36 research outputs found
Genome sequencing analysis of Streptomyces coelicolor mutants that overcome the phosphate-depending vancomycin lethal effect
Abstract Background Glycopeptide antibiotics inhibit bacterial cell-wall synthesis, and are important for the treatment of infections caused by multi drug-resistant strains of enterococci, streptococci and staphylococci. The main mechanism by which bacteria resist the action of glycopeptides is by producing a modified cell-wall in which the dipeptide D-Alanine-D-Alanine is substituted by D-Alanine-D-Lactate or D-Alanine-D-Serine. Recently, it has been shown that inorganic phosphate (Pi) induces hypersensitivity to vancomycin in Streptomyces coelicolor (which is highly resistant to the antibiotic in low-Pi media). This finding was surprising because the bacterium possesses the entire set of genes responsible for vancomycin resistance (VR); including those coding for the histidine kinase/response regulator pair VanS/VanR that activates the system. Results This work shows that high Pi amounts in the medium hamper the activation of the van promoters and consequently inhibit VR in S. coelicolor; i.e. the repression effect being stronger when basic or acidic forms of the nutrient are used. In addition, this work shows that lysozyme resistance is also highly regulated by the Pi concentration in the medium. At least five different mutations contribute to the overcoming of this repression effect over VR (but not over lysozyme resistance). Therefore, the interconnection of VR and lysozyme resistance mechanisms might be inexistent or complex. In particular, two kinds of mutant in which Pi control of VR has been lost (one class expresses the van genes in a constitutive manner; the other retains inducibility by vancomycin) have been isolated and further characterized in this study. Sequencing revealed that the first class of mutation conferred a single amino acid substitution in the second transmembrane helix of the VanS protein; whereas the other class hampered the expression or activity of a putative homolog (SCO1213) to the staphylococcal GatD protein. Complementation, phenotypic and bioinformatics analyses identified SCO1213, and its upstream gene (i.e. murT), as relevant genetic determinants involved with VR in S. coelicolor. Conclusion The genomic approach of this study together with other genetic and phenotypic analyses has allowed the identification of the uncharacterized murT-gatD Streptomyces genes and the characterization of their involvement with the Pi control of VR in S. coelicolor
Mecanismo molecular del control por fosfato de la cascada de expresión de los genes de biosíntesis de metabolitos secundarios en Streptomyces coelicolor
216 p.Se analiza la estructura operadora de pitH2, glpQ1, glpQ2 y afsS al conocimiento de la caja PHO en Streptomyces coelicolor; La inducción y represión génica por la proteína PhoP; La respuesta primaria ejercida por PhoP ante la escasez de fosfato en el medio; La interacción de las regulaciones por carbono, nitrógeno y fósforo y el efecto de PhoP en el metabolismo secundari
Characterization of the keratinolytic activity of three streptomyces strains and impact of their co-cultivation on this activity
Producción CientíficaIn this study, we describe the characterization of three efficient chicken feather-degrading Streptomyces bacteria isolated from honeybee samples and assess the impact of their co-cultivation on this activity and antistaphylococcal activity. Streptomyces griseoaurantiacus AD2 was the strain showing the highest keratinolytic activity (4000 U × mL−1), followed by Streptomyces albidoflavus AN1 and Streptomyces drozdowiczii AD1, which both generated approximately 3000 U × mL−1. Moreover, a consortium constituted of these three strains was able to use chicken feathers as its sole nutrient source and growth in such conditions led to a significant increase in antibiotic production. S. griseoaurantiacus AD2 was the only strain that exhibited weak antimicrobial activity against Staphylococcus aureus. UPLC analyses revealed that a significant number of peaks detected in the extracts of co-cultures of the three strains were missing in the extracts of individual cultures. In addition, the production of specialized metabolites, such as undecylprodigiosin and manumycin A, was clearly enhanced in co-culture conditions, in agreement with the results of the antimicrobial bioassays against S. aureus. Our results revealed the benefits of co-cultivation of these bacterial species in terms of metabolic wealth and antibiotic production. Our work could thus contribute to the development of novel microbial-based strategies to valorize keratin waste.Fondo Europeo de Desarrollo Regional (FEDER), (grant TCUE 2021–2023) - (project 067/229111)Unión Europea, H2020-MSCA-IF-2016 - (grant UE-18-VANRESTREP-740080
