Submitted to: Journal of Applied Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/15/2005
Publication Date: 4/1/2005
Citation: Ehrlich, K.C., Yu, J., Cotty, P.J. 2005. Aflatoxin biosynthesis gene clusters and flanking regions. Journal of Applied Microbiology. 99:518-527. Interpretive Summary: The aflatoxins and sterigmatocystins are toxic and cancer causing products of a common type of fungus (Aspergillus) that is found in soils of warm growing regions. Preharvest aflatoxin contamination of crops is widespread in temperate growing areas when conditions are favorable and is believed to be caused by only some of the known Aspergillus species capable of aflatoxin production. Pre-harvest production of aflatoxins on plants is known to be dependent on climate conditions, soil type and soil moisture, and degree of susceptibility of the plant. Our long term research strategy is to reduce aflatoxin contamination of cottonseed, corn, peanuts, and other susceptible crops. In order to find acceptable ways to do this, we need to understand the conditions that favor aflatoxin production by the fungi and to develop methods to interrupt the production by interfering with the machinery that the fungus uses to make aflatoxins. To do this, we compared the aflatoxin- and sterigmatocystin-producing machinery (the gene cluster containing 27 genes) in six different Aspergillus species. Some species are not a problem for agriculture. We found that the species that are a problem for agriculture make aflatoxins under very different growth conditions than those that have not been found to be a problem. In particular, our comparison shows that soil nitrogen should affect aflatoxin differently in the different species. We also identified two new genes in the gene cluster machinery. Only one of these new genes is present in the cluster of genes that is needed to make sterigmatocystins. Based on this work we hope to identify target sites in the aflatoxin-producing machinery to prevent aflatoxin production by the fungus using inexpensive inhibitors that could be applied to a growing susceptible crop.
Technical Abstract: Aflatoxin biosynthesis gene clusters were sequenced from isolates of A. nomius, two sclerotial morphotypes of A. flavus, and a strain of Aspergillus (SBG) which resembles A. flavus but produces both B and G aflatoxins. The aflatoxin clusters in the two types of A. flavus have more than 99 percent sequence identity, while the A. flavus cluster is 96 percent homologous to that of A. parasiticus, 93 percent to that of the SBG species, and 91 percent to that of A. nomius. Sequence alignment revealed that portions of genes encoding a short chain alcohol dehydrogenase and a cytochrome P450 monooxygenase were deleted in the A. flavus cluster. The latter gene is necessary for formation of aflatoxin G1. Except for these genes, the other aflatoxin cluster genes are conserved in all species. Two additional open reading frames were discovered which appear to be co-regulated by the pathway specific transcription factor, AflR. Both have no known protein functional domains. One gene is in the pksA/nor-1 intergenic region, while the other, in the verB/avfA intergenic region, is a homolog of the first. The promoter regions of four cluster genes (aflR, avfA, aflT, and aflJ in A. nomius) lack binding sites for AflR. Differences in the number and position in intergenic regions of binding sites for certain globally acting transcription factors suggests a divergence in regulation of aflatoxin gene expression among the Aspergillus species. Some of the genes in the aflatoxin cluster (hexA, hexB, aflR, aflJ, aflY, nor-1, and estA), have 12 to 20 percent lower homology to A. nidulans stc cluster orthologs than do the other cluster genes. Such variability suggests that individual genes in the cluster evolved under different selective pressures, a result inconsistent with the hypothesis that horizontal transfer plays an important role in cluster radiation and evolution.