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.     

Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus

Cyclopiazonic acid (CPA), an indole-tetramic acid mycotoxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins. Aflatoxin biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. flavus lacking aflatoxin production due to the loss of the entire aflatoxin gene cluster and portions of the subtelomeric region are often unable to produce CPA, which suggests a physical link of genes involved in CPA biosynthesis to the aflatoxin gene cluster. Examining the subtelomeric region in A. flavus isolates of different chemotypes revealed a region possibly associated with CPA production. Disruption of three of the four genes present in this region predicted to encode a monoamine oxidase, a dimethylallyl tryptophan synthase, and a hybrid polyketide non-ribosomal peptide synthase abolished CPA production in an aflatoxigenic A. flavus strain. Therefore, some of the CPA biosynthesis genes are organized in a mini-gene cluster that is next to the aflatoxin gene cluster in A. flavus.     

Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR

The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5 kb from 20 kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A. flavus not captured with other methods.

Significance and Impact of the Study

Aflatoxins, potent naturally‐occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A. flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A. flavus populations.     

Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates

Aspergillus flavus populations are genetically diverse. Isolates that produce either, neither, or both aflatoxins and cyclopiazonic acid (CPA) are present in the field. We investigated defects in the aflatoxin gene cluster in 38 nonaflatoxigenic A. flavus isolates collected from southern United States. PCR assays using aflatoxin-gene-specific primers grouped these isolates into eight (A-H) deletion patterns. Patterns C, E, G, and H, which contain 40 kb deletions, were examined for their sequence breakpoints. Pattern C has one breakpoint in the cypA 3' untranslated region (UTR) and another in the verA coding region. Pattern E has a breakpoint in the amdA coding region and another in the ver1 5'UTR. Pattern G contains a deletion identical to the one found in pattern C and has another deletion that extends from the cypA coding region to one end of the chromosome as suggested by the presence of telomeric sequence repeats, CCCTAATGTTGA. Pattern H has a deletion of the entire aflatoxin gene cluster from the hexA coding region in the sugar utilization gene cluster to the telomeric region. Thus, deletions in the aflatoxin gene cluster among A. flavus isolates are not rare, and the patterns appear to be diverse. Genetic drift may be a driving force that is responsible for the loss of the entire aflatoxin gene cluster in nonaflatoxigenic A. flavus isolates when aflatoxins have lost their adaptive value in nature.     

Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.

Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.     

5-Azacytidine inhibits aflatoxin biosynthesis in Aspergillus flavus

Aflatoxins, mainly produced by Aspergillus flavus and A. parasiticus, are a group of potent mycotoxins with carcinogenic, hepatotoxic, and immunosuppressive properties. Many studies have been devoted to investigating their biosynthesis mechanism since they were discovered half a century ago. 5-Azacytidine (5-AC), a derivative of the nucleoside cytidine and an inactivator of DNA methyltransferase, is widely used for studies in epigenetics and cancer biology, and has also been used for studying secondary metabolism in fungi. In this study, 5-AC was applied to investigate its effect on the development and aflatoxin biosynthesis of A. flavus. The results indicate that 5-AC inhibits the ability to produce aflatoxin and also causes a fluffy aconidial phenotype. Further studies revealed that 5-AC affects gene expression of A. flavus to a limited degree, and the unique homolog of DNA methyltransferase gene (DmtA) expressed constitutively during different developmental stages of A. flavus irrespective of 5-AC. This work may provide some basic data to elucidate the role of 5-AC in aflatoxin biosynthesis and the development of A. flavus.     

Recombination and lineage‐specific gene loss in the aflatoxin gene cluster of Aspergillus flavus

