Molecular characterization of a fungal secondary metabolism promoter: transcription of the Aspergillus nidulans isopenicillin N synthetase gene is modulated by upstream negative elements

Abstract

The Aspergillus nidulans IPNS gene, encoding isopenicillin N synthetase, is a secondary metabolism gene. It is contiguous to, but divergently transcribed from, the ACVS gene at the penicillin gene cluster. The untranslated region between both ORFs is 872bp long. Here we present the physical and functional characterization of the IPNS transcriptional unit. Transcriptional start point (tsp) mapping reveals heterogeneity at the 5'-end of the mRNA, with a major start at -106 relative to the initiation codon. This indicates that the actual length of the non-transcribed intergenic region is 525bp. Functional elements in the IPNS upstream region have been defined by assaying beta-galactosidase activity in extracts from recombinant strains carrying deletion derivatives of the IPNS promoter fused to lacZ, integrated in single copy at the argB locus. Strains were grown in penicillin production broth under carbon catabolite repressing or derepressing conditions. The results of deletion analysis indicate that: (i) the IPNS promoter is mostly regulated by negative controls that act upon a high basal activity; (ii) sequential deletion of three of the negative cis-acting elements results in a mutated promoter that is 40 times (sucrose broth) or 12 times (lactose broth) more active than the wild type; (iii) one of these negative cis-acting elements is involved in sucrose repression. Strikingly, it is located outside the non-transcribed 525bp intergenic region and maps to the coding region of the divergently transcribed ACVS gene; (iv) a 5'-deletion up to -56 (relative to the major tsp) contains information to provide almost half of the maximal promoter activity and allows initiation of transcription at the correct site. By using total-protein extracts from mycelia grown under penicillin producing conditions we have detected a DNA-binding activity that specifically shifts a promoter fragment located between -654 and -455 (relative to IPNS tsp). Deletions covering this region partially abolish IPNS promoter activity. The fragment in question overlaps the ACVS tsp.

On the Mechanism by which Alkaline pH Prevents Expression of an Acid-Expressed Gene

“…Protein extraction followed the protocol described above but using the buffer described previously (31). ␤-Galactosidase activities were determined as described previously (31). Values given are the average of at least three independent experiments for each transformant, and standard errors are indicated.…”

“…After 16 h of growth at 30°C, mycelia were harvested, washed with distilled water, dried, and frozen in liquid nitrogen. Protein extraction followed the protocol described above but using the buffer described previously (31). ␤-Galactosidase activities were determined as described previously (31).…”

“…All promoters were sequenced to ensure the absence of unintended mutations, and the presence of a single copy of the construct integrated at argB was confirmed by Southern blotting as described previously (31).…”

A lacZ reporter fusion method for the genetic analysis of regulatory mutations in pathways of fungal secondary metabolism and its application to the Aspergillus nidulans penicillin pathway

“…We and others have previously used lacZ fusions for functional analysis of the ipnA promoter (5,27). As in bacteria, expression of these fusions can be scored in plate assays with the chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside).…”

“…Carbon regulation in response to the availability of a preferred carbon source modulates ipnA expression through the action of a yet undefined negative-acting regulatory gene (5,16,17,27). pH regulation positively regulates ipnA transcription in response to alkaline ambient pH (18) through the action of the zinc finger protein PacC (29).…”