Early Cephamycin Biosynthetic Genes Are Expressed from a Polycistronic Transcript in Streptomyces clavuligerus

Abstract

ABSTRACT A polycistronic transcript that is initiated at the lat promoter has been implicated in the expression of the genes involved in early steps of cephamycin C biosynthesis in Streptomyces clavuligerus. pcbC is also expressed as a monocistronic transcript from its own promoter. However, an alternative interpretation involving expression via three separate yet interdependent transcripts has also been proposed. To distinguish between these possibilities, mutants lacking the lat promoter and containing a transcription terminator within the lat gene (Δ lat :: tsr /term mutants) were created. This mutation eliminated the production of lysine-ɛ-aminotransferase (the lat gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of δ-( l -α-aminoadipyl)- l -cysteinyl- d -valine synthetase (ACVS) (the pcbAB gene product) was eliminated in Δ lat :: tsr /term mutants, while production of isopenicillin N synthase (IPNS) (the pcbC gene product) was greatly reduced. The provision of α-aminoadipate to the Δ lat :: tsr /term mutants, either via exogenous feeding or via lat gene complementation, did not restore production of ACVS or IPNS. Analysis of RNA isolated from the Δ lat :: tsr /term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic pcbC transcript levels were greatly decreased. In contrast, Δ lat mutants created by in-frame internal deletion of lat maintained the polycistronic transcript and allowed production of wild-type levels of both ACVS and IPNS.