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1.
J Microbiol Biotechnol ; 32(9): 1134-1145, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36116920

RESUMO

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993+. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.


Assuntos
Streptomyces coelicolor , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Antraquinonas/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA , Desoxirribonucleases/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Regiões Promotoras Genéticas , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética
2.
Sci Rep ; 12(1): 15579, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114335

RESUMO

A genomic and bioactivity informed analysis of the metabolome of the extremophile Amycolatopsis sp. DEM30355 has allowed for the discovery and isolation of the polyketide antibiotic tatiomicin. Identification of the biosynthetic gene cluster was confirmed by heterologous expression in Streptomyces coelicolor M1152. Structural elucidation, including absolute stereochemical assignment, was performed using complementary crystallographic, spectroscopic and computational methods. Tatiomicin shows antibiotic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Cytological profiling experiments suggest a putative antibiotic mode-of-action, involving membrane depolarisation and chromosomal decondensation of the target bacteria.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Policetídeos , Streptomyces coelicolor , Amycolatopsis , Antibacterianos/química , Staphylococcus aureus Resistente à Meticilina/genética , Streptomyces coelicolor/genética
3.
Arch Microbiol ; 204(9): 582, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36042049

RESUMO

Streptomyces coelicolor is a model organism for studying streptomycetes. This genus possesses relevant medical and economical roles, because it produces many biologically active metabolites of pharmaceutical interest, including the majority of commercialized antibiotics. In this bioinformatic study, the transcriptome of S. coelicolor has been analyzed to identify novel RNA species and quantify the expression of both annotated and novel transcripts in solid and liquid growth medium cultures at different times. The major characteristics disclosed in this study are: (i) the diffuse antisense transcription; (ii) the great abundance of transfer-messenger RNAs (tmRNA); (iii) the abundance of rnpB transcripts, paramount for the RNase-P complex; and (iv) the presence of abundant fragments derived from pre-ribosomal RNA leader sequences of unknown biological function. Overall, this study extends the catalogue of ncRNAs in S. coelicolor and suggests an important role of non-coding transcription in the regulation of biologically active molecule production.


Assuntos
Streptomyces coelicolor , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico , Ribonuclease P/metabolismo
4.
Biomolecules ; 12(8)2022 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-36009001

RESUMO

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the ß subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.


Assuntos
Flavodoxina , Streptomyces coelicolor , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flavodoxina/química , Flavodoxina/genética , Flavodoxina/metabolismo , Oxirredução , Solo , Streptomyces coelicolor/metabolismo
5.
Org Biomol Chem ; 20(28): 5525-5534, 2022 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-35786703

RESUMO

Uncovering the comprehensive catalytic mechanism for the activation of triplet O2 through metal-free and cofactor-free oxidases and oxygenases remains one of the most challenging problems in the area of enzymatic catalysis. Herein, we performed multiscale simulation with molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) techniques to reveal the detailed mechanism of ActVA-Orf6 monooxygenase catalyzed oxygenation of phenols to quinones from Streptomyces coelicolor, such as the oxidation of 6-deoxydihydrocarafungin (DDHK) to dihydrocarafungin (DHK). The entire catalytic mechanism consists of three steps: (1) proton-coupled electron transfer (PCET) from the substrate DDHK to triplet O2 with the aid of an explicit water molecule, (2) the formation of a C-O bond via an open-shell singlet diradical complexation pathway, and (3) dehydration via a six-membered ring mode assisted by one water molecule. The complete energetic profiles show that the rate-determining step is the dehydration with an energy barrier of 20.7 kcal mol-1, which is close to that of 19.7 kcal mol-1 derived from experimental kinetic data. Our mechanistic study not only helps to deeply understand the fundamental mechanism of metal-free and cofactor-free oxidase and oxygenase catalyzed different reactions, but also discloses a new route that proceeds through the processes of PCET and the open-shell singlet transition state.


