Sterigmatocystin Biosynthesis Research Articles

The novel nematicide chiricanine A suppresses Bursaphelenchus xylophilus pathogenicity in Pinus massoniana by inhibiting Aspergillus and its secondary metabolite, sterigmatocystin.

Pine wilt disease (PWD) is responsible for extensive economic and ecological damage to Pinus spp. forests and plantations worldwide. PWD is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and transmitted into pine trees by a vector insect, the Japanese pine sawyer (JPS, Monochamus alternatus). Host infection by PWN will attract JPS to spawn, which leads to the co-existence of PWN and JPS within the host tree, an essential precondition for PWD outbreaks. Through the action of their metabolites, microbes can manipulate the co-existence of PWN and JPS, but our understanding on how key microorganisms engage in this process remains limited, which severely hinders the exploration and utilization of promising microbial resources in the prevention and control of PWD. In this study we investigated how the PWN-associated fungus Aspergillus promotes the co-existence of PWN and JPS in the host trees (Pinus massoniana) via its secondary metabolite, sterigmatocystin (ST), by taking a multi-omics approach (phenomics, transcriptomics, microbiome, and metabolomics). We found that Aspergillus was able to promote PWN invasion and pathogenicity by increasing ST biosynthesis in the host plant, mainly by suppressing the accumulation of ROS (reactive oxygen species) in plant tissues that could counter PWN. Further, ST accumulation triggered the biosynthesis of VOC (volatile organic compounds) that attracts JPS and drives the coexistence of PWN and JPS in the host plant, thereby encouraging the local transmission of PWD. Meanwhile, we show that application of an Aspergillus inhibitor (chiricanine A treatment) results in the absence of Aspergillus and decreases the in vivo ST amount, thereby sharply restricting the PWN development in host. This further proved that Aspergillus is vital and sufficient for promoting PWD transmission. Altogether, these results document, for the first time, how the function of Aspergillus and its metabolite ST is involved in the entire PWD transmission chain, in addition to providing a novel and long-term effective nematicide for better PWD control in the field.

Monitoring of sterigmatocystin biosynthesis using RT-qPCR in airborne Aspergillus species of the series Versicolores

Aspergilli series Versicolores have been shown to be explanatory variables for different symptoms like coughing and dizziness experienced by residents of mold-damaged homes. Among these species, eight are particularly recurrent in bioaerosols: Aspergillus amoenus, A. creber, A. fructus, A. jensenii, A. protuberus, A. puulaauensis, A. sydowii and A. tabacinus. In order to monitor the biosynthesis of sterigmatocystin (a mycotoxin associated with a risk of cancer development) and the development of these molds, we developed an RT-qPCR tool by targeting the aflR and rho1 genes. A total of 30 fungal isolates representing these eight species were included. For each of them, sterigmatocystin was quantified by UPLC-HRMS and (1 → 3)-β-D-glucan by visible spectrophotometry using Endosafe®-PTS™-Glucan Cartridges. After validation of our method by RT-qPCR, the direct assay was compared to the amount of aflR and rho1 cDNA. The sterigmatocystin and aflR assays showed a significant correlation between these two approaches (p < 0.0001), demonstrated for the first time the production of sterigmatocystin by A. tabacinus and suggested the ability of A. sydowii to synthesize sterigmatocystin. Assays conducted on (1 → 3)-β-D-glucan and rho1 did not show a correlation, supporting the multiplicity of functions performed in fungal cells by the RHO1 GTPase. The proposed tool could allow monitoring of sterigmatocystin biosynthesis by Aspergillus of the series Versicolores under different culture and climatic conditions.

First report on the metabolic characterization of Sterigmatocystin production by select Aspergillus species from the Nidulantes section in Foeniculum vulgare.

