Species-specific effects of the introduction of Aspergillus nidulans gfdB in osmophilic aspergilli.

Industrial fungi need a strong environmental stress tolerance to ensure acceptable efficiency and yields. Previous studies shed light on the important role that Aspergillus nidulans gfdB, putatively encoding a NAD+-dependent glycerol-3-phosphate dehydrogenase, plays in the oxidative and cell wall integrity stress tolerance of this filamentous fungus model organism. The insertion of A. nidulans gfdB into the genome of Aspergillus glaucus strengthened the environmental stress tolerance of this xerophilic/osmophilic fungus, which may facilitate the involvement of this fungus in various industrial and environmental biotechnological processes. On the other hand, the transfer of A. nidulans gfdB to Aspergillus wentii, another promising industrial xerophilic/osmophilic fungus, resulted only in minor and sporadic improvement in environmental stress tolerance and meanwhile partially reversed osmophily. Because A. glaucus and A. wentii are phylogenetically closely related species and both fungi lack a gfdB ortholog, these results warn us that any disturbance of the stress response system of the aspergilli may elicit rather complex and even unforeseeable, species-specific physiological changes. This should be taken into consideration in any future targeted industrial strain development projects aiming at the fortification of the general stress tolerance of these fungi. KEY POINTS: • A. wentii c' gfdB strains showed minor and sporadic stress tolerance phenotypes. • The osmophily of A. wentii significantly decreased in the c' gfdB strains. • Insertion of gfdB caused species-specific phenotypes in A. wentii and A. glaucus.

Physiological background of the remarkably high Cd2+ tolerance of the Aspergillus fumigatus Af293 strain.

The physiological background of the unusually high cadmium tolerance (MIC50 > 2 mM) of Aspergillus fumigatus Af293 was investigated. The cadmium tolerance of the tested environmental and clinical A. fumigatus strains varied over a wide range (0.25 mM < MIC50 < 1 mM). Only the Af293 strain showed a MIC50 value of >2 mM, and this phenotype was accompanied by increased in vivo virulence in mice. A strong correlation was found between the cadmium tolerance and the transcription of the pcaA gene, which encodes a putative cadmium efflux pump. The cadmium tolerance also correlated with the iron tolerance and the extracellular siderophore production of the strains. In addition to these findings, Af293 did not show the synergism between iron toxicity and cadmium toxicity that was detected in the other strains. Based on these results, we suggest that the primary function of PcaA should be acting as a ferrous iron pump and protecting cells from iron overload. Nevertheless, the heterologous expression of pcaA may represent an attractive strain improvement strategy to construct fungal strains for use in biosorption or biomining processes or to prevent accumulation of this toxic metal in crops.

Fungal Stress Database (FSD)--a repository of fungal stress physiological data.

Abstract: The construction of the Fungal Stress Database (FSD) was initiated and fueled by two major goals. At first, some outstandingly important groups of filamentous fungi including the aspergilli possess remarkable capabilities to adapt to a wide spectrum of environmental stress conditions but the underlying mechanisms of this stress tolerance have remained yet to be elucidated. Furthermore, the lack of any satisfactory interlaboratory standardization of stress assays, e.g. the widely used stress agar plate experiments, often hinders the direct comparison and discussion of stress physiological data gained for various fungal species by different research groups. In order to overcome these difficulties and to promote multilevel, e.g. combined comparative physiology-based and comparative genomics-based, stress research in filamentous fungi, we constructed FSD, which currently stores 1412 photos taken on Aspergillus colonies grown under precisely defined stress conditions. This study involved altogether 18 Aspergillus strains representing 17 species with two different strains for Aspergillus niger and covered six different stress conditions. Stress treatments were selected considering the frequency of various stress tolerance studies published in the last decade in the aspergilli and included oxidative (H2O2, menadione sodium bisulphite), high-osmolarity (NaCl, sorbitol), cell wall integrity (Congo Red) and heavy metal (CdCl2) stress exposures. In the future, we would like to expand this database to accommodate further fungal species and stress treatments. URL: http://www.fung-stress.org/

Transcriptome-Based Modeling Reveals that Oxidative Stress Induces Modulation of the AtfA-Dependent Signaling Networks in Aspergillus nidulans.

