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.

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.

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.

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.

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.

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.

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.

Isolation and characterization of extracellular vesicles from biotechnologically important fungus Aureobasidium pullulans.

Extracellular vesicles (EVs) are increasingly recognized as an important mechanism for cell-cell interactions. Their role in fungi is still poorly understood and they have been isolated from only a handful of species. Here, we isolated and characterized EVs from Aureobasidium pullulans, a biotechnologically important black yeast-like fungus that is increasingly used for biocontrol of phytopathogenic fungi and bacteria. After optimization of the isolation protocol, characterization of EVs from A. pullulans by transmission electron microscopy (TEM) revealed a typical cup-shaped morphology and different subpopulations of EVs. These results were confirmed by nanoparticle tracking analysis (NTA), which revealed that A. pullulans produced 6.1 × 108 nanoparticles per milliliter of culture medium. Proteomic analysis of EVs detected 642 proteins. A small fraction of them had signal peptides for secretion and transmembrane domains. Proteins characteristic of different synthesis pathways were found, suggesting that EVs are synthesized by multiple pathways in A. pullulans. Enrichment analysis using Gene Ontology showed that most of the proteins found in the EVs were associated with primary metabolism. When sequencing the small RNA fraction of A. pullulans EVs, we found two hypothetical novel mil-RNAs. Finally, we tested the biocontrol potential of EVs from A. pullulans. The EVs did not inhibit the germination of spores of three important phytopathogenic fungi-Botrytis cinerea, Colletotrichum acutatum, and Penicillium expansum. However, exposure of grown cultures of C. acutatum and P. expansum to A. pullulans EVs resulted in visible changes in morphology of colonies. These preliminary results suggest that EVs may be part of the antagonistic activity of A. pullulans, which is so far only partially understood. Thus, the first isolation and characterization of EVs from A. pullulans provides a starting point for further studies of EVs in the biotechnologically important traits of the biocontrol black fungus A. pullulans in particular and in the biological role of fungal EVs in general.

Clonality, inbreeding, and hybridization in two extremotolerant black yeasts.

BACKGROUND: The great diversity of lifestyles and survival strategies observed in fungi is reflected in the many ways in which they reproduce and recombine. Although a complete absence of recombination is rare, it has been reported for some species, among them 2 extremotolerant black yeasts from Dothideomycetes: Hortaea werneckii and Aureobasidium melanogenum. Therefore, the presence of diploid strains in these species cannot be explained as the product of conventional sexual reproduction. RESULTS: Genome sequencing revealed that the ratio of diploid to haploid strains in both H. werneckii and A. melanogenum is about 2:1. Linkage disequilibrium between pairs of polymorphic loci and a high degree of concordance between the phylogenies of different genomic regions confirmed that both species are clonal. Heterozygosity of diploid strains is high, with several hybridizing genome pairs reaching the intergenomic distances typically seen between different fungal species. The origin of diploid strains collected worldwide can be traced to a handful of hybridization events that produced diploids, which were stable over long periods of time and distributed over large geographic areas. CONCLUSIONS: Our results, based on the genomes of over 100 strains of 2 black yeasts, show that although they are clonal, they occasionally form stable and highly heterozygous diploid intraspecific hybrids. The mechanism of these apparently rare hybridization events, which are not followed by meiosis or haploidization, remains unknown. Both extremotolerant yeasts, H. werneckii and even more so A. melanogenum, a close relative of the intensely recombining and biotechnologically relevant Aureobasidium pullulans, provide an attractive model for studying the role of clonality and ploidy in extremotolerant fungi.

Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return.

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.

Coevolution of the Ess1-CTD axis in polar fungi suggests a role for phase separation in cold tolerance.