Cross‐talk between two global regulators in Streptomyces: PhoP and AfsR interact in the control of afsS, pstS and phoRP transcription
[EN] The regulatory proteins AfsR and PhoP control expression of the biosynthesis of actinorhodin and undecylprodigiosin in Streptomyces coelicolor. Electrophoretic mobility shift assays showed that PhoPDBD does not bind directly to the actII-ORF4, redD and atrA promoters, but it binds to the afsS promoter, in a region overlapping with the AfsR operator. DNase I footprinting studies revealed a PhoP protected region of 26 nt (PHO box; two direct repeats of 11 nt) that overlaps with the AfsR binding sequence. Binding experiments indicated a competition between AfsR and PhoP; increasing concentrations of PhoPDBD resulted in the disappearance of the AfsR-DNA complex. Expression studies using the reporter luxAB gene coupled to afsS promoter showed that PhoP downregulates afsS expression probably by a competition with the AfsR activator. Interestingly, AfsR binds to other PhoP-regulated promoters including those of pstS (a component of the phosphate transport system) and phoRP (encoding the two component system itself). Analysis of the AfsR-protected sequences in each of these promoters allowed us to distinguish the AfsR binding sequence from the overlapping PHO box. The reciprocal regulation of the phoRP promoter by AfsR and of afsS by PhoP suggests a fine interplay of these regulators on the control of secondary metabolismSIThis project was supported by grants of the ‘Comisión Interministerial de Ciencia y Tecnología’ (BIO2003-01489, BIO2006-14853-C02-01); Ministry of Education, Science and Technology, Madrid (GEN2003-20245-C09-01); the ERA-NET SySMO Project (GEN2006-27745-E/SYS) and the European Union (ACTINOGEN LSHM-CT-2004-005224). F. Santos-Beneit received a fellowship of the FPI programme (Ministry of Education, Spain). We thank S. Horinouchi for the plasmids containing the afsR gene, M. Bibb for strain S. coelicolor M513 (ΔafsR) and B. Martín, J. Merino, A. Casenave and B. Aguado for excellent technical assistanc
Recent trends and advances in biogas upgrading and methanotrophs-based valorization
Producción CientíficaThe global quest for sustainability in industrial activities and waste management has recently boosted biogas production worldwide. However, the rapid decrease in the levelized cost of electricity of renewable energies will soon entail electricity prices from biogas much higher than those from solar or wind power. In this context, the upgrading of biogas into biomethane represents an alternative to on-site biogas combustion. Membrane separation technology is rapidly dominating the biogas upgrading market and displacing scrubbing and adsorption technologies driven by the recent breakthroughs in material science. Similarly, biogas biorefineries have recently emerged as an innovative platform for biogas valorization capable of biotransforming methane into added value products. The limited number of bioproducts naturally synthesized by methanotrophs can be boosted via metabolic engineering of methanotrophs, while novel bioreactor configurations capable of supporting a cost-effective methane mass transfer from the gas phase to the methanotrophic broth are currently under investigation to facilitate the full scale implementation of biogas biorefineries.Junta de Castilla y Leon - FEDER (program CLU 2017–09, CL-EI-2021–07 and UIC 315)European Commission-H2020-MSCA-IF-2019 project UP-GRAD (894515)Ministerio de Ciencia, Innovación y Universidades (project IJC2019–040495-I
Genome‐wide transcriptomic and proteomic analysis of the primary response to phosphate limitation in Streptomyces coelicolor M145 and in a ΔphoP mutant
[EN] Phosphate limitation in Streptomyces and in other bacteria triggers expression changes of a large number of genes. This response is mediated by the two-component PhoR-PhoP system. A Streptomyces coelicolor ΔphoP mutant (lacking phoP) has been obtained by gene replacement. A genome-wide analysis of the primary response to phosphate limitation using transcriptomic and proteomic studies has been made in the parental S. coelicolor M145 and in the ΔphoP mutant strains. Statistical analysis of the contrasts between the four sets of data generated (two strains under two phosphate conditions) allowed the classification of all genes into 12 types of profiles. The primary response to phosphate limitation involves upregulation of genes encoding scavenging enzymes needed to obtain phosphate from different phosphorylated organic compounds and overexpression of the high-affinity phosphate transport system pstSCAB. Clear interactions have been found between phosphate metabolism and expression of nitrogen-regulated genes and between phosphate and nitrate respiration genes. PhoP-dependent repressions of