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1.
Curr Microbiol ; 76(9): 1045-1054, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31214822

RESUMO

ATP-dependent Lon protease plays important roles in different physiological processes, including cellular differentiation of the bacteria and is a part of an important stress response regulon (HspR/HAIR). In Streptomyces, biosynthesis of secondary metabolites starts with cellular differentiation and stress is one of the factor that affect metabolite production. To clarify the effect of Lon protease on secondary metabolite production, we constructed a recombinant strain of Streptomyces coelicolor A3(2) that has one extra copy of lon gene with its own promoter and transcriptional terminator in its genome. Expression of lon gene in the recombinant strain was determined by quantitative real time (RT-qPCR). Actinorhodin and undecylprodigiosin production of the recombinant cell was measured in liquid R2YE and it was found to produce about 34 times more actinorhodin and 9 times more undecylprodigiosin than the wild-type at 168 h of growth. Development of stable Streptomyces strains capable of producing high amounts of secondary metabolites is valuable for biotechnology industry. One extra copy of lon gene is enough to boost antibiotic production by S. coelicolor A3(2) and this change do not cause any metabolic burden in the cell.


Assuntos
Antibacterianos/biossíntese , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Antraquinonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Regulon , Streptomyces coelicolor/crescimento & desenvolvimento
2.
Mar Drugs ; 17(2)2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795576

RESUMO

Aborycin is a ribosomally synthesized member of the type I lasso peptide natural products. In the present study, aborycin was isolated and identified from the deep-sea-derived microbe Streptomyces sp. SCSIO ZS0098. The aborycin biosynthetic gene cluster (abo) was identified on the basis of genome sequence analyses and then heterologously expressed in Streptomyces coelicolor M1152 to effectively produce aborycin. Aborycin generated in this fashion exhibited moderate antibacterial activity against 13 Staphylococcus aureus strains from various sources with minimum inhibitory concentrations MICs = 8.0~128 µg/mL, against Enterococcus faecalis ATCC 29212 with an MIC = 8.0 µg/mL, and against Bacillus thuringiensis with MIC = 2.0 µg/mL. Additionally, aborycin displayed potent antibacterial activity (MIC = 0.5 µg/mL) against the poultry pathogen Enterococcus gallinarum 5F52C. The reported abo cluster clearly has the potential to provide a means of expanding the repertoire of anti-infective type I lasso peptides.


Assuntos
Peptídeos/farmacologia , Streptomyces/genética , Sequência de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Bacillus thuringiensis/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Enterococcus/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/metabolismo , Família Multigênica , Peptídeos/química , Peptídeos/genética , Peptídeos/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Streptomyces/metabolismo , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/metabolismo
3.
Chemistry ; 25(14): 3675-3684, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30600851

RESUMO

Nitric oxide (NO) can function as both a cytotoxin and a signalling molecule. In both cases, reaction with iron-sulfur (Fe-S) cluster proteins plays an important role because Fe-S clusters are reactive towards NO and so are a primary site of general NO-induced damage (toxicity). This sensitivity to nitrosylation is harnessed in the growing group of regulatory proteins that function in sensing of NO via an Fe-S cluster. Although information about the products of cluster nitrosylation is now emerging, detection and identification of intermediates remains a major challenge, due to their transient nature and the difficulty in distinguishing spectroscopically similar iron-NO species. Here we report studies of the NO-sensing Fe-S cluster regulators NsrR and WhiD using non-denaturing mass spectrometry, in which non-covalent interactions between the protein and Fe/S/NO species are preserved. The data provide remarkable insight into the nitrosylation reactions, permitting identification, for the first time, of protein-bound mono-, di- and tetranitrosyl [4Fe-4S] cluster complexes ([4Fe-4S](NO), [4Fe-4S])(NO)2 and [4Fe-4S](NO)4 ) as intermediates along pathways to formation of product Roussin's red ester (RRE) and Roussin's black salt (RBS)-like species. The data allow the nitrosylation mechanisms of NsrR and WhiD to be elucidated and clearly distinguished.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Mycobacterium tuberculosis/metabolismo , Óxido Nítrico/metabolismo , Streptomyces coelicolor/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/química , Proteínas com Ferro-Enxofre/química , Modelos Moleculares , Mycobacterium tuberculosis/química , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Streptomyces coelicolor/química , Fatores de Transcrição/química
4.
Biotechnol J ; 14(4): e1800180, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30525286

RESUMO

Streptomyces coelicolor is a model organism for the Actinobacteria, a phylum known to produce an extensive range of different bioactive compounds that include antibiotics currently used in the clinic. Biosynthetic gene clusters discovered in genomes of other Actinobacteria can be transferred to and expressed in S. coelicolor, making it a factory for heterologous production of secondary metabolites. Genome-scale metabolic reconstructions have successfully been used in several biotechnology applications to facilitate the over-production of target metabolites. Here, the authors present iKS1317, the most comprehensive and accurate reconstructed genome-scale metabolic model (GEM) for S. coelicolor. The model reconstruction is based on previous models, publicly available databases, and published literature and includes 1317 genes, 2119 reactions, and 1581 metabolites. It correctly predicts wild-type growth in 96.5% of the evaluated growth environments and gene knockout predictions in 78.4% when comparing with observed mutant growth phenotypes, with a total accuracy of 83.3%. However, using a minimal nutrient environment for the gene knockout predictions, iKS1317 has an accuracy of 87.1% in predicting mutant growth phenotypes. Furthermore, we used iKS1317 and existing strain design algorithms to suggest robust gene-knockout strategies to increase the production of acetyl-CoA. Since acetyl-CoA is the most important precursor for polyketide antibiotics, the suggested strategies may be implemented in vivo to improve the function of S. coelicolor as a heterologous expression host.


Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Engenharia Metabólica/métodos , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Metaboloma/genética , Modelos Biológicos , Streptomyces coelicolor/genética
5.
Nucleic Acids Res ; 47(2): 621-633, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30371884

RESUMO

HrdB in streptomycetes is a principal sigma factor whose deletion is lethal. This is also the reason why its regulon has not been investigated so far. To overcome experimental obstacles, for investigating the HrdB regulon, we constructed a strain whose HrdB protein was tagged by an HA epitope. ChIP-seq experiment, done in 3 repeats, identified 2137 protein-coding genes organized in 337 operons, 75 small RNAs, 62 tRNAs, 6 rRNAs and 3 miscellaneous RNAs. Subsequent kinetic modeling of regulation of protein-coding genes with HrdB alone and with a complex of HrdB and a transcriptional cofactor RbpA, using gene expression time series, identified 1694 genes that were under their direct control. When using the HrdB-RbpA complex in the model, an increase of the model fidelity was found for 322 genes. Functional analysis revealed that HrdB controls the majority of gene groups essential for the primary metabolism and the vegetative growth. Particularly, almost all ribosomal protein-coding genes were found in the HrdB regulon. Analysis of promoter binding sites revealed binding motif at the -10 region and suggested the possible role of mono- or di-nucleotides upstream of the -10 element.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulon , Fator sigma/metabolismo , Streptomyces coelicolor/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Imunoprecipitação da Cromatina , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes de RNAr , Cinética , Modelos Genéticos , Regiões Promotoras Genéticas , RNA Bacteriano/genética , RNA de Transferência/genética , Análise de Sequência de DNA , Streptomyces coelicolor/metabolismo
6.
Acta Biochim Biophys Sin (Shanghai) ; 51(1): 97-103, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30452545

RESUMO

Natural genetic materials contain many biosynthetic gene clusters encoding potentially valuable natural products, many of which can be used directly without codon optimization or other manipulations. With the development of synthetic biology, several DNA assembly standards have been proposed, conveniently facilitating the reuse of natural materials. Among these standards, the iBrick assembly standard was developed by our laboratory to manipulate large DNA fragments, employing two homing endonucleases. Considering the difficulty of cloning large iBrick parts using conventional endonuclease-mediated restriction and ligation methods, we herein present a new method, known as iCatch, which readily captures biosynthetic gene clusters. As the clusters cloned by iCatch have the prefix and suffix of the iBrick standard, they serve as new iBrick parts and are therefore conducive to further editing and assembly with the iBrick standard. iCatch employs the natural homologous recombination system to flank the region of interest with I-SceI and PI-PspI recognition sites, after which the genome is digested with I-SceI or PI-PspI and the fragments are then self-ligated to clone the target DNA fragments. We used this method to successfully capture the actinorhodin biosynthetic cluster from Streptomyces coelicolor and then heterologously expressed this cluster in a thermophilic Streptomyces strain. We propose that iCatch can be used for the cloning of DNA sequences that are dozens of kilobases in length, facilitating the heterologous expression of microbial natural products. Moreover, this cloning methodology can be a complementary tool for the iBrick standard, especially in applications requiring the manipulation of large DNA fragments.


Assuntos
Clonagem Molecular/métodos , DNA/genética , DNA/metabolismo , Endonucleases/metabolismo , Antraquinonas/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Genes Bacterianos/genética , Recombinação Homóloga , Família Multigênica , Reprodutibilidade dos Testes , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
8.
PLoS One ; 13(11): e0207278, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30440014

RESUMO

Flavonols are a flavonoid subfamily widely distributed in plants, including several ones of great importance in human and animal diet (apple, tomato, broccoli, onion, beans, tea). These polyphenolic nutraceuticals exert potent antimicrobial (membrane potential disruptors), antioxidant (free-radical scavengers), pharmacokinetic (CYP450 modulators), anti-inflammatory (lipoxygenase inhibitors), antiangiogenic (VEGF inhibitors) and antitumor (cyclin inhibitors) activities. Biotechnological production of these nutraceuticals, for example via heterologous biosynthesis in industrial actinomycetes, is favored since in plants these polyphenols appear as inactive glycosylated derivatives, in low concentrations or as part of complex mixtures with other polyphenolic compounds. In this work, we describe the de novo biosynthesis of three important flavonols, myricetin, kaempferol and quercetin, in the industrially relevant actinomycetes Streptomyces coelicolor and S. albus. De novo biosynthesis of kaempferol, myricetin and quercetin in actinomycetes has not been described before.


Assuntos
Suplementos Nutricionais , Flavonoides , Quempferóis , Microrganismos Geneticamente Modificados , Quercetina , Streptomyces coelicolor , Flavonoides/biossíntese , Flavonoides/genética , Quempferóis/biossíntese , Quempferóis/genética , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Quercetina/biossíntese , Quercetina/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
9.
Sci Rep ; 8(1): 16524, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30410115

RESUMO

Coiled-coil domains of intermediate filaments (IF) and prokaryotic IF-like proteins enable oligomerisation and filamentation, and no additional function is ascribed to these coiled-coil domains. However, an IF-like protein from Streptomyces reticuli was reported to display cellulose affinity. We demonstrate that cellulose affinity is an intrinsic property of the IF-like proteins FilP and Scy and the coiled-coil protein DivIVA from the genus Streptomyces. Furthermore, IF-like proteins and DivIVA from other prokaryotic species and metazoan IF display cellulose affinity despite having little sequence homology. Cellulose affinity-based purification is utilised to isolate native FilP protein from the whole cell lysate of S. coelicolor. Moreover, cellulose affinity allowed for the isolation of IF and IF-like protein from the whole cell lysate of C. crescentus and a mouse macrophage cell line. The binding to cellulose is mediated by certain combinations of coiled-coil domains, as demornstrated for FilP and lamin. Fusions of target proteins to cellulose-binding coiled-coil domains allowed for cellulose-based protein purification. The data presented show that cellulose affinity is a novel function of certain coiled-coil domains of IF and IF-like proteins from evolutionary diverse species.


Assuntos
Bactérias/metabolismo , Celulose/metabolismo , Filamentos Intermediários/química , Filamentos Intermediários/metabolismo , Macrófagos/metabolismo , Animais , Bactérias/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Escherichia coli/química , Escherichia coli/metabolismo , Macrófagos/citologia , Espectrometria de Massas , Camundongos , Ligação Proteica , Domínios Proteicos , Homologia de Sequência , Streptomyces coelicolor/química , Streptomyces coelicolor/metabolismo
10.
Microbiol Res ; 217: 14-22, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30384905

RESUMO

Chitin is the second most abundant carbohydrate biopolymer present in soils and is utilized by antibiotic-producing Streptomyces species. Its monomer, N-acetylglucosamine (GlcNAc), regulates the developmental program of the model organism Streptomyces coelicolor. GlcNAc blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. However, it is unclear if the same GlcNAc regulatory profile observed in S. coelicolor applies also to other industrially important Streptomyces species. We report here the negative effect of GlcNAc on differentiation and tacrolimus (FK506) production by Streptomyces tsukubaensis NRRL 18488. Using microarrays technology, we found that GlcNAc represses the transcription of fkbN, encoding the main transcriptional activator of the tacrolimus biosynthetic cluster, and of ppt1, encoding a phosphopantheteinyltransferase involved in tacrolimus biosynthesis. On the contrary, GlcNAc stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis and pyruvate metabolism. The results obtained support those previously reported for S. coelicolor, but some important differences were observed; for example genes involved in GlcNAc transport and metabolism and genes encoding transcriptional regulators such as crr, ptsI, nagE1, nagE2, nagB, chiA, chiJ, ngcE, dasR or atrA are not significantly induced in S. tsukubaensis by GlcNAc addition. Differences in the GlcNAc transport systems, in the physiology of S. tsukubaensis and S. coelicolor and/or the different composition of the culture media used are likely to be responsible for the discrepancies observed between these species.


Assuntos
Acetilglucosamina/genética , Acetilglucosamina/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Tacrolimo/metabolismo , Transcriptoma/genética , Acetilglucosamina/farmacologia , Proteínas de Bactérias/genética , Sítios de Ligação , Vias Biossintéticas/genética , Carbono/metabolismo , Proteínas de Transporte/genética , Meios de Cultura , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Nitrogênio/metabolismo , Regulon , Alinhamento de Sequência , Streptomyces/efeitos dos fármacos , Streptomyces/crescimento & desenvolvimento , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Fatores de Transcrição/efeitos dos fármacos
11.
Curr Opin Chem Biol ; 47: 134-141, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30447488

RESUMO

The recently discovered futalosine-dependent menaquinone biosynthesis pathway employs radical chemistry for the naphthoquinol core assembly. Mechanistic studies on this pathway have resulted in the discovery of novel reaction motifs. MqnA is the first example of a chorismate dehydratase. MqnE is the first example of a radical SAM enzyme that catalyzes the addition of the 5'-deoxyadenosyl radical to the substrate double bond rather than hydrogen atom abstraction. Both MqnE and MqnC reaction sequences involve radical additions to a benzene ring followed by formation of an aryl radical anion intermediate. The enzymology of the tailoring reactions after dihydroxynaphthoic acid formation remains to be elucidated. Since the futalosine-dependent menaquinone biosynthesis pathway is absent in humans, mechanistic studies on this pathway may promote the development of new antibiotics.


Assuntos
Hidrolases/metabolismo , Nucleosídeos/metabolismo , Vitamina K 2/metabolismo , Ácido Corísmico/metabolismo , Humanos , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/metabolismo
12.
Appl Microbiol Biotechnol ; 102(24): 10623-10643, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30327831

RESUMO

With the rising threat of anti-microbial resistance (AMR), there is an urgent need to enhance efficacy of existing antibiotics. Understanding the myriad mechanisms through which bacteria evade these drugs would be of immense value to designing novel strategies against them. Streptomyces coelicolor A3(2) M145 belongs to the actinomyctes species that are responsible for more than two-thirds of antibiotics. This group of bacteria therefore encodes for various mechanisms that can resist both endogenous and non-endogenous antibiotics. In an earlier study, we had studied the transcriptomic response of these bacteria to ciprofloxacin, when cultured in a minimal media. In this work, we investigate why the minimum inhibitory concentration of the drug increases by fourfold when the bacteria are grown in a nutrient-rich media. Through transcriptomic, biochemical, and microscopic studies, we show that S. coelicolor responds to ciprofloxacin in a concentration-dependent manner. While, sub-inhibitory concentration of the drug primarily causes oxidative stress, the inhibitory concentration of ciprofloxacin evokes a more severe genome-wide response in the cell, which ranges from the familiar upregulation of the SOS response and DNA repair pathways to the widespread alterations in the central metabolism pathway to accommodate the increased needs of nucleotides and other precursors. Further, the upregulation of peptidoglycan synthesis genes, along with microscopy images, suggest alterations in the cell morphology to increase fitness of the bacteria during the antibiotic stress. The data also points to the enhanced efflux activity in cells cultured in rich media that contributes significantly towards reducing intracellular drug concentration and thus promotes survival.


Assuntos
Ciprofloxacino/farmacologia , Meios de Cultura/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Meios de Cultura/química , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Testes de Sensibilidade Microbiana , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Streptomyces coelicolor/metabolismo
13.
PLoS Comput Biol ; 14(10): e1006541, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30335785

RESUMO

RAVEN is a commonly used MATLAB toolbox for genome-scale metabolic model (GEM) reconstruction, curation and constraint-based modelling and simulation. Here we present RAVEN Toolbox 2.0 with major enhancements, including: (i) de novo reconstruction of GEMs based on the MetaCyc pathway database; (ii) a redesigned KEGG-based reconstruction pipeline; (iii) convergence of reconstructions from various sources; (iv) improved performance, usability, and compatibility with the COBRA Toolbox. Capabilities of RAVEN 2.0 are here illustrated through de novo reconstruction of GEMs for the antibiotic-producing bacterium Streptomyces coelicolor. Comparison of the automated de novo reconstructions with the iMK1208 model, a previously published high-quality S. coelicolor GEM, exemplifies that RAVEN 2.0 can capture most of the manually curated model. The generated de novo reconstruction is subsequently used to curate iMK1208 resulting in Sco4, the most comprehensive GEM of S. coelicolor, with increased coverage of both primary and secondary metabolism. This increased coverage allows the use of Sco4 to predict novel genome editing targets for optimized secondary metabolites production. As such, we demonstrate that RAVEN 2.0 can be used not only for de novo GEM reconstruction, but also for curating existing models based on up-to-date databases. Both RAVEN 2.0 and Sco4 are distributed through GitHub to facilitate usage and further development by the community (https://github.com/SysBioChalmers/RAVEN and https://github.com/SysBioChalmers/Streptomyces_coelicolor-GEM).


Assuntos
Biologia Computacional/métodos , Redes e Vias Metabólicas/genética , Software , Streptomyces coelicolor/genética , Simulação por Computador , Bases de Dados Genéticas , Edição de Genes , Modelos Genéticos , Streptomyces coelicolor/metabolismo
14.
J Zhejiang Univ Sci B ; 19(9): 708-717, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30178637

RESUMO

otrA resembles elongation factor G (EF-G) and is considered to be an oxytetracycline (OTC)-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE* promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coelicolor M145 by intergeneric conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M145-OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin (Act) production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the transcriptional level of pathway-specific regulatory gene actII-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.


Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Streptomyces coelicolor/efeitos dos fármacos , Antraquinonas/metabolismo , Farmacorresistência Bacteriana/genética , Streptomyces coelicolor/citologia , Streptomyces coelicolor/metabolismo
15.
Sci Rep ; 8(1): 13686, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30209340

RESUMO

Streptomyces coelicolor is a Gram-positive microorganism often used as a model of physiological and morphological differentiation in streptomycetes, prolific producers of secondary metabolites with important biological activities. In the present study, we analysed Streptomyces coelicolor growth and differentiation in the presence of the hypo-methylating agent 5'-aza-2'-deoxycytidine (5-aza-dC) in order to investigate whether cytosine methylation has a role in differentiation. We found that cytosine demethylation caused a delay in spore germination, aerial mycelium development, sporulation, as well as a massive impairment of actinorhodin production. Thus, we searched for putative DNA methyltransferase genes in the genome and constructed a mutant of the SCO1731 gene. The analysis of the SCO1731::Tn5062 mutant strain demonstrated that inactivation of SCO1731 leads to a strong decrease of cytosine methylation and almost to the same phenotype obtained after 5-aza-dC treatment. Altogether, our data demonstrate that cytosine methylation influences morphological differentiation and actinorhodin production in S. coelicolor and expand our knowledge on this model bacterial system.


Assuntos
Diferenciação Celular/fisiologia , Metiltransferases/metabolismo , Streptomyces coelicolor/metabolismo , Antraquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Micélio/metabolismo , Esporos Bacterianos/metabolismo
16.
Methods Mol Biol ; 1841: 249-260, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30259491

RESUMO

The extension and biological role of Ser/Thr/Tyr phosphorylation in prokaryotes have been only scarcely studied. In this chapter, we describe the state of the art of microbial phosphoproteomics, focusing on protocols used for studying the phosphoproteome of Streptomyces coelicolor, one of the bacteria encoding the largest number of eukaryote-like Ser/Thr/Tyr kinases.


Assuntos
Proteínas de Bactérias/metabolismo , Fosfoproteínas/metabolismo , Proteoma , Proteômica , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida , Fosfoproteínas/isolamento & purificação , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
17.
Methods Enzymol ; 608: 83-95, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30173774

RESUMO

Sesquiterpene synthases catalyze the conversion of farnesyl diphosphate to more than 300 different hydrocarbon and alcohol natural products which often contain multiple fused rings and stereocenters. Recent work has taken advantage of the exquisite stereospecificity of these enzymes to synthesize complex novel sesquiterpenoids using nonnatural substrates. In this chapter, we describe the expression, purification, and use of one such synthase to convert a nonnatural substrate to a novel cyclic ether, thereby expanding the terpenome.


Assuntos
Alquil e Aril Transferases/metabolismo , Microbiologia Industrial/métodos , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Streptomyces coelicolor/enzimologia , Biologia Sintética/métodos , Alquil e Aril Transferases/genética , Biocatálise , Ciclização , Escherichia coli/genética , Éteres/química , Éteres/metabolismo , Vetores Genéticos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
18.
J Nat Prod ; 81(8): 1745-1751, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30106291

RESUMO

Two new staurosporine derivatives, staurosporines M1 and M2 (4 and 5), in addition to five previously reported metabolites (1-3, 6, and 7), were generated by the heterologous expression of engineered spc gene clusters in Streptomyces coelicolor M1146. The structures of these derivatives were determined by a combination of spectroscopic methods and CD measurement. Compounds 1, 2, 4, and 5 showed effective activities against three tumor cell lines (HCT-116, K562, and Huh 7.5), and 3 was active against HCT-116 and K562 cells. In addition, compounds 3 and 5 showed undetectable toxicity up to 100 µM toward the normal hepatic cell line LO2. Based on the IC50 values, their structure and activity relationships are discussed.


Assuntos
Antibióticos Antineoplásicos/síntese química , Estaurosporina/análogos & derivados , Estaurosporina/síntese química , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Humanos , Estrutura Molecular , Família Multigênica/genética , Estaurosporina/farmacologia , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Relação Estrutura-Atividade
19.
Microbes Environ ; 33(3): 272-281, 2018 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-30089751

RESUMO

In the model species Streptomyces coelicolor A3(2), the uptake of chitin-degradation byproducts, mainly N,N'- diacetylchitobiose ([GlcNAc]2) and N-acetylglucosamine (GlcNAc), is performed by the ATP-binding cassette (ABC) transporter DasABC-MsiK and the sugar-phosphotransferase system (PTS), respectively. Studies on the S. coelicolor chromosome have suggested the occurrence of additional uptake systems of GlcNAc-related compounds, including the SCO6005-7 cluster, which is orthologous to the ABC transporter NgcEFG of S. olivaceoviridis. However, despite conserved synteny between the clusters in S. coelicolor and S. olivaceoviridis, homology between them is low, with only 35% of residues being identical between NgcE proteins, suggesting different binding specificities. Isothermal titration calorimetry experiments revealed that recombinant NgcESco interacts with GlcNAc and (GlcNAc)2, with Kd values (1.15 and 1.53 µM, respectively) that were higher than those of NgcE of S. olivaceoviridis (8.3 and 29 nM, respectively). The disruption of ngcESco delayed (GlcNAc)2 consumption, but did not affect GlcNAc consumption ability. The ngcESco-dasA double mutation severely decreased the ability to consume (GlcNAc)2 and abolished the induction of chitinase production in the presence of (GlcNAc)2, but did not affect the GlcNAc consumption rate. The results of these biochemical and reverse genetic analyses indicate that NgcESco acts as a (GlcNAc)2- binding protein of the ABC transporter NgcEFGSco-MsiK. Transcriptional and biochemical analyses of gene regulation demonstrated that the ngcESco gene was slightly induced by GlcNAc, (GlcNAc)2, and chitin, but repressed by DasR. Therefore, a model was proposed for the induction of the chitinolytic system and import of (GlcNAc)2, in which (GlcNAc)2 generated from chitin by chitinase produced leakily, is mainly transported via NgcEFG-MsiK and induces the expression of chitinase genes and dasABCD.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dissacarídeos/metabolismo , Streptomyces coelicolor/metabolismo , Acetilglucosamina/metabolismo , Transporte Biológico , Quitina/metabolismo , Quitinases/metabolismo , Meios de Cultura , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Família Multigênica/genética , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genética
20.
Appl Microbiol Biotechnol ; 102(19): 8437-8446, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30105571

RESUMO

The marine actinomycete genus Salinispora is a remarkably prolific source of structurally diverse and biologically active secondary metabolites. Herein, we select the model organism Salinispora tropica CNB-440 for development as a heterologous host for the expression of biosynthetic gene clusters (BGCs) to complement well-established Streptomyces host strains. In order to create an integratable host with a clean background of secondary metabolism, we replaced three genes (salA-C) essential for salinosporamide biosynthesis with a cassette containing the Streptomyces coelicolor ΦC31 phage attachment site attB to generate the mutant S. tropica CNB-4401 via double-crossover recombination. This mutagenesis not only knocks-in the attachment site attB in the genome of S. tropica CNB-440 but also abolishes production of the salinosporamides, thereby simplifying the strain's chemical background. We validated this new heterologous host with the successful integration and expression of the thiolactomycin BGC that we recently identified in several S. pacifica strains. When compared to the extensively engineered superhost S. coelicolor M1152, the production of thiolactomycins from S. tropica CNB-4401 was approximately 3-fold higher. To the best of our knowledge, this is the first example of using a marine actinomycete as a heterologous host for natural product BGC expression. The established heterologous host may provide a useful platform to accelerate the discovery of novel natural products and engineer biosynthetic pathways.


Assuntos
Produtos Biológicos/metabolismo , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Família Multigênica/genética , Actinobacteria/genética , Sítios de Ligação Microbiológicos/genética , Vias Biossintéticas/genética , Metabolismo Secundário/genética , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Tiofenos/metabolismo
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