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Applied and Environmental Microbiology, March 2005, p. 1539-1545, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1539-1545.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of cis-Acting Sites NorL, a TATA Box, and AflR1 in nor-1 Transcriptional Activation in Aspergillus parasiticus

Michael J. Miller,^1,2, Ludmila V. Roze,¹ Frances Trail,³ and John E. Linz^1,2,4*

Department of Food Science and Human Nutrition,¹ Center for Integrative Toxicology,² Department of Plant Pathology,³ National Center for Food Safety and Toxicology, Michigan State University, East Lansing, Michigan⁴

Received 26 April 2004/ Accepted 28 September 2004

The transcription factor AflR is required for up-regulation of specific pathway genes involved in aflatoxin biosynthesis in the filamentous fungus Aspergillus. nor-1 encodes an early aflatoxin pathway enzyme; its promoter contains a consensus AflR binding site (AflR1). Proteins in Aspergillus parasiticus cell extracts and AflR expressed in Escherichia coli do not bind to A. parasiticus AflR1 in vitro, so it was not clear if this site was required for nor-1 expression or if other transcription factors contributed to gene regulation. In this study we defined the role of AflR1 in nor-1 expression in A. parasiticus and identified additional cis-acting sites required for maximum nor-1 transcriptional activation. Deletion and substitution of AflR1 in the nor-1 promoter in A. parasiticus nor-1::GUS reporter strains showed that this site is required for nor-1 transcriptional activation in vivo. Substitution of a putative TATA box in the nor-1 promoter resulted in nondetectable ß-glucuronidase (GUS) activity, demonstrating that this TATA box is functional in vivo. We also identified a novel cis-acting site, designated NorL, between residues –210 and –238 that was required for maximum nor-1 transcriptional activation in A. parasiticus grown in liquid medium and on solid medium. Using an electrophoretic mobility shift assay, we identified a specific NorL-dependent DNA-protein complex that relies on a functional AflR, either directly or indirectly, for maximum binding capacity. Because the NorL site appears only once in the aflatoxin gene cluster, its association with the nor-1 promoter may have important implications for the overall regulatory scheme for the aflatoxin pathway.

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* Corresponding author. Mailing address: 234B GM Trout Food Science & Human Nutrition Bldg., Michigan State University, East Lansing, MI 48824. Phone: (517) 355-8474, ext. 126. Fax: (517) 353-8963. E-mail: jlinz{at}msu.edu.

Present address: Department of Food Science, North Carolina State University, Raleigh, N.C.

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Applied and Environmental Microbiology, March 2005, p. 1539-1545, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1539-1545.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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