Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster.

The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

Characterization of the Aspergillus ochraceoroseus aflatoxin/sterigmatocystin biosynthetic gene cluster.

Production of carcinogenic aflatoxins has been reported from members of Aspergillus section Flavi, Aspergillus section Nidulantes and a newly proposed Aspergillus section Ochraceorosei that consists of Aspergillus ochraceoroseus and A. rambellii. Unlike members of section Flavi, A. ochraceoroseus and A. rambellii have been shown to accumulate both aflatoxin (AF) and the aflatoxin precursor sterigmatocystin (ST). Alhough morphologically distinct from A. nidulans, molecular characterization of A. ochraceoroseus AF/ST genes and physiological characteristics of AF/ST production indicated that A. ochraceoroseus is more closely related to A. nidulans than to A. flavus. Knowing that the A. nidulans ST gene cluster is organized differently from the A. flavus AF gene cluster, we determined the genetic organization of the AF/ST biosynthetic cluster in A. ochraceoroseus. Sequencing of overlapping lambda clones and genomic PCR fragments obtained by gene-walking techniques demonstrated that the A. ochraceoroseus AF/ST gene cluster is organized much like the A. nidulans ST gene cluster except that the region from aflN to aflW is located directly upstream of aflC and in reverse orientation such that aflW represents the distal end and aflY the proximal end of the cluster. The A. ochraceoroseus cluster genes demonstrated 62-76% nucleotide identity to their A. nidulans ST cluster gene homologs. Transformation of an A. nidulans aflR mutant with the A. ochraceoroseus aflR restored ST production in A. nidulans transformants. PCR amplification of A. rambellii genomic DNA demonstrated that the AF/ST gene cluster is organized in the same manner as that of A. ochraceoroseus.

Evolution of the aflatoxin gene cluster.

WhyAspergillus species produce aflatoxin remains an unsolved question. In this report we suggest that evolution of the aflatoxin biosynthesis gene cluster has been a multistep process. More than 300 million years ago a primordial cluster of genes allowed production of anthraquinones that may have served as insect attractants to facilitate spore dispersal. Later adaptive evolutionary steps introduced genes into the cluster that encoded enzymes associated with fungal virulence. These genes may have allowed the otherwise saprophytic fungi to be better able to colonize living plants. Later, genes for production of aflatoxins B1 and G1 were added to the basal cluster. Loss of the ability to produce aflatoxin G1 occurred with the divergence ofA. flavus, a species that, perhaps, was more successful than its ancestors at colonizing plants. This logical progression in evolutionary development of the aflatoxin biosynthetic cluster fits the phylogenetic data as well as known chemical reactivity of the initially formed anthraquinone polyketide metabolites.

Regulatory elements in aflatoxin biosynthesis.

This review provides a synopsis of factors involved in the regulation of aflatoxin inAspergillus species at the molecular level. Much of the knowledge available today on the regulation of secondary metabolite production in fungi has been gleaned from studies of the aflatoxin gene cluster inA. flavus andA. parasiticus and the sterigmatocystin gene cluster inA. nidulans. Regulation of these two gene clusters is under the control of both pathway-specific transcription factors such as AflR and AflJ and global or broad-domain transcription factors such as AreA and PacC. Study of secondary metabolite (sec-) mutants inA. parasiticus first identified an association between mycotoxin production and fungal development. This linkage has been extended at the molecular level by the characterization of a G-protein/cAMP/Protein kinase A signaling pathway that regulates sporulation via the transcription factor BrlA and aflatoxin/sterigmatocystin production via AflR. Another global regulator of mycotoxin production, VeA, mediates a developmental light-response inA. nidulans andA. flavus. Though not similar to any known fungal transcriptional regulators, VeA controls aflatoxin/sterigmatocystin production via transcriptional control of AflR and it also regulates development of sexual structures such as cleistothecia inA. nidulans and sclerotia inA. flavus.

Aflatoxin biosynthesis gene clusters and flanking regions.

