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The contribution of stress protein duplication and deletion events to the evolution of the Aspergilli was studied. We performed a large-scale homology analysis of stress proteins and generated and analysed three stress defence system models based on Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Although both yeast-based and A. nidulans-based models were suitable to trace evolutionary changes, the A. nidulans-based model performed better in mapping stress protein radiations. The strong Mantel correlation found between the positions of species in the phylogenetic tree on the one hand and either in the A. nidulans-based or S. cerevisiae-based models on the other hand demonstrated that stress protein expansions and reductions contributed significantly to the evolution of the Aspergilli. Interestingly, stress tolerance attributes correlated well with the number of orthologs only for a few stress proteins. Notable examples are Ftr1 iron permease and Fet3 ferro-O2-oxidoreductase, elements of the reductive iron assimilation pathway, in the S. cerevisiae-based model, as well as MpkC, a HogA-like mitogen activated protein kinase in the A. nidulans-based model. In the case of the iron assimilation proteins, the number of orthologs showed a positive correlation with H2O2-induced stress tolerance while the number of MpkC orthologs correlated positively with Congo Red induced cell wall stress, sorbitol induced osmotic stress and H2O2 induced oxidative stress tolerances. For most stress proteins, changes in the number of orthologs did not correlate well with any stress tolerance attributes. As a consequence, stress tolerance patterns of the studied Aspergilli did not correlate with either the sets of stress response proteins in general or with the phylogeny of the species studied. These observations suggest that stress protein duplication and deletion events significantly contributed to the evolution of stress tolerance attributes of Aspergilli. In contrast, there are other processes, which may counterbalance the effects of stress gene duplications or deletions including (i) alterations in the structures of stress proteins leading to changes in their biological activities, (ii) varying biosynthesis of stress proteins, (iii) rewiring stress response regulatory networks or even (iv) acquiring new stress response genes by horizontal gene transfer. All these multilevel changes are indispensable for the successful adaptation of filamentous fungi to altering environmental conditions, especially when these organisms are entering new ecological niches.

N-Acetyl- d-glucosamine, chito-oligomers and carbon starvation regulated chiA, chiB, and nagA gene expressions in Aspergillus nidulans cultures. The gene expression patterns of the main extracellular endochitinase ChiB and the N-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-related chiA or the autolysis-coupled chiB genes although both of them were down-regulated under diamide stress. The transcription of the chiC gene with unknown physiological function was repressed by increased intracellular superoxide concentration. [ABSTRACT FROM AUTHOR]

é. leiter, f. marx, t. pusztahelyi, h. haas and i. pócsi. 2004.Purification and characterization of the high molecular massCandida albicans-killing protein secreted byPenicillium chrysogenum.The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (Mr) = 155 000, subunit structureα2 withMr,α = 76 000, isoelectric point (pI) = 5·4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1·0 mg ml−1) prevented oxidative cell injuries triggered by 0·004 U GOX inEmericella nidulanscultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0·004 : 200 U. A secondary inhibition of growth inE. nidulanscultures by the oxygen-depleting GOX–catalase system was likely to replace the primary inhibition exerted by H2O2.Penicillium chrysogenumGOX possesses similar enzymological features to those described earlier for otherPenicilliumGOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms.Penicillium chrysogenumGOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent. [ABSTRACT FROM AUTHOR]

Physiological and morphological changes in carbon-limited autolyzing cultures of Aspergillus nidulans were described. The carbon starvation arrested conidiation while the formation of filamentous and “yeast-like” hyphal fragments with profoundly altered metabolism enabled the fungus to survive the nutritional stress. The morphological and physiological stress responses, which maintained the cellular integrity of surviving hyphal fragments at the expense of autolyzing cells, were highly concerted and regulated. Moreover, sublethal concentrations of the protein synthesis inhibitor cycloheximide or the mitochondrial uncoupler 2,4-dinitrophenol completely blocked the autolysis. In accordance with the propositions of the free-radical theory of ageing reactive oxygen species accumulated in the surviving fragments with a concomitant increase in the specific superoxide dismutase activity and a continuous decrease in cell viability. Glutathione was degraded extensively in carbon-starving cells due to the action of γ-glutamyltranspeptidase, which resulted in a glutathione-glutathione disulfide redox imbalance during autolysis. [ABSTRACT FROM AUTHOR]

