Multi-omics Study of Planobispora rosea, Producer of the Thiopeptide Antibiotic GE2270A.

Planobispora rosea is the natural producer of the potent thiopeptide antibiotic GE2270A. Here, we present the results of a metabolomics and transcriptomics analysis of P. rosea during production of GE2270A. The data generated provides useful insights into the biology of this genetically intractable bacterium. We characterize the details of the shutdown of protein biosynthesis and the respiratory chain associated with the end of the exponential growth phase. We also provide the first description of the phosphate regulon in P. rosea. Based on the transcriptomics data, we show that both phosphate and iron are limiting P. rosea growth in our experimental conditions. Additionally, we identified and validated a new biosynthetic gene cluster associated with the production of the siderophores benarthin and dibenarthin in P. rosea. Together, the metabolomics and transcriptomics data are used to inform and refine the very first genome-scale metabolic model for P. rosea, which will be a valuable framework for the interpretation of future studies of the biology of this interesting but poorly characterized species. IMPORTANCE Planobispora rosea is a genetically intractable bacterium used for the production of GE2270A on an industrial scale. GE2270A is a potent thiopeptide antibiotic currently used as a precursor for the synthesis of two compounds under clinical studies for the treatment of Clostridium difficile infection and acne. Here, we present the very first systematic multi-omics investigation of this important bacterium, which provides a much-needed detailed picture of the dynamics of metabolism of P. rosea while producing GE2270A.

Coupled Transcriptomics for Differential Expression Analysis and Determination of Transcription Start Sites: Design and Bioinformatics.

The term coupled transcriptomics is coined to describe a design of an RNA-seq experiment intended for both differential expression analysis and genome-wide determination of the transcription start sites (TSS). The minimal requirements for the first analysis are two experimental conditions with at least two biological replicates enabling statistical tests. The second analysis involves the bioinformatics comparison of the data generated from a control RNA-seq library with another library enriched in primary transcripts using Terminator™ 5'-phosphate-dependent exonuclease, in an experiment denominated differential RNA-seq (dRNA-seq). Usually, dRNA-seq is carried out with specific protocols for library construction, different of those used for common differential expression analysis. Our experimental design allows to use the same data for both analyses, reducing the number of libraries to be generated and sequenced. This is a guide for designing a coupled transcriptomics experiment and for the subsequent bioinformatics procedures. The proposed methods can be applied to the detection and study of small RNA genes.

ArgR of Streptomyces coelicolor Is a Pleiotropic Transcriptional Regulator: Effect on the Transcriptome, Antibiotic Production, and Differentiation in Liquid Cultures.

ArgR is a well-characterized transcriptional repressor controlling the expression of arginine and pyrimidine biosynthetic genes in bacteria. In this work, the biological role of Streptomyces coelicolor ArgR was analyzed by comparing the transcriptomes of S. coelicolor ΔargR and its parental strain, S. coelicolor M145, at five different times over a 66-h period. The effect of S. coelicolor ArgR was more widespread than that of the orthologous protein of Escherichia coli, affecting the expression of 1544 genes along the microarray time series. This S. coelicolor regulator repressed the expression of arginine and pyrimidine biosynthetic genes, but it also modulated the expression of genes not previously described to be regulated by ArgR: genes involved in nitrogen metabolism and nitrate utilization; the act, red, and cpk genes for antibiotic production; genes for the synthesis of the osmotic stress protector ectoine; genes related to hydrophobic cover formation and sporulation (chaplins, rodlins, ramR, and whi genes); all the cwg genes encoding proteins for glycan cell wall biosynthesis; and genes involved in gas vesicle formation. Many of these genes contain ARG boxes for ArgR binding. ArgR binding to seven new ARG boxes, located upstream or near the ectA-ectB, afsS, afsR, glnR, and redH genes, was tested by DNA band-shift assays. These data and those of previously assayed fragments permitted the construction of an improved model of the ArgR binding site. Interestingly, the overexpression of sporulation genes observed in the ΔargR mutant in our culture conditions correlated with a sporulation-like process, an uncommon phenotype.