Assuntos
Oxigenases de Função Mista , Prótons , Catálise , Desidratação , Elétrons , Oxigenases de Função Mista/metabolismo , Simulação de Dinâmica Molecular , Teoria Quântica , Streptomyces coelicolor , Água
6.
PLoS One ; 17(7): e0270379, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35834474

RESUMO

Streptomyces lividans TK23 interacts with mycolic acid-containing bacteria (MACB), such as Tsukamurella pulmonis TP-B0596, and this direct cell contact activates its secondary metabolism (e.g., the production of undecylprodigiosin: RED). Here, we employed carbon (12C5+) ion beam-induced mutagenesis to investigate the signature of induced point mutations and further identify the gene(s) responsible for the production of secondary metabolites induced by T. pulmonis. We irradiated spores of the Streptomyces coelicolor strain JCM4020 with carbon ions to generate a mutant library. We screened the RED production-deficient mutants of S. coelicolor by mixing them with T. pulmonis TP-B0596 on agar plates, identifying the red/white phenotype of the growing colonies. Through this process, we selected 59 RED-deficient mutants from around 152,000 tested spores. We resequenced the genomes of 16 mutants and identified 44 point mutations, which revealed the signatures induced by 12C5+-irradiation. Via gene complementation experiments, we also revealed that two genes-glutamate synthase (gltB) and elongation factor G (fusA)-are responsible for the reduced production of RED.


Assuntos
Streptomyces coelicolor , Antibacterianos/metabolismo , Carbono/metabolismo , Íons/metabolismo , Mutagênese , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces lividans/metabolismo
7.
Commun Biol ; 5(1): 769, 2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-35908109

RESUMO

Several transcription factors of the Rrf2 family use an iron-sulfur cluster to regulate DNA binding through effectors such as nitric oxide (NO), cellular redox status and iron levels. [4Fe-4S]-NsrR from Streptomyces coelicolor (ScNsrR) modulates expression of three different genes via reaction and complex formation with variable amounts of NO, which results in detoxification of this gas. Here, we report the crystal structure of ScNsrR complexed with an hmpA1 gene operator fragment and compare it with those previously reported for [2Fe-2S]-RsrR/rsrR and apo-IscR/hyA complexes. Important structural differences reside in the variation of the DNA minor and major groove widths. In addition, different DNA curvatures and different interactions with the protein sensors are observed. We also report studies of NsrR binding to four hmpA1 variants, which indicate that flexibility in the central region is not a key binding determinant. Our study explores the promotor binding specificities of three closely related transcriptional regulators.


Assuntos
Proteínas Ferro-Enxofre , Streptomyces coelicolor , Proteínas de Bactérias/metabolismo , DNA/genética , DNA/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Óxido Nítrico/metabolismo , Streptomyces coelicolor/genética , Fatores de Transcrição/metabolismo
8.
Nucleic Acids Res ; 50(14): 8363-8376, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35871291

RESUMO

Streptomyces coelicolor (Sc) is a model organism of actinobacteria to study morphological differentiation and production of bioactive metabolites. Sc zinc uptake regulator (Zur) affects both processes by controlling zinc homeostasis. It activates transcription by binding to palindromic Zur-box sequences upstream of -35 elements. Here we deciphered the molecular mechanism by which ScZur interacts with promoter DNA and Sc RNA polymerase (RNAP) by cryo-EM structures and biochemical assays. The ScZur-DNA structures reveal a sequential and cooperative binding of three ScZur dimers surrounding a Zur-box spaced 8 nt upstream from a -35 element. The ScRNAPσHrdB-Zur-DNA structures define protein-protein and protein-DNA interactions involved in the principal housekeeping σHrdB-dependent transcription initiation from a noncanonical promoter with a -10 element lacking the critical adenine residue at position -11 and a TTGCCC -35 element deviating from the canonical TTGACA motif. ScZur interacts with the C-terminal domain of ScRNAP α subunit (αCTD) in a complex structure trapped in an active conformation. Key ScZur-αCTD interfacial residues accounting for ScZur-dependent transcription activation were confirmed by mutational studies. Together, our structural and biochemical results provide a comprehensive model for transcription activation of Zur family regulators.


Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA , Streptomyces coelicolor , Ativação Transcricional , Proteínas de Bactérias/metabolismo , DNA Bacteriano/química , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Streptomyces coelicolor/metabolismo , Zinco/metabolismo
9.
Nat Commun ; 13(1): 3502, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35715393

RESUMO

Transcriptional regulation is a critical process to ensure expression of genes necessary for growth and survival in diverse environments. Transcription is mediated by multiple transcription factors including activators, repressors and sigma factors. Accurate computational prediction of the regulon of target genes for transcription factors is difficult and experimental identification is laborious and not scalable. Here, we demonstrate regulon identification by in vitro transcription-sequencing (RIViT-seq) that enables systematic identification of regulons of transcription factors by combining an in vitro transcription assay and RNA-sequencing. Using this technology, target genes of 11 sigma factors were identified in Streptomyces coelicolor A3(2). The RIViT-seq data expands the transcriptional regulatory network in this bacterium, discovering regulatory cascades and crosstalk between sigma factors. Implementation of RIViT-seq with other transcription factors and in other organisms will improve our understanding of transcriptional regulatory networks across biology.


Assuntos
Regulon , Streptomyces coelicolor , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Regulon/genética , Fator sigma/genética , Fator sigma/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Nat Commun ; 13(1): 2700, 2022 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-35577776

RESUMO

Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of RNR genes and, often, its own, in most bacteria and some archaea. NrdR senses the concentration of nucleotides through its ATP-cone, an evolutionarily mobile domain that also regulates the enzymatic activity of many RNRs, while a Zn-ribbon domain mediates binding to NrdR boxes upstream of and overlapping the transcription start site of RNR genes. Here, we combine biochemical and cryo-EM studies of NrdR from Streptomyces coelicolor to show, at atomic resolution, how NrdR binds to DNA. The suggested mechanism involves an initial dodecamer loaded with two ATP molecules that cannot bind to DNA. When dATP concentrations increase, an octamer forms that is loaded with one molecule each of dATP and ATP per monomer. A tetramer derived from this octamer then binds to DNA and represses transcription of RNR. In many bacteria - including well-known pathogens such as Mycobacterium tuberculosis - NrdR simultaneously controls multiple RNRs and hence DNA synthesis, making it an excellent target for novel antibiotics development.


Assuntos
Ribonucleotídeo Redutases , Streptomyces coelicolor , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica , Nucleotídeos/química , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Streptomyces coelicolor/metabolismo
12.
Environ Microbiol ; 24(7): 3081-3096, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35384219

RESUMO

Our previous study using transposon mutagenesis indicated that disruption of the putative response regulator gene orrA impacted antibiotic production in Streptomyces coelicolor. In this study, the role of OrrA was further characterized by comparing the phenotypes and transcriptomic profiles of the wild-type S. coelicolor strain M145 and ΔorrA, a strain with an inactivated orrA gene. Chromatin immunoprecipitation using a strain expressing OrrA fused with FLAG showed that OrrA binds the promoter of wblA, whose expression was downregulated in ΔorrA. The interaction of OrrA with the wblA promoter was further validated by a pull-down assay. Similar to ΔorrA, the deletion mutant of wblA (ΔwblA) was defective in development, and developmental genes were expressed at similar levels in ΔorrA and ΔwblA. Although both OrrA and WblA downregulated actinorhodin and undecylprodigiosin, their roles in regulation of the calcium-dependent antibiotic and yellow-pigmented type I polyketide differed. sco1375, a gene of unknown function, was identified as another OrrA target, and overexpression of either sco1375 or wblA in ΔorrA partially restored the wild-type phenotype, indicating that these genes mediate some of the effects of OrrA. This study revealed targets of OrrA and provided more insights into the role of the orphan response regulator OrrA in Streptomyces.


Assuntos
Streptomyces coelicolor , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Reguladores/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
13.
Bioorg Med Chem Lett ; 66: 128727, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35413414

RESUMO

Actinorhodin (ACT) is a benzoisochromanequinone antibiotic produced by Streptomyces coelicolor A3(2), which has served as a favored model organism for comprehensive studies of antibiotic biosynthesis and its regulation. (S)-DNPA undergoes various modifications as an intermediate in the ACT biosynthetic pathway, including enoyl reduction to DDHK. It has been suggested that actVI-ORF2 encodes an enoyl reductase (ER). However, its function has not been characterized in vitro. In this study, biochemical analysis of recombinant ActVI-ORF2 revealed that (S)-DNPA is converted to DDHK in a stereospecific manner with NADPH acting as a cofactor. (R)-DNPA was also reduced to 3-epi-DDHK with the comparable efficacy as (S)-DNPA, suggesting that the stereospecificity of ActVI-ORF2 was not affected by the stereochemistry at the C-3 of DNPA. ActVI-ORF2 is a new example of a discrete ER, which is distantly related to known ERs according to phylogenetic analysis.


