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Carbohydrate metabolism and carbon fixation in Roseobacter denitrificans OCh114.

Tang KH, Feng X, Tang YJ, Blankenship RE - PLoS ONE (2009)

Bottom Line: We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B(12) (cyanocobalamin).Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination.Through measurements of (13)C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10-15% of protein carbon from CO(2); (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biology and Chemistry, Washington University in St. Louis, St. Louis, Missouri, United States of America.

ABSTRACT
The Roseobacter clade of aerobic marine proteobacteria, which compose 10-25% of the total marine bacterial community, has been reported to fix CO(2), although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO(2) assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B(12) (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of (13)C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10-15% of protein carbon from CO(2); (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source.

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Proposed carbohydrate metabolism and amino acid biosynthesis pathways in R. denitrificans.Predicted 13C-labeling distributions using D-[1-13C]glucose and D-[6-13C]glucose in the EMP and ED pathways are shown (a), and the cleavage of a hexose molecule in the EMP vs. ED pathway (b). Abbreviations: EMP, Embden-Meyerhof-Parnas (glycolysis); ED, Entner-Doudoroff; and PP, pentose phosphate. Experimentally identified 13C-labeling patterns are reported in the context.
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pone-0007233-g002: Proposed carbohydrate metabolism and amino acid biosynthesis pathways in R. denitrificans.Predicted 13C-labeling distributions using D-[1-13C]glucose and D-[6-13C]glucose in the EMP and ED pathways are shown (a), and the cleavage of a hexose molecule in the EMP vs. ED pathway (b). Abbreviations: EMP, Embden-Meyerhof-Parnas (glycolysis); ED, Entner-Doudoroff; and PP, pentose phosphate. Experimentally identified 13C-labeling patterns are reported in the context.

Mentions: In the cultures grown in the minimal medium containing either D-[1-13C]glucose or D-[6-13C]glucose, the isotopomer labeling data of serine (the precursor is 3-phosphoglycerate) and alanine (the precursor is pyruvate) were different (Table S1e, S1f, S1i, and S3). Three metabolic pathways can be employed for sugar utilization by R. denitrificans and need to be considered to account for the isotopomer abundance in these protein-derived amino acids: (1) the Embden-Meyerhof-Parnas (EMP) pathway (glycolysis), by which one [1-13C]glucose molecule is cleaved into two glyceraldehyde-3-phosphate (GAP) molecules: one is [3-13C]-labeled and the other is unlabeled using either D-[1-13C]glucose or D-[6-13C]glucose; (2) the Entner-Doudoroff (ED) pathway [18], by which one molecule of glucose generates one molecule of GAP, in which the third carbon is labeled using D-[6-13C]glucose, and one molecule of pyruvate, where the first carbon is labeled using D-[1-13C]glucose (Figure 2a); (3) the pentose phosphate (PP) pathway, the first carbon of glucose is removed as CO2, if the oxidative PP pathway is active, to generate ribose-5-phosphate, which can be converted to GAP through the non-oxidative PP pathway (Figure S2). As shown in Figure 2a, regardless of the pathways, GAP is converted into 3-phosphoglycerate (3-PGA), the precursor of glycine, serine, and cysteine, and then to pyruvate, the precursor of alanine, valine, leucine, and isoleucine.


Carbohydrate metabolism and carbon fixation in Roseobacter denitrificans OCh114.

Tang KH, Feng X, Tang YJ, Blankenship RE - PLoS ONE (2009)

Proposed carbohydrate metabolism and amino acid biosynthesis pathways in R. denitrificans.Predicted 13C-labeling distributions using D-[1-13C]glucose and D-[6-13C]glucose in the EMP and ED pathways are shown (a), and the cleavage of a hexose molecule in the EMP vs. ED pathway (b). Abbreviations: EMP, Embden-Meyerhof-Parnas (glycolysis); ED, Entner-Doudoroff; and PP, pentose phosphate. Experimentally identified 13C-labeling patterns are reported in the context.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749216&req=5

pone-0007233-g002: Proposed carbohydrate metabolism and amino acid biosynthesis pathways in R. denitrificans.Predicted 13C-labeling distributions using D-[1-13C]glucose and D-[6-13C]glucose in the EMP and ED pathways are shown (a), and the cleavage of a hexose molecule in the EMP vs. ED pathway (b). Abbreviations: EMP, Embden-Meyerhof-Parnas (glycolysis); ED, Entner-Doudoroff; and PP, pentose phosphate. Experimentally identified 13C-labeling patterns are reported in the context.
Mentions: In the cultures grown in the minimal medium containing either D-[1-13C]glucose or D-[6-13C]glucose, the isotopomer labeling data of serine (the precursor is 3-phosphoglycerate) and alanine (the precursor is pyruvate) were different (Table S1e, S1f, S1i, and S3). Three metabolic pathways can be employed for sugar utilization by R. denitrificans and need to be considered to account for the isotopomer abundance in these protein-derived amino acids: (1) the Embden-Meyerhof-Parnas (EMP) pathway (glycolysis), by which one [1-13C]glucose molecule is cleaved into two glyceraldehyde-3-phosphate (GAP) molecules: one is [3-13C]-labeled and the other is unlabeled using either D-[1-13C]glucose or D-[6-13C]glucose; (2) the Entner-Doudoroff (ED) pathway [18], by which one molecule of glucose generates one molecule of GAP, in which the third carbon is labeled using D-[6-13C]glucose, and one molecule of pyruvate, where the first carbon is labeled using D-[1-13C]glucose (Figure 2a); (3) the pentose phosphate (PP) pathway, the first carbon of glucose is removed as CO2, if the oxidative PP pathway is active, to generate ribose-5-phosphate, which can be converted to GAP through the non-oxidative PP pathway (Figure S2). As shown in Figure 2a, regardless of the pathways, GAP is converted into 3-phosphoglycerate (3-PGA), the precursor of glycine, serine, and cysteine, and then to pyruvate, the precursor of alanine, valine, leucine, and isoleucine.

Bottom Line: We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B(12) (cyanocobalamin).Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination.Through measurements of (13)C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10-15% of protein carbon from CO(2); (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biology and Chemistry, Washington University in St. Louis, St. Louis, Missouri, United States of America.

ABSTRACT
The Roseobacter clade of aerobic marine proteobacteria, which compose 10-25% of the total marine bacterial community, has been reported to fix CO(2), although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO(2) assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B(12) (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of (13)C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10-15% of protein carbon from CO(2); (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source.

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