Spices are typically grown in climates that support the growth of toxigenic fungi and the production of mycotoxins. The Aspergilli described in this study, as well as the sterigmatocystin (STC) detected, are causes for concern due to their potential to induce food poisoning. One of the most well-known producers of the carcinogenic STC is Aspergillus nidulans. This research explores the occurrence of STC-producing fungi in Foeniculum vulgare, a spice that is marketed in India and other parts of the world. This innovative study details the mycotoxigenic potential of five Aspergilli belonging to Section Nidulantes, namely Aspergillus latus (02 isolates), Emericella quadrilineata (02 isolates), and Aspergillus nidulans (01 isolate), with respect to STC contamination. These five isolates of Aspergilli were screened to produce STC on yeast extract sucrose (YES) medium in a controlled environment with regard to light, temperature, pH, and humidity, among other variables. The expression patterns of regulatory genes, namely, aflR, laeA, pacC, fluG, flbA, pksA, and mtfA were studied on the Czapek–Dox agar (CDA) medium. STC biosynthesis by the test isolates was done in potato dextrose broth (PDB) under optimum conditions, followed by the extraction and purification of the broth using ethyl acetate. High-performance liquid chromatography (HPLC) with an ultraviolet (UV) detector was utilized to detect compounds in eluted samples. F. vulgare contains Aspergilli that have been shown to have mycotoxigenic potential, which can accumulate in the spice during its active growth and thereby cause the elaboration of mycotoxins.

Involvement of VIVID in white light-responsive pigmentation, sexual development and sterigmatocystin biosynthesis in the filamentous fungus Podospora anserina.

Light serves as a source of information and regulates diverse physiological processes in living organisms. Fungi perceive and respond to light through a complex photosensory system. Fungi have evolved the desensitization mechanism to adapt to the changing light signal in a natural environment. White light exerts multiple essential impacts on the model filamentous fungus Podospora anserina. However, the light sensing and response in this species has not been investigated. In this study, we demonstrated that the loss of function of the light desensitization protein VIVID (VVD) in P. anserina triggered exacerbated light responses and therefore led to drastic morphological and physiological changes. The white light-sensitive mutant Δvvd showed growth reduction, spermatia overproduction, enhanced hyphae pigmentation and reduced oxidative stress tolerance. We observed the decreased expression level of sterigmatocystin gene cluster by transcriptome analysis and finally detected the reduced production of sterigmatocystin in Δvvd in response to white light. Our data indicate that VVD acts as a repressor of white collar complex. This study exhibits a vital role of VVD in governing white light-responsive gene expression and secondary metabolite production and contributes to a better understanding of the photoreceptor VVD in P. anserina.

Hyperosmotic medium partially restores the growth defect and the impaired production of sterigmatocystin of an Aspergillus nidulans ΔpmtC mutant in a HogA-independent manner

The protein O-mannosyltransferase catalyzes O-mannosylation in the endoplasmic reticulum by transferring mannose to the seryl or threonyl residues of substrate proteins. We previously reported a deletion mutant of O-mannosyltransferase C (ΔpmtC) in Aspergillus nidulans with impaired vegetative growth and sterigmatocystin (ST) production. In this study, we investigated whether osmotic conditions contribute to the developmental processes and ST biosynthesis of the ΔpmtC deletion mutant. We found that hyphal growth and ST production partially improved in the presence of NaCl, KCl or sorbitol as osmotic stabilizers. Conidiation of the ΔpmtC deletion mutant was not restored under osmotic stress conditions when the hogA gene was deleted. The hogA gene encodes a protein required for the cellular response to osmotic pressure. However, the yield of ST and the vegetative growth of the ΔhogA ΔpmtC double deletant was restored by high osmolarity in a HogA-independent manner.

Involvement of PaSNF1 in Fungal Development, Sterigmatocystin Biosynthesis, and Lignocellulosic Degradation in the Filamentous Fungus Podospora anserina.

