Márton Miskei

The 2008 update of the Aspergillus nidulans genome annotation: A community effort

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

Comparison of gene expression signatures of diamide, H2O2 and menadione exposed Aspergillus nidulans cultures – linking genome-wide transcriptional changes to cellular physiology

BackgroundIn addition to their cytotoxic nature, reactive oxygen species (ROS) are also signal molecules in diverse cellular processes in eukaryotic organisms. Linking genome-wide transcriptional changes to cellular physiology in oxidative stress-exposed Aspergillus nidulans cultures provides the opportunity to estimate the sizes of peroxide (O22-), superoxide (O2•-) and glutathione/glutathione disulphide (GSH/GSSG) redox imbalance responses.ResultsGenome-wide transcriptional changes triggered by diamide, H2O2 and menadione in A. nidulans vegetative tissues were recorded using DNA microarrays containing 3533 unique PCR-amplified probes. Evaluation of LOESS-normalized data indicated that 2499 gene probes were affected by at least one stress-inducing agent. The stress induced by diamide and H2O2 were pulse-like, with recovery after 1 h exposure time while no recovery was observed with menadione. The distribution of stress-responsive gene probes among major physiological functional categories was approximately the same for each agent. The gene group sizes solely responsive to changes in intracellular O22-, O2•- concentrations or to GSH/GSSG redox imbalance were estimated at 7.7, 32.6 and 13.0 %, respectively. Gene groups responsive to diamide, H2O2 and menadione treatments and gene groups influenced by GSH/GSSG, O22- and O2•- were only partly overlapping with distinct enrichment profiles within functional categories. Changes in the GSH/GSSG redox state influenced expression of genes coding for PBS2 like MAPK kinase homologue, PSK2 kinase homologue, AtfA transcription factor, and many elements of ubiquitin tagging, cell division cycle regulators, translation machinery proteins, defense and stress proteins, transport proteins as well as many enzymes of the primary and secondary metabolisms. Meanwhile, a separate set of genes encoding transport proteins, CpcA and JlbA amino acid starvation-responsive transcription factors, and some elements of sexual development and sporulation was ROS responsive.ConclusionThe existence of separate O22-, O2•- and GSH/GSSG responsive gene groups in a eukaryotic genome has been demonstrated. Oxidant-triggered, genome-wide transcriptional changes should be analyzed considering changes in oxidative stress-responsive physiological conditions and not correlating them directly to the chemistry and concentrations of the oxidative stress-inducing agent.

Fuzzy complexes: Specific binding without complete folding

Specific molecular recognition is assumed to require a well‐defined set of contacts and devoid of conformational and interaction ambiguities. Growing experimental evidence demonstrates however, that structural multiplicity or dynamic disorder can be retained in protein complexes, termed as fuzziness. Fuzzy regions establish alternative contacts between specific partners usually via transient interactions. Nature often tailors the dynamic properties of these segments via post‐translational modifications or alternative splicing to fine‐tune affinity. Most experimentally characterized fuzzy complexes are involved in regulation of gene‐expression, signal transduction and cell‐cycle regulation. Fuzziness is also characteristic to viral protein complexes, cytoskeleton structure, and surprisingly in a few metabolic enzymes. A plausible role of fuzzy complexes in increasing half‐life of intrinsically disordered proteins is also discussed.

Transcriptome changes initiated by carbon starvation in Aspergillus nidulans.

Carbon starvation is a common stress for micro-organisms both in nature and in industry. The carbon starvation stress response (CSSR) involves the regulation of several important processes including programmed cell death and reproduction of fungi, secondary metabolite production and extracellular hydrolase formation. To gain insight into the physiological events of CSSR, DNA microarray analyses supplemented with real-time RT-PCR (rRT-PCR) experiments on 99 selected genes were performed. These data demonstrated that carbon starvation induced very complex changes in the transcriptome. Several genes contributing to protein synthesis were upregulated together with genes involved in the unfolded protein stress response. The balance between biosynthesis and degradation moved towards degradation in the case of cell wall, carbohydrate, lipid and nitrogen metabolism, which was accompanied by the production of several hydrolytic enzymes and the induction of macroautophagy. These processes provide the cultures with long-term survival by liberating nutrients through degradation of the cell constituents. The induced synthesis of secondary metabolites, antifungal enzymes and proteins as well as bacterial cell wall-degrading enzymes demonstrated that carbon-starving fungi should have marked effects on the micro-organisms in their surroundings. Due to the increased production of extracellular and vacuolar enzymes during carbon starvation, the importance of the endoplasmic reticulum increased considerably.

AtfA bZIP-type transcription factor regulates oxidative and osmotic stress responses in Aspergillus nidulans

The aim of the study was to demonstrate that the bZIP-type transcription factor AtfA regulates different types of stress responses in Aspergillus nidulans similarly to Atf1, the orthologous ‘all-purpose’ transcription factor of Schizosaccharomyces pombe. Heterologous expression of atfA in a S. pombe Δatf1 mutant restored the osmotic stress tolerance of fission yeast in surface cultures to the same level as recorded in complementation studies with the atf1 gene, and a partial complementation of the osmotic and oxidative-stress-sensitive phenotypes was also achieved in submerged cultures. AtfA is therefore a true functional ortholog of fission yeast’s Atf1. As demonstrated by RT-PCR experiments, elements of both oxidative (e.g. catalase B) and osmotic (e.g. glycerol-3-phosphate dehydrogenase B) stress defense systems were transcriptionally regulated by AtfA in a stress-type-specific manner. Deletion of atfA resulted in oxidative-stress-sensitive phenotypes while the high-osmolarity stress sensitivity of the fungus was not affected significantly. In A. nidulans, the glutathione/glutathione disulfide redox status of the cells as well as apoptotic cell death and autolysis seemed to be controlled by regulatory elements other than AtfA. In conclusion, the orchestrations of stress responses in the aspergilli and in fission yeast share several common features, but further studies are needed to answer the important question of whether a fission yeast-like core environmental stress response also operates in the euascomycete genus Aspergillus.

