Transcriptional analysis of the isopenicillin N synthase gene of Streptomyces clavuligerus

Transcriptional regulation of the isopenicillin N synthase (IPNS) gene (pcbC) of Streptomyces clavuligerus was analyzed using two experimental procedures. Promoter probe vector analyses and mRNA analyses were performed to characterize the DNA region directly upstream of pcbC and this led to an examination of the transcriptional regulation of the two genes found immediately upstream of pcbC; lat and pcbAB. These genes encode the cephamycin biosynthetic enzymes lysine {dollar}varepsilon{dollar}-amino transferase and {dollar}delta{dollar}-(scL-{dollar} alpha{dollar}-aminoadipyl)- scL-cysteinyl- scD-valine synthetase respectively, which act prior to IPNS in the biosynthetic pathway.; The promoter probe vector, pIJ4083, which contains the promoter-less catechol-2,3-dioxygenase (C23O) gene (xylE) as a reporter gene, was used to analyze the sequence upstream of pcbC for promoter activity. Introduction of an SphI endonuclease restriction site at the start codon of pcbC by site-directed mutagenesis allowed the cloning of a DNA fragment containing 307 bp of sequence immediately upstream of 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. Deletions of the fragment were generated to further localize sequences important for the initiation of transcription. A series of DNA fragments that varied in the amount of sequence remaining upstream of the pcbC start codon were inserted upstream of the xylE gene in pIJ4083 and examined for C23O activity. Deletion analysis indicated that DNA sequence important for promoter activity was at least 152 bp upstream of the pcbC coding region.; Northern blot transfer and hybridization of total RNA extracted from S. clavuligerus with a pcbC-specific probe identified a monocistronic 1.2 kb pcbC transcript that was first detected during stationary phase. Analysis of the pcbC transcript by primer extension located the transcription start point to a C nucleotide 91 bp upstream of the pcbC start codon. S1 nuclease protection assays of the pcbC transcript detected not only the transcript initiating 91 bp upstream of pcbC but also a second larger transcript suggesting possible cotranscription with the upstream pcbAB gene. When the DNA region immediately upstream of pcbAB was examined by promoter probe analysis, no promoter activity was detected and S1 nuclease protection experiments failed to identify a tsp directly upstream of pcbAB. Northern blot analysis showed no distinct pcbAB transcript and indicated severely degraded mRNA. Similar results were obtained when Northern blot analysis was used to search for a lat transcript. Promoter probe analysis indicated the presence of an active promoter within a DNA fragment that extended 227 bp upstream of lat structural gene. S1 nuclease mapping of the 5{dollar}spprime{dollar} end of the lat transcript identified the tsp to be a T residue 88 bp upstream of the lat start codon. Comparisons of the S1 nuclease protected DNA fragments generated using RNA isolated at various stages of growth indicated a similar transcription pattern for all the genes, suggesting the possibility that pcbC is transcribed as part of an operon together with the pcbAB and lat genes and also as a single monocistronic message.