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Title: Analysis of aflatoxin regulatory factors in serial transfer-induced non-aflatoxigenic Aspergillus parasiticus

item Cary, Jeffrey
item Bhatnagar, Deepak
item Yu, Jiujiang
item Cleveland, Thomas

Submitted to: Journal of Food Additives & Contaminants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/10/2007
Publication Date: 10/1/2007
Citation: Kale, S.P., Cary, J.W., Hollis, N., Wilkinson, J.R., Bhatnagar, D., Yu, J., Cleveland, T.E., Bennett, J.W. 2007. Analysis of aflatoxin regulatory factors in serial transfer-induced non-aflatoxigenic Aspergillus parasiticus. Journal of Food Additives & Contaminants. 24(10):1061-1069.

Interpretive Summary: This work describes experiments that have been conducted in an effort to better understand the mechanisms by which aflatoxins are produced by fungi. Aflatoxins are toxic and carcinogenic compounds often produced by the fungi, Aspergillus flavus and Aspergillus parasiticus, during growth on crops such as corn, peanuts, cottonseed, and treenuts. Because of the potential health risks, aflatoxin contamination of food and feed crops is also of great economic importance to farmers who cannot sell their crops due to strict domestic and international regulatory guidelines with regards to aflatoxin contamination. We have succeeded in developing strains of Aspergillus parasiticus that no longer produce aflatoxins. This study was focused on characterizing a variant of A. parasiticus termed sec' that stands for secondary metabolism negative. These strains do not make aflatoxin. We were able to show that molecular factors that are known to be involved in control of aflatoxin production in the fungus are not responsible for loss of aflatoxin production in the sec' strain. This led us to theorize that another factor(s) in the sec' strains are responsible for regulation of aflatoxin biosynthesis. These strains will serve as tools to aid in unraveling the mechanisms responsible for production of aflatoxin which will in turn help to devise strategies for eliminating aflatoxin contamination of food and feed crops.

Technical Abstract: Aflatoxins (AFs) are carcinogenic secondary metabolites of Aspergillus parasiticus. In previous studies, non-toxigenic A. parasiticus sec' (for secondary metabolism negative) variants were generated from their toxigenic sec+ (for secondary metabolism positive) parents for genetic and physiological analysis for understanding regulation of AF biosynthesis. In this study, aflR and aflJ, positive regulators of AF production and laeA, a global regulator of secondary metabolism, were analyzed to determine if they play a role in establishment of the sec' phenotype. Previous studies have shown no difference in aflR DNA sequence in the sec+ and sec' strains. The current study showed that while this sequence identity extended to the aflJ as well as the aflJ-aflR intergenic region, expression of aflR in the sec- strain was several fold lower than that observed in the sec+ strain while aflJ expression was barely detectable in the sec- strain. Western blot analysis indicated that despite AflR protein being present in the sec- strain, no toxin production resulted. Introduction of exogenous aflR into the sec- strain increased aflR expression, but did not restore AF production. Lastly, reverse transcription-PCR analysis revealed that laeA was expressed in both sec+ and sec' strains. These results suggest that although aflR, aflJ, and laeA are necessary for AF production, they are not sufficient. We propose that the aflR and aflJ expression may be regulated by element(s) downstream from laeA or from pathways not influenced by laeA.