RESUMEN
Stapylococcus aureus colonises the nose of healthy individuals but can also cause a wide range of infections. Amino acid (AA) synthesis and their availability is crucial to adapt to conditions encountered in vivo. Most S. aureus genomes comprise all genes required for AA biosynthesis. Nevertheless, different strains require specific sets of AAs for growth. In this study we show that regulation inactivates pathways under certain conditions which result in these observed auxotrophies. We analyzed in vitro and modeled in silico in a Boolean semiquantitative model (195 nodes, 320 edges) the regulatory impact of stringent response (SR) on AA requirement in S. aureus HG001 (wild-type) and in mutant strains lacking the metabolic regulators RSH, CodY and CcpA, respectively. Growth in medium lacking single AAs was analyzed. Results correlated qualitatively to the in silico predictions of the final model in 92% and quantitatively in 81%. Remaining gaps in our knowledge are evaluated and discussed. This in silico model is made fully available and explains how integration of different inputs is achieved in SR and AA metabolism of S. aureus. The in vitro data and in silico modeling stress the role of SR and central regulators such as CodY for AA metabolisms in S. aureus.
Asunto(s)
Aminoácidos Esenciales/metabolismo , Staphylococcus aureus/crecimiento & desarrollo , Aminoácidos Esenciales/biosíntesis , Aminoácidos Esenciales/deficiencia , Simulación por Computador , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas , Modelos Biológicos , Mutación , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
Ruminants and humans are unable to synthesize essential amino acids (EAAs) and conditionally essential amino acids (CEAAs) under normal conditions and need to acquire them from plant sources. Maize plays, as a major crop, a central role in global food security. However, maize is deficient in several EAAs and CEAAs. Genetic engineering has been successfully used to enrich the EAA content of maize to some extent, including the content of Lys, Trp, and Met. However, research on other EAAs is lacking. Genetic engineering provides several viable approaches for increasing the EAA content in maize, including transformation of a single gene, transformation of multiple genes in a single cassette, overexpression of putative amino acid transporters, engineering the amino acid biosynthesis pathway including silencing of feedback inhibition enzymes, and overexpression of major enzymes in this pathway. These challenging processes require a deep understanding of the biosynthetic and metabolic pathways of individual amino acids, and the interaction of individual amino acids with other metabolic pathways.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos Esenciales/biosíntesis , Vías Biosintéticas , Ingeniería Genética/métodos , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Zea mays/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos Esenciales/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Zea mays/genéticaRESUMEN
Ingesting protein-containing supplements and foods provides essential amino acids (EAA) necessary to increase muscle and whole-body protein synthesis (WBPS). Large variations exist in the EAA composition of supplements and foods, ranging from free-form amino acids to whole protein foods. We sought to investigate how changes in peripheral EAA after ingesting various protein and free amino acid formats altered muscle and whole-body protein synthesis. Data were compiled from four previous studies that used primed, constant infusions of L-(ring-2H5)-phenylalanine and L-(3,3-2H2)-tyrosine to determine fractional synthetic rate of muscle protein (FSR), WBPS, and circulating EAA concentrations. Stepwise regression indicated that max EAA concentration (EAACmax; R2 = 0.524, p < 0.001), EAACmax (R2 = 0.341, p < 0.001), and change in EAA concentration (ΔEAA; R = 0.345, p < 0.001) were the strongest predictors for postprandial FSR, Δ (change from post absorptive to postprandial) FSR, and ΔWBPS, respectively. Within our dataset, the stepwise regression equation indicated that a 100% increase in peripheral EAA concentrations increases FSR by ~34%. Further, we observed significant (p < 0.05) positive (R = 0.420-0.724) correlations between the plasma EAA area under the curve above baseline, EAACmax, ΔEAA, and rate to EAACmax to postprandial FSR, ΔFSR, and ΔWBPS. Taken together our results indicate that across a large variety of EAA/protein-containing formats and food, large increases in peripheral EAA concentrations are required to drive a robust increase in muscle and whole-body protein synthesis.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Aminoácidos Esenciales/farmacología , Proteínas Musculares/biosíntesis , Proteínas Musculares/farmacocinética , Biosíntesis de Proteínas , Envejecimiento/fisiología , Aminoácidos/metabolismo , Aminoácidos/farmacocinética , Suplementos Dietéticos , Ingestión de Alimentos , Alimentos , Humanos , Cinética , Masculino , Metabolismo , Músculo Esquelético/metabolismo , Fenilalanina , Periodo Posprandial , Proteína de Suero de LecheRESUMEN
