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A two-component flavin-dependent monooxygenase involved in actinorhodin biosynthesis in Streptomyces coelicolor  [PDF]
Julien Valton,Laurent Filisetti,Marc Fontecave,Vincent Nivière
Quantitative Biology , 2015, DOI: 10.1074/jbc.M407722200
Abstract: The two-component flavin-dependent monooxygenases belong to an emerging class of enzymes involved in oxidation reactions in a number of metabolic and biosynthetic pathways in microorganisms. One component is a NAD(P)H:flavin oxidoreductase, which provides a reduced flavin to the second component, the proper monooxygenase. There, the reduced flavin activates molecular oxygen for substrate oxidation. Here, we study the flavin reductase ActVB and ActVA-ORF5 gene product, both reported to be involved in the last step of biosynthesis of the natural antibiotic actinorhodin in Streptomyces coelicolor. For the first time we show that ActVA-ORF5 is a FMN-dependent monooxygenase that together with the help of the flavin reductase ActVB catalyzes the oxidation reaction. The mechanism of the transfer of reduced FMN between ActVB and ActVA-ORF5 has been investigated. Dissociation constant values for oxidized and reduced flavin (FMNox and FMNred) with regard to ActVB and ActVA-ORF5 have been determined. The data clearly demonstrate a thermodynamic transfer of FMNred from ActVB to ActVA-ORF5 without involving a particular interaction between the two protein components. In full agreement with these data, we propose a reaction mechanism in which FMNox binds to ActVB, where it is reduced, and the resulting FMNred moves to ActVA-ORF5, where it reacts with O2 to generate a flavinperoxide intermediate. A direct spectroscopic evidence for the formation of such species within ActVA-ORF5 is reported.
LAL Regulators SCO0877 and SCO7173 as Pleiotropic Modulators of Phosphate Starvation Response and Actinorhodin Biosynthesis in Streptomyces coelicolor  [PDF]
Susana M. Guerra, Antonio Rodríguez-García, Javier Santos-Aberturas, Cláudia M. Vicente, Tamara D. Payero, Juan F. Martín, Jesús F. Aparicio
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031475
Abstract: LAL regulators (Large ATP-binding regulators of the LuxR family) constitute a poorly studied family of transcriptional regulators. Several regulators of this class have been identified in antibiotic and other secondary metabolite gene clusters from actinomycetes, thus they have been considered pathway-specific regulators. In this study we have obtained two disruption mutants of LAL genes from S. coelicolor (Δ0877 and Δ7173). Both mutants were deficient in the production of the polyketide antibiotic actinorhodin, and antibiotic production was restored upon gene complementation of the mutants. The use of whole-genome DNA microarrays and quantitative PCRs enabled the analysis of the transcriptome of both mutants in comparison with the wild type. Our results indicate that the LAL regulators under study act globally affecting various cellular processes, and amongst them the phosphate starvation response and the biosynthesis of the blue-pigmented antibiotic actinorhodin. Both regulators act as negative modulators of the expression of the two-component phoRP system and as positive regulators of actinorhodin biosynthesis. To our knowledge this is the first characterization of LAL regulators with wide implications in Streptomyces metabolism.
