Analysis of the ionic interaction between the hydrophobin RodA and two cutinases of Aspergillus nidulans obtained via an Aspergillus oryzae expression system

Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. In the fungus Aspergillus oryzae, the hydrophobin RolA and the polyesterase CutL1 are co-expressed when the sole available carbon source is the biodegradable polyester polybutylene succinate-co-adipate (PBSA). RolA promotes the degradation of PBSA by attaching to the particle surface, changing its structure and interacting with CutL1 to concentrate CutL1 on the PBSA surface. We previously reported that positively charged residues in RolA and negatively charged residues in CutL1 are cooperatively involved in the ionic interaction between RolA and CutL1. We also reported that hydrophobin RodA of the model fungus Aspergillus nidulans, which was obtained via an A. oryzae expression system, interacted via ionic interactions with CutL1. In the present study, phylogenetic and alignment analyses revealed that the N-terminal regions of several RolA orthologs contained positively charged residues and that the corresponding negatively charged residues on the surface of CutL1 that were essential for the RolA–CutL1 interaction were highly conserved in several CutL1 orthologs. A PBSA microparticle degradation assay, a pull-down assay using a dispersion of Teflon particles, and a kinetic analysis using a quartz crystal microbalance revealed that recombinant A. nidulans RodA interacted via ionic interactions with two recombinant A. nidulans cutinases. Together, these results imply that ionic interactions between hydrophobins and cutinases may be common among aspergilli and other filamentous fungi.

     

The evolutionary history of Cytochrome P450 genes in four filamentous Ascomycetes

Background  

The Cytochrome P450 system is important in fungal evolution for adapting to novel ecological niches. To elucidate the evolutionary process of cytochrome P450 genes in fungi with different life styles, we studied the patterns of gene gains and losses in the genomes of four filamentous Ascomycetes, including two saprotrophs (Aspergillus nidulans (AN) and Neurospora crassa (NC)) and two plant pathogens (Fusarium graminearum (FG) and Magnaporthe grisea (MG)).     

Effect of ozone on spore germination,spore production and biomass production in two <Emphasis Type="Italic">Aspergillus</Emphasis> species

The ability of ozone gas to reduce food spoilage is relatively well documented, but the developmental effects of the gas on food spoilage fungi are not well known. In this study two model aspergilli, Aspergillus nidulans and Aspergillus ochraceus were used to study the effects of ozone on spore germination, sporulation and biomass production. These effects were examined under three levels of ozone; two high level ozone exposures (200 and 300 μmol mol⁻¹) and one low level exposure (0.2 μmol mol⁻¹). The two species behaved noticeably different to each other. Ozone was more effective in reducing growth from spore inocula than mycelia. No spore production could be detected in A. nidulans exposed to continuous low level O₃, whereas the same treatment reduced spores produced in A. ochraceus by 94%. Overall the work suggests that ozone exposure is an effective method to prevent spread of fungal spores in a food storage situation.     

Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

The genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans by deleting the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). The csnE/CSN5 gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs.     

Sporogenic Effect of Polyunsaturated Fatty Acids on Development of Aspergillus spp.

Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in some A. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intact veA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.     

Recent advances in genome mining of secondary metabolite biosynthetic gene clusters and the development of heterologous expression systems in Aspergillus nidulans

Fungi are prolific producers of secondary metabolites (SMs) that show a variety of biological activities. Recent advances in genome sequencing have shown that fungal genomes harbor far more SM gene clusters than are expressed under conventional laboratory conditions. Activation of these “silent” gene clusters is a major challenge, and many approaches have been taken to attempt to activate them and, thus, unlock the vast treasure chest of fungal SMs. This review will cover recent advances in genome mining of SMs in Aspergillus nidulans. We will also discuss current updates in gene annotation of A. nidulans and recent developments in A. nidulans as a molecular genetic system, both of which are essential for rapid and efficient experimental verification of SM gene clusters on a genome-wide scale. Finally, we will describe advances in the use of A. nidulans as a heterologous expression system to aid in the analysis of SM gene clusters from other fungal species that do not have an established molecular genetic system.     

Evidence for an arginine-dependent route for the synthesis of NO in the model filamentous fungus Aspergillus nidulans

Nitric oxide (NO) is a signalling molecule in eukaryotic and prokaryotic organisms. NO levels transiently boost upon induction of conidiation in Aspergillus nidulans. Only one pathway for NO synthesis involving nitrate reductase has been reported in filamentous fungi so far, but this does not satisfy all the NO produced in fungal cells. Here we provide evidence for at least one additional biosynthetic pathway in A. nidulans involving l -arginine or an intermediate metabolite as a substrate. Under certain growth conditions, the addition of l -arginine to liquid media elicited a burst of NO that was not dependent on any of the urea cycle genes. The NO levels were controlled by the metabolically available arginine, which was regulated by mobilization from the vacuoles and during development. In vitro assays with protein extracts and amino acid profiling strongly suggested the existence of an arginine-dependent NO pathway analogous to the mammalian NO synthase. Addition of polyamines induced NO synthesis, and mutations in the polyamine synthesis genes puA and spdA reduced the production of NO. In conclusion, here we report an additional pathway for the synthesis of NO in A. nidulans using urea cycle intermediates.     

The extracellular β‐1,3‐endoglucanase EngA is involved in autolysis of Aspergillus nidulans

Aims: To elucidate the roles of the β‐1,3‐endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. Methods and Results: A β‐1,3‐endoglucanase was purified from carbon‐starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene‐expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. Conclusions: The β‐1,3‐endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall–degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. Significance and Impact of the Study: No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.     

Monitoring novel metabolic pathways using metabolomics and machine learning: induction of the phosphoketolase pathway in Aspergillus nidulans cultivations

The physiological phenotype of Aspergillus nidulans was determined under different environmental conditions through the quantification of the intracellular and extracellular metabolite pools and clear evidence of the presence of a novel fungal metabolic pathway, the phosphoketolase pathway, was obtained. Induction of the phosphoketolase pathway resulted after blocking the EMP pathway through the deactivation of glyceraldehyde-3-P dehydrogenase (G3PD). Deactivation of G3PD in cultivations of A. nidulans on glucose and xylose led to a 10-fold decrease in the specific growth rate; however, growth could still be sustained solely through the phosphoketolase pathway. Metabolomics and machine learning tools were successfully used to monitor the alteration caused by the inhibition of G3PD in the metabolism of A. nidulans grown on glucose, xylose, acetate as well as mixtures of glucose or xylose with acetate. This is the first study that demonstrates in vivo that the fungal central carbon metabolism includes an active phosphoketolase pathway.     

Methionine Requirements of Rats in Various Body Weights

A niaD gene encoding nitrate reductase was isolated from Aspergillus oryzae KBN616 and sequenced. The structural gene comprises 2973 bp and 868 amino acids, which showed a high degree of similarity to nitrate reductases from other filamentous fungi. The coding sequence is interrupted by six introns varying in size from 48 to 98 bp. The intron positions are all conserved among the niaD genes from A. oryzae, Aspergillus nidulans, and Aspergillus niger. A homologous transformation system was developed for an industrial shoyu koji mold, A. oryzae KBN616, based on the nitrate reductase (niaD) of the nitrate assimilation pathway.     

Multicellular Ascomycetous Fungal Genomes Contain More Than 8000 Genes

Fungi comprise a large monophyletic group of uni- and multicellular eukaryotic organisms in which many species are of economic or medical importance. Fungal genomes are variable in size (13–42 Mb), and multicellular species support true spatial and temporal cell-type-specific regulation of gene expression. In a 38.8-kbAspergillus nidulanscontiguous genomic DNA region, a transposable element and 12 potential genes were identified, 7 similar to genes in other organisms. This observation is consistent with the prediction that multicellular ascomycetous fungi harbor 8000–9000 genes in a 36-Mb average genome. Thus, the genomic DNA sequence of filamentous fungi will provide substantial amounts of genetic and functional information that is not available in yeast, for the human and other metazoan minimal gene complement.     

Screening of antifungal activity of various plant leaves extracts from Indian plants

The present study was designated to evaluate the antifungal activity and to root out the antifungal plant leaf extracts from this Indian folk-flore. The in vitro antifungal assay was performed by agar diffusion test and minimum inhibitory concentration (MIC) for hexane, ethyl acetate, methanol and distilled water plant leaf extracts. Extraction of 17 different plant leaves was carried out in different solvents such as hexane, ethyl acetate, methanol and distilled water. Among them extractive yield of methanol was maximum than the rest of the three solvents. These extracts were screened for their antifungal activity against nine different fungi. Among these ethyl acetate extracts of Adhatoda vasica, Ocimum sanctum and Holoptelea integrifolia exhibited maximum antifungal activity against Alternaria sp., Aspergillus parasi, Aspergillus nidulans, Trichoderma harzianum and Aspergillus flavus with MIC of 80, 40 and 20 ppm against Aspergillus nidulans and Alternaria sp. Ethyl acetate extracts showed promising antifungal activity against Adhatoda vasica, Ocimum sanctum and Holoptelea integrifolia against Aspergillus nidulans, and Alternaria sp. might be applicable as fungicide against fungal plants disease.