To feed the world's growing population, increasing the yield of crops is not the only important factor, improving crop quality is also important, and it presents a significant challenge. Among the important crops, horticultural crops (particularly fruits and vegetables) provide numerous health compounds, such as vitamins, antioxidants, and amino acids. Essential amino acids are those that cannot be produced by the organism and, therefore, must be obtained from diet, particularly from meat, eggs, and milk, as well as a variety of plants. Extensive efforts have been devoted to increasing the levels of essential amino acids in plants. Yet, these efforts have been met with very little success due to the limited genetic resources for plant breeding and because high essential amino acid content is generally accompanied by limited plant growth. With a deep understanding of the biosynthetic pathways of essential amino acids and their interactions with the regulatory networks in plants, it should be possible to use genetic engineering to improve the essential amino acid content of horticultural plants, rendering these plants more nutritionally favorable crops. In the present report, we describe the recent advances in the enhancement of essential amino acids in horticultural plants and possible future directions towards their bio-fortification.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Aminoácidos Esenciales/genética , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Vías Biosintéticas/genética , Cruzamiento , Productos Agrícolas/enzimología , Alimentos Fortificados , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Ingeniería Genética , Valor Nutritivo , Desarrollo de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Various animals are associated with specific endosymbiotic microorganisms that provide the host with essential nutrients or confer protection against natural enemies. Genomic analyses of the many endosymbioses that are found in plant sap-feeding hemipteran insects have revealed independent acquisitions - and occasional replacements - of endosymbionts, such that many of these endosymbioses involve two or more microbial partners. In this Review, I discuss how partitioning of the genetic capacity for metabolic function between different endosymbionts has sustained nutritional function in multi-partner endosymbioses, and how the phenotypic traits of these endosymbionts can be shaped by co-evolutionary interactions with both co-occurring microbial taxa and the host, which often operate over long evolutionary timescales.
Asunto(s)
Aminoácidos Esenciales/metabolismo , Fenómenos Fisiológicos Bacterianos , Hemípteros/microbiología , Hemípteros/fisiología , Simbiosis , Aminoácidos Esenciales/biosíntesis , Animales , Evolución Biológica , Vías Biosintéticas , Hemípteros/genética , Fenotipo , Filogenia , Plantas/química , Simbiosis/genéticaRESUMEN
Although amino acids are critical for all forms of life, only proteogenic amino acids that humans and animals cannot synthesize de novo and therefore must acquire in their diets are classified as essential. Nine amino acids-lysine, methionine, threonine, phenylalanine, tryptophan, valine, isoleucine, leucine, and histidine-fit this definition. Despite their nutritional importance, several of these amino acids are present in limiting quantities in many of the world's major crops. In recent years, a combination of reverse genetic and biochemical approaches has been used to define the genes encoding the enzymes responsible for synthesizing, degrading, and regulating these amino acids. In this review, we describe recent advances in our understanding of the metabolism of the essential amino acids, discuss approaches for enhancing their levels in plants, and appraise efforts toward their biofortification in crop plants.
Asunto(s)
Aminoácidos Esenciales/metabolismo , Productos Agrícolas/metabolismo , Genes de Plantas , Aminoácidos Esenciales/biosíntesis , Productos Agrícolas/enzimología , Productos Agrícolas/genética , Ingeniería Genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Aspartate-semialdehyde dehydrogenase (ASADH; EC 1.2.1.11) is a key enzyme in the biosynthesis of essential amino acids in prokaryotes and fungi, inhibition of ASADH leads to the development of novel antitubercular agents. In the present work, a combined structure and ligand-based pharmacophore modeling, molecular docking, and molecular dynamics (MD) approaches were employed to identify potent inhibitors of mycobacterium tuberculosis (Mtb)-ASADH. The structure-based pharmacophore hypothesis consists of three hydrogen bond acceptor (HBA), two negatively ionizable, and one positively ionizable center, while ligand-based pharmacophore consists of additional one HBA and one hydrogen bond donor features. The validated pharmacophore models were used to screen the chemical databases (ZINC and NCI). The screened hits were subjected to ADME and toxicity filters, and subsequently to the molecular docking analysis. Best-docked 25 compounds carry the characteristics of highly electronegative functional groups (-COOH and -NO2) on both sides and exhibited the H-bonding interactions with highly conserved residues Arg99, Arg249, and His256. For further validation of docking results, MD simulation studies were carried out on two representative compounds NSC51108 and ZINC04203124. Both the compounds remain bound to the key active residues of Mtb-ASADH during the MD simulations. These identified hits can be further used for lead optimization and in the design more potent inhibitors against Mtb-ASADH.
