scholarly journals CcpN Controls Central Carbon Fluxes in Bacillus subtilis

2008 ◽  
Vol 190 (18) ◽  
pp. 6178-6187 ◽  
Author(s):  
Simon Tännler ◽  
Eliane Fischer ◽  
Dominique Le Coq ◽  
Thierry Doan ◽  
Emmanuel Jamet ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Tricarboxylic Acid Cycle ◽  
Pentose Phosphate ◽  
Growth Defect ◽  
Protein Coding ◽  
Arginine Catabolism ◽  
Futile Cycling ◽  
Mutant Phenotypes ◽  
Tricarboxylic Acid Cycle Intermediates

ABSTRACT The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes.

scholarly journals Metabolic phases during the development of granulation tissue

1967 ◽  
Vol 105 (1) ◽  
pp. 333-341 ◽  
Author(s):  
Kirsti Lampiaho ◽  
E. Kulonen
Keyword(s):  
Granulation Tissue ◽  
Collagen Synthesis ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Pentose Phosphate ◽  
Acid Cycle ◽  
Short Period ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Observation Period

1. The metabolism of incubated slices of sponge-induced granulation tissue, harvested 4–90 days after the implantation, was studied with special reference to the capacity of collagen synthesis and to the energy metabolism. Data are also given on the nucleic acid contents during the observation period. Three metabolic phases were evident. 2. The viability of the slices for the synthesis of collagen was studied in various conditions. Freezing and homogenization destroyed the capacity of the tissue to incorporate proline into collagen. 3. Consumption of oxygen reached the maximum at 30–40 days. There was evidence that the pentose phosphate cycle was important, especially during the phases of the proliferation and the involution. The formation of lactic acid was maximal at about 20 days. 4. The capacity to incorporate proline into collagen hydroxyproline in vitro was limited to a relatively short period at 10–30 days. 5. The synthesis of collagen was dependent on the supply of oxygen and glucose, which latter could be replaced in the incubation medium by other monosaccharides but not by the metabolites of glucose or tricarboxylic acid-cycle intermediates.

scholarly journals Improving the Production of Riboflavin by Introducing a Mutant Ribulose 5-Phosphate 3-Epimerase Gene in Bacillus Subtilis

2021 ◽  
Author(s):  
Bin Yang ◽  
Yiwen Sun ◽  
Shouying Fu ◽  
Miaomiao Xia ◽  
Chuan Liu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pentose Phosphate Pathway ◽  
De Novo ◽  
Fold Increase ◽  
Fermentation Medium ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Growth Defect ◽  
Riboflavin Production ◽  
Expression Of Genes

Abstract Background: Ribulose 5-phosphate (Ru5P) and guanosine 5'-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP (purine de novo synthesis pathway). As a product of the oxidative pentose phosphate pathway (OPPP), Ru5P can flow back into the Embden-Meyerhof-Parnas pathway (EMP) through the non-oxidative pentose phosphate pathway (NOPPP). Major enzymes of the NOPPP include transaldolase (Tal), transketolase (Tkt), and ribulose 5-phosphate 3-epimerase (Rpe), which catalyze the conversion of Ru5P into xylulose 5-phosphate (X5P). Thus, inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. However, there are no studies on the mutation or deletion of Rpe for improving riboflavin production. Results: We mutated the genomic copy of rpe, in the riboflavin-producing Bacillus subtilis BSLY. The resulting strain BSR produced 320.50±31.28 mg/L of riboflavin in a fermentation medium containing 40 g/L sucrose as the carbon source, representing a 5-fold increase over the parental strain. We also constructed an rpe nonsense mutation strain BSRN, which produced 365.69±27.53 mg/L riboflavin in fermentation medium. However, BSRN had a growth defect compared with BSR and BSLY, which was also present in media containing glucose, maltose, and fructose. The growth of BSRN was restored when mutant or wild-type rpe was overexpressed. Notably, BSR exhibited respective increases of the inosine and guanine titers by 163% and 40%. Furthermore, a 35.96-fold increase of inosine 5’-monophosphate (IMP) titer was detected in BSRN. The transcription levels of most OPPP, purine, and GTP synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were respectively 49% lower and 310% higher than in BSLY. The production of riboflavin was increased to 479.90±33.21 mg/L when ribA was expressed in BSR. The expression of zwf, gntZ, prs, and purF, respectively enhanced the riboflavin production by 48%, 31%, 26%, and 35%. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29±63.44 mg/L).Conclusions: This paper describes a mutation of the ribulose 5-phosphate 3-epimerase and its influence on riboflavin production. The results of qRT-PCR and HPLC analyses indicated that the rpe mutant showed a different pattern of purine metabolism, while the cells maintained generally normal levels of the transcription of genes related to PPP and purine de novel synthesis pathways. With the increased expression of genes in the OPPP, purine, and riboflavin synthesis pathways, the production of riboflavin was effectively enhanced, showing the potential for further engineering of this strain.

