A Novel cAMP-response Element, CRE1, Modulates Expression of nor-1 in Aspergillus parasiticus

Abstract

The level of aflatoxin accumulation in the filamentous fungus Aspergillus parasiticus is modulated by a variety of environmental cues. The presence of glucose (a preferred carbon source) in liquid and solid glucose minimal salts (GMS) growth media strongly stimulated aflatoxin accumulation. Peptone (a non-preferred carbon source) in peptone minimal salts (PMS) media stimulated only low levels of aflatoxin accumulation. Glucose stimulated transcription of the aflatoxin structural genes ver-1 and nor-1 to similar intermediate levels in liquid GMS, while on solid media, ver-1 transcription was stimulated to 20-fold higher levels than nor-1. PMS liquid and solid media stimulated very low or non-detectable levels of transcription of both genes. Electrophoretic mobility shift analysis using a nor-1 promoter fragment (norR) and A. parasiticus cell protein extracts revealed specific DNA-protein complexes of different mobility on GMS and PMS solid and liquid media. An imperfect cAMP-response element, CRE1, was identified in norR that mediated formation of the specific DNA-protein complexes. Mutation in CRE1 or AflR1 (AflR cis-acting site) caused up to a 3-fold decrease in cAMP-mediated stimulation of nor-1 promoter activity on GMS agar. South-Western blot analysis identified a 32-kDa protein that specifically bound to norR. p32 could be co-immunoprecipitated by anti-AflR antibody and co-purified with an AflR-maltose-binding protein fusion demonstrating a physical interaction between AflR and p32 in vitro. We hypothesize that p32 assists AflR in binding to the nor-1 promoter, thereby modulating nor-1 gene expression in response to environmental cues.Aflatoxins are secondary metabolites produced by Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus on food and feed crops (1). Aflatoxins have a broad range of toxic effects on humans and animals including carcinogenicity, neurotoxicity, and reproductive and developmental toxicity (for a review, see Ref.2). Therefore, aflatoxin contamination raises serious concerns related to environmental safety, food quality, and human and animal health.

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

“…A GUS activity analysis was performed with two independent single-copy 3Ј integrants from each nor-1::GUS reporter construct grown on YES solid medium as described in Materials and Methods. Representative colonies are shown as follows: in panel A, isolate 1 carrying the wild-type 332-bp promoter fragment (332), deletion mutants 76 and 64, isolate 1 carrying the 332-bp fragment with an AflR1 substitution mutation (AflRm-1), and the control strain NR-1 (C) (no plasmid integrated into the genome); in panel B, isolates 1 and 2 carrying the wild-type 332-bp promoter fragment (332-1 and 332-2), isolates 1 and 2 carrying the 332-bp fragment with a TATA substitution mutation (TATAm-1 and TATAm-2), and the same control strain (C) as in panel A; and in panel C, isolates 1 and 2 carrying the wild-type 332-bp promoter fragment (332-1 and 332-2), deletion mutants 298, 268, 238, and 210, isolate 1 carrying the 332-bp fragment with a NorL substitution mutation (NorLm-1), and the same control strain (C) as in panel A. relative ability of the nor-1 promoter to respond to exogenous cAMP was not affected in either NorLm-1 or NorLm-2, which was consistent with the observation that the cAMP response is mediated by CRE-1 (27). Thus, while a functional NorL site is not sufficient for nor-1 transcriptional activation, it is necessary for maximum nor-1 transcriptional activation under a variety of growth conditions.…”

“…Substitution of NorL reduced nor-1 promoter activity two-to sixfold in liquid growth medium and on solid growth medium, confirming the biological significance of this site. Mutation of NorL did not influence the relative ability of the nor-1 promoter to respond to cAMP, which is consistent with the role of CRE-1 in mediating the cAMP regulatory effect (27). EMSA identified two specific complexes containing DNA binding proteins, designated NorLbp and protein A, that formed when a 40-bp oligonucleotide probe carrying the NorL site was used; both complexes were effectively competed with wild-type probe, but only the protein A/DNA complex was competed with a 40-bp mutant oligonucleotide probe sequence.…”

“…This effect was mediated at least in part by a novel cis-acting site designated CRE-1 (26,27). We found that the FIG.…”

VeA Is Associated with the Response to Oxidative Stress in the Aflatoxin Producer Aspergillus flavus

“…The cultures were further incubated for 24 h. Preparation of cell extracts enriched in nuclear proteins from the A. flavus 70S, psl82, and ⌬veA strains for electrophoretic mobility shift assays (EMSAs) was performed as previously described (28). Briefly, the mycelia cultured as detailed above were harvested by filtration through Miracloth, ground in liquid nitrogen with a mortar and pestle, and resuspended in lysis buffer as described previously (28). The proteins were precipitated with ammonium sulfate (10% and then 70%) and collected by centrifugation.…”

Aflatoxins Biochemistry and Molecular Biology - Biotechnological Approaches for Control in Crops

“…Nevertheless, lactose exhibited a significant enhancing effect on OTA biosynthesis both in restrictive and conducive media, whilst glucose can show a repressive effect on OTA synthesis ). This negative effect may be partially explained by the involvement of CreA, the regulator of the carbon repression system which also acts as a controller of the secondary metabolism in many fungal species (Roze et al, 2004). Other environmental factors, such as temperature, water activity and pH, strongly influence mycotoxin biosynthesis.…”