Asunto(s)
Aminoácidos Esenciales/química , Aspartato-Semialdehído Deshidrogenasa/química , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimología , Aminoácidos Esenciales/biosíntesis , Aspartato-Semialdehído Deshidrogenasa/genética , Aspartato-Semialdehído Deshidrogenasa/metabolismo , Enlace de Hidrógeno , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Células Procariotas/enzimología , Conformación Proteica , Relación Estructura-ActividadRESUMEN
BACKGROUND: The lentil plant, Lens culinaris L., is a member of the Leguminoceae family and constitutes one of the most important traditional dietary components. The purpose of the current study was to investigate the effects of sprouting for 3, 4, 5 and 6 days on proximate, bioactive compounds and antioxidative characteristics of lentil (Lens culinaris) sprouts. MATERIAL AND METHODS: Lentil seeds were soaked in distilled water (1:10, w/v) for 12 h at room temperature (~25°C), then kept between thick layers of cotton cloth and allowed to germinate in the dark for 3, 4, 5 and 6 days. The nutritional composition, protein solubility, free amino acids, antinutritional factors, bioactive compounds and antioxidant activity of raw and germinated samples were determined using standard official procedures. RESULTS: Sprouting process caused significant (P ≤ 0.05) increases in moisture, protein, ash, crude fiber, protein solubility, free amino acids, total, reducing and nonreducing sugars. However, oil content, antinutritional factors (tannins and phytic acid) significantly (P ≤ 0.05) decreased. Results indicated that total essential amino acids of lentil seeds protein formed 38.10% of the total amino acid content. Sulfur-containing amino acids were the first limiting amino acid, while threonine was the second limiting amino acid in raw and germinated lentil seeds. Sprouting process has a positive effect on the essential amino acid contents and protein efficiency ratio (PER) of lentil sprouts. Phenolics content increased from 1341.13 mg/100 g DW in raw lentil seeds to 1411.50, 1463.00, 1630.20 and 1510.10 in those samples germinated for 3, 4, 5 and 6 days, respectively. Sprouted seeds had higher DPPH radical scavenging and reducing power activities. CONCLUSIONS: Based on these results, sprouting process is recommended to increase nutritive value, and antioxidant activity of lentil seeds.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Antioxidantes/metabolismo , Germinación , Lens (Planta)/metabolismo , Ácido Fítico/biosíntesis , Plantones/metabolismo , Taninos/biosíntesis , Aminoácidos Esenciales/análisis , Antioxidantes/análisis , Antioxidantes/química , Carbohidratos/química , Carbohidratos de la Dieta/análisis , Carbohidratos de la Dieta/metabolismo , Grasas de la Dieta/análisis , Fibras de la Dieta/análisis , Egipto , Flavonoides/análisis , Flavonoides/biosíntesis , Flavonoides/química , Humanos , Lens (Planta)/química , Lens (Planta)/crecimiento & desarrollo , Valor Nutritivo , Ácido Fítico/análisis , Proteínas de Vegetales Comestibles/análisis , Proteínas de Vegetales Comestibles/biosíntesis , Proteínas de Vegetales Comestibles/química , Polifenoles/análisis , Polifenoles/biosíntesis , Polifenoles/química , Plantones/química , Plantones/crecimiento & desarrollo , Semillas/química , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Solubilidad , Taninos/análisis , Factores de TiempoRESUMEN
Essential amino acids (EAA) consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS) and betaine-homocysteine S-methyltransferase (BHMT) diverged from the expected Tree of Life (ToL) relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Secuencia Conservada/genética , Acetolactato Sintasa/genética , Acetolactato Sintasa/metabolismo , Secuencia de Aminoácidos , Animales , Betaína-Homocisteína S-Metiltransferasa/genética , Betaína-Homocisteína S-Metiltransferasa/metabolismo , Evolución Biológica , Hongos/enzimología , Hongos/genética , Humanos , Filogenia , Plantas/enzimología , Plantas/genética , Sacaropina Deshidrogenasas/genética , Sacaropina Deshidrogenasas/metabolismoRESUMEN
