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Antimicrobial Agents and Chemotherapy, May 1999, p. 1215-1224, Vol. 43, No. 5
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Genes Specific for the Biosynthesis of Clavam Metabolites Antipodal to Clavulanic Acid Are Clustered with the Gene for Clavaminate Synthase 1 in Streptomyces clavuligerus

Roy H. Mosher,1,dagger Ashish S. Paradkar,1,Dagger Cecilia Anders,1 Barry Barton,2 and Susan E. Jensen1,*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9,1 and SmithKline Beecham Pharmaceuticals, Worthing, West Sussex BN14 8QH, United Kingdom2

Received 4 October 1998/Returned for modification 11 January 1999/Accepted 20 February 1999

Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames, cvm4 and cvm5, located directly downstream of cas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis.

* Corresponding author. Mailing address: Department of Biological Sciences, CW405 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: Biology Program, University of Illinois at Springfield, Springfield, IL 62794-9243.

Dagger Present address: Diversa Corporation, San Diego, CA 92121.

Antimicrobial Agents and Chemotherapy, May 1999, p. 1215-1224, Vol. 43, No. 5
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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