Irene Santamarta

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.

Regulatory mechanisms controlling antibiotic production in Streptomyces clavuligerus

Streptomyces clavuligerus produces a large array of natural compounds with antibiotic, antitumor, β-lactamase inhibition or inmunomodulating activities. The production of cephamycin C, clavulanic acid and other compounds with a clavam structure has been studied for many years. A network of regulatory mechanisms is present in S. clavuligerus to control the formation of different compounds by pathway-specific regulators or pleiotropic regulators. The possible existence of a γ-butyrolactone signaling system in this streptomycete is emerging. In addition, S. clavuligerus possesses a stringent control mechanism somehow different from those previously reported in other Streptomyces species.

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.

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.

Transcriptional analysis and proteomics of the holomycin gene cluster in overproducer mutants of Streptomyces clavuligerus.

Expression of the holomycin biosynthesis genes (hlm) has been studied in the wild type strain Streptomyces clavuligerus ATCC 27064 and holomycin overproducer mutants. RT-PCR transcription analysis of S. clavuligerus oppA2::aph showed a higher transcription of the hlmA, B, C, D, E, F, G, H, I and hlmL genes, a slightly lower expression for hlmK and no significant differences for the transcription of the two putative regulatory genes, hlmM and hlmJ, in relation to the wild type strain. Accordingly, protein spots corresponding to HlmD, HlmF and HlmG, which were barely detectable in the wild type strain, were present in high amounts in the holomycin overproducer S. clavuligerus oppA2::aph proteome. Transcription start point analysis of the hlm genes revealed that the annotated sequences in the databases for several hlm genes were incorrect. The hlm cluster was introduced into Streptomyces coelicolor M1154 and holomycin production by S. coelicolor M1154 [pVR-hol1] was validated by bioassays and confirmed by HPLC analysis and mass spectrometry. Heterologous holomycin production by the S. coelicolor transformant is 500-fold lower than in S. clavuligerus oppA2::aph. The transformant S. coelicolor M1154 [pVR-hol1] shows holomycin sensitivity to 100 μg/ml, similar to that of the parental S. coelicolor M1154 strain, suggesting that heterologous expression in S. coelicolor might be toxic due to the lack of an holomycin resistance gene in this host strain.

A rhodanese-like protein is highly overrepresented in the mutant S. clavuligerus oppA2::aph: effect on holomycin and other secondary metabolites production

A protein highly overrepresented in the proteome of Streptomyces clavuligerus oppA2::aph was characterized by MS/MS as a rhodanese‐like enzyme. The rhlA gene, encoding this protein, was deleted from strains S. clavuligerus ATCC 27064 and S. clavuligerus oppA2::aph to characterized the RhlA enzyme activity, growth on different sulfur sources and antibiotic production by the mutants. Whereas total thiosulfate sulfurtransferase activity in cell extracts was not affected by the rhlA deletion, growth, cephamycin C and clavulanic acid production were impaired in the rhlA mutants. Holomycin production was drastically reduced (66–90%) in the rhlA mutants even when using S. clavuligerusΔrhlA pregrown cells, suggesting that this enzyme might be involved in the formation of the cysteine precursor for this sulfur‐containing antibiotic. While growth on thiosulfate as the sole sulfur source was particularly low the volumetric and specific antibiotic production of the three antibiotics increased in all the strains in the presence of thiosulfate. This stimulatory effect of thiosulfate on antibiotic production was confirmed by addition of thiosulfate to pre‐grown cells and appears to be a general effect of thiosulfate on oxidative stress as was also evident in the production of staurosporin by S. clavuligerus.

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.