Various animals derive nutrients from symbiotic microorganisms with much-reduced genomes, but it is unknown whether, and how, the supply of these nutrients is regulated. Here, we demonstrate that the production of essential amino acids (EAAs) by the bacterium Buchnera aphidicola in the pea aphid Acyrthosiphon pisum is elevated when aphids are reared on diets from which that EAA are omitted, demonstrating that Buchnera scale EAA production to host demand. Quantitative proteomics of bacteriocytes (host cells bearing Buchnera) revealed that these metabolic changes are not accompanied by significant change in Buchnera or host proteins, suggesting that EAA production is regulated post-translationally. Bacteriocytes in aphids reared on diet lacking the EAA methionine had elevated concentrations of both methionine and the precursor cystathionine, indicating that methionine production is promoted by precursor supply and is not subject to feedback inhibition by methionine. Furthermore, methionine production by isolated Buchnera increased with increasing cystathionine concentration. We propose that Buchnera metabolism is poised for EAA production at certain maximal rates, and the realized release rate is determined by precursor supply from the host. The incidence of host regulation of symbiont nutritional function via supply of key nutritional inputs in other symbioses remains to be investigated.
Asunto(s)
Aminoácidos Esenciales/metabolismo , Áfidos/microbiología , Áfidos/fisiología , Buchnera/metabolismo , Proteoma , Aminoácidos Esenciales/biosíntesis , Animales , Áfidos/genética , Áfidos/crecimiento & desarrollo , Dieta , Metionina/biosíntesis , Metionina/metabolismo , Ninfa/genética , Ninfa/crecimiento & desarrollo , Ninfa/microbiología , Ninfa/fisiologíaRESUMEN
Threonine is a nutritionally essential amino acid (EAA) for the growth and development of humans and other nonruminant animals and must be provided in diets to sustain life. The aim of this study was to synthesize threonine in mammalian cells through transgenic techniques. To achieve this goal, we combined the genes involved in bacterial threonine biosynthesis pathways into a single open reading frame separated by self-cleaving peptides (2A) and then linked it into a transposon system (piggyBac). The plasmids pEF1a-IRES-GFP-E2F-his and pEF1a-IRES-GFP-M2F-his expressed Escherichia coli homoserine kinase and threonine synthase efficiently in mouse cells and enabled cells to synthesize threonine from homoserine. This biosynthetic pathway occurred with a low level of efficiency in transgenic mice. Three transgenic mice were identified by Southern blot from 72 newborn mice, raising the possibility that a high level of expression of these genes in mouse embryos might be lethal. The results indicated that it is feasible to synthesize threonine in animal cells using genetic engineering technology. Further work is required to improve the efficiency of this method for introducing genes into mammals. We propose that the transgenic technology provides a promising means to enhance the synthesis of nutritionally EAAs in farm animals and to eliminate or reduce supplementation of these nutrients in diets for livestock, poultry and fish.
Asunto(s)
Aminoácidos Esenciales , Regulación Enzimológica de la Expresión Génica , Aminoácidos Esenciales/biosíntesis , Aminoácidos Esenciales/genética , Animales , Escherichia coli/enzimología , Escherichia coli/genética , Ratones , Ratones Transgénicos , Células 3T3 NIHRESUMEN
Amino acids play important roles in both human and animal nutrition and in the maintenance of health. Here, amino acids are classified into three groups: first, essential amino acids, which are essential to nutrition; second, functional amino acids, recently found to be important in the promotion of physiological functions; and third, dipeptides, which are used to resolve problematic features of specific free amino acids, such as their instability or insolubility. This review focusses on recent researches concerning the microbial production of essential amino acids (lysine and methionine), functional amino acids (histidine and ornithine), and a dipeptide (L-alanyl-L-glutamine).
