Identification of a major cis-acting DNA element controlling the bidirectionally transcribed penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) of Aspergillus nidulans

Abstract

The ␤-lactam antibiotic penicillin is produced as a secondary metabolite by some filamentous fungi. In this study, the molecular regulation of the Aspergillus (Emericella) nidulans penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) was analyzed. acvA and ipnA are divergently oriented and separated by an intergenic region of 872 bp. Translational fusions of acvA and ipnA with the two Escherichia coli reporter genes lacZ and uidA enabled us to measure the regulation of both genes simultaneously. A moving-window analysis of the 872-bp intergenic region indicated that the divergently oriented promoters are, at least in part, overlapping and share common regulatory elements. Removal of nucleotides ؊353 to ؊432 upstream of the acvA gene led to a 10-fold increase of acvA-uidA expression and simultaneously to a reduction of ipnA-lacZ expression to about 30%. Band shift assays and methyl interference analysis using partially purified protein extracts revealed that a CCAAT-containing DNA element within this region was specifically bound by a protein (complex), which we designated PENR1, for penicillin regulator. Deletion of 4 bp within the identified protein binding site caused the same contrary effects on acvA and ipnA expression as observed for all of the deletion clones which lacked nucleotides ؊353 to ؊432. The PENR1 binding site thus represents a major cis-acting DNA element. The intergenic regions of the corresponding genes of the ␤-lactam-producing fungi Penicillium chrysogenum and Acremonium chrysogenum also diluted the complex formed between the A. nidulans probe and PENR1 in vitro, suggesting that these ␤-lactam biosynthesis genes are regulated by analogous DNA elements and proteins.

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

“…After 24 h of cultivation, the coculture was prepared for HPLC analysis. For penicillin production, fermentation media were applied as previously described (39). Penicillin bioassay was carried out with Bacillus calidolactis C953 as an indicator organism (40).…”

The Aspergillus nidulans Penicillin‐Biosynthesis Gene aat (penDE) is Controlled by a CCAAT‐Containing DNA Element

“…The protein binding site identified here was nearly identical to that between acvA and ipnA which is bound by the putative regulatory protein PENRl (Then Bergh et al, 1996). Hence, cross-competition experiments were carried out using both the 50-base DNA fragment (WT) spanning the binding motif in the aat promoter, and a DNA fragment (AI) spanning the PENRl site between acvA and ipnA.…”

“…By means of band-shift analysis, a cis-acting DNA sequence containing a CCAAT core sequence was identified in the ant promoter which was specifically bound by a protein (complex). Partial purification of the binding protein and cross-competition experiments indicated that the identified CCAAT sequence was bound by the putative penicillinregulatory protein (complex) PENRI which was shown to bind to a similar CCAAT site between acvA and ipnA (Then Bergh et al, 1996). Mutation of the PENRI-binding site within the aat promoter led to a reduction of aat expression, providing evidence for the functionality of the PENRI-binding site in vivo.…”

“…In recent years, it has become evident that at the molecular level, regulation of penicillin biosynthesis is complex. Several protein factors seem to be involved (Brakhage and Van den Brulle, 1995;Chu et al, 1995;Feng et al, 1995;Haas and Marzluf, 1995; P6rez-Esteban et al, 1995;Tilburn et al, 1995;Then Bergh et al, 1996).…”

“…In recent years, it has become evident that at the molecular level, regulation of penicillin biosynthesis is complex. Several protein factors seem to be involved (Brakhage and Van den Brulle, 1995;Chu et al, 1995;Feng et al, 1995;Haas and Marzluf, 1995; P6rez-Esteban et al, 1995;Tilburn et al, 1995;Then Bergh et al, 1996).For the studies presented here, A. nidu1an.s was used because this fungus has, in contrast to the deuteromycete P chrysogenum which is employed for industrial production of penicillin, a welldefined sexual cycle facilitating genetic analyses (MacDonald and Holt, 1976;Timberlake and Marshall, 1988;Davis and Hynes, 1989; Clutterbuck, 1993).The A. nidulans nut @enDE) gene lies downstream of the ipnA gene, separated by an intergenic region of 825 bp (Ramon et al, 1987;Montenegro et al, 1990;Tobin et al, 1990; Fig. 1).…”