RESUMO
We previously identified the aur1 biosynthetic gene cluster (BGC) in Streptomyceslavendulae subsp. lavendulae CCM 3239 (formerly Streptomycesaureofaciens CCM 3239), which is responsible for the production of the unusual angucycline-like antibiotic auricin. Auricin is produced in a narrow interval of the growth phase after entering the stationary phase, after which it is degraded due to its instability at the high pH values reached after the production phase. The complex regulation of auricin BGC is responsible for this specific production by several regulators, including the key activator Aur1P, which belongs to the family of atypical response regulators. The aur1P gene forms an operon with the downstream aur1O gene, which encodes an unknown protein without any conserved domain. Homologous aur1O genes have been found in several BGCs, which are mainly responsible for the production of angucycline antibiotics. Deletion of the aur1O gene led to a dramatic reduction in auricin production. Transcription from the previously characterized Aur1P-dependent biosynthetic aur1Ap promoter was similarly reduced in the S. lavendulaeaur1O mutant strain. The aur1O-specific coactivation of the aur1Ap promoter was demonstrated in a heterologous system using a luciferase reporter gene. In addition, the interaction between Aur1O and Aur1P has been demonstrated by a bacterial two-hybrid system. These results suggest that Aur1O is a specific coactivator of this key auricin-specific positive regulator Aur1P. Bioinformatics analysis of Aur1O and its homologues in other BGCs revealed that they represent a new family of transcriptional coactivators involved in the regulation of secondary metabolite biosynthesis. However, they are divided into two distinct sequence-specific subclasses, each of which is likely to interact with a different family of positive regulators.
Assuntos
Streptomyces aureofaciens , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Regiões Promotoras Genéticas , Streptomyces aureofaciens/genética , Streptomyces aureofaciens/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
6-Demethylchlortetracycline (6-DCT), a tetracycline antibiotic produced by Streptomyces aureofaciens, is a crucial precursor employed for the semi-synthesis of tigecycline, minocycline, and amadacyclin (PTK 0796). In this study, the 6-DCT biosynthetic gene cluster (BGC) was cloned from genomic DNA of a high 6-DCT-producing strain, S. aureofaciens DM-1, using the transformation-associated recombination method. An extra copy of the 6-DCT BGC was introduced and integrated into the chromosome of S. aureofaciens DM-1. Duplication of the 6-DCT BGC resulted in a maximum increase of the 6-DCT titer by 34%.
Assuntos
Antibacterianos/biossíntese , Demeclociclina/biossíntese , Família Multigênica , Streptomyces aureofaciens/genética , Recombinação Genética , Streptomyces aureofaciens/metabolismoRESUMO
We previously identified the aur1 gene cluster which produces the angucycline antibiotic auricin. Preliminary characterisation of auricin revealed that it is modified by a single aminodeoxysugar, D-forosamine. Here we characterise the D-forosamine-specific genes. The four close tandem genes, aur1TQSV, encoding enzymes involved in the initial steps of the deoxysugar biosynthesis, were located on a large operon with other core auricin biosynthetic genes. Deleting these genes resulted in the absence of auricin and the production of deglycosylated auricin intermediates. The two final D-forosamine biosynthetic genes, sa59, an NDP-hexose aminotransferase, and sa52, an NDP-aminohexose N-dimethyltransferase, are located in a region rather distant from the core auricin genes. A deletion analysis of these genes confirmed their role in D-forosamine biosynthesis. The Δsa59 mutant had a phenotype similar to that of the cluster deletion mutant, while the Δsa52 mutant produced an auricin with a demethylated D-forosamine. Although auricin contains a single deoxyhexose, two glycosyltransferase genes were found to participate in the attachment of D-forosamine to the auricin aglycon. An analysis of the expression of the D-forosamine biosynthesis genes revealed that the initial D-forosamine biosynthetic genes aur1TQSV are regulated together with the other auricin core genes by the aur1Ap promoter under the control of the auricin-specific activator Aur1P. The expression of the other D-forosamine genes, however, is governed by promoters differentially dependent upon the two SARP family auricin-specific activators Aur1PR3 and Aur1PR4. These promoters contain direct repeats similar to the SARP consensus sequence and are involved in the interaction with both regulators.