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Aminoácidos Esenciales/provisión & distribución , Biotecnología/métodos , Dipéptidos/biosíntesis , Dipéptidos/provisión & distribución , Alimentos Funcionales/provisión & distribución , Aminoácidos Esenciales/metabolismo , Animales , Corynebacterium glutamicum/metabolismo , Dipéptidos/metabolismo , Fermentación , Humanos , Ingeniería Metabólica , Péptido Sintasas/metabolismoRESUMEN
Metabolic crossfeeding is an important process that can broadly shape microbial communities. However, little is known about specific crossfeeding principles that drive the formation and maintenance of individuals within a mixed population. Here, we devised a series of synthetic syntrophic communities to probe the complex interactions underlying metabolic exchange of amino acids. We experimentally analyzed multimember, multidimensional communities of Escherichia coli of increasing sophistication to assess the outcomes of synergistic crossfeeding. We find that biosynthetically costly amino acids including methionine, lysine, isoleucine, arginine, and aromatics, tend to promote stronger cooperative interactions than amino acids that are cheaper to produce. Furthermore, cells that share common intermediates along branching pathways yielded more synergistic growth, but exhibited many instances of both positive and negative epistasis when these interactions scaled to higher dimensions. In more complex communities, we find certain members exhibiting keystone species-like behavior that drastically impact the community dynamics. Based on comparative genomic analysis of >6,000 sequenced bacteria from diverse environments, we present evidence suggesting that amino acid biosynthesis has been broadly optimized to reduce individual metabolic burden in favor of enhanced crossfeeding to support synergistic growth across the biosphere. These results improve our basic understanding of microbial syntrophy while also highlighting the utility and limitations of current modeling approaches to describe the dynamic complexities underlying microbial ecosystems. This work sets the foundation for future endeavors to resolve key questions in microbial ecology and evolution, and presents a platform to develop better and more robust engineered synthetic communities for industrial biotechnology.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Bacterias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Interacciones Microbianas , Aminoácidos/biosíntesis , Bacterias/metabolismo , Técnicas de Cocultivo , Ecología , Ecosistema , Regulación Bacteriana de la Expresión Génica , Modelos Biológicos , Filogenia , TemperaturaRESUMEN
BACKGROUND: Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont's contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here. RESULTS: Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes. CONCLUSION: We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Betaproteobacteria/genética , Transferencia de Gen Horizontal , Simbiosis , Trypanosomatina/genética , Trypanosomatina/microbiología , Betaproteobacteria/fisiología , Evolución Biológica , Genoma Bacteriano , Filogenia , Trypanosomatina/clasificación , Trypanosomatina/metabolismoRESUMEN
Humans, as well as farm animals, cannot synthesize a number of essential amino acids, which are critical for their survival. Hence, these organisms must obtain these essential amino acids from their diets. Cereal and legume crops, which represent the major food and feed sources for humans and livestock worldwide, possess limiting levels of some of these essential amino acids, particularly Lys and Met. Extensive efforts were made to fortify crop plants with these essential amino acids using traditional breeding and mutagenesis. However, aside from some results obtained with maize, none of these approaches was successful. Therefore, additional efforts using genetic engineering approaches concentrated on increasing the synthesis and reducing the catabolism of these essential amino acids and also on the expression of recombinant proteins enriched in them. In the present review, we discuss the basic biological aspects associated with the synthesis and accumulation of these amino acids in plants and also describe recent developments associated with the fortification of crop plants with essential amino acids by genetic engineering approaches.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Productos Agrícolas/metabolismo , Alimentos Fortificados , Lisina/biosíntesis , Metionina/biosíntesis , Vías Biosintéticas , Cruzamiento , Regulación de la Expresión Génica de las Plantas , Ingeniería Genética , Valor Nutritivo , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes/biosíntesisRESUMEN
