Heterologous production of bioactive xenoacremone analogs in Aspergillus nidulans.

Tyrosine-decahydrofluorene derivatives are a class of hybrid compounds that integrate the properties of polyketides and nonribosomal peptides. These compounds feature a [6.5.6] tricarbocyclic core and a para-cyclophane ether moiety in their structures and exhibit anti-tumor and anti-microbial activities. In this study, we constructed the biosynthetic pathway of xenoacremones from Xenoacremonium sinensis ML-31 in the Aspergillus nidulans host, resulting in the identification of four novel tyrosine-decahydrofluorene analogs, xenoacremones I-L (1-4), along with two known analogs, xenoacremones A and B. Remarkably, compounds 3 and 4 contained a 12-membered para-cyclophane ring system, which is unprecedented among tyrosine-decahydrofluorene analogs in X. sinensis. The successful reconstruction of the biosynthetic pathway and the discovery of novel analogs demonstrate the utility of heterologous expression strategy for the generation of structurally diverse natural products with potential biological activities.

Urease and Carbonic Anhydrase Inhibitory Effect of Xanthones from Aspergillus nidulans, an Endophytic Fungus of Nyctanthes arbor-tristis.

Urease plays a major role in the pathogenesis of peptic and gastric ulcer and also causes acute pyelonephritis and development of infection-induced reactive arthritis. Carbonic anhydrases (CA) cause pathological disorders such as epilepsy (CA I), glaucoma, gastritis, renal, pancreatic carcinomas, and malignant brain tumors (CA II). Although various synthetic urease and carbonic anhydrase inhibitors are known, these have many side effects. Hence, present studies were undertaken on ethyl acetate extract of Aspergillus nidulans, an endophytic fungus separated from the leaves of Nyctanthes arbor-tristis Linn. and led to the isolation of five furanoxanthones, sterigmatin (1: ), sterigmatocystin (3: ), dihydrosterigmatocystin (4: ), oxisterigmatocystin C (5: ), acyl-hemiacetal sterigmatocystin (6: ), and a pyranoxanthone (2: ). Acetylation of 3: gave compound O-acetyl sterigmatocystin (7: ). Their chemical structures were elucidated by 1H and 13C NMR and MS. The inhibitory effect of isolated compounds was evaluated on urease and carbonic anhydrase (bCA II) enzymes in vitro. Compounds 3: and 6: showed significant urease inhibition (IC50 19 and 21 µM), while other compounds exhibited varying degrees of urease inhibition (IC50 33 - 51 µM). Compounds 4, 6: and 7: exhibited significant inhibition of bCA II (IC50 values 21, 25 and 18 µM respectively), compounds 1: -3: displayed moderate inhibition (IC50 61, 76 and 31 µM respectively) while 5: showed no inhibition. A mechanistic study of the most active urease inhibitors was also performed using enzyme kinetics and molecular docking. All compounds were found non-toxic on the NIH-3T3 cell line.

Biochemical Characterization of a Pectate Lyase AnPL9 from Aspergillus nidulans.

Pectinolytic enzymes have diverse industrial applications. Among these, pectate lyases act on the internal α-1,4-linkage of the pectate backbone, playing a critical role in pectin degradation. While most pectate lyases characterized thus far are of bacterial origin, fungi can also be excellent sources of pectinolytic enzymes. In this study, we performed biochemical characterization of the pectate lyase AnPL9 belonging to the polysaccharide lyase family 9 (PL9) from the filamentous fungus Aspergillus nidulans. Recombinant AnPL9 was produced using a Pichia pastoris expression system and purified. AnPL9 exhibited high activity on homogalacturonan (HG), pectin from citrus peel, pectin from apple, and the HG region in rhamnogalacturonan-I. Although digalacturonic acid and trigalacturonic acid were not degraded by AnPL9, tetragalacturonic acid was converted to 4,5-unsaturated digalacturonic acid and digalacturonic acid. These results indicate that AnPL9 degrades HG oligosaccharides with a degree of polymerization > 4. Furthermore, AnPL9 was stable within a neutral-to-alkaline pH range (pH 6.0-11.0). Our findings suggest that AnPL9 is a candidate pectate lyase for biotechnological applications in the food, paper, and textile industries. This is the first report on a fungal pectate lyase belonging to the PL9 family.

l-Arabinose induces d-galactose catabolism via the Leloir pathway in Aspergillus nidulans.