AIMS: To compare the biosynthetic gene cluster sequences of the main aflatoxin (AF)-producing Aspergillus species. METHODS AND RESULTS: Sequencing was on fosmid clones selected by homology to Aspergillus parasiticus sequence. Alignments revealed that gene order is conserved among AF gene clusters of Aspergillus nomius, A. parasiticus, two sclerotial morphotypes of Aspergillus flavus, and an unnamed Aspergillus sp. Phylogenetic relationships were established using the maximum likelihood method implemented in PAUP. Based on the Eurotiomycete/Sordariomycete divergence time, the A. flavus-type cluster has been maintained for at least 25 million years. Such conservation of the genes and gene order reflects strong selective constraints on rearrangement. Phylogenetic comparison of individual genes in the cluster indicated that ver-1, which has homology to a melanin biosynthesis gene, experienced selective forces distinct from the other pathway genes. Sequences upstream of the polyketide synthase-encoding gene vary among the species, but a four-gene sugar utilization cluster at the distal end is conserved, indicating a functional relationship between the two adjacent clusters. CONCLUSIONS: The high conservation of cluster components needed for AF production suggests there is an adaptive value for AFs in character-shaping niches important to those taxa. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first comparison of the complete nucleotide sequences of gene clusters harbouring the AF biosynthesis genes of the main AF-producing species. Such a comparison will aid in understanding how AF biosynthesis is regulated in experimental and natural environments.

Molecular genetic analysis and regulation of aflatoxin biosynthesis.

Aflatoxins, produced by some Aspergillus species, are toxic and extremely carcinogenic furanocoumarins. Recent investigations of the molecular mechanism of AFB biosynthesis showed that the genes required for biosynthesis are in a 70 kb gene cluster. They encode a DNA-binding protein functioning in aflatoxin pathway gene regulation, and other enzymes such as cytochrome p450-type monooxygenases, dehydrogenases, methyltransferases, and polyketide and fatty acid synthases. Information gained from these studies has led to a better understanding of aflatoxin biosynthesis by these fungi. The characterization of genes involved in aflatoxin formation affords the opportunity to examine the mechanism of molecular regulation of the aflatoxin biosynthetic pathway, particularly during the interaction between aflatoxin-producing fungi and plants.

Variability in nitrogen regulation of aflatoxin production by Aspergillus flavus strains.

Aflatoxins are toxic and carcinogenic metabolites of several Aspergillus species. The effect of nitrate on aflatoxin production and expression of the key regulatory genes involved in aflatoxin biosynthesis, aflR and aflJ, were compared among isolates of the S(B) and S(BG) strains of A. flavus. Aflatoxin production by two of the three strain S(B) isolates did not differ significantly between the two media tested, whereas for S(BG) A. flavus isolates, the level of aflatoxins in buffered nitrate medium was as much as 20-fold lower than in ammonium salts medium. Expression of aflR was not significantly affected by growth of cultures in nitrate medium for most of the isolates. However, on nitrate medium, expression of aflJ was 2.6-fold higher for the S(B) isolates than it was on ammonium salts medium, whereas for the S(BG) isolates aflJ expression was 2-fold lower on nitrate than on ammonium salts medium. This difference may result from the presence in the aflJ/aflR intergenic region of S(BG) isolates of fewer putative binding sites (HGATAR sites) for AreA, the positive-acting, wide domain transcription factor involved in regulation of nitrogen metabolism.

DNA from Aspergillus flavus contains 5-methylcytosine.

DNA from Aspergillus sp. has been reported not to contain 5-methylcytosine. However, it has been found that Aspergillus nidulans responds to 5-azacytidine, a drug that is a strong inhibitor of DNA methyltransferases. Therefore, we have re-examined the occurrence of 5-methylcytosine in DNA from Aspergillus flavus by using a highly sensitive and specific method for detection of modified bases in genomic DNA comprising high-performance liquid chromatography separation of nucleosides, labeling of the nucleoside with deoxynucleoside kinase and two-dimensional thin-layer chromatography. Our results show that 5-methylcytosine is present in DNA from A. flavus. We estimate the relative amounts of 5-methylcytosine to cytosine to be approximately 1/400.

adhA in Aspergillus parasiticus is involved in conversion of 5'-hydroxyaverantin to averufin.

Two routes for the conversion of 5'-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5'-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene, adhA, from the aflatoxin gene cluster of Aspergillus parasiticus SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases-a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD(+)-NADP(+), and the motif YXXXK, which is found at the active site. A. parasiticus SU-1, which produces aflatoxins, has two copies of adhA (adhA1), whereas A. parasiticus SRRC 2043, a strain that accumulates O-methylsterigmatocystin (OMST), has only one copy. Disruption of adhA in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those adhA disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture.