Most of the world's biodiversity lives in cold (-2° to 4°C) and hypersaline environments. To understand how cells adapt to such conditions, we isolated two key components of the transcription machinery from fungal species that live in extreme polar environments: the Ess1 prolyl isomerase and its target, the carboxy-terminal domain (CTD) of RNA polymerase II. Polar Ess1 enzymes are conserved and functional in the model yeast, Saccharomyces cerevisiae. By contrast, polar CTDs diverge from the consensus (YSPTSPS)26 and are not fully functional in S. cerevisiae. These CTDs retain the critical Ess1 Ser-Pro target motifs, but substitutions at Y1, T4, and S7 profoundly affected their ability to undergo phase separation in vitro and localize in vivo. We propose that environmentally tuned phase separation by the CTD and other intrinsically disordered regions plays an adaptive role in cold tolerance by concentrating enzymes and substrates to overcome energetic barriers to metabolic activity.

Cultivable Skin Mycobiota of Healthy and Diseased Blind Cave Salamander (Proteus anguinus).

Proteus anguinus is a neotenic cave salamander, endemic to the Dinaric Karst and a symbol of world natural heritage. It is classified as "vulnerable" by the International Union for Conservation of Nature (IUCN) and is one of the EU priority species in need of strict protection. Due to inaccessibility of their natural underground habitat, scientific studies of the olm have been conducted mainly in captivity, where the amphibians are particularly susceptible to opportunistic microbial infections. In this report, we focused on the diversity of cultivable commensal fungi isolated from the skin of asymptomatic and symptomatic animals obtained from nature (20 specimens) and captivity (22 specimens), as well as from underground water of two karstic caves by direct water filtration and by exposure of keratin-based microbial baits and subsequent isolation from them. In total 244 fungal isolates were recovered from the animals and additional 153 isolates were obtained from water samples. Together, these isolates represented 87 genera and 166 species. Symptomatic animals were colonized by a variety of fungal species, most of them represented by a single isolate, including genera known for their involvement in chromomycosis, phaeohyphomycosis and zygomycosis in amphibians: Acremonium, Aspergillus, Cladosporium, Exophiala, Fusarium, Mucor, Ochroconis, Phialophora and Penicillium. One symptomatic specimen sampled from nature was infected by the oomycete Saprolegnia parasitica, the known causative agent of saprolegniosis. This is the first comprehensive report on cultivable skin mycobiome of this unique amphibian in nature and in captivity, with an emphasis on potentially pathogenic fungi and oomycetes.

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.

From Glaciers to Refrigerators: the Population Genomics and Biocontrol Potential of the Black Yeast Aureobasidium subglaciale.

Apples are affected by numerous fungi known as storage rots, which cause significant losses before and after harvest. Concerns about increasing antimicrobial resistance, bans on various fungicides, and changing consumer preferences are motivating the search for safer means to prevent fruit rot. The use of antagonistic microbes has been shown to be an efficient and environmentally friendly alternative to conventional phytopharmaceuticals. Here, we investigate the potential of Aureobasidium subglaciale for postharvest rot control. We tested the antagonistic activity of 9 strains of A. subglaciale and 7 closely related strains against relevant phytopathogenic fungi under conditions simulating low-temperature storage: Botrytis cinerea, Penicillium expansum, and Colletotrichum acutatum. We also investigated a selection of phenotypic traits of all strains and sequenced their whole genomes. The tested strains significantly reduced postharvest rot of apples at low temperatures caused by B. cinerea, C. acutatum (over 60%), and P. expansum (about 40%). Several phenotypic traits were observed that may contribute to this biocontrol capacity: growth at low temperatures, tolerance to high temperatures and elevated solute concentrations, and strong production of several extracellular enzymes and siderophores. Population genomics revealed that 7 of the 15 strains originally identified as A. subglaciale most likely belong to other, possibly undescribed species of the same genus. In addition, the population structure and linkage disequilibrium of the species suggest that A. subglaciale is strictly clonal and therefore particularly well suited for use in biocontrol. Overall, these data suggest substantial biological control potential for A. subglaciale, which represents another promising biological agent for disease control in fresh fruit. IMPORTANCE After harvest, fruits are often stored at low temperatures to prolong their life. However, despite the low temperatures, much of the fruit is lost to rot caused by a variety of fungi, resulting in major economic losses and food safety risks. An increasingly important environmentally friendly alternative to conventional methods of mitigating the effects of plant diseases is the use of microorganisms that act similarly to probiotics-occupying the available space, producing antimicrobial compounds, and consuming the nutrients needed by the rot-causing species. To find a new microorganism for biological control that is particularly suitable for cold storage of fruit, we tested different isolates of the cold-loving yeast Aureobasidium subglaciale and studied their phenotypic characteristics and genomes. We demonstrated that A. subglaciale can significantly reduce rotting of apples caused by three rot-causing molds at low temperatures and thus has great potential for preventing fruit rot during cold storage.