Assuntos
Streptomyces coelicolor , Streptomyces , Antraquinonas/química , Antibacterianos/metabolismo , Oxirredutases/metabolismo , Filogenia , Piranos/metabolismo , Streptomyces/metabolismo , Streptomyces coelicolor/metabolismo
14.
Nat Commun ; 13(1): 2266, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35477578

RESUMO

In colonies of the filamentous multicellular bacterium Streptomyces coelicolor, a subpopulation of cells arises that hyperproduces metabolically costly antibiotics, resulting in a division of labor that increases colony fitness. Because these cells contain large genomic deletions that cause massive reductions to individual fitness, their behavior is similar to altruistic worker castes in social insects or somatic cells in multicellular organisms. To understand these mutant cells' reproductive and genomic fate after their emergence, we use experimental evolution by serially transferring populations via spore-to-spore transfer for 25 cycles, reflective of the natural mode of bottlenecked transmission for these spore-forming bacteria. We show that in contrast to wild-type cells, putatively altruistic mutant cells continue to decline in fitness during transfer while they lose more fragments from their chromosome ends. In addition, the base-substitution rate in mutants increases roughly 10-fold, possibly due to mutations in genes for DNA replication and repair. Ecological damage, caused by reduced sporulation, coupled with DNA damage due to point mutations and deletions, leads to an inevitable and irreversible type of mutational meltdown in these cells. Taken together, these results suggest the cells arising in the S. coelicolor division of labor are analogous to altruistic reproductively sterile castes of social insects.


Assuntos
Streptomyces coelicolor , Diploide , Mutação , Mutação Puntual , Esporos Bacterianos/genética , Streptomyces coelicolor/genética
15.
mBio ; 13(3): e0386221, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35467418

RESUMO

The transcription factor AdpA is a key regulator controlling both secondary metabolism and morphological differentiation in Streptomyces. Due to its critical functions, its expression undergoes multilevel regulations at transcriptional, posttranscriptional, and translational levels, yet no posttranslational regulation has been reported. Sulfane sulfur, such as hydro polysulfide (HSnH, n ≥ 2) and organic polysulfide (RSnH, n ≥ 2), is common inside microorganisms, but its physiological functions are largely unclear. Here, we discovered that sulfane sulfur posttranslationally modifies AdpA in Streptomyces coelicolor via specifically reacting with Cys62 of AdpA to form a persulfide (Cys62-SSH). This modification decreases the affinity of AdpA to its self-promoter PadpA, allowing increased expression of adpA, further promoting the expression of its target genes actII-4 and wblA. ActII-4 activates actinorhodin biosynthesis, and WblA regulates morphological development. Bioinformatics analyses indicated that AdpA-Cys62 is highly conserved in Streptomyces, suggesting the prevalence of such modification in this genus. Thus, our study unveils a new type of regulation on the AdpA activity and sheds a light on how sulfane sulfur stimulates the production of antibiotics in Streptomyces. IMPORTANCE Streptomyces species produce a myriad of natural products with (potential) clinical applications. While the database of biosynthetic gene clusters is quickly expanding, their regulation mechanisms are rarely known. Sulfane sulfur species are commonly present in microorganisms with unclear functions. Here, we discovered that sulfane sulfur increases actinorhodin (ACT) production in S. coelicolor. The underlying mechanism is that sulfane sulfur specifically reacts with AdpA, a global transcription factor controlling both ACT gene cluster and morphological differentiation-related genes, to form sulfhydrated AdpA. This modification changes the dynamics of AdpA-controlled gene networks and leads to high expression of ACT biosynthetic genes. Given the wide prevalence of AdpA and sulfane sulfur in Streptomyces, this mechanism may represent a common regulating pattern of all AdpA-controlled biosynthetic pathways. Thus, this finding provides a new strategy for mining and activating valuable biosynthetic gene clusters.