Novel Two-Component Systems Implied in Antibiotic Production in Streptomyces coelicolor  [PDF]
Ana Yepes,Sergio Rico,Antonio Rodríguez-García,Ramón I. Santamaría,Margarita Díaz
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0019980
Abstract: The abundance of two-component systems (TCSs) in Streptomyces coelicolor A3(2) genome indicates their importance in the physiology of this soil bacteria. Currently, several TCSs have been related to antibiotic regulation, and the purpose in this study was the characterization of five TCSs, selected by sequence homology with the well-known absA1A2 system, that could also be associated with this important process. Null mutants of the five TCSs were obtained and two mutants (ΔSCO1744/1745 and ΔSCO4596/4597/4598) showed significant differences in both antibiotic production and morphological differentiation, and have been renamed as abr (antibiotic regulator). No detectable changes in antibiotic production were found in the mutants in the systems that include the ORFs SCO3638/3639, SCO3640/3641 and SCO2165/2166 in any of the culture conditions assayed. The system SCO1744/1745 (AbrA1/A2) was involved in negative regulation of antibiotic production, and acted also as a negative regulator of the morphological differentiation. By contrast, the system SCO4596/4597/4598 (AbrC1/C2/C3), composed of two histidine kinases and one response regulator, had positive effects on both morphological development and antibiotic production. Microarray analyses of the ΔabrC1/C2/C3 and wild-type transcriptomes revealed downregulation of actII-ORF4 and cdaR genes, the actinorhodin and calcium-dependent antibiotic pathway-specific regulators respectively. These results demonstrated the involvement of these new two-component systems in antibiotic production and morphological differentiation by different approaches. One is a pleiotropic negative regulator: abrA1/A2. The other one is a positive regulator composed of three elements, two histidine kinases and one response regulator: abrC1/C2/C3.
Metabolic modeling and analysis of the metabolic switch in Streptomyces coelicolor
Mohammad T Alam, Maria E Merlo, The STREAM Consortium, David A Hodgson, Elizabeth MH Wellington, Eriko Takano, Rainer Breitling
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-202
Abstract: We reconstructed the stoichiometric matrix of S. coelicolor, including the major antibiotic biosynthesis pathways, and performed flux balance analysis to predict flux changes that occur when the cell switches from biomass to antibiotic production. We defined the model input based on observed fermenter culture data and used a dynamically varying objective function to represent the metabolic switch. The predicted fluxes of many genes show highly significant correlation to the time series of the corresponding gene expression data. Individual mispredictions identify novel links between antibiotic production and primary metabolism.Our results show the usefulness of constraint-based modeling for providing a detailed interpretation of time course gene expression data.The transition from exponential growth to stationary phase is a major event in microbial physiology [1]. During the exponential phase of growth, bacterial cells produce metabolites necessary for growth and grow rapidly. Once essential nutrients have been depleted, cells switch to stationary phase, stop growing, reorganize their energy metabolism and often start producing a new set of secondary metabolites, including antibiotics [2].In this study, we have explored the metabolic switch in Streptomyces coelicolor, the model organism of the antibiotics producing genus Streptomyces. The genome of this soil bacterium has been sequenced and contains about 7825 genes, one of the largest numbers for any bacterium [3]. More than 20 clusters coding for the 4 known and several predicted antibiotics or related compounds have been identified in the genome [4]. To optimize the production of valuable secondary metabolites, understanding the shift from primary to secondary metabolism during the transition phase will play a key role.We constructed a constraints-based genome-scale stoichiometric model of S. coelicolor metabolism, based on earlier similar models [5,6], and integrated the model predictions with a large gene expres
The dynamic architecture of the metabolic switch in Streptomyces coelicolor
Kay Nieselt, Florian Battke, Alexander Herbig, Per Bruheim, Alexander Wentzel, ?yvind M Jakobsen, H?vard Sletta, Mohammad T Alam, Maria E Merlo, Jonathan Moore, Walid AM Omara, Edward R Morrissey, Miguel A Juarez-Hermosillo, Antonio Rodríguez-García, Merle Nentwich, Louise Thomas, Mudassar Iqbal, Roxane Legaie, William H Gaze, Gregory L Challis, Ritsert C Jansen, Lubbert Dijkhuizen, David A Rand, David L Wild, Michael Bonin, Jens Reuther, Wolfgang Wohlleben, Margaret CM Smith, Nigel J Burroughs, Juan F Martín