The aim of the present study was to test whether the dietary non-essential/conditionally essential amino acid composition has an effect on growth and protein utilisation and on δ13C of individual amino acids in rainbow trout (Oncorhynchus mykiss). Trout were reared on six purified diets containing only synthetic amino acids in place of protein. Diet 1 mimicked the amino acid composition of fishmeal, in diet 2, cysteine (Cys), glycine (Gly), proline (Pro) and tyrosine (Tyr) were isonitrogenously replaced by their precursor amino acids serine (Ser), glutamic acid (Glu) and phenylalanine (Phe), and in diet 3, alanine (Ala), asparagine and aspartate, Cys, Gly, Pro, Ser and Tyr were isonitrogenously replaced by Glu. Diets 4, 5 and 6 resembled diets 1, 2 and 3 except that Glu contained 0·1 % 13C-enriched Glu. A control group was reared on a fishmeal-based diet. A total of forty-two trout (4·7 (sd 0·57) g) were fed one of the diets at a level of 3·5 % body mass for 10 weeks in a flow-through system. Dietary non-essential amino acid composition significantly influenced protein gain (P < 0·025) and δ13C of Ala, arginine (Arg), Gly, histidine (His), Phe and Tyr. Non-enriched Glu was predominantly found in trout fed 13C-enriched Glu, which is consistent with the fact that Glu has been shown to be used extensively in the gut as an energy source but is less consistent with the enrichment of Pro in fish fed diet 6 compared with fish fed diet 3. Further research is required to better understand the mechanisms that lead to the alteration of amino acid δ13C between diet and body tissues.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Alimentación Animal , Acuicultura/métodos , Isótopos de Carbono/metabolismo , Proteínas en la Dieta/metabolismo , Oncorhynchus mykiss/crecimiento & desarrollo , Animales , Cromatografía Liquida , Oncorhynchus mykiss/metabolismoRESUMEN
Protein synthesis requires a continuous supply of all of the indispensable (essential) amino acids (IAAs). If any IAA is deficient, animals must obtain the limiting amino acid by diet selection. Sensing of IAA deficiency requires an intact anterior piriform cortex (APC), but does it act alone? Shortly after rats begin eating an IAA-deficient diet, the meal ends and EPSPs are activated in the APC; from there, neurons project to feeding circuits; the meal ends within 20 min. Within the APC in vivo, uncharged tRNA activates the general amino acid control non-derepressing 2 (GCN2) enzyme system increasing phosphorylation of eukaryotic initiation factor (P-eIF2α), which blocks general protein synthesis. If this paleocortex is sufficient for sensing IAA depletion, both neuronal activation and P-eIF2α should occur in an isolated APC slice. We used standard techniques for electrophysiology and immunohistochemistry. After rats ate IAA-devoid or -imbalanced diets, their depleted slices responded to different stimuli with increased EPSP amplitudes. Slices from rats fed a control diet were bathed in artificial CSF replete with all amino acids with or without the IAA, threonine, or a tRNA synthetase blocker, l-threoninol, or its inactive isomer, d-threoninol. Thr depletion in vitro increased both EPSP amplitudes and P-eIF2α. l (but not d)-threoninol also increased EPSP amplitudes relative to control. Thus, we show independent excitation of the APC with responses parallel to those known in vivo. These data suggest a novel idea: in addition to classical processing of peripheral sensory input, direct primary sensing may occur in mammalian cortex.
Asunto(s)
Aminoácidos Esenciales/deficiencia , Corteza Cerebral/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Neuronas/metabolismo , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , Aminoácidos Esenciales/biosíntesis , Aminoácidos Esenciales/metabolismo , Animales , Corteza Cerebral/fisiología , Dieta , Electrofisiología , Inmunohistoquímica , Masculino , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Fosforilación , Ratas , Ratas Sprague-Dawley , Treonina/deficienciaRESUMEN
The evolution of intimate symbiosis requires the coordination of gene expression and content between the distinct partner genomes; this coordination allows the fusion of capabilities of each organism into a single integrated metabolism. In aphids, the 10 essential amino acids are scarce in the phloem sap diet and are supplied by the obligate bacterial endosymbiont (Buchnera), which lives inside specialized cells called bacteriocytes. Although Buchnera's genome encodes most genes for essential amino acid biosynthesis, several genes in essential amino acid pathways are missing, as are most genes for production of nonessential amino acids. Additionally, it is unresolved whether the supply of nitrogen for amino acid biosynthesis is supplemented by recycling of waste ammonia. We compared pea aphid gene expression between bacteriocytes and other body tissues using RNA sequencing and pathway analysis and exploiting the genome sequences available for both partners. We found that 26 genes underlying amino acid biosynthesis were up-regulated in bacteriocytes. Seven of these up-regulated genes fill the gaps of Buchnera's essential amino acid pathways. In addition, genes underlying five nonessential amino acid pathways lost from Buchnera are up-regulated in bacteriocytes. Finally, our results reveal that two genes, glutamine synthetase and glutamate synthase, which potentially work together in the incorporation of ammonium nitrogen into glutamate (GOGAT) cycle to assimilate ammonia into glutamate, are up-regulated in bacteriocytes. Thus, host gene expression and symbiont capabilities are closely integrated within bacteriocytes, which function as specialized organs of amino acid production. Furthermore, the GOGAT cycle may be a key source of nitrogen fueling the integrated amino acid metabolism of the aphid-Buchnera partnership.