The sucrose non-fermenting 1/AMP-activated protein kinase (SNF1/AMPK) is a central regulator of carbon metabolism and energy production in the eukaryotes. In this study, the functions of the Podospora anserina SNF1 (PaSNF1) ortholog were investigated. The ΔPaSNF1 mutant displays a delayed development of mycelium and fruiting bodies and fails to form ascospores. The expression of the PaSNF1 gene in the strain providing female organs in a cross is sufficient to ensure fertility, indicating a maternal effect. Results of environmental stress showed that ΔPaSNF1 was hypersensitive to stress, such as osmotic pressure and heat shock, and resistant to fluconazole. Interestingly, the knockout of PaSNF1 significantly promoted sterigmatocystin (ST) synthesis but suppressed cellulase [filter paperase (FPA), endoglucanase (EG), and β-glucosidase (BG)] activity. Further, transcriptome analysis indicated that PaSNF1 made positive regulatory effects on the expression of genes encoding cellulolytic enzymes. These results suggested that PaSNF1 may function in balancing the operation of primary and secondary metabolism. This study suggested that SNF1 was a key regulator concerting vegetative growth, sexual development, and stress tolerance. Our study provided the first genetic evidence that SNF1 was involved in the ST biosynthesis and that it may also be a major actor of lignocellulose degradation in P. anserina.

The putative C2H2 transcription factor RocA is a novel regulator of development and secondary metabolism in Aspergillus nidulans.

Multiple transcriptional regulators play important roles in the coordination of developmental processes, including asexual and sexual development, and secondary metabolism in the filamentous fungus Aspergillus nidulans. In the present study, we characterized a novel putative C2H2-type transcription factor (TF), RocA, in relation to development and secondary metabolism. Deletion of rocA increased conidiation and caused defective sexual development. In contrast, the overexpression of rocA exerted opposite effects on both phenotypes. Additionally, nullifying rocA resulted in enhanced brlA expression and reduced nsdC expression, whereas its overexpression exerted the opposite effects. These results suggest that RocA functions as a negative regulator of asexual development by repressing the expression of brlA encoding a key asexual development activator, but as a positive regulator of sexual development by enhancing the expression of nsdC encoding a pivotal sexual development activator. Deletion of rocA increased the production of sterigmatocystin (ST), as well as the expression of its biosynthetic genes, aflR and stcU. Additionally, the expression of the biosynthetic genes for penicillin (PN), ipnA and acvA, and for terrequinone (TQ), tdiB and tdiE, was increased by rocA deletion. Thus, it appears that RocA functions as a negative transcriptional modulator of the secondary metabolic genes involved in ST, PN, and TQ biosynthesis. Taken together, we propose that RocA is a novel transcriptional regulator that may act either positively or negatively at multiple target genes necessary for asexual and sexual development and secondary metabolism.

Reduction of sterigmatocystin biosynthesis and growth of food-borne fungi by lactic acid.

Food contamination by fungi and mycotoxins presents a problem for food safety even today. Since lactic acid (LA) has Generally Recognized As Safe (GRAS) status, the aim of this research was to determine its potential in protection of food against mycological and mycotoxicological contamination. In this study, LA showed an inhibitory effect on the growth of food-borne fungi (Penicillium aurantiogriseum K51, Aspergillus parasiticus KB31, Aspergillus versicolor S72, and Aspergillus niger K95) and on biosynthesis of sterigmatocystin (STE). For the antifungal effect of LA on the growth of food-borne fungi, the disc diffusion and microdilution methods were performed. The effect of LA on the STE biosynthesis by A. versicolor was determined using an LC-MS/MS technique. The largest inhibition zone was observed for A. versicolor (inhibition zone of 24 ± 0.35 mm), while there were no inhibition zones for A. niger and A. parasiticus at all tested LA concentrations. The minimal inhibitory concentration (MIC) of LA on fungi ranged from 25.0 mg/mL to 50.0 mg/mL, while the minimum fungicidal concentrations (MFCs) ranged from 50.0 mg/mL to 100.0 mg/mL. Complete inhibition of STE biosynthesis by A. versicolor was observed at an LA concentration of 50.0 mg/mL. The obtained results showed that LA could be efficient for protection of food against mycological and STE contamination.

GPCR-mediated glucose sensing system regulates light-dependent fungal development and mycotoxin production.

Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production.

Disruption of stcA blocks sterigmatocystin biosynthesis and improves echinocandin B production in Aspergillus delacroxii.