l-Arabinose and d-galactose are the principal constituents of l-arabinogalactan, and also co-occur in other hemicelluloses and pectins. In this work we hypothesized that similar to the induction of relevant glycoside hydrolases by monomers liberated from these plant heteropolymers, their respective catabolisms in saprophytic and phytopathogenic fungi may respond to the presence of the other sugar to promote synergistic use of the complex growth substrate. We showed that these two sugars are indeed consumed simultaneously by Aspergillus nidulans, while l-arabinose is utilised faster in the presence than in the absence of d-galactose. Furthermore, the first two genes of the Leloir pathway for d-galactose catabolism - encoding d-galactose 1-epimerase and galactokinase - are induced more rapidly by l-arabinose than by d-galactose eventhough deletion mutants thereof grow as well as a wild type strain on the pentose. d-Galactose 1-epimerase is hyperinduced by l-arabinose, d-xylose and l-arabitol but not by xylitol. The results suggest that in A. nidulans, l-arabinose and d-xylose - both requiring NADPH for their catabolisation - actively promote the enzyme infrastructure necessary to convert ß-d-galactopyranose via the Leloir pathway with its α-anomer specific enzymes, into ß-d-glucose-6-phosphate (the starting substrate of the oxidative part of the pentose phosphate pathway) even in the absence of d-galactose.

Production of an emericellin and its analogues as fungal biological responses for Shimbu-to extract.

This research examined the production of fungal metabolites as a biological response to Kampo medicines. Shimbu-to (SMB) is a Kampo medicine composed of five herbal components: peony root (Shakuyaku), ginger (Shokyo), processed aconite root (Bushi), Poria sclerotium (Bukuryo), and Atractylodes lancea rhizomes (Sojutsu). High-performance liquid chromatography (HPLC) analysis of the fungus Aspergillus nidulans CBS 112.46 incubated in potato dextrose broth supplemented with SMB extract revealed emericellin (2) as the major peak and new xanthone analogues 24-hydroxyshamixanthone (1), shamixanthone (3), epishamixanthone (4), pre-shamixanthone (5), and variecoxanthone A (6) as minor peaks. The structure of 1 was determined by detailed analysis of 1D-NMR, 2D-NMR, and MS data. The results suggest that SMB extract regulates the biosynthesis of emericellin and its analogues in A. nidulans. Further investigations revealed that glucose induces the biosynthesis of emericellin and its analogues in A. nidulans in a concentration-dependent manner.

Transcriptomic and metabolomic profiling of ionic liquid stimuli unveils enhanced secondary metabolism in Aspergillus nidulans.

BACKGROUND: The inherent potential of filamentous fungi, especially of Ascomycota, for producing diverse bioactive metabolites remains largely silent under standard laboratory culture conditions. Innumerable strategies have been described to trigger their production, one of the simplest being manipulation of the growth media composition. Supplementing media with ionic liquids surprisingly enhanced the diversity of extracellular metabolites generated by penicillia. This finding led us to evaluate the impact of ionic liquids' stimuli on the fungal metabolism in Aspergillus nidulans and how it reflects on the biosynthesis of secondary metabolites (SMs). RESULTS: Whole transcriptional profiling showed that exposure to 0.7 M cholinium chloride or 1-ethyl-3-methylimidazolium chloride dramatically affected expression of genes encoding both primary and secondary metabolism. Both ionic liquids apparently induced stress responses and detoxification mechanisms but response profiles to each stimulus were unique. Primary metabolism was up-regulated by choline, but down-regulated by 1-ethyl-3-methylimidazolium chloride; both stimulated production of acetyl-CoA (key precursor to numerous SMs) and non proteinogenic amino acids (building blocks of bioactive classes of SMs). In total, twenty one of the sixty six described backbone genes underwent up-regulation. Accordingly, differential analysis of the fungal metabolome showed that supplementing growth media with ionic liquids resulted in ca. 40 differentially accumulated ion masses compared to control conditions. In particular, it stimulated production of monodictyphenone and orsellinic acid, otherwise cryptic. Expression levels of genes encoding corresponding polyketide biosynthetic enzymes (i.e. backbone genes) increased compared to control conditions. The corresponding metabolite extracts showed increased cell polarity modulation potential in an ex vivo whole tissue assay (The lial Live Targeted Epithelia; theLiTE™). CONCLUSIONS: Ionic liquids, a diverse class of chemicals composed solely of ions, can provide an unexpected means to further resolve the diversity of natural compounds, guiding discovery of fungal metabolites with clinical potential.