scholarly journals Impact of Activation of the Neotrehalosadiamine/Kanosamine Biosynthetic Pathway on the Metabolism of Bacillus subtilis

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Natsumi Saito ◽  
Huong Minh Nguyen ◽  
Takashi Inaoka
Keyword(s):  
Bacillus Subtilis ◽  
Energy Metabolism ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Growth Defect ◽  
Metabolome Analysis ◽  
Content Type ◽  
Tca Cycle Intermediates

ABSTRACT The pentose phosphate (PP) pathway is one of the major sources of cellular NADPH. A Bacillus subtilis zwf mutant that lacks glucose-6-phosphate dehydrogenase (the enzyme that catalyzes the first step of the PP pathway) showed inoculum-dose-dependent growth. This growth defect was suppressed by glcP disruption, which causes the upregulation of the autoinducer neotrehalosadiamine (NTD)/kanosamine biosynthetic pathway. A metabolome analysis showed that the stimulation of NTD/kanosamine biosynthesis caused significant accumulation of tricarboxylic acid (TCA) cycle intermediates and NADPH. Because the major malic enzyme YtsJ concomitantly generates NADPH through malate-to-pyruvate conversion, de novo NTD/kanosamine biosynthesis can result in an increase in the intracellular NADPH pool via the accumulation of malate. In fact, a zwf mutant grew in malate-supplemented medium. Artificial induction of glcP in the zwf mutant caused a reduction in the intracellular NADPH pool. Moreover, the correlation between the expression level of the NTD/kanosamine biosynthesis operon ntdABC and the intracellular NADPH pool was confirmed. Our results suggest that NTD/kanosamine has the potential to modulate carbon energy metabolism through an autoinduction mechanism. IMPORTANCE Autoinducers enable bacteria to sense cell density and to coordinate collective behavior. NTD/kanosamine is an autoinducer produced by B. subtilis and several close relatives, although its physiological function remains unknown. The most important finding of this study was the significance of de novo NTD/kanosamine biosynthesis in the modulation of the central carbon metabolism in B. subtilis. We showed that NTD/kanosamine biosynthesis caused an increase in the NADPH pool via the accumulation of TCA cycle intermediates. These results suggest a possible role for NTD/kanosamine in carbon energy metabolism. As Bacillus species are widely used for the industrial production of various useful enzymes and compounds, the NTD/kanosamine biosynthetic pathway might be utilized to control metabolic pathways in these industrial strains.

scholarly journals Improving the Production of Riboflavin by Introducing a Mutant Ribulose 5-Phosphate 3-Epimerase Gene in Bacillus subtilis

2021 ◽  
Vol 9 ◽  
Author(s):  
Bin Yang ◽  
Yiwen Sun ◽  
Shouying Fu ◽  
Miaomiao Xia ◽  
Yuan Su ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Flux ◽  
Parent Strain ◽  
Fold Increase ◽  
Pentose Phosphate ◽  
Growth Defect ◽  
Riboflavin Biosynthesis ◽  
Riboflavin Production ◽  
Single Nucleotide ◽  
Metabolic Phenotypes