Aflatoxins produced by Aspergillus flavus are potent carcinogens that contaminate agricultural crops. Recent efforts to reduce aflatoxin concentrations in crops have focused on biological control using nonaflatoxigenic A. flavus strains AF36 (=NRRL 18543) and NRRL 21882 (the active component of afla‐guard^®). However, the evolutionary potential of these strains to remain nonaflatoxigenic in nature is unknown. To elucidate the underlying population processes that influence aflatoxigenicity, we examined patterns of linkage disequilibrium (LD) spanning 21 regions in the aflatoxin gene cluster of A. flavus. We show that recombination events are unevenly distributed across the cluster in A. flavus. Six distinct LD blocks separate late pathway genes aflE, aflM, aflN, aflG, aflL, aflI and aflO, and there is no discernable evidence of recombination among early pathway genes aflA, aflB, aflC, aflD, aflR and aflS. The discordance in phylogenies inferred for the aflW/aflX intergenic region and two noncluster regions, tryptophan synthase and acetamidase, is indicative of trans‐species evolution in the cluster. Additionally, polymorphisms in aflW/aflX divide A. flavus strains into two distinct clades, each harbouring only one of the two approved biocontrol strains. The clade with AF36 includes both aflatoxigenic and nonaflatoxigenic strains, whereas the clade with NRRL 21882 comprises only nonaflatoxigenic strains and includes all strains of A. flavus missing the entire gene cluster or with partial gene clusters. Our detection of LD blocks in partial clusters indicates that recombination may have played an important role in cluster disassembly, and multilocus coalescent analyses of cluster and noncluster regions indicate lineage‐specific gene loss in A. flavus. These results have important implications in assessing the stability of biocontrol strains in nature.     

Isolation of an Aspergillus terreus mutant impaired in arginine biosynthesis and its complementation with the argB gene from Aspergillus nidulans

Using filtration enrichment techniques, an Aspergillus terreus arginine auxotrophic strain which contains a mutation that abolishes ornithine transcarbamylase (OTCase) activity has been isolated. This mutant has been genetically transformed with the cloned Aspergillus nidulans OTCase gene. Prototrophic transformants arose at a frequency of about 50 transformants per microgram of plasmid DNA. Southern blot analysis of DNA from the transformants showed that the transforming DNA was ectopically integrated at different locations in the A. terreus genome, often in multiple tandem copies. The transformants were phenotypically stable for several mitotic divisions and retained their capacity to produce extracellular enzymes.     

Degradation of aflatoxin by Aspergillus flavus

Aflatoxin degradative activity was demonstrated in 6- to 12-d-old intact mycelium and cell-free extracts of Aspergillus flavus. The addition of cycloheximide, SKF 525-A or metyrapone to cultures of A. flavus prevented subsequent degradation of the aflatoxins, while in cell-free extracts degradation was inhibited by SKF 525-A, metyrapone and cytochrome c but not by KCN. In cell-free extracts, aflatoxin degradation was enhanced by NADPH and NaIO4. The results suggest the involvement of cytochrome P-450 monooxygenases in the aflatoxin degradative activity of A. flavus.     

Effect of lithium on aflatoxin biosynthesis by certain strains of Aspergillus flavus group

Summary The effect of lithium on growth and aflatoxin production in chemically defined medium was studied on three aflatoxigenic strains of Asperigillus flavus group. The three strains used differed in their aflatoxin producing capacities. The possible mechanism of lithium induced stimulation of aflatoxin biosynthesis is discussed.     

Behaviour of Aspergillus flavus in presence of Aspergillus niger during biosynthesis of aflatoxin B1

Aspergillus niger or Aspergillus tamarii when grown as mixed cultures with toxigenic A. flavus inhibits biosynthesis of aflatoxin by A. flavus, owing primarily to its ability to produce inhibitors of aflatoxin biosynthesis and to their ability to degrade aflatoxin. Gluconic acid partly prevents aflatoxin production. The other factors such as changes in pH of the medium and the effect on the growth of A. flavus have no role in imparting capabilities to these cultures to inhibit aflatoxin production by A. flavus.     

Participation in aflatoxin biosynthesis by a reductase enzyme encoded by vrdA gene outside the aflatoxin gene cluster

Three reactions from hydroxyversicolorone to versicolorone, from versiconal hemiacetal acetate to versiconol acetate, and from versiconal to versiconol are involved in a metabolic grid in aflatoxin biosynthesis. This work demonstrated that the same reductase of Aspergillus parasiticus catalyzes the three reactions. The gene (named vrdA) encoding the reductase was cloned, and its sequence did not show homology to any regions in aflatoxin gene cluster. Its cDNA encoding a 38,566 Da protein was separated by three introns in the genome. Deletion of the vrdA gene in A. parasiticus caused a significant decrease in enzyme activity, but did not affect aflatoxin productivity of the fungi. Although the vrdA gene was expressed in culture conditions conducive to aflatoxin production, it was expressed even in the aflR deletion mutant. These results suggest that the vrdA is not an aflatoxin biosynthesis gene, although it actually participates in aflatoxin biosynthesis in cells.