Using NMR Metabolomics to Investigate Tricarboxylic Acid Cycle-dependent Signal Transduction in Staphylococcus epidermidis

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to “sense” environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is “sensing” disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are “sensed” by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.     

Synthesis and deformylation of Staphylococcus aureus delta-toxin are linked to tricarboxylic acid cycle activity

In bacteria, translation initiates with formyl-methionine; however, the N-terminal formyl group is usually removed by peptide deformylase, an enzymatic activity requiring iron. Staphylococcus aureus delta-toxin is a 26-amino-acid polypeptide secreted predominantly with a formylated N-terminal methionine, which led us to investigate regulation of delta-toxin deformylation. We observed that during exponential and early postexponential growth, delta-toxin accumulated in the culture medium in formylated and deformylated forms. In contrast, only formylated delta-toxin accumulated after the early postexponential phase. The transition from producing both species of delta-toxin to producing only formyl-methionine-containing delta-toxin coincided with increased tricarboxylic acid (TCA) cycle activity. The TCA cycle contains several iron-requiring enzymes, which led us to hypothesize that TCA cycle induction depletes the iron in the culture medium, thereby inhibiting peptide deformylase activity. As expected, S. aureus depletes the iron in the culture medium between the postexponential and stationary phases of growth. Inhibition of delta-toxin deformylation was relieved by TCA cycle inactivation or by addition of supplemental iron to the culture medium. Of interest, peptides containing formyl-methionine are potent chemoattractants for neutrophils, suggesting that delta-toxin deformylation may have functional consequences. We found neutrophil chemotactic activity only with formylated delta-toxin. The S. aureus TCA cycle is derepressed upon depletion of rapidly catabolizable carbon sources; this coincides with the transition to producing only formylated delta-toxin and results in an increased inflammatory response. The proinflammatory response should increase host cell damage and result in the release of nutrients. Taken together, these results establish that there is an important linkage between bacterial metabolism and pathogenesis.     

Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under nitrate conditions using different levels of omics data

It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.     

Circadian rhythm of a TCA cycle enzyme is apparently regulated at the translational level in the dinoflagellate Lingulodinium polyedrum

Previously, the authors have reported that intracellular amounts of several metabolic-related enzymes from the photosynthetic dinoflagellate Lingulodinium polyedrum(formerly Gonyaulax polyedra) showed a daily rhythm under a 12:12 h LD cycle. This led the authors to hypothesize that a circadian clock controls metabolism, including the tricarboxylic acid (TCA) cycle. In this study, the authors investigated daily changes in the levels of mRNA, protein, and enzyme activity of several metabolic enzymes during 12:12 h LD, 8:16 h LD, and constant light conditions. The NADP-dependent isocitrate dehydrogenase (NADPICDH) in the TCA cycle exhibited circadian changes of protein abundance and enzyme activity under all conditions, whereas its mRNA level remained constant throughout the cycle. These results indicate that the rhythm of NADPICDH is regulated by a circadian control of protein synthesis or modification rather than by message levels and suggest that the TCA cycle may be controlled by the circadian clock system.     

Enhancement of recombinant protein production in Escherichia coli by coproduction of aspartase

As commonly recognized, the excretion of acetate by the aerobic growth of Escherichia coli on glucose is a manifestation of imbalanced flux between glycolysis and the tricarboxylic acid (TCA) cycle. Accordingly, this may restrict the production of recombinant proteins in E. coli, due to the limited amounts of precursor metabolites produced in TCA cycle. To approach this issue, an extra supply of intermediate metabolites in TCA cycle was made by conversion of aspartate to fumarate, a reaction mediated by the activity of L-aspartate ammonia-lyase (aspartase). As a result, in the glucose minimal medium containing aspartate, the production of two recombinant proteins, beta-galactosidase and green fluorescent protein, in the aspartase-producing strain was substantially increased by 5-fold in association with 30-40% more biomass production. This preliminary study illustrates the great promise of this approach used to enhance the production of these two recombinant proteins.     

Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus.

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.     

Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis

Superoxide dismutases (SODs) are key components of the plant antioxidant defense system. While plastidic and cytosolic isoforms have been extensively studied, the importance of mitochondrial SOD at a cellular and whole-plant level has not been established. To address this, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated in which expression of AtMSD1, encoding the mitochondrial manganese (Mn)SOD, was suppressed by antisense. The strongest antisense line showed retarded root growth even under control growth conditions. There was evidence for a specific disturbance of mitochondrial redox homeostasis in seedlings grown in liquid culture: a mitochondrially targeted redox-sensitive green fluorescent protein was significantly more oxidized in the MnSOD-antisense background. In contrast, there was no substantial change in oxidation of cytosolically targeted redox-sensitive green fluorescent protein, nor changes in antioxidant defense components. The consequences of altered mitochondrial redox status of seedlings were subtle with no widespread increase of mitochondrial protein carbonyls or inhibition of mitochondrial respiratory complexes. However, there were specific inhibitions of tricarboxylic acid (TCA) cycle enzymes (aconitase and isocitrate dehydrogenase) and an inhibition of TCA cycle flux in isolated mitochondria. Nevertheless, total respiratory CO2 output of seedlings was not decreased, suggesting that the inhibited TCA cycle enzymes can be bypassed. In older, soil-grown plants, redox perturbation was more pronounced with changes in the amount and/or redox poise of ascorbate and glutathione. Overall, the results demonstrate that reduced MnSOD affects mitochondrial redox balance and plant growth. The data also highlight the flexibility of plant metabolism with TCA cycle inhibition having little effect on overall respiratory rates.     

Effects of arcA and arcB genes knockout on the metabolism in Escherichia coli under anaerobic and microaerobic conditions

The Arc system is a two-component regulatory system composed of ArcA and ArcB in Escherichia coli. In the present study, the effects of arcA and arcB genes knockout on the TCA cycle activation in E. coli were investigated for the anaerobic and microaerobic conditions. Under anaerobic condition, the TCA cycle was up-regulated along with high lactate production, together with up-regulation of LDH for arcB mutant as compared with the parent strain. Due to down-regulation of aceE, aceF and lpdA genes which code for PDHc and low activity of Pfl in arcB mutant, the glycolysis as well as oxidative pentose phosphate pathway was down-regulated under anaerobic condition. The TCA cycle enzymes were further up-regulated when nitrate was added by modifying the redox state along with lower lactate production for arcB mutant. Different from the case of anaerobic condition, the glycolysis was activated under microaerobic condition, which may be partly due to the increased activity of PDHc encoded by aceE, F and lpdA genes. Under microaerobic condition, the TCA cycle genes together with their corresponding enzymes were up-regulated for arcB mutant as compared with the parent strain. These characteristics were further enhanced in arcA mutant as compared with the case of arcB mutant. The up-regulation of the TCA cycle together with down-regulation of cydB gene expression caused higher redox state in the arcA/B mutants, which in turn repressed the TCA cycle. Then the TCA cycle could be further increased by the addition of nicotinic acid (NA).     

Metabolic analysis of antibody producing CHO cells in fed‐batch production

Chinese hamster ovary (CHO) cells are commonly used for industrial production of recombinant proteins in fed batch or alternative production systems. Cells progress through multiple metabolic stages during fed‐batch antibody (mAb) production, including an exponential growth phase accompanied by lactate production, a low growth, or stationary phase when specific mAb production increases, and a decline when cell viability declines. Although media composition and cell lineage have been shown to impact growth and productivity, little is known about the metabolic changes at a molecular level. Better understanding of cellular metabolism will aid in identifying targets for genetic and metabolic engineering to optimize bioprocess and cell engineering. We studied a high expressing recombinant CHO cell line, designated high performer (HP), in fed‐batch productions using stable isotope tracers and biochemical methods to determine changes in central metabolism that accompany growth and mAb production. We also compared and contrasted results from HP to a high lactate producing cell line that exhibits poor growth and productivity, designated low performer (LP), to determine intrinsic metabolic profiles linked to their respective phenotypes. Our results reveal alternative metabolic and regulatory pathways for lactate and TCA metabolite production to those reported in the literature. The distribution of key media components into glycolysis, TCA cycle, lactate production, and biosynthetic pathways was shown to shift dramatically between exponential growth and stationary (production) phases. We determined that glutamine is both utilized more efficiently than glucose for anaplerotic replenishment and contributes more significantly to lactate production during the exponential phase. Cells shifted to glucose utilization in the TCA cycle as growth rate decreased. The magnitude of this metabolic switch is important for attaining high viable cell mass and antibody titers. We also found that phosphoenolpyruvate carboxykinase (PEPCK1) and pyruvate kinase (PK) are subject to differential regulation during exponential and stationary phases. The concomitant shifts in enzyme expression and metabolite utilization profiles shed light on the regulatory links between cell metabolism, media metabolites, and cell growth. Biotechnol. Bioeng. 2013; 110: 1735–1747. © 2013 Wiley Periodicals, Inc.     

