Characterization of DNA‐binding sequences for CcaR in the cephamycin–clavulanic acid supercluster of Streptomyces clavuligerus

Abstract

SummaryRT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000-to 10 000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

“…asoensis (222), cmcI and ceaS2-II in S. clavuligerus (223), and fdmR2 in S. griseus (219). Degeneracy of the repeats may make the structure of the targets difficult to characterize, perhaps explaining why the spacers appear to vary from 4 to 15 nt in different targets (216,217,219). Alternatively, these differences could perhaps be related to variations in the structure of SARPs.…”

“…The specific sequences recognized by SARPs generally overlap the Ϫ35 regions of their targets, but sometimes the sequences are far from the transcriptional start point (tsp) of their target genes. Some SARP-binding sites contain three discernible heptamers, such as those upstream of actII-ORF1 and actVI-ORF1 in S. coelicolor (29), claR, cefD, and cefF in S. clavuligerus (216), vlmJ and vlmA-vlmH in Streptomyces viridifaciens (217), dnrD in S. peucetius (218), fdmD in S. griseus (219), pimM in S. natalensis (220), and so on. Other sites contain only two obvious heptamers, such as sanO-sanN in S. ansochromogenes (221), polB and polC in Streptomyces cacaoi subsp.…”

“…Like ActII-ORF4 and RedD, many other SARPs are less than 300 residues long, and some of these have been shown, mainly by mutant phenotypes, to function as pathway-specific activators, including CcaR (cephamycin-clavulanic acid supercluster in S. clavuligerus) (216), DnrI (daunorubicin biosynthesis in S. peucetius) (218,224), Aur1PR2 and Aur1PR3 (auricin biosynthesis in S. aureofaciens) (225), TylS and TylT (which activate the transcription of the tylosin CSR gene tylR in S. fradiae) (226), FdmR1 (fredericamycin biosynthesis in S. griseus) (219), ThnU (cephamycin C biosynthesis in Streptomyces cattleya) (227), SrrY and SrrZ (lankamycin biosynthetic regulatory cascade in Streptomyces rochei) (228), and VlmI (valanimycin biosynthesis in Streptomyces viridifaciens) (217). These ActII-ORF4-like SARPs are referred to here as "small SARPs."…”

Genetic engineering of an industrial strain of Streptomyces clavuligerusfor further enhancement of clavulanic acid production

“…In addition, CcaR binding to the promoter of ceaS2 positively affected cas2 expression (Santamarta et al, 2011;Paradkar, 2013). Medema et al (2011a) conducted a genome-wide gene expression analysis of a randomly mutated industrial strain of S. clavuligerus compared to the wild type and showed that the expression of cas2, claR, and ccaR increased by 5.47-fold, 2.23-fold, and 5.6-fold, respectively, in the industrial one.…”

Induction of Holomycin Production and Complex Metabolic Changes by the argR Mutation in Streptomyces clavuligerus NP1

“…Both the CA supercluster and the clavam cluster lie on the chromosome, whereas the paralog cluster is located on the pSCL4 plasmid (37). The complexity of CA-clavam biosynthesis is also reflected at the level of regulation, and at least 6 regulatory genes (ccaR [45,52], claR [43], cvm7P [59], and orf21 to orf23 [25,55]) were identified among the three gene clusters. Intricate cross-regulation between the arginine and CA (12, 51), cephamycin C and CA (44,45), and CA and holomycin (11) pathways were also reported.…”