Ribulose 5-phosphate (Ru5P) and guanosine 5′-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP. Ribulose 5-phosphate 3-epimerase (Rpe) catalyzes the conversion of ribulose 5-phosphate into xylulose 5-phosphate. Inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. Here we investigated the effect of mutation of rpe and other related genes on riboflavin production, physiological and metabolic phenotypes in Bacillus subtilis LY (BSLY). Introducing single nucleotide deletion (generated BSR) or nonsense mutation (generated BSRN) on the genomic copy of rpe, resulting in more than fivefold increase of riboflavin production over the parental strain. BSR process 62% Rpe activity, while BSRN lost the entire Rpe activity and had a growth defect compared with the parent strain. BSR and BSRN exhibited increases of the inosine and guanine titers, in addition, BSRN exhibited an increase of inosine 5′-monophosphate titer in fermentation. The transcription levels of most oxidative pentose phosphate pathway and purine synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were higher than in BSLY. The production of riboflavin was increased to 479.90 ± 33.21 mg/L when ribA was overexpressed in BSR. The overexpression of zwf, gntZ, prs, and purF also enhanced the riboflavin production. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29 ± 63.44 mg/L), showing the potential for further engineering of this strain.

scholarly journals Intracellular Carbon Fluxes in Riboflavin-Producing Bacillussubtilis during Growth on Two-Carbon Substrate Mixtures

2002 ◽  
Vol 68 (4) ◽  
pp. 1760-1771 ◽  
Author(s):  
Michael Dauner ◽  
Marco Sonderegger ◽  
Michel Hochuli ◽  
Thomas Szyperski ◽  
Kurt Wüthrich ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Energy Content ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Fluxes ◽  
Pentose Phosphate ◽  
Carbon Substrate ◽  
Split Ratio ◽  
Nuclear Magnetic Resonance Spectra ◽  
Substrate Mixtures

ABSTRACT Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional 13C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.

scholarly journals Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats

1977 ◽  
Vol 166 (3) ◽  
pp. 583-592 ◽  
Author(s):  
Frederick C. Kauffman ◽  
Roxanne K. Evans ◽  
Ronald G. Thurman
Keyword(s):  
Malic Enzyme ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Tricarboxylic Acid Cycle ◽  
Pentose Phosphate ◽  
Phosphogluconate Dehydrogenase ◽  
Oxidation Reduction ◽  
Fructose 1,6 Bisphosphate ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Glucose 6 Phosphate Dehydrogenase

The contents of adenine nucleotides as well as steady-state concentrations of a number of glycolytic, pentose phosphate-pathway and tricarboxylic acid-cycle intermediates were measured in extracts of livers from normal and phenobarbital-treated rats that were perfused with p-nitroanisole. Metabolites were measured in livers that were freeze-clamped during periods of maximal rates of drug metabolism. Treatment of rats with phenobarbital increased rates of p-nitroanisole O-demethylation approx. fivefold. The concentrations of lactate, xylulose 5-phosphate and ribulose 5-phosphate were increased by phenobarbital treatment, whereas that of fructose 1,6-bisphosphate declined. Perfusion of livers with p-nitroanisole produced significant increases in 6-phosphogluconate and ribulose 5-phosphate in livers from phenobarbital-treated rats, but not in livers from control rats. Treatment of rats with phenobarbital caused [NADP+]/[NADPH] to change in the direction of more oxidation, as calculated from measured concentrations of 6-phosphogluconate and ribulose 5-phosphate; however, the [NADP+]/[NADPH] ratio calculated from ‘malic’ enzyme was not changed. Additions of p-nitroanisole produced a reduction of NADP+ as calculated from 6-phosphogluconate dehydrogenase activity, but did not alter the [NADP+]/[NADPH] ratio calculated from substrates assumed to be in equilibrium with ‘malic’ enzyme. Activities of both glucose 6-phosphate dehydrogenase and ‘malic’ enzyme were increased by phenobarbital treatment. NAD+ became more reduced as a result of phenobarbital treatment; however, perfusion of livers with p-nitroanisole did not cause a change in the oxidation–reduction state of this nucleotide. Concentrations of adenine nucleotides in livers were not altered significantly by treatment of rats with phenobarbital; however, a significant decline in the [ATP]/[ADP] ratio occurred during mixed-function oxidation of p-nitroanisole in livers from phenobarbital-treated rats, but not in livers from normal rats. Perfusion of livers with two other substrates for mixed-function oxidation, hexobarbital and aminopyrine, produced an increase in the [NADP+]/[NADPH] ratio calculated from ‘malic’ enzyme. In contrast with livers perfused with p-nitroanisole, there was no significant change in adenine nucleotides in livers exposed to hexobarbital or aminopyrine. Addition of 2,4-dinitrophenol (25μm) to the perfusate containing aminopyrine decreased the [ATP]/[ADP] ratio and tended to prevent the oxidation of NADPH observed with aminopyrine alone. Thus in the presence of an uncoupler of oxidative phosphorylation, NADPH generation may exceed its utilization via mixed-function oxidation.

