RESUMO
Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.
Assuntos
Cianobactérias/citologia , Cianobactérias/metabolismo , Ferroptose , Ferro/metabolismo , Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Cálcio/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Citosol/metabolismo , Glutationa/metabolismo , Resposta ao Choque Térmico , Lipidômica , Lipídeos/química , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Synechocystis/metabolismo , Tilacoides/metabolismoRESUMO
In anaerobic digestion of agro-industrial effluents and livestock wastes, concentrations of ammoniacal nitrogen above 800 mg L-1 are reported to lead to the eutrophication of water bodies. Through the metabolic versatility of microalgae, this nitrogen source can be used and removed, producing carotenoids, phycobiliproteins, polyhydroxyalkanoates, and fatty acids of industrial interest. The challenge of making it feasible is the toxicity of ammoniacal nitrogen to microalgae. Therefore, three strategies were evaluated. The first one was to find species of cyanobacteria with high ammoniacal nitrogen removal efficiency comparing Arthrospira platensis, Synechocystis D202, and Spirulina labyrinthiformis cultivations. The most promising species was cultivated in the second strategy, where cell acclimatization and increasing of the inoculum were evaluated. The cultivation condition that culminated in the best efficiency of ammoniacal nitrogen removal was combined with the third strategy, which consisted of conducting the fed-batch bioprocess. In the batch mode, ammoniacal nitrogen was supplied only once in one fed and was present in high initial concentrations. In fed-batch, multiple feedings with low concentrations of ammoniacal nitrogen were used to decrease the inhibitory effect of ammoniacal nitrogen. Arthrospira platensis showed high potential for ammoniacal nitrogen removal. Using the highest initial cell concentration of Arthrospira platensis cultivated by fed-batch, an increase in the consumption of NH3 to 165.1 ± 1.8 mg L-1 and an ammoniacal nitrogen removal efficiency close to 90% were observed. Under this condition, 180.52 ± 11.67 mg g-1 of phycocyanin was attained. Also, the fed-batch cultivations have the potential to reduce the biomass cost production by 33% in comparison to batch experiments.
Assuntos
Amônia/metabolismo , Cianobactérias/crescimento & desenvolvimento , Nitrogênio/metabolismo , Técnicas de Cultura Celular por Lotes , Biodegradação Ambiental , Cianobactérias/metabolismo , Ficocianina/metabolismo , Spirulina/crescimento & desenvolvimento , Spirulina/metabolismo , Synechocystis/crescimento & desenvolvimento , Synechocystis/metabolismoRESUMO
Cyanobacteria have been considered as promising candidates for sustainable bioproduction from inexpensive raw materials, as they grow on light, carbon dioxide, and minimal inorganic nutrients. In this study, we present a genome-scale metabolic network model for Synechocystis sp. PCC 6803 and study the optimal design of the strain for ethanol production by using a mixed integer linear problem reformulation of a bilevel programming problem that identifies gene knockouts which lead to coupling between growth and product synthesis. Five mutants were found, where the in silico model predicts coupling between biomass growth and ethanol production in photoautotrophic conditions. The best mutant gives an in silico ethanol production of 1.054 mmol·gDW -1 ·h -1 .
Assuntos
Biocombustíveis , Etanol/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Técnicas de Inativação de Genes , Microbiologia Industrial , Redes e Vias Metabólicas , Microrganismos Geneticamente Modificados , Modelos BiológicosRESUMO
The LrtA protein of Synechocystis sp. PCC 6803 intervenes in cyanobacterial post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family of proteins, involved in protein synthesis. In this work, we studied the conformational preferences and stability of isolated LrtA in solution. At physiological conditions, as shown by hydrodynamic techniques, LrtA was involved in a self-association equilibrium. As indicated by Nuclear Magnetic Resonance (NMR), circular dichroism (CD) and fluorescence, the protein acquired a folded, native-like conformation between pH 6.0 and 9.0. However, that conformation was not very stable, as suggested by thermal and chemical denaturations followed by CD and fluorescence. Theoretical studies of its highly-charged sequence suggest that LrtA had a Janus sequence, with a context-dependent fold. Our modelling and molecular dynamics (MD) simulations indicate that the protein adopted the same fold observed in other members of the HPF family (ß-α-ß-ß-ß-α) at its N-terminal region (residues 1100), whereas the C terminus (residues 100197) appeared disordered and collapsed, supporting the overall percentage of overall secondary structure obtained by CD deconvolution. Then, LrtA has a chameleonic sequence and it is the first member of the HPF family involved in a self-association equilibrium, when isolated in solution.
