Transcriptional analysis of the isopenicillin N synthase-encoding gene of Streptomyces clavuligerus

The gene (pcbC) encoding isopenicillin N synthase of Streptomyces clavuligerus is separated from an upstream open reading frame (ORF) by a 31-bp intergenic region. Inspection of the sequence of this intergenic region did not identify a promoter sequence. The promoter probe plasmid, pIJ4083, which contains the promoter-less catechol-2,3-dioxygenase (C23O)-encoding gene (xylE) as a reporter gene, was used to analyze the sequence upstream from the pcbC gene for promoter activity. Introduction of an SphI site at the start codon of pcbC by site-directed mutagenesis allowed the cloning of a 335-bp fragment (-334 to +1 in relation to the pcbC start codon) immediately upstream from xylE in pIJ4083. C23O activity was detected in both Streptomyces lividans and S. clavuligerus cultures that contained the upstream fragment, suggesting the presence of a promoter sequence. Northern analysis of total RNA extracted from S. clavuligerus identified a monocistronic 1.2-kb transcript hybridizing to a pcbC-specific probe. When RNA was isolated at various times during growth in liquid culture, the presence of a transcript was first detected during stationary phase. Analysis of the pcbC transcript by primer extension located the transcription start point to a C residue within the upstream ORF, 91 bp upstream from the pcbC start codon.