Kenneth C. Ehrlich

Clustered Pathway Genes in Aflatoxin Biosynthesis

Aflatoxins, a group of polyketide-derived furanocoumarins (Fig. [1][1]), are the most toxic and carcinogenic compounds among the known mycotoxins. Among the at least 16 structurally related aflatoxins characterized, however, there are only four major aflatoxins, B1, B2, G1, and G2 (AFB1, AFG1, AFB2

Toxins of filamentous fungi.

Mycotoxins are low-molecular-weight secondary metabolites of fungi. The most significant mycotoxins are contaminants of agricultural commodities, foods and feeds. Fungi that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate (i.e. conditions favorable for fungal growth), they can also be found in temperate conditions. Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions and microbial, insect or other animal interactions. Although outbreaks of mycotoxicoses in humans have been documented, several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo. Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age and nutritional status. Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs. Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities. This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest. Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems. Current research is designed to develop natural biocontrol competitive fungi and to enhance host resistance against fungal growth or toxin production. These efforts could prevent toxin formation entirely. Rigorous programs for reducing the risk of human and animal exposure to contaminated foods and feed also include economically feasible and safe detoxification processes and dietary modifications. Although risk assessment has been made for some mycotoxins, additional, systematic epidemological data for human exposure is needed for establishing toxicological parameters for mycotoxins and the safe dose for humans. It is unreasonable to expect complete elimination of the mycotoxin problem. But multiple approaches will be needed to minimize the economic impact of the toxins on the entire agriculture industry and their harmfulness to human and animal health.

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

ABSTRACT Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.

CpG methylation inhibits binding of several sequence-specific DNA-binding proteins from pea, wheat, soybean and cauliflower

To elucidate how methylation of specific sites in plant DNA might control transcription, we examined the effect of DNA methylation at CpG sequences on the binding of plant nuclear factors to an oligonucleotide duplex containing the consensus sequence for mammalian CREB (cAMP response element binding protein). CREB is part of the ATF (activating transcription factor) family of mammalian proteins specifically binding to 5′-TGACGTCA-3′ and related sequences. Proteins recognizing the CREB-specific ligand were identified in nuclear extracts of pea seeds wheat germ, cauliflower, and soybean leaves using electrophoretic mobility shift assays. Cytosine methylation inhibited binding of this protein in all these extracts, and so this sequence-specific DNA-binding activity is referred to as methylation-inhibited binding protein 1 (MIB-1). Sites somewhat similar to that of the CREB ligand are found in the upstream regions of a wheat histone H3 gene and tomato and pea ribulose 1,5-bisphosphate carboxylase genes. These sites were bound preferentially by distinct proteins that may be related to the previously described plant proteins HBP-1, HSBF, ASF-1, or GBF. Methylation of cytosine residues at these sites and at a site for MIB-1 located upstream of a soybean proline-rich protein gene also reduced specific binding with all the nuclear extracts tested. Similarly, substitution of the central CpG dinucleotide with TpG decreased binding.

An isolate of Aspergillus flavus used to reduce aflatoxin contamination in cottonseed has a defective polyketide synthase gene

Contamination of certain foods and feeds with the highly toxic and carcinogenic family of Aspergillus mycotoxins, the aflatoxins, can place a severe economic burden on farmers. As one strategy to reduce aflatoxin contamination, the non-aflatoxin-producing A. flavus isolate AF36 is currently being applied to agricultural fields to competitively exclude aflatoxin-producing Aspergillus species. We now show that the polyketide synthase gene (pksA) required for aflatoxin biosynthesis in AF36, and in other members of the same vegetative compatibility group, possesses a nucleotide polymorphism near the beginning of the coding sequence. This nucleotide change introduces a premature stop codon into the coding sequence, thereby preventing enzyme production and aflatoxin accumulation.

Aflatoxin biosynthesis gene clusters and flanking regions

Aims:  To compare the biosynthetic gene cluster sequences of the main aflatoxin (AF)‐producing Aspergillus species.

Sequence comparison of aflR from different Aspergillus species provides evidence for variability in regulation of aflatoxin production

Aflatoxin contamination of foods and feeds is a world-wide agricultural problem. Aflatoxin production requires expression of the biosynthetic pathway regulatory gene, aflR, which encodes a Cys6Zn2-type DNA-binding protein. Homologs of aflR from Aspergillus nomius, bombycis, parasiticus, flavus, and pseudotamarii were compared to investigate the molecular basis for variation among aflatoxin-producing taxa in the regulation of aflatoxin production. Variability was found in putative promoter consensus elements and coding region motifs, including motifs involved in developmental regulation (AbaA, BrlA), regulation of nitrogen source utilization (AreA), and pH regulation (PacC), and in coding region PEST domains. Some of these elements may affect expression of aflJ, a gene divergently transcribed from aflR, that also is required for aflatoxin accumulation. Comparisons of phylogenetic trees obtained with either aligned aflR intergenic region sequence or coding region sequence and the observed divergence in regulatory features among the taxa provide evidence that regulatory signals for aflatoxin production evolved to respond to a variety of environmental stimuli under differential selective pressures. Phylogenetic analyses also suggest that isolates currently assigned to the A. flavus morphotype SBG represent a distinct species and that A. nomius is a diverse paraphyletic assemblage likely to contain several species.

Aflatoxigenicity in Aspergillus: molecular genetics, phylogenetic relationships and evolutionary implications

Aflatoxins (AFs) are toxic and carcinogenic secondary metabolites produced by isolates of Aspergillus section Flavi as well as a number of Aspergillus isolates that are classified outside of section Flavi. Characterization of the AF and sterigmatocystin (ST) gene clusters and analysis of factors governing regulation of their biosynthesis has resulted in these two mycotoxins being the most extensively studied of fungal secondary metabolites. This wealth of information has allowed the determination of the molecular basis for non-production of AF in natural isolates of A. flavus and domesticated strains of A. oryzae. This review provides an overview of the molecular analysis of the AF and ST gene clusters as well as new information on an AF gene cluster identified in the non-section Flavi isolate, Aspergillus ochraceoroseus. Additionally, molecular phylogenetic analysis using AF biosynthetic gene sequences as well as ribosomal DNA internal transcribed spacer (ITS) sequences between various section Flavi and non-section Flavi species has enabled determination of the probable evolutionary history of the AF and ST gene clusters. A model for the evolution of the AF and ST gene clusters as well as possible biological roles for AF are discussed.

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 genes 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 Aspergillus parasiticus niaD and niiA gene cluster

Abstract 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.