Methionine increased the intracellular glutathione (reduced) (GSH) pool and the specific γ-glutamyltransferase ( γ-GT) activity in the cephalosporin C (CPC) producer Acremonium chrysogenum. The accelerated turnover of GSH might be indicative of the existence of a functioning γ-glutamate cycle, and might supply the CPC biosynthetic machinery with l-cysteine. The γ-GT was not subject to nitrogen metabolic repression but was more active in cells exposed to different oxidative-stress-generating agents. Exogenous cysteine hindered both the uptake of methionine and the induction of γ-GT, and was not beneficial for CPC production. There was no causal relationship between the redox status of the cells and the observed cell morphology. [ABSTRACT FROM AUTHOR]

Cyclodextrin glycosyltransferase (CGTase) activity was monitored in Bacillus macerans culture fluids up to 56 h incubation time using glucose (G1), maltose (G2), maltotriose (G3), maltoheptaose (G7), α-,β-,γ-cyclodextrins (CDs) and soluble starch as carbon sources. Highest maximum specific growth rates (µmax) were observed with glucose, γ-CD and soluble starch (µmax values were 0.86, 0.74 and 0.69/h, respectively) while the maximum viable cell numbers were always within the range of 2.3–7.1×1011 CFU/mL independently of the carbon source used. Highest CGTase production was found in the presence of soluble starch and G7 (55.0 and 35.4 nkat/mL, respectively), these saccharides being easily transformed to CDs by CGTase. Moreover, when culture media were supplemented with cyclic malto-oligosaccharides the CGTase activities were about twice higher (19.6–20.6 nkat/mL) than those obtained with the linear G2 and G3 saccharides (8.9 and 11.3 nkat/mL, respectively) which give rise only to negligible quantities of CDs. CDs, which are the major end products of the action of CGTase, are regarded thus as the likely physiological inducers of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract Background In 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. Results Genome-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. Conclusion The 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.

Xerophilic fungi occupy versatile environments owing to their rich arsenal helping them successfully adapt to water constraints as a result of low relative humidity, high-osmolarity, and high-salinity conditions. The general term xerophilic fungi relates to organisms that tolerate and/or require reduced water activity, while halophilic and osmophilic are applied to specialized groups that require high salt concentrations or increased osmotic pressure, respectively. Species belonging to the family Aspergillaceae, and especially those classified in Aspergillus subgenus Aspergillus (sections Restricti and Aspergillus) and Polypaecilum, are particularly enriched in the group of osmophilic and salt-tolerant filamentous fungi. They produce an unprecedently wide spectrum of salt tolerant enzymes including proteases, peptidases, glutaminases, γ-glutamyl transpeptidases, various glycosidases such as cellulose-decomposing and starch-degrading hydrolases, lipases, tannases, and oxidareductases. These extremophilic fungi also represent a huge untapped treasure chest of yet-to-be-discovered, highly valuable, biologically active secondary metabolites. Furthermore, these organisms are indispensable agents in decolorizing textile dyes, degrading xenobiotics and removing excess ions in high-salt environments. They could also play a role in fermentation processes at low water activity leading to the preparation of daqu, meju, and tea. Considering current and future agricultural applications, salt-tolerant and osmophilic Aspergilli may contribute to the biosolubilization of phosphate in soil and the amelioration salt stress in crops. Transgenes from halophile Aspergilli may find promising applications in the engineering of salt stress and drought-tolerant agricultural crops. Aspergilli may also spoil feed and food and raise mycotoxin concentrations above the permissible doses and, therefore, the development of novel feed and food preservation technologies against these Aspergillus spp. is also urgently needed. On the other hand, some xerophilic Aspergilli have been shown to be promising biological control agents against mites. KEY POINTS: • Salt tolerant and osmophilic Aspergilli can be found in versatile environments • These fungi are rich resources of valuable enzymes and secondary metabolites • Biotechnological and agricultural applications of these fungi are expanding., Competing Interests: Declarations Ethical statement This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare no competing interests., (© 2024. The Author(s).)

