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Journal of Bacteriology, January 2000, p. 348-356, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Dylan C. Alexander,dagger Michael J. Brumlik, Linda Lee,Dagger and Susan E. Jensen*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Received 28 June 1999/Accepted 28 October 1999

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 (Delta lat::tsr/term mutants) were created. This mutation eliminated the production of lysine-varepsilon -aminotransferase (the lat gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of delta -(L-alpha -aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) (the pcbAB gene product) was eliminated in Delta lat::tsr/term mutants, while production of isopenicillin N synthase (IPNS) (the pcbC gene product) was greatly reduced. The provision of alpha -aminoadipate to the Delta 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 Delta lat::tsr/term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic pcbC transcript levels were greatly decreased. In contrast, Delta 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.

* Corresponding author. Mailing address: Department of Biological Sciences, CW 405 Biological Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Phone: (780) 492-0672. Fax: (780) 492-2216. E-mail: susan.jensen{at}ualberta.ca.

dagger Present address: Schering-Plough Research Institute, Kenilworth, New Jersey.

Dagger Present address: Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada.

Journal of Bacteriology, January 2000, p. 348-356, Vol. 182, No. 2
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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