Energy metabolism and alginate biosynthesis in Pseudomonas aeruginosa: role of the tricarboxylic acid cycle.

Infection with mucoid, alginate-producing strains of Pseudomonas aeruginosa is the leading cause of mortality among patients with cystic fibrosis. Alginate production by P. aeruginosa is not constitutive but is triggered by stresses such as starvation. The algR2 (also termed algQ) gene has been previously identified as being necessary for mucoidy; an algR2 mutant strain is unable to produce alginate when grown at 37 degrees C. We show here that the levels of phosphorylated succinyl coenzyme A synthetase (Scs) and nucleoside diphosphate kinase (Ndk), which form a complex in P. aeruginosa, are reduced in the algR2 mutant. We were able to correlate the lower level of phosphorylated Scs with a decrease in Scs activity. Western blots (immunoblots) also showed a decreased level of Ndk in the algR2 mutant, but the presence of another kinase activity sensitive to Tween 20 provides the missing Ndk function. The effect of AlgR2 on tricarboxylic acid (TCA) cycle enzymes appears to be specific for Scs, since none of the other TCA cycle enzymes measured showed a significant decrease in activity. Furthermore, the ability of the algR2 mutant to grow on TCA cycle intermediates, but not glucose, is impaired. These data indicate that AlgR2 is responsible for maintaining proper operation of the TCA cycle and energy metabolism.     

Intra- and extra-cellular flux distributions of TCA and glyoxalate cycle and vancomycin production of Amycolatopsis orieantalis grown in different glycerol concentration

The relationship between tricarboxylic acid (TCA) and glyoxalate cycle and the effect of their metabolites levels on the vancomycin production of Amycolatopsis orientalis were investigated in different concentration of glycerol (2.5-20 g/l). Intracellular glycerol levels increased with respect to increases in glycerol concentrations of the growth medium. Extracellular glycerol levels decreased slowly up to 24 h while uptake rates were increased during 36-48 h for 10 and 15 g/l and during 36-60 h at 20 g/l of glycerol. Intracellular citrate, alpha-ketoglutarate, fumarate levels increased up to 10 g/l glycerol concentration. However, intracellular succinate and malate levels were increased up to 15 g/l glycerol. Extracellular citrate, alpha-ketoglutarate, succinate and malate levels increased with respect to increases in glycerol concentration. The highest alpha-ketoglutarate dehydrogenase activity was determined at 15 g/l glycerol. Isocitrate lyase activity showed a positive correlation with the increases in glycerol concentration of the growth medium. Vancomycin production increased with the increases in glycerol concentration from 5 to 10 g/l. These results showed that A. orientalis grown in glycerol containing medium used glyoxalate shunt actively instead of TCA cycle which supports precursors of many amino acid which are effective on the antibiotic production.     

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS.

We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13C] and [U-13C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the Entner-Doudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.     

Kinetic characterization of vero cell metabolism in a serum‐free batch culture process

A global kinetic study of the central metabolism of Vero cells cultivated in a serum‐free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum‐free medium which is a valuable help for the implementation of this cell line in serum‐free industrial production processes. Biotechnol. Bioeng. 2010;107: 143–153. © 2010 Wiley Periodicals, Inc.     

Oxygen supply strongly influences metabolic fluxes,the production of poly(3-hydroxybutyrate) and alginate,and the degree of acetylation of alginate in Azotobacter vinelandii

The aim of this study was to evaluate carbon flux in Azotobacter vinelandii using metabolic flux analysis (MFA) under high and low aeration conditions to achieve an improved understanding of how these changes could be related to alginate acetylation and PHB production. Changes in oxygen availability had a considerable impact on the metabolic fluxes and were reflected in the growth rate, the specific glucose consumption rate, and the alginate and PHB yields. The main differences at the metabolic flux level were observed in three important pathways. The first important difference was consistent with respiratory protection; an increase in the flux generated through the tricarboxylic acid (TCA) cycle for cultures grown under high aeration conditions (up to 2.61 times higher) was observed. In the second important difference, the fluxes generated through pyruvate dehydrogenase, phosphoenol pyruvate carboxykinase and pyruvate kinase, all of which are involved in acetyl-CoA metabolism, increased by 10, 43.9 and 17.5%, respectively, in cultures grown under low aeration conditions compared with those grown under high aeration conditions. These changes were related to alginate acetylation, which was 2.6 times higher in the cultures with limited oxygen, and the changes were also related to a drastic increase in PHB production. Finally, the glyoxylate shunt was active under both of the conditions that were tested, and a 2.79-fold increase was observed in cultures that were grown under the low aeration condition.