Assuntos
Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Hexosaminas/biossíntese , Macrolídeos/metabolismo , Streptomyces aureofaciens/genética , Transaminases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Deleção de Genes , Família Multigênica , Óperon , Regiões Promotoras Genéticas , Metabolismo Secundário/genética , Alinhamento de Sequência , Streptomyces aureofaciens/metabolismo , Transaminases/metabolismoRESUMO
Contamination of food and feed by Aflatoxin B1 (AFB1) is a cause of serious economic and health problems. Different processes have been used to degrade AFB1. In this study, biological degradation of AFB1 was carried out using three Actinomycete species, Rhodococcus erythropolis ATCC 4277, Streptomyces lividans TK 24, and S. aureofaciens ATCC 10762, in liquid cultures. Biodegradation of AFB1 was optimised under a range of temperatures from 25 to 40 °C and pH values of 4.0 to 8.0. An initial concentration of 20 µg/mL of AFB1 was used in this study. The amount of AFB1 remaining was measured against time by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC), coupled with UV and mass spectrometry (LC-MS). All species were able to degrade the AFB1, and no significant difference was found between them. AFB1 remained in the liquid culture for R. erythropolis, S. lividans and S. aureofaciens were 0.81 µg/mL, 2.41 µg/mL and 2.78 µg/mL respectively, at the end of the first 24 h. Degradation occurred at all incubation temperatures and the pH with the optimal conditions for R. erythropolis was achieved at 30 °C and pH 6, whereas for S. lividans and S. aureofaciens the optimum conditions for degradation were 30 °C and pH 5. Analysis of the degradative route indicated that each microorganism has a different way of degrading AFB1. The metabolites produced by R. erythropolis were significantly different from the other two microorganisms. Products of degradation were identified through metabolomic studies by utilizing high-resolution mass spectral data. Mass spectrometric analysis indicated that the degradation of AFB1 was associated with the appearance of a range of lower molecular weight compounds. The pathway of degradation or chemical alteration of AFB1 was followed by means of high resolution Fourier transform mass spectrometry (HR-FTMS) analysis as well as through the MS2 fragmentation to unravel the degradative pathway for AFB1. AFB1 bio-degradation was coupled with the accumulation of intermediates of fatty acid metabolism and glycolysis. A plausible mechanism of degradation of AFB1 by Rhodococcus was hypothesized.
Assuntos
Aflatoxina B1/metabolismo , Contaminação de Alimentos/prevenção & controle , Metabolômica/métodos , Rhodococcus/metabolismo , Streptomyces aureofaciens/metabolismo , Streptomyces lividans/metabolismo , Biodegradação Ambiental , Temperatura Alta , Concentração de Íons de HidrogênioRESUMO
The γ-butyrolactone (GBL) autoregulator-receptor systems play a role in controlling secondary metabolism and/or morphological differentiation in many Streptomyces species. We previously identified the aur1 gene cluster, located on the Streptomyces aureofaciens CCM 3239 large linear plasmid pSA3239, which is responsible for the production of the angucycline antibiotic auricin. Here, we describe the characterisation of two genes, sagA and sagR, encoding GBL autoregulatory signalling homologues, which lie in the upstream part of the aur1 cluster. SagA was similar to GBL synthases and SagR to GBL receptors. The expression of each gene is directed by its own promoter, sagAp for sagA and sagRp for sagR. Both genes were active mainly during the exponential phase, and their transcription was interdependent. The disruption of sagA abolished auricin production, while the disruption of sagR resulted in precocious but dramatically reduced auricin production. Transcription from the aur1Pp and aur1Rp promoters, which direct the expression of auricin-specific cluster-situated regulators (CSRs), was also precocious and increased in the sagR mutant strain. In addition, SagR was also shown to specifically bind both promoters in vitro. These results indicated that the SagA-SagR GBL system regulates auricin production. Unlike many other GBL receptors, SagR does not bind its own promoter, but Aur1R, an auricin-specific repressor from the family of pseudo GBL receptors, does bind both sagAp and sagRp promoters. Moreover, the expression of both promoters was deregulated in an aur1R mutant, indicating that the SagA-SagR GBL system is regulated by a feedback mechanism involving the auricin-specific CSR Aur1R, which regulates downstream.
