Decoding the biological information contained in two ancient Slavonic parchment codices: an added historical value.

This study provides an example in the emerging field of biocodicology showing how metagenomics can help answer relevant questions that may contribute to a better understanding of the history of ancient manuscripts. To this end, two Slavonic codices dating from the 11th century were investigated through shotgun metagenomics. Endogenous DNA enabled to infer the animal origin of the skins used in the manufacture of the two codices, while nucleic sequences recovered from viruses were investigated for the first time in this material, opening up new possibilities in the field of biocodicology. In addition, the microbiomes colonizing the surface of the parchments served to determine their conservation status and their latent risk of deterioration. The saline environment provided by the parchments selected halophilic and halotolerant microorganisms, which are known to be responsible for the biodegradation of parchment. Species of Nocardiopsis, Gracilibacillus and Saccharopolyspora, but also members of the Aspergillaceae family were detected in this study, all possessing enzymatic capabilities for the biodeterioration of this material. Finally, a relative abundance of microorganisms originating from the human skin microbiome were identified, most probably related to the intensive manipulation of the manuscripts throughout the centuries, which should be taken with caution as they can be potential pathogens.

Gene expression profiling identifies pathways involved in seed maturation of Jatropha curcas.

BACKGROUND: Jatropha curcas, a tropical shrub, is a promising biofuel crop, which produces seeds with high content of oil and protein. To better understand the maturation process of J. curcas seeds and to improve its agronomic performance, a two-step approach was performed in six different maturation stages of seeds: 1) generation of the entire transcriptome of J. curcas seeds using 454-Roche sequencing of a cDNA library, 2) comparison of transcriptional expression levels using a custom Agilent 8x60K oligonucleotide microarray. RESULTS: A total of 793,875 high-quality reads were assembled into 19,382 unique full-length contigs, of which 13,507 could be annotated with Gene Ontology (GO) terms. Microarray data analysis identified 9111 probes (out of 57,842 probes), which were differentially expressed between the six maturation stages. The expression results were validated for 75 selected transcripts based on expression levels, predicted function, pathway, and length. Result from cluster analyses showed that transcripts associated with fatty acid, flavonoid, and phenylpropanoid biosynthesis were over-represented in the early stages, while those of lipid storage were over-represented in the late stages. Expression analyses of different maturation stages of J. curcas seed showed that most changes in transcript abundance occurred between the two last stages, suggesting that the timing of metabolic pathways during seed maturation in J. curcas occurs in late stages. The co-expression results showed that the hubs (CB5-D, CDR1, TT8, DFR, HVA22) with the highest number of edges, associated with fatty acid and flavonoid biosynthesis, are showing a decrease in their expression during seed maturation. Furthermore, seed development and hormone pathways are significantly well connected. CONCLUSION: The obtained results revealed differentially expressed sequences (DESs) regulating important pathways related to seed maturation, which could contribute to the understanding of the complex regulatory network during seed maturation with the focus on lipid, flavonoid and phenylpropanoid biosynthesis. This study provides detailed information on transcriptional changes during J. curcas seed maturation and provides a starting point for a genomic survey of seed quality traits. The results highlighted specific genes and processes relevant to the molecular mechanisms involved in Jatropha seed maturation. These data can also be utilized regarding other Euphorbiaceae species.

Back to the Salt Mines: Genome and Transcriptome Comparisons of the Halophilic Fungus Aspergillus salisburgensis and Its Halotolerant Relative Aspergillus sclerotialis.

Salt mines are among the most extreme environments as they combine darkness, low nutrient availability, and hypersaline conditions. Based on comparative genomics and transcriptomics, we describe in this work the adaptive strategies of the true halophilic fungus Aspergillus salisburgensis, found in a salt mine in Austria, and compare this strain to the ex-type halotolerant fungal strain Aspergillus sclerotialis. On a genomic level, A. salisburgensis exhibits a reduced genome size compared to A. sclerotialis, as well as a contraction of genes involved in transport processes. The proteome of A. sclerotialis exhibits an increased proportion of alanine, glycine, and proline compared to the proteome of non-halophilic species. Transcriptome analyses of both strains growing at 5% and 20% NaCl show that A. salisburgensis regulates three-times fewer genes than A. sclerotialis in order to adapt to the higher salt concentration. In A. sclerotialis, the increased osmotic stress impacted processes related to translation, transcription, transport, and energy. In contrast, membrane-related and lignolytic proteins were significantly affected in A. salisburgensis.

