INVESTIGATION OF THE STREPTOMYCES-CLAVULIGERUS CEPHAMYCIN-C GENE-CLUSTER AND ITS REGULATION BY THE CCAR PROTEIN

As part of a search for transcriptional regulatory genes, sequence ana lysis of several previously unsequenced gaps in the cephamycin biosynt hetic cluster has revealed the presence in Streptomyces clavuligerus o f seven genes not previously described. These include genes encoding a n apparent penicillin binding protein and a transport or efflux protei n, as well as the CmcI and CmcJ proteins, which catalyze late reaction s in the cephamycin biosynthetic pathway. In addition, we discovered a gene, designated pcd, which displays significant homology to genes en coding semialdehyde dehydrogenases and may represent the gene encoding the long-sought-after dehydrogenase involved in the conversion of lys ine to alpha-aminoadipate. Finally, two genes, sclU and rhsA, with no obvious function in cephamycin biosynthesis may define the end of the cluster. The previously described CcaR protein displays homology to a number of Streptomyces pathway-specific transcriptional activators. Th e ccaR gene was shown to be essential for the biosynthesis of cephamyc in, clavulanic acid, and non-clavulanic acid clavams. Complementation of a deletion mutant lacking ccaR and the adjacent orf11 and blp genes showed that only ccaR was essential for the biosynthesis of cephamyci n, clavulanic acid, and clavams and that mutations in orf11 or blp had no discernible effects. The lack of cephamycin production in ccaR mut ants was directly attributable to the absence of biosynthetic enzymes responsible for the early and middle steps of the cephamycin biosynthe tic pathway. Complementation of the ccaR deletion mutant resulted in t he return of these biosynthetic enzymes and the restoration of cephamy cin production.