To better understand the molecular functions of the master stress-response regulator AtfA in Aspergillus nidulans, transcriptomic analyses of the atfA null mutant and the appropriate control strains exposed to menadione sodium bisulfite- (MSB-), t-butylhydroperoxide- and diamide-induced oxidative stresses were performed. Several elements of oxidative stress response were differentially expressed. Many of them, including the downregulation of the mitotic cell cycle, as the MSB stress-specific upregulation of FeS cluster assembly and the MSB stress-specific downregulation of nitrate reduction, tricarboxylic acid cycle, and ER to Golgi vesicle-mediated transport, showed AtfA dependence. To elucidate the potential global regulatory role of AtfA governing expression of a high number of genes with very versatile biological functions, we devised a model based on the comprehensive transcriptomic data. Our model suggests that an important function of AtfA is to modulate the transduction of stress signals. Although it may regulate directly only a limited number of genes, these include elements of the signaling network, for example, members of the two-component signal transduction systems. AtfA acts in a stress-specific manner, which may increase further the number and diversity of AtfA-dependent genes. Our model sheds light on the versatility of the physiological functions of AtfA and its orthologs in fungi.

Study on the glutathione metabolism of the filamentous fungus Aspergillus nidulans.

Yeast protein sequence-based homology search for glutathione (GSH) metabolic enzymes and GSH transporters demonstrated that Aspergillus nidulans has a robust GSH uptake and metabolic system with several paralogous genes. In wet laboratory experiments, two key genes of GSH metabolism, gcsA, and glrA, encoding γ-l-glutamyl-l-cysteine synthetase and glutathione reductase, respectively, were deleted. The gene gcsA was essential, and the ΔgcsA mutant required GSH supplementation at considerably higher concentration than the Saccharomyces cerevisiae gsh1 mutant (8-10 mmol l-1 vs. 0.5 µmol l-1). In addition to some functions known previously, both genes were important in the germination of conidiospores, and both gene deletion strains required the addition of extra GSH to reach wild-type germination rates in liquid cultures. Nevertheless, the supplementation of cultures with 10 mmol l-1 GSH was toxic for the control and ΔglrA strains especially during vegetative growth, which should be considered in future development of high GSH-producer fungal strains. Importantly, the ΔglrA strain was characterized by increased sensitivity toward a wide spectrum of osmotic, cell wall integrity and antimycotic stress conditions in addition to previously reported temperature and oxidative stress sensitivities. These novel phenotypes underline the distinguished functions of GSH and GSH metabolic enzymes in the stress responses of fungi.

Stress tolerances of nullmutants of function-unknown genes encoding menadione stress-responsive proteins in Aspergillus nidulans.

A group of menadione stress-responsive function-unkown genes of Aspergillus nidulans (Locus IDs ANID_03987.1, ANID_06058.1, ANID_10219.1, and ANID_10260.1) was deleted and phenotypically characterized. Importantly, comparative and phylogenetic analyses of the tested A. nidulans genes and their orthologs shed light only on the presence of a TANGO2 domain with NRDE protein motif in the translated ANID_06058.1 gene but did not reveal any recognizable protein-encoding domains in other protein sequences. The gene deletion strains were subjected to oxidative, osmotic, and metal ion stress and, surprisingly, only the ΔANID_10219.1 mutant showed an increased sensitivity to 0.12 mmol l(-1) menadione sodium bisulfite. The gene deletions affected the stress sensitivities (tolerances) irregularly, for example, some strains grew more slowly when exposed to various oxidants and/or osmotic stress generating agents, meanwhile the ΔANID_10260.1 mutant possessed a wild-type tolerance to all stressors tested. Our results are in line with earlier studies demonstrating that the deletions of stress-responsive genes do not confer necessarily any stress-sensitivity phenotypes, which can be attributed to compensatory mechanisms based on other elements of the stress response system with overlapping functions.

Core oxidative stress response in Aspergillus nidulans.

BACKGROUND: The b-Zip transcription factor AtfA plays a key role in regulating stress responses in the filamentous fungus Aspergillus nidulans. To identify the core regulons of AtfA, we examined genome-wide expression changes caused by various stresses in the presence/absence of AtfA using A. nidulans microarrays. We also intended to address the intriguing question regarding the existence of core environmental stress response in this important model eukaryote. RESULTS: Examination of the genome wide expression changes caused by five different oxidative stress conditions in wild type and the atfA null mutant has identified a significant number of stereotypically regulated genes (Core Oxidative Stress Response genes). The deletion of atfA increased the oxidative stress sensitivity of A. nidulans and affected mRNA accumulation of several genes under both unstressed and stressed conditions. The numbers of genes under the AtfA control appear to be specific to a stress-type. We also found that both oxidative and salt stresses induced expression of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of additional clusters. Moreover, certain clusters were down-regulated by the stresses tested. CONCLUSION: Our data suggest that the observed co-regulations were most likely consequences of the overlapping physiological effects of the stressors and not of the existence of a general environmental stress response. The function of AtfA in governing various stress responses is much smaller than anticipated and/or other regulators may play a redundant or overlapping role with AtfA. Both stress inducible and stress repressive regulations of secondary metabolism seem to be frequent features in A. nidulans.