Assuntos
Proteínas de Bactérias/química , Proteínas Ribossômicas/química , Ribossomos/química , Synechocystis/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Soluções , Synechocystis/metabolismo , TermodinâmicaRESUMO
Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Bactérias , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Transporte de Cátions , Proteínas de Escherichia coli , Escherichia coli , Synechocystis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/isolamento & purificação , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/isolamento & purificação , Proteínas de Transporte de Cobre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Synechocystis/metabolismoRESUMO
Cyanobacteria represent the largest and most diverse group of prokaryotes capable of performing oxygenic photosynthesis and are frequently found in environments contaminated with heavy metals. Several studies have been performed in these organisms in order to better understand the effects of metals such as Zn, Cd, Cu, Ni and Co. In Synechocystis sp. PCC 6803, genes involved in Ni, Co, Cu and Zn resistance have been reported. However, proteomic studies for the identification of proteins modulated by heavy metals have not been carried out. In the present work, we have analyzed the proteomic pattern alterations of the cyanobacterium Synechocystis sp. PCC 6803 in response to Ni, Co and Cd in order to identify the metabolic processes affected by these metals. We show that some proteins are commonly regulated in response to the different metal ions, including ribulose1,5-bisphosphate carboxylase and the periplasmic iron-binding protein FutA2, while others, such as chaperones, were specifically induced by each metal. We also show that the main processes affected by the metals are carbon metabolism and photosynthesis, since heavy metals affect proteins required for the correct functioning of these activities. BIOLOGICAL SIGNIFICANCE: This is the first report on the proteomic profile of Synechocystis sp. PCC 6803 wild type and mutant strains for the identification of proteins affected by the heavy metals Ni, Co and Cd. We have identified proteins commonly responsive to all three metals and also chaperones specifically modulated by each metal. Our data also supports previous studies that suggest the existence of additional sensor systems for Co.
Assuntos
Cádmio/química , Cobalto/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Níquel/química , Synechocystis/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Citosol/metabolismo , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Homeostase , Mutação , Proteoma/metabolismoRESUMO
Nowadays, the reconstruction of genome-scale metabolic models is a nonautomatized and interactive process based on decision making. This lengthy process usually requires a full year of one person's work in order to satisfactory collect, analyze, and validate the list of all metabolic reactions present in a specific organism. In order to write this list, one manually has to go through a huge amount of genomic, metabolomic, and physiological information. Currently, there is no optimal algorithm that allows one to automatically go through all this information and generate the models taking into account probabilistic criteria of unicity and completeness that a biologist would consider. This work presents the automation of a methodology for the reconstruction of genome-scale metabolic models for any organism. The methodology that follows is the automatized version of the steps implemented manually for the reconstruction of the genome-scale metabolic model of a photosynthetic organism, Synechocystis sp. PCC6803. The steps for the reconstruction are implemented in a computational platform (COPABI) that generates the models from the probabilistic algorithms that have been developed. For validation of the developed algorithm robustness, the metabolic models of several organisms generated by the platform have been studied together with published models that have been manually curated. Network properties of the models, like connectivity and average shortest mean path of the different models, have been compared and analyzed.
Assuntos
Automação/métodos , Biologia Computacional/métodos , Genoma , Metabolismo , Modelos Biológicos , Synechocystis/genética , Synechocystis/metabolismo , AlgoritmosAssuntos
Proteínas de Bactérias/genética , Etanol/farmacologia , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Proteoma/genética , Synechocystis/efeitos dos fármacos , Adaptação Fisiológica , Motivos de Aminoácidos , Biocombustíveis , Proteínas de Bactérias/metabolismo , Etanol/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Fotossíntese/efeitos dos fármacos , Proteoma/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento , Synechocystis/metabolismo , Transcrição GênicaRESUMO
Microorganisms, plants and animals regulate the synthesis of unsaturated fatty acids (UFAs) during changing environmental conditions as well as in response to nutrients. Unsaturation of fatty acid chains has important structural roles in cell membranes: a proper ratio of saturated to UFAs contributes to membrane fluidity. Alterations in this ratio have been implicated in various disease states including cardiovascular diseases, immune disorders, cancer and obesity. They are also the major components of triglycerides and intermediates in the synthesis of biologically active molecules such as eicosanoids, which mediates fever, inflammation and neurotransmission. UFAs homeostasis in many organisms is achieved by feedback regulation of fatty acid desaturases gene transcription. Here, we review recently discovered components and mechanisms of the regulatory machinery governing the transcription of fatty acid desaturases in bacteria, yeast and animals.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/fisiologia , Transdução de Sinais/fisiologia , Animais , Bacillus subtilis/metabolismo , Regulação da Expressão Gênica , Humanos , Insulina/fisiologia , Leptina/fisiologia , Fluidez de Membrana/fisiologia , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Pseudomonas aeruginosa/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Synechocystis/metabolismoRESUMO
The role of sucrose in cyanobacteria is still not fully understood. It is generally considered a salt-response molecule, and particularly, in Synechocystis sp. strain PCC 6803, it is referred as a secondary osmolyte. We showed that sucrose accumulates transiently in Synechocystis cells at early stages of a salt shock, which could be ascribed to salt activation of sucrose-phosphate synthase (SPS, UDP-glucose: D-fructose-6-phosphate 2-alpha-D-glucosyltransferase; EC 2.4.1.14), the key enzyme in sucrose synthesis pathway, and to an increase of the expression of the SPS encoding gene. Experiments with a mutant strain impaired in sucrose biosynthesis showed that sucrose is essential in stationary phase cells to overcome a later salt stress. Taken together, these results led us to suggest a more intricate function for sucrose than to be an osmoprotectant compound.