Expression ofccaR, Encoding the Positive Activator of Cephamycin C and Clavulanic Acid Production inStreptomyces clavuligerus, Is Dependent onbldG

Abstract

In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.Streptomyces spp. are unique among prokaryotic organisms because their life cycle involves filamentous, vegetative mycelia and a series of complex morphological changes resulting in the formation of aerial hyphae and chains of unigenomic spores.In Streptomyces coelicolor, the genetically best-characterized streptomycete, a number of genes have been characterized that function in the global regulation of morphological differentiation. Such genes are referred to as bld (bald) genes, since mutants lack the characteristic fuzzy coating of aerial hyphae seen in the wild-type strain. Interestingly, some of the bld mutants that have been isolated are also defective in the production of secondary metabolites, such as antibiotics, suggesting that the corresponding bld genes are involved in the regulation of both differentiation processes.To date, 14 S. coelicolor bld mutants have been isolated and eight of the bld genes have been cloned and characterized. The bld gene products are diverse in nature (7,16,28,33,35,36,43,53) and include a putative anti-anti-sigma factor, BldG (11) that is the subject of this study. Characterization of the bldG locus (11) revealed both a bldG-complementing ge...

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

“…Although an understanding of the higher level regulatory system that controls the expression of pathway-specific regulatory genes for individual biosynthetic gene clusters should be essential to control and improve the secondary metabolite production, only a few cases have been studied to date (9,10,20). The results with the bdpA disruptants of S. avermitilis suggest that the BdpA protein, which is an AdpA homolog, has the ability to activate the expression of strR.…”

“…4), thereby confirming the role of CcaR in the regulation of cephamycin C biosynthesis in recombinant S. avermitilis. It has also been reported that the expression of ccaR in S. clavuligerus was dependent on the anti-sigma factor antagonist encoded by bldG (20). It was, therefore, of some interest whether or not the corresponding bldG ortholog in S. avermitilis, rsbV (sav4614), could control the expression of ccaR.…”

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

“…A bldG probe was included as an internal control in every reaction; that for claR (shown in Fig. 7c) was repeated without the bldG probe to allow visualization of the claR-tsp2 protected fragment, which differs from that of the bldG-tsp1 protected segment by only one nucleotide (Bignell et al, 2005). Reactions for each strain were performed concomitantly, loaded on the same gel and exposed on the same film, allowing quantitative comparison of different time points and different strains for each probe.…”

“…In parallel, transcription of the regulatory genes ccaR (Pérez-Llarena et al, 1997) and bldG (Bignell et al, 2005) (both involved in the regulation of cephamycin C and clavulanic acid production), and of claR (Pérez-Redondo et al, 1998, Paradkar & Jensen, 1998 (involved in regulating clavulanic acid biosynthesis), was determined, as was that of relA.…”

The plant pathogen Streptomyces scabies 87-22 has a functional pyochelin biosynthetic pathway that is regulated by TetR- and AfsR-family proteins

“…To complement the DSCAB1401 mutant, a~1.7 kb fragment containing the SCAB1401 coding sequence and 1 kb of upstream DNA was amplified by PCR and cloned into the EcoRI site of the QC31 integrative plasmid, pAU3-45 (Bignell et al, 2005). The resulting plasmid, pRFSRL34, was moved to the DSCAB1401 strain by conjugation.…”