Comparison of transcriptional and translational changes caused by long-term menadione exposure in Aspergillus nidulans

Under long-term oxidative stress caused by menadione sodium bisulfite, genome-wide transcriptional and proteome-wide translational changes were compared in Aspergillus nidulans vegetative cells. The comparison of proteomic and DNA microarray expression data demonstrated that global gene expression changes recorded with either flip-flop or dendrimer cDNA labeling techniques supported proteome changes moderately with 40% and 34% coincidence coefficients, respectively. Enzyme levels in the glycolytic pathway were alternating, which was a direct consequence of fluctuating gene expression patterns. Surprisingly, enzymes in the vitamin B2 and B6 biosynthetic pathways were repressed concomitantly with the repression of some protein folding chaperones and nuclear transport elements. Under long-term oxidative stress, the peroxide-detoxifying peroxiredoxins and cytochrome c peroxidase were replaced by thioredoxin reductase, a nitroreductase and a flavohemoprotein, and protein degradation became predominant to eliminate damaged proteins.

Comparative studies of differential expression of chitinolytic enzymes encoded by chiA, chiB, chiC and nagA genes in Aspergillus nidulans

N-Acetyl-d-glucosamine, chito-oligomers and carbon starvation regulatedchiA, chiB, andnagA gene expressions inAspergillus nidulans cultures. The gene expression patterns of the main extracellular endochitinase ChiB and theN-acetyl-β-d-glucosaminidase NagA were similar, and the ChiB-NagA enzyme system may play a morphological and/or nutritional role during autolysis. Alterations in the levels of reactive oxygen species or in the glutathione-glutathione disulfide redox balance, characteristic physiological changes developing in ageing and autolyzing fungal cultures, did not affect the regulation of either the growth-relatedchiA or the autolysis-coupledchiB genes although both of them were down-regulated under diamide stress. The transcription of thechiC gene with unknown physiological function was repressed by increased intracellular superoxide concentration.

FuzDB: Database of fuzzy complexes, a tool to develop stochastic structure-function relationships for protein complexes and higher-order assemblies

FuzDB (http://protdyn-database.org) compiles experimentally observed fuzzy protein complexes, where intrinsic disorder (ID) is maintained upon interacting with a partner (protein, nucleic acid or small molecule) and directly impacts biological function. Entries in the database have both (i) structural evidence demonstrating the structural multiplicity or dynamic disorder of the ID region(s) in the partner bound form of the protein and (ii) in vitro or in vivo biological evidence that indicates the significance of the fuzzy region(s) in the formation, function or regulation of the assembly. Unlike the other intrinsically disordered or unfolded protein databases, FuzDB focuses on ID regions within a biological context, including higher-order assemblies and presents a detailed analysis of the structural and functional data. FuzDB also provides interpretation of experimental results to elucidate the molecular mechanisms by which fuzzy regions—classified on the basis of topology and mechanism—interfere with the structural ensembles and activity of protein assemblies. Regulatory sites generated by alternative splicing (AS) or post-translational modifications (PTMs) are also collected. By assembling all this information, FuzDB could be utilized to develop stochastic structure–function relationships for proteins and could contribute to the emergence of a new paradigm.

FSRD: fungal stress response database

Adaptation to different types of environmental stress is a common part of life for today’s fungi. A deeper understanding of the organization, regulation and evolution of fungal stress response systems may lead to the development of novel antifungal drugs and technologies or the engineering of industrial strains with elevated stress tolerance. Here we present the Fungal Stress Response Database (http://internal.med.unideb.hu/fsrd) aimed to stimulate further research on stress biology of fungi. The database incorporates 1985 fungal stress response proteins with verified physiological function(s) and their orthologs identified and annotated in 28 species including human and plant pathogens, as well as important industrial fungi. The database will be extended continuously to cover other fully sequenced fungal species. Our database, as a starting point for future stress research, facilitates the analysis of literature data on stress and the identification of ortholog groups of stress response proteins in newly sequenced fungal genomes. Database URL: http://internal.med.unideb.hu/fsrd

Polyphasic characterization of “Aspergillus nidulans var. roseus” ATCC 58397

Polyphasic characterization of the echinocandin B producer Aspergillus nidulans var. roseus ATCC 58397 strain was carried out to elucidate its taxonomical status. According to its carbon source utilization and secondary metabolite spectrum as well as the partial β-tubulin, calmodulin, and γ-actin gene sequences, A. nidulans var. roseus belongs to the Emericella rugulosa species. Auxotroph mutants of A. nidulans var. roseus ATCC 58397 and E. rugulosa CBS 171.71 and CBS 133.60 formed stable heterokaryons on minimal medium with several A. nidulans strains, and in the case of A. nidulans var. roseus, even cleistothecia were developed.