Microbiology 305: Critical Analyses in Microbiology
Microbiology 810: Current Issues in Microbiology
Microbiology/Oncology 545: Topics in Biotechnology
Food Research Institute
Professor, Department of Genetics
Editorial Board Member of Scientific Reports
Graduate Trainer, Genetics, Molecular and Environmental Toxicology Center, Plant Pathology, Food Science.
Faculty of 1000 Biology, Faculty Member for Microbiology, Microbial Growth & Development Section
Microbiology Teaching Fellows Program
In the filamentous fungus Aspergillus nidulans, the velvet family protein VeA and the global regulator of secondary metabolism LaeA govern development and secondary metabolism mostly by acting as the VelB/VeA/LaeA heterotrimeric complex. While functions of these highly conserved controllers have been well studied, the genome-wide regulatory networks governing cellular and chemical development remain to be uncovered. Here, by integrating transcriptomic analyses, protein-DNA interactions, and the known A. nidulans gene/protein interaction data, we have unraveled the gene regulatory networks governed by VeA and LaeA. Within the networks, VeA and LaeA directly control the expression of numerous genes involved in asexual/sexual development and primary/secondary metabolism in A. nidulans. Totals of 3,190 and 1,834 potential direct target genes of VeA and LaeA were identified, respectively, including several important developmental and metabolic regulators such as flbA · B · C , velB · C , areA , mpkB , and hogA . Moreover, by analyzing over 8,800 ChIP-seq peaks, we have revealed the predicted common consensus sequences 5'-TGATTGGCTG-3' and 5'-TCACGTGAC-3' that VeA and LaeA might bind to interchangeably. These findings further expand the biochemical and genomic studies of the VelB/VeA/LaeA complex functionality in the gene regulation. In summary, this study unveils genes that are under the regulation of VeA and LaeA, proposes the VeA- and LaeA-mediated gene regulatory networks, and demonstrates their genome-wide developmental and metabolic regulations in A. nidulans. IMPORTANCE Fungal development and metabolism are genetically programmed events involving specialized cellular differentiation, cellular communication, and temporal and spatial regulation of gene expression. In genus Aspergillus, the global regulators VeA and LaeA govern developmental and metabolic processes by affecting the expression of downstream genes, including multiple transcription factors and signaling elements. Due to their vital roles in overall biology, functions of VeA and LaeA have been extensively studied, but there still has been a lack of knowledge about their genome-wide regulatory networks. In this study, employing the model fungus A. nidulans, we have identified direct targets of VeA and LaeA and their gene regulatory networks by integrating transcriptome, protein-DNA interaction, and protein-protein interaction analyses. Our results demonstrate the genome-wide regulatory mechanisms of these global regulators, thereby advancing the knowledge of fungal biology and genetics.
Post-translational modifications of chromatin structure by histone acetyltransferase (HATs) play a pivotal role in the regulation of gene expression and diverse biological processes. However, the function of GNAT family HATs, especially Elp3, in the opportunistic human pathogenic fungus Aspergillus fumigatus is largely unknown. To investigate the roles of the GNAT family HATs Elp3 and GcnE in the A. fumigatus , we have generated and characterized individual null Δ elp3 and Δ gcnE mutants. The radial growth of fungal colonies was significantly decreased by the loss of elp3 or gcnE , and the number of asexual spores (conidia) in the Δ gcnE mutant was significantly reduced. Moreover, the mRNA levels of the key asexual development regulators were also significantly low in the Δ gcnE mutant compared to wild type (WT). Whereas both the Δ elp3 and Δ gcnE mutants were markedly impaired in the formation of adherent biofilms, the Δ gcnE mutant showed a complete loss of surface structure and of intercellular matrix. The Δ gcnE mutant responded differently to oxidative stressors and showed significant susceptibility to triazole antifungal agents. Furthermore, Elp3 and GcnE function oppositely in the production of secondary metabolites, and the Δ gcnE mutant showed attenuated virulence. In conclusion, Elp3 and GcnE are associated with diverse biological processes and can be potential targets for controlling the pathogenic fungus.
The bZIP transcription factors (TFs) govern regulation of development, secondary metabolism, and various stress responses in filamentous fungi. In this work, we carried out genome-wide expression studies employing Illumina RNAseq to understand the roles of the two bZIP transcription factors AtfA and AtfB in Aspergillus nidulans . Comparative analyses of transcriptomes of control, Δ atfA , Δ atfB , and Δ atfA Δ atfB mutant strains were performed. Dependence of a gene on AtfA (AtfB) was decided by its differential downregulation both between the reference and Δ atfA (Δ atfB ) strains and between the Δ atfB (Δ atfA ) and the Δ atfA Δ atfB strains in vegetatively grown cells (mycelia) and asexual spores (conidia) of menadione sodium bisulfite (MSB)-treated or untreated cultures. As AtfA is the primary bZIP TF governing stress-response in A. nidulans , the number of differentially expressed genes for Δ atfA was significantly higher than for Δ atfB in both mycelial and conidial samples, and most of the AtfB-dependent genes showed AtfA dependence, too. Moreover, the low number of genes depending on AtfB but not on AtfA can be a consequence of Δ atfA leading to downregulation of atfB expression. Conidial samples showed much higher abundance of atfA and atfB mRNAs and more AtfA- and AtfB-affected genes than mycelial samples. In the presence of MSB, the number of AtfB- (but not of AtfA-) affected genes decreased markedly, which was accompanied with decreased mRNA levels of atfB in MSB-treated mycelial (reference strain) and conidial (Δ atfA mutant) samples. In mycelia, the overlap between the AtfA-dependent genes in MSB-treated and in untreated samples was low, demonstrating that distinct genes can be under AtfA control under different conditions. Carbohydrate metabolism genes were enriched in the set of AtfA-dependent genes. Among them, AtfA-dependence of glycolytic genes in conidial samples was the most notable. Levels of transcripts of certain secondary metabolitic gene clusters, such as the Emericellamide cluster, also showed AtfA-dependent regulation. Genes encoding catalase and histidine-containing phosphotransfer proteins showed AtfA-dependence under all experimental conditions. There were 23 AtfB-dependent genes that did not depend on AtfA under any of our experimental conditions. These included a putative α-glucosidase ( agdB ), a putative α-amylase, calA, which is involved in early conidial germination, and an alternative oxidase. In summary, in A. nidulans there is a complex interaction between the two bZIP transcription factors, where AtfA plays the primary regulatory role.
In filamentous fungal Aspergillus species, growth, development, and secondary metabolism are genetically programmed biological processes, which require precise coordination of diverse signaling elements, transcription factors (TFs), upstream and downstream regulators, and biosynthetic genes. For the last few decades, regulatory roles of these controllers in asexual/sexual development and primary/secondary metabolism of Aspergillus species have been extensively studied. Among a wide spectrum of regulators, a handful of global regulators govern upstream regulation of development and metabolism by directly and/or indirectly affecting the expression of various genes including TFs. In this review, with the model fungus Aspergillus nidulans as the central figure, we summarize the most well-studied main upstream regulators and their regulatory roles. Specifically, we present key functions of heterotrimeric G proteins and G protein-coupled receptors in signal transduction), the velvet family proteins governing development and metabolism, LaeA as a global regulator of secondary metabolism, and NsdD, a key GATA-type TF, affecting development and secondary metabolism and provide a snapshot of overall upstream regulatory processes underlying growth, development, and metabolism in Aspergillus fungi.
The VosA-VelB hetero-dimeric complex plays a pivotal role in regulating development and secondary metabolism in Aspergillus nidulans . In this work, we characterize a new VosA/VelB-activated gene called vadH , which is predicted to encode a 457-amino acid length protein containing four adjacent CH zinc-finger domains. Mutational inactivation of vosA or velB led to reduced mRNA levels of vadH throughout the lifecycle, suggesting that VosA and VelB have a positive regulatory effect on the expression of vadH . The deletion of vadH resulted in decreased asexual development (conidiation) but elevated production of sexual fruiting bodies (cleistothecia), indicating that VadH balances asexual and sexual development in A. nidulans . Moreover, the vadH deletion mutant exhibited elevated susceptibility to hyperosmotic stress compared to wild type and showed elevated production of the mycotoxin sterigmatocystin (ST). Genome-wide expression analyses employing RNA-Seq have revealed that VadH is likely involved in regulating more genes and biological pathways in the developmental stages than those in the vegetative growth stage. The brlA , abaA , and wetA genes of the central regulatory pathway for conidiation are downregulated significantly in the vadH null mutant during asexual development. VadH also participates in regulating the genes, mat2 , ppgA and lsdA , etc., related to sexual development, and some of the genes in the ST biosynthetic gene cluster. In summary, VadH is a putative transcription factor with four CH finger domains and is involved in regulating asexual/sexual development, osmotic stress response, and ST production in A. nidulans .
Aflatoxins (AFs) are the most important toxic, mutagenic, and carcinogenic fungal toxins that routinely contaminate food and feed. While more than 20 AFs have been identified to date, aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), and M1 (AFM1) are the most common. Over 25 species of Aspergillus have been shown to produce AFs, with Aspergillus flavus , Aspergillus parasiticus , and Aspergillus nomius being the most important and well-known AF-producing fungi. These ubiquitous molds can propagate on agricultural commodities to produce AFs in fields and during harvesting, processing, transportation, and storage. Countries with warmer climates and that produce foods susceptible to AF contamination shoulder a substantial portion of the global AF burden. Pakistan's warm climate promotes the growth of toxigenic fungi, resulting in frequent AF contamination of human foods and animal feeds. The potential for contamination in Pakistan is exacerbated by improper storage conditions and a lack of regulatory limits and enforcement mechanisms. High levels of AFs in common commodities produced in Pakistan are a major food safety problem, posing serious health risks to the population. Furthermore, aflatoxin contamination contributes to economic losses by limiting exports of these commodities. In this review, recent information regarding the fungal producers of AFs, prevalence of AF contamination of foods and feed, current regulations, and AF prevention and removal strategies are summarized, with a major focus on Pakistan.
Transcription factors (TFs) with the basic leucin zipper domain are key elements of the stress response pathways in filamentous fungi. In this study, we functionally characterized the two bZIP type TFs AtfA and AtfB by deletion ( Δ ) and overexpression (OE) of their encoding genes in all combination: ΔatfA, ΔatfB, ΔatfAΔatfB, ΔatfAatfB OE, ΔatfBatfA OE, atfA OE, atfB OE and atfA OE atfB OE in Aspergillus nidulans . Based on our previous studies, ΔatfA increased the sensitivity of the fungus to oxidative stress mediated by menadione sodium bisulfite (MSB) and tert-butylhydroperoxide ( t BOOH), while ΔatfB was not sensitive to any oxidative stress generating agents, namely MSB, t BOOH and diamide at all. Contrarily, the ΔatfB mutant was sensitive to NaCl, but tolerant to sorbitol. Overexpression of atfB was able to compensate the MSB sensitivity of the ΔatfA mutant. Heavy metal stress elicited by CdCl reduced diameter of the atfB OE and atfA OE atfB OE mutant colonies to about 50% of control colony, while the cell wall stress generating agent CongoRed increased the tolerance of the ΔatfA mutant. When we tested the heat stress sensitivity of the asexual spores (conidiospores) of the mutants, we found that conidiospores of ΔatfAatfB OE and ΔatfBatfA OE showed nearly 100% tolerance to heat stress. Asexual development was negatively affected by ΔatfA , while atfA OE and atfA OE coupled with ΔatfB increased the number of conidiospores of the fungus approximately 150% compared to the control. Overexpression of atfB led to a 25% reduction in the number of conidiospores, but increased levels of abaA mRNA and size of conidiospores. Sexual fruiting body (cleistothecium) formation was diminished in the ΔatfA and the ΔatfAΔatfB mutants, while relatively elevated in the ΔatfB and the ΔatfBatfA OE mutants. Production of the mycotoxin sterigmatocystin (ST) was decreased to undetectable levels in the ΔatfA mutant, yet ST production was restored in the ΔatfAΔatfB mutant, suggesting that ΔatfB can suppress ST production defect caused by ΔatfA . Levels of ST were also significantly decreased in the ΔatfAatfB OE, ΔatfBatfA OE and atfA OE atfB OE mutants.
Aspergillus flavus is a representative fungal species in the Aspergillus section Flavi and has been used as a model system to gain insights into fungal development and toxin production. A. flavus has several adverse effects on humans, including the production of the most carcinogenic mycotoxin aflatoxins and causing aspergillosis in immune-compromised patients. In addition, A. flavus infection of crops results in economic losses due to yield loss and aflatoxin contamination. A. flavus is a saprophytic fungus that disperses in the ecosystem mainly by producing asexual spores (conidia), which also provide long-term survival in the harsh environmental conditions. Conidia are composed of the rodlet layer, cell wall, and melanin and are produced from an asexual specialized structure called the conidiophore. The production of conidiophores is tightly regulated by various regulators, including the central regulatory cascade composed of BrlA-AbaA-WetA, the fungi-specific velvet regulators, upstream regulators, and developmental repressors. In this review, we summarize the findings of a series of recent studies related to asexual development in A. flavus and provide insights for a better understanding of other fungal species in the section Flavi.
The NF-ƙB-type VosA-VelB velvet complex acts as a global regulator governing development and metabolism in fungi. One of the VosA-VelB-activated developmental (VAD) genes called vadZ is predicted to encode a 557-amino acid protein containing a highly conserved GAL4-type Zn(II)Cys (or C zinc) binuclear cluster DNA-binding domain in Aspergillus nidulans. In this report, we characterize the function of the vadZ gene in controlling development and sterigmatocystin (ST) production in A. nidulans. To verify VosA-VelB mediated activation of vadZ, we checked relative mRNA levels of vadZ in wild-type (WT), ΔvosA, and ΔvelB mutant strains during vegetative, asexual, and sexual development phases. At the beginning of asexual development, the absence of vosA led to a 66.2-fold lowered vadZ mRNA levels, whereas ΔvelB resulted in a 3.6-fold decrease in vadZ mRNA levels. The deletion of vadZ resulted in significantly restricted colony growth coupled with reduced asexual development, but increased formation of sexual fruiting bodies called cleistothecia. In addition, nullifying vadZ caused elevated mRNA levels of the two key sexual developmental activators esdC and nsdD throughout the lifecycle. Moreover, the ΔvadZ mutant showed elevated production of ST and enhanced mRNA levels of ST biosynthetic genes. In summary, the putative C transcription factor VadZ promotes asexual development and suppresses the sexual development and the ST production in A. nidulans.
No abstract available.
Sesame Sesamum indicum L. is a major oil-based seed crop that has been widely cultivated and consumed in Pakistan. Unfortunately, sesame is highly prone to Aspergillus fungal growth in the field, and under inappropriate storage conditions can become contaminated with aflatoxins, the most potent carcinogen found in nature. Here, we have isolated a high number of Aspergillus isolates from sesame seeds in fresh and stored conditions obtained from rainfed and irrigated zones of Punjab, Pakistan, and characterized them for aflatoxigenic potentials. Using morphological identification techniques, 260 isolates were grouped as potential Aspergillus section Flavi, with 126 and 134 originating from the rainfed and irrigated zones, respectively. Out of 260 in total, 188 isolates were confirmed to produce aflatoxins. There were no significant differences in potential aflatoxigenic isolates with respect to the rainfed and irrigated zones. However, the number of potential aflatoxigenic isolates was significantly higher ( p < 0.05) in stored samples than that of those from fresh sesame seeds in the rainfed and irrigated zone. Whole genome sequencing and comparative analyses of 12 select isolates have revealed that one of the A. flavus isolates, which produced very low aflatoxins (AFP10), has an elevated missense variant rate, numerous high impact mutations, and a 600 base pair deletion in the norB gene. In summary, our study provides insights into aflatoxigenic potential and the associated genetic diversity of indigenous Aspergillus section Flavi isolates and potential management strategies for reducing aflatoxin contamination levels in a major crop consumed in Punjab, Pakistan.