Echinocandin B (ECB) is an important lipohexapeptide used for chemical manufacture of the antifungal agent anidulafungin. Sterigmatocystin (ST) is a polyketide mycotoxin produced by certain species of Aspergillus such as Aspergillus delacroxii SIPIW15, which could produce both ECB and ST. However, the presence of the potent carcinogen ST will greatly affect the quality and safety of ECB production. Therefore, it is essential to eliminate the ST biosynthesis and increase ECB titers in Asp. delacroxii SIPIW15. In this study, the polyketide synthase gene (stcA) required for biosynthesis of ST and its flanking region in Asp. delacroxii SIPIW15 were cloned, sequenced and analyzed firstly. Based on Agrobacterium-mediated transformation, the ΔstcA mutant AMT-1 was obtained and its yield of ECB was increased by 40% without ST detected at the same time as compared to the original strain. The results of the fed-batch experiments showed that the ECB yield of the ΔstcA strain AMT-1 was increased to 2163 ± 31mg/l and no ST was detected in the 50l bioreactor. This work suggested that the ΔstcA strain AMT-1 has the potential for application in ECB production improvement, and more importantly, to eliminate ST-related environmental pollution in ECB fermentation industry.

Functional characterization of the sterigmatocystin secondary metabolite gene cluster in the filamentous fungus Podospora anserina: involvement in oxidative stress response, sexual development, pigmentation and interspecific competitions

Filamentous fungi are known as prolific untapped reservoirs of diverse secondary metabolites, where genes required for their synthesis are organized in clusters. The bioactive properties of these compounds are closely related to their functions in fungal biology, which are not well understood. In this study, we focused on the Podospora anserina gene cluster responsible for the biosynthesis of sterigmatocystin (ST). Deletion of the PaStcA gene encoding the polyketide synthase and overexpression (OE) of the PaAflR gene encoding the ST-specific transcription factor in P. anserina were performed. We showed that growth of PaStcAΔ was inhibited in the presence of methylglyoxal, while OE-PaAflR showed a little inhibition, indicating that ST production may enhance oxidative stress tolerance in P. anserina. We also showed that the OE-PaAflR strain displayed an overpigmented thallus mediated by the melanin pathway. Overexpression of PaAflR also led to sterility. Interspecific confrontation assays showed that ST-overexpressed strains produced a high level of peroxides and possessed a higher competitiveness against other fungi. Comparative metabolite profiling demonstrated that PaStcAΔ strain was unable to produce ST, while OE-PaAflR displayed a ST overproduction. This study contributes to a better understanding of ST in P. anserina, especially with regard to its involvement in fungal physiology.

Sterigmatocystin production is restricted to hyphae located in the proximity of hülle cells.

Aspergillus nidulans produces sterigmatocystin, a secondary metabolite mycotoxin, for the protection of its reproductive structures. Previous studies on grazing behavior of fungivore arthropods, regulation of sexual development, and secondary metabolite biosynthesis have revealed the association of sterigmatocystin biosynthesis with sexual reproduction, but the spatial distribution of sterigmatocystin producing hyphae within the colony has never been investigated. In this work, we aimed to locate the site of sterigmatocystin production within the colony by employing a yCFP reporter system. We demonstrated that the stcO promoter is active only in vegetative hyphae that surround groups of hülle cells and the activity decreases and eventually ceases as the distance between the hypha and the hülle cells increases. This phenomenon indicates that the vegetative mycelium might consist of morphologically uniform, but functionally different hyphae.

Protein O-mannosyltransferases are required for sterigmatocystin production and developmental processes in Aspergillus nidulans.

