RESUMEN
Systems-level metabolic network reconstructions and the derived constraint-based (CB) mathematical models are efficient tools to explore bacterial metabolism. Approximately one-fourth of the Mycobacterium tuberculosis (Mtb) genome contains genes that encode proteins directly involved in its metabolism. These represent potential drug targets that can be systematically probed with CB models through the prediction of genes essential (or the combination thereof) for the pathogen to grow. However, gene essentiality depends on the growth conditions and, so far, no in vitro model precisely mimics the host at the different stages of mycobacterial infection, limiting model predictions. These limitations can be circumvented by combining expression data from in vivo samples with a validated CB model, creating an accurate description of pathogen metabolism in the host. To this end, we present here a thoroughly curated and extended genome-scale CB metabolic model of Mtb quantitatively validated using 13C measurements. We describe some of the efforts made in integrating CB models and high-throughput data to generate condition specific models, and we will discuss challenges ahead. This knowledge and the framework herein presented will enable to identify potential new drug targets, and will foster the development of optimal therapeutic strategies.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Redes y Vías Metabólicas/genética , Modelos Estadísticos , Mycobacterium tuberculosis/metabolismo , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Isótopos de Carbono , Farmacorresistencia Bacteriana Múltiple/genética , Redes Reguladoras de Genes , Interacciones Huésped-Patógeno , Humanos , Terapia Molecular Dirigida , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Biología de Sistemas , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/metabolismo , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/patología , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/metabolismo , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
BACKGROUND: Ethylene glycol (EG) is a bulk chemical that is mainly used as an anti-freezing agent and a raw material in the synthesis of plastics. Production of commercial EG currently exclusively relies on chemical synthesis using fossil resources. Biochemical production of ethylene glycol from renewable resources may be more sustainable. RESULTS: Herein, a synthetic pathway is described that produces EG in Escherichia coli through the action of (D)-xylose isomerase, (D)-xylulose-1-kinase, (D)-xylulose-1-phosphate aldolase, and glycolaldehyde reductase. These reactions were successively catalyzed by the endogenous xylose isomerase (XylA), the heterologously expressed human hexokinase (Khk-C) and aldolase (Aldo-B), and an endogenous glycolaldehyde reductase activity, respectively, which we showed to be encoded by yqhD. The production strain was optimized by deleting the genes encoding for (D)-xylulose-5 kinase (xylB) and glycolaldehyde dehydrogenase (aldA), and by overexpressing the candidate glycolaldehyde reductases YqhD, GldA, and FucO. The strain overproducing FucO was the best EG producer reaching a molar yield of 0.94 in shake flasks, and accumulating 20 g/L EG with a molar yield and productivity of 0.91 and 0.37 g/(L.h), respectively, in a controlled bioreactor under aerobic conditions. CONCLUSIONS: We have demonstrated the feasibility to produce EG from (D)-xylose via a synthetic pathway in E. coli at approximately 90 % of the theoretical yield.
Asunto(s)
Escherichia coli/metabolismo , Glicol de Etileno/metabolismo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas , Xilosa/metabolismo , Reactores BiológicosRESUMEN
Mycolic acids, very long-chain α-alkyl, ß-hydroxylated fatty acids, occur in the members of the order Corynebacteriales where their chain lengths (C(26)-C(88)) and structural features (oxygen functions, cis or trans double bonds, cyclopropane rings and methyl branches) are genus- and species-specific. The molecular composition and structures of the mycolic acids of two species belonging to the genus Segniliparus were determined by a combination of modern analytical chemical techniques, which include MS and NMR. They consist of mono-ethylenic C(62-)C(64) (α'), di-ethylenic C(77)-C(79) (α) and extremely long-chain mycolic acids (α(+)) ranging from 92 to 98 carbon atoms and containing three unsaturations, cis and/or trans double bonds and/or cyclopropanes. The double bonds in each class of mycolic acids were positioned by oxidative cleavage and exhibit locations similar to those of α- and α'-mycolic acids of mycobacteria. For the ultralong chain α-mycolic acids, the three double bonds were located at equally spaced carbon intervals (C(13)-C(16)), with the methyl branches adjacent to the proximal and distal trans double bonds. Examination of the Segniliparus rotundus genome compared with those of other members of the Corynebacteriales indicated two obvious differences in genes encoding the elongation fatty acid (FAS-II) enzymes involved in the biosynthesis of mycolic acids: the organization of 3-ketoacyl-ACP synthases (KasA and KasB) and (3R)-hydroxyacyl-ACP dehydratases (HadAB/BC), on one hand, and the presence of two copies of the hadB gene encoding the catalytic domain of the latter enzyme type, on the other. This observation is discussed in light of the most recent data accumulated on the biosynthesis of this hallmark of Corynebacteriales.