Helvafuranone Produced by the Fungus Aspergillus nidulans BF0142 Isolated from Hot Spring-derived Soil.

The fungus, Aspergillus nidulans BFO 142, was isolated from hot spring-derived soil collected at Hell Valley in Noboribetsu, Hokkaido, Japan. A new furanone compound designated helvafuranone (1) was isolated along with microperfuranone (2), 9-hydroxymicroperfuranone (3), diorcinol (4), emestrin (5), and sterigmatocystin (6), from a culture broth of A. nidulans BF0142. The structure of 1 was elucidated as 5-hydroxy-4-(4-hydroxybenzyl)-3-(4- hydroxybenzyl)furanone based on various NMR experiments and chemical modifications.

Efeito da Aloe arborescens Miller e da Aloe barbadensis Miller sobre o desenvolvimento vegetativo em Aspergillus nidulans / Effects of the Aloe arborescens Millerand Aloe barbadensis Milleron over vegetative growth in the Aspergillus nidulans

RESUMOA pesquisa de produtos naturais benéficos à saúde humana vem crescendo nos últimos 20 anos. Considerando que as plantas de Aloe são amplamente utilizadas pela população humana, em geral de maneira terapêutica, o objetivo deste estudo foi avaliar os efeitos de Aloearborescens Miller e Aloe barbadensis Miller, sobre o desenvolvimento vegetativo de linhagens normais e mutantes de Aspergillus nidulans. Conídios da linhagem biA1methG1, MSE e CLB3 de A. nidulans, foram inoculados em meio completo sem (Controle) e com extratos das duas espécies incubados por 2, 4, 6 e 8 horas a 37ºC, no escuro. Foi analisado em microscópio óptico, 200 conídios de cada tratamento. Para o desenvolvimento das colônias, as linhagens foram inoculadas no centro das placas juntamente com o meio de cultura sólido e sobre a membrana de diálise, visando a medição do diâmetro e do peso. A análise estatística foi baseada no teste de Tukey e todos os procedimentos experimentais foram conduzidos em triplicata. Todas as linhagens apresentaram interferências positivas quando expostas às plantas de Aloe, porém, de maneira variada. Ambas as espécies aceleraram a germinação em todas as linhagens testadas e atuaram na redução significativa de conídios mortos e/ou malformados. Em relação ao desenvolvimento vegetativo, todos os dados referentes ao peso úmido e diâmetro corrigido dos tratamentos demonstraram progressos, contudo, a razão diâmetro/peso apresentou somente na linhagem MSE, ação favorável dos tratamentos naturais. As informações deste estudo sugerem benefícios de A. arborescens e A. barbadensis, justificando a importância e continuidade da investigação, para melhor elucidar os mecanismos de ação dessas plantas.

ABSTRACTThe researches about natural products that arebeneficial to human health have been growing over the past 20 years. Since Aloe plants are broadly used by the general population, frequently due to therapeutic reasons, the objective of this study was to evaluate the effects of Aloe arborescens Millerand Aloe barbadensis Miller on the vegetative growth of normal and mutant strains of Aspergillus nidulans. The conidia of thebiA1methG1, MSE and CLB3 strains of A. nidulans were inoculated in complete environment without (control) and with extracts of two species of Aloeincubated for 2, 4, 6 and 8 hours at 37˚C. 200 conidia were analyzed by optical microscopy. For the development of the colonies, the strains were inoculated in the center of the plates together with the solid environment of the cultivation and over the dialysis membrane for measuring the diameter and weighing. The statistical analysis was based on the Tukey test and all experimental procedures were performed in triplicate. All strains showed positive interference when exposed to Aloe plants, however, through different manners. Both species have accelerated the germination in all tested strains and acted in the significant reduction of dead and / or malformed conidia. Regarding the vegetative growth, all data related to wet weight and corrected diameter of the treatments revealed progress, however, the ratio diameter/weightpresented improvement only in the MSE lineage, favorable action of natural treatments. The information from this study suggest that A. arborescens and A. barbadensis are beneficial, thus justifying the importance of research maintenance in order to better elucidate the action mechanisms of these plants.

Inhibition of Tau aggregation by three Aspergillus nidulans secondary metabolites: 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde.