Genome Sequencing of Steroid Producing Bacteria Using Ion Torrent Technology and a Reference Genome.

The Next-Generation Sequencing technology has enormously eased the bacterial genome sequencing and several tens of thousands of genomes have been sequenced during the last 10 years. Most of the genome projects are published as draft version, however, for certain applications the complete genome sequence is required.In this chapter, we describe the strategy that allowed the complete genome sequencing of Mycobacterium neoaurum NRRL B-3805, an industrial strain exploited for steroid production, using Ion Torrent sequencing reads and the genome of a close strain as the reference. This protocol can be applied to analyze the genetic variations between closely related strains; for example, to elucidate the point mutations between a parental strain and a random mutagenesis-derived mutant.

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.

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.

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 Delta relA strains.

Connecting primary and secondary metabolism: AreB, an IclR-like protein, binds the ARE(ccaR) 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. clavuligerusDeltaareB, 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. clavuligerusDeltaareB, because AreB binds the areB-leuCD intergenic region acting as a positive modulator. Clavulanic acid and cephamycin C production are improved in the DeltaareB mutant although no drastic difference in ccaR expression was observed. Pure recombinant AreB protein does not bind the ARE(ccaR) 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 ARE(ccaR) sequence is not observed when filtered extracts are obtained from the DeltaareB mutant, suggesting that biosynthesis of the small-molecular-weight effector is also controlled by AreB.

Characterization of a two-gene operon epeRA involved in multidrug resistance in Streptomyces clavuligerus.

Two genes, epeR and epeA, are located downstream of argH in the Streptomyces clavuligerus genome. EpeR belongs to the TetR family of transcriptional regulators. It is homologous to PqrA of Streptomyces coelicolor (74.3% identity) and to NfxB of Pseudomonas aeruginosa (30.9% identity). EpeA encodes a protein with 14 transmembrane spanning domains (TMS) of the major facilitator superfamily. It shares 68.9% identity to PqrB of S. coelicolor and 46.5% identity to LfrA, conferring resistance to fluoroquinolones in Mycobacterium smegmatis. Disruption of epeR results in a S. clavuligerus epeR::aph mutant which shows increased resistance to ethidium bromide and proflavine (16- and 32-fold higher than the wild type). Taking into consideration the sensitivity to drugs of different transformants carrying functional copies of either epeR or epeA, it might be concluded that both genes appear to be co-transcribed, with epeR encoding a regulatory protein which controls the expression of epeA.

Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces.

Bacteria in the genus Streptomyces are major producers of antibiotics and other pharmacologically active compounds. Genetic and physiological manipulations of these bacteria are important for new drug discovery and production development. An essential part of any 'genetic toolkit' is the availability of regulatable promoters. We have adapted the tetracycline (Tc) repressor/operator (TetR/tetO) regulatable system from transposon Tn10 for use in Streptomyces. The synthetic Tc controllable promoter (tcp), tcp830, was active in a wide range of Streptomyces species, and varying levels of induction were observed after the addition of 1-100 ng/ml of anhydrotetracycline (aTc). Streptomyces coelicolor contained an innate Tc-controllable promoter regulated by a TetR homologue (SCO0253). Both natural and synthetic promoters were active and inducible throughout growth. Using the luxAB genes expressing luciferase as a reporter system, we showed that induction factors of up to 270 could be obtained for tcp830. The effect of inducers on the growth of S.coelicolor was determined; addition of aTc at concentrations where induction is optimal, i.e. 0.1-1 microg/ml, ranged from no effect on growth rate to a small increase in the lag period compared with cultures with no inducer.

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 (ARE(ccaR)). This sequence is [corrected] located upstream of the ccaR gene, encoding [corrected] 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 (ARE(brp)) 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 Deltabrp::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 Deltabrp::neo1 lacked a high-mobility band-shift due to Brp but still showed a [corrected] 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 C [corrected] biosynthesis by its action on ccaR gene expression.

Two oligopeptide-permease-encoding genes in the clavulanic acid cluster of Streptomyces clavuligerus are essential for production of the beta-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.

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.