Cadmium is an exceptionally toxic industrial and environmental pollutant classified as a human carcinogen. In order to provide insight into how we can keep our environment safe from cadmium contamination and prevent the accumulation of it in the food chain, we aim to elucidate how Aspergillus nidulans , one of the most abundant fungi in soil, survives and handles cadmium stress. As AtfA is the main transcription factor governing stress responses in A. nidulans , we examined genome-wide expression responses of wild-type and the atfA null mutant exposed to CdCl. Both strains showed up-regulation of the crpA Cu/Cd pump gene and AN7729 predicted to encode a putative bis(glutathionato)-cadmium transporter, and transcriptional changes associated with elevated intracellular Cys availability leading to the efficient adaptation to Cd. Although the deletion of atfA did not alter the cadmium tolerance of the fungus, the cadmium stress response of the mutant differed from that of a reference strain. Promoter and transcriptional analyses of the "Phospho-relay response regulator" genes suggest that the AtfA-dependent regulation of these genes can be relevant in this phenomenon. We concluded that the regulatory network of A. nidulans has a high flexibility allowing the fungus to adapt efficiently to stress both in the presence and absence of this important transcription factor.
Microbes (bacteria, yeasts, molds), in addition to plants and animals, were domesticated for their roles in food preservation, nutrition and flavor. Aspergillus oryzae is a domesticated filamentous fungal species traditionally used during fermentation of Asian foods and beverage, such as sake, soy sauce, and miso. To date, little is known about the extent of genome and phenotypic variation of A. oryzae isolates from different clades. Here, we used long-read Oxford Nanopore and short-read Illumina sequencing to produce a highly accurate and contiguous genome assemble of A. oryzae 14160, an industrial strain from China. To understand the relationship of this isolate, we performed phylogenetic analysis with 90 A. oryzae isolates and 1 isolate of the A. oryzae progenitor, Aspergillus flavus . This analysis showed that A. oryzae 14160 is a member of clade A, in comparison to the RIB 40 type strain, which is a member of clade F. To explore genome variation between isolates from distinct A. oryzae clades, we compared the A. oryzae 14160 genome with the complete RIB 40 genome. Our results provide evidence of independent evolution of the alpha-amylase gene duplication, which is one of the major adaptive mutations resulting from domestication. Synteny analysis revealed that both genomes have three copies of the alpha-amylase gene, but only one copy on chromosome 2 was conserved. While the RIB 40 genome had additional copies of the alpha-amylase gene on chromosomes III, and V, 14160 had a second copy on chromosome II and an third copy on chromosome VI. Additionally, we identified hundreds of lineage specific genes, and putative high impact mutations in genes involved in secondary metabolism, including several of the core biosynthetic genes. Finally, to examine the functional effects of genome variation between strains, we measured amylase activity, proteolytic activity, and growth rate on several different substrates. RIB 40 produced significantly higher levels of amylase compared to 14160 when grown on rice and starch. Accordingly, RIB 40 grew faster on rice, while 14160 grew faster on soy. Taken together, our analyses reveal substantial genome and phenotypic variation within A. oryzae .
The VosA-VelB heterocomplex governs expression of several genes associated with fungal development and secondary metabolism. In this study, we have investigated the functions of one of the VosA-VelB-activated developmental genes vadJ in development and production of the mycotoxin sterigmatocystin in the model fungus Aspergillus nidulans. The vadJ gene is predicted to encode a 957-amino acid length protein containing a highly conserved sensor histidine kinase domain. The deletion of vosA or velB resulted in decreased mRNA levels of vadJ throughout the life cycle, suggesting that VosA and VelB are necessary for proper expression of vadJ. Nullifying vadJ led to highly restricted colony growth, lowered formation of asexual spores, and about two-fold reduction in conidial viability. Conversely, the deletion of vadJ resulted in elevated production of sexual fruiting bodies and sterigmatocystin. These suggest that VadJ is necessary for proper coordination of asexual and sexual development, and sterigmatocystin production. In accordance with this idea, the deletion of vadJ led to elevated mRNA levels of the two key sexual developmental activators esdC and nsdD. In summary, the putative sensor histidine kinase VadJ represses sexual development and sterigmatocystin production, but activates asexual development in A. nidulans.
Monascus spp. are widely used in the production of monacolin K and food- grade pigments in East Asia. In Aspergillus species, the three transcription factors BrlA → AbaA → WetA sequentially function as the central activators of asexual development (conidiation), leading to the formation of conidiophores. Unlike their close relative Aspergillus spp., Monascus spp. produce basipetospora-type asexual spores (conidia), and their genomes contain homologs of brlA and wetA but not abaA. In the present study, to investigate their roles in Monascus conidiation, MrbrlA and MrwetA were functionally characterized by gene knockout and overexpression in Monascus ruber M7. The results revealed that the deletion and overexpression of MrbrlA and/or MrwetA caused no apparent changes in the morphology, size, number, structure, or germination of conidia. However, deletion and overexpression of MrwetA severely repressed sexual development and affected the production of secondary metabolites. Taken together, these results suggest that the well-established central regulatory model of conidiation in Aspergillus is not applicable in their Monascus relatives. The results of the present study could enrich our understanding of the asexual development regulatory networks in filamentous fungi.
The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus , five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (Δ) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA , brlA, and wetA . Moreover, Δ mbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the Δ mbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the Δ mbsA conidium. Comparative transcriptomic analyses revealed that the Δ mbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the Δ mbsA mutant produced higher amount of GT, Δ mbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway.
Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance for deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigated functions of the dugB and dugC genes in the model fungus Aspergillus nidulans These genes are orthologues of dug2 and dug3 , which are involved in cytosolic GSH degradation in Saccharomyces cerevisiae The deletion of dugB , dugC , or both resulted in a moderate increase in the GSH content in mycelia grown on glucose, reduced conidium production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding mitogen-activated protein (MAP) kinase pathway elements (e.g., steC , sskB , hogA , and mkkA ) or regulatory proteins of conidiogenesis and sexual differentiation (e.g., flbA , flbC , flbE , nosA , rosA , nsdC , and nsdD ) were downregulated in the Δ dugB Δ dugC mutant. Deletion of dugB and/or dugC slowed the depletion of GSH pools during carbon starvation. It also reduced accumulation of reactive oxygen species and decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions-in contrast to mycotoxin production-were controlled at the posttranscriptional level. We suggest that GSH connects starvation and redox regulation to each other: cells utilize GSH as a stored carbon source during starvation. The reduction of GSH content alters the redox state, activating regulatory pathways responsible for carbon starvation stress responses. IMPORTANCE Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character, and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, and secondary metabolite production (including mycotoxin formation), as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH-producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity, or oxidative stress tolerance of a fungal strain is manipulated.
In filamentous fungi, asexual development involves cellular differentiation and metabolic remodeling leading to the formation of intact asexual spores. The development of asexual spores (conidia) in Aspergillus is precisely coordinated by multiple transcription factors (TFs), including VosA, VelB, and WetA. Notably, these three TFs are essential for the structural and metabolic integrity, i.e., proper maturation, of conidia in the model fungus Aspergillus nidulans To gain mechanistic insight into the complex regulatory and interdependent roles of these TFs in asexual sporogenesis, we carried out multi-omics studies on the transcriptome, protein-DNA interactions, and primary and secondary metabolism employing A. nidulans conidia. RNA sequencing and chromatin immunoprecipitation sequencing analyses have revealed that the three TFs directly or indirectly regulate the expression of genes associated with heterotrimeric G-protein signal transduction, mitogen-activated protein (MAP) kinases, spore wall formation and structural integrity, asexual development, and primary/secondary metabolism. In addition, metabolomics analyses of wild-type and individual mutant conidia indicate that these three TFs regulate a diverse array of primary metabolites, including those in the tricarboxylic acid (TCA) cycle, certain amino acids, and trehalose, and secondary metabolites such as sterigmatocystin, emericellamide, austinol, and dehydroaustinol. In summary, WetA, VosA, and VelB play interdependent, overlapping, and distinct roles in governing morphological development and primary/secondary metabolic remodeling in Aspergillus conidia, leading to the production of vital conidia suitable for fungal proliferation and dissemination. IMPORTANCE Filamentous fungi produce a vast number of asexual spores that act as efficient propagules. Due to their infectious and/or allergenic nature, fungal spores affect our daily life. Aspergillus species produce asexual spores called conidia; their formation involves morphological development and metabolic changes, and the associated regulatory systems are coordinated by multiple transcription factors (TFs). To understand the underlying global regulatory programs and cellular outcomes associated with conidium formation, genomic and metabolomic analyses were performed in the model fungus Aspergillus nidulans Our results show that the fungus-specific WetA/VosA/VelB TFs govern the coordination of morphological and chemical developments during sporogenesis. The results of this study provide insights into the interdependent, overlapping, or distinct genetic regulatory networks necessary to produce intact asexual spores. The findings are relevant for other Aspergillus species such as the major human pathogen Aspergillus fumigatus and the aflatoxin producer Aspergillus flavus .
The most common, toxic, and carcinogenic mycotoxins found in human food and animal feed are the aflatoxins (AFs). The United States is a leading exporter of various nuts, with a marketing value of $9.1 billion in 2019; the European Union countries are the major importers of U.S. nuts. In the past few years, border rejections and notifications for U.S. tree nuts and peanuts exported to the E.U. countries have increased due to AF contamination. In this work, we analyzed notifications from the "Rapid Alert System for Food and Feed (RASFF)" on U.S. food and feed products contaminated with mycotoxins, primarily AFs, for the 10-year period 2010-2019. Almost 95% of U.S. mycotoxin RASFF notifications were reported for foods and only 5% for feeds. We found that 98.9% of the U.S. food notifications on mycotoxins were due to the AF contamination in almond, peanut, and pistachio nuts. Over half of these notifications (57.9%) were due to total AF levels greater than the FDA action level in food of 20 ng g. The Netherlands issued 27% of the AF notifications for U.S. nuts. Border rejection was reported for more than 78% of AF notifications in U.S. nuts. All U.S. feed notifications on mycotoxins occurred due to the AF contamination. Our research contributes to better understanding the main reasons behind RASFF mycotoxins notifications of U.S. food and feed products destined to E.U. countries. Furthermore, we speculate possible causes of this problem and provide a potential solution that could minimize the number of notifications for U.S. agricultural export market.
Compared to the traditional heterogeneous assays, a homogeneous immunoassay is a preferred format for its simplicity. By cloning and isolating luminescent proteins from bioluminescent organisms, bioluminescence has been widely used for various biological applications. In this study, we present the development of a homogeneous luminescence immunoassay (FNanoBiT assay) for detection of fumonisin B1 (FB1), based on the binding of two subunits of an engineered luminescent protein (NanoLuc). For the detection of the mycotoxin FB1 in foods, the anti-fumonisin antibody was conjugated to the large subunit of NanoLuc (FLgBiT), and the FB1 was conjugated to the small subunit (FSmBiT). The conjugates were used for the detection of FB1 in a competitive immunoassay format without the need of a secondary antibody, or washing steps. The developed FNanoBiT assay revealed high specificity toward FB1 with no cross-reactivity with other mycotoxins, and it demonstrated acceptable recovery (higher than 94%) and relative standard deviation from spiked maize samples. Further, the assay was successfully applied for the detection of FB1 in naturally contaminated maize, with a dynamic range of 0.533-6.81 ng mL-1 and a detection limit of 0.079 ng mL-1. The results derived with FNanoBiT assay of all spiked samples showed a strong correlation to those obtained by the High-performance liquid chromatography method. Thus, the FNanoBiT based homogeneous immunoassay could be used as a rapid, and simple tool for the analysis of mycotoxin-contaminated foods.
Afla-Guard is a commercial non-toxigenic Aspergillus flavus strain used to decrease aflatoxin contamination level in field. Its mitochondrial genome was sequenced, showing that its length is 29,208 bp with typical configuration of Aspergillus mitochondrial genome. 17 SNPs and 27 INDELs were identified by comparing with previous A. flavus mitochondrial genome. Phylogenetic trees present that A. flavus of Afla-Guard was clustered with the previous A. flavus mitochondrial genome.
The mitogenome of Aspergillus flavus SRRC1009 was sequenced to investigate intraspecific variations on mitochondrial genomes of A. flavus . It shows 29,202 bp with a typical configuration of Aspergillus mitogenome. Sixteen SNPs and 22 INDELs and 17 SNPs and 27 INDELs were identified against AflaGuard and JQ355000, respectively. Phylogenetic trees present in the three A. flavus mitochondrial genomes were clustered with A. oryzae mitochondrial genome in one clade.
Aspergillus flavus is an agriculturally and medically important filamentous fungus that produces mycotoxins, including aflatoxins, which are potent carcinogens. Here, we generated short- and long-read transcript sequence data from the growth of A. flavus strain NRRL 3357 under both typical and stress conditions to produce a new annotation of its genome.
The APSES transcription factor (TF) in Aspergillus species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the rgdA gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus Aspergillus fumigatus The rgdA deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators abaA , brlA , and wetA were decreased in the Δ rgdA mutant compared to those in the wild type (WT). Moreover, Δ rgdA resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the Δ rgdA mutant. Comparative transcriptomic analyses revealed that the Δ rgdA mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the Δ rgdA mutant exhibited elevated production of GT, Δ rgdA strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the Δ rgdA mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling. IMPORTANCE Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus Aspergillus fumigatus This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in A. fumigatus Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development.
The heterotrimeric G-protein (G-protein) signaling pathway is one of the most important signaling pathways that transmit external signals into the inside of the cell, triggering appropriate biological responses. The external signals are sensed by various G-protein-coupled receptors (GPCRs) and transmitted into G-proteins consisting of the α, β, and γ subunits. Regulators of G-protein signaling (RGSs) are the key controllers of G-protein signaling pathways. GPCRs, G-proteins, and RGSs are the primary upstream components of the G-protein signaling pathway, and they are highly conserved in most filamentous fungi, playing diverse roles in biological processes. Recent studies characterized the G-protein signaling components in the opportunistic pathogenic fungus Aspergillus fumigatus . In this review, we have summarized the characteristics and functions of GPCRs, G-proteins, and RGSs, and their regulatory roles in governing fungal growth, asexual development, germination, stress tolerance, and virulence in A. fumigatus .
McrA is a key transcription factor that functions as a global repressor of fungal secondary metabolism in Aspergillus species. Here, we report that mcrA is one of the VosA-VelB target genes and McrA governs the cellular and metabolic development in Aspergillus nidulans. The deletion of mcrA resulted in a reduced number of conidia and decreased mRNA levels of brlA, the key asexual developmental activator. In addition, the absence of mcrA led to a loss of long-term viability of asexual spores (conidia), which is likely associated with the lack of conidial trehalose and increased β-(1,3)-glucan levels in conidia. In supporting its repressive role, the mcrA deletion mutant conidia contain more amounts of sterigmatocystin and an unknown metabolite than the wild type conidia. While overexpression of mcrA caused the fluffy-autolytic phenotype coupled with accelerated cell death, deletion of mcrA did not fully suppress the developmental defects caused by the lack of the regulator of G-protein signaling protein FlbA. On the contrary to the cellular development, sterigmatocystin production was restored in the ΔflbA ΔmcrA double mutant, and overexpression of mcrA completely blocked the production of sterigmatocystin. Overall, McrA plays a multiple role in governing growth, development, spore viability, and secondary metabolism in A. nidulans.
Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms' primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing Mycobacterium tuberculosis H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different in silico approaches to understand CYP128 P450 family's distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 CYP128 P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the CYP128 gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between M. tuberculosis complex (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacterial category species. Of CYP128 P450s, 78% was a part of BGCs with CYP124A1 , or together with CYP124A1 and CYP121A1 . The CYP128 family ranked fifth in the conservation ranking. Unique amino acid patterns are present at the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that the CYP128A1 bind to MK9 with the highest affinity compared to the azole drugs analyzed. This study provides comprehensive comparative analysis and structural insights of CYP128A1 in M. tuberculosis .
Unraveling the role of cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins present in living and non-living entities, in secondary metabolite synthesis is gaining momentum. In this direction, in this study, we analyzed the genomes of 203 Streptomyces species for P450s and unraveled their association with secondary metabolism. Our analyses revealed the presence of 5460 P450s, grouped into 253 families and 698 subfamilies. The CYP107 family was found to be conserved and highly populated in Streptomyces and Bacillus species, indicating its key role in the synthesis of secondary metabolites. Streptomyces species had a higher number of P450s than Bacillus and cyanobacterial species. The average number of secondary metabolite biosynthetic gene clusters (BGCs) and the number of P450s located in BGCs were higher in Streptomyces species than in Bacillus , mycobacterial, and cyanobacterial species, corroborating the superior capacity of Streptomyces species for generating diverse secondary metabolites. Functional analysis via data mining confirmed that many Streptomyces P450s are involved in the biosynthesis of secondary metabolites. This study was the first of its kind to conduct a comparative analysis of P450s in such a large number (203) of Streptomyces species, revealing the P450s' association with secondary metabolite synthesis in Streptomyces species. Future studies should include the selection of Streptomyces species with a higher number of P450s and BGCs and explore the biotechnological value of secondary metabolites they produce.
