urease activity. Xerotolerant and moderately xerophilic species with the highest degradation potential belong to the genus Penicillium, while Aspergillus penicillioides and A. salinicola represent obligately xerophilic species with the most diverse degradation potential in low aw environments.

A screening method for plastic-degrading fungi.

The growing amount of plastic waste requires new ways of disposal or recycling. Research into the biodegradation of recalcitrant plastic polymers is gathering pace. Despite some progress, these efforts have not yet led to technologically and economically viable applications. In this study, we show that respirometric screening of environmental fungal isolates in combination with scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy can be used to identify new strains with the potential for the degradation of plastic polymers. We screened 146 fungal strains, 71 isolated from car repair shops, an environment rich in long-chain hydrocarbons, and 75 isolated from hypersaline water capable of growing at high concentrations of NaCl. When grown in a minimal medium with no carbon source, some strains produced significantly more CO2 when a pure plastic polymer was added to the medium, some only at high salinity. A selection of these strains was shown by FTIR and Raman spectroscopy to alter the properties of plastic polymers: Cladosporium sp. EXF-13502 on polyamide, Rhodotorula dairenensis EXF-13500 on polypropylene, Rhodotorula sp. EXF-10630 on low-density polyethylene and Wickerhamomyces anomalus EXF-6848 on polyethylene terephthalate. Respirometry in combination with specific spectroscopic methods is an efficient method for screening microorganisms capable of at least partial plastic degradation and can be used to expand the repertoire of potential plastic degraders. This is of particular importance as our results also show that individual strains are only active against certain polymers and under certain conditions. Therefore, efficient biodegradation of plastics is likely to depend on a collection of specialized microorganisms rather than a single universal plastic degrader.

High-throughput screening of non-conventional yeasts for conversion of organic waste to microbial oils via carboxylate platform.

Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse yeasts for microbial oil production by utilizing short-chain fatty acids (SCFAs) that can be produced from organic waste and focuses on identifying strains with the best SCFA utilisation, tolerance and lipid production. A collection of 1434 yeast strains was cultivated with SCFAs as the sole carbon source. Eleven strains emerged as candidates with promising growth rates and high lipid accumulation. Subsequent fermentation experiments in liquid SCFA-rich media, which focused on optimizing lipid accumulation by adjusting the carbon to nitrogen (C/N) ratio, showed an increase in lipid content at a C/N ratio of 200:1, but with a concurrent reduction in biomass. Two strains were characterized by their superior ability to produce lipids compared to the reference strain Yarrowia lipolytica CECT124: Y. lipolytica EXF-17398 and Pichia manshurica EXF-7849. Characterization of these two strains indicated that they exhibit a biotechnologically relevant balance between maximizing lipid yield and maintaining growth at high SCFA concentrations. These results emphasize the potential of using SCFAs as a sustainable feedstock for oleochemical production, offering a dual benefit of waste valorisation and microbial oil production.

Genome and transcriptome sequencing of the halophilic fungus Wallemia ichthyophaga: haloadaptations present and absent.

BACKGROUND: The basidomycete Wallemia ichthyophaga from the phylogenetically distinct class Wallemiomycetes is the most halophilic fungus known to date. It requires at least 10% NaCl and thrives in saturated salt solution. To investigate the genomic basis of this exceptional phenotype, we obtained a de-novo genome sequence of the species type-strain and analysed its transcriptomic response to conditions close to the limits of its lower and upper salinity range. RESULTS: The unusually compact genome is 9.6 Mb large and contains 1.67% repetitive sequences. Only 4884 predicted protein coding genes cover almost three quarters of the sequence. Of 639 differentially expressed genes, two thirds are more expressed at lower salinity. Phylogenomic analysis based on the largest dataset used to date (whole proteomes) positions Wallemiomycetes as a 250-million-year-old sister group of Agaricomycotina. Contrary to the closely related species Wallemia sebi, W. ichthyophaga appears to have lost the ability for sexual reproduction. Several protein families are significantly expanded or contracted in the genome. Among these, there are the P-type ATPase cation transporters, but not the sodium/ hydrogen exchanger family. Transcription of all but three cation transporters is not salt dependent. The analysis also reveals a significant enrichment in hydrophobins, which are cell-wall proteins with multiple cellular functions. Half of these are differentially expressed, and most contain an unusually large number of acidic amino acids. This discovery is of particular interest due to the numerous applications of hydrophobines from other fungi in industry, pharmaceutics and medicine. CONCLUSIONS: W. ichthyophaga is an extremophilic specialist that shows only low levels of adaptability and genetic recombination. This is reflected in the characteristics of its genome and its transcriptomic response to salt. No unusual traits were observed in common salt-tolerance mechanisms, such as transport of inorganic ions or synthesis of compatible solutes. Instead, various data indicate a role of the cell wall of W. ichthyophaga in its response to salt. Availability of the genomic sequence is expected to facilitate further research into this unique species, and shed more light on adaptations that allow it to thrive in conditions lethal to most other eukaryotes.