Generation of aflR disruption mutants of Aspergillus parasiticus.

The aflR gene of Aspergillus parasiticus and A. flavus encodes a binuclear zinc-finger, DNA-binding protein, AflR, responsible for activating the transcription of all known aflatoxin biosynthetic genes including itself. Studies to determine how environmental and nutritional factors affect aflR expression and hence aflatoxin production in A. parasiticus have been difficult to perform due to the lack of aflR "knockout" mutants. Transformation of an O-methylsterigmatocystin (OMST)-accumulating strain of A. parasiticus with an aflR-niaD gene disruption vector resulted in clones harboring a recombinationally inactivated aflR gene which no longer produced OMST or aflR transcript. By transformation of this aflR disruptant strain with constructs containing mutated versions of the aflR promoter, we identified three cis-acting sites that were necessary for aflR function: an AflR-binding site, a PacC-binding site, and a G + A-rich site near the transcription start site of aflR.

Induction of the soybean phytoalexins coumestrol and glyceollin by Aspergillus.

Several isoflavonoid phytoalexins produced by soybeans are known to be estrogenic, with potential beneficial health effects in humans. Increased production of phytoalexins by the soybean plant will facilitate research efforts in this area. In this study, phytoalexin induction and accumulation in soybean cotyledon tissue was observed using four species of Aspergillus: A. sojae, A. oryzae, A. niger, and A. flavus. All four Aspergillus species tested elicited phytoalexin accumulation in living soybean cotyledons. Results from a time course study indicated that maximum concentrations of the phytoalexin glyceollin, 955 microg/g fresh weight (fw), occurred at day 3 in soybean cotyledon tissue inoculated with A. sojae. Other Aspergillus species caused an accumulation of glyceollin at significantly lower levels. A maximum concentration of coumestrol of 27.2 microg/g fw was obtained from soybean cotyledons inoculated with A. niger. Soybean phytoalexins induced by food-grade A. sojae and A. oryzae allowed the collection of higher concentrations of phytoalexins for further examination in several in vitro and in vivo biological studies conducted to determine potential estrogenic activities.

Promoter elements involved in the expression of the Aspergillus parasiticus aflatoxin biosynthesis pathway gene avnA.

One of the early genes in aflatoxin biosynthesis, avnA, encodes a pathway-specific cytochrome P-450 monooxygenase that catalyzes the hydroxylation of the polyketide anthraquinone, averantin. Based on beta-glucuronidase (GUS) reporter and electrophoretic mobility shift assays, promoter sites upstream of -118 bp in the 367-bp verB-avnA intergenic region are not required for avnA gene activity. Therefore, only the -100 to -110 site of the four putative binding sites for AFLR, the aflatoxin biosynthetic pathway transcription regulatory protein (consensus binding sequence: 5'-TCGN(5)CGR-3') was required for elevated avnA expression.

Isolation and characterization of experimentally induced, aflatoxin biosynthetic pathway deletion mutants of Aspergillus parasiticus.

A plasmid vector (pDEL2) was engineered for the purpose of introducing a deletion within the aflatoxin (AF) biosynthetic gene cluster of Aspergillus parasiticus. The vector was constructed by PCR amplification of a region of the AF gene cluster from an A. parasiticus isolate that had undergone an aberrant recombinational event during transformation with a nor A-niaD gene disruption vector. This recombinational event resulted in the deletion of an approximately 6-kb region of the AF gene cluster and accumulation of the AF precursor averantin (AVN). Northern hybridization analysis confirmed that the deletion event resulted in no detectable transcription of the norA gene or the AF biosynthetic genes, avnA, verA, and ver-1. Transformation of A. parasiticus RHN1 with pDEL2 resulted in 16% of the transformants accumulating AVN. Southern hybridization analysis of randomly selected AVN-accumulating transformants indicated that all had undergone a double-crossover homologous, recombinational event resulting in the 6-kb norA to avnA deletion within the AF gene cluster. Aflatoxin precursor feeding studies performed on one of the AVN-accumulating, RHN1(pDEL2) transformants confirmed that the enzyme activities associated with the deleted genes were no longer present.

