The Carboxy-Terminal Portion of the Aflatoxin Pathway Regulatory Protein AFLR of Aspergillus parasiticus Activates GAL1 :: lacZ Gene Expression in Saccharomyces cerevisiae

Abstract

AFLR, a DNA-binding protein of 444 amino acids, transactivates the expression of aflatoxin biosynthesis genes in Aspergillus parasiticus and Aspergillus flavus, as well as the sterigmatocystin synthesis genes in Aspergillus nidulans. We show here by fusion of various aflR coding regions to the GAL4 DNA-binding coding region that the AFLR carboxyl terminus contained a region that activatedGAL1::lacZ gene expression inSaccharomyces cerevisiae and that the AFLR internal region was required for the activation activity. Compared to the AFLR carboxy-terminal fusion protein (AFLRC), a mutant AFLRC retained approximately 75% of the activation activity after deletion of three acidic amino acids, Asp365, Glu366, and Glu367, in a previously identified acidic stretch. Removal of the carboxy-terminal amino acid, Glu444, did not affect the activation activity. Substitutions of acidic Glu423, Asp439, or Asp436/Asp439 with basic amino acids, Lys and His, resulted in 10- to 15-fold-lower activation activities. Strikingly, the Asp436His mutation abolished the activation activity. Substitutions of basic His428 and His442 with acidic Asp resulted in 20 and 40% decreases in the activation activities, respectively. Simultaneous substitutions of Arg427, Arg429, and Arg431 with Leu also significantly decreased the activation activity; the decrease was approximately 50-fold. Results suggest that the AFLR carboxy-terminal region is involved in transcription activation and that total acidity in this region is not a major determinant of AFLR’s activation ability inS. cerevisiae.

Differentiation of aflatoxin-producing and non-producing strains of Aspergillus flavus group

“…This gene plays an important role in the aflatoxin biosynthetic pathway by regulating the activity of other structural genes such as omt‐A , ver‐1 and nor‐1 (Woloshuk et al . 1994; Chang et al . 1999).…”

Phytochemical Inhibition of Aflatoxigenicity in Aspergillus flavus by Constituents of Walnut (Juglans regia)

“…The aflatoxin biosynthesis genes occur in a 75 kb cluster, and the effect of gallic acid could be upstream of this gene cluster. Alternatively, the regulatory gene, aflR, expression of which is essential for the functioning of other genes in the cluster (30), encodes for a zinc-containing, DNA-binding protein, and it is conceivable that the gallic acid acts as a chelator of this metal ion through sequestration by catecholic hydroxyl groups. Other metal ions essential for fungal growth, such as copper and iron, could be complexed in a similar manner (31).…”

Population genomics ofAspergillus sojaeis shaped by the food environment

“…The aflR premature stop codon mutation in AS strains has been characterized (Chang et al 2007), and a mutant lacking the AP functional copy, but expressing the AS copy was unable to produce versicolorin A, an aflatoxin intermediate (Takahashi et al 2002). Other chimeric constructs revealed that, while the AS aflR promoter and 5’ region are functional, constructs containing the AS truncated aflR were unable to produce aflatoxin (Chang et al 1999). Additionally, the AS allele of pksA contains a premature stop codon resulting in a truncated protein lacking the 3’ thioesterase domain.…”