Transcriptome Study of an Exophiala dermatitidis PKS1 Mutant on an ex Vivo Skin Model: Is Melanin Important for Infection?

The black yeast Exophiala dermatitidis is a polyextremophilic human pathogen, especially known for growing in man-made extreme environments. Reported diseases caused by this fungus range from benign cutaneous to systemic infections with 40% fatality rate. While the number of cases steadily increases in both immunocompromised and immunocompetent people, detailed knowledge about infection mechanisms, virulence factors and host response are scarce. To understand the impact of the putative virulence factor melanin on the infection, we generated a polyketide synthase (PKS1) mutant using CRISPR/Cas9 resulting in a melanin deficient strain. The mutant and the wild-type fungus were inoculated onto skin explants using an ex vivo skin organ culture model to simulate in vivo cutaneous infection. The difference between the mutant and wild-type transcriptional landscapes, as assessed by whole RNA-sequencing, were small and were observed in pathways related to the copper homeostasis, cell wall genes and proteases. Seven days after inoculation the wild-type fungus completely colonized the stratum corneum, invaded the skin and infected keratinocytes while the mutant had only partially covered the skin and showed no invasiveness. Our results suggest that melanin dramatically improves the invasiveness and virulence of E. dermatitidis during the first days of the skin infection.

Big Sound and Extreme Fungi-Xerophilic, Halotolerant Aspergilli and Penicillia with Low Optimal Temperature as Invaders of Historic Pipe Organs.

Recent investigations have shown that xerophilic fungi may pose a biodeterioration risk by threatening objects of cultural heritage including many types of materials, including wood, paint layers, organic glues or leather and even metal. Historic—and also new built—pipe organs combine all those materials. In this study, halotolerant aspergilli and penicillia with low optimal temperatures were shown to be the most frequent invaders of pipe organs. The fungi form white mycelia on the organic components of the organs with a clear preference for the bolus paint of the wooden pipes, the leather-made hinges of the stop actions and all parts fixed by organic glue. Physiological tests showed that the strains isolated from the instruments all show a halotolerant behavior, although none was halophilic. The optimum growth temperature is below 20 °C, thus the fungi are perfectly adapted to the cool and relatively dry conditions in the churches and organs respectively. The de-novo genome sequences analyses of the strains are currently ongoing and will reveal the genomic basis for the halotolerant behavior of the fungi.

Draft Genome Sequence of the Saccharomyces cerevisiae × Saccharomyces kudriavzevii HA1836 Interspecies Hybrid Yeast.

Saccharomyces cerevisiae × Saccharomyces kudriavzevii interspecies hybrid yeasts have frequently been isolated from alcoholic fermentation environments. Here, we report the draft genome sequence of the S. cerevisiae × S. kudriavzevii HA1836 strain isolated from grapes from an Austrian vineyard.

Draft Genome Sequence of the Interspecies Hybrid Saccharomyces pastorianus Strain HA2560, Isolated from a Municipal Wastewater Treatment Plant.

Saccharomyces pastorianus is an industrially relevant yeast frequently isolated from brewing environments. Here, we report the draft genome sequence of the S. pastorianus HA2560 strain isolated from a municipal wastewater treatment plant.

Draft Genome Sequences of the Black Rock Fungus Knufia petricola and Its Spontaneous Nonmelanized Mutant.

The fungal genus Knufia mostly comprises extremotolerant species from environmental sources, especially rock surfaces. The draft genome sequence of the rock fungus Knufia petricola presented here is the first whole-genome sequence of the only species among black fungi known to have a nonmelanized spontaneous mutant.

Genomic and transcriptomic analysis of the toluene degrading black yeast Cladophialophora immunda.

Cladophialophora immunda is an ascomycotal species belonging to the group of the black yeasts. These fungi have a thick and melanized cell wall and other physiological adaptations that allows them to cope with several extreme physical and chemical conditions. Member of the group can colonize some of the most extremophilic environments on Earth. Cladophialophora immunda together with a few other species of the order Chaetothyriales show a special association with hydrocarbon polluted environments. The finding that the fungus is able to completely mineralize toluene makes it an interesting candidate for bioremediation purposes. The present study is the first transcriptomic investigation of a fungus grown in presence of toluene as sole carbon and energy source. We could observe the activation of genes involved in toluene degradatation and several stress response mechanisms which allowed the fungus to survive the toluene exposure. The thorough comparative genomics analysis allowed us to identify several events of horizontal gene transfer between bacteria and Cladophialophora immunda and unveil toluene degradation steps that were previously reported in bacteria. The work presented here aims to give new insights into the ecology of Cladophialophora immunda and its adaptation strategies to hydrocarbon polluted environments.