antibiotic biosynthesis and of the morphological differentiation genes correlated with the observed ΔphoP mutant phenotype. Bioinformatic analysis of the presence of PHO boxes (PhoP-binding sequences) in the upstream regions of PhoP-controlled genes were validated by binding of PhoP, as shown by electrophoretic mobility shift assaysSIThis work was supported by grants of the CICYT (Madrid,Spain) to J. F. M. (BIO2003-01489 and GEN2003-20245-C09-01) and of the European Union (Project ActinoGEN OJ 2003/C164). A. Rodríguez was supported by a Torres Quevedo contract (PTQ2002-0468), and F. Santos received a FPI fellowship from the Ministry of Education (Madrid, Spain). We thank J. Merino, B. Martín, A. Casenave, and B. Aguado for excellent technical assistance, and V. Mersinias, O. Domínguez, L. Lombardía, and J.C. Oliveros for helpful discussion of microarray procedure
Phosphate control over nitrogen metabolism in Streptomyces coelicolor: direct and indirect negative control of glnR, glnA, glnII and amtB expression by the response regulator PhoP
[EN] Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxescomposed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB - glnK - glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. 'Information theory' quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussedSIFunding by Comisión Interministerial de Ciencia y Tecnología [BIO2003-01489, BIO2006-14853-C02-01]; the ‘Ministerio de Ciencia e Innovación’, Madrid [GEN2003-20245-C09-01]; the AECID (Agencia Española de Cooperación Internacional para el Desarrollo), ‘Ministerio de Asuntos Exteriores y de Cooperación’, Madrid [A/010257/07]; the ERA-NET SySMO Project [GEN2006-27745-E/SYS]; and the European Union (ACTINOGEN LSHM-CT-2004-005224). F.P.U. fellowship of the Ministerio de Ciencia e Innovación (Spain) (to K.A.); fellowship of the F.P.I. program (Ministerio de Ciencia e Innovación, Spain) (to F.S.B.). Funding for open access charge: Institute of Biotechnology of Leó
Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor
Streptomyces tsukubaensis as a new model for carbon repression: transcriptomic response to tacrolimus repressing carbon sources
[EN] In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces tsukubaensis. A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster. In addition, we detected important differences in the transcriptional response to glucose between S. tsukubaensis and the model species Streptomyces coelicolor. A number of genes encoding key players of morphological and biochemical differentiation were strongly and permanently downregulated by the carbon sources. Finally, we identified several genes showing transcriptional profiles highly correlated to that of the tacrolimus biosynthetic pathway regulator FkbN that might be potential candidates for the improvement of tacrolimus productionSIThis study was funded by the Government of Spain (grant number PIM2010-EEI00677). María Ordóñez-Robles received a FPU grant from the Government of Spain (grant number AP2009-4508)
Modulation of multiple gene clusters’ expression by the PAS-LuxR transcriptional regulator PteF
[EN] PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from Streptomyces avermitilis. Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator. Transcriptomic analyses were performed in S. avermitilis ΔpteF and its parental strain. Both strains were grown in a YEME medium without sucrose, and the samples were taken at exponential and stationary growth phases. A total of 257 genes showed an altered expression in the mutant, most of them at the exponential growth phase. Surprisingly, despite PteF being considered an activator, most of the genes affected showed overexpression, thereby suggesting a negative modulation. The affected genes were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation, among others, but were particularly related to secondary metabolite biosynthesis. Notably, 10 secondary metabolite gene clusters out of the 38 encoded by the genome showed altered expression profiles in the mutant, suggesting a regulatory role for PteF that is wider than expected. The transcriptomic results were validated by quantitative reverse-transcription polymerase chain reaction. These findings provide important clues to understanding the intertwined regulatory machinery that modulates antibiotic biosynthesis in StreptomycesSIThis research was funded by the Spanish Ministerio de Economía, Industria y Competitividad (grants BIO2013-42983-P and PCIN-2016-190 to J.F.A.), FPU contracts of the Ministerio de Educación, Cultura y Deporte (AP2007-02055 to T.D.P., FPU13/01537 to A.d.P.), a contract from the Junta de Castilla y León co-financed by the European Social Fund (to E.G.B.), and a fellowship from the Portuguese Fundação para a Ciência e a Tecnologia (SFRH/BD/64006/2009 to C.M.V.)