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-10
Abstract: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.The switch from primary metabolism (exponential growth) to secondary metabolism (stationary growth) upon nutrient starvation is commonly found in most microorganisms [1]. The phenomenon has been known for a long time, but new details of function and regulation of the "metabolic switch" continue to emerge as we begin to apply postgenomic technology to the analysis. Understanding the switch to secondary metabolism is of major importance in biotechnology, where it can contribute to the optimized production of commercially relevant secondary metabolites, such as antibiotics.Here we have used the soil bacterium Streptomyces coelicolor, the model organism of the antibiotics producing genus Streptomyces, to dissect its metabolic switch in unprecedented detail. Reproducible growth of the filamentous Streptomyces species has been a challenge, and especially producing the same quantity of antibiotics in each fermentation has been a major hurdle for conducting systems biology experiments in these species. Some short gene expression time
Genome-wide inference of regulatory networks in Streptomyces coelicolor
Marlene Castro-Melchor, Salim Charaniya, George Karypis, Eriko Takano, Wei-Shou Hu
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-578
Abstract: In this study, more than 500 samples of genome-wide temporal transcriptome data, comprising wild-type and more than 25 regulatory gene mutants of Streptomyces coelicolor probed across multiple stress and medium conditions, were investigated. Information based on transcript and functional similarity was used to update a previously-predicted whole-genome operon map and further applied to predict transcriptional networks constituting modules enriched in diverse functions such as secondary metabolism, and sigma factor. The predicted network displays a scale-free architecture with a small-world property observed in many biological networks. The networks were further investigated to identify functionally-relevant modules that exhibit functional coherence and a consensus motif in the promoter elements indicative of DNA-binding elements.Despite the enormous experimental as well as computational challenges, a systems approach for integrating diverse genome-scale datasets to elucidate complex regulatory networks is beginning to emerge. We present an integrated analysis of transcriptome data and genomic features to refine a whole-genome operon map and to construct regulatory networks at the cistron level in Streptomyces coelicolor. The functionally-relevant modules identified in this study pose as potential targets for further studies and verification.Streptomycetes are soil-living organisms with a complex life cycle that includes formation of aerial mycelia and spores. Members of this genus have large genomes and the capability of producing multiple secondary metabolites, many of which have uses as antibiotics, anti-tumor agents, and immunosuppressants [1]. The genome of Streptomyces coelicolor, the model organism for this high G+C genus, contains 7825 genes. The genome contains more than 20 secondary metabolite clusters and 965 genes encoding proteins predicted to have a regulatory role [2].With more genes than lower eukaryotes and an unusually high number of regulators, dec
Functional analyses of TcrA-a TPR-containing regulatory protein in Streptomyces coelicolor A3 (2)

LIU Jin-man,YANG Ke-qian,

微生物学报 , 2006,
Abstract: In Streptomyces coelicolor A3(2),SCO5433 encodes a TPR domain containing protein designated TcrA(TPR containing regulator A).TcrA is similar in amino acid sequence to AfsR,a well-characterized global regulatory protein in S.coelicolor A3(2).Disruption of tcrA enhanced the production of spore pigment on MM containing glucose or mannitol and also resulted in more diffusible pigment production on MM containing mannitol or MS agar medium,but no significant effects on morphological differentiation were observed.Complementation of tcrA mutation restored the phenotype of tcrA mutant to that of the wild-type strain. These results suggest that tcrA is involved in the regulation of secondary metabolism under defined conditions.In S.coelicolor A3(2),the AfsK/AfsR system positively regulates the production of secondary metabolites while the results of our work suggest that there might be a TcrA-dependent pathway that negatively regulates secondary metabolism.