The aggregation of the microtubule-associated protein tau is a significant event in many neurodegenerative diseases including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activity. We have screened Aspergillus nidulans secondary metabolites containing aromatic ring structures for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol and the previously identified aggregation inhibitor emodin as a positive control. While several compounds showed some activity, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde were potent aggregation inhibitors as determined by both a filter trap assay and electron microscopy. In this study, these three compounds were stronger inhibitors than emodin, which has been shown in a prior study to inhibit the heparin induction of tau aggregation with an IC50 of 1-5 µM. Additionally, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde reduced, but did not block, tau stabilization of microtubules. 2,ω-Dihydroxyemodin and asperthecin have similar structures to previously identified tau aggregation inhibitors, while asperbenzaldehyde represents a new class of compounds with tau aggregation inhibitor activity. Asperbenzaldehyde can be readily modified into compounds with strong lipoxygenase inhibitor activity, suggesting that compounds derived from asperbenzaldehyde could have dual activity. Together, our data demonstrates the potential of 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde as lead compounds for further development as therapeutics to inhibit tau aggregation in Alzheimer's disease and neurodegenerative tauopathies.

The Aspergillus giganteus antifungal protein AFPNN5353 activates the cell wall integrity pathway and perturbs calcium homeostasis.

BACKGROUND: The antifungal protein AFPNN5353 is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger. RESULTS: We determined an AFPNN5353 hypersensitive phenotype of non-functional A. nidulans mutants in the protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the α-glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall integrity pathway (CWIP) and the remodelling of the cell wall in response to AFPNN5353. The activation of the CWIP by AFPNN5353, however, operates independently from RhoA which is the central regulator of CWIP signal transduction in fungi.Furthermore, we provide evidence that calcium (Ca2+) signalling plays an important role in the mechanistic function of this antifungal protein. AFPNN5353 increased about 2-fold the cytosolic free Ca2+ ([Ca2+]c) of a transgenic A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl2 counteracted AFPNN5353 toxicity, ameliorated the perturbation of the [Ca2+]c resting level and prevented protein uptake into Aspergillus sp. cells. CONCLUSIONS: The present study contributes new insights into the molecular mechanisms of action of the A. giganteus antifungal protein AFPNN5353. We identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the CWIP and the Ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. This knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture.

The effect of resource quantity and resource stoichiometry on microbial carbon-use-efficiency.

The carbon-use-efficiency (CUE) of microorganisms is an important parameter in determining ecosystem-level carbon (C) cycling; however, little is known about how variance in resources affects microbial CUE. To elucidate how resource quantity and resource stoichiometry affect microbial CUE, we cultured four microorganisms - two fungi (Aspergillus nidulans and Trichoderma harzianum) and two bacteria (Pectobacterium carotovorum and Verrucomicrobium spinosum) - under 12 unique C, nitrogen (N) and phosphorus (P) ratios. Whereas the CUE of A. nidulans was strongly affected by C, bacterial CUE was more strongly affected by mineral nutrients (N and P). Specifically, CUE in P. carotovorum was positively correlated with P, while CUE of V. spinosum primarily depended on N. This resulted in a positive relationship between fungal CUE and resource C : nutrient stoichiometry and a negative relationship between bacterial CUE and resource C : nutrient stoichiometry. The difference in the direction of the relationship between CUE and C : nutrient for fungi vs. bacteria was consistent with differences in biomass stoichiometry and suggested that fungi have a higher C demand than bacteria. These results suggest that the links between biomass stoichiometry, resource demand and CUE may provide a mechanism for commonly observed temporal and spatial patterns in microbial community structure and function in natural habitats.

Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans.

The function of seven paralogues phylogenetically related to the Saccharomyces cerevisiae Fur4p together with a number of functionally related transporters present in Aspergillus nidulans has been investigated. After deletion of the cognate genes we checked the incorporation of radiolabelled substrates, utilization of nitrogen sources, resistance to toxic analogues and supplementation of auxotrophies. FurA and FurD encode allantoin and uracil transporters respectively. No function was found for FurB, FurC, FurE, FurF and FurG. As we failed to identify Fur-related transporters for uridine, pyridoxine or thiamine, we deleted other possible candidates for these functions. A FCY2-like gene carrying in its 5' UTR a putative thiamine pyrophosphate riboswitch, and which encodes a protein similar to the pyridoxine transporter of yeast (Tpn1p), does not encode either a major thiamine or a pyridoxine transporter. CntA, a member of the concentrative nucleoside transporter family, is a general nucleoside permease, while no function was found for PnpA, a member of the equilibrative transporter family. Phylogenetic analysis shows that within the ascomycetes, the same transport activity could be catalysed by totally unrelated proteins and that within the Fur subfamily convergent evolution towards uracil and allantoin transport activity has occurred at least three and two independent times respectively.