Assuntos
Streptomyces coelicolor , Streptomyces , Antraquinonas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptomyces/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Enxofre/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Int J Mol Sci ; 23(7)2022 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-35409114

RESUMO

Streptomyces coelicolor is a soil bacterium living in a habitat with very changeable nutrient availability. This organism possesses a complex nitrogen metabolism and is able to utilize the polyamines putrescine, cadaverine, spermidine, and spermine and the monoamine ethanolamine. We demonstrated that GlnA2 (SCO2241) facilitates S. coelicolor to survive under high toxic polyamine concentrations. GlnA2 is a gamma-glutamylpolyamine synthetase, an enzyme catalyzing the first step in polyamine catabolism. The role of GlnA2 was confirmed in phenotypical studies with a glnA2 deletion mutant as well as in transcriptional and biochemical analyses. Among all GS-like enzymes in S. coelicolor, GlnA2 possesses the highest specificity towards short-chain polyamines (putrescine and cadaverine), while its functional homolog GlnA3 (SCO6962) prefers long-chain polyamines (spermidine and spermine) and GlnA4 (SCO1613) accepts only monoamines. The genome-wide RNAseq analysis in the presence of the polyamines putrescine, cadaverine, spermidine, or spermine revealed indication of the occurrence of different routes for polyamine catabolism in S. coelicolor involving GlnA2 and GlnA3. Furthermore, GlnA2 and GlnA3 are differently regulated. From our results, we can propose a complemented model of polyamine catabolism in S. coelicolor, which involves the gamma-glutamylation pathway as well as other alternative utilization pathways.


Assuntos
Streptomyces coelicolor , Cadaverina , Ligases , Poliaminas/metabolismo , Putrescina/metabolismo , Espermidina/metabolismo , Espermina/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
17.
Metallomics ; 14(3)2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35238926

RESUMO

Copper modulates secondary metabolism in Streptomyces. Although the cytosolic copper concentration is controlled by several chaperones and transporters, the formation of copper nanoparticles (NPs) and its relation to the antibiotic production has never been established in the model Streptomyces coelicolor. In this work, state-of-the-art analytical tools are used to evaluate the incorporation of copper in individual spores of S. coelicolor at different exposure concentrations (40, 80, and 160 µM Cu). Among them, the use of single cell-inductively coupled plasma-mass spectrometry revealed incorporation levels in the range of 2 to 2.5 fg/spore (median) increasing up to 4.75 fg/spore at the upper exposure concentrations. The copper storage within the spores in the form of NPs was evaluated using a combination of single particle-inductively coupled plasma-mass spectrometry and transmission electron microscopy. The obtained data confirmed the presence of NPs in the range of 8 to 40 (mean size 21 nm) inside S. coelicolor spores. The presence of the NPs was correlated with the actinorhodin production in liquid non-sporulating cultures amended with up to 80 µM Cu. However, further increase to 160 µM Cu, yielded to a significant decrease in antibiotic production. Secondary metabolism is activated under stressful conditions and cytosolic copper seems to be one of the signals triggering antibiotic production. Particularly, NP formation might contribute to modulate the secondary metabolism and prevent for copper toxicity. This work describes, for first time, the formation of endogenous copper NPs in S. coelicolor and reveals their correlation with the secondary metabolism.


Assuntos
Nanopartículas , Streptomyces coelicolor , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Cobre/metabolismo , Metabolismo Secundário , Esporos Bacterianos/metabolismo , Streptomyces coelicolor/metabolismo
18.
mBio ; 13(2): e0045622, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35357207

RESUMO

Peptidoglycan is a major constituent of the bacterial cell wall and an important determinant for providing protection to cells. In addition to peptidoglycan, many bacteria synthesize other glycans that become part of the cell wall. Streptomycetes grow apically, where they synthesize a glycan that is exposed at the outer surface, but how it gets there is unknown. Here, we show that deposition of the apical glycan at the cell surface of Streptomyces coelicolor depends on two key enzymes, the glucanase CslZ and the lytic polysaccharide monooxygenase LpmP. Activity of these enzymes allows localized remodeling and degradation of the peptidoglycan, and we propose that this facilitates passage of the glycan. The absence of both enzymes not only prevents morphological development but also sensitizes strains to lysozyme. Given that lytic polysaccharide monooxygenases are commonly found in microbes, this newly identified biological role in cell wall remodeling may be widespread. IMPORTANCE Lytic polysaccharide monooxygenases are used in industry for the efficient degradation of recalcitrant polysaccharide substrates. Only recently, we have begun to appreciate some of their important biological roles. In this article, we provide evidence that these enzymes are involved in remodeling peptidoglycan, which is a conserved component of the bacterial cell wall. Given that lytic polysaccharide monooxygenases are commonly found in microbes, this newly identified biological role in cell wall remodeling may be widespread.