The P-type ATPase CrpA is an important Cu /Cd pump in the Aspergilli, significantly contributing to the heavy metal stress tolerance of these ascomycetous fungi. As expected, the deletion of crpA resulted in Cu /Cd -sensitive phenotypes in Aspergillus nidulans on stress agar plates inoculated with conidia. Nevertheless, paradoxical growth stimulations were observed with the ΔcrpA strain in both standard Cu stress agar plate experiments and cellophane colony harvest (CCH) cultures, when exposed to Cd . These observations reflect efficient compensatory mechanisms for the loss of CrpA operating under these experimental conditions. It is remarkable that the ΔcrpA strain showed a 2.7 times higher Cd biosorption capacity in CCH cultures, which may facilitate the development of new, fungal biomass-based bioremediation technologies to extract harmful Cd ions from the environment. The nullification of crpA also significantly changed the spatial distribution of Cu and Cd in CCH cultures, as demonstrated by the combined particle-induced X-ray emission and scanning transmission ion microscopy technique. Most important, the centers of gravity for Cu and Cd accumulations of the ΔcrpA colonies shifted toward the older regions as compared with wild-type surface cultures.
The DnaJ family of proteins (or J-proteins) are molecular chaperones that govern protein folding, degradation, and translocation in many organisms. Although J-proteins play key roles in eukaryotic and prokaryotic biology, the role of J-proteins in Aspergillus species is currently unknown. In this study, we characterized the dnjA gene, which encodes a putative DnaJ protein, in two Aspergillus species: Aspergillus nidulans and Aspergillus flavus. Expression of the dnjA gene is inhibited by the velvet regulator VosA, which plays a pivotal role in spore survival and metabolism in Aspergillus. The deletion of dnjA decreased the number of asexual spores (conidia), produced abnormal conidiophores, and reduced sexual fruiting bodies (cleistothecia) or sclerotia. In addition, the absence of dnjA caused increased sterigmatocystin or aflatoxin production in A. nidulans and A. flavus, respectively. These results suggest that DnjA plays a conserved role in asexual and sexual development and mycotoxin production in Aspergillus species. However, DnjA also plays a species-specific role; AniDnjA but not AflDnjA, affects conidial viability, trehalose contents, and thermal tolerance of conidia. In plant virulence assay, the infection ability of the ΔAfldnjA mutant decreased in the kernels, suggesting that DnjA plays a crucial role in the pathogenicity of A. flavus. Taken together, these results demonstrate that DnjA is multifunctional in Aspergillus species; it is involved in diverse biological processes, including fungal differentiation and secondary metabolism.
Culture methods supplemented with high-performance liquid chromatography (HPLC) technique provide a rapid and simple tool for detecting levels of aflatoxins (AFs) produced by fungi. This study presents a robust method for simultaneous quantification of aflatoxin (AF) B1, B2, G1, and G2 levels in several fungal cultivation states: submerged shake culture, liquid slant culture, and solid-state culture. The recovery of the method was evaluated by spiking a mixture of AFs at several concentrations to the test medium. The applicability of the method was evaluated by using aflatoxigenic and non-aflatoxigenic Aspergilli . A HPLC coupled with the diode array (DAD) and fluorescence (FLD) detectors was used to determine the presence and amounts of AFs. Both detectors showed high sensitivity in detecting spiked AFs or AFs produced in situ by toxigenic fungi. Our methods showed 76%-88% recovery from medium spiked with 2.5, 10, 50, 100, and 500 ng/mL AFs. The limit of quantification (LOQ) for AFs were 2.5 to 5.0 ng/mL with DAD and 0.025 to 2.5 ng/mL with FLD. In this work, we described in detail a protocol, which can be considered the foremost and only verified method, to extract, detect, and quantify AFs employing both aflatoxigenic and non-toxigenic Aspergilli .
The short-lived hydrophobic gas nitric oxide (NO) is a broadly conserved signaling molecule in all domains of life, including the ubiquitous and versatile filamentous fungi (molds). Several studies have suggested that NO plays a vast and diverse signaling role in molds. In this review, we summarize NO-mediated signaling and the biosynthesis and degradation of NO in molds, and highlight the recent advances in understanding the NO-mediated regulation of morphological and physiological processes throughout the fungal life cycle. In particular, we describe the role of NO in molds as a signaling molecule that modulates asexual and sexual development, the formation of infection body appressorium, and the production of secondary metabolites (SMs). In addition, we also summarize NO detoxification and protective mechanisms against nitrooxidative stress.
The prokaryotic phylum Cyanobacteria are some of the oldest known photosynthetic organisms responsible for the oxygenation of the earth. Cyanobacterial species have been recognised as a prosperous source of bioactive secondary metabolites with antibacterial, antiviral, antifungal and/or anticancer activities. Cytochrome P450 monooxygenases (CYPs/P450s) contribute to the production and diversity of various secondary metabolites. To better understand the metabolic potential of cyanobacterial species, we have carried out comprehensive analyses of P450s, predicted secondary metabolite biosynthetic gene clusters (BGCs), and P450s located in secondary metabolite BGCs. Analysis of the genomes of 114 cyanobacterial species identified 341 P450s in 88 species, belonging to 36 families and 79 subfamilies. In total, 770 secondary metabolite BGCs were found in 103 cyanobacterial species. Only 8% of P450s were found to be part of BGCs. Comparative analyses with other bacteria Bacillus , Streptomyces and mycobacterial species have revealed a lower number of P450s and BGCs and a percentage of P450s forming part of BGCs in cyanobacterial species. A mathematical formula presented in this study revealed that cyanobacterial species have the highest gene-cluster diversity percentage compared to Bacillus and mycobacterial species, indicating that these diverse gene clusters are destined to produce different types of secondary metabolites. The study provides fundamental knowledge of P450s and those associated with secondary metabolism in cyanobacterial species, which may illuminate their value for the pharmaceutical and cosmetics industries.
The velvet regulator VosA plays a pivotal role in asexual sporulation in the model filamentous fungus Aspergillus nidulans . In the present study, we characterize the roles of VosA in sexual spores (ascospores) in A . nidulans . During ascospore maturation, the deletion of vosA causes a rapid decrease in spore viability. The absence of vosA also results in a lack of trehalose biogenesis and decreased tolerance of ascospores to thermal and oxidative stresses. RNA-seq-based genome-wide expression analysis demonstrated that the loss of vosA leads to elevated expression of sterigmatocystin (ST) biosynthetic genes and a slight increase in ST production in ascospores. Moreover, the deletion of vosA causes upregulation of additional gene clusters associated with the biosynthesis of other secondary metabolites, including asperthecin, microperfuranone, and monodictyphenone. On the other hand, the lack of vosA results in the downregulation of various genes involved in primary metabolism. In addition, vosA deletion alters mRNA levels of genes associated with the cell wall integrity and trehalose biosynthesis. Overall, these results demonstrate that the velvet regulator VosA plays a key role in the maturation and the cellular and metabolic integrity of sexual spores in A. nidulans .
In the comparative transcriptomic studies of wild type (WT) and rax1 null mutant strains, we obtained an average of 22,222,727 reads of 101 bp per sample and found that 183 genes showed greater than 2.0-fold differential expression, where 92 and 91 genes were up-and down-regulated in rax1 compared to WT, respectively. In accordance with the significantly reduced levels of gliM and casB transcripts in the absence of rax1, the rax1 mutant exhibited increased sensitivity to exogenous gliotoxin (GT) without affecting levels of GT production. Moreover, rax1 resulted in significantly restricted colony growth and reduced viability under endoplasmic reticulum stress condition. In summary, Rax1 positively affects expression of gliM and metacaspase genes.
The regulator of G-protein signaling (RGS) proteins play an important role in upstream control of heterotrimeric G-protein signaling pathways. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus , six RGS protein-encoding genes are present. To characterize the rgsA gene predicted to encode a protein with an RGS domain, we generated an rgsA null mutant and observed the phenotypes of the mutant. The deletion (Δ) of rgsA resulted in increased radial growth and enhanced asexual sporulation in both solid and liquid culture conditions. Accordingly, transcripts levels of the key asexual developmental regulators abaA , brlA, and wetA are elevated in the Δ rgsA mutant. Moreover, Δ rgsA resulted in elevated spore germination rates in the absence of a carbon source. The activity of cAMP-dependent protein kinase A (PKA) and mRNA levels of genes encoding PKA signaling elements are elevated by Δ rgsA . In addition, mRNA levels of genes associated with stress-response signaling increased with the lack of rgsA , and the Δ rgsA spores showed enhanced tolerance against oxidative stressors. Comparative transcriptomic analyses revealed that the Δ rgsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes. Accordingly, the rgsA null mutant exhibited increased production of GT and elevated virulence in the mouse. Conversely, the majority of genes encoding glucan degrading enzymes were down-regulated by Δ rgsA, and endoglucanase activities were reduced. In summary, RgsA plays multiple roles, governing growth, development, stress responses, virulence, and external polymer degradation-likely by attenuating PKA signaling.
Fungal development is regulated by a variety of transcription factors in Aspergillus nidulans. Previous studies demonstrated that the NF-κB type velvet transcription factors regulate certain target genes that govern fungal differentiation and cellular metabolism. In this study, we characterize one of the VosA/VelB-inhibited developmental genes called vidA, which is predicted to encode a 581-amino acid protein with a CH zinc finger domain at the C-terminus. Levels of vidA mRNA are high during the early and middle phases of asexual development and decrease during the late phase of asexual development and asexual spore (conidium) formation. Deletion of either vosA or velB results in increased vidA mRNA accumulation in conidia, suggesting that vidA transcript accumulation in conidia is repressed by VosA and VelB. Phenotypic analysis demonstrated that deletion of vidA causes decreased colony growth, reduced production of asexual spores, and abnormal formation of sexual fruiting bodies. In addition, the vidA deletion mutant conidia contain more trehalose and β-glucan than wild type. Overall, these results suggest that VidA is a putative transcription factor that plays a key role in governing proper fungal growth, asexual and sexual development, and conidia formation in A. nidulans.
Strains of Monascus filamentous fungal species have been used to produce fermented foods in Asian countries, such as China, Japan, and The Korean Peninsula, for nearly 2,000 years. At present, their fermented products are widely used as food additives and nutraceutical supplements worldwide owing to their production of beneficial secondary metabolites. Heterotrimeric G-protein signaling pathways participate in regulating multiple biological processes in fungi. Previously, we identified three Monascus ruber M7 G-protein α subunits (Mga1-3) and demonstrated that Mga1 can regulate growth, reproduction and some secondary metabolites' production. Here, we systematically analyzed and compared the roles of mga 1-3 by combining single- and double-gene(s) knockouts and their transcriptomic data. First, mga 2 and mga 3 knock-out mutants and pairwise combinations of mga 1-3 deletion strains were generated. Then the changes in growth, development and the main secondary metabolites, Monascus pigments and citrinin, in these mutants were systematically compared with M. ruber M7. Moreover, RNA-Seq analyses of these mutants were performed. All three Gα subunits worked together to regulate biological processes in M. ruber M7, with Mga1 playing a major role, while Mga2 and Mga3 playing supplemental roles. According to the existing literatures which we can find, gene knock-out mutants of the pairwise combination of mga 1-3 and their transcriptome analysis are first reported in this study. The current results have clearly demonstrated the functional division of Mga1-3 in M. ruber M7, and could provide a deeper understanding of the effects of different Gα subunits on growth, development and secondary metabolism in other filamentous fungi.
Tremellomycetes , a fungal class in the subphylum Agaricomycotina , contain well-known opportunistic and emerging human pathogens. The azole drug fluconazole, used in the treatment of diseases caused by some species of Tremellomycetes , inhibits cytochrome P450 monooxygenase CYP51, an enzyme that converts lanosterol into an essential component of the fungal cell membrane ergosterol. Studies indicate that mutations and over-expression of CYP51 in species of Tremellomycetes are one of the reasons for fluconazole resistance. Moreover, the novel drug, VT-1129, that is in the pipeline is reported to exert its effect by binding and inhibiting CYP51. Despite the importance of CYPs, the CYP repertoire in species of Tremellomycetes has not been reported to date. This study intends to address this research gap. Comprehensive genome-wide CYP analysis revealed the presence of 203 CYPs (excluding 16 pseudo-CYPs) in 23 species of Tremellomycetes that can be grouped into 38 CYP families and 72 CYP subfamilies. Twenty-three CYP families are new and three CYP families (CYP5139, CYP51 and CYP61) were conserved across 23 species of Tremellomycetes . Pathogenic cryptococcal species have 50% fewer CYP genes than non-pathogenic species. The results of this study will serve as reference for future annotation and characterization of CYPs in species of Tremellomycetes .
Tuberculosis (TB) is one of the top infectious diseases causing numerous human deaths in the world. Despite enormous efforts, the physiology of the causative agent, Mycobacterium tuberculosis , is poorly understood. To contribute to better understanding the physiological capacity of these microbes, we have carried out extensive in silico analyses of the 1111 mycobacterial species genomes focusing on revealing the role of the orphan cytochrome P450 monooxygenase (CYP) CYP139 family. We have found that CYP139 members are present in 894 species belonging to three mycobacterial groups: M. tuberculosis complex (850-species), Mycobacterium avium complex (34-species), and non-tuberculosis mycobacteria (10-species), with all CYP139 members belonging to the subfamily "A". CYP139 members have unique amino acid patterns at the CXG motif. Amino acid conservation analysis placed this family in the 8th among CYP families belonging to different biological domains and kingdoms. Biosynthetic gene cluster analyses have revealed that 92% of CYP139As might be associated with producing different secondary metabolites. Such enhanced secondary metabolic potentials with the involvement of CYP139A members might have provided mycobacterial species with advantageous traits in diverse niches competing with other microbial or viral agents, and might help these microbes infect hosts by interfering with the hosts' metabolism and immune system.
Soil-dwelling fungal species possess the versatile metabolic capability to degrade complex organic compounds that are toxic to humans, yet the mechanisms they employ remain largely unknown. Benzo[ a ]pyrene (BaP) is a pervasive carcinogenic contaminant, posing a significant concern for human health. Here, we report that several Aspergillus species are capable of degrading BaP. Exposing Aspergillus nidulans cells to BaP results in transcriptomic and metabolic changes associated with cellular growth and energy generation, implying that the fungus utilizes BaP as a growth substrate. Importantly, we identify and characterize the conserved bapA gene encoding a cytochrome P450 monooxygenase that is necessary for the metabolic utilization of BaP in Aspergillus We further demonstrate that the fungal NF-κB-type velvet regulators VeA and VelB are required for proper expression of bapA in response to nutrient limitation and BaP degradation in A. nidulans Our study illuminates fundamental knowledge of fungal BaP metabolism and provides novel insights into enhancing bioremediation potential. IMPORTANCE We are increasingly exposed to environmental pollutants, including the carcinogen benzo[ a ]pyrene (BaP), which has prompted extensive research into human metabolism of toxicants. However, little is known about metabolic mechanisms employed by fungi that are able to use some toxic pollutants as the substrates for growth, leaving innocuous by-products. This study systemically demonstrates that a common soil-dwelling fungus is able to use benzo[ a ]pyrene as food, which results in expression and metabolic changes associated with growth and energy generation. Importantly, this study reveals key components of the metabolic utilization of BaP, notably a cytochrome P450 monooxygenase and the fungal NF-κB-type transcriptional regulators. Our study advances fundamental knowledge of fungal BaP metabolism and provides novel insight into designing and implementing enhanced bioremediation strategies.