Spider webs as eDNA samplers: Biodiversity assessment across the tree of life.

The concept of environmental DNA (eDNA) utilizes nucleic acids of organisms directly from the environment. Recent breakthrough studies have successfully detected a wide spectrum of prokaryotic and eukaryotic eDNA from a variety of environments, ranging from ancient to modern, and from terrestrial to aquatic. With their diversity and ubiquity in nature, spider webs might act as powerful biofilters and could thus represent a promising new source of eDNA, but their utility under natural field conditions is severely understudied. Here, we bridge this knowledge gap to establish spider webs as a source of eDNA with far reaching implications. First, we conducted a field study to track specific arthropod targets from different spider webs. We then used high-throughput amplicon sequencing of taxonomic barcodes to investigate the utility of spider web eDNA for biodiversity monitoring of animals, fungi and bacteria. Our results show that genetic remains on spider webs allow the detection of even the smallest target organisms. We also demonstrate that eDNA from spider webs is useful in research of community compositions across the different domains of life, with potentially highly detailed temporal and spatial information.

Small-Molecule Mn Antioxidants in Caenorhabditis elegans and Deinococcus radiodurans Supplant MnSOD Enzymes during Aging and Irradiation.

Denham Harman's oxidative damage theory identifies superoxide (O2•-) radicals as central agents of aging and radiation injury, with Mn2+-dependent superoxide dismutase (MnSOD) as the principal O2•--scavenger. However, in the radiation-resistant nematode Caenorhabditis elegans, the mitochondrial antioxidant enzyme MnSOD is dispensable for longevity, and in the model bacterium Deinococcus radiodurans, it is dispensable for radiation resistance. Many radiation-resistant organisms accumulate small-molecule Mn2+-antioxidant complexes well-known for their catalytic ability to scavenge O2•-, along with MnSOD, as exemplified by D. radiodurans. Here, we report experiments that relate the MnSOD and Mn-antioxidant content to aging and oxidative stress resistances and which indicate that C. elegans, like D. radiodurans, may rely on Mn-antioxidant complexes as the primary defense against reactive oxygen species (ROS). Wild-type and ΔMnSOD D. radiodurans and C. elegans were monitored for gamma radiation sensitivities over their life spans while gauging Mn2+-antioxidant content by electron paramagnetic resonance (EPR) spectroscopy, a powerful new approach to determining the in vivo Mn-antioxidant content of cells as they age. As with D. radiodurans, MnSOD is dispensable for radiation survivability in C. elegans, which hyperaccumulates Mn-antioxidants exceptionally protective of proteins. Unexpectedly, ΔMnSOD mutants of both the nematodes and bacteria exhibited increased gamma radiation survival compared to the wild-type. In contrast, the loss of MnSOD renders radiation-resistant bacteria sensitive to atmospheric oxygen during desiccation. Our results support the concept that the disparate responses to oxidative stress are explained by the accumulation of Mn-antioxidant complexes which protect, complement, and can even supplant MnSOD. IMPORTANCE The current theory of cellular defense against oxidative damage identifies antioxidant enzymes as primary defenders against ROS, with MnSOD being the preeminent superoxide (O2•-) scavenger. However, MnSOD is shown to be dispensable both for radiation resistance and longevity in model organisms, the bacterium Deinococcus radiodurans and the nematode Caenorhabditis elegans. Measured by electron paramagnetic resonance (EPR) spectroscopy, small-molecule Mn-antioxidant content was shown to decline in unison with age-related decreases in cell proliferation and radioresistance, which again are independent of MnSOD presence. Most notably, the Mn-antioxidant content of C. elegans drops precipitously in the last third of its life span, which links with reports that the steady-state level of oxidized proteins increases exponentially during the last third of the life span in animals. This leads us to propose that global responses to oxidative stress must be understood through an extended theory that includes small-molecule Mn-antioxidants as potent O2•--scavengers that complement, and can even supplant, MnSOD.