Optimized submerged batch fermentation strategy for systems scale studies of metabolic switching in Streptomyces coelicolor A3(2)
Alexander Wentzel, Per Bruheim, Anders ?verby, ?yvind M Jakobsen, H?vard Sletta, Walid A M Omara, David A Hodgson, Trond E Ellingsen
BMC Systems Biology , 2012, DOI: 10.1186/1752-0509-6-59
Abstract: By a step-wise approach, cultivation conditions and two fully defined cultivation media were developed and evaluated using strain M145 of S. coelicolor A3(2), providing a high degree of cultivation reproducibility and enabling reliable studies of the effect of phosphate depletion and L-glutamate depletion on the metabolic transition to antibiotic production phase. Interestingly, both of the two carbon sources provided, D-glucose and L-glutamate, were found to be necessary in order to maintain high growth rates and prevent secondary metabolite production before nutrient depletion. Comparative analysis of batch cultivations with (i) both L-glutamate and D-glucose in excess, (ii) L-glutamate depletion and D-glucose in excess, (iii) L-glutamate as the sole source of carbon and (iv) D-glucose as the sole source of carbon, reveal a complex interplay of the two carbon sources in the bacterium's central carbon metabolism.The present study presents for the first time a dedicated cultivation strategy fulfilling the requirements for systems biology studies of metabolic switching in S. coelicolor A3(2). Key results from labelling and cultivation experiments on either or both of the two carbon sources provided indicate that in the presence of D-glucose, L-glutamate was the preferred carbon source, while D-glucose alone appeared incapable of maintaining culture growth, likely due to a metabolic bottleneck at the oxidation of pyruvate to acetyl-CoA.Streptomyces coelicolor A3(2) is the best studied member of the genus Streptomyces[1], which provides the source of numerous antibiotic compounds in clinical use today. The genome sequence of S. coelicolor A3(2) was published in 2002 [2] revealing its genome as one of the largest bacterial genomes known to date. Like most members of the genus, it exhibits a complex life-cycle including the differentiation of substrate mycelium to aerial mycelium and the formation of spores [3]. Upon nutrient limitation, S. coelicolor A3(2) responds with
ArgR of Streptomyces coelicolor Is a Versatile Regulator  [PDF]
Rosario Pérez-Redondo, Antonio Rodríguez-García, Alma Botas, Irene Santamarta, Juan F. Martín, Paloma Liras
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0032697
Abstract: ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSA.
Transcriptomic Analysis of Liquid Non-Sporulating Streptomyces coelicolor Cultures Demonstrates the Existence of a Complex Differentiation Comparable to That Occurring in Solid Sporulating Cultures  [PDF]
Paula Yagüe, Antonio Rodríguez-García, María Teresa López-García, Beatriz Rioseras, Juan Francisco Martín, Jesús Sánchez, Angel Manteca
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0086296
Abstract: Streptomyces species produce many clinically relevant secondary metabolites and exhibit a complex development that includes hyphal differentiation and sporulation in solid cultures. Industrial fermentations are usually performed in liquid cultures, conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that no differentiation took place. The aim of this work was to compare the transcriptomes of S. coelicolor growing in liquid and solid cultures, deepening the knowledge of Streptomyces differentiation. Microarrays demonstrated that gene expression in liquid and solid cultures were comparable and data indicated that physiological differentiation was similar for both conditions. Eighty-six percent of all transcripts showed similar abundances in liquid and solid cultures, such as those involved in the biosynthesis of actinorhodin (actVA, actII-4) and undecylprodigiosin (redF); activation of secondary metabolism (absR1, ndsA); genes regulating hydrophobic cover formation (aerial mycelium) (bldB, bldC, bldM, bldN, sapA, chpC, chpD, chpE, chpH, ramA, ramC, ramS); and even some genes regulating early stages of sporulation (wblA, whiG, whiH, whiJ). The two most important differences between transcriptomes from liquid and solid cultures were: first, genes related to secondary metabolite biosynthesis (CDA, CPK, coelichelin, desferrioxamine clusters) were highly up-regulated in liquid but not in solid cultures; and second, genes involved in the final stages of hydrophobic cover/spore maturation (chpF, rdlA, whiE, sfr) were up-regulated in solid but not in liquid cultures. New information was also provided for several non-characterized genes differentially expressed in liquid and solid cultures which might be regulating, at least in part, the metabolic and developmental differences observed between liquid and solid cultures.
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