Identification of the sites of action for regulatory genes controlling the amdS gene of Aspergillus nidulans.

Abstract

The amdS gene of Aspergillus nidulans, which encodes an acetamidase enzyme, is positively regulated by the trans-acting genes amdR, facB, amdA, and areA. Sequence changes in several cis-acting mutations in the 5' region of the gene which specifically affect amdS regulation were determined. The amdI9 mutation, which results in increased facB-dependent acetate induction, is due to a single-base change at base pair -210 relative to the start point of translation. The amdI93 mutation, which abolishes amdR-dependent omega-amino acid induction, is a deletion of base pairs -181 to -151. The amdI66 mutation, which causes increased gene activation in strains carrying amdA regulatory gene mutations, is a duplication of base pairs -107 to -90. Transformation of A. nidulans can generate transformants containing multiple integrated copies of plasmid sequences. When these plasmids carry a potential binding site for a regulatory gene product, growth on substrates whose catabolism requires genes activated by that regulatory gene can be reduced, apparently because of titration of the regulatory gene product. Introduction of 5' amdS sequences via cotransformation into strains of various genotypes was used to localize sequences apparently involved in binding of the products of the amdR, amdA, and facB genes. The position of these sequences is in agreement with the positions of the specific cis-acting mutations. Consistent with these results, a transformant of A. nidulans derived from a plasmid deleted for sequences upstream from -111 was found to have lost amdR- and facB-mediated control but was still regulated by the amdA gene. In addition, amdS expression in this transformant was still dependent on the areA gene.

Isolation and analysis of the acetate regulatory gene, facB, from Aspergillus nidulans.

“…The T56A19 strain was derived from transformation of a strain carrying an amdI9 mutation. In T56A19, the high acetamidase levels resulting from the amdI9 mutation are reduced, presumably because of facB product titration by multiple copies of the 108-bp fragment containing the amdI9 sequence (15). T56A19 also exhibits poor growth on 2-pyrrolidinone medium because of amdR product titration resulting in decreased activation of the amdR-controlled lamA or lamB genes or both (1, 16a).…”

“…Sites of action for two regulatory genes controlling amdS expression have been localized to a 108-bp PstI-SmaI restriction fragment in the 5' region of the gene (15). Two cis-acting mutations affecting amdS expression have been located in this fragment: a single base-pair change (amdI9) leading to increased facB-dependent acetamide utilization and a 30-bp deletion (amdI93) resulting in the loss of amdR-mediated induction by w-amino acids (15). Deletion of the entire 108-bp fragment results in loss offacB and amdR regulation of amdS expression (15).…”

“…It has been demonstrated that A. nidulans transformants containing multiple copies of the 108-bp fragment show reduced activation of genes subject to amdR and facB control, presumably because of titration of the regulatory gene products (15). The T56A19 strain was derived from transformation of a strain carrying an amdI9 mutation.…”

“…Acetamidase, the amdS gene product, catalyzes the breakdown of acetamide to acetate and ammonium. A single base-pair change in the 5' region of amdS (the amdI9 mutation) greatly increasesfacB-dependent induction of amdS expression (13,15). The high level offacB-dependent acetamide utilization in amdI9 strains is reduced when multiple copies of the amdI9 sequence are introduced via transformation (15,18).…”

“…A single base-pair change in the 5' region of amdS (the amdI9 mutation) greatly increasesfacB-dependent induction of amdS expression (13,15). The high level offacB-dependent acetamide utilization in amdI9 strains is reduced when multiple copies of the amdI9 sequence are introduced via transformation (15,18). Utilization of acetate is also affected in transformants receiving a very large number of copies (>70) of the sequence (18 MATERIALS AND METHODS Strains.…”