RESUMEN
Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine ß-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from Microcystis aeruginosa PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structurally uncharacterized N-terminal region. We provide evidence that CBS-CP12, while combining properties of both regulatory domains, behaves different from CP12 and plant CBSX. It does not form a ternary complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase. Instead, CBS-CP12 decreases the activity of PRK in an AMP-dependent manner. We propose that the novel domain architecture and oligomeric state of CBS-CP12 expand its regulatory function beyond those of CP12 in cyanobacteria.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Cloroplastos/genética , Cistationina betasintasa/genética , Microcystis/genética , Familia de Multigenes , Proteínas Bacterianas/metabolismo , Proteínas de Cloroplastos/metabolismo , Cistationina betasintasa/metabolismo , Microcystis/metabolismo , Dominios ProteicosRESUMEN
The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.
Asunto(s)
ADN de Hongos/química , Proteínas Fúngicas/química , Ácidos Nucleicos Heterodúplex/química , Pichia/enzimología , ARN de Hongos/química , Telomerasa/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , ADN de Hongos/genética , ADN de Hongos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Calor , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Pichia/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Using metabolic and transcriptomic phenotyping, we studied acclimation of cyanobacteria to low inorganic carbon (LC) conditions and the requirements for coordinated alteration of metabolism and gene expression. To analyse possible metabolic signals for LC sensing and compensating reactions, the carboxysome-less mutant ΔccmM and the photorespiratory mutant ΔglcD1/D2 were compared with wild-type (WT) Synechocystis. Metabolic phenotyping revealed accumulation of 2-phosphoglycolate (2PG) in ΔccmM and of glycolate in ΔglcD1/D2 in LC- but also in high inorganic carbon (HC)-grown mutant cells. The accumulation of photorespiratory metabolites provided evidence for the oxygenase activity of RubisCO at HC. The global gene expression patterns of HC-grown ΔccmM and ΔglcD1/D2 showed differential expression of many genes involved in photosynthesis, high-light stress and N assimilation. In contrast, the transcripts of LC-specific genes, such as those for inorganic carbon transporters and components of the carbon-concentrating mechanism (CCM), remained unchanged in HC cells. After a shift to LC, ΔglcD1/D2 and WT cells displayed induction of many of the LC-inducible genes, whereas ΔccmM lacked similar changes in expression. From the coincidence of the presence of 2PG in ΔccmM without CCM induction and of glycolate in ΔglcD1/D2 with CCM induction, we regard a direct role for 2PG as a metabolic signal for the induction of CCM during LC acclimation as less likely. Instead, our data suggest a potential role for glycolate as a signal molecule for enhanced expression of CCM genes.
Asunto(s)
Proteínas Bacterianas/genética , Carbono/metabolismo , Fotosíntesis , Synechocystis/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Luz , Mutación , Fotosíntesis/efectos de la radiación , Synechocystis/genética , Synechocystis/efectos de la radiaciónRESUMEN
Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N(2)-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N(2)-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N(2) fixation was more sensitive to O(2) in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O(2)-scavenging enzyme to protect nitrogenase in extant N(2)-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Oxigenasas de Función Mixta/metabolismo , Nostoc/enzimología , Nostoc/genética , Oxidorreductasas de Alcohol/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Respiración de la Célula , Chlamydomonas reinhardtii/fisiología , Chlamydomonas reinhardtii/efectos de la radiación , Cianobacterias/enzimología , Cianobacterias/genética , Cianobacterias/fisiología , Cianobacterias/efectos de la radiación , Glicolatos/metabolismo , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutación , Fijación del Nitrógeno/fisiología , Nitrogenasa/genética , Nitrogenasa/metabolismo , Nostoc/fisiología , Nostoc/efectos de la radiación , Oxidación-Reducción , Oxígeno/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alineación de SecuenciaRESUMEN