Aspergillus nidulans produces sterigmatocystin (ST), a precursor of a carcinogenic secondary metabolite aflatoxin (AF), during its developmental process. ST biosynthesis has been shown to be affected by various regulatory factors. In this study, we investigated the involvement of O-mannosyltransferases (PmtA, PmtB, PmtC), in ST production and morphological development. Deletion of pmtA (ΔpmtA), pmtB (ΔpmtB) or pmtC (ΔpmtC) caused no spore production and a significant decline of vegetative growth. A tremendous decline of ST level was observed in all Δpmt mutants at the third day after inoculation. By extending the growth period, ST production of ΔpmtA and ΔpmtB increased to the wild-type level 7days after inoculation. On the other hand, ST was not detected from 7- or 14-day cultures in ΔpmtC. Expression levels of aflR gene, an essential regulator of the ST biosynthesis pathway, were also down-regulated in the Δpmt strains. By introducing the aflR overexpression cassette, ST production in the ΔpmtA and ΔpmtB significantly increased to levels comparable to the wild type. However, the presence of the aflR overexpression cassette could not improve ST production in the ΔpmtC mutant. These data suggest that the PMT family is a new endogenous factor that is required for ST biosynthesis in A. nidulans. These findings provide better understanding of the regulatory mechanisms of AF/ST biosynthesis, which can ultimately contribute to our ability to control aflatoxin contamination.

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus.

ABSTRACTThe study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin’s carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique.

Sirtuin E is a fungal global transcriptional regulator that determines the transition from the primary growth to the stationary phase

In response to limited nutrients, fungal cells exit the primary growth phase, enter the stationary phase, and cease proliferation. Although fundamental to microbial physiology in many environments, the regulation of this transition is poorly understood but likely involves many transcriptional regulators. These may include the sirtuins, which deacetylate acetyllysine residues of histones and epigenetically regulate global transcription. Therefore, we investigated the role of a nuclear sirtuin, sirtuin E (SirE), from the ascomycete fungus Aspergillus nidulans An A. nidulans strain with a disrupted sirE gene (SirEΔ) accumulated more acetylated histone H3 during the stationary growth phase when sirE was expressed at increased levels in the wild type. SirEΔ exhibited decreased mycelial autolysis, conidiophore development, sterigmatocystin biosynthesis, and production of extracellular hydrolases. Moreover, the transcription of the genes involved in these processes was also decreased, indicating that SirE is a histone deacetylase that up-regulates these activities in the stationary growth phase. Transcriptome analyses indicated that SirE repressed primary carbon and nitrogen metabolism and cell-wall synthesis. Chromatin immunoprecipitation demonstrated that SirE deacetylates acetylated Lys-9 residues in histone H3 at the gene promoters of α-1,3-glucan synthase (agsB), glycolytic phosphofructokinase (pfkA), and glyceraldehyde 3-phosphate (gpdA), indicating that SirE represses the expression of these primary metabolic genes. In summary, these results indicate that SirE facilitates the metabolic transition from the primary growth phase to the stationary phase. Because the observed gene expression profiles in stationary phase matched those resulting from carbon starvation, SirE appears to control this metabolic transition via a mechanism associated with the starvation response.

CpsA regulates mycotoxin production, morphogenesis and cell wall biosynthesis in the fungus Aspergillus nidulans.

The model fungus Aspergillus nidulans synthesizes numerous secondary metabolites, including sterigmatocystin (ST). The production of this toxin is positively controlled by the global regulator veA. In the absence of veA (ΔveA), ST biosynthesis is blocked. Previously, we performed random mutagenesis in a ΔveA strain and identified revertant mutants able to synthesize ST, among them RM1. Complementation of RM1 with a genomic library revealed that the mutation occurred in a gene designated as cpsA. While in the ΔveA genetic background cpsA deletion restores ST production, in a veA wild-type background absence of cpsA reduces and delays ST biosynthesis decreasing the expression of ST genes. Furthermore, cpsA is also necessary for the production of other secondary metabolites, including penicillin, affecting the expression of PN genes. In addition, cpsA is necessary for normal asexual and sexual development. Chemical and microscopy analyses revealed that CpsA is found in cytoplasmic vesicles and it is required for normal cell wall composition and integrity, affecting adhesion capacity and oxidative stress sensitivity. The conservation of cpsA in Ascomycetes suggests that cpsA homologs might have similar roles in other fungal species.

