Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi.

The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used.     

Divergent regulation of aflatoxin production at acidic pH by two <Emphasis Type="Italic">Aspergillus</Emphasis> strains

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A. flavus isolates produced more AF as the external pH became increasingly acidic, an atypical strain from West Africa produced less. The lower AF production was not well correlated with decreases in expression of the aflatoxin pathway regulatory gene, aflR, or of two other biosynthesis genes.     

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.     

veA is required for toxin and sclerotial production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

Characterization of the function of the ver-1A and ver-1B genes, involved in aflatoxin biosynthesis in Aspergillus parasiticus.

The ver-1A gene was cloned and its nucleotide sequence was determined as part of a previous study on aflatoxin B1 (AFB1) biosynthesis in the filamentous fungus Aspergillus parasiticus SU-1. A second copy of this gene, ver-1B, was tentatively identified in this fungal strain. In this study, ver-1B was cloned by screening an A. parasiticus cosmid library with a ver-1A probe. The nucleotide sequence of ver-1B was determined. The predicted amino acid sequence of ver-1B had 95% identity with ver-1A. A translational stop codon, found in the ver-1B gene coding region, indicated that it encodes a truncated polypeptide. To confirm the function of the ver-1 genes in AFB1 synthesis, a plasmid (pDV-VA) was designed to disrupt ver-1A and/or ver-1B by transformation of the AFB1 producer A. parasiticus NR-1. One disruptant, VAD-102, which accumulated the pathway intermediate versicolorin A was obtained. Southern hybridization analysis of VAD-102 revealed that ver-1A but not ver-1B was disrupted. A functional ver-1A gene was transformed back into strain VAD-102. Transformants which received ver-1A produced AFB1, confirming that ver-1A is the only functional ver-1 gene in A. parasiticus SU-1 and that its gene product is involved in the conversion of versicolorin A to sterigmatocystin in AFB1 biosynthesis. A duplicated chromosomal region (approximately 12 kb) was identified upstream from ver-1A and ver-1B by Southern hybridization analysis. This duplicated region contained the aflR gene, which is proposed to be one regulator of AFB1, synthesis. A similar gene duplication was also identified in several other strains of A. parasiticus.     

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species.

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus

A complex regulatory network governs the biosynthesis of aflatoxin. While several genes involved in aflatoxin production are known, their action alone cannot account for its regulation. Arrays of clones from an Aspergillus flavus cDNA library and glass slide microarrays of ESTs were screened to identify additional genes. An initial screen of the cDNA clone arrays lead to the identification of 753 unique ESTs. Many showed sequence similarity to known metabolic and regulatory genes; however, no function could be ascribed to over 50% of the ESTs. Gene expression analysis of Aspergillus parasiticus grown under conditions conducive and non-conductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. Twenty-four genes were more highly expressed during aflatoxin biosynthesis and 18 genes were more highly expressed prior to aflatoxin biosynthesis. No predicted function could be ascribed to 18 of the 24 genes whose elevated expression was associated with aflatoxin biosynthesis.