Assuntos
Streptomyces coelicolor , Bactérias/metabolismo , Parede Celular/metabolismo , Carboidratos da Dieta/metabolismo , Oxigenases de Função Mista/metabolismo , Peptidoglicano/metabolismo , Polissacarídeos/metabolismo , Streptomyces coelicolor/metabolismo
19.
mBio ; 13(2): e0042522, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35357210

RESUMO

The bacterial response to antibiotics eliciting resistance is one of the key challenges in global health. Despite many attempts to understand intrinsic antibiotic resistance, many of the underlying mechanisms still remain elusive. In this study, we found that iron supplementation promoted antibiotic resistance in Streptomyces coelicolor. Iron-promoted resistance occurred specifically against bactericidal antibiotics, irrespective of the primary target of antibiotics. Transcriptome profiling revealed that some genes in the central metabolism and respiration were upregulated under iron-replete conditions. Iron supported the growth of S. coelicolor even under anaerobic conditions. In the presence of potassium cyanide, which reduces aerobic respiration of cells, iron still promoted respiration and antibiotic resistance. This suggests the involvement of a KCN-insensitive type of respiration in the iron effect. This phenomenon was also observed in another actinobacterium, Mycobacterium smegmatis. Taken together, these findings provide insight into a bacterial resistance strategy that mitigates the activity of bactericidal antibiotics whose efficacy accompanies oxidative damage by switching the respiration mode. IMPORTANCE A widely investigated mode of antibiotic resistance occurs via mutations and/or by horizontal acquisition of resistance genes. In addition to this acquired resistance, most bacteria exhibit intrinsic resistance as an inducible and adaptive response to different classes of antibiotics. Increasing attention has been paid recently to intrinsic resistance mechanisms because this may provide novel therapeutic targets that help rejuvenate the efficacy of the current antibiotic regimen. In this study, we demonstrate that iron promotes the intrinsic resistance of aerobic actinomycetes Streptomyces coelicolor and Mycobacterium smegmatis against bactericidal antibiotics. A surprising role of iron to increase respiration, especially in a mode of using less oxygen, appears a fitting strategy to cope with bactericidal antibiotics known to kill bacteria through oxidative damage. This provides new insights into developing antimicrobial treatments based on the availability of iron and oxygen.


Assuntos
Actinobacteria , Streptomyces coelicolor , Actinobacteria/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/genética , Resistência Microbiana a Medicamentos , Ferro/metabolismo , Oxigênio/metabolismo , Respiração , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
20.
Sci Total Environ ; 826: 154151, 2022 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-35231524

RESUMO

The environmental and human health risks posed by nanoplastics have attracted considerable attention; however, research on the combined toxicity of nanoplastics and plasticizers is limited. This study analyzed the combined effects of nanoplastics and dibutyl phthalate (DBP) on Streptomyces coelicolor M145 (herein referred to as M145) and its mechanism. The results demonstrated that when the concentration of both nanoplastics and DBP was 1 mg/L, the co-addition was not toxic to M145. When the DBP concentration increased to 5 mg/L, the combined toxicity of 1 mg/L nanoplastics and 5 mg/L DBP reduced when compared to the 5 mg/L DBP treatment group. Similarly, the combined toxicity of 10 mg/L nanoplastics and 1 mg/L DBP on M145 was also lower than that of only 10 mg/L nanoplastics. The co-addition of 10 mg/L nanoplastics and 5 mg/L DBP resulted in the lowest survival rate (41.3%). The key reason for differences in cytotoxicity were variations in the agglomeration of nanoplastics and the adsorption of DBP on nanoplastics. The combination of 10 mg/L nanoplastics and 5 mg/L DBP maximized the production of antibiotics; actinorhodin and undecylprodigiosin yields were 3.5 and 1.8-fold higher than that of the control, respectively. This indicates that the excessive production of antibiotics may be a protective mechanism for bacteria. This study provides a new perspective for assessing the risk of co-exposure to nanoplastics and organic contaminants on microorganisms in nature.


Assuntos
Streptomyces coelicolor , Antibacterianos/toxicidade , Dibutilftalato/toxicidade , Humanos , Microplásticos , Plastificantes
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