An evolutionary conserved d-galacturonic acid metabolic pathway operates across filamentous fungi capable of pectin degradation.

Transcriptome analysis of Aspergillus niger transfer cultures grown on galacturonic acid media identified a highly correlating cluster of four strongly induced hypothetical genes linked with a subset set of genes encoding pectin degrading enzymes. Three of the encoded hypothetical proteins now designated GAAA to GAAC are directly involved in further galacturonic acid catabolism. Functional and biochemical analysis revealed that GAAA is a novel d-galacturonic acid reductase. Two non-allelic Aspergillus nidulans strains unable to utilize galacturonic acid are mutated in orthologs of gaaA and gaaB, respectively. The A. niger gaaA and gaaC genes share a common promoter region. This feature appears to be strictly conserved in the genomes of plant cell wall degrading fungi from subphylum Pezizomycotina. Combined with the presence of homologs of the gaaB gene in the same set of fungi, these strongly suggest that a common d-galacturonic acid utilization pathway is operative in these species.

Convergent evolution of hydroxylation mechanisms in the fungal kingdom: molybdenum cofactor-independent hydroxylation of xanthine via alpha-ketoglutarate-dependent dioxygenases.

The xanthine oxidases and dehydrogenases are among the most conserved enzymes in all living kingdoms. They contain the molybdopterin cofactor Moco. We show here that in the fungi, in addition to xanthine dehydrogenase, a completely different enzyme is able to catalyse the oxidation of xanthine to uric acid. In Aspergillus nidulans this enzyme is coded by the xanA gene. We have cloned the xanA gene and determined its sequence. A deletion of the gene has the same phenotype as the previously known xanA1 miss-sense mutation. Homologues of xanA exist only in the fungal kingdom. We have inactivated the cognate gene of Schizosaccharomyces pombe and this results in strongly impaired xanthine utilization as a nitrogen source. We have shown that the Neurospora crassa homologue is functionally equivalent to xanA. The enzyme coded by xanA is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase which shares a number of properties with other enzymes of this group. This work shows that only in the fungal kingdom, an alternative mechanism of xanthine oxidation, not involving Moco, has evolved using the dioxygenase scaffold.

Endogenous lipogenic regulators of spore balance in Aspergillus nidulans.

The ability of fungi to produce both meiospores and mitospores has provided adaptive advantages in survival and dispersal of these organisms. Here we provide evidence of an endogenous mechanism that balances meiospore and mitospore production in the model filamentous fungus Aspergillus nidulans. We have discovered a putative dioxygenase, PpoC, that functions in association with a previously characterized dioxygenase, PpoA, to integrate fatty acid derived oxylipin and spore production. In contrast to PpoA, deletion of ppoC significantly increased meiospore production and decreased mitospore development. Examination of the PpoA and PpoC mutants indicate that this ratio control is associated with two apparent feedback loops. The first loop shows ppoC and ppoA expression is dependent upon, and regulates the expression of, nsdD and brlA, genes encoding transcription factors required for meiospore or mitospore production, respectively. The second loop suggests Ppo oxylipin products antagonistically signal the generation of Ppo substrates. These data support a case for a fungal "oxylipin signature-profile" indicative of relative sexual and asexual spore differentiation.

Effect of vitamin E on autolysis and sporulation of Aspergillus nidulans.

The morphologic and physiologic effects of vitamin E, a powerful antioxidant, on the autolysis and sporulation of Aspergillus nidulans FGSC26 were studied. In carbon-depleted submerged cultures, reactive oxygen species (ROS) accumulated in the cells and, concomitantly, progressing autolysis was observed, which was characterized by decreasing dry cell masses and pellet diameters as well as by increasing extracellular chitinase activities. Vitamin E supplemented at a concentration of 1 g/L hindered effectively the intracellular accumulation of ROS, the autolytic loss of biomass, the disintegration of pellets, and the release of chitinase activities. In surface cultures, vitamin E inhibited autolysis of both A. nidulans FGSC26 and a loss-of-function FlbA autolytic phenotype mutant. In addition, supplementation of the culture medium with this antioxidant also had a negative effect on the sporulation of strain FGSC26 and the FadAG203R hypersporulating phenotype mutant. These results suggest that accumulation of ROS was involved in the initiation of both sporulation and autolysis in this filamentous fungus, but that FadA/FlbA signaling was not involved in this vitamin E-dependent regulation. Vitamin E can be recommended as a supplement in fermentations in which the disintegration of pellets and gross autolysis should be avoided.

