RESUMEN
One of the key ways in which microbes are thought to regulate their metabolism is by modulating the availability of enzymes through transcriptional regulation. However, the limited success of efforts to manipulate metabolic fluxes by rewiring the transcriptional network has cast doubt on the idea that transcript abundance controls metabolic fluxes. In this study, we investigate control of metabolic flux in the model bacterium Bacillus subtilis by quantifying fluxes, transcripts, and metabolites in eight metabolic states enforced by different environmental conditions. We find that most enzymes whose flux switches between on and off states, such as those involved in substrate uptake, exhibit large corresponding transcriptional changes. However, for the majority of enzymes in central metabolism, enzyme concentrations were insufficient to explain the observed fluxes--only for a number of reactions in the tricarboxylic acid cycle were enzyme changes approximately proportional to flux changes. Surprisingly, substrate changes revealed by metabolomics were also insufficient to explain observed fluxes, leaving a large role for allosteric regulation and enzyme modification in the control of metabolic fluxes.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas , ARN Mensajero/genética , Isótopos de Carbono , Cinética , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Integration host factor (IHF) sites are largely absent from intergenic regions of ORFs encoding central metabolic functions in Pseudomonas putida mt-2. To gain an insight into this unequal distribution of otherwise abundant IHF-binding sequences, the transcriptome of IHF-plus and IHF-minus cells growing exponentially on glucose as sole carbon source was examined. In parallel, the cognate metabolic fluxes of the wild-type P. putida strain and its ihfA derivative were determined by culturing cells to a steady-state physiological regime with (13)C-labelled glucose. While expression of many transcripts was altered by the lack of IHF, flux balance analysis revealed that the ihfA mutation did not influence central carbon metabolism. Identification of multiple IHF sites adjacent to genes responsive to the factor allowed a refinement of the consensus and the mapping of the preferred binding positions for activation or repression of associated promoters. That few (if any) of the genes affected by IHF involved core pathways suggested that the central carbon metabolism tolerates the loss of the factor. Instead, IHF controlled various cell surface-related functions and downregulated genes encoding ribosomal proteins, the alpha subunit of RNA polymerase and components of the ATP synthase. These results were confirmed with lacZ fusions to a suite of promoters detected in the transcriptome as affected by IHF. Taken together, the data suggest that IHF plays a role in the physiological shift that sets P. putida for entering stationary phase.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Integración del Huésped/genética , Factores de Integración del Huésped/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Regulón/genética , Adenosina Trifosfato/biosíntesis , Biopelículas , Isótopos de Carbono/análisis , Regulación Bacteriana de la Expresión Génica , Pseudomonas putida/crecimiento & desarrollo , TranscriptomaRESUMEN
UNLABELLED: Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTS(Fru)), while the other (N-related PTS, or PTS(Ntr)) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTS(Fru) or PTS(Ntr) genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIA(Ntr)) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIA(Ntr) protein of the PTS(Ntr) thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. IMPORTANCE: This study demonstrates that EIIA(Ntr) influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIA(Ntr) protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Metabolismo Energético , Regulación Bacteriana de la Expresión Génica , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Pseudomonas putida/enzimología , Pseudomonas putida/metabolismo , Proteínas Bacterianas/genética , Fructosa/metabolismo , Glucosa/metabolismo , Redes y Vías Metabólicas/genética , Modelos Biológicos , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genéticaRESUMEN
Despite our increasing topological knowledge on regulation networks in model bacteria, it is largely unknown which of the many co-occurring regulatory events actually control metabolic function and the distribution of intracellular fluxes. Here, we unravel condition-dependent transcriptional control of Escherichia coli metabolism by large-scale (13)C-flux analysis in 91 transcriptional regulator mutants on glucose and galactose. In contrast to the canonical respiro-fermentative glucose metabolism, fully respiratory galactose metabolism depends exclusively on the phosphoenol-pyruvate (PEP)-glyoxylate cycle. While 2/3 of the regulators directly or indirectly affected absolute flux rates, the partitioning between different pathways remained largely stable with transcriptional control focusing primarily on the acetyl-CoA branch point. Flux distribution control was achieved by nine transcription factors on glucose, including ArcA, Fur, PdhR, IHF A and IHF B, but was exclusively mediated by the cAMP-dependent Crp regulation of the PEP-glyoxylate cycle flux on galactose. Five further transcription factors affected this flux only indirectly through cAMP and Crp by increasing the galactose uptake rate. Thus, E. coli actively limits its galactose catabolism at the expense of otherwise possible faster growth.
Asunto(s)
Acetilcoenzima A/metabolismo , Carbono/metabolismo , Galactosa/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucosa/metabolismo , Isótopos de Carbono/metabolismo , Ciclo del Ácido Cítrico , AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucólisis , Glioxilatos/metabolismo , Marcaje Isotópico , Oxígeno/metabolismo , Fosfoenolpiruvato/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
In several Gram-positive bacterial species, the global transcriptional regulatory protein CodY adjusts the expression of many metabolic genes, apparently in response to changes in the pools of specific metabolites, i.e., the branched-chain amino acids (BCAAs) isoleucine, leucine, and valine (ILV) and the nucleoside triphosphate GTP. CodY not only responds to these metabolites as measured in vitro but also regulates the genes that direct their synthesis. We have constructed a set of strains lacking binding sites for the CodY protein in cis at loci coding for the ILV biosynthetic machinery, effectively overexpressing these genes in an attempt to modulate the ILV input signal to CodY. Metabolite analyses of strains derepressed for genes needed for ILV synthesis revealed more than a 6-fold increase in the valine pool and a 2-fold increase in the isoleucine and leucine pools. Accumulation of the branched-chain amino acids was accompanied by a 24-fold induction of the bkd operon (required for branched-chain fatty acid synthesis) and 6-fold hyperrepression of the CodY-regulated yhdG and yufN genes, demonstrating that CodY perceives intracellular fluctuations in at least one if its input signals. We conclude that changes in the rate of endogenous ILV synthesis serve as an important signal for CodY-mediated gene regulation.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Bacillus subtilis/clasificación , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Aminoácidos de Cadena Ramificada/química , Aminoácidos de Cadena Ramificada/genética , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Genotipo , Mutación , Operón , Transcripción Genética , Regulación hacia ArribaRESUMEN
Commonly glucose is considered to be the only preferred substrate in Bacillus subtilis whose presence represses utilization of other alternative substrates. Because recent data indicate that malate might be an exception, we quantify here the carbon source utilization hierarchy. Based on physiology and transcriptional data during co-utilization experiments with eight carbon substrates, we demonstrate that malate is a second preferred carbon source for B. subtilis, which is rapidly co-utilized with glucose and strongly represses the uptake of alternative substrates. From the different hierarchy and degree of catabolite repression exerted by glucose and malate, we conclude that both substrates might act through different molecular mechanisms. To obtain a quantitative and functional network view of how malate is (co)metabolized, we developed a novel approach to metabolic flux analysis that avoids error-prone, intuitive, and ad hoc decisions on (13)C rearrangements. In particular, we developed a rigorous approach for deriving reaction reversibilities by combining in vivo intracellular metabolite concentrations with a thermodynamic feasibility analysis. The thus-obtained analytical model of metabolism was then used for network-wide isotopologue balancing to estimate the intracellular fluxes. These (13)C-flux data revealed an extraordinarily high malate influx that is primarily catabolized via the gluconeogenic reactions and toward overflow metabolism. Furthermore, a considerable NADPH-producing malic enzyme flux is required to supply the biosynthetically required NADPH in the presence of malate. Co-utilization of glucose and malate resulted in a synergistic decrease of the respiratory tricarboxylic acid cycle flux.
Asunto(s)
Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Carbono/metabolismo , Malatos/farmacología , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Isótopos de Carbono , Genes Bacterianos/genética , Glucosa/metabolismo , Glucosa/farmacología , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Marcaje Isotópico , Malatos/metabolismo , Especificidad por Sustrato , Termodinámica , Transcripción Genética/efectos de los fármacosRESUMEN
This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production.
Asunto(s)
Antibacterianos/biosíntesis , Citosol/metabolismo , NADP/metabolismo , Penicilina G/metabolismo , Penicillium chrysogenum/metabolismo , Vía de Pentosa FosfatoRESUMEN
This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and (13)C-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS). Because flux sensitivity around several important metabolic nodes proved to be dependent on the applied technique, the combination of the three (13)C quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and (13)C-labelling data provided evidence that a combination of assimilatory metabolism and pentose phosphate pathway activity diverted some of the carbon away from glycerol formation. Metabolite balancing indicated that this results in excess cytosolic NADH, suggesting the presence of a cytosolic NADH sink in addition to those that were deleted. The exchange flux of four-carbon dicarboxylic acids across the mitochondrial membrane, as measured by the (13)C-labelling data, supports a possible role of a malate/aspartate or malate/oxaloacetate redox shuttle in the transfer of these redox equivalents from the cytosol to the mitochondrial matrix.
Asunto(s)
Glicerol/metabolismo , Redes y Vías Metabólicas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulación hacia Arriba , Carbono/metabolismo , Isótopos de Carbono/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Eliminación de Gen , Glicerolfosfato Deshidrogenasa/genética , Espectroscopía de Resonancia Magnética , NADH Deshidrogenasa/genética , Oxidación-Reducción , Saccharomyces cerevisiae/enzimología , Triosa-Fosfato Isomerasa/genéticaRESUMEN
In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.
Asunto(s)
Gluconatos/metabolismo , Micología/métodos , Penicillium chrysogenum/metabolismo , Vía de Pentosa Fosfato/fisiología , Isótopos de Carbono/metabolismo , Medios de Cultivo , Glucosa , Glucólisis , Penicillium chrysogenum/crecimiento & desarrollo , Reproducibilidad de los ResultadosRESUMEN
The currently applied reaction structure in stoichiometric flux balance models for the nonoxidative branch of the pentose phosphate pathway is not in accordance with the established ping-pong kinetic mechanism of the enzymes transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). Based upon the ping-pong mechanism, the traditional reactions of the nonoxidative branch of the pentose phosphate pathway are replaced by metabolite specific, reversible, glycolaldehyde moiety (C(2)) and dihydroxyacetone moiety (C(3)) fragments producing and consuming half-reactions. It is shown that a stoichiometric model based upon these half-reactions is fundamentally different from the currently applied stoichiometric models with respect to the number of independent C(2) and C(3) fragment pools in the pentose phosphate pathway and can lead to different label distributions for (13)C-tracer experiments. To investigate the actual impact of the new reaction structure on the estimated flux patterns within a cell, mass isotopomer measurements from a previously published (13)C-based metabolic flux analysis of Saccharomyces cerevisiae were used. Different flux patterns were found. From a genetic point of view, it is well known that several micro-organisms, including Escherichia coli and S. cerevisiae, contain multiple genes encoding isoenzymes of transketolase and transaldolase. However, the extent to which these gene products are also actively expressed remains unknown. It is shown that the newly proposed stoichiometric model allows study of the effect of isoenzymes on the (13)C-label distribution in the nonoxidative branch of the pentose phosphate pathway by extending the half-reaction based stoichiometric model with two distinct transketolase enzymes instead of one. Results show that the inclusion of isoenzymes affects the ensuing flux estimates.
Asunto(s)
Vía de Pentosa Fosfato/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Isótopos de Carbono , Cinética , Modelos Biológicos , Oxidación-ReducciónRESUMEN
Metabolic-flux analyses in microorganisms are increasingly based on (13)C-labeling data. In this paper a new approach for the measurement of (13)C-label distributions is presented: rapid sampling and quenching of microorganisms from a cultivation, followed by extraction and detection by liquid chromatography-mass spectrometry of free intracellular metabolites. This approach allows the direct assessment of mass isotopomer distributions of primary metabolites. The method is applied to the glycolytic and pentose phosphate pathways of Saccharomyces cerevisiae strain CEN.PK113-7D grown in an aerobic, glucose-limited chemostat culture. Detailed investigations of the measured mass isotopomer distributions demonstrate the accuracy and information-richness of the obtained data. The mass fractions are fitted with a cumomer model to yield the metabolic fluxes. It is estimated that 24% of the consumed glucose is catabolized via the pentose phosphate pathway. Furthermore, it is found that turnover of storage carbohydrates occurs. Inclusion of this turnover in the model leads to a large confidence interval of the estimated split ratio.