Asunto(s)
Aminoácidos Esenciales/biosíntesis , Áfidos/genética , Áfidos/microbiología , Buchnera/metabolismo , Evolución Molecular , Regulación de la Expresión Génica/genética , Simbiosis , Aminoácidos Esenciales/genética , Animales , Áfidos/metabolismo , Secuencia de Bases , Glutamato Sintasa/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Datos de Secuencia Molecular , Compuestos de Amonio Cuaternario/metabolismo , Análisis de Secuencia de ARN , Especificidad de la EspecieRESUMEN
The robustness and stability of complex cellular networks is often attributed to the redundancy of components, including genes, enzymes and pathways. Estimation of redundancy is still an open question in systems biology. Current theoretical tools to measure redundancy have various strengths and shortcomings in providing a comprehensive description of metabolic networks. Specially, there is a lack of effective measures to cover different perturbation situations. Here we present a pathway knockout algorithm to improve quantitative measure of redundancy in metabolic networks grounded on the elementary flux mode (EFM) analysis. The proposed redundancy measure is based on the average ratio of remaining EFMs after knockout of one EFM in the unperturbed state. We demonstrated with four example systems that our algorithm overcomes limits of previous measures, and provides additional information about redundancy in the situation of targeted attacks. Additionally, we compare existing enzyme knockout and our pathway knockout algorithm by the mean-field analysis, which provides mathematical expression for the average ratio of remaining EFMs after both types of knockout. Our results prove that multiple-enzymes knockout does not always yield more information than single-enzyme knockout for evaluating redundancy. Indeed, pathway knockout considers additional effects of structural asymmetry. In the metabolic networks of amino acid anabolism in Escherichia coli and human hepatocytes, and the central metabolism in human erythrocytes, we validate our mean-field solutions and prove the capacity of pathway knockout algorithm. Moreover, in the E. coli model the two sub-networks synthesizing amino acids that are essential and those that are non-essential for humans are studied separately. In contrast to previous studies, we find that redundancy of two sub-networks is similar with each other, and even sub-networks synthesizing essential amino acids can be more redundant.
Asunto(s)
Algoritmos , Redes y Vías Metabólicas/fisiología , Biología de Sistemas/métodos , Aminoácidos/biosíntesis , Aminoácidos Esenciales/biosíntesis , Enzimas/genética , Enzimas/metabolismo , Eritrocitos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Hepatocitos/metabolismo , HumanosRESUMEN
BACKGROUND: In silico analyses provide valuable insight into the biology of obligately intracellular pathogens and symbionts with small genomes. There is a particular opportunity to apply systems-level tools developed for the model bacterium Escherichia coli to study the evolution and function of symbiotic bacteria which are metabolically specialised to overproduce specific nutrients for their host and, remarkably, have a gene complement that is a subset of the E. coli genome. RESULTS: We have reconstructed and analysed the metabolic network of the gamma-proteobacterium Buchnera aphidicola (symbiont of the pea aphid) as a model for using systems-level approaches to discover key traits of symbionts with small genomes. The metabolic network is extremely fragile with > 90% of the reactions essential for viability in silico; and it is structured so that the bacterium cannot grow without producing the essential amino acid, histidine, which is released to the insect host. Further, the amount of essential amino acid produced by the bacterium in silico can be controlled by host supply of carbon and nitrogen substrates. CONCLUSION: This systems-level analysis predicts that the fragility of the bacterial metabolic network renders the symbiotic bacterium intolerant of drastic environmental fluctuations, whilst the coupling of histidine production to growth prevents the bacterium from exploiting host nutrients without reciprocating. These metabolic traits underpin the sustained nutritional contribution of B. aphidicola to the host and, together with the impact of host-derived substrates on the profile of nutrients released from the bacteria, point to a dominant role of the host in controlling the symbiosis.