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Cytochrome P450 monooxygenases (P450s) found in all domains of life are known for their catalytic versatility and stereo- and regio-specific activity. While the impact of lifestyle on P450 evolution was reported in many eukaryotes, this remains to be addressed in bacteria. In this report, Streptomyces and Mycobacterium, belonging to the phylum Actinobacteria, were studied owing to their contrasting lifestyles and impacts on human. Analyses of all P450s and those predicted to be associated with secondary metabolism have revealed that different lifestyles have affected the evolution of P450s in these bacterial genera. We have found that while species in both genera have essentially the same number of P450s in the genome, Streptomyces P450s are much more diverse than those of Mycobacterium. Moreover, despite both belonging to Actinobacteria, only 21 P450 families were common, and 123 and 56 families were found to be unique to Streptomyces and Mycobacterium, respectively. The presence of a large and diverse number of P450s in Streptomyces secondary metabolism contributes to antibiotic diversity, helping to secure the niche. Conversely, based on the currently available functional data, types of secondary metabolic pathways and associated P450s, mycobacterial P450s seem to play a role in utilization or synthesis of lipids.
The regulator of G protein signaling (RGS) domain proteins generally attenuate heterotrimeric G protein signaling, thereby fine-tune the duration and strength of signal transduction. In this study, we characterize the functions of RgsD, one of the six RGS domain proteins present in the human pathogenic fungus Aspergillus fumigatus. The deletion (Δ) of rgsD results in enhanced asexual sporulation coupled with increased mRNA levels of key developmental activators. Moreover, ΔrgsD leads to increased spore tolerance to UV and oxidative stress, which might be associated with the enhanced expression of melanin biosynthetic genes and increased amount of melanin. Yeast two-hybrid assays reveal that RgsD can interact with the three Gα proteins GpaB, GanA, and GpaA, showing the highest interaction potential with GpaB. Importantly, the ΔrgsD mutant shows elevated expression of genes in the cAMP-dependent protein kinase A (PKA) pathway and PKA catalytic activity. The ΔrgsD mutant also display increased gliotoxin production and elevated virulence toward Galleria mellonella wax moth larvae. Transcriptomic analyses using RNA-seq reveal the expression changes associated with the diverse phenotypic outcomes caused by ΔrgsD. Collectively, we conclude that RgsD attenuates cAMP-PKA signaling pathway and negatively regulates asexual development, toxigenesis, melanin production, and virulence in A. fumigatus.
The formation of sexual fruiting bodies for plant pathogenic fungi is a key strategy to propagate their progenies upon environmental stresses. Stemphylium eturmiunum is an opportunistic plant pathogen fungus causing blight in onion. This self-fertilizing filamentous ascomycete persists in the soil by forming pseudothecia, the sexual fruiting body which helps the fungus survive in harsh environments. However, the regulatory mechanism of pseudothecial formation remains unknown. To uncover the mechanism for pseudothecial formation so as to find a practical measure to control the propagation of this onion pathogen, we tentatively used DNA methyltransferase inhibitor 5-azacytidine (5-AC) to treat S. eturmiunum. 5-AC treatment silenced the gene-encoding monoacylglycerol lipase (magl) concomitant with the presence of the inheritable fluffy phenotype and defectiveness in pseudothecial development. Moreover, the silence of magl also resulted in a reduction of arachidonic acid (AA) formation from 27 ± 3.1 µg/g to 9.5 ± 1.5 µg/g. To correlate the biosynthesis of AA and pseudothecial formation, we created magl knockdown and overexpression strains. Knockdown of magl reduced AA to 11 ± 2.4 µg/g, which subsequently disabled pseudothecial formation. In parallel, overexpression of magl increased AA to 37 ± 3.4 µg/g, which also impaired pseudothecial formation. Furthermore, exogenous addition of AA to the culture of magl-silenced or magl knockdown strains rescued the pseudothecial formation but failed in the gpr1 knockdown strain of S. eturmiunum, which implicates the involvement of AA in signal transduction via a putative G protein-coupled receptor 1. Thus, AA at a cellular level of 27 ± 3.1 µg/g is essential for sexual development of S. eturmiunum. Disturbance in the biosynthesis of AA by up- and down-regulating the expression of magl disables the pseudothecial development. The specific requirement for AA in pseudothecial development by S. eturmiunum provides a hint to curb this onion pathogen: to impede pseudothecial formation by application of AA.
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has long-been used as a model organism to gain insights into the genetic basis of asexual and sexual developmental processes both in other members of the genus , and filamentous fungi in general. Paradigms have been established concerning the regulatory mechanisms of conidial development. However, recent studies have shown considerable genome divergence in the fungal kingdom, questioning the general applicability of findings from , and certain longstanding evolutionary theories have been questioned. The phylogenetic distribution of key regulatory elements of asexual reproduction in was investigated in a broad taxonomic range of fungi. This revealed that some proteins were well conserved in the ( AbaA, FlbA, FluG, NsdD, MedA, and some velvet proteins), suggesting similar developmental roles. However, other elements ( BrlA) had a more restricted distribution solely in the , and it appears that the genetic control of sporulation seems to be more complex in the aspergilli than in some other taxonomic groups of the . The evolution of the velvet protein family is discussed based on the history of expansion and contraction events in the early divergent fungi. Heterologous expression of the gene in failed to induce development of complete conidiophores as seen in the aspergilli, but did result in increased conidial production. The absence of many components of the asexual developmental pathway from members of the supports the hypothesis that differences in the complexity of their spore formation is due in part to the increased diversity of the sporulation machinery evident in the Pezizomycotina. Investigations were also made into the evolution of sex and sexuality in the aspergilli. loci were identified from the heterothallic () and () and the homothallic (=). A consistent architecture of the locus was seen in these and other heterothallic aspergilli whereas much variation was seen in the arrangement of loci in homothallic aspergilli. This suggested that it is most likely that the common ancestor of the aspergilli exhibited a heterothallic breeding system. Finally, the supposed prevalence of asexuality in the aspergilli was examined. Investigations were made using as a representative 'asexual' species. It was possible to induce a sexual cycle in given the correct and partners and environmental conditions, with recombination confirmed utilising molecular markers. This indicated that sexual reproduction might be possible in many supposedly asexual aspergilli and beyond, providing general insights into the nature of asexuality in fungi.
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Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria -42; fungi -19; plant -28; animal -22; plant and animal -1 and common P450 family -1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study's results offer new understanding of the dynamic structural nature of P450s.
The G-protein-coupled receptor (GPCR) family represents the largest and most varied collection of membrane embedded proteins that are sensitized by ligand binding and interact with heterotrimeric G proteins. Despite their presumed critical roles in fungal biology, the functions of the GPCR family members in the opportunistic human pathogen Aspergillus fumigatus are largely unknown, as only two (GprC and GprD) of the 15 predicted GPCRs have been studied. Here, we characterize the gprK gene, which is predicted to encode a hybrid GPCR with both 7-transmembrane and regulator of G-protein signaling (RGS) domains. The deletion of gprK causes severely impaired asexual development coupled with reduced expression of key developmental activators. Moreover, ΔgprK results in hyper-activation of germination even in the absence of carbon source, and elevated expression and activity of the protein kinase A PkaC1. Furthermore, proliferation of the ΔgprK mutant is restricted on the medium when pentose is the sole carbon source, suggesting that GprK may function in external carbon source sensing. Notably, the absence of gprK results in reduced tolerance to oxidative stress and significantly lowered mRNA levels of the stress-response associated genes sakA and atfA. Activities of catalases and SODs are severely decreased in the ΔgprK mutant, indicating that GprK may function in proper activation of general stress response. The ΔgprK mutant is also defective in gliotoxin (GT) production and slightly less virulent toward the greater wax moth, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters are down-regulated by ΔgprK. In summary, GprK is necessary for proper development, GT production, and oxidative stress response, and functions in down-regulating the PKA-germination pathway.
The aryl hydrocarbon receptor (AhR) is a ligand activated transcriptional regulator, which governs key biological processes including detoxification of carcinogens. β-Naphthoflavone (β-NF) is a non-toxic flavonoid, and a potent AhR agonist. Thus, β-NF can induce the representative detoxifying enzyme cytochrome P4501A1, thereby enhancing the detoxification potential. However, its low water solubility hampers the use. We found that supramolecular complexation of β-NF with the synthetic 6,6'-thiobis(methylene)-β-cyclodextrin (β-CD-S) dimer significantly enhanced β-NF's role as an AhR agonist. The water solubility of β-NF was increased to 469 fold by effective supramolecular complexation with the β-CD-S dimer, and caused significant induction of cytochrome P4501A1. Stable formation of the supramolecular complex of β-NF with β-CD-S-dimer was verified by various analyses. In summary, supramolecular complexation of β-NF with β-CD-S dimer greatly enhanced bio-availability of β-NF as an AhR agonist. Our findings provide an easy, non-destructive, and alternative approach to enhance the bio-availability of therapeutics.
Aspergillus fumigatus reproduces and infects host by forming a high number of small asexual spores (conidia). The velvet proteins are global transcriptional regulators governing the complex process of conidiogenesis in this fungus. Here, to further understand the velvet-mediated regulation, we carried out comparative proteomic analyses of conidia of wild type (WT) and three velvet mutants (ΔveA, ΔvelB and ΔvosA). Cluster analysis of 184 protein spots showing at least 1.5-fold differential accumulation between WT and mutants reveal the clustering of WT- ΔveA and ΔvelB-ΔvosA. Among 43 proteins identified by Nano-LC-ESI-MS/MS, 23 including several heat shock proteins showed more than two-fold reduction in both the ∆velB and ∆vosA conidia. On the contrary, three proteins exhibited more than five-fold increase in ∆veA only, including the putative RNA polymerase II degradation factor DefA. The deletion of defA resulted in a reduced number of conidia and restricted colony growth. In addition, the defA deletion mutant conidia showed hypersensitivity against the DNA damaging agents NQO and MMS, while the ΔveA mutant conidia were more resistant against to NQO. Taken together, we propose that VeA controls protein level of DefA in conidia, which are dormant and equipped with multiple layers of protection against environmental cues.
Asexual development (conidiation) in the filamentous fungus Aspergillus nidulans is governed by orchestrated gene expression. The three key negative regulators of conidiation SfgA, VosA, and NsdD act at different control point in the developmental genetic cascade. Here, we have revealed that NsdD is a key repressor affecting the quantity of asexual spores in Aspergillus. Moreover, nullifying both nsdD and vosA results in abundant formation of the development specific structure conidiophores even at 12 h of liquid culture, and near constitutive activation of conidiation, indicating that acquisition of developmental competence involves the removal of negative regulation exerted by both NsdD and VosA. NsdD's role in repressing conidiation is conserved in other aspergilli, as deleting nsdD causes enhanced and precocious activation of conidiation in Aspergillus fumigatus or Aspergillus flavus. In vivo NsdD-DNA interaction analyses identify three NsdD binding regions in the promoter of the essential activator of conidiation brlA, indicating a direct repressive role of NsdD in conidiation. Importantly, loss of flbC or flbD encoding upstream activators of brlA in the absence of nsdD results in delayed activation of brlA, suggesting distinct positive roles of FlbC and FlbD in conidiation. A genetic model depicting regulation of conidiation in A. nidulans is presented.
The filamentous fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing severe and usually fatal invasive aspergillosis in immunocompromised patients. This fungus produces a large number of small hydrophobic asexual spores called conidia as the primary means of reproduction, cell survival, propagation, and infectivity. The initiation, progression, and completion of asexual development (conidiation) is controlled by various regulators that govern expression of thousands of genes associated with formation of the asexual developmental structure conidiophore, and biogenesis of conidia. In this review, we summarize key regulators that directly or indirectly govern conidiation in this important pathogenic fungus. Better understanding these developmental regulators may provide insights into the improvement in controlling both beneficial and detrimental aspects of various Aspergillus species.
Excess adipogenesis is a characteristic of obesity, which is associated with serious health problem, including type 2 diabetes. Here, to better understand the mechanisms for the development of adipocytes, we investigated the regulatory role of 15-(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in adipogenesis by treating 3T3-L1 murine preadipocytes and human bone marrow mesenchymal stem cells (hBMSCs) with 15(S)-HETE. In the 3T3-L1 study, 15(S)-HETE stimulated lipid accumulation and enhanced expression of adipogenic markers such as peroxisome proliferator-activated receptor gamma (PPARγ), yet reduced the activity of factor negatively controlling adipogenesis, such as the 5'-AMP-activated protein kinase. In the early stage of adipogenesis, 15(S)-HETE increased activation of protein kinase B and expression of CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ. Finally, 15(S)-HETE promoted adipogenesis in hBMSCs, and resulted in increased lipid accumulation and expression of adipogenic markers. In conclusion, 15(S)-HETE functions as a natural PPARγ agonist and enhances adipogenesis. Our findings may provide the basis for the development of novel therapeutic measures to treat obesity.
Mitochondria play key roles in cellular energy generation and lifespan of most eukaryotes. To understand the functions of four nuclear-encoded genes predicted to be related to the maintenance of mitochondrial morphology and function in Aspergillus nidulans, systematic characterization was carried out. The deletion and overexpression mutants of aodA, dnmA, mnSOD and pimA encoding alternative oxidase, dynamin related protein, manganese superoxide dismutase and Lon protease, respectively, were generated and examined for their growth, stress tolerances, respiration, autolysis, cell death, sterigmatocystin production, hyphal morphology and size, and mitochondrial superoxide production as well as development. Overall, genetic manipulation of these genes had less effect on cellular physiology and ageing in A. nidulans than that of their homologs in another fungus Podospora anserina with a well-characterized senescence. The observed interspecial phenotypic differences can be explained by the dissimilar intrinsic stabilities of the mitochondrial genomes in A. nidulans and P. anserina. Furthermore, the marginally altered phenotypes observed in A. nidulans mutants indicate the presence of effective compensatory mechanisms for the complex networks of mitochondrial defense and quality control. Importantly, these findings can be useful for developing novel platforms for heterologous protein production, or on new biocontrol and bioremediation technologies based on Aspergillus species.
Paclitaxel (PTX) is a commonly used drug to treat diverse cancer types. However, its treatment can generate resistance and the mechanisms of PTX-resistance in lung cancers are still unclear. We demonstrated that non-small cell lung cancers (NSCLCs) survive PTX treatment. Compared with the progenitor NSCLC A549 cells, the PTX-resistant A549 cells (A549/PTX) displayed enhanced sphere-formation ability. The proportion of the cancer stem cell marker, aldehyde dehydrogenase-positive cells, and epithelial-mesenchymal transition signaling protein levels were also elevated in A549/PTX. Importantly, the levels of oncoproteins phosphoinositide-3 kinase/Akt, mucin 1 cytoplasmic domain (MUC1-C) and β-catenin were also significantly elevated in A549/PTX. Furthermore, nuclear translocation of MUC1-C and β-catenin increased in A549/PTX. The c-SRC protein, an activator of MUC1-C, was also overexpressed in A549/PTX. These observations led to the hypothesis that enhanced expression of MUC1-C is associated with stemness and PTX resistance in NSCLCs. To test this, we knocked down or overexpressed MUC1-C in A549/PTX and found that inhibition of MUC1-C expression coupled with PTX treatment was sufficient to reduce the sphere-forming ability and survival of A549/PTX. In summary, our in vitro and in vivo studies have revealed a potential mechanism of MUC1-C-mediated PTX resistance and provided insights into a novel therapeutic measure for lung cancers.
We present the synthesis of novel magnetic nanoparticles functionalized by benzene- and β-cyclodextrin-derivatized dextran. The grafting strategy was based on the [alkynyl-iron] cluster in the modified dextrans, which were prepared by click reaction from alkyne-modified dextran and benzyl azide or mono-6-O-deoxy-monoazido β-cyclodextrin. Characterization was then carried out by thermogravimetric analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and vibrating sample magnetometry. Using the developed magnetic nanoparticles, the potential for removing polycyclic aromatic hydrocarbons such as phenanthrene and pyrene by sorption onto the nanomaterials was assessed. In the sorption, pi-stacking interactions of the benzene-derivatized dextran and host-guest chemistry of the β-cyclodextrin-derivatized dextran were considered to be significant. Furthermore, the polysaccharide derivative-coated magnetic adsorbents could be recovered by an external magnet for reuse.
Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host/ecological niche can influence shaping the P450 content of an organism. The present study initiates our understanding of P450 family patterns in basidiomycete biotrophic plant pathogens.
