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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 pentose phosphate pathway in R. denitrificans.All of the steps are reversible, except expulsion of CO2 catalyzed by 6-phosphogluconate dehydrogenase. Predicted 13C-labeling distributions using D-[1-13C]glucose and D-[6-13C]glucose in the oxidative PP and non-oxidative PP pathways are shown. Experimentally identified 13C-labeling patterns are reported in the context.
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pone-0007233-g004: Proposed pentose phosphate pathway in R. denitrificans.All of the steps are reversible, except expulsion of CO2 catalyzed by 6-phosphogluconate dehydrogenase. Predicted 13C-labeling distributions using D-[1-13C]glucose and D-[6-13C]glucose in the oxidative PP and non-oxidative PP pathways are shown. Experimentally identified 13C-labeling patterns are reported in the context.

Mentions: The ED pathway also leads to different predicted labeling in the aromatic amino acids. Phosphoenolpyruvate (PEP), synthesized from 3-PGA, and erythrose-4-phosphate (E4P), the intermediate in the non-oxidative PP pathway, are the precursors of phenylalanine, tyrosine and tryptophan. Labeled E4P in the non-oxidative PP pathway (Figure S2) leads to lower M+0 value in the aromatic amino acids (0.50±0.03) compared to serine (0.74) using D-[1-13C]glucose. The ED and PP pathways can explain much higher M+0 value in phenylalanine (0.48) and tyrosine (0.53) using D-[1-13C]glucose than the value in these amino acids (0.04) using D-[6-13C]glucose, and more labeled carbon can be incorporated into PEP and E4P using D-[6-13C]glucose (Figure 2a and 4).


Carbohydrate metabolism and carbon fixation in Roseobacter denitrificans OCh114.

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

Proposed pentose phosphate pathway in R. denitrificans.All of the steps are reversible, except expulsion of CO2 catalyzed by 6-phosphogluconate dehydrogenase. Predicted 13C-labeling distributions using D-[1-13C]glucose and D-[6-13C]glucose in the oxidative PP and non-oxidative PP pathways are shown. Experimentally identified 13C-labeling patterns are reported in the context.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749216&req=5

pone-0007233-g004: Proposed pentose phosphate pathway in R. denitrificans.All of the steps are reversible, except expulsion of CO2 catