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1.
Int J Mol Sci ; 22(18)2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34576306

RESUMO

Streptomycetes are important biotechnological bacteria that produce several clinically bioactive compounds. They have a complex development, including hyphae differentiation and sporulation. Cytosolic copper is a well-known modulator of differentiation and secondary metabolism. The interruption of the Streptomyces coelicolor SCO2730 (copper chaperone, SCO2730::Tn5062 mutant) blocks SCO2730 and reduces SCO2731 (P-type ATPase copper export) expressions, decreasing copper export and increasing cytosolic copper. This mutation triggers the expression of 13 secondary metabolite clusters, including cryptic pathways, during the whole developmental cycle, skipping the vegetative, non-productive stage. As a proof of concept, here, we tested whether the knockdown of the SCO2730/31 orthologue expression can enhance secondary metabolism in streptomycetes. We created a SCO2730/31 consensus antisense mRNA from the sequences of seven key streptomycetes, which helped to increase the cytosolic copper in S. coelicolor, albeit to a lower level than in the SCO2730::Tn5062 mutant. This antisense mRNA affected the production of at least six secondary metabolites (CDA, 2-methylisoborneol, undecylprodigiosin, tetrahydroxynaphtalene, α-actinorhodin, ε-actinorhodin) in the S. coelicolor, and five (phenanthroviridin, alkylresorcinol, chloramphenicol, pikromycin, jadomycin G) in the S. venezuelae; it also helped to alter the S. albus metabolome. The SCO2730/31 consensus antisense mRNA designed here constitutes a tool for the knockdown of SCO2730/31 expression and for the enhancement of Streptomyces' secondary metabolism.


Assuntos
Proteínas de Bactérias/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Chaperonas Moleculares/metabolismo , Metabolismo Secundário , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/genética , Cobre/metabolismo , ATPases Transportadoras de Cobre/genética , Chaperonas Moleculares/genética , Streptomyces coelicolor/genética
2.
Curr Microbiol ; 78(10): 3696-3707, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34426858

RESUMO

We previously reported that the two-component system MacRS regulates morphogenesis and production of the blue-pigmented antibiotic actinorhodin (ACT) in Streptomyces coelicolor. In this study, the role of MacRS was further extended to include control of the production of the red-pigmented antibiotic undecylprodigiosin (RED) and the calcium-dependent antibiotic (CDA), and control of other important cellular activities. Our data indicated that disruption of the MacRS TCS reduced production not only of ACT but also of RED and CDA. RNA-Seq analysis revealed that genes involved in both secondary metabolism and primary metabolism are differentially expressed in the MacRS deletion mutant ΔmacRS. Moreover, we found that genes of the Zur regulon are also markedly downregulated in ΔmacRS, suggesting a role for macRS in zinc homeostasis. In addition to previously identified MacR sites with strong matches to the MacR consensus recognition sequence, a genome-wide search revealed over one hundred less-stringent matches, including potential sites upstream of absR1, crgA, and smeA. Electrophoretic mobility shift assays demonstrated that MacR binds some of these sites in vitro. Although there is no strong MacR site upstream of the ACT regulatory gene actII-orf4 (sco5085), we showed that an engineered MacR site enhanced ACT production, providing an approach for modulating production of useful compounds. Altogether, our work suggests an important role for MacRS in a range of cellular activities in Streptomyces and its potential application in strain engineering.


Assuntos
Streptomyces coelicolor , Antraquinonas , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulon , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
3.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360615

RESUMO

In contrast to Bacillus subtilis, Streptomyces coelicolor A3(2) contains nine homologues of stress response sigma factor SigB with a major role in differentiation and osmotic stress response. The aim of this study was to further characterize these SigB homologues. We previously established a two-plasmid system to identify promoters recognized by sigma factors and used it to identify promoters recognized by the three SigB homologues, SigF, SigG, and SigH from S. coelicolor A3(2). Here, we used this system to identify 14 promoters recognized by SigB. The promoters were verified in vivo in S. coelicolor A3(2) under osmotic stress conditions in sigB and sigH operon mutants, indicating some cross-recognition of these promoters by these two SigB homologues. This two-plasmid system was used to examine the recognition of all identified SigB-, SigF-, SigG-, and SigH-dependent promoters with all nine SigB homologues. The results confirmed this cross-recognition. Almost all 24 investigated promoters were recognized by two or more SigB homologues and data suggested some distinguishing groups of promoters recognized by these sigma factors. However, analysis of the promoters did not reveal any specific sequence characteristics for these recognition groups. All promoters showed high similarity in the -35 and -10 regions. Immunoblot analysis revealed the presence of SigB under osmotic stress conditions and SigH during morphological differentiation. Together with the phenotypic analysis of sigB and sigH operon mutants in S. coelicolor A3(2), the results suggest a dominant role for SigB in the osmotic stress response and a dual role for SigH in the osmotic stress response and morphological differentiation. These data suggest a complex regulation of the osmotic stress response in relation to morphological differentiation in S. coelicolor A3(2).


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras Genéticas , Fator sigma/genética , Streptomyces coelicolor/genética , Transcrição Genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Fator sigma/metabolismo , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/metabolismo
4.
Appl Microbiol Biotechnol ; 105(14-15): 5905-5914, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34287659

RESUMO

In the model actinomycete strain, Streptomyces coelicolor, an orphan histidine kinase (HK) named OhkA (encoded by SCO1596), which belongs to bacterial two-component regulatory systems (TCSs), has been identified as being involved in the regulation of both antibiotic biosynthesis and morphological development. However, its cognate response regulator (RR) remains unknown due to its isolated genetic location on the genome, which impedes the elucidation of the mechanism underlying OhkA-mediated regulation. Here, we identified the orphan RR OrrA (encoded by SCO3008) as the cognate RR of OhkA according to mutant phenotypic changes, transcriptomics analysis, and bacterial two-hybrid experiment. Considering that the partner RR of the orphan HK is also orphan, a library of mutants with in-frame individual deletion of these functionally unknown orphan RR-encoding genes were generated. Through phenotypic analysis, it was found that the ∆orrA mutant exhibited similar phenotypic changes as that of the ∆ohkA mutant, showing increased production of actinorhodin (ACT) and undecylprodigiosin (RED), and pink colony surface. Further transcriptomics analysis showed these two mutants exhibited highly similar transcriptomics profiles. Finally, the direct interaction between OhkA and OrrA was revealed by bacterial two-hybrid system. The identification of the partner RR of OhkA lays a good foundation for an in-depth elucidation of the molecular mechanism underlying OhkA-mediated regulation of development and antibiotic biosynthesis in Streptomyces. KEY POINTS: • OrrA was identified as the partner RR of the orphan histidine kinase OhkA. • The ∆orrA and ∆ohkA mutants showed similar phenotype and transcriptomic profiling. • Specific interaction of OrrA and OhkA was revealed by bacterial two-hybrid system.


Assuntos
Streptomyces coelicolor , Streptomyces , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/genética , Histidina Quinase/metabolismo , Metabolismo Secundário/genética , Streptomyces/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
5.
FEBS Lett ; 595(14): 1914-1919, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34080704

RESUMO

Biological structures with highly curved membranes, such as caveolae and transport vesicles, are essential for signal transduction and membrane trafficking. Although membrane proteins in these structures are subjected to physical stress due to the curvature of the lipid bilayers, the effect of this membrane curvature on protein structure and function remains unclear. In this study, we established an experimental procedure to evaluate membrane curvature-induced structural changes in the prototypical potassium channel KcsA. The effect of a large membrane curvature was estimated using fluorescently labeled KcsA by incorporating it into liposomes with a small diameter (< 30 nm). We found that a large membrane curvature significantly affects the activation gate conformation of the KcsA channel.


Assuntos
Proteínas de Bactérias/química , Lipossomos/química , Fosfatidilcolinas/química , Canais de Potássio/química , Potássio/química , Coloração e Rotulagem/métodos , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quimotripsina/química , Corantes Fluorescentes/química , Expressão Gênica , Transporte de Íons , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Potássio/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Rodaminas/química , Streptomyces coelicolor/genética
6.
Appl Environ Microbiol ; 87(14): e0048021, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33990302

RESUMO

Nitric oxide (NO) is an important signaling molecule in eukaryotic and prokaryotic cells. A previous study revealed an NO synthase-independent NO production metabolic cycle in which the three nitrogen oxides, nitrate (NO3-), nitrite (NO2-), and NO, were generated in the actinobacterium Streptomyces coelicolor A3(2). NO was suggested to act as a signaling molecule, functioning as a hormone that regulates secondary metabolism. Here, we demonstrate the NO-mediated regulation of the production of the blue-pigmented antibiotic actinorhodin (ACT), via the heme-based DevS/R two-component system (TCS). Intracellular NO controls the stabilization or inactivation of DevS, depending on the NO concentration. An electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR analysis revealed the direct binding between DevR and the promoter region of actII-ORF4, resulting in gene expression. Our results indicate that NO regulates the DevS/R TCS, thereby strictly controlling the secondary metabolism of S. coelicolor A3(2). IMPORTANCE Diverse organisms, such as mammals, plants, and bacteria, utilize NO via well-known signal transduction mechanisms. Many useful secondary metabolite-producing bacteria of the Streptomyces genus had been also suggested for the metabolism regulated by endogenously produced NO; however, the regulatory mechanisms remain to be elucidated. In this study, we demonstrated the molecular mechanism by which endogenously produced NO regulates antibiotic production via the DevS/R TCS in S. coelicolor A3(2). NO serves as both a stabilizer and a repressor in the regulation of antibiotic production. This report shows the mechanism by which Streptomyces utilizes endogenously produced NO to modulate its normal life cycle. Moreover, this study implies that studying NO signaling in actinobacteria can help in the development of both clinical strategies against pathogenic actinomycetes and the actinobacterial industries.


Assuntos
Óxido Nítrico/metabolismo , Streptomyces coelicolor/metabolismo , Actinas/genética , Antraquinonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Helminto/genética , Regiões Promotoras Genéticas , Metabolismo Secundário , Streptomyces coelicolor/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
J Microbiol Biotechnol ; 31(5): 756-763, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-33820885

RESUMO

Agarose is a linear polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG). It is a major component of the red algal cell wall and is gaining attention as an abundant marine biomass. However, the inability to ferment AHG is considered an obstacle in the large-scale use of agarose and could be addressed by understanding AHG catabolism in agarolytic microorganisms. Since AHG catabolism was uniquely confirmed in Vibrio sp. EJY3, a gram-negative marine bacterial species, we investigated AHG metabolism in Streptomyces coelicolor A3(2), an agarolytic gram-positive soil bacterium. Based on genomic data, the SCO3486 protein (492 amino acids) and the SCO3480 protein (361 amino acids) of S. coelicolor A3(2) showed identity with H2IFE7.1 (40% identity) encoding AHG dehydrogenase and H2IFX0.1 (42% identity) encoding 3,6-anhydro-L-galactonate cycloisomerase, respectively, which are involved in the initial catabolism of AHG in Vibrio sp. EJY3. Thin layer chromatography and mass spectrometry of the bioconversion products catalyzed by recombinant SCO3486 and SCO3480 proteins, revealed that SCO3486 is an AHG dehydrogenase that oxidizes AHG to 3,6-anhydro-L-galactonate, and SCO3480 is a 3,6-anhydro-L-galactonate cycloisomerase that converts 3,6-anhydro-L-galactonate to 2-keto-3-deoxygalactonate. SCO3486 showed maximum activity at pH 6.0 at 50°C, increased activity in the presence of iron ions, and activity against various aldehyde substrates, which is quite distinct from AHG-specific H2IFE7.1 in Vibrio sp. EJY3. Therefore, the catabolic pathway of AHG seems to be similar in most agar-degrading microorganisms, but the enzymes involved appear to be very diverse.


Assuntos
Galactose/análogos & derivados , NADPH Desidrogenase/metabolismo , Racemases e Epimerases/metabolismo , Streptomyces coelicolor/enzimologia , Aldeídos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Galactose/metabolismo , Concentração de Íons de Hidrogênio , Ferro , Redes e Vias Metabólicas , NADPH Desidrogenase/genética , Racemases e Epimerases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodófitas/química , Sefarose/metabolismo , Streptomyces coelicolor/metabolismo , Especificidade por Substrato , Temperatura
8.
Nature ; 590(7846): 463-467, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33536618

RESUMO

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.


Assuntos
Antibacterianos/biossíntese , Furanos/metabolismo , Streptomyces coelicolor/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Furanos/química , Hormônios/química , Hormônios/classificação , Hormônios/metabolismo , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/classificação , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Transdução de Sinais , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Relação Estrutura-Atividade
9.
J Biosci Bioeng ; 131(5): 525-536, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33549493

RESUMO

The previously reported Streptomyces coelicolor M1146 is commonly used as a host strain for engineering of secondary metabolite production. In this study, absolute quantification of intracellular and extracellular metabolites of M1146 was performed in mid-log phase and stationary phase to observe major metabolites and the changes that occurred during growth. Decreased levels of central carbon metabolites (glycolysis, TCA cycle, and pentose phosphate pathway) and increased levels of amino acids were observed in stationary phase compared to mid-log phase. Furthermore, comparative metabolome analyses of M1146 upon expression of the actinorhodin biosynthetic gene cluster (M1146+ACT), a point mutation on the rpoB gene encoding RNA polymerase beta-subunit (M1152), and both expression of actinorhodin biosynthetic gene cluster and a rpoB point mutation (M1152+ACT) were performed. M1146+ACT showed higher levels of important cofactors, such as ATP, NADPH, and FMN while M1152 led to higher levels of intracellular S-adenosyl-methionine, acyl-CoAs, and extracellular nucleosides compared to M1146. M1152+ACT exhibited the highest levels of actinorhodin with elevated bases, nucleosides, and nucleotides, such as intracellular PRPP (phosphoribosyl phosphate), ATP, along with extracellular inosine, uridine, and guanine compared to the other three strains, which were considered to be combined effects of actinorhodin gene cluster expression and a rpoB point mutation. Metabolites analysis by means of absolute quantification demonstrated changes in precursors of secondary metabolites before and after phosphate depletion in M1146. Comparative metabolome analysis provided further insights into the effects of actinorhodin gene cluster expression along with a rpoB point mutation on the metabolome of S. coelicolor.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Metaboloma , Família Multigênica/genética , Mutação Puntual , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Antraquinonas/metabolismo
10.
ACS Synth Biol ; 10(2): 243-251, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33471506

RESUMO

Synthetic biology-based approaches have been employed to generate advanced natural product (NP) pathway intermediates to overcome obstacles in NP drug discovery and production. Type II polyketides (PK-IIs) comprise a major subclass of NPs that provide attractive structures for antimicrobial and anticancer drug development. Herein, we have assembled five biosynthetic pathways using a generalized operon design strategy in Streptomyces coelicolor M1152 to allow comparative analysis of metabolite production in an improved heterologous host. The work resulted in production of four distinct PK-II core structures, namely benzoisochromanequinone, angucycline, tetracenomycin, and pentangular compounds, which serve as precursors to diverse pharmaceutically important NPs. Our bottom-up design strategy provided evidence that the biosynthetic pathway of BE-7585A proceeds via an angucycline core structure, instead of rearrangement of an anthracycline aglycone, and led to the discovery of a novel 26-carbon pentangular polyketide. The synthetic biology platform presented here provides an opportunity for further controlled production of diverse PK-IIs in a heterologous host.


Assuntos
Produtos Biológicos/metabolismo , Descoberta de Drogas/métodos , Policetídeos/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Genes Bacterianos , Engenharia Metabólica/métodos , Naftacenos/metabolismo , Plasmídeos/genética , Tioaçúcares/metabolismo
11.
Biochemistry ; 60(5): 365-372, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33482062

RESUMO

LnmK stereospecifically accepts (2R)-methylmalonyl-CoA, generating propionyl-S-acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure-function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2R)-methylmalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl-O-LnmK acyl-enzyme intermediate. Due to the inherent reactivity of LnmK and methylmalonyl-CoA, a substrate-bound structure could not be obtained. To gain insight into substrate specificity, stereospecificity, and catalytic mechanism, we determined the structures of LnmK with bound substrate analogues that bear malonyl-thioester isosteres where the carboxylate is represented by a nitro or sulfonate group. The nitro-bearing malonyl-thioester isosteres bind in the nitronate form, with specific hydrogen bonds that allow modeling of the (2R)-methylmalonyl-CoA substrate and rationalization of stereospecificity. The sulfonate isosteres bind in multiple conformations, suggesting the large active site of LnmK allows multiple binding modes. Considering the smaller malonyl group has more conformational freedom than the methylmalonyl group, we hypothesized the active site can entropically screen against catalysis with the smaller malonyl-CoA substrate. Indeed, our kinetic analysis reveals malonyl-CoA is accepted at 1% of the rate of methylmalonyl-CoA. This study represents another example of how our nitro- and sulfonate-bearing methylmalonyl-thioester isosteres are of use for elucidating enzyme-substrate binding interactions and revealing insights into catalytic mechanism. Synthesis of a larger panel of analogues presents an opportunity to study enzymes with complicated structure-function relationships such as acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases, and ß-ketoacylsynthases.


Assuntos
Aciltransferases/química , Carboxiliases/química , Proteína de Transporte de Acila/metabolismo , Acil Coenzima A/química , Carbono-Carbono Ligases/química , Catálise , Domínio Catalítico , Malonil Coenzima A/metabolismo , Streptomyces/metabolismo , Streptomyces coelicolor/metabolismo , Especificidade por Substrato
12.
Appl Environ Microbiol ; 87(7)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33483304

RESUMO

Overexpression of efflux pumps is one of the major determinants of resistance in bacteria. Streptomyces species harbor a large array of efflux pumps that are transcriptionally silenced under laboratory conditions. However, their dissemination results in multidrug resistance in different clinical pathogens. In this study, we have identified an efflux pump from Streptomyces coelicolor, SCO4121, belonging to the major facilitator superfamily (MFS) family of transporters and characterized its role in antibiotic resistance. SCO4121 provided resistance to multiple dissimilar drugs upon overexpression in both native and heterologous hosts. Further, deletion of SCO4121 resulted in increased sensitivity toward ciprofloxacin and chloramphenicol, suggesting the pump to be a major transporter of these substrates. Apart from providing multidrug resistance, SCO4121 imparted increased tolerance against the strong oxidant HOCl. In wild-type Streptomyces coelicolor cells, these drugs were found to transcriptionally regulate the pump in a concentration-dependent manner. Additionally, we identified SCO4122, a MarR regulator that positively regulates SCO4121 in response to various drugs and the oxidant HOCl. Thus, through these studies we present the multiple roles of SCO4121 in S. coelicolor and highlight the intricate mechanisms via which it is regulated in response to antibiotics and oxidative stress.IMPORTANCE One of the key mechanisms of drug resistance in bacteria is overexpression of efflux pumps. Streptomyces species are a reservoir of a large number of efflux pumps, potentially to provide resistance to both endogenous and nonendogenous antibiotics. While many of these pumps are not expressed under standard laboratory conditions, they result in resistance to multiple drugs when spread to other bacterial pathogens through horizontal gene transfer. In this study, we have identified a widely conserved efflux pump SCO4121 from Streptomyces coelicolor with roles in both multidrug resistance and oxidative stress tolerance. We also report the presence of an adjacent MarR regulator, SCO4122, which positively regulates SCO4121 in the presence of diverse substrates in a redox-responsive manner. This study highlights that soil bacteria such as Streptomyces can reveal novel mechanisms of antibiotic resistance that may potentially emerge in clinically important bacteria.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Membrana Transportadoras/genética , Estresse Oxidativo/genética , Streptomyces coelicolor/genética , Proteínas de Bactérias/metabolismo , Cloranfenicol/farmacologia , Ciprofloxacina/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/metabolismo
13.
Appl Microbiol Biotechnol ; 104(23): 10075-10089, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33057789

RESUMO

Although the genome of the Streptomyces model strain S. coelicolor was sequenced nearly two decades ago, the function of many annotated genes has not been verified, including that of gene sco1979, which was predicted to encode a transcriptional regulator of the xenobiotic response element (XRE) family. In this study, we showed that SCO1979 represses its own transcription and that deletion of sco1979 from S. coelicolor markedly enhanced production of three antibiotics, which are actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA), suggesting that SCO1979 represses their biosynthesis. We demonstrated that transcription of genes in the ACT, RED, and CDA pathways was generally increased in the mutant strain Δ1979 compared with levels in the wild-type strain M145. Additionally, purified recombinant SCO1979 interacted with DNA sequences upstream of sco1979 and actII-orf4, redZ, and cdaR, the pathway-specific regulators for the three pathways, implying that SCO1979 potentially regulates the ACT, RED, and CDA pathways via their specific regulators. In addition, disruption of sco1979 led to the notably delayed formation of aerial mycelium and spores, and consistent with this, transcription of genes associated with aerial hyphae and spore formation, such as chp and rdl, and ram, was reduced in Δ1979, implying the involvement of SCO1979 in cellular development control as well. In summary, our findings demonstrated that SCO1979 is a pleiotropic regulator with roles in both secondary metabolism and morphological development in S. coelicolor. KEY POINTS: • SCO1979 is a novel Streptomyces regulator of the XRE family. • SCO1979 regulates its own transcription. • SCO1979 regulates antibiotic production and cellular development.


Assuntos
Streptomyces coelicolor , Animais , Antraquinonas , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Masculino , Elementos de Resposta , Ovinos , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Xenobióticos
14.
Mar Drugs ; 18(9)2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867397

RESUMO

The actinomycete strain Streptomyces coelicolor LY001 was purified from the sponge Callyspongia siphonella. Fractionation of the antimicrobial extract of the culture of the actinomycete afforded three new natural chlorinated derivatives of 3-phenylpropanoic acid, 3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid (1), 3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid methyl ester (2), and 3-(3-chloro-4-hydroxyphenyl)propanoic acid (3), together with 3-phenylpropanoic acid (4), E-cinnamic acid (5), and the diketopiperazine alkaloids cyclo(l-Phe-trans-4-OH-l-Pro) (6) and cyclo(l-Phe-cis-4-OH-d-Pro) (7) were isolated. Interpretation of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) data of 1-7 supported their assignments. Compounds 1-3 are first candidates of the natural chlorinated phenylpropanoic acid derivatives. The production of the chlorinated derivatives of 3-phenylpropionic acid (1-3) by S. coelicolor provides insight into the biosynthetic capabilities of the marine-derived actinomycetes. Compounds 1-3 demonstrated significant and selective activities towards Escherichia. coli and Staphylococcus aureus, while Candida albicans displayed more sensitivity towards compounds 6 and 7, suggesting a selectivity effect of these compounds against C. albicans.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Callyspongia/microbiologia , Fenilpropionatos/farmacologia , Streptomyces coelicolor/metabolismo , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antifúngicos/química , Antifúngicos/isolamento & purificação , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Oceano Índico , Testes de Sensibilidade Microbiana , Estrutura Molecular , Fenilpropionatos/química , Fenilpropionatos/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Relação Estrutura-Atividade
15.
Appl Environ Microbiol ; 86(20)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32801172

RESUMO

Regulation of antibiotic production by Streptomyces is complex. We report that the response regulator MtrA is a master regulator for antibiotic production in Streptomyces Deletion of MtrA altered production of actinorhodin, undecylprodigiosin, calcium-dependent antibiotic, and the yellow-pigmented type I polyketide and resulted in altered expression of the corresponding gene clusters in S. coelicolor Integrated in vitro and in vivo analyses identified MtrA binding sites upstream of cdaR, actII-orf4, and redZ and between cpkA and cpkD MtrA disruption also led to marked changes in chloramphenicol and jadomycin production and in transcription of their biosynthetic gene clusters (cml and jad, respectively) in S. venezuelae, and MtrA sites were identified within cml and jad MtrA also recognized predicted sites within the avermectin and oligomycin pathways in S. avermitilis and in the validamycin gene cluster of S. hygroscopicus The regulator GlnR competed for several MtrA sites and impacted production of some antibiotics, but its effects were generally less dramatic than those of MtrA. Additional potential MtrA sites were identified in a range of other antibiotic biosynthetic gene clusters in Streptomyces species and other actinobacteria. Overall, our study suggests a universal role for MtrA in antibiotic production in Streptomyces and potentially other actinobacteria.IMPORTANCE In natural environments, the ability to produce antibiotics helps the producing host to compete with surrounding microbes. In Streptomyces, increasing evidence suggests that the regulation of antibiotic production is complex, involving multiple regulatory factors. The regulatory factor MtrA is known to have additional roles beyond controlling development, and using bioassays, transcriptional studies, and DNA-binding assays, our study identified MtrA recognition sequences within multiple antibiotic pathways and indicated that MtrA directly controls the production of multiple antibiotics. Our analyses further suggest that this role of MtrA is evolutionarily conserved in Streptomyces species, as well as in other actinobacterial species, and also suggest that MtrA is a major regulatory factor in antibiotic production and in the survival of actinobacteria in nature.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Streptomyces coelicolor/genética , Streptomyces/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Genes Bacterianos/genética , Família Multigênica/genética , Streptomyces/metabolismo , Streptomyces coelicolor/metabolismo
16.
PLoS Comput Biol ; 16(7): e1008039, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32649676

RESUMO

Antibiotic production is coordinated in the Streptomyces coelicolor population through the use of diffusible signaling molecules of the γ-butyrolactone (GBL) family. The GBL regulatory system involves a small, and not completely defined two-gene network which governs a potentially bi-stable switch between the "on" and "off" states of antibiotic production. The use of this circuit as a tool for synthetic biology has been hampered by a lack of mechanistic understanding of its functionality. We here present the creation and analysis of a versatile and adaptable ensemble model of the Streptomyces GBL system (detailed information on all model mechanisms and parameters is documented in http://www.systemsbiology.ls.manchester.ac.uk/wiki/index.php/Main_Page). We use the model to explore a range of previously proposed mechanistic hypotheses, including transcriptional interference, antisense RNA interactions between the mRNAs of the two genes, and various alternative regulatory activities. Our results suggest that transcriptional interference alone is not sufficient to explain the system's behavior. Instead, antisense RNA interactions seem to be the system's driving force, combined with an aggressive scbR promoter. The computational model can be used to further challenge and refine our understanding of the system's activity and guide future experimentation.


Assuntos
4-Butirolactona/metabolismo , Streptomyces coelicolor/metabolismo , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Simulação por Computador , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Regiões Promotoras Genéticas , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , Streptomyces coelicolor/genética , Biologia Sintética
17.
Gene ; 755: 144883, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32565321

RESUMO

The anti-anti-sigma factor BldG has a pleiotropic function in Streptomyces coelicolor A3(2), regulating both morphological and physiological differentiation. Together with the anti-sigma factor UshX, it participates in a partner-switching activation of the sigma factor σH, which has a dual role in the osmotic stress response and morphological differentiation in S. coelicolor A3(2). In addition to UshX, BldG also interacts with the anti-sigma factor ApgA, although no target sigma factor has yet been identified. However, neither UshX nor ApgA phosphorylates BldG. This phosphorylation is provided by the anti-sigma factor RsfA, which is specific for the late developmental sigma factor σF. However, BldG is phosphorylated in the rsfA mutant, suggesting that some other anti-sigma factors containing HATPase_c kinase domain are capable to phosphorylate BldG in vivo. Bacterial two-hybrid system (BACTH) was therefore used to investigate the interactions of all suitable anti-sigma factors of S. coelicolor A3(2) with BldG. At least 15 anti-sigma factors were found to interact with BldG. These interactions were confirmed by native PAGE. In addition to RsfA, BldG is specifically phosphorylated on the conserved phosphorylation Ser57 residue by at least seven additional anti-sigma factors. However, only one of them, SCO7328, has been shown to interact with three sigma factors, σG, σK and σM. A mutant with deleted SCO7328 gene was prepared in S. coelicolor A3(2), however, no specific function of SCO7328 in growth, differentiation or stress response could be attributed to this anti-sigma factor. These results suggest that BldG is specifically phosphorylated by several anti-sigma factors and it plays a role in the regulation of several sigma factors in S. coelicolor A3(2). This suggests a complex regulation of the stress response and differentiation in S. coelicolor A3(2) through this pleiotropic anti-sigma factor.