The Transcriptome of Exophiala dermatitidis during Ex-vivo Skin Model Infection.

The black yeast Exophiala dermatitidis is a widespread polyextremophile and human pathogen, that is found in extreme natural habitats and man-made environments such as dishwashers. It can cause various diseases ranging from phaeohyphomycosis and systemic infections, with fatality rates reaching 40%. While the number of cases in immunocompromised patients are increasing, knowledge of the infections, virulence factors and host response is still scarce. In this study, for the first time, an artificial infection of an ex-vivo skin model with Exophiala dermatitidis was monitored microscopically and transcriptomically. Results show that Exophiala dermatitidis is able to actively grow and penetrate the skin. The analysis of the genomic and RNA-sequencing data delivers a rich and complex transcriptome where circular RNAs, fusion transcripts, long non-coding RNAs and antisense transcripts are found. Changes in transcription strongly affect pathways related to nutrients acquisition, energy metabolism, cell wall, morphological switch, and known virulence factors. The L-Tyrosine melanin pathway is specifically upregulated during infection. Moreover the production of secondary metabolites, especially alkaloids, is increased. Our study is the first that gives an insight into the complexity of the transcriptome of Exophiala dermatitidis during artificial skin infections and reveals new virulence factors.

Genome sequence of the filamentous soil fungus Chaetomium cochliodes reveals abundance of genes for heme enzymes from all peroxidase and catalase superfamilies.

BACKGROUND: The ascomycetous family Chaetomiaceae (class Sordariomycetes) includes numerous soilborn, saprophytic, endophytic and pathogenic fungi which can adapt to various growth conditions and living niches by providing a broad armory of oxidative and antioxidant enzymes. RESULTS: We release the 34.7 Mbp draft genome of Chaetomium cochliodes CCM F-232 consisting of 6036 contigs with an average size of 5756 bp and reconstructed its phylogeny. We show that this filamentous fungus is closely related but not identical to Chaetomium globosum and Chaetomium elatum. We screened and critically analysed this genome for open reading frames coding for essential antioxidant enzymes. It is demonstrated that the genome of C. cochliodes contains genes encoding putative enzymes from all four known heme peroxidase superfamilies including bifunctional catalase-peroxidase (KatG), cytochrome c peroxidase (CcP), manganese peroxidase, two paralogs of hybrid B peroxidases (HyBpox), cyclooxygenase, linoleate diol synthase, dye-decolorizing peroxidase (DyP) of type B and three paralogs of heme thiolate peroxidases. Both KatG and DyP-type B are shown to be introduced into ascomycetes genomes by horizontal gene transfer from various bacteria. In addition, two putative large subunit secretory and two small-subunit typical catalases are found in C. cochliodes. We support our genomic findings with quantitative transcription analysis of nine peroxidase & catalase genes. CONCLUSIONS: We delineate molecular phylogeny of five distinct gene superfamilies coding for essential heme oxidoreductases in Chaetomia and from the transcription analysis the role of this antioxidant enzymatic armory for the survival of a peculiar soil ascomycete in various harsh environments.

Pathogenic Yet Environmentally Friendly? Black Fungal Candidates for Bioremediation of Pollutants.

A collection of 163 strains of black yeast-like fungi from the CBS Fungal Biodiversity Center (Utrecht, The Netherlands), has been screened for the ability to grow on hexadecane, toluene and polychlorinated biphenyl 126 (PCB126) as the sole carbon and energy source. These compounds were chosen as representatives of relevant environmental pollutants. A microtiter plate-based culture assay was set up in order to screen the fungal strains for growth on the selected xenobiotics versus glucose, as a positive control. Growth was observed in 25 strains on at least two of the tested substrates. Confirmation of substrate assimilation was performed by cultivation on closed vials and analysis of the headspace composition with regard to the added volatile substrates and the generated carbon dioxide. Exophiala mesophila (CBS 120910) and Cladophialophora immunda (CBS 110551), both of the order Chaetothyriales and isolated from a patient with chronic sinusitis and a polluted soil sample, respectively, showed the ability to grow on toluene as the sole carbon and energy source. Toluene assimilation has previously been described for C. immunda but this is the first account for E. mesophila. Also, this is the first time that the capacity to grow on alkylbenzenes has been demonstrated for a clinical isolate. Assimilation of toluene could not be demonstrated for the human opportunistic pathogen Pseudoallescheria boydii (CBS 115.59, Microascales), but the results from microtiter plate assays suggest that strains of this species are promising candidates for further studies. The outstanding abilities of black yeast-like fungi to thrive in extreme environments makes them ideal agents for the bioremediation of polluted soils, and for the treatment of contaminated gas streams in biofilters. However, interrelations between hydrocarbonoclastic and potentially pathogenic strains need to be elucidated in order to avoid the possibility of biohazards occurring.