Cyanobacteria invented oxygenic photosynthesis about 3.5 billion years ago. The by-product molecular oxygen initiated the oxygenase reaction of RubisCO, the main carboxylating enzyme in photosynthetic organisms. During oxygenase reaction, the toxic side product 2-phosphoglycolate (2-PG) is produced and must be quickly metabolized. Photorespiratory 2-PG metabolism is used for this purpose by higher plants. The existence of an active 2-PG metabolism in cyanobacteria has been the subject of controversy since these organisms have evolved an efficient carbon-concentrating mechanism (CCM), which should considerably reduce the oxygenase activity of RubisCO. Based on emerging cyanobacterial genomic information, we have found clear indications for the existence of many genes possibly involved in the photorespiratory 2-PG metabolism. Using a genetic approach with the model Synechocystis sp. strain PCC 6803, we generated and characterized defined mutants in these genes to verify their function. Our results show that cyanobacteria perform an active photorespiratory 2-PG metabolism, which employs three routes in Synechocystis: a plant-like cycle, a bacterial-like glycerate pathway, and a complete decarboxylation branch. In addition to the detoxification of 2-PG, this essential metabolism helps cyanobacterial cells acclimate to high light conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Cianobacterias/metabolismo , Glicolatos/metabolismo , Fotosíntesis , Transducción de Señal , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión GénicaRESUMEN
In cyanobacteria, photorespiratory 2-phosphoglycolate (2PG) metabolism is mediated by three different routes, including one route involving the glycine decarboxylase complex (Gcv). It has been suggested that, in addition to conversion of 2PG into non-toxic intermediates, this pathway is important for acclimation to high-light. The photoreduction of O(2) (Mehler reaction), which is mediated by two flavoproteins Flv1 and Flv3 in cyanobacteria, dissipates excess reductants under high-light by the four electron-reduction of oxygen to water. Single and double mutants defective in these processes were constructed to investigate the relation between photorespiratory 2PG-metabolism and the photoreduction of O(2) in the cyanobacterium Synechocystis sp. PCC 6803. The single mutants Deltaflv1, Deltaflv3, and DeltagcvT, as well as the double mutant Deltaflv1/DeltagcvT, were completely segregated but not the double mutant Deltaflv3/DeltagcvT, suggesting that the T-protein subunit of the Gcv (GcvT) and Flv3 proteins cooperate in an essential process. This assumption is supported by the following results: (1) The mutant Deltaflv3/DeltagcvT showed a considerable longer lag phase and sometimes bleached after shifts from slow (low light, air CO(2)) to rapid (standard light, 5% CO(2)) growing conditions. (2) Photoinhibition experiments indicated a decreased ability of the mutant Deltaflv3/DeltagcvT to cope with high-light. (3) Fluorescence measurements showed that the photosynthetic electron chain is reduced in this mutant. Our data suggest that the photorespiratory 2PG-metabolism and the photoreduction of O(2), particularly that catalyzed by Flv3, cooperate during acclimation to high-light stress in cyanobacteria.
Asunto(s)
Aclimatación/efectos de la radiación , Glicolatos/metabolismo , Luz , Oxígeno/metabolismo , Synechocystis/metabolismo , Synechocystis/efectos de la radiación , Aclimatación/efectos de los fármacos , Aerobiosis/efectos de los fármacos , Aerobiosis/efectos de la radiación , Dióxido de Carbono/farmacología , Clorofila/metabolismo , Fluorescencia , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genotipo , Immunoblotting , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Complejo de Proteína del Fotosistema II/metabolismo , Synechocystis/genética , Synechocystis/crecimiento & desarrolloRESUMEN
Vascular endothelial growth factor (VEGF) is known to be upregulated by hypoxia in vitro. However, in vivo data about VEGF regulation in chronic hypoxic diseases are conflicting. We investigated the effects of hypoxia on plasma VEGF concentration in healthy subjects. To control known confounders, such as insulin, glucose concentrations, or exercise, hypoxic effects on VEGF were studied during experimentally clamping glucose concentrations at rest. In a double-blind crossover study design, we induced hypoxia for 30 min by decreasing oxygen saturation to 75% (vs. normoxic control) in 14 healthy men. Plasma VEGF concentration was determined at baseline, immediately after hypoxia had ended, and after a further 150 min. Levels of its soluble (s)Flt-1 receptor were assessed at baseline and at the end of the clamp. In parallel, catecholamine and cortisol levels were monitored. To investigate potential effects of glucose administration on the release of VEGF, we performed a third session, reducing glucose infusion for 30 min while serum insulin was held stable thereby inducing hypoglycemia. Hypoxia decreased VEGF levels compared with the normoxic control (P<0.05). VEGF concentrations increased during hypoglycemia (P<0.02) but were comparable to the normoglycemic control at the end of the clamp (P>0.80). sFlt-1 receptor concentration remained unchanged during hypoxia and hypoglycemia compared with control (both P>0.4). Epinephrine concentration (P<0.01) increased upon hypoxia, whereas norepinephrine and cortisol did not change. Contrary to in vitro studies, in healthy humans hypoxia decreases plasma VEGF concentration, suggesting that systemic VEGF concentration may be differently regulated than the expression on cellular basis.