Background: Along bacteria, yeasts are common in forages and forage fermentations as spoilage microbes or as additives, yet few studies exist with species-level data on these fungi's occurrence in feedstuff. Active dry yeast and other yeast-based products are also common feed additives in animal husbandry. Here, we aimed to characterize both fermented and non-fermented milking cow feedstuff samples from Hungary to assess their microbial diversity in the first such study from Central Europe., Results: We applied long-read bacterial metabarcoding to 10 fermented and 25 non-fermented types of samples to assess bacterial communities and their characteristics, surveyed culturable mold and yeast abundance, and identified culturable yeast species. Fermented forages showed the abundance of Aerococcaceae, Bacillaceae, Brucellaceae, Lactobacillaceae, Staphylococcaceae, and Thermoactinomycetaceae, non-fermented ones had Cyanothecaceae, Enterobacteriaceae, Erwiniaceae, Gomontiellaceae, Oxalobacteraceae, Rhodobiaceae, Rickettsiaceae, and Staphylococcaceae. Abundances of bacterial families showed mostly weak correlation with yeast CFU numbers, only Microcoleaceae (positive) and Enterococcaceae and Alcaligenaceae (negative correlation) showed moderate correlation. We identified 14 yeast species, most commonly Diutina rugosa, Pichia fermentans, P. kudriavzevii, and Wickerhahomyces anomalus. We recorded S. cerevisiae isolates only from animal feed mixes with added active dry yeast, while the species was completely absent from fermented forages. The S. cerevisiae isolates showed high genetic uniformity., Conclusion: Our results show that both fermented and non-fermented forages harbor diverse bacterial microbiota, with higher alpha diversity in the latter. The bacterial microbiome had an overall weak correlation with yeast abundance, but yeasts were present in the majority of the samples, including four new records for forages as a habitat for yeasts. Yeasts in forages mostly represented common species including opportunistic pathogens, along with a single strain of Saccharomyces used as a feed mix additive., (© 2024. The Author(s).)

The genome of the osmophilic Aspergillus wentii , unlike that of the osmotolerant Aspergillus nidulans , contains only the gfdA , but not the gfdB , glycerol 3-phosphate dehydrogenase gene. Here, we studied transcriptomic changes of A. nidulans (reference strain and Δ gfdB gene deletion mutant) and A. wentii (reference strain and An-gfdB expressing mutant) elicited by high osmolarity. A. nidulans showed a canonic hyperosmotic stress response characterized by the upregulation of the trehalose and glycerol metabolism genes (including gfdB ), as well as the genes of the high-osmolarity glycerol (HOG) map kinase pathway. The deletion of gfdB caused only negligible alterations in the transcriptome, suggesting that the glycerol metabolism was flexible enough to compensate for the missing GfdB activity in this species. A. wentii responded differently to increased osmolarity than did A. nidulans , e.g., the bulk upregulation of the glycerol and trehalose metabolism genes, along with the HOG pathway genes, was not detected. The expression of An-gfdB in A. wentii did not abolish osmophily, but it reduced growth and caused much bigger alterations in the transcriptome than did the missing gfdB gene in A. nidulans . Flexible glycerol metabolism and hence, two differently regulated gfd genes, may be more beneficial for osmotolerant (living under changing osmolarity) than for osmophilic (living under constantly high osmolarity) species.