scholarly journals Malonyl-proteome profiles of Staphylococcus aureus reveal lysine malonylation modification in enzymes involved in energy metabolism

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yanan Shi ◽  
Jingjing Zhu ◽  
Yan Xu ◽  
Xiaozhao Tang ◽  
Zushun Yang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Energy Metabolism ◽  
Active Sites ◽  
Current Knowledge ◽  
Tricarboxylic Acid Cycle ◽  
Protein A ◽  
Pentose Phosphate ◽  
Post Translational Modification ◽  
Bacterial Physiology ◽  
Dihydrolipoyl Dehydrogenase

Abstract Background Protein lysine malonylation, a novel post-translational modification (PTM), has been recently linked with energy metabolism in bacteria. Staphylococcus aureus is the third most important foodborne pathogen worldwide. Nonetheless, substrates and biological roles of malonylation are still poorly understood in this pathogen. Results Using anti-malonyl-lysine antibody enrichment and high-resolution LC-MS/MS analysis, 440 lysine-malonylated sites were identified in 281 proteins of S. aureus strain. The frequency of valine in position − 1 and alanine at + 2 and + 4 positions was high. KEGG pathway analysis showed that six categories were highly enriched, including ribosome, glycolysis/gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), valine, leucine, isoleucine degradation, and aminoacyl-tRNA biosynthesis. In total, 31 malonylated sites in S. aureus shared homology with lysine-malonylated sites previously identified in E. coli, indicating malonylated proteins are highly conserved among bacteria. Key rate-limiting enzymes in central carbon metabolic pathways were also found to be malonylated in S. aureus, namely pyruvate kinase (PYK), 6-phosphofructokinase, phosphoglycerate kinase, dihydrolipoyl dehydrogenase, and F1F0-ATP synthase. Notably, malonylation sites were found at or near protein active sites, including KH domain protein, thioredoxin, alanine dehydrogenase (ALD), dihydrolipoyl dehydrogenase (LpdA), pyruvate oxidase CidC, and catabolite control protein A (CcpA), thus suggesting that lysine malonylation may affect the activity of such enzymes. Conclusions Data presented herein expand the current knowledge on lysine malonylation in prokaryotes and indicate the potential roles of protein malonylation in bacterial physiology and metabolism.

scholarly journals Triheptanoin partially restores levels of tricarboxylic acid cycle intermediates in the mouse pilocarpine model of epilepsy

2013 ◽  
Vol 129 (1) ◽  
pp. 107-119 ◽  
Author(s):  
Mussie G. Hadera ◽  
Olav B. Smeland ◽  
Tanya S. McDonald ◽  
Kah Ni Tan ◽  
Ursula Sonnewald ◽  
...  
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Pilocarpine Model ◽  
Tricarboxylic Acid Cycle Intermediates
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