Two Oligopeptide-Permease-Encoding Genes in the Clavulanic Acid Cluster of Streptomyces clavuligerus Are Essential for Production of the β-Lactamase Inhibitor

Abstract

ABSTRACT orf7 ( oppA1 ) and orf15 ( oppA2 ) are located 8 kb apart in the clavulanic acid gene cluster of Streptomyces clavuligerus and encode proteins which are 48.0% identical. These proteins show sequence similarity to periplasmic oligopeptide-binding proteins. Mutant S. clavuligerus oppA1 :: acc , disrupted in oppA1 , lacks clavulanic acid production. Clavulanic acid production is restored by transformation with plasmid pIJ699- oppA1 , which carries oppA1 , but not with the multicopy plasmid pIJ699- oppA2 , which carries oppA2 . The mutant S. clavuligerus oppA2 :: aph also lacks clavulanic acid production, shows a bald phenotype, and overproduces holomycin (5). Clavulanic acid production at low levels is restored in the oppA2 -disrupted mutants by transformation with plasmid pIJ699- oppA2 , but it is not complemented by the multicopy plasmid pIJ699- oppA1 . Both genes encode oligopeptide permeases with different substrate specificities. The disrupted S. clavuligerus oppA2 :: aph is not able to grow on RPPGFSPFR (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; bradykinin), but both mutants grow on VAPG (Val-Ala-Pro-Gly) as the only nitrogen source, indicating differences in the peptide bound by the proteins encoded by both genes. The null S. clavuligerus oppA1 :: acc and S. clavuligerus oppA2 :: aph mutants are more resistant to the toxic tripeptide phosphinothricyl-alanyl-alanine (also named bialaphos) than the wild-type strain, suggesting that this peptide might be transported by these peptide-binding proteins.