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus.

AFLR is a Zn2Cys6-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5'-TCGN5CGA-3'. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5'-TCGN5CGA-3' binding site within 200bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5'-TCGCAGCCCGG-3' at -110bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7bp of the 5'-TCGN5CGA-3' motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5'-TCGSWNNSCGR-3'.

Characterization of the promoter for the gene encoding the aflatoxin biosynthetic pathway regulatory protein AFLR.

Most genes in the aflatoxin biosynthetic pathway in Aspergillus parasiticus are regulated by the binuclear zinc cluster DNA-binding protein AFLR. The aflR promoter was analyzed in beta-glucuronidase reporter assays to elucidate some of the elements involved in the gene's transcription control. Truncation at 118 bp upstream of the translational start site increased promoter activity 5-fold, while truncation at -100 reduced activity about 20-fold. These findings indicate the presence of an important positive regulatory element between -100 and -118 and a negative regulatory region further upstream. Electrophoretic mobility shift assays on nuclear extracts from A. parasiticus induced for aflatoxin expression suggest that AFLR and another, possibly more abundant, protein bind to the -100/-118 region. Another protein binds to a sequence at position -159 to -164 that matches the consensus binding site for the transcription factor involved in pH-dependent gene regulation, PACC.

Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P-450 monooxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B1, G1, B2, and G2.

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400-->Leu-400, Ala-143-->Ser-143, and Ile-528-->Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 in A. parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.

Alteration of different domains in AFLR affects aflatoxin pathway metabolism in Aspergillus parasiticus transformants.

AFLR, a zinc binuclear cluster DNA-binding protein, is required for activation of genes comprising the aflatoxin biosynthetic pathway in Aspergillus spp. Transformation of Aspergillus parasiticus with plasmids containing the intact aflR gene gave clones that produced fivefold more aflatoxin pathway metabolites than did the untransformed strain. When a 13-bp region in the aflR promoter (position -102 to -115 with respect to the ATG) was deleted, including a portion of a palindromic site previously shown to bind recombinant AFLR, metabolite production was 40% that of transformants with intact aflR. This result provides further evidence that this site may be involved in the autoregulation of aflR. Overexpression of pathway genes could also result from increased quantities of AFLR titrating out a putative repressor protein. In AFLR, a 20-amino-acid acidic region near its carboxy-terminus resembles the region in yeast GAL4 required for GAL80 repressor binding. When 3 of the acidic amino acids in this region were deleted, levels of metabolites were even higher than those produced by transformants with intact aflR, as would be expected if repressor binding was suppressed in transformants containing this altered protein. Transformation with plasmids mutated at the AFLR zinc cluster (Cys to Trp at amino acid position 49) or at a putative nuclear localization signal region (RRARK deleted) gave clones with one-fifth the metabolite production of the untransformed fungus in spite of the transformants making the same or more aflR mRNA. Since these transformants retained a copy of intact aflR, the latter results can be explained best by assuming that AFLR activates genes involved in aflatoxin production as a dimeric protein and that heterodimers containing both mutant and intact AFLR strands are inactive.

Characterization of the Aspergillus parasiticus niaD and niiA gene cluster.

The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus.

The Aspergillus niger (ficuum) aphA gene encodes a pH 6.0-optimum acid phosphatase.

We have used the Aspergillus niger (An) aphA gene as a probe and cloned the A. ficuum (Af) SRRC 265 gene encoding an extracellular pH 6.0-optimum acid phosphatase (APase6) from a genomic library. The identity of the Af aphA gene was confirmed and its nucleotide (nt) sequence verified by comparing its deduced amino acid (aa) sequence to that of purified Af APase6. A comparison of the nt sequences of the An and Af genes suggested that errors were made in the previously reported An aphA sequence. Several regions of the An aphA were resequenced and the mistakes corrected. With its nt sequence corrected, the An aphA is nearly identical to the cloned Af gene encoding APase6, and in 90.4% agreement in the coding regions. Both genes have three conserved introns and when translated, both nt sequences code for a polypeptide of 614 aa. There is now evidence that the two cloned genes are homologous and code for acid phosphatases that are 96% identical.