Assuntos
Fator sigma/genética , Streptomyces coelicolor/imunologia , Streptomyces coelicolor/metabolismo , Sequência de Aminoácidos/genética , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases/genética , Regulação Bacteriana da Expressão Gênica/genética , Pleiotropia Genética/genética , Fosforilação/genética , Fosfotransferases/metabolismo , Regiões Promotoras Genéticas/genética , Fator sigma/imunologia , Fator sigma/metabolismo , Streptomyces/genética , Streptomyces coelicolor/genética , Transcrição Genética/genética
18.
Sci Rep ; 10(1): 8492, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444655

RESUMO

Most currently used antibiotics originate from Streptomycetes and phosphate limitation is an important trigger of their biosynthesis. Understanding the molecular processes underpinning such regulation is of crucial importance to exploit the great metabolic diversity of these bacteria and get a better understanding of the role of these molecules in the physiology of the producing bacteria. To contribute to this field, a comparative proteomic analysis of two closely related model strains, Streptomyces lividans and Streptomyces coelicolor was carried out. These strains possess identical biosynthetic pathways directing the synthesis of three well-characterized antibiotics (CDA, RED and ACT) but only S. coelicolor expresses them at a high level. Previous studies established that the antibiotic producer, S. coelicolor, is characterized by an oxidative metabolism and a reduced triacylglycerol content compared to the none producer, S. lividans, characterized by a glycolytic metabolism. Our proteomic data support these findings and reveal that these drastically different metabolic features could, at least in part, due to the weaker abundance of proteins of the two component system PhoR/PhoP in S. coelicolor compared to S. lividans. In condition of phosphate limitation, PhoR/PhoP is known to control positively and negatively, respectively, phosphate and nitrogen assimilation and our study revealed that it might also control the expression of some genes of central carbon metabolism. The tuning down of the regulatory role of PhoR/PhoP in S. coelicolor is thus expected to be correlated with low and high phosphate and nitrogen availability, respectively and with changes in central carbon metabolic features. These changes are likely to be responsible for the observed differences between S. coelicolor and S. lividans concerning energetic metabolism, triacylglycerol biosynthesis and antibiotic production. Furthermore, a novel view of the contribution of the bio-active molecules produced in this context, to the regulation of the energetic metabolism of the producing bacteria, is proposed and discussed.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteoma/análise , Regulon , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/genética , Glicólise , Nitrogênio , Fosfatos , Proteoma/metabolismo , Streptomyces coelicolor/crescimento & desenvolvimento
19.
J Ind Microbiol Biotechnol ; 47(4-5): 413-423, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32367443

RESUMO

CRISPR-Cas9 has proven as a very powerful gene editing tool for Actinomyces, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes Streptomyces coelicolor M145 and Streptomyces lividans TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in Actinomyces.


Assuntos
Sistemas CRISPR-Cas , Recombinação Genética , Streptomyces/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Família Multigênica , Plasmídeos/genética , Streptomyces/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
20.
Sci Rep ; 10(1): 5727, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32235931

RESUMO

The glycopeptide antibiotic vancomycin has been widely used to treat infections of Gram-positive bacteria including Clostridium difficile and methicillin-resistant Staphylococcus aureus. However, since its introduction, high level vancomycin resistance has emerged. The genes responsible require the action of the two-component regulatory system VanSR to induce expression of resistance genes. The mechanism of detection of vancomycin by this two-component system has yet to be elucidated. Diverging evidence in the literature supports activation models in which the VanS protein binds either vancomycin, or Lipid II, to induce resistance. Here we investigated the interaction between vancomycin and VanS from Streptomyces coelicolor (VanSSC), a model Actinomycete. We demonstrate a direct interaction between vancomycin and purified VanSSC, and traced these interactions to the extracellular region of the protein, which we reveal adopts a predominantly α-helical conformation. The VanSSC-binding epitope within vancomycin was mapped to the N-terminus of the peptide chain, distinct from the binding site for Lipid II. In targeting a separate site on vancomycin, the effective VanS ligand concentration includes both free and lipid-bound molecules, facilitating VanS activation. This is the first molecular description of the VanS binding site within vancomycin, and could direct engineering of future therapeutics.


Assuntos
Proteínas de Bactérias/metabolismo , Streptomyces coelicolor/metabolismo , Fatores de Transcrição/metabolismo , Resistência a Vancomicina/genética , Vancomicina/farmacologia , Proteínas de Bactérias/genética , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/genética , Fatores de Transcrição/genética
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