Although tyrosol is a quorum-sensing molecule of Candida species, it has antifungal activity at supraphysiological concentrations. Here, we studied the effect of tyrosol on the physiology and genome-wide transcription of Aspergillus nidulans to gain insight into the background of the antifungal activity of this compound. Tyrosol efficiently reduced germination of conidia and the growth on various carbon sources at a concentration of 35 mM. The growth inhibition was fungistatic rather than fungicide on glucose and was accompanied with downregulation of 2199 genes related to e.g. mitotic cell cycle, glycolysis, nitrate and sulphate assimilation, chitin biosynthesis, and upregulation of 2250 genes involved in e.g. lipid catabolism, amino acid degradation and lactose utilization. Tyrosol treatment also upregulated genes encoding glutathione-S-transferases (GSTs), increased specific GST activities and the glutathione (GSH) content of the cells, suggesting that A. nidulans can detoxify tyrosol in a GSH-dependent manner even though this process was weak. Tyrosol did not induce oxidative stress in this species, but upregulated "response to nutrient levels", "regulation of nitrogen utilization", "carbon catabolite activation of transcription" and "autophagy" genes. Tyrosol may have disturbed the regulation and orchestration of cellular metabolism, leading to impaired use of nutrients, which resulted in growth reduction., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pathogens have to cope with oxidative, iron- and carbon(glucose)-limitation stresses in the human body. To understand how combined iron-carbon limitation alters oxidative stress responses, Aspergillus fumigatus was cultured in glucose-peptone or peptone containing media supplemented or not with deferiprone as an iron chelator. Changes in the transcriptome in these cultures were recorded after H 2 O 2 treatment. Responses to oxidative stress were highly dependent on the availability of glucose and iron. Out of the 16 stress responsive antioxidative enzyme genes, only the cat2 catalase-peroxidase gene was upregulated in more than two culturing conditions. The transcriptional responses observed in iron metabolism also varied substantially in these cultures. Only extracellular siderophore production appeared important regardless of culturing conditions in oxidative stress protection, while the enhanced synthesis of Fe-S cluster proteins seemed to be crucial for oxidative stress treated iron-limited and fast growing (glucose rich) cultures. Although pathogens and host cells live together in the same place, their culturing conditions (e.g., iron availability or occurrence of oxidative stress) can be different. Therefore, inhibition of a universally important biochemical process, like Fe-S cluster assembly, may selectively inhibit the pathogen growth in vivo and represent a potential target for antifungal therapy.

Mycotoxins produced by Fusarium species are secondary metabolites with low molecular weight formed by filamentous fungi generally resistant to different environmental factors and, therefore, undergo slow degradation. Contamination by Fusarium mycotoxins in cereals and millets is the foremost quality challenge the food and feed industry faces across the globe. Several types of chemical preservatives are employed in the mitigation process of these mycotoxins, and they help in long-term storage; however, chemical preservatives can be used only to some extent, so the complete elimination of toxins from foods is still a herculean task. The growing demand for green-labeled food drives to evade the use of chemicals in the production processes is getting much demand. Thus, the biocontrol of food toxins is important in the developing food sector. Fusarium mycotoxins are world-spread contaminants naturally occurring in commodities, food, and feed. The major mycotoxins Fusarium species produce are deoxynivalenol, fumonisins, zearalenone, and T2/HT2 toxins. Lactic acid bacteria (LAB), generally regarded as safe (GRAS), is a well-explored bacterial community in food preparations and preservation for ages. Recent research suggests that LAB are the best choice for extenuating Fusarium mycotoxins. Apart from Fusarium mycotoxins, this review focuses on the latest studies on the mechanisms of how LAB effectively detoxify and remove these mycotoxins through their various bioactive molecules and background information of these molecules., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Krishnan, Nampoothiri, Suresh, Linh, Balakumaran, Pócsi and Pusztahelyi.)

