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

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 peptidebinding proteins.

The Pathway-Specific Regulator ClaR of Streptomyces clavuligerus Has a Global Effect on the Expression of Genes for Secondary Metabolism and Differentiation

“…The parA gene in pSCL2 and 11 genes located in pSCL4 (SCLAV_p0032, p0126, p0353, p0528, p0713, p0828, p1090, p1250, p1328, p1452, and p1539) were quantified by qPCR. Levels of the linear plasmids similar to those of the wild-type strain were found in strains S. clavuligerus ccaR::aph, S. clavuligerus cyp::tsr, S. clavuligerus orf13::apr, S. clavuligerus orf14::aph, S. clavuligerus oppA1::acc, S. clavuligerus relA:: neo, and S. clavuligerus ⌬adpA (22,32,33,34,35,36). However, the old S. clavuligerus claR::aph strain, obtained by transformation of protoplasts to disrupt the claR gene (16), was found to give a very low qPCR DNA amplification of genes located in either pSCL2 or pSCL4 (Fig.…”

“…The S. clavuligerus oppA2::aph strain used previously in our laboratory (16,32) was shown by qPCR amplification and transcriptomic analysis to carry in the mycelium 25,000-fold fewer copies of plasmid pSCL2 and 10,000-fold fewer copies of plasmid pSCL4 than the wild-type strain (21). The lack of sporulation of S. clavuligerus oppA2::aph, previously believed to be due to the oppA2 mutation (32), was shown to be dependent on the lack of pSCL4 (21). To discriminate whether the effects described for the mutations in the clavulanic acid gene cluster are real or due to the absence of plasmids in the mutant strains, the levels of plasmids pSCL2 and pSCL4 were quantified in our collection of mutant strains and compared to those of the wild-type strain.…”

Biosynthesis of clavam metabolites

“…Both of these genes encode predicted oligopeptide permeases-type binding proteins and both contain a domain found in bacterial extracellular solute-binding proteins. Mutants defective in either gene cannot produce any clavulanic acid [41]. Analysis of oppA1 and oppA2 mutants provided evidence that both are involved in peptide transport, but how this translates into their having essential roles in clavulanic acid production is still unclear.…”

Regulatory mechanisms controlling antibiotic production in Streptomyces clavuligerus

“…These authors identiWed the claR-dependent transcription of genes located immediately upstream and downstream of claR that have been later characterized as essential for clavulanic acid production. They encode an oligopeptide permease (oppA1) [42], the clavulanic-9-aldehyde reductase (car) [54] and a cytochrome P450 (cyp) [39,43]. Expression of claR in ccaR-disrupted mutants is null as measured by Northern hybridization or S1-analysis.…”