Extremophilic and extremotolerant fungi.

There are few places on Earth that are truly aseptic. Even environments that we may consider 'extreme', such as glaciers, deserts, or hypersaline bodies of water (Figure 1), can harbour life. The organisms that thrive in such environments - mostly microbes - are often referred to as 'extremophiles'. However, what constitutes extreme is in the eye of the beholder. Extremophilic organisms are so adapted to their environment that they perceive extreme conditions as optimal for their growth and can sometimes even be stressed by what we perceive as moderate. Stress is therefore not an optimal criterion for defining what is extreme. Instead, extreme conditions can be seen as those in which the majority of species cannot grow or even survive.

Degradation of polypropylene by fungi Coniochaeta hoffmannii and Pleurostoma richardsiae.

The urgent need for better disposal and recycling of plastics has motivated a search for microbes with the ability to degrade synthetic polymers. While microbes capable of metabolizing polyurethane and polyethylene terephthalate have been discovered and even leveraged in enzymatic recycling approaches, microbial degradation of additive-free polypropylene (PP) remains elusive. Here we report the isolation and characterization of two fungal strains with the potential to degrade pure PP. Twenty-seven fungal strains, many isolated from hydrocarbon contaminated sites, were screened for degradation of commercially used textile plastic. Of the candidate strains, two identified as Coniochaeta hoffmannii and Pleurostoma richardsiae were found to colonize the plastic fibers using scanning electron microscopy (SEM). Further experiments probing degradation of pure PP films were performed using C. hoffmannii and P. richardsiae and analyzed using SEM, Raman spectroscopy and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). The results showed that the selected fungi were active against pure PP, with distinct differences in the bonds targeted and the degree to which each was altered. Whole genome and transcriptome sequencing was conducted for both strains and the abundance of carbohydrate active enzymes, GC content, and codon usage bias were analyzed in predicted proteomes for each. Enzymatic assays were conducted to assess each strain's ability to degrade naturally occurring compounds as well as synthetic polymers. These investigations revealed potential adaptations to hydrocarbon-rich environments and provide a foundation for further investigation of PP degrading activity in C. hoffmannii and P. richardsiae.

Xerophilic fungi contaminating historically valuable easel paintings from Slovenia.

Historically valuable canvas paintings are often exposed to conditions enabling microbial deterioration. Painting materials, mainly of organic origin, in combination with high humidity and other environmental conditions, favor microbial metabolism and growth. These preconditions are often present during exhibitions or storage in old buildings, such as churches and castles, and also in museum storage depositories. The accumulated dust serves as an inoculum for both indoor and outdoor fungi. In our study, we present the results on cultivable fungi isolated from 24 canvas paintings, mainly exhibited in Slovenian sacral buildings, dating from the 16th to 21st centuries. Fungi were isolated from the front and back of damaged and undamaged surfaces of the paintings using culture media with high- and low-water activity. A total of 465 isolates were identified using current taxonomic DNA markers and assigned to 37 genera and 98 species. The most abundant genus was Aspergillus, represented by 32 species, of which 9 xerophilic species are for the first time mentioned in contaminated paintings. In addition to the most abundant xerophilic A. vitricola, A. destruens, A. tardicrescens, and A. magnivesiculatus, xerophilic Wallemia muriae and W. canadensis, xerotolerant Penicillium chrysogenum, P. brevicompactum, P. corylophilum, and xerotolerant Cladosporium species were most frequent. When machine learning methods were used to predict the relationship between fungal contamination, damage to the painting, and the type of material present, proteins were identified as one of the most important factors and cracked paint was identified as a hotspot for fungal growth. Aspergillus species colonize paintings regardless of materials, while Wallemia spp. can be associated with animal fat. Culture media with low-water activity are suggested in such inventories to isolate and obtain an overview of fungi that are actively contaminating paintings stored indoors at low relative humidity.