Three binding sites for the Aspergillus nidulans PacC zinc-finger transcription factor are necessary and sufficient for regulation by ambient pH of the isopenicillin N synthase gene promoter.

The isopenicillin N synthase (ipnA) gene, encoding a key penicillin biosynthetic enzyme in Aspergillus nidulans, represents a prototype of an alkaline-expressed gene. ipnA is under ambient pH regulation, and its promoter (ipnA(p)) contains binding sites for the zinc-finger transcription factor PacC. We show here that three of these sites, denoted ipnA2, ipnA3, and ipnA4AB, are efficiently recognized by the protein in an isolated sequence context. Single, double, and triple inactivation of these sites in any possible combination reduced promoter activity under alkaline conditions but had no effect under acidic conditions (under which promoter activity was low), as measured by the expression of wildtype and mutant ipnA(p)::lacZ fusion genes integrated in single copy into a common chromosomal location. This establishes a physiological role for these PacC binding sites and demonstrates a direct role for PacC in ambient pH regulation of ipnA gene expression. In addition, this confirms our previous proposal that PacC is an activator for alkaline-expressed genes. Notably, our experiments show that ipnA2, the highest affinity site for PacC in the ipnA(p), contributes relatively modestly to PacC-mediated activation. By contrast, the lower affinity sites ipnA3 and ipnA4AB contribute more substantially to regulation by ambient pH. Inactivation of these three binding sites reduced promoter activity under alkaline conditions to that observed under acidic conditions, showing that these three PacC sites at ipnA(p) are sufficient to account for its activation by alkaline ambient pH.