Assuntos
Antibacterianos/metabolismo , Regulação Bacteriana da Expressão Gênica , Macrolídeos/metabolismo , Streptomyces aureofaciens/genética , Streptomyces aureofaciens/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Transcrição GênicaAssuntos
Glicosídeos/química , Bactérias Gram-Positivas/efeitos dos fármacos , Polienos/química , Policetídeo Sintases/metabolismo , Streptomyces aureofaciens/metabolismo , Bacillus subtilis/efeitos dos fármacos , Descoberta de Drogas , Escherichia coli/efeitos dos fármacos , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Imageamento por Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Polienos/isolamento & purificação , Polienos/farmacologia , Policetídeo Sintases/análise , Policetídeo Sintases/química , Streptomyces aureofaciens/enzimologiaRESUMO
The gene product of orf50 from actinophage µ1/6 of Streptomyces aureofaciens is a putative endolysin, Lyt µ1/6. It has a two-domain modular structure, consisting of an N-terminal catalytic and a C-terminal cell wall binding domain (CBD). Comparative analysis of Streptomyces phage endolysins revealed that they all have a modular structure and contain functional C-terminal domains with conserved amino acids, probably associated with their binding function. A blast analysis of Lyt µ1/6 in conjunction with secondary and tertiary structure prediction disclosed the presence of a PG_binding_1 domain within the CBD. The sequence of the C-terminal domain of lyt µ1/6 and truncated forms of it were cloned and expressed in Escherichia coli. The ability of these CBD variants fused to GFP to bind to the surface of S. aureofaciens NMU was shown by specific binding assays.
Assuntos
Bacteriófagos/genética , Endopeptidases/química , Streptomyces aureofaciens/virologia , Sequência de Aminoácidos , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Streptomyces aureofaciens/metabolismoRESUMO
Streptomyces bacteria are major producers of bioactive natural products, including many antibiotics. We identified a gene cluster, aur1, in a large linear plasmid of Streptomyces aureofaciens CCM3239. The cluster is responsible for the production of a new angucycline polyketide antibiotic auricin. Several tailoring biosynthetic genes were scatted in rather distant aur1 flanking regions. Auricin was produced in a very narrow growth phase interval of several hours after entry into stationary phase, after which it was degraded to non-active metabolites because of its instability at the high pH values reached after the production stage. Strict transcriptional regulation of the auricin biosynthetic gene cluster has been demonstrated, including feed-forward and feedback control by auricin intermediates via several of the huge number of regulatory genes present in the aur1 cluster. The complex mechanism may ensure strict confinement of auricin production to a specific growth stage.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Vias Biossintéticas/genética , Regulação Bacteriana da Expressão Gênica , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Streptomyces aureofaciens/metabolismo , Família Multigênica , Plasmídeos , Streptomyces aureofaciens/genética , Streptomyces aureofaciens/crescimento & desenvolvimentoRESUMO
Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants of Streptomyces aureofaciens and the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0g/l CTC predominated along with 1.2g/l TC, and discovered that chlorination by ctcP (an FADH2-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the ΔctcP strain accumulated about 18.9g/l "clean" TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with low kcat of 0.51±0.01min(-1), while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression of ctcP in S. aureofaciens and improved CTC production to a final titer of 25.9g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms.
Assuntos
Proteínas de Bactérias/biossíntese , Clortetraciclina/biossíntese , Halogenação , Engenharia Metabólica , Oxirredutases/biossíntese , Streptomyces aureofaciens/metabolismo , Proteínas de Bactérias/genética , Oxirredutases/genética , Streptomyces aureofaciens/genéticaRESUMO
Three regulators, Aur1P, Aur1R and a SARP-family Aur1PR3, have been previously found to control expression of the aur1 cluster for the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Here, we describe an additional regulatory gene, aur1PR4, encoding a homologue from the SARP-family regulators. Its role in auricin regulation was confirmed by its disruption that dramatically affected auricin production. However, transcription from the aur1Ap promoter, directing expression of 22 auricin biosynthetic genes, was not substantially affected in the Δaur1PR4 mutant. A new promoter, sa13p, directing transcription of four putative auricin tailoring genes, was found to be dependent on aur1PR4. Moreover, analysis of the sa13p promoter region revealed the presence of three heptameric repeat sequences corresponding to putative SARP-binding sites. Expression of aur1PR4 is directed by a single promoter, aur1PR4p, which is induced after entry into stationary phase. Transcription from aur1PR4p was absent in a S. aureofaciens Δaur1P mutant strain, and Aur1P was shown to bind specifically to the aur1PR4p promoter. These results indicate a complex network of regulation of the auricin gene cluster. Both Aur1P and Aur1PR3 are involved in regulation of the core aur1A-U biosynthetic genes, and Aur1PR4 in regulation of putative auricin tailoring genes.
