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United States Department of Agriculture

Agricultural Research Service

Title: Regulatory Elements in Aflatoxin Biosynthesis

item Cary, Jeffrey
item Ehrlich, Kenneth
item Bhatnagar, Deepak
item Cleveland, Thomas

Submitted to: Mycotoxin Research
Publication Type: Review Article
Publication Acceptance Date: January 10, 2006
Publication Date: March 1, 2006
Citation: Cary, J.W., Ehrlich, K., Kale, S.P., Calvo, A.M., Bhatnagar, D., Cleveland, T.E. 2006. Regulatory Elements in Aflatoxin Biosynthesis. Mycotoxin Research. 22(2):105-109.

Technical Abstract: Aflatoxin (AF) biosynthesis in fungi is responsive to environmental cues, such as carbon and nitrogen source, stress, plant constituents (i.e. volatiles and tannins), and physical factors such as pH and temperature. These environmental stimuli are transduced via complex signaling cascades that control the expression of both global-acting and AF pathway-specific transcription factors. Two known AF pathway-specific regulator genes, aflR and aflJ, have been localized to the AF biosynthetic gene cluster in Aspergillus flavus and A. parasiticus. These genes are also present in the sterigmatocystin (ST) cluster of A. nidulans. The aflR gene was first characterized in A. flavus by Payne et al. and in A. parasiticus by Chang et al. AflR protein is a Gal4-type, Zn-finger binuclear cluster, positive-acting transcription factor that is required for expression of all known AF biosynthetic genes. AflR activates transcription of AF pathway genes following binding to the conserved 11 bp palindromic sequence, 5’-TCGN5CGR-3’, present in the promoter regions of most AF genes. Fungal isolates in which the aflR gene has been inactivated no longer produce AF nor express AF pathway genes while fungi engineered to over-express aflR produce increased levels of AF transcripts leading to increased levels of AF production. AflJ is a second gene in the AF pathway cluster that appears to play a role in regulation of AF biosynthesis. The aflJ gene was first characterized by Meyers et al. AflJ resides adjacent to aflR in the AF gene cluster and the two genes are divergently transcribed. AflJ protein does not demonstrate significant identity to other proteins of known function in the databases. Interestingly, it was shown that aflJ mutants do not produce AF (or only very minute amounts) but they still express AF pathway genes though at reduced levels. So it appears that AflJ is not functioning like AflR as a transcriptional activator of AF pathway genes. Chang et al. used a yeast two-hybrid system to show that AflJ can bind to the C-terminal region of AflR. Based on AflJ’s ability to bind AflR, it has been proposed that AflJ is either acting as a transcriptional enhancer or co-activator of AflR. However, more studies will be needed to determine the exact role of AflJ in AF biosynthesis. An intergenic region of 758 bp is located between the bidirectionally transcribed aflR and aflJ genes. To analyze promoter function of the aflR gene, the entire 758-bp intergenic region as well as truncated forms of this region were used to drive expression of the Escherichia coli-' glucuronidase (GUS)-encoding gene uidA. Removal of sequences in the promoter from nucleotide -758 to -280 (with respect to the aflR translational start site) had no apparent effect on promoter activity, but further truncation to -118 enhanced gene expression nearly 5-fold. Therefore, there appears to be a negative regulatory element in the region from -280 to -118. Further removal of bases -118 to -100 almost entirely eliminated GUS gene expression. When the region from -118 to -107 was deleted, two-thirds of this activity was lost. Therefore, sequences in the 18-bp region from -100 to -118 appear to be critical for aflR promoter activity. This region overlaps a 10-bp palindrome (-120 to -111) and a purine-rich region. EMSA using nuclear extracts from A. parasiticus and oligonucleotide ligands covering the region from -81 to -173 revealed the presence of a putative PacC-binding site (5’-GCCARG-3’). Binding to the aflR PacC site is consistent with the function of this protein in repressing transcription of acid-expressed genes under alkaline conditions. AF biosynthesis in A. flavus occurs in acidic media, but is inhibited in alkaline media. It is possible that PacC binding to the -148 to -173 site has a negative effect on aflR expression. In a different study Chang et al. found that the nitrogen regulatory protein, AreA, bound to GATA sites in the aflR-aflJ intergenic region, suggesting that nitrogen regulation of AF production could be linked to AreA control of aflR and aflJ expression. Since sites in the aflR-aflJ intergenic region are recognized by transcription factors that are themselves regulated by environmental signals (pH regulates the activity of PacC and nitrate regulates AreA), it is probable that, at least for nitrate, the effects on AF pathway gene transcription may, in part, be directly caused by changes in the expression of aflR or aflJ resulting from activation by these factors. To support this observation, Ehrlich et al. found that certain strains of AF-producing Aspergilli respond differently to nitrate than do other strains, and that the differences could be correlated with differences in the number of possible GATA sites (ranging from five to nine) near the aflJ start site. In addition, there is also variability with respect to the presence and location of binding sites for the transcriptional regulators PacC and BrlA (involved in conidiation) in the aflR-aflJ intergenic region. The variability in binding sites may be the result of each species need to adapt to different environmental stimuli inherent to their particular ecological niche. Other genes in the AF biosynthetic cluster have AreA and PacC binding sites at key positions in their promoters that may affect their expression. For example, the 1.7 kb intergenic region separating the nor-1 and pksA genes has two adjacent PacC sites nearly in the middle that, from site-directed mutagenesis studies, affect expression of pksA, which encodes the pathway-specific polyketide synthase necessary for the first steps in formation of the polyketide backbone. In A. nidulans, the promoter region of the gene stcU, which is necessary for conversion of VERA to DMST, contains a PacC-binding site immediately upstream of its AflR-binding site and is probably involved in expression of this gene. The role of carbon utilization in the regulation of expression of genes involved in AF biosynthesis is not, as yet, well understood. Unlike the biosynthesis of many other secondary metabolites, AF gene expression is induced by the presence of simple carbohydrates, for example glucose, sucrose, or maltose, but not by peptone, sorbose, or lactose. It should be noted that all of the AF pathway genes so far studied lack CreA sites in their promoters and, therefore, would not be expected to be subject to carbon catabolite repression mediated by the transcription factor CreA. However, an interesting possible role for CreA in aflR expression could be control of expression of the antisense aflR mRNA transcript, since two tandem CreA-binding sites, GCGGGGaGTGGGG, are present at the start of this reported transcript. If carbon catabolite repression prevents the expression of this transcript, no decrease in the amount of AflR protein could occur. A close relationship between AF production and fungal development was first observed by Bennett when they noted “strain degeneration” in A. parasiticus upon repeated subculturing of macerated mycelia. This technique produced morphological variants that gave “fan” and “fluff” phenotypes due to increased mycelial growth. These variants also demonstrated decreased conidiation, as well as AF production, compared to the wild-type strain. Kale et al. continued studies on “strain degeneracy” and found that these variants, termed sec- strains for secondary metabolite negative, were stable and did not revert back to wild-type levels of AF production or conidiation. Expanding on the studies of Bennett and Kale, Hicks et al. identified the genetic mechanism in A. nidulans that linked sterigmatocystin (ST) (and AF production in A. parasiticus/flavus) to fungal development. The mechanism involved a G-protein/cAMP/protein kinase A (PKA) signaling pathway. FadA is the alpha subunit of the A. nidulans heterotrimeric G protein. When FadA is bound to GTP, i.e. in its active form, ST/AF production (and sporulation

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