Nutrient sensing and utilisation are fundamental for all life forms. As heterotrophs, fungi have evolved a diverse range of mechanisms for sensing and taking up various nutrients. Despite its importance, only a limited number of nutrient receptors and their corresponding ligands have been identified in fungi. G-protein coupled receptors (GPCRs) are the largest family of transmembrane receptors. The Aspergillus nidulans genome encodes 16 putative GPCRs, but only a few have been functionally characterised. Our previous study showed the increased expression of an uncharacterised putative GPCR, gprH, during carbon starvation. GprH appears conserved throughout numerous filamentous fungi. Here, we reveal that GprH is a putative receptor involved in glucose and tryptophan sensing. The absence of GprH results in a reduction in cAMP levels and PKA activity upon adding glucose or tryptophan to starved cells. GprH is pre-formed in conidia and is increasingly active during carbon starvation, where it plays a role in glucose uptake and the recovery of hyphal growth. GprH also represses sexual development under conditions favouring sexual fruiting and during carbon starvation in submerged cultures. In summary, the GprH nutrient-sensing system functions upstream of the cAMP-PKA pathway, influences primary metabolism and hyphal growth, while represses sexual development in A. nidulans.
Orchestration of cellular growth and development occurs during the life cycle of Aspergillus nidulans. A multi-copy genetic screen intended to unveil novel regulators of development identified the AN6578 locus predicted to encode a protein with the WOPR domain, which is a broadly present fungi-specific DNA-binding motif. Multi-copy of AN6578 disrupted the normal life cycle of the fungus leading to enhanced proliferation of vegetative cells, whereas the deletion resulted in hyper-active sexual fruiting with reduced asexual development (conidiation), thus named as osaA (Orchestrator of Sex and Asex). Further genetic studies indicate that OsaA balances development mainly by repressing sexual development downstream of the velvet regulator VeA. The absence of osaA is sufficient to suppress the veA1 allele leading to the sporulation levels comparable to veA+ wild type (WT). Genome-wide transcriptomic analyses of WT, veA1, and ΔosaA veA1 strains by RNA-Seq further corroborate that OsaA functions in repressing sexual development downstream of VeA. However, OsaA also plays additional roles in controlling development, as the ΔosaA veA1 mutant exhibits precocious and enhanced formation of Hülle cells compared to WT. The OsaA orthologue of Aspergillus flavus is able to complement the osaA null phenotype in A. nidulans, suggesting a conserved role of this group of WOPR domain proteins. In summary, OsaA is an upstream orchestrator of morphological and chemical development in Aspergillus that functions downstream of VeA.
The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus.
The b-Zip transcription factor AtfA plays a key role in regulating stress responses in the filamentous fungus Aspergillus nidulans. To identify the core regulons of AtfA, we examined genome-wide expression changes caused by various stresses in the presence/absence of AtfA using A. nidulans microarrays. We also intended to address the intriguing question regarding the existence of core environmental stress response in this important model eukaryote. Examination of the genome wide expression changes caused by five different oxidative stress conditions in wild type and the atfA null mutant has identified a significant number of stereotypically regulated genes (Core Oxidative Stress Response genes). The deletion of atfA increased the oxidative stress sensitivity of A. nidulans and affected mRNA accumulation of several genes under both unstressed and stressed conditions. The numbers of genes under the AtfA control appear to be specific to a stress-type. We also found that both oxidative and salt stresses induced expression of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of additional clusters. Moreover, certain clusters were down-regulated by the stresses tested. Our data suggest that the observed co-regulations were most likely consequences of the overlapping physiological effects of the stressors and not of the existence of a general environmental stress response. The function of AtfA in governing various stress responses is much smaller than anticipated and/or other regulators may play a redundant or overlapping role with AtfA. Both stress inducible and stress repressive regulations of secondary metabolism seem to be frequent features in A. nidulans.
Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members' duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes.
Beta-glucans are a heterologous group of fibrous glucose polymers that are a major constituent of cell walls in Ascomycetes and Basidiomycetes fungi. Synthesis of β (1,3)- and (1,6)-glucans is coordinated with fungal cell growth and development, thus, is under tight genetic regulation. Here, we report that β-glucan synthesis in both asexual and sexual spores is turned off by the NF-kB like fungal regulators VosA and VelB in Aspergillus nidulans. Our genetic and genomic analyses have revealed that both VosA and VelB are necessary for proper down-regulation of cell wall biosynthetic genes including those associated with β-glucan synthesis in both types of spores. The deletion of vosA or velB results in elevated accumulation of β-glucan in asexual spores. Double mutant analyses indicate that VosA and VelB play an inter-dependent role in repressing β-glucan synthesis in asexual spores. In vivo chromatin immuno-precipitation analysis shows that both VelB and VosA bind to the promoter region of the β-glucan synthase gene fksA in asexual spores. Similarly, VosA is required for proper repression of β-glucan synthesis in sexual spores. In summary, the VosA-VelB hetero-complex is a key regulatory unit tightly controlling proper levels of β-glucan synthesis in asexual and sexual spores.
Aspergillus nidulans exhibited high γ-glutamyl transpeptidase (γGT) activity in both carbon-starved and carbon-limited cultures. Glucose repressed, but casein peptone increased γGT production. Null mutation of creA did not influence γGT formation, but the functional meaB was necessary for the γGT induction. Deletion of the AN10444 gene (ggtA) completely eliminated the γGT activity, and the mRNA levels of ggtA showed strong correlation with the observed γGT activities. While ggtA does not contain a canonical signal sequence, the γGT activity was detectable both in the fermentation broth and in the hyphae. Deletion of the ggtA gene did not prevent the depletion of glutathione observed in carbon-starved and carbon-limited cultures. Addition of casein peptone to carbon-starved cultures lowered the formation of reactive species (RS). Deletion of ggtA could hinder this decrease and resulted in elevated RS formation. This effect of γGT on redox homeostasis may explain the reduced cleistothecia formation of ΔggtA strains in surface cultures.
The removal of polycyclic aromatic hydrocarbons by soil washing using water is extremely difficult due to their intrinsic hydrophobic nature. In this study, the effective aqueous solubility enhancements of seven polycyclic aromatic hydrocarbons by chemically modified hydroxypropyl rhizobial cyclic β-(1 → 2)-D-glucans and epichlorohydrin β-cyclodextrin dimer have been investigated for the first time. In the presence of hydroxypropyl cyclic β-(1 → 2)-D-glucans, the solubility of benzo[a]pyrene is increased up to 38 fold of its native solubility. The solubility of pyrene and phenanthrene dramatically increased up to 160 and 359. Coronene, chrysene, perylene, and fluoranthene also show an increase of 11, 23, 23, and 97 fold, respectively, of enhanced solubility by complexation with synthetic epichlorohydrin β-cyclodextrin dimer. The physicochemical properties of the complex are characterized by Fourier-transform infrared spectra and differential scanning calorimetry. Utilizing a scanning electron microscopy, the morphological structures of native benzo[a]pyrene, pyrene, phenanthrene, coronene, chrysene, perylene, fluoranthene and their complex with novel carbohydrate-solubilizers are studied. These results elucidate that polycyclic aromatic hydrocarbons are able to form an efficient complex with hydroxypropyl cyclic β-(1 → 2)-D-glucans and β-cyclodextrin dimer, suggesting the potential usage of chemically modified novel carbohydrate-solubilizers.
Aspergillus fumigatus is one of the most common fungi found in the environment. It is an opportunistic human pathogen causing invasive pulmonary aspergillosis with a high mortality rate in immunocompromised patients. Conidia, the asexual spores, serve as the main dispersal and infection agent allowing entrance of the fungus into the host through the respiratory tract. Therefore, understanding the asexual developmental process that gives rise to the conidia is of great interest to the scientific community and is currently the focus of an immense load of research being conducted. We have been studying the genetic basis that controls asexual development and gliotoxin biosynthesis in A. fumigatus. In this review, we discuss the genetic regulatory system that dictates conidiation in this important fungus by covering the roles of crucial genetic factors from the upstream heterotrimeric G-protein signaling components to the more specific downstream central activators of the conidiation pathway. In addition, other key asexual regulators including the velvet regulators, the Flb proteins and their associated regulatory factors are discussed.
Cytochrome P450 (CYP) monooxygenase superfamily contributes a broad array of biological functions in living organisms. In fungi, CYPs play diverse and pivotal roles in versatile metabolism and fungal adaptation to specific ecological niches. In this report, CYPomes in the 47 genomes of fungi belong to the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota have been studied. The comparison of fungal CYPomes suggests that generally fungi possess abundant CYPs belonging to a variety of families with the two global families CYP51 and CYP61, indicating individuation of CYPomes during the evolution of fungi. Fungal CYPs show highly conserved characteristic motifs, but very low overall sequence similarities. The characteristic motifs of fungal CYPs are distinguishable from those of CYPs in animals, plants, and especially archaea and bacteria. The four representative motifs contribute to the general function of CYPs. Fungal CYP51s and CYP61s can be used as the models for the substrate recognition sites analysis. The CYP proteins are clustered into 15 clades and the phylogenetic analyses suggest that the wide variety of fungal CYPs has mainly arisen from gene duplication. Two large duplication events might have been associated with the booming of Ascomycota and Basidiomycota. In addition, horizontal gene transfer also contributes to the diversification of fungal CYPs. Finally, a possible evolutionary scenario for fungal CYPs along with fungal divergences is proposed. Our results provide the fundamental information for a better understanding of CYP distribution, structure and function, and new insights into the evolutionary events of fungal CYPs along with the evolution of fungi.
The low-molecular-weight succinoglycans isolated from Sinorhizobium meliloti are repeating octasaccharide units consisting of monomers, dimers, and trimers. Pindolol is a beta-blocker used to treat cardiovascular disorders. We investigated the formation of complexes between pindolol and low-molecular-weight succinoglycan monomers (SGs). Even though SGs have a linear structure, the solubility of pindolol in the presence of SGs was increased up to 7-fold compared with methyl-β-cyclodextrin reported as the best solubilizer of pindolol. Complexation of SGs with pindolol was confirmed by nuclear magnetic resonance, Fourier-transform infrared spectroscopy, differential scanning calorimetry, and scanning electron microscopy. Formation constants of complexes were determined from phase solubility diagrams. Conformation of complex was suggested based on a molecular docking study. The present study indicated that formation of pindolol/SGs complexes not only resulted in increased pindolol solubility but also could be useful for improving its clinical application as it did not affect cell viability.
Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (-) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (-) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening >100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA∼flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the ΔfluG, ΔflbA, and ΔflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A. nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA.
Fungal development and secondary metabolism is intimately associated via activities of the fungi-specific velvet family proteins including VeA, VosA, VelB and VelC. Among these, VelC has not been characterized in Aspergillus nidulans. In this study, we characterize the role of VelC in asexual and sexual development in A. nidulans. The velC mRNA specifically accumulates during the early phase of sexual development. The deletion of velC leads to increased number of conidia and reduced production of sexual fruiting bodies (cleistothecia). In the velC deletion mutant, mRNA levels of the brlA, abaA, wetA and vosA genes that control sequential activation of asexual sporulation increase. Overexpression of velC causes increased formation of cleistothecia. These results suggest that VelC functions as a positive regulator of sexual development. VelC is one of the five proteins that physically interact with VosA in yeast two-hybrid and GST pull down analyses. The ΔvelC ΔvosA double mutant produced fewer cleistothecia and behaved similar to the ΔvosA mutant, suggesting that VosA is epistatic to VelC in sexual development, and that VelC might mediate control of sex through interacting with VosA at specific life stages for sexual fruiting.
Morphological development of fungi and their combined production of secondary metabolites are both acting in defence and protection. These processes are mainly coordinated by velvet regulators, which contain a yet functionally and structurally uncharacterized velvet domain. Here we demonstrate that the velvet domain of VosA is a novel DNA-binding motif that specifically recognizes an 11-nucleotide consensus sequence consisting of two motifs in the promoters of key developmental regulatory genes. The crystal structure analysis of the VosA velvet domain revealed an unforeseen structural similarity with the Rel homology domain (RHD) of the mammalian transcription factor NF-κB. Based on this structural similarity several conserved amino acid residues present in all velvet domains have been identified and shown to be essential for the DNA binding ability of VosA. The velvet domain is also involved in dimer formation as seen in the solved crystal structures of the VosA homodimer and the VosA-VelB heterodimer. These findings suggest that defence mechanisms of both fungi and animals might be governed by structurally related DNA-binding transcription factors.
Mycotoxins are natural contaminants of food and feed products, posing a substantial health risk to humans and animals throughout the world. A plethora of filamentous fungi has been identified as mycotoxin producers and most of these fungal species belong to the genera Aspergillus, Fusarium, and Penicillium. A number of studies have been conducted to better understand the molecular mechanisms of biosynthesis of key mycotoxins and the regulatory cascades controlling toxigenesis. In many cases, the mycotoxin biosynthetic genes are clustered and regulated by one or more pathway-specific transcription factor(s). In addition, as biosynthesis of many secondary metabolites is coordinated with fungal growth and development, there are a number of upstream regulators affecting biosynthesis of mycotoxins in fungi. This review presents a concise summary of the regulation of mycotoxin biosynthesis, focusing on the roles of the upstream regulatory elements governing biosynthesis of aflatoxin and sterigmatocystin in Aspergillus.
A facile one-step strategy is reported to synthesize nanocomposites of chitosan-reduced graphene oxide-nickel nanoparticles (CS-RGO-NiNPs) onto a screen-printed electrode (SPE). The synthesis is initiated by electrostatic and hydrophobic interactions and formation of self-assembled nanocomposite precursors of negatively charged graphene oxide (GO) and positively charged CS and nickel cations (Ni(2+)). The intrinsic mechanism of co-depositions from the nanocomposite precursor solution under cathodic potentials is based on simultaneous depositions of CS at high localized pH and in situ reduced hydrophobic RGO from GO as well as cathodically reduced metal precursors into nanoparticles. There is no need for any pre- or post-reduction of GO due to the in situ electrochemical reduction and the removal of oxygenated functionalities, which lead to an increase in hydrophobicity of RGO and successive deposition on the electrode surface. The as-prepared CS-RGO-NiNPs-modified SPE sensor exhibited outstanding performance for enzymeless glucose (Glc) sensing in alkaline media with high sensitivity (318.4µAmM(-1)cm(-2)), wide linear range (up to 9mM), low detection limit (4.1µM), acceptable selectivity against common interferents in physiological fluids, and excellent stability. A microfluidic device was fabricated incorporating the SPE sensor for real-time Glc detection in human urine samples; the results obtained were comparable to those obtained using a high-performance liquid chromatography (HPLC) coupled with an electrochemical detector. The excellent sensing performance, operational characteristics, ease of fabrication, and low cost bode well for this electrochemical microfluidic device to be developed as a point-of-care healthcare monitoring unit.
Vegetative growth signaling of the opportunistic human pathogenic fungus Aspergillus fumigatus is mediated by GpaA (Gα). FlbA is a regulator of G protein signaling, which attenuates GpaA-mediated growth signaling in this fungus. The flbA deletion (ΔflbA) and the constitutively active GpaA (GpaA(Q204L)) mutants exhibit enhanced proliferation, precocious autolysis, and reduced asexual sporulation. In this study, we demonstrate that both mutants also show enhanced tolerance against H2O2 and their radial growth was approximately 1.6 fold higher than that of wild type (WT) in medium with 10 mM H2O2. We performed quantitative PCR (qRT-PCR) for examination of mRNA levels of three catalase encoding genes (catA, cat1, and cat2) in WT and the two mutants. According to the results, while levels of spore-specific catA mRNA were comparable among the three strains, cat1 and cat2 mRNA levels were significantly higher in the two mutants than in WT. In particular, the ΔflbA mutant showed significantly enhanced and prolonged expression of cat1 and precocious expression of cat2. In accordance with this result, activity of the Cat1 protein in the ΔflbA mutant was higher than that of gpaA (Q204L) and WT strains. For activity of the Cat2 protein, both mutants began to show enhanced activity at 48 and 72 hr of growth compared to WT. These results lead to the conclusion that GpaA activates expression and activity of cat1 and cat2, whereas FlbA plays an antagonistic role in control of catalases, leading to balanced responses to neutralizing the toxicity of reactive oxygen species.
Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented.
FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (â
Asexual sporulation (conidiation) in the ascomycetous filamentous fungi involves the formation of conidia, formed on specialized structures called conidiophores. Conidiation in filamentous fungi involves many common themes including spatial and temporal regulation of gene expression, specialized cellular differentiation, intra-/inter-cellular communications, and response to environmental factors. The commencement, progression and completion of conidiation are regulated by multiple positive and negative genetic elements that direct expression of genes required for proper vegetative growth and the assembly of the conidiophore and spore maturation. Light is one of the key environmental factors affecting conidiation. Developmental mechanisms in Aspergillus nidulans and Neurospora crassa have been intensively studied, leading to important outlines. Here, we summarize genetic control of conidiation including the light-responding mechanisms in the two model fungi.
Fungal development and secondary metabolism is intimately associated via activities of the fungi-specific velvet family proteins. Here we characterize the four velvet regulators in the opportunistic human pathogen Aspergillus fumigatus. The deletion of AfuvosA, AfuveA and AfuvelB causes hyperactive asexual development (conidiation) and precocious and elevated accumulation of AfubrlA during developmental progression. Moreover, the absence of AfuvosA, AfuveA or AfuvelB results in the abundant formation of conidiophores and highly increased AfubrlA mRNA accumulation in liquid submerged culture, suggesting that they act as repressors of conidiation. The deletion of AfuvosA or AfuvelB causes a reduction in conidial trehalose amount, long-term spore viability, conidial tolerance to oxidative and UV stresses, and accelerated and elevated conidial germination regardless of the presence or absence of an external carbon source, suggesting an interdependent role of them in many aspects of fungal biology. Genetic studies suggest that AfuAbaA activates AfuvosA and AfuvelB expression during the mid to late phase of conidiation. Finally, the AfuveA null mutation can be fully complemented by Aspergillus nidulans VeA, which can physically interact with AfuVelB and AfuLaeA in vivo. A model depicting the similar yet different roles of the velvet regulators governing conidiation and sporogenesis in A. fumigatus is presented.
Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the ΔAnricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in ΔAnricA. Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAnricA, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the ΔricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore development in the ΔAnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans.
The multifunctional regulator VelB physically interacts with other velvet regulators and the resulting complexes govern development and secondary metabolism in the filamentous fungus Aspergillus nidulans. Here, we further characterize VelB's role in governing asexual development and conidiogenesis in A. nidulans. In asexual spore formation, velB deletion strains show reduced number of conidia, and decreased and delayed mRNA accumulation of the key asexual regulatory genes brlA, abaA, and vosA. Overexpression of velB induces a two-fold increase of asexual spore production compared to wild type. Furthermore, the velB deletion mutant exhibits increased conidial germination rates in the presence of glucose, and rapid germination of conidia in the absence of external carbon sources. In vivo immuno-pull-down analyses reveal that VelB primarily interacts with VosA in both asexual and sexual spores, and VelB and VosA play an inter-dependent role in spore viability, focal trehalose biogenesis and control of conidial germination. Genetic and in vitro studies reveal that AbaA positively regulates velB and vosA mRNA expression during sporogenesis, and directly binds to the promoters of velB and vosA. In summary, VelB acts as a positive regulator of asexual development and regulates spore maturation, focal trehalose biogenesis and germination by interacting with VosA in A. nidulans.
Understanding in vivo protein-protein interactions is critical to dissect precise functions of the regulatory proteins of fungal secondary metabolites. As many fungi differentially produce a diverse array of secondary metabolites during their lifecycle, it is important to understand the cell-type specific regulation of secondary metabolism. However, due to the difficulty of sample preparation of biologically active proteins in fungal spores, protein-protein interaction studies have been generally restricted. While some outstanding studies revealed protein-protein interactions of selected regulators, including the velvet proteins in vegetative cells, a detailed protocol for investigating the protein-protein interactions in the fungal spores has not yet been reported. Here, we describe a working protocol for the purification and identification of interacting protein partners of the spores of Aspergillus nidulans employing the VelB protein as an example.
With the completion of genomes of various Aspergillus species, large-scale genome-wide expression studies can be carried out. Genomics, however, is more powerful and efficient when combined with genetics. A multi-copy-based gain-of-function screen is a complementary method to loss-of-function genetic screen and can identify novel genes that may not be easily identifiable through loss-of-function-type screens. Particularly, gain-of-function genetic screens would identify novel activators or repressors of fungal development and secondary metabolism. Here, we describe a working protocol for the identification of novel regulators in Aspergillus nidulans.
Carbon-starving Aspergillus nidulans cultures produce high activities of versatile hydrolytic enzymes and, among these, ChiB endochitinase and EngA β-1,3-endoglucanase showed significant antifungal activity against various fungal species. Double deletion of engA and chiB diminished the antifungal activity of the fermentation broths and increased conidiogenesis and long-term viability of A. nidulans, but decreased the growth rate on culture media containing weak carbon sources. Production of ChiB and EngA can influence fungal communities either directly due to their antifungal properties or indirectly through their effects on vegetative growth. Our data suggest saprophytic fungi as promising future candidates to develop novel biocontrol technologies.
Extracellular proteinase formation in carbon depleted cultures of the model filamentous fungus Aspergillus nidulans was studied to elucidate its regulation and possible physiological function. As demonstrated by gene deletion, culture optimization, microbial physiological and enzymological experiments, the PrtA and PepJ proteinases of A. nidulans did not appear to play a decisive role in the autolytic decomposition of fungal cells under the conditions we tested. However, carbon starvation induced formation of the proteinases observable in autolytic cultures. Similar to other degradative enzymes, production of proteinase was regulated by FluG-BrlA asexual developmental signaling and modulated by PacC-dependent pH-responsive signaling. Under the same carbon starved culture conditions, alterations of CreA, MeaB or heterotrimeric G protein mediated signaling pathways caused less significant changes in the formation of extracellular proteinases. Taken together, these results indicate that while the accumulation of PrtA and PepJ is tightly coupled to the initiation of autolysis, they are not essential for autolytic cell wall degradation in A. nidulans. Thus, as Aspergillus genomes contain a large group of genes encoding proteinases with versatile physiological functions, selective control of proteinase production in fungal cells is needed for the improved industrial use of fungi.
The opportunistic human pathogen Aspergillus fumigatus produces a massive number of asexual spores (conidia) as the primary means of dispersal, survival, genome protection and infection of hosts. In this report, we investigate the functions of two developmental regulators, AfuAbaA and AfuWetA, in A. fumigatus. The AfuabaA gene is predicted to encode an ATTS/TEA DNA-binding domain protein and is activated by AfuBrlA during the middle stage of A. fumigatus asexual development (conidiation). The deletion of AfuabaA results in the formation of aberrant conidiophores exhibiting reiterated cylinder-like terminal cells lacking spores. Furthermore, the absence of AfuabaA causes delayed autolysis and cell death, whereas the overexpression of AfuabaA accelerates these processes, indicating an additional role for AfuAbaA. The AfuwetA gene is sequentially activated by AfuAbaA in the late phase of conidiation. The deletion of AfuwetA causes the formation of defective spore walls and a lack of trehalose biogenesis, leading to a rapid loss of spore viability and reduced tolerance to various stresses. This is the first report to demonstrate that WetA is essential for trehalose biogenesis in conidia. Moreover, the absence of AfuwetA causes delayed germ-tube formation and reduced hyphal branching, suggesting a role of AfuWetA in the early phase of fungal growth. A genetic model depicting the regulation of conidiation in A. fumigatus is proposed.
Members of the genus Aspergillus are the most common fungi and all reproduce asexually by forming long chains of conidiospores (or conidia). The impact of various Aspergillus species on humans ranges from beneficial to harmful. For example, several species including Aspergillus oryzae and Aspergillus niger are used in industry for enzyme production and food processing. In contrast, Aspergillus flavus produce the most potent naturally present carcinogen aflatoxins, which contaminate various plant- and animal-based foods. Importantly, the opportunistic human pathogen Aspergillus fumigatus has become the most prevalent airborne fungal pathogen in developed countries, causing invasive aspergillosis in immunocompromised patients with a high mortality rate. A. fumigatus produces a massive number of small hydrophobic conidia as the primary means of dispersal, survival, genome-protection, and infecting hosts. Large-scale genome-wide expression studies can now be conducted due to completion of A. fumigatus genome sequencing. However, genomics becomes more powerful and informative when combined with genetics. We have been investigating the mechanisms underlying the regulation of asexual development (conidiation) and gliotoxin biosynthesis in A. fumigatus, primarily focusing on a characterization of key developmental regulators identified in the model fungus Aspergillus nidulans. In this review, I will summarize our current understanding of how conidiation in two aspergilli is regulated.
Several upstream developmental activators control asexual development (conidiation) in Aspergillus. In this study, we characterize one of such activators called flbE in Aspergillus fumigatus and Aspergillus nidulans. The predicted FlbE protein is composed of 222 and 201 aa in A. fumigatus and A. nidulans, respectively. While flbE is transiently expressed during early phase of growth in A. nidulans, it is somewhat constitutively expressed during the lifecycle of A. fumigatus. The deletion of flbE causes reduced conidiation and delayed expression of brlA and vosA in both species. Moreover, FlbE is necessary for salt-induced development in liquid submerged culture in A. fumigatus. The A. nidulans flbE null mutation is fully complemented by A. fumigatus flbE, indicating a functional conservancy of FlbE in Aspergillus. Both the deletion and overexpression of flbE in A. nidulans result in developmental defects, enhanced autolysis, precocious cell death, and delayed expression of brlA/vosA, suggesting that balanced activity of FlbE is crucial for proper growth and development. Importantly, the N-terminal portion of FlbE exhibits the trans-activation ability in yeast, whereas the C-terminal half negatively affects its activity. Site-directed mutagenesis of certain conserved N-terminal amino acids abolishes the ability of trans-activation, overexpression-induced autolysis, and complementing the null mutation. Finally, overexpression of flbD, but not flbB or flbC, restores conidiation in A. nidulans ΔflbE, generally supporting the current genetic model for developmental regulation.
VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells), which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators.
The opportunistic human pathogen Aspergillus fumigatus reproduces asexually by forming a massive number of mitospores called conidia. In this study, we characterize the upstream developmental regulator A. fumigatus flbB (AfuflbB). Northern blotting and cDNA analyses reveal that AfuflbB produces two transcripts predicted to encode two basic leucine zipper domain (bZIP) polypeptides, AfuFlbBβ (420 amino acids [aa]) and AfuFlbBα (390 aa). The deletion of AfuflbB results in delayed/reduced sporulation, precocious cell death, the lack of conidiophore development in liquid submerged culture, altered expression of AfubrlA and AfuabaA, and blocked production of gliotoxin. While introduction of the wild-type (WT) AfuflbB allele fully complemented these defects, disruption of the ATG start codon for either one of the AfuFlbB polypeptides leads to a partial complementation, indicating the need of both polypeptides for WT levels of asexual development and gliotoxin biogenesis. Consistent with this, Aspergillus nidulans flbB(+) encoding one polypeptide (426 aa) partially complements the AfuflbB null mutation. The presence of 0.6 M KCl in liquid submerged culture suppresses the defects caused by the lack of one, but not both, of the AfuFlbB polypeptides, suggesting a genetic prerequisite for AfuFlbB in A. fumigatus development. Finally, Northern blot analyses reveal that both AfuflbB and AfuflbE are necessary for expression of AfuflbD, suggesting that FlbD functions downstream of FlbB/FlbE in aspergilli.
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. 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. 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. 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.
Asexual development (conidiation) in Aspergillus is governed by multiple regulators. Here, we characterize the upstream developmental activator FlbC in Aspergillus nidulans. flbC mRNA is detectable throughout the life cycle, at relatively high levels during vegetative growth, early asexual and late sexual developmental phases. The deletion of flbC causes a delay/reduction in conidiation, brlA and vosA expression, and conidial germination. While overexpression of flbC (OEflbC) does not elaborate conidiophores, it inhibits hyphal growth and activates expression of brlA, abaA and vosA, but not wetA. FlbC is conserved in filamentous Ascomycetes containing two C(2) H(2) zinc fingers at the C-terminus and a putative activation domain at the N-terminus. FlbC localizes in the nuclei of both hyphae and developmental cells. Localization and expression of FlbC are not affected by the absence of FlbB or FlbE, and vice versa. Importantly, overexpression of flbC causes growth inhibition and activation of abaA and vosA in the absence of brlA and abaA respectively. In vitro DNA-binding assay reveals that FlbC binds to the brlA, abaA and vosA, but not the wetA, promoters. In summary, FlbC is a putative nuclear transcription factor necessary for proper activation of conidiation, and its balanced activity is crucial for governing growth and development in A. nidulans.
Phosphatidylcholines (PCs) are a class of major cell membrane phospholipids that participate in many physiological processes. Three genes, choA, choB and choC, have been proposed to function in the endogenous biosynthesis of PC in Aspergillus nidulans. In this study, we characterize the choC gene encoding a putative highly conserved phospholipid methyltransferase. The previously reported choC3 mutant allele results from a mutation leading to the E177K amino acid substitution. The transcript of choC accumulates at high levels during vegetative growth and early asexual developmental phases. The deletion of choC causes severe impairment of vegetative growth, swelling of hyphal tips and the lack of both asexual and sexual development, suggesting the requirement of ChoC and PC in growth and development. Noticeably, supplementation of the mutant with the penultimate precursor of PC N, N-dimethylaminoethanol leads to full recovery of vegetative growth, but incomplete progression of asexual and sexual development, implying differential roles of PC and its intermediates in fungal growth and development. Importantly, while the choC deletion mutant shows reduced vegetative growth and precocious cell death until day 4, it regains hyphal proliferation and cell viability from day 5, indicating the presence of an alternative route for cellular membrane function in A. nidulans.
The roles of the Gbetagamma subunits of the opportunistic human pathogen Aspergillus fumigatus were investigated. The predicted AfuSfaD (Gbeta) protein consists of 353 amino acids and shows 94-98% similarity with other Aspergillus Gbeta subunits. AfuGpgA consists of 90 amino acids showing 95-98% identity with other fungal G-protein gamma subunits. The deletion (Delta) of AfusfaD or AfugpgA resulted in severe impairment in vegetative growth, conidial germination and conidial trehalose breakdown. While the total number of conidia produced by DeltaAfusfaD and DeltaAfugpgA strains on solid medium was only about 1% of wild type, the growth-adjusted conidiation levels were twofold higher than those of wild type. Enhanced formation of conidiophores and elevated AfubrlA mRNA levels were observable in DeltaAfusfaD or DeltaAfugpgA strains in liquid submerged culture. Moreover, overexpression of AfusfaD or AfugpgA caused reduced levels of submerged culture conidiation, indicating that Gbetagamma is involved in negative regulation of conidiation. Gliotoxin and other metabolites were not detected in the chloroform extracts of DeltaAfusfaD and DeltaAfugpgA culture filtrates. Northern blot analyses revealed that, while AfulaeA mRNA levels unchanged, accumulation of gliZ mRNA was delayed by DeltaAfusfaD or DeltaAfugpgA. A model summarizing the roles of AfusfaD and AfugpgA in A. fumigatus is presented.
Elucidation of the regulation of ChiB production in Aspergillus nidulans. Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures. The endochitinase ChiB plays an important role in autolysis of A. nidulans, and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling. Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.
Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans. Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (DeltaflbA) mutant autolyzed completely after a 3-day incubation, the DeltaflbA DeltachiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the DeltaflbA-induced autolysis. However, importantly, both DeltaflbA and DeltaflbA DeltachiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the DeltaflbA and DeltaflbA DeltachiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, beta-N-acetylhexosaminidase (NagA), and myo-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.
Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans, light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites.
The fungal colony is a complex multicellular unit consisting of various cell types and functions. Asexual spore formation (conidiation) is integrated through sensory and regulatory elements into the general morphogenetic plan, in which the activation of the transcription factor BrlA is the first determining step. A number of early regulatory elements acting upstream of BrlA (fluG and flbA-E) have been identified, but their functional relations remain to be further investigated. In this report we describe FlbB as a putative basic-zipper-type transcription factor restricted to filamentous fungi. FlbB accumulates at the hyphal apex during early vegetative growth but is later found in apical nuclei, suggesting that an activating modification triggers nuclear import. Moreover, proper temporal and quantitative expression of FlbB is a prerequisite for brlA transcription, and misscheduled overexpression inhibits conidiation. We also present evidence that FlbB activation results in the production of a second diffusible signal, acting downstream from the FluG factor, to induce conidiation.