Assuntos
Antibacterianos/metabolismo , Regulação Bacteriana da Expressão Gênica , Macrolídeos/metabolismo , Streptomyces aureofaciens/genética , Streptomyces aureofaciens/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sítios de Ligação , Vias Biossintéticas/genética , DNA Bacteriano/genética , Técnicas de Inativação de Genes , Dados de Sequência Molecular , Policetídeos/metabolismo , Regiões Promotoras Genéticas , Alinhamento de Sequência , Fatores de Transcrição/genéticaRESUMO
We previously identified a polyketide synthase gene cluster, aur1, responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. A sequence analysis of the aur1 flanking regions revealed the presence of several genes encoding proteins homologous to those for Streptomyces linear plasmid replication, partitioning and telomere-binding. Pulse-field gel electrophoresis detected the single, 240-kb linear plasmid, pSA3239, in S. aureofaciens CCM3239. The presence of the auricin cluster in pSA3239 was confirmed by several approaches. In addition to aur1, pSA3239 also carries a large number of regulatory genes, and two gene clusters involved in the production of secondary metabolites: the aur2 cluster for an unknown secondary metabolite and the bpsA cluster for the blue pigment indigoidine.
Assuntos
Antibacterianos/metabolismo , Vias Biossintéticas/genética , Macrolídeos/metabolismo , Família Multigênica , Plasmídeos , Streptomyces aureofaciens/genética , Streptomyces aureofaciens/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The polyketide gene cluster aur1 is responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is regulated in a complex manner involving several regulators, including a key pathway-specific positive regulator Aur1P that belongs to the family of 'atypical' response regulators. Production of auricin is induced after entry into stationary phase. However, auricin was produced in only a short time interval of several hours. We found that the decrease of auricin production was due to a strict regulation of auricin biosynthetic genes at the transcriptional level by a feedback mechanism; auricin and/or its intermediate(s) inhibited binding of Aur1P to its cognate biosynthetic promoter aur1Ap and consequently stopped its activation. In addition, we also determined that synthesised auricin is unstable during growth of S. aureofaciens CCM3239 in the production medium even though purified auricin is stable for days in various organic solvents. The critical parameter affecting its stability was pH. Auricin is stable at acid pH and unstable at neutral and alkaline pH. The drop in auricin concentration was due to an increase of pH shortly after induction of auricin production during cultivation of S. aureofaciens CCM3239.
Assuntos
Antibacterianos/metabolismo , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Macrolídeos/metabolismo , Streptomyces aureofaciens/genética , Streptomyces aureofaciens/metabolismo , Vias Biossintéticas/genética , Transcrição GênicaRESUMO
The polyketide gene cluster aur1 is responsible for the production of the antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is low and strictly regulated by two regulators, Aur1P and Aur1R. To improve auricin yield, we genetically manipulated S. aureofaciens CCM 3239 strain to overcome this strict regulation. A regulatory region including aur1R, aur1P, aur1O and the target biosynthetic aur1Ap promoter were replaced by the strong constitutive ermEp* promoter. However, auricin production was decreased in such a genetically manipulated strain. In the second strategy we placed the aur1P gene for auricin pathway-specific activator under the control of the ermEp* promoter. The resulting strain has been shown to produce 2.8-fold higher amount of auricin compared with the WT strain.
Assuntos
Antibacterianos/biossíntese , Macrolídeos/metabolismo , Streptomyces aureofaciens/genética , Cromatografia Líquida de Alta Pressão , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Plasmídeos/genética , Regiões Promotoras Genéticas , Mapeamento por Restrição , Streptomyces aureofaciens/metabolismoRESUMO
Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.
Assuntos
Regulação Bacteriana da Expressão Gênica , Macrolídeos/metabolismo , Família Multigênica/genética , Proteínas Repressoras/metabolismo , Streptomyces aureofaciens/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Família Multigênica/fisiologia , Mutação , Regiões Promotoras Genéticas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Sequência de DNA , Transdução de Sinais , Streptomyces aureofaciens/genética , Streptomyces aureofaciens/crescimento & desenvolvimento , Transativadores/química , Transativadores/genéticaRESUMO
Poly(epsilon-L-lysine) (epsilon-PL) is a naturally-occurring L-lysine homopolymer having antimicrobial activity. A newly-isolated strain of Streptomyces aureofaciens produced a short chain length epsilon-PL consisting of 5-20 residues at the highest production level of 4.5 g l(-1). This epsilon-PL had different spectra in terms of antimicrobial activity from the epsilon-PL that is now used as a food preservative. The high productivity was based on multiple metabolic pathways for L-lysine synthesis, and a great flux from L-lysine to epsilon-PL. The usefulness of this new epsilon-PL and its producing strain was discussed.
Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Polilisina/biossíntese , Polilisina/farmacologia , Streptomyces aureofaciens/metabolismo , Aminoácidos/análise , Antibacterianos/química , Cromatografia Líquida de Alta Pressão , Redes e Vias Metabólicas/genética , Modelos Biológicos , Polilisina/químicaRESUMO
An incomplete oligoketide (PK; 'polyketide') gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Pigmentos Biológicos/biossíntese , Piperidonas/metabolismo , Streptomyces aureofaciens/genética , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Streptomyces aureofaciens/metabolismoRESUMO
Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.
Assuntos
Proteínas de Bactérias/metabolismo , Genes Reguladores , Macrolídeos/metabolismo , Família Multigênica , Streptomyces aureofaciens/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , RNA Bacteriano/genética , Streptomyces aureofaciens/metabolismo , Transcrição GênicaRESUMO
Analysis of the alpha-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to the identification of five putative regulatory genes, which are congregated into a subcluster. Analysis of the lipReg1-4 and lipX1 showed that they encode components of two-component signal transduction systems (LipReg1 and LipReg2), multiple antibiotics resistance-type regulator (LipReg3), large ATP-binding regulators of the LuxR family-type regulator (LipReg4), and small ribonuclease (LipRegX1), respectively. A combination of targeted gene disruptions, complementation experiments, lipomycin production studies, and gene expression analysis via RT-PCR suggests that all regulatory lip genes are involved in alpha-lipomycin production. On the basis of the obtained data, we propose that LipReg2 controls the activity of LipReg1, which in its turn govern the expression of the alpha-lipomycin pathway-specific regulatory gene lipReg4. The ribonuclease gene lipX1 and the transporter regulator lipReg3 appear to work independently of genes lipReg1, lipReg2, and lipReg4.
Assuntos
Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Glicosídeos/genética , Streptomyces aureofaciens/genética , Antibacterianos/química , Antibacterianos/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Escherichia coli/genética , Genes Reguladores , Teste de Complementação Genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Família Multigênica , Mutagênese , Polienos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Streptomyces aureofaciens/metabolismoRESUMO
In vitro phosphorylation of EF-Tu was shown in cell-free extract from dormant spores of Streptomyces coelicolor by a protein kinase present in spores. EF-Tu phosphorylation was observed on both intrinsic S. coelicolor factor and externally added purified EF-Tu from S. aureofaciens, on two isoforms. Putative serine and threonine residues as potential phosphorylation targets were determined in primary sequence and demonstrated on 3D structure model of EF-Tu.
Assuntos
Fator Tu de Elongação de Peptídeos/metabolismo , Proteínas Quinases/metabolismo , Esporos Bacterianos/metabolismo , Streptomyces coelicolor/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional , Imageamento Tridimensional , Modelos Moleculares , Dados de Sequência Molecular , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/isolamento & purificação , Fosforilação , Estrutura Terciária de Proteína , Alinhamento de Sequência , Esporos Bacterianos/enzimologia , Streptomyces aureofaciens/metabolismoRESUMO
Transition from exponential phase of growth to stationary phase in Streptomyces aureofaciens is characterized by a decrease in the rate of translation and induction of tetracycline (Ttc) biosynthesis. In exponential phase, no significant changes were found in the activity of ribosomes at binding of ternary complex Phe-tRNA.EF-Tu.GTP to the A-site on ribosomes. Overexpression of Ttc in stationary phase is accompanied by a decrease in the binding of the ternary complex Phe-tRNA.EF-Tu.GTP to the A-site of ribosome and a formation of an aggregate with Ttc by part of the ribosomes. Antibiotics that cause ribosome to stall or pause could increase the requirement for tmRNA in the process called trans-translation. We found differences in the level of tmRNA during the development of S. aureofaciens. Subinhibitory concentrations of Ttc, streptomycin and chloramphenicol induced an increase in the tmRNA level in cells from the exponential phase of growth. In vitro trans-translation system of S. aureofaciens was sensitive to Ttc at a concentration of > 15 micromol/L; the trans-translation system can thus be considered to contribute to resistance against Ttc produced only at sublethal concentrations. These experiments suggest that the main role of the rising tmRNA level at the beginning of the Ttc production is connected with ribosome rescue.