Manganese superoxide dismutases (MnSODs) play a pivotal role in the preservation of mitochondrial integrity and function in fungi under various endogenous and exogenous stresses. Deletion of Aspergillus nidulans mnSOD/SodB increased oxidative stress sensitivity and apoptotic cell death rates as well as affected antioxidant enzyme and sterigmatocystin productions, respiration, conidiation and the stress tolerance of conidiospores. The physiological consequences of the lack of sodB were more pronounced during carbon starvation than in the presence of glucose. Lack of SodB also affected the changes in the transcriptome, recorded by high-throughput RNA sequencing, in menadione sodium bisulfite (MSB)-exposed, submerged cultures supplemented with glucose. Surprisingly, the difference between the global transcriptional changes of the ΔsodB mutant and the control strain were relatively small, indicating that the SodB-dependent maintenance of mitochondrial integrity was not essential under these experimental conditions. Owing to the outstanding physiological flexibility of the Aspergilli, certain antioxidant enzymes and endogenous antioxidants together with the reduction in mitochondrial functions compensated well for the lack of SodB. The lack of sodB reduced the growth of surface cultures more than of the submerged culture, which should be considered in future development of fungal disinfection methods.

Several P 1B -type ATPases are important Cd 2+ /Cu 2+ pumps in Aspergillus species, and they are tightly associated with the heavy metal stress tolerance of these ascomycetous fungi. To better understand the roles of the two P 1B -type ATPases, Aspergillus nidulans CrpA Cd 2+ /Cu 2+ pump (orthologue of the Candida albicans Crp1 Cd 2+ /Cu 2+ pump) and Aspergillus fumigatus PcaA Cd 2+ pump (orthologue of the Saccharomyces cerevisiae Pca1 Cd 2+ pump), we have generated individual mutants and characterized their heavy metal susceptibilities. The deletion of CrpA in A. nidulans has led to the increased sensitivity of the fungus to stresses induced by Zn 2+ , Fe 2+ , or the combination of oxidative-stress-inducing menadione sodium bisulfite and Fe 3+ . Heterologous expression of A. fumigatus PcaA in the S. cerevisiae pca1 deletion mutant has resulted in enhanced tolerance of the yeast to stresses elicited by Cd 2+ or Zn 2+ but not by Fe 2+ /Fe 3+ or Cu 2+ . Mammalian host immune defense can attack microbes by secreting Zn 2+ or Cu 2+ , and the oxidative stress induced by host immune systems can also disturb metal (Cu 2+ , Fe 2+ , and Zn 2+ ) homeostasis in microbes. In summary, PcaA and CrpA can protect fungal cells from these complex stresses that contribute to the virulence of the pathogenic Aspergillus species. Moreover, due to their presence on the fungal cell surface, these P 1B -type ATPases may serve as a novel drug target in the future. IMPORTANCE Mammalian host immune defense disrupts heavy metal homeostasis of fungal pathogens. P1B-type ATPase of Aspergillus fumigatus and Aspergillus nidulans may help to cope with this stress and serve as virulence traits. In our experiments, both A. nidulans Cd2+/Cu2+ pump CrpA and A. fumigatus Cd2+ pump PcaA protected fungal cells from toxic Zn2+, and CrpA also decreased Fe2+ susceptibility most likely indirectly. In addition, CrpA protected cells against the combined stress induced by the oxidative stressor menadione and Fe3+. Since P1B-type ATPases are present on the fungal cell surface, these proteins may serve as a novel drug target in the future.

Basic leucine zipper (bZIP) transcription factors are crucial components of differentiation, cellular homeostasis and the environmental stress defense of eukaryotes. In this work, we further studied the consequence of gene deletion and overexpression of two bZIP transcription factors, NapA and RsmA, on superoxide production, mitochondrial morphology and hyphal diameter of Aspergillus nidulans. We have found that reactive oxygen species production was influenced by both gene deletion and overexpression of napA under tert-butylhydroperoxide (tBOOH) elicited oxidative stress. Furthermore, gene expression of napA negatively correlated with mitochondrial volumetric ratio as well as sterigmatocystin production of A. nidulans. High rsmA expression was accompanied with elevated relative superoxide ratio in the second hyphal compartment. A negative correlation between the expression of rsmA and catalase enzyme activity or mitochondrial volumetric ratio was also confirmed by statistical analysis. Hyphal diameter was independent on either rsmA and napA expression as well as 0.2 mM tBOOH treatment., (© 2023. The Author(s).)