Differential Control of Asexual Development and Sterigmatocystin Biosynthesis by a Novel Regulator in Aspergillus nidulans

The filamentous fungus Aspergillus nidulans primarily reproduces by forming asexual spores called conidia and produces the mycotoxin sterigmatocystin (ST), the penultimate precursor of aflatoxins. It has been known that asexual development and ST production are tightly co-regulated by various regulatory inputs. Here, we report that the novel regulator AslA with a C2H2 domain oppositely regulates development and ST biosynthesis. Nullifying aslA resulted in defective conidiation and reduced expression of brlA encoding a key activator of asexual development, which indicates that AslA functions as an upstream activator of brlA expression. aslA deletion additionally caused enhanced ST production and expression of aflR encoding a transcriptional activator for ST biosynthetic genes, suggesting that AslA functions as an upstream negative regulator of aflR. Cellular and molecular studies showed that AslA has a trans-activation domain and is localized in the nuclei of vegetative and developing cells but not in spores, indicating that AslA is likely a transcription factor. Introduction of the aslA homologs from distantly-related aspergilli complemented the defects caused by aslA null mutation in A. nidulans, implying a functional conservancy of AslA. We propose that AslA is a novel regulator that may act at the split control point of the developmental and metabolic pathways.

A phosphorylation code of the Aspergillus nidulans global regulator VelvetA (VeA) determines specific functions.

The velvet protein VeA is a global fungal regulator for morphogenetic pathways as well as for the control of secondary metabolism. It is found exclusively in filamentous fungi, where it fulfills conserved, but also unique functions in different species. The involvement of VeA in various morphogenetic and metabolic pathways is probably due to spatially and timely controlled specific protein-protein interactions with other regulators such as phytochrome (FphA) or velvet-like proteins (VelB). Here we present evidence that Aspergillus nidulans VeA is a multi-phosphorylated protein and hypothesize that at least four specific amino acids (T167, T170, S183 and Y254) undergo reversible phosphorylation to trigger development and sterigmatocystin biosynthesis. Double mutation of T167 to valine and T170 to glutamic acid exerted the largest effects with regards to sexual development and veA gene expression. In comparison with wild-type VeA, which shuttles out of the nuclei after illumination this VeA variant showed stronger nuclear accumulation than the wild type, independent of the light conditions. The interaction between VeA and VelB or FphA, respectively, was affected in the T167V-T170E mutant. Our results suggest complex regulation of the phosphorylation status of the VeA protein.

A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

Secondary metabolites (SMs) produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

Role of the zinc finger transcription factor SltA in morphogenesis and sterigmatocystin biosynthesis in the fungus Aspergillus nidulans.

Potassium, a widely accepted macronutrient, is vital for many physiological processes such as regulation of cell volume, maintenance of intracellular pH, synthesis of proteins and activation of enzymes in filamentous fungi. Another cation, calcium, plays an essential role in many signaling processes from lower to higher eukaryotes. Imbalance in the intracellular ionic levels of potassium or calcium causes adverse effects on cell growth, morphology and development, and eventually death. Previous studies on the adaptation of Aspergillus nidulans to salt and osmotic stress conditions have revealed the role of SltA, a C2H2 zinc finger transcription factor in cation homeostasis. SltA is highly conserved in the Ascomycota phylum with no identifiable homolog in S. cerevisiae and other yeast-like fungi, and prevents toxicity by the cations Na+, K+, Li+, Cs+ and Mg2+, but not by Ca2+. However its role in morphology and biosynthesis of natural products such as mycotoxins remained unknown. This study shows the first characterization of the role of calcium and SltA fungal homologs in morphogenesis using the model system A. nidulans. Addition of potassium to sltA deletion mutants resulted in decreased levels of sterigmatocystin production. A similar phenotype was observed for both types of mutants in veA1 and veA + genetic background. Expression of the sterigmatocystin genes aflR and stcU was strongly reduced in sltA deletion mutant when K+ was added. Additionally, increased concentrations of K+ drastically reduced sexual and asexual development, as well as radial growth in deletion sltA colonies. This reduction was accompanied by lower expression of the morphology related genes nsdD, steA and brlA. Interestingly, addition of calcium was able to stimulate asexual and sexual development and remediate the deletion sltA phenotype, including defects in morphology and toxin production.