Trehalose is a compatible osmolyte produced by bacteria, fungi, insects and plants to protect the integrity of cells against various environmental stresses. Spores, the reproductive, survival and infection bodies of fungi require high amounts of trehalose for long-term survival. Here, via a gain-of-function genetic screen, we identify the novel regulator VosA that couples the formation of spores and focal trehalose biogenesis in the model fungus Aspergillus nidulans. The vosA gene is expressed specifically during the formation of both sexual and asexual spores (conidia). Levels of vosA mRNA and protein are high in both types of spore. The deletion of vosA results in the lack of trehalose in spores, a rapid loss of the cytoplasm, organelles and viability of spores, and a dramatic reduction in tolerance of conidia to heat and oxidative stress. Moreover, the absence of vosA causes uncontrolled activation of asexual development, whereas the enhanced expression of vosA blocks sporulation, suggesting that VosA also functions in negative-feedback regulation of sporogenesis. VosA localizes in the nucleus of mature conidia and its C-terminal region contains a potential transcription activation domain, indicating that it may function as a transcription factor primarily controlling the late process of sporulation including trehalose biogenesis. VosA is conserved in most fungi and may define a new fungus-specific transcription factor family.
The opportunistic human pathogen Aspergillus fumigatus produces a large quantity of asexual spores (conidia), which are the primary agent causing invasive aspergillosis in immunocompromised patients. We investigated the mechanisms controlling asexual sporulation (conidiation) in A. fumigatus via examining functions of four key regulators, GpaA (Galpha), AfFlbA (RGS), AfFluG, and AfBrlA, previously studied in Aspergillus nidulans. Expression analyses of gpaA, AfflbA, AffluG, AfbrlA, and AfwetA throughout the life cycle of A. fumigatus revealed that, while transcripts of AfflbA and AffluG accumulate constantly, the latter two downstream developmental regulators are specifically expressed during conidiation. Both loss-of-function AfflbA and dominant activating GpaA(Q204L) mutations resulted in reduced conidiation with increased hyphal proliferation, indicating that GpaA signaling activates vegetative growth while inhibiting conidiation. As GpaA is the primary target of AfFlbA, the dominant interfering GpaA(G203R) mutation suppressed reduced conidiation caused by loss of AfflbA function. These results corroborate the hypothesis that functions of G proteins and RGSs are conserved in aspergilli. We then examined functions of the two major developmental activators AfFluG and AfBrlA. While deletion of AfbrlA eliminated conidiation completely, null mutation of AffluG did not cause severe alterations in A. fumigatus sporulation in air-exposed culture, implying that, whereas the two aspergilli may have a common key downstream developmental activator, upstream mechanisms activating brlA may be distinct. Finally, both AffluG and AfflbA mutants showed reduced conidiation and delayed expression of AfbrlA in synchronized developmental induction, indicating that these upstream regulators contribute to the proper progression of conidiation.
No abstract available.
We have carried out an in silico exploration of the genomes of Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus oryzae, and identified components of G-protein/cAMP-mediated signaling. Putative G-protein coupled receptors (GPCRs) were distributed over nine classes. The GPCRs within classes were well conserved among aspergilli but varied in other ascomycetes. As previously observed in A. nidulans and other fungi, three Galpha, one Gbeta, and one Ggamma subunits of G proteins were identified in A. fumigatus, whereas an additional likely non-functional Galpha subunit was present in A. oryzae. While most fungal species had five proteins containing the regulator of G-protein signaling (RGS) domain predicted to participate in attenuation of G-protein signaling, A. fumigatus and A. oryzae had an additional RGS protein (RgsD) related to RgsA of A. nidulans. Genes encoding adenylate cyclase, a regulatory subunit and two catalytic subunits of the cAMP-dependent protein kinase, were also identified in the three aspergilli. Finally, regulators of cAMP signaling including low- and high-affinity phosphodiesterases were identified. Taken together, our data indicate a striking diversity at the GPCR level, but little diversity of components at the G-protein and cAMP-signaling level. This may reflect the abilities of these fungi to adapt to various ecological niches and to integrate diverse environmental cues into highly conserved cellular processes.
Heterotrimeric G proteins (G proteins) are conserved in all eukaryotes and are crucial components sensing and relaying external cues into the cells to elicit appropriate physiological and biochemical responses. Basic units of the heterotrimeric G protein signaling system include a G protein-coupled receptor (GPCR), a G protein composed of alpha, beta, and gamma subunits, and variety of effectors. Sequential sensitization and activation of these G protein elements translates external signals into gene expression changes, resulting in appropriate cellular behaviors. Regulators of G protein signaling (RGSs) constitute a crucial element of appropriate control of the intensity and duration of G protein signaling. For the past decade, G protein signaling and its regulation have been intensively studied in a number of model and/or pathogenic fungi and outcomes of the studies provided better understanding on the upstream regulation of vegetative growth, mating, development, virulence/pathogenicity establishment, and biosynthesis of secondary metabolites in fungi. This review focuses on the characteristics of the basic upstream G protein components and RGS proteins, and their roles controlling various aspects of biological processes in the model filamentous ascomycete fungus Aspergillus nidulans. In particular, their functions in controlling hyphal proliferation, asexual spore formation, sexual fruiting, and the mycotoxin sterigmatocystin production are discussed.
The asexual spore is one of the most crucial factors contributing to the fecundity and fitness of filamentous fungi. Although the developmental activator FluG was shown to be necessary for activation of asexual sporulation (conidiation) and production of the carcinogenic mycotoxin sterigmatocystin (ST) in the model filamentous fungus Aspergillus nidulans, the molecular mechanisms underlying the developmental switch have remained elusive. In this study, we report that the FluG-mediated conidiation in A. nidulans occurs via derepression. Suppressor analyses of fluG led to the identification of the sfgA gene encoding a novel protein with the Gal4-type Zn(II)2Cys6 binuclear cluster DNA-binding motif at the N terminus. Deletion (delta) and 31 other loss-of-function sfgA mutations bypassed the need for fluG in conidiation and production of ST. Moreover, both delta sfgA and delta sfgA delta fluG mutations resulted in identical phenotypes in growth, conidiation, and ST production, indicating that the primary role of FluG is to remove repressive effects imposed by SfgA. In accordance with the proposed regulatory role of SfgA, overexpression of sfgA inhibited conidiation and delayed/reduced expression of conidiation- and ST-specific genes. Genetic analyses demonstrated that SfgA functions downstream of FluG but upstream of transcriptional activators (FlbD, FlbC, FlbB, and BrlA) necessary for normal conidiation.
Phosducin or phosducin-like protein (PhLP) is a positive regulator of Gbetagamma activity. The Gbeta (SfaD) and Ggamma (GpgA) subunits function in vegetative growth and developmental control in the model filamentous fungus Aspergillus nidulans. To better understand the nature of Gbetagamma-mediated signaling, phnA, encoding an A. nidulans PhLP, has been studied. Deletion of phnA resulted in phenotypes almost identical to those caused by deletion of sfaD, i.e., reduced biomass, asexual sporulation in liquid submerged culture, and defective fruiting body formation, suggesting that PhnA is necessary for Gbeta function. The requirement for the RGS protein FlbA in asexual sporulation could be bypassed by the DeltaphnA mutation, indicating that PhnA functions in FlbA-controlled vegetative growth signaling, primarily mediated by the heterotrimeric G protein composed of FadA (Galpha), SfaD, and GpgA. However, whereas deletion of fadA restored both asexual sporulation and the production of sterigmatocystin (ST), deletion of sfaD, gpgA, or phnA failed to restore ST production in the DeltaflbA mutant. Further studies revealed that SfaD, GpgA, and PhnA are necessary for the expression of aflR, encoding the transcriptional activator for the ST biosynthetic genes, and subsequent ST biosynthesis. Overexpression of aflR bypassed the need for SfaD in ST production, indicating that the results of SfaD-mediated signaling may include transcriptional activation of aflR. Potential differential roles of FadA, Gbetagamma, and FlbA in controlling ST biosynthesis are further discussed.
Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.
No abstract available.
The role of heterotrimeric G-proteins in cAMP-dependent germination of conidia was investigated in the filamentous ascomycete Aspergillus nidulans. We demonstrate that the G alpha-subunit GanB mediates a rapid and transient activation of cAMP synthesis in response to glucose during the early period of germination. Moreover, deletion of individual G-protein subunits resulted in defective trehalose mobilization and altered germination kinetics, indicating that GanB(alpha)-SfaD(beta)-GpgA(gamma) constitutes a functional heterotrimer and controls cAMP/PKA signaling in response to glucose as well as conidial germination. Further genetic analyses suggest that GanB plays a primary role in cAMP/PKA signaling, whereas the SfaD-GpgA (G betagamma) heterodimer is crucial for proper activation of GanB signaling sensitized by glucose. In addition, the RGS protein RgsA is also involved in regulation of the cAMP/PKA pathway and germination via attenuation of GanB signaling. Genetic epistatic analyses led us to conclude that all controls exerted by GanB(alpha)-SfaD(beta)-GpgA(gamma) on conidial germination are mediated through the cAMP/PKA pathway. Furthermore, GanB may function in sensing various carbon sources and subsequent activation of downstream signaling for germination.
Vegetative growth signaling in the filamentous fungus Aspergillus nidulans is primarily mediated by the heterotrimeric G-protein composed of FadA (G alpha), SfaD (G beta), and a presumed G gamma. Analysis of the A. nidulans genome identified a single gene named gpgA encoding a putative G gamma-subunit. The predicted GpgA protein consists of 90 amino acids showing 72% similarity with yeast Ste18p. Deletion (delta) of gpgA resulted in restricted vegetative growth and lowered asexual sporulation. Moreover, similar to the delta sfaD mutant, the delta gpgA mutant was unable to produce sexual fruiting bodies (cleistothecia) in self-fertilization and was severely impaired with cleistothecial development in outcross, indicating that both SfaD and GpgA are required for fruiting body formation. Developmental and morphological defects caused by deletion of flbA encoding an RGS protein negatively controlling FadA-mediated vegetative growth signaling were suppressed by delta gpgA, indicating that GpgA functions in FadA-SfaD-mediated vegetative growth signaling. However, deletion of gpgA could not bypass the need for the early developmental activator FluG in asexual sporulation, suggesting that GpgA functions in a separate signaling pathway. We propose that GpgA is the only A. nidulans G gamma-subunit and is required for normal vegetative growth as well as proper asexual and sexual developmental progression.
Filamentous fungal genomes contain two distantly related cyclic AMP-dependent protein kinase A catalytic subunits (PKAs), but only one PKA is found to play a principal role. In Aspergillus nidulans, PkaA is the primary PKA that positively functions in vegetative growth and spore germination but negatively controls asexual sporulation and production of the mycotoxin sterigmatocystin. In this report, we present the identification and characterization of pkaB, encoding the secondary PKA in A. nidulans. Although deletion of pkaB alone does not cause any apparent phenotypic changes, the absence of both pkaB and pkaA is lethal, indicating that PkaB and PkaA are essential for viability of A. nidulans. Overexpression of pkaB enhances hyphal proliferation and rescues the growth defects caused by DeltapkaA, indicating that PkaB plays a role in vegetative growth signaling. However, unlike DeltapkaA, deletion of pkaB does not suppress the fluffy-autolytic phenotype resulting from DeltaflbA. While upregulation of pkaB rescues the defects of spore germination resulting from DeltapkaA in the presence of glucose, overexpression of pkaB delays spore germination. Furthermore, upregulation of pkaB completely abolishes spore germination on medium lacking a carbon source. In addition, upregulation of pkaB enhances the level of submerged sporulation caused by DeltapkaA and reduces hyphal tolerance to oxidative stress. In conclusion, PkaB is the secondary PKA that has a synthetic lethal interaction with PkaA, and it plays an overlapping role in vegetative growth and spore germination in the presence of glucose but an opposite role in regulating asexual sporulation, germination in the absence of a carbon source, and oxidative stress responses in A. nidulans.
Fungal secondary metabolites are of intense interest to humankind due to their pharmaceutical (antibiotics) and/or toxic (mycotoxins) properties. In the past decade, tremendous progress has been made in understanding the genes that are associated with production of various fungal secondary metabolites. Moreover, the regulatory mechanisms controlling biosynthesis of diverse groups of secondary metabolites have been unveiled. In this review, we present the current understanding of the genetic regulation of secondary metabolism from clustering of biosynthetic genes to global regulators balancing growth, sporulation, and secondary metabolite production in selected fungi with emphasis on regulation of metabolites of agricultural concern. Particularly, the roles of G protein signaling components and developmental regulators in the mycotoxin sterigmatocystin biosynthesis in the model fungus Aspergillus nidulans are discussed in depth.
Gene replacement via homologous double crossover in filamentous fungi requires relatively long (preferentially >0.5 kb) flanking regions of the target gene. For this reason, gene replacement cassettes are usually constructed through multiple cloning steps. To facilitate gene function studies in filamentous fungi avoiding tedious cloning steps, we have developed a PCR-assisted DNA assembly procedure and applied it to delete genes in filamentous fungi. While the principle of this procedure is essentially the same as other recently reported PCR-based tools, our technique has been effectively used to delete 31 genes in three fungal species. Moreover, this PCR-based method was used to fuse more than 10 genes to a controllable promoter. In this report, a detailed protocol for this easy to follow procedure and examples of genes deleted or over-expressed are presented. In conjunction with the availability of genome sequences, the application of this technique should facilitate functional characterization of genes in filamentous fungi. To stream line the analysis of the transformants a relatively simple procedure for genomic DNA or total RNA isolation achieving approximately 100 samples/person/day is also presented.
The aflT gene resides between the polyketide synthase gene pksA and the P450-encoding cypA gene in the aflatoxin gene cluster of Aspergillus parasiticus. It is a single copy gene in the genome of A. parasiticus SRRC 2043 and SU-1 and was also found at the same relative position in the genome of Aspergillus flavus isolates. The predicted AFLT protein contained 14 transmembrane domains and had various degrees of the amino acid identity (34-56%) to fungal transporters belonging to the major facilitator superfamily. Targeted deletion of aflT in A. parasiticus SU-1 yielded transformants that were morphologically similar to SU-1. These aflT-deleted mutants produced and secreted aflatoxins comparable to the parental strain although they lost the production of the aflT transcript. Real-time RT-PCR analysis showed that the expression of aflT was controlled neither by the aflatoxin pathway-specific activator AFLR nor by the co-activator AFLJ, which differed from the regulation of the aflatoxin biosynthetic genes pksA, nor1, ver1, and omtA. The FadA-dependent G-protein signaling pathway previously shown to govern aflatoxin biosynthesis and sporulation plays a role in the regulation of aflT expression.
The filamentous fungus Aspergillus nidulans possesses both asexual and sexual reproductive cycles. Sexual fruiting bodies (cleistothecia) can be formed in both homothallic (self) and heterothallic (outcross) conditions. In this study, we characterized two genes, gprA and gprB, that are predicted to encode putative G protein-coupled receptors (GPCRs) similar to fungal pheromone receptors. Deletion (Delta) of gprA or gprB resulted in the production of a few small cleistothecia carrying a reduced number of ascospores, whereas DeltagprADeltagprB eliminated fruiting body formation in homothallic conditions. However, nullifying gprA and/or gprB did not affect vegetative growth, asexual sporulation, Hülle cell formation or even cleistothecia formation in outcross, indicating that GprA and GprB are specifically required for self-fertilization. The gprA and gprB genes encode two transcripts and, for both genes, larger transcripts are detectable during vegetative growth and asexual development whereas smaller transcripts accumulate during sexual development. Upregulation of nsdD encoding a key sexual developmental activator resulted in the production of barren cleistothecia in the DeltagprADeltagprB mutant, suggesting that NsdD can partially rescue the developmental defects caused by deletion of GPCRs and that GprA/B-mediated signalling may activate other genes necessary for maturation of cleistothecia and ascosporogenesis. Deletion of gprA and/or gprB suppressed growth defects caused by DeltagprD, implying that GprA/B function downstream of GprD-mediated negative control of sexual development.
