Physical and transcriptional map of an aflatoxin gene cluster in Aspergillus parasiticus and functional disruption of a gene involved early in the aflatoxin pathway

Abstract

Two genes involved in aflatoxin B 1 (AFB1) biosynthesis in Aspergillus parasiticus, nor-1 and ver-1, were localized to a 35-kb region on one A. parasiticus chromosome and to the genomic DNA fragment carried on a single cosmid, NorA. A physical and transcriptional map of the 35-kb genomic DNA insert in cosmid NorA was prepared to help determine whether other genes located in the nor-1-ver-1 region were involved in aflatoxin synthesis. Northern (RNA) analysis performed on RNA isolated from A. parasiticus SU1 grown in aflatoxininducing medium localized 14 RNA transcripts encoded by this region. Eight of these transcripts, previously unidentified, showed a pattern of accumulation similar to that of nor-1 and ver-1, suggesting possible involvement in AFB1 synthesis. To directly test this hypothesis, gene-1, encoding one of the eight transcripts, was disrupted in A. parasiticus CS10, which accumulates the aflatoxin precursor versicolorin A, by insertion of plasmid pAPNVES4. Thin-layer chromatography revealed that gene-1 disruptant clones no longer accumulated versicolorin A. Southern hybridization analysis of these clones indicated that gene-1 had been disrupted by insertion of the disruption vector. These data confirmed that gene-1 is directly involved in AFB1 synthesis. The predicted amino acid sequence of two regions of gene-1 showed a high degree of identity and similarity with the ␤-ketoacyl-synthase and acyltransferase functional domains of polyketide synthases, consistent with a proposed role for gene-1 in polyketide backbone synthesis.

Genetic organization and function of the aflatoxin B1 biosynthetic genes

“…Analyses of nucleotide sequences indicate that motifs common to known PKS and FAS are conserved in the gene products, namely L-ketoacyl synthase, acyltransferase and acyl carrier protein [12,23^25]. The FAS genes also contain conserved domains for L-ketoacyl reductase, enoyl reductase and enoyl hydrase [12,23,25]. The absence of ketoreductase in the PKS sequence is consistent with a lack of a reductive step in the PKS reaction that produces noranthrone.…”

“…The ¢rst gene was isolated and described in 1992 [9,10], and since the last review on the molecular biology of a£atoxin biosynthesis [11], rapid and signi¢cant progress followed the discovery that genes involved in a£atoxin biosynthesis in both A. £avus and A. parasiticus are clustered [12,13]. The cluster in A.…”

The initiation and pattern of spread of histone H4 acetylation parallel the order of transcriptional activation of genes in the aflatoxin cluster

“…The aflatoxin genes, separated by 0.2–1.5 kb intergenic regions, align within the cluster in an order that roughly correlates with ‘early’, ‘middle’ and ‘late’ enzyme activities in the aflatoxin biosynthetic pathway. Transcription of the aflatoxin genes is activated during a transition from exponential growth to stationary growth in response to declining glucose or sucrose concentration, as well as to other environmental and nutritional cues (Buchanan and Lewis, 1984; Skory et al ., 1993; Trail et al ., 1995a; Yu et al ., 2000a). In early studies we hypothesized that clustering of aflatoxin genes enabled such regulation to occur, although no conclusive data were reported (Trail et al ., 1995b).…”

Completed sequence of aflatoxin pathway gene cluster in Aspergillus parasiticus1

“…In the last decade, signi¢cant progress has been made in deciphering the a£atoxin biosynthetic pathway and its genetic control [5^11]. The linkage between the nor-1 and ver-1 genes with the regulatory gene a£R in cosmid clones [12,13] and analysis of overlapping cosmid clones in A. parasiticus and A. £avus led to the discovery of the a£atoxin pathway gene cluster [6,10,11].…”