Toward the massive genome of Proteus anguinus-illuminating longevity, regeneration, convergent evolution, and metabolic disorders.

Deciphering the genetic code of organisms with unusual phenotypes can help answer fundamental biological questions and provide insight into mechanisms relevant to human biomedical research. The cave salamander Proteus anguinus (Urodela: Proteidae), also known as the olm, is an example of a species with unique morphological and physiological adaptations to its subterranean environment, including regenerative abilities, resistance to prolonged starvation, and a life span of more than 100 years. However, the structure and sequence of the olm genome is still largely unknown owing to its enormous size, estimated at nearly 50 gigabases. An international Proteus Genome Research Consortium has been formed to decipher the olm genome. This perspective provides the scientific and biomedical rationale for exploring the olm genome and outlines potential outcomes, challenges, and methodological approaches required to analyze and annotate the genome of this unique amphibian.

Seven Years at High Salinity-Experimental Evolution of the Extremely Halotolerant Black Yeast Hortaea werneckii.

The experimental evolution of microorganisms exposed to extreme conditions can provide insight into cellular adaptation to stress. Typically, stress-sensitive species are exposed to stress over many generations and then examined for improvements in their stress tolerance. In contrast, when starting with an already stress-tolerant progenitor there may be less room for further improvement, it may still be able to tweak its cellular machinery to increase extremotolerance, perhaps at the cost of poorer performance under non-extreme conditions. To investigate these possibilities, a strain of extremely halotolerant black yeast Hortaea werneckii was grown for over seven years through at least 800 generations in a medium containing 4.3 M NaCl. Although this salinity is well above the optimum (0.8-1.7 M) for the species, the growth rate of the evolved H. werneckii did not change in the absence of salt or at high concentrations of NaCl, KCl, sorbitol, or glycerol. Other phenotypic traits did change during the course of the experimental evolution, including fewer multicellular chains in the evolved strains, significantly narrower cells, increased resistance to caspofungin, and altered melanisation. Whole-genome sequencing revealed the occurrence of multiple aneuploidies during the experimental evolution of the otherwise diploid H. werneckii. A significant overrepresentation of several gene groups was observed in aneuploid regions. Taken together, these changes suggest that long-term growth at extreme salinity led to alterations in cell wall and morphology, signalling pathways, and the pentose phosphate cycle. Although there is currently limited evidence for the adaptive value of these changes, they offer promising starting points for future studies of fungal halotolerance.

Virulence Traits and Population Genomics of the Black Yeast Aureobasidium melanogenum.

The black yeast-like fungus Aureobasidium melanogenum is an opportunistic human pathogen frequently found indoors. Its traits, potentially linked to pathogenesis, have never been systematically studied. Here, we examine 49 A. melanogenum strains for growth at 37 °C, siderophore production, hemolytic activity, and assimilation of hydrocarbons and human neurotransmitters and report within-species variability. All but one strain grew at 37 °C. All strains produced siderophores and showed some hemolytic activity. The largest differences between strains were observed in the assimilation of hydrocarbons and human neurotransmitters. We show for the first time that fungi from the order Dothideales can assimilate aromatic hydrocarbons. To explain the background, we sequenced the genomes of all 49 strains and identified genes putatively involved in siderophore production and hemolysis. Genomic analysis revealed a fairly structured population of A.melanogenum, raising the possibility that some phylogenetic lineages have higher virulence potential than others. Population genomics indicated that the species is strictly clonal, although more than half of the genomes were diploid. The existence of relatively heterozygous diploids in an otherwise clonal species is described for only the second time in fungi. The genomic and phenotypic data from this study should help to resolve the non-trivial taxonomy of the genus Aureobasidium and reduce the medical hazards of exploiting the biotechnological potential of other, non-pathogenic species of this genus.