Streptomyces clavuligerus reIA-null mutants overproduce clavulanic acid and cephamycin C:: negative regulation of secondary metabolism by (p)ppGpp

The (p)ppGpp synthetase gene, relA, of Streptomyces clavuligerus was cloned, sequenced and shown to be located in a genomic region that is highly conserved in other Streptomyces species. reIA-disrupted and reIA-deleted mutants of S. clavuligerus were constructed, and both were unable to form aerial mycelium or to sporulate, but regained these abilities when complemented with wild-type reIA. Neither ppGpp nor pppGpp was detected in the S. clavuligerus reIA-deletion mutant. In contrast to another study, clavulanic acid and cephamycin C production increased markedly in the mutants compared to the wild-type strain; clavulanic acid production increased three- to fourfold, while that of cephamycin C increased about 2.5-fold. Complementation of the reIA-null mutants with wild-type reIA decreased antibiotic yields to approximately wild-type levels. Consistent with these observations, transcription of genes involved in clavulanic acid (ceaS2) or cephamycin C (cefD) production increased dramatically in the reIA-deleted mutant when compared to the wild-type strain. These results are entirely consistent with the growth-associated production of both cephamycin C and clavulanic acid, and demonstrate, apparently for the first time, negative regulation of secondary metabolite biosynthesis by (p)ppGpp in a Streptomyces species of industrial interest.