Asunto(s)
Actinomycetales/química , Actinomycetales/genética , Vías Biosintéticas/genética , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Actinomycetales/metabolismo , Enzimas/genética , Espectroscopía de Resonancia Magnética , Espectrometría de MasasRESUMEN
Corynebacterineae are characterized by the presence of long-chain lipids, notably mycolic acids (α-alkyl, ß-hydroxy fatty acids), the structures of which are genus-specific. Mycolic acids from two environmental strains, Amycolicicoccus subflavus and Hoyosella altamirensis, were isolated and their structures were established using a combination of mass spectrometry analysis, (1)H-NMR spectroscopy and chemical degradations. The C(2)-C(3) cleavage of these C(30)-C(36) acids led to the formation of two fragments: saturated C(9)-C(11) acids, and saturated and unsaturated C(20)-C(25) aldehydes. Surprisingly, the fatty acids at the origin of the two fragments making up these mycolic acids were present in only minute amounts in the fatty acid pool. Moreover, the double bond in the main C(24) aldehyde fragment was located at position ω16, whereas that found in the ethylenic fatty acids of the bacteria was at ω9. These data question the biosynthesis of these new mycolic acids in terms of the nature of the precursors, chain elongation and desaturation. Nevertheless, they are consistent with the occurrence of the key genes of mycolic acid biosynthesis, including those encoding proteins of the fatty acid synthase II system, identified in the genome of A. subflavus. Altogether, while the presence of mycolic acids and analysis of their 16S rDNA sequences would suggest that these strains belong to the Mycobacteriaceae family, the originality of their structures reinforces the recent description of the novel genera Amycolicicoccus and Hoyosella.
Asunto(s)
Actinomycetales/química , Actinomycetales/clasificación , Microbiología Ambiental , Ácidos Micólicos/análisis , Actinomycetales/aislamiento & purificación , Vías Biosintéticas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ácidos Micólicos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
The very high content of structurally diverse and biologically active lipids of exotic structures is the hallmark of Mycobacteria. As such the lipid composition is commonly used to characterize mycobacterial strains at the species and type-species levels. The present chapter describes the methods that allow the purification of the most commonly isolated biologically active lipids and those used for analyzing extractable lipids and their constituents, cell wall-linked mycolic acids (MA), and lipoarabinomannan (LAM). These involve various chromatographic techniques and analytical procedures necessary for structural and metabolic studies of mycobacterial lipids. In addition, as the use of physical methods has brought important overhang on chemical structures of the very-long-chain MA, which typify mycobacteria, NMR and mass spectrometry data of these specific fatty acids are included.
Asunto(s)
Pared Celular/metabolismo , Lípidos/análisis , Lípidos/aislamiento & purificación , Lipopolisacáridos/análisis , Lipopolisacáridos/aislamiento & purificación , Mycobacterium/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de MasasRESUMEN
The yeast trehalose-6-phosphate synthase (Tps1) catalyzes the formation of trehalose-6-phosphate (T6P) in trehalose synthesis. Besides, Tps1 plays a key role in carbon and energy homeostasis in this microbial cell, as shown by the well documented loss of ATP and hyper accumulation of sugar phosphates in response to glucose addition in a mutant defective in this protein. The inability of a Saccharomyces cerevisiae tps1 mutant to cope with fermentable sugars is still a matter of debate. We reexamined this question through a quantitative analysis of the capability of TPS1 homologues from different origins to complement phenotypic defects of this mutant. Our results allowed to classify this complementation in three groups. A first group enclosed TPS1 of Klyveromyces lactis with that of S. cerevisiae as their expression in Sctps1 cells fully recovered wild type metabolic patterns and fermentation capacity in response to glucose. At the opposite was the group with TPS1 homologues from the bacteria Escherichia coli and Ralstonia solanacearum, the plant Arabidopsis thaliana and the insect Drosophila melanogaster whose metabolic profiles were comparable to those of a tps1 mutant, notably with almost no accumulation of T6P, strong impairment of ATP recovery and potent reduction of fermentation capacity, albeit these homologous genes were able to rescue growth of Sctps1 on glucose. In between was a group consisting of TPS1 homologues from other yeast species and filamentous fungi characterized by 5 to 10 times lower accumulation of T6P, a weaker recovery of ATP and a 3-times lower fermentation capacity than wild type. Finally, we found that glucose repression of gluconeogenic genes was strongly dependent on T6P. Altogether, our results suggest that the TPS protein is indispensable for growth on fermentable sugars, and points to a critical role of T6P as a sensing molecule that promotes sugar fermentation and glucose repression.
RESUMEN
Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (<17kDa) of single oocytes, cumulus cells (CC) and granulosa cells (GC), which shared a total of 439 peaks. By comparing the ICM-MS profiles of single oocytes and CC before and after in vitro maturation (IVM), 71 different peaks were characterised, and their relative abundance was found to vary depending on the stage of oocyte meiotic maturation. To identify these endogenous biomolecules, top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. BIOLOGICAL SIGNIFICANCE: Intact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases.
