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Applied and Environmental Microbiology, January 2002, p. 306-315, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.306-315.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in Aspergillus parasiticus

Ching-Hsun Chiou,1 Michael Miller,1 David L. Wilson,1,2 Frances Trail,3 and John E. Linz1,2,4*

Department of Food Science and Human Nutrition,1 Department of Microbiology and Molecular Genetics,4 National Food Safety and Toxicology Center,2 Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan3

Received 3 May 2001/ Accepted 22 October 2001

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-ß-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.

* Corresponding author. Mailing address: Department of Food Science and Human Nutrition, 234B GM Trout Food Science and Human Nutrition Building, Michigan State University, East Lansing, MI 48824. Phone: (517) 353-9624. Fax: (517) 353-8963. E-mail: jlinz{at}msu.edu.

Applied and Environmental Microbiology, January 2002, p. 306-315, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.306-315.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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