Draft Genome of Debaryomyces fabryi CBS 789T, Isolated from a Human Interdigital Mycotic Lesion.

The yeast genus Debaryomyces comprises species isolated from various natural habitats, man-made environments, and clinical materials. Here, the draft genome of D. fabryi CBS 789(T), isolated from a human interdigital mycotic lesion, is presented.

Proteome of tolerance fine-tuning in the human pathogen black yeast Exophiala dermatitidis.

UNLABELLED: The black yeast Exophiala dermatitidis is a worldwide distributed agent of primary and secondary diseases in both immunocompromised and healthy humans, with a high prevalence in human-made environments. Since thermo-tolerance has a crucial role in the fungus persistence in man-dominated habitat and in its pathogenicity, three incubation temperatures (37, 45, 1 °C) and two time spans (1 h, 1 week) were selected to simulate different environmental conditions and to investigate the effect of temperature on the proteome of E. dermatitidis CBS 525.76. Using a novel protocol for protein extraction from black yeasts, 2-D DIGE could be applied for characterization of changes in total protein spot abundance among the experimental conditions. A total of 32 variable proteins were identified by mass spectrometry. Data about protein functions, localization and pathways were also obtained. A typical stress response under non-optimal temperature could not be observed at the proteome level, whereas a reduction of the metabolic activity, mostly concerning processes as the general carbon metabolism, was detected after exposure to cold. These results suggest that a fine protein modulation takes place following temperature treatment and a repertoire of stable protein might be at the base of E. dermatitidis adaptation to altered growth conditions. SIGNIFICANCE: E. dermatitidis is a pathogenic black yeast causing neurotropic infections, systemic and subcutaneous disease in a wide range of hosts, including humans. The discovery of the fungus high prevalence in man-made habitats, including sauna facilities, drinking water and dishwashers, generated concern and raised questions about the infection route. In the present work - which is the first contribution on E. dermatitidis proteome - the effect of different temperature conditions on the fungus protein pattern have been analyzed by using a gel-based approach and the temperature responsive proteins have been identified. The absence of a typical stress response following the exposure to non-optimal temperature was detected at the proteome level, along with a general reduction of the metabolic activity after exposure to cold. These results suggest that a very fine regulation of the protein expression as well as adaptations involving a basic set of stable proteins may be at the base of E. dermatitidis enormous ecological plasticity, which plays a role in the fungus distribution, also enabling the transition from natural to human habitat and to the human host.

From Glacier to Sauna: RNA-Seq of the Human Pathogen Black Fungus Exophiala dermatitidis under Varying Temperature Conditions Exhibits Common and Novel Fungal Response.

Exophiala dermatitidis (Wangiella dermatitidis) belongs to the group of the so-called black yeasts. Thanks in part to its thick and strongly melanized cell walls, E. dermatitidis is extremely tolerant to various kinds of stress, including extreme pH, temperature and desiccation. E. dermatitidis is also the agent responsible for various severe illnesses in humans, such as pneumonia and keratitis, and might lead to fatal brain infections. Due to its association with the human environment, its poly-extremophilic lifestyle and its pathogenicity in humans, E. dermatitidis has become an important model organism. In this study we present the functional analysis of the transcriptional response of the fungus at 1°C and 45°C, in comparison with that at 37°C, for two different exposition times, i.e. 1 hour and 1 week. At 1°C, E. dermatitidis uses a large repertoire of tools to acclimatize, such as lipid membrane fluidization, trehalose production or cytoskeleton rearrangement, which allows the fungus to remain metabolically active. At 45°C, the fungus drifts into a replicative state and increases the activity of the Golgi apparatus. As a novel finding, our study provides evidence that, apart from the protein coding genes, non-coding RNAs, circular RNAs as well as fusion-transcripts are differentially regulated and that the function of the fusion-transcripts can be related to the corresponding temperature condition. This work establishes that E. dermatitidis adapts to its environment by modulating coding and non-coding gene transcription levels and through the regulation of chimeric and circular RNAs.

