Rosario Pérez-Redondo

The claR gene of Streptomyces clavuligerus, encoding a LysR-type regulatory protein controlling clavulanic acid biosynthesis, is linked to the clavulanate-9-aldehyde reductase (car) gene.

Two genes, claR and car, encoding proteins involved in clavulanic acid biosynthesis, have been found in a 2.8-kb BglII-EcoRI DNA fragment of Streptomyces clavuligerus adjacent to the region containing the cephamycin and clavulanic acid biosynthesis gene cluster. claR encoded a protein of 431 amino acids (deduced Mr 47080), that showed a significant degree of homology with several transcriptional activators of the LysR family. The ClaR protein contained two helix-turn-helix (HTH) motifs in the amino and carboxyl terminal regions. The second gene, car, encoded a protein of 247 amino acids (Mr 26629) that showed a strong similarity to oxydoreductases of the SDR family. Twelve amino acids of the amino-terminal region were identical to those previously obtained by Edman degradation of the purified clavulanic-9-aldehyde reductase of S. clavuligerus. Amplification of the claR gene in multicopy plasmids resulted in a threefold increase in clavulanic acid production and in a five- to sixfold increase of alanylclavam biosynthesis, whereas cephamycin production was significantly reduced both in defined and in complex media. By contrast, amplification of the car gene had no significant effect on clavulanic acid and alanylclavam or cephamycin production. Both claR and car are expressed as monocistronic transcripts; the level of transcript declined rapidly after 48h in complex media, but low sustained levels of both transcripts were observed in defined GSPG medium until 96h. claR and car were not significantly expressed in mutants disrupted in the ccaR gene, a regulatory gene that controls positively clavulanic acid and cephamycin biosynthesis. These results indicate that clavulanic acid and cephamycin biosynthesis in S. clavuligerus is controlled by a cascade of regulatory proteins that include CcaR and ClaR.

CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin c-clavulanic acid cluster in Streptomyces clavuligerus

The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.

Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus

Cell‐free extracts from Streptomyces clavuligerus, purified by elution from heparin‐agarose with an ARE‐containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (AREccaR). This sequence, located upstream of the ccaR gene, encodes the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus. Sixty‐one nucleotides upstream of brp another ARE sequence (AREbrp) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp. A brp‐deleted mutant, S. clavuligerus Δbrp::neo1, produced 150–300% clavulanic acid and 120–220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Δbrp::neo1 lacked a high‐mobility band‐shift due to Brp but still showed the slow‐mobility band‐shift observed in the wild‐type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin biosynthesis by its action on ccaR gene expression.

Connecting primary and secondary metabolism: AreB, an IclR‐like protein, binds the AREccaR sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production

A protein binding to the autoregulatory element (ARE) upstream of the regulatory ccaR gene of Streptomyces clavuligerus was isolated previously by DNA affinity binding. The areB gene, encoding this protein, is located upstream and in opposite orientation to the leuCD operon of S. clavuligerus; it encodes a 239‐amino‐acid protein of the IclR family with a helix–turn–helix motif at the N‐terminal region. An areB‐deleted mutant, S. clavuligerusΔareB, has been constructed by gene replacement. This strain requires leucine for optimal growth in defined media. Expression of the leuCD operon is retarded in S. clavuligerusΔareB, because AreB binds the areB‐leuCD intergenic region acting as a positive modulator. Clavulanic acid and cephamycin C production are improved in the ΔareB mutant although no drastic difference in ccaR expression was observed. Pure recombinant AreB protein does not bind the AREccaR sequence (as shown by EMSA) unless filtered extracts from S. clavuligerus ATCC 27064‐containing molecules of Mr lower than 10 kDa are added to the binding reaction. Restoration of binding to the AREccaR sequence is not observed when filtered extracts are obtained from the ΔareB mutant, suggesting that biosynthesis of the small‐molecular‐weight effector is also controlled by AreB.

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

RT‐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.

Draft Genome of Streptomyces tsukubaensis NRRL 18488, the Producer of the Clinically Important Immunosuppressant Tacrolimus (FK506)

The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.

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

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.

ArgR of Streptomyces coelicolor Is a Versatile Regulator

ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSA.

The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene.

Streptomyces clavuligerus ATCC 27064 is unable to use glucose but has genes for a glucose permease (glcP) and a glucose kinase (glkA). Transformation of S. clavuligerus 27064 with the Streptomyces coelicolor glcP1 gene with its own promoter results in a strain able to grow on glucose. The glcP gene of S. clavuligerus encodes a 475 amino acid glucose permease with 12 transmembrane segments. GlcP is a functional protein when expressed from the S. coelicolor glcP1 promoter and complements two different glucose transport-negative Escherichia coli mutants. Transcription studies indicate that the glcP promoter is very weak and does not allow growth on glucose. These results suggest that S. clavuligerus initially contained a functional glucose permease gene, like most other Streptomyces species, and lost the expression of this gene by adaptation to glucose-poor habitats.

Glycerol utilization gene cluster in Streptomyces clavuligerus.

ABSTRACT The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus ΔrelA strains.