Indole-diterpene gene cluster from Aspergillus flavus

Abstract

Aflatrem is a potent tremorgenic mycotoxin produced by the soil fungus Aspergillus flavus and is a member of a large structurally diverse group of secondary metabolites known as indole-diterpenes. By using degenerate primers for conserved domains of fungal geranylgeranyl diphosphate synthases, we cloned two genes, atmG and ggsA (an apparent pseudogene), from A. flavus. Adjacent to atmG are two other genes, atmC and atmM. These three genes have 64 to 70% amino acid sequence similarity and conserved synteny with a cluster of orthologous genes, paxG, paxC, and paxM, from Penicillium paxilli which are required for indole-diterpene biosynthesis. atmG, atmC, and atmM are coordinately expressed, with transcript levels dramatically increasing at the onset of aflatrem biosynthesis. A genomic copy of atmM can complement a paxM deletion mutant of P. paxilli, demonstrating that atmM is a functional homolog of paxM. Thus, atmG, atmC, and atmM are necessary, but not sufficient, for aflatrem biosynthesis by A. flavus. This provides the first genetic evidence for the biosynthetic pathway of aflatrem in A. flavus.

FIG. 1.

A. flavus contains two GGPP synthases. (A) P. paxilli paxG gene map showing conserved domains (I to V), intron positions (inverted triangles), and consensus polypeptide sequences used to design degenerate primers to amplify the region between domains III and V. (B) PCR products amplified with degenerate primers ggpps27 and ggpps29 (lanes 2 and 4) and ggpps27 and ggpps28 (lanes 3 and 5) from genomic DNAs of P. paxilli (lanes 2 and 3) and A. flavus (lanes 4 and 5). The products shown in lanes 3 and 5 were from nested PCR amplifications of 1/100 dilutions of the products shown in lanes 2 and 4, respectively. Lane 1 contains a 1-kb DNA ladder (Invitrogen).

FIG. 2.

Southern analysis of A. flavus genomic DNA probed with atmG and ggsA. The autoradiograph shows Southern blots of A. flavus genomic DNA (1 μg) digested with five different restriction enzymes and probed with ³²P-labeled 262-bp atmG and 218-bp ggsA probes. The numbers on the left and right correspond to the sizes of the restriction fragments that hybridized to the probes.

FIG. 3.

Physical gene map of A. flavus atm locus and conserved synteny with A. nidulans and M. grisea. (A) Physical map of A. flavus insert DNA from cosmid pSZ-20 showing restriction enzyme sites for BamHI, EcoRI, and HindIII and the sizes of fragments of >3 kb. The 16 putative genes identified within this 38,895-bp sequence are labeled AF101 to AF116. Orthologs of P. paxilli indole-diterpene biosynthetic genes are labeled as atm (aflatrem biosynthesis) genes in accordance with A. nidulans nomenclature, as described by Bennett and Lasure (4). Exons of each gene are indicated by blocks, and the arrows indicate the direction of transcription. Tick marks are at 1-kb intervals. Syntenous regions for the A. flavus atm locus are shown for A. nidulans (B) and M. grisea (C). Exons with the same color are orthologous.

FIG. 4.

Comparison of gene structure of A. flavus GGPP synthase gene with those of other fungal GGPP synthase genes. Gene structures are given for F. fujikuroi primary (Ffggs) and secondary (Ffggs2), P. paxilli primary (Ppggs1) and secondary (PppaxG), and A. flavus atmG (AfatmG) GGPP synthases. The positions of introns (inverted triangles) and conserved domains I to V (shading) are identified.

FIG. 5.

Gene expression analysis. (A) Growth of A. flavus showing changes in dry weight (□) and aflatrem production (▪). (B) Autoradiographs of Northern blots of A. flavus total RNAs (10 μg) probed with ³²P-labeled 568-bp atmG, 400-bp atmC, 372-bp atmM, 562-bp act1, and 358-bp tub2 cDNAs. The ethidium bromide-stained 16S rRNA region of each gel is shown for comparison. The numbers on the right correspond to the sizes of the hybridized transcripts by comparison to a set of RNA standards (Promega). (C) RT-PCR analysis from 24 to 96 h, with mRNA as the template together with the genomic product (gDNA). Fragment sizes are indicated in base pairs.

FIG. 6.

HPLC analysis of indole-diterpenes from extracts of a P. paxilli paxM mutant containing the A. flavus atmM gene. (A) Wild type. (B) LMM-100 (paxM mutant). (C) LMM-100 containing atmM (pSZ-43). (D) Paxilline standard (5 μg). The units on the y axis are milli-absorbance units at 230 nm, and the retention time on the x axis is given in minutes.