Transcriptional mapping of the genes encoding the early enzymes of the cephamycin biosynthetic pathway of Streptomyces clavuligerus.
Isopenicillin-N synthase (IPNS) of Streptomyces clavuligerus is encoded by the pcbC gene which is found within the cephamycin biosynthetic gene cluster. pcbC is located directly downstream from lat and pcbAB, which encode the enzymes, lysine epsilon-amino transferase and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, respectively. These enzymes act prior to IPNS in the biosynthetic pathway, and the three genes are transcribed in the same direction. Previous pcbC transcriptional studies involving recombinant promoter probe plasmids, Northern analysis and 5' primer extension indicated the presence of a monocistronic 1.2-kb transcript that initiated within pcbAB, 92-bp upstream from the pcbC start codon. S1 nuclease mapping studies have now shown, not only the transcript initiating 92 bp upstream from pcbC, but also a transcript initiating further upstream, possibly including the entire pcbAB gene. Promoter probe analysis and S1 nuclease mapping failed to detect promoter activity or a transcription start point (tsp) directly upstream from pcbAB, suggesting that pcbAB transcripts initiated within or upstream from lat. Northern analysis, to search for a pcbAB transcript, showed no distinct transcript and indicated severely degraded mRNA. Similar results were obtained when Northern analysis was used to search for lat transcripts. Promoter probe analysis to locate the lat promoter indicated that a sequence promoting transcription was present in a 330-bp DNA fragment that extended from 227-bp upstream from the lat structural gene to 103 bp inside the gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Petrich
A K
AK
Department of Microbiology, University of Alberta, Edmonton, Canada.
Leskiw
B K
BK
Paradkar
A S
AS
Jensen
S E
SE
D001483:Base Sequence; D015152:Blotting, Northern; D002513:Cephamycins / Q000096:biosynthesis*; D002874:Chromosome Mapping; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D051306:L-Lysine 6-Transaminase; D008969:Molecular Sequence Data; D005810:Multigene Family; D010088:Oxidoreductases; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D002513:Cephamycins; D004269:DNA, Bacterial; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D000637:Transaminases; D051306:L-Lysine 6-Transaminase; D010453:Peptide Synthases