Regulators of G-protein signalling play a crucial role in controlling the degree of heterotrimeric G-protein signalling. In addition to the previously studied flbA, we have identified three genes (rgsA, rgsB and rgsC) encoding putative RGS proteins in the genome of Aspergillus nidulans. Characterization of the rgsA gene revealed that RgsA downregulates pigment production and conidial germination, but stimulates asexual sporulation (conidiation). Deletion of rgsA (DeltargsA) resulted in reduced colony size with increased aerial hyphae, elevated accumulation of brown pigments as well as enhanced tolerance of conidia and vegetative hyphae against oxidative and thermal stress. Moreover, DeltargsA resulted in conidial germination in the absence of a carbon source. Deletion of both flbA and rgsA resulted in an additive phenotype, suggesting that the G-protein pathways controlled by FlbA and RgsA are different. Morphological and metabolic alterations caused by DeltargsA were suppressed by deletion of ganB encoding a Galpha subunit, indicating that the primary role of RgsA is to control negatively GanB-mediated signalling. Overexpression of rgsA caused inappropriate conidiation in liquid submerged culture, supporting the idea that GanB signalling represses conidiation. Our findings define a second and specific RGS-Galpha pair in A. nidulans, which may govern upstream regulation of fungal cellular responses to environmental changes.
G protein-coupled receptors (GPCRs) are key components of heterotrimeric G protein-mediated signalling pathways that detect environmental signals and confer rapid cellular responses. To broaden our understanding of signalling mechanisms in the filamentous fungus Aspergillus nidulans, intensive analyses of the Aspergillus nidulans genome have been carried out and nine genes (gprA approximately gprI) that are predicted to encode seven transmembrane spanning GPCRs have been identified. Six of nine putative GPCRs have been disrupted and the gprD gene was found to play a central role in coordinating hyphal growth and sexual development. Deletion of gprD (Delta gprD) causes extremely restricted hyphal growth, delayed conidial germination and uncontrolled activation of sexual development resulting in a small colony covered by sexual fruiting bodies. Genetic studies indicate that GprD may not signal through the FadA (G alpha)-protein kinase A (PKA) pathway. Elimination of sexual development rescues both growth and developmental abnormalities caused by Delta gprD, suggesting that the primary role of GprD is to negatively regulate sexual development. This is supported by the fact that environmental conditions inhibiting sexual development suppress growth defects of the Delta gprD mutant. We propose that the GprD-mediated signalling cascade negatively regulates sexual development, which is required for proper proliferation of A. nidulans.
Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.
The ability to reproduce both sexually and asexually is one of the characteristics of the homothalic ascomycete Aspergillus nidulans. Unlike the other Aspergillus species, A. nidulans undergoes sexual development that seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction of A. nidulans we previously isolated and characterized several NSD (never in sexual development) mutants that failed to produce any sexual reproductive organs, and identified four complementation groups, nsdA, nsdB, nsdC, and nsdD. The nsdD gene has been isolated, and it is predicted to encode a GATA-type transcription factor with the type IVb zinc finger DNA-binding domain. The mRNA of the nsdD gene started to accumulate in the early phase of vegetative growth, and the level increased as sexual development proceeded. However, it decreased during asexual sporulation and no nsdD mRNA was detected in conidia. Deletion of nsdD resulted in no cleistothecia (fruiting bodies) formation, even under the conditions that preferentially promoted sexual development, indicating that nsdD is necessary for sexual development. In contrast, when the nsdD gene was over-expressed, sexual-specific organ (Hülle cell) was formed even in submerged culture, which normally completely blocked sexual development, and the number of cleistothecia was also dramatically increased on solid medium. These results lead us to propose that the nsdD gene functions in activating sexual development of A. nidulans. Multiple copies of the nsdD gene could suppress nsdB5 and veA1, indicating that either nsdD acts downstream of these genes or possibly functions in overlapping pathway(s).
flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that antagonizes FadA (G(i)alpha-subunit of heterotrimeric G protein)-mediated growth signaling to allow asexual development. We previously defined and characterized five suppressors of flbA (sfa) loss-of-function mutations and showed that one suppressor (sfaB) resulted from a novel dominant-negative allele of fadA. In this report we show that a second suppressor gene (sfaD) is predicted to encode the beta subunit of a heterotrimeric G protein. Deletion of sfaD suppressed all defects resulting from complete loss-of-flbA function mutations, caused a hyperactive sporulation phenotype and severely reduced vegetative growth. However, the sfaD deletion could not suppress the growth activation caused by dominant-activating fadA alleles, indicating that constitutively active FadA can cause proliferative growth in the absence of Gbetagamma signaling. We propose that SfaD and FadA are both positive growth regulators with partially overlapping functions and that FlbA has an important role in controlling the activities of both proteins. Inactivation of signaling events stimulated by both components of the heterotrimeric G protein is essential for both sexual and asexual sporulation.
We showed previously that two genes, fl bA and fadA, have a major role in determining the balance between growth, sporulation, and mycotoxin (sterigmatocystin; ST) production by the filamentous fungus Aspergillus nidulans. fadA encodes the alpha subunit for a heterotrimeric G-protein, and continuous activation of FadA blocks sporulation and ST production while stimulating growth. fl bA encodes an A. nidulans regulator of G-protein signaling (RGS) domain protein that antagonizes FadA-mediated signaling to allow development. To better understand FlbA function and other aspects of FadA-mediated growth control, we have isolated and characterized mutations in four previously undefined genes designated as sfaA, sfaC, sfaD, and sfaE (suppressors of flbA), and a new allele of fadA (fadAR205H), all of which suppress a fl bA loss-of-function mutation ( fl bA98). These suppressors overcome fl bA losses of function in both sporulation and ST biosynthesis. fadAR205H, sfaC67, sfaD82, and sfaE83 mutations are dominant to wild type whereas sfaA1 is semidominant. sfaA1 also differs from other suppressor mutations in that it cannot suppress a fl bA deletion mutation (and is therefore allele specific) whereas all the dominant suppressors can bypass complete loss of fl bA. Only sfaE83 suppressed dominant activating mutations in fadA, indicating that sfaE may have a unique role in fadA- fl bA interactions. Finally, none of these suppressor mutations bypassed fl uG loss-of-function mutations in development-specific activation.
Microbial secondary metabolite production is frequently associated with developmental processes such as sporulation, but there are few cases where this correlation is understood. Recent work with the filamentous fungus Aspergillus nidulans has provided new insights into the mechanisms coordinating production of the toxic secondary metabolite sterigmatocystin with asexual sporulation. These processes have been shown to be linked through a common need to inactivate a heterotrimeric G protein dependent signaling pathway that, when active, serves to stimulate growth while blocking both sporulation and sterigmatocystin biosynthesis.
The formation of mitotically derived spores, called conidia, is a common reproductive mode in filamentous fungi, particularly among the large fungal class Ascomycetes. Asexual sporulation strategies are nearly as varied as fungal species; however, the formation of conidiophores, specialized multicellular reproductive structures, by the filamentous fungus Aspergillus nidulans has emerged as the leading model for understanding the mechanisms that control fungal sporulation. Initiation of A. nidulans conidiophore formation can occur either as a programmed event in the life cycle in response to intrinsic signals or to environmental stresses such as nutrient deprivation. In either case, a development-specific set of transcription factors is activated and these control the expression of each other as well as genes required for conidiophore morphogenesis. Recent progress has identified many of the earliest-acting genes needed for initiating conidiophore development and shown that there are at least two antagonistic signaling pathways that control this process. One pathway is modulated by a heterotrimeric G protein that when activated stimulates growth and represses both asexual and sexual sporulation as well as production of the toxic secondary metabolite, sterigmatocystin. The second pathway apparently requires an extracellular signal to induce sporulation-specific events and to direct the inactivation of the first pathway, removing developmental repression. A working model is presented in which the regulatory interactions between these two pathways during the fungal life cycle determine whether cells grow or develop.
The initiation of conidiophore development in the filamentous fungus Aspergillus nidulans is a complex process requiring the activities of several genes including fluG, flbA, flbB, flbC, flbD, and flbE. Recessive mutations in any one of these genes result in greatly reduced expression of the brlA developmental regulatory gene and a colony morphology described as fluffy. These fluffy mutants have somewhat diverse phenotypes but generally grow as undifferentiated masses of vegetative hyphae to form large cotton-like colonies. In this paper we describe a genetic screen to identify dominant mutations resulting in similar fluffy colony morphologies. We have identified 36 dominant fluffy mutant strains and shown that 29 of these mutants have greatly reduced brlA expression as compared to wild-type. In addition, we have found that 19 of these mutants are not only developmentally altered but also fail to produce the toxic, carcinogenic, secondary metabolite sterigmatocystin. At least three of the mutants isolated result from dominant activating mutations in fadA which encodes the G alpha subunit of a heterotrimeric G-protein. Another of the mutants results from a dominant interfering mutation in brlA. We discuss the approaches taken to characterize these potentially important regulators of growth, development and secondary metabolism.
The filamentous fungus Aspergillus nidulans contains a cluster of 25 genes that encode enzymes required to synthesize a toxic and carcinogenic secondary metabolite called sterigmatocystin (ST), a precursor of the better known fungal toxin aflatoxin (AF). One ST Cluster (stc) gene, aflR, functions as a pathway-specific transcriptional regulator for activation of other genes in the ST pathway. However, the mechanisms controlling activation of aflR and synthesis of ST and AF are not understood. Here we show that one important level for control of stc gene expression requires genes that were first identified as early acting regulators of asexual sporulation. Specifically, we found that loss-of-function mutations in flbA, which encodes a RGS domain protein, or dominant activating mutations in fadA, which encodes the alpha subunit of a heterotrimeric G protein, block both ST production and asexual sporulation. Moreover, overexpression of flbA or dominant interfering fadA mutations cause precocious stc gene expression and ST accumulation, as well as unscheduled sporulation. The requirement for flbA in sporulation and ST production could be suppressed by loss-of-function fadA mutations. The ability of flbA to activate stc gene expression was dependent upon another early acting developmental regulator, fluG, and AflR, the stc gene-specific transcription factor. These results are consistent with a model in which both asexual sporulation and ST production require inactivation of proliferative growth through inhibition of FadA-dependent signaling. This regulatory mechanism is conserved in AF-producing fungi and could therefore provide a means of controlling AF contamination.
flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that is required for control of mycelial proliferation and activation of asexual sporulation. We identified a dominant mutation in a second gene, fadA, that resulted in a very similar phenotype to flbA loss-of-function mutants. Analysis of fadA showed that it encodes the alpha-subunit of a heterotrimeric G protein, and the dominant phenotype resulted from conversion of glycine 42 to arginine (fadA(G42R)). This mutation is predicted to result in a loss of intrinsic GTPase activity leading to constitutive signaling, indicating that activation of this pathway leads to proliferation and blocks sporulation. By contrast, a fadA deletion and a fadA dominant-interfering mutation (fadA(G203R)) resulted in reduced growth without impairing sporulation. In fact, the fadA(G203R) mutant was a hyperactive asexual sporulator and produced elaborate sporulation structures, called conidiophores, under environmental conditions that blocked wild-type sporulation. Both the fadA(G203R) and the fadA deletion mutations suppressed the flbA mutant phenotype as predicted if the primary role of FlbA in sporulation is in blocking activation of FadA signaling. Because overexpression of flbA could not suppress the fadA(G42R) mutant phenotype, we propose that FlbA's role in modulating the FadA proliferation signal is dependent upon the intrinsic GTPase activity of wild-type FadA.
Under limiting growth conditions, Aspergillus nidulans produces a carcinogenic secondary metabolite related to aflatoxin and called sterigmatocystin (ST). The genes for ST biosynthesis are co-ordinately regulated and are all found within an approximately 60-kilobase segment of DNA. One of the genes within this region is predicted to encode a CX2CX6CX6CX2CX6CX2 zinc binuclear cluster DNA-binding protein that is related to the Aspergillus flavus and Aspergillus parasiticus aflatoxin regulatory gene aflR. Deletion of the A. nidulans aflR homolog resulted in an inability to induce expression of genes within the ST gene cluster and a loss of ST production. Because A. nidulans aflR mRNA accumulates specifically under conditions that favor ST production we expect that activation of ST biosynthetic genes is determined by A. nidulans aflR. In support of this hypothesis, we demonstrated that induced expression of the A. flavus aflR gene in A. nidulans, under conditions that normally suppress ST gene expression, resulted in activation of genes in the ST biosynthetic pathway. This result demonstrates that AflR function is conserved between Aspergillus spp. and that aflR expression is sufficient to activate genes in the ST pathway.
Sterigmatocystin (ST) and the aflatoxins (AFs), related fungal secondary metabolites, are among the most toxic, mutagenic, and carcinogenic natural products known. The ST biosynthetic pathway in Aspergillus nidulans is estimated to involve at least 15 enzymatic activities, while certain Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius strains contain additional activities that convert ST to AF. We have characterized a 60-kb region in the A. nidulans genome and find it contains many, if not all, of the genes needed for ST biosynthesis. This region includes verA, a structural gene previously shown to be required for ST biosynthesis, and 24 additional closely spaced transcripts ranging in size from 0.6 to 7.2 kb that are coordinately induced only under ST-producing conditions. Each end of this gene cluster is demarcated by transcripts that are expressed under both ST-inducing and non-ST-inducing conditions. Deduced polypeptide sequences of regions within this cluster had a high percentage of identity with enzymes that have activities predicted for ST/AF biosynthesis, including a polyketide synthase, a fatty acid synthase (alpha and beta subunits), five monooxygenases, four dehydrogenases, an esterase, an 0-methyltransferase, a reductase, an oxidase, and a zinc cluster DNA binding protein. A revised system for naming the genes of the ST pathway is presented.
A filamentous fungus, Aspergillus nidulans, produces the carcinogenic mycotoxin sterigmatocystin (ST), which is a polyketide-derived secondary metabolite. A gene (pksST) encoding the ST polyketide synthase (PKSst) in A. nidulans was cloned, sequenced, and characterized. Large induced deletion mutants, which did not make ST or any ST intermediates, were used to identify genes associated with ST biosynthesis. Among the transcripts detected within the deletion region, which showed developmental expression with ST production, was a 7.2-kb transcript. Functional inactivation of the gene encoding the 7.2-kb transcript blocked production of ST and all ST intermediate substrates but did not affect transcription of the pathway genes, indicating that this gene was involved in a very early step of ST biosynthesis. These results also indicate that PKSst was not associated with activation of other ST genes. Sequencing of the region spanning this gene revealed that it encoded a polypeptide with a deduced length of 2,181 amino acids that had high levels of similarity to many of the known polyketide synthases and FASs. This gene, pksST, encodes a multifunctional novel type I polyketide synthase which has as active sites a beta-ketoacyl acyl carrier protein synthase, an acyltransferase, duplicated acyl carrier proteins, and a thioesterase, all of these catalytic sites may be multiply used. In addition, a 1.9-kb transcript, which also showed developmental expression, was mapped adjacent to pksST, and the sequence of this gene revealed that it encoded a cytochrome P-450 monooxygenase-like peptide.
The present paper describes an enzyme-linked immunoassay (ELISA) used in combination with thin-layer chromatography (TLC) and liquid chromatography (LC) for determination of fusarochromanone (TDP) mycotoxins in barley, wheat, and a Fusarium culture grown in rice and corn. The mycotoxins were first extracted from the sample with 100% methanol and subjected to TLC or LC without additional cleanup treatment. Individual fractions eluted from TLC or LC were acetylated, then analyzed by ELISA. Determinations of TDP toxins at levels as low as 0.1 and 0.5 ng were achieved by ELISA in combination with LC and TLC, respectively. The detection limit for TDP-1 in barley and wheat was about 20 ppb by ELISA alone as compared with a detection limit of 5 ppb by a combination of ELISA with either TLC or LC. Overall analytical recovery (% of added) of TDP-1 added to barley and wheat at 5, 10, and 20 ppb of TDP-1 was 106.9 +/- 15.3 and 113.2 +/- 11.6 by LC-ELISA and 108.8 +/- 9.1 and 110.4 +/- 4.9 by TLC-ELISA, respectively. Analysis of extracts obtained from Fusarium equiseti R6137 grown in corn and rice by the combination of TLC and ELISA revealed that diacetyl-TDP was also produced by this fungus in addition to TDP-1 and TDP-2. Comparable results were obtained when fungal extracts were subjected to ELISA, LC, and immunochromatography (i.e., combination of ELISA with either TLC or LC).