Asunto(s)
Oocitos/citología , Folículo Ovárico/citología , Proteómica/métodos , Análisis de la Célula Individual/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Biomarcadores/análisis , Bovinos , Células del Cúmulo/química , Femenino , Células de la Granulosa/química , Meiosis , Oocitos/química , Folículo Ovárico/químicaRESUMEN
The endogenous peptides and small proteins extracted from bovine ovarian follicular cells (oocytes, cumulus and granulosa cells) were identified by Top-down High Resolution Mass Spectrometry (TD-HR-MS/MS) in order to annotate peptido- and proteoforms detected using qualitative and quantitative profiling method based on ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). The description and analysis of these Top-down MS data in the context of oocyte quality biomarkers research are available in the original research article of Labas et al. (2017) http://dx.doi.org/10.1016/j.jprot.2017.03.027[1]. Raw data derived from this peptidomic/proteomic analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD004892). Here, we described the inventory of all identified peptido- and proteoforms including their biochemical and structural features, and functional annotation of correspondent proteins. This peptide/protein inventory revealed that TD-HR-MS/MS was appropriate method for both global and targeted proteomic analysis of ovarian tissues, and it can be further employed as a reference for other studies on follicular cells including single oocytes.
RESUMEN
Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two ß subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Carbono/metabolismo , Mycobacterium tuberculosis/metabolismo , Sintasas Poliquetidas/metabolismo , Subunidades de Proteína/metabolismo , Acetilcoenzima A/metabolismo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/genética , Ligasas de Carbono-Carbono/genética , Clonación Molecular , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Malonil Coenzima A/metabolismo , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Sintasas Poliquetidas/genética , Ingeniería de Proteínas , Subunidades de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
BACKGROUND: Glycolic acid (GA) is a two-carbon hydroxyacid with applications in the cosmetic, textile, and medical industry. Microbial GA production from all sugars can be achieved by engineering the natural glyoxylate shunt. The synthetic (d)-xylulose-1 phosphate (X1P) pathway provides a complementary route to produce GA from (d)-xylose. The simultaneous operation of the X1P and glyoxylate pathways increases the theoretical GA yield from xylose by 20 %, which may strongly improve GA production from hemicellulosic hydrolysates. RESULTS: We herein describe the construction of an E. coli strain that produces GA via the glyoxylate pathway at a yield of 0.31 , 0.29 , and 0.37 g/g from glucose, xylose, or a mixture of glucose and xylose (mass ratio: 33:66 %), respectively. When the X1P pathway operates in addition to the glyoxylate pathway, the GA yields on the three substrates are, respectively, 0.39 , 0.43 , and 0.47 g/g. Upon constitutive expression of the sugar permease GalP, the GA yield of the strain which simultaneously operates the glyoxylate and X1P pathways further increases to 0.63 g/g when growing on the glucose/xylose mixture. Under these conditions, the GA yield on the xylose fraction of the sugar mixture reaches 0.75 g/g, which is the highest yield reported to date. CONCLUSIONS: These results demonstrate that the synthetic X1P pathway has a very strong potential to improve GA production from xylose-rich hemicellulosic hydrolysates.
RESUMEN
The fertilization ability of male gametes is achieved after their transit through the epididymis where important post-gonadal differentiation occurs in different cellular compartments. Most of these maturational modifications occur at the protein level. The epididymal sperm maturation process was investigated using the ICM-MS (Intact Cell MALDI-TOF MS) approach on boar spermatozoa isolated from four different epididymal regions (immature to mature stage). Differential and quantitative MALDI-TOF profiling for whole cells or sub-cellular fractions was combined with targeted top-down MS in order to identify endogenous biomolecules. Using this approach, 172m/z peaks ranging between 2 and 20kDa were found to be modified during maturation of sperm. Using top-down MS, 62m/z were identified corresponding to peptidoforms/proteoforms with post-translational modifications (MS data are available via ProteomeXchange with identifier PXD001303). Many of the endogenous peptides were characterized as N-, C-terminal sequences or internal fragments of proteins presenting specific cleavages, suggesting the presence of sequential protease activities in the spermatozoa. This is the first time that such proteolytic activities could be evidenced for various sperm proteins through quantification of their proteolytic products. ICM-MS/top-down MS thus proved to be a valid approach for peptidome/degradome studies and provided new contributions to understanding of the maturation process of the male gamete involved in the development of male fertility. BIOLOGICAL SIGNIFICANCE: This peptidomic study (i) characterized the peptidome of epididymal spermatozoa from boar (Sus scrofa); (ii) established characteristic molecular phenotypes distinguishing degrees of maturation of spermatozoa during epididymal transit, and (iii) revealed that protease activities were at the origin of numerous peptides from known and unknown proteins involved in sperm maturation and/or fertility processes.
Asunto(s)
Fertilidad/fisiología , Proteolisis , Proteoma/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espermatozoides/metabolismo , Animales , Epidídimo/metabolismo , Masculino , Maduración del Esperma , Espermatozoides/citología , PorcinosRESUMEN
The final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several epididymal maturation states could be an informative approach to finding sperm modifications related to maturation and fertility. Here we show the data from differential peptidomic/proteomic analyses on spermatozoa isolated from 4 epididymal regions (immature to mature stage) using a profiling approach based on MALDI-TOF mass spectrometry and, combined to top-down MS in order to identify peptidoforms and proteoforms. By this way, 172m/z peaks ranging between 2 and 20 kDa were found to be modified during maturation of sperm. A total of 62m/z were identified corresponding to 32 different molecular species. The interpretation of these data can be found in the research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1].