Amid the possible causes of a very famous foxing: molecular and microscopic insight into Leonardo da Vinci's self-portrait.

Leonardo da Vinci's self-portrait is affected by foxing spots. The portrait has no fungal or bacterial infections in place, but is contaminated with airborne spores and fungal material that could play a role in its disfigurement. The knowledge of the nature of the stains is of great concern because future conservation treatments should be derived from scientific investigations. The lack of reliable scientific data, due to the non-culturability of the microorganisms inhabiting the portrait, prompted the investigation of the drawing using non-invasive and micro-invasive sampling, in combination with scanning electron microscope (SEM) imaging and molecular techniques. The fungus Eurotium halophilicum was found in foxing spots using SEM analyses. Oxalates of fungal origin were also documented. Both findings are consistent with the hypothesis that tonophilic fungi germinate on paper metabolizing organic acids, oligosaccharides and proteic compounds, which react chemically with the material at a low water activity, forming brown products and oxidative reactions resulting in foxing spots. Additionally, molecular techniques enabled a screening of the fungi inhabiting the portrait and showed differences when different sampling techniques were employed. Swabs samples showed a high abundance of lichenized Ascomycota, while the membrane filters showed a dominance of Acremonium sp. colonizing the drawing.

Draft Genome Sequence of Exophiala mesophila, a Black Yeast with High Bioremediation Potential.

The fungal genus Exophiala comprises both pathogen species, which cause severe infections in humans, and environmental species, which are able to degrade alkylbenzene compounds. The draft genome sequence of Exophiala mesophila presented here is the first genome assembly of an alkylbenzene-degrading organism belonging to the genus Exophiala.

Developmentally regulated expression and expression strategies of Drosophila snoRNAs.

Small nucleolar RNAs constitute a significant portion of the eukaryotic small ncRNA transcriptome and guide site-specific methylation or pseudouridylation of target RNAs. In addition, they can play diverse regulatory roles on gene expression, acting as precursors of smaller fragments able to modulate alternative splicing or operate as microRNAs. Defining their expression strategies and the full repertory of their biological functions is a critical, but still ongoing, process in most organisms. Considering that Drosophila melanogaster is one of the most advantageous model organism for genetic, functional and developmental studies, we analysed the whole genomic organization of its annotated snoRNAs - whose vast majority is known to be embedded in an intronic context - and show by GO term enrichment analysis that protein-coding genes involved in cell division and cytoskeleton organization are those mostly preferred as hosts. This finding was unexpected, and delineates an unpredicted link between snoRNA host genes and cell proliferation that might be of general relevance. We also defined by quantitative RT-PCR the expression of a representative subset of annotated specimens throughout the life cycle, providing a first overview on developmental profiling of the fly snoRNA transcriptome. We found that most of the tested specimens, rather than acting as housekeeping genes with uniform expression, exhibit dynamic developmental expression patterns; moreover, intronic snoRNAs harboured by the same host gene often exhibit distinct temporal profiles, indicating that they can be expressed uncoordinatedly. In addition to provide an updated outline of the fly snoRNA transcriptome, our data highlight that expression of these versatile ncRNAs can be finely regulated.

Draft Genome of Cladophialophora immunda, a Black Yeast and Efficient Degrader of Polyaromatic Hydrocarbons.

The fungal genus Cladophialophora comprises many species which cause severe and even fatal infections in humans as well as environmental strains able to degrade polyaromatic hydrocarbons. The draft genome of Cladophialophora immunda presented here is the first whole-genome sequence within this important genus.

Unusual Novel SnoRNA-Like RNAs in Drosophila melanogaster.

A computational screen for novel small nucleolar RNAs in Drosophila melanogaster uncovered 15 novel snoRNAs and snoRNA-like long non-coding RNAs. In contrast to earlier surverys, the novel sequences are mostly poorly conserved and originate from unusual genomic locations. The majority derive from precurors antisense to well-known protein-coding genes, and four of the candidates are produced from exon-coding regions. Only a minority of the new sequences appears to have canonical target sites in ribosomal or small nuclear RNAs. Taken together, these evolutionary young, poorly conserved, and genomically atypical sequences point at a class of snoRNA-like transcripts with predominantly regulatory functions in the fruit fly genome.