The kinesin family member BimC contains a second microtubule binding region attached to the N terminus of the motor domain.

The kinesin family member BimC has a highly positively charged domain of approximately 70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by approximately 7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mm potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in kcat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity.

4'-phosphopantetheinyl transferase-encoding npgA is essential for siderophore biosynthesis in Aspergillus nidulans.

Aspergillus nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin as an intracellular iron-storage compound. Siderophore biosynthesis involves the enzymatic activity of nonribosomal peptide synthetases (NRPS). NRPS contain 4'-phosphopantetheine as an essential prosthetic group, which is attached by 4'-phosphopantetheinyl transferases. A. nidulans appears to possess at least one gene, npgA, encoding such an enzyme. Using a strain carrying a temperature-sensitive allele, cfwA2, we showed that NpgA is essential for biosynthesis of both the peptide bond-containing ferricrocin and the ester bond-containing triacetylfusarinene C. The cfwA2 strain was found to be iron-starved at the restrictive temperature during iron-replete conditions, consistent with the siderophore system being the major iron-uptake system-as we recently demonstrated. Northern analysis indicated that, in contrast to other genes which are involved in siderophore biosynthesis and uptake, expression of npgA is not controlled by the GATA-transcription factor SreA. It was shown previously that NpgA is required for biosynthesis of penicillin, pigment, and potentially lysine via the alpha-aminoadipate pathway. Supplementation with lysine plus triacetylfusarinine C restored normal growth of the cfwA2 strain at the restrictive temperature, suggesting that the growth defect of the mutant is mainly due to impaired biosynthesis of siderophores and lysine.

The Aspergillus nidulans metR gene encodes a bZIP protein which activates transcription of sulphur metabolism genes.

The identification, isolation and characterization of a new Aspergillus nidulans positive-acting gene metR, which encodes a transcriptional activator of sulphur metabolism, is reported. metR mutants are tight auxotrophs requiring methionine or homocysteine for growth. Mutations in the metR gene are epistatic to mutations in the negative-acting sulphur regulatory scon genes. The metR coding sequence is interrupted by a single intron of 492 bp which is unusually long for fungi. Aspergillus nidulans METR is a member of bZIP family of DNA-binding proteins. The bZIP domains of METR and the Neurospora crassa CYS3 transcriptional activator of sulphur genes are highly similar. Although Neurospora cys-3 gene does not substitute for the metR function, a chimeric metR gene with a cys-3 bZIP domain is able to transform the DeltametR mutant to methionine prototrophy. This indicates that METR recognizes the same regulatory sequence as CYS3. The metR gene is not essential, as deletion mutants are viable and have similar phenotype as point mutants. In contrast to the Neurospora cys-3, transcription of the metR gene was found to be regulated neither by METR protein nor by sulphur source. Transcription of metR gene is derepressed in the sconB2 mutant. Transcription of genes encoding sulphate permease, homocysteine synthase, cysteine synthase, ATP-sulphurylase, and sulphur controller--sconB is strongly regulated by the metR gene product and depends on the character of the metR mutation and sulphur supplementation.

NMR solution structure of AlcR (1-60) provides insight in the unusual DNA binding properties of this zinc binuclear cluster protein.

The three-dimensional structure of the DNA-binding domain (residues 1-60) of the ethanol regulon transcription factor AlcR from Aspergillus nidulans has been solved by NMR. This domain belongs to the zinc binuclear cluster class. Although the core of the protein is similar to previously characterized structures, consisting of two helices organized around a Zn(2)Cys(6 )motif, the present structure presents important variations, among them the presence of two supplementary helices. This structure gives new insight into the understanding of the AlcR specificities in DNA binding such as longer consensus half-sites, in vitro monomeric binding but in vivo multiple repeat transcriptional activation, either in direct or inverse orientations. The presence of additional contacts of the protein with its DNA target can be predicted from a model proposed for the interaction with the consensus DNA target. The clustering of accessible negative charges on helix 2 delineates a possible interaction site for other determinants of the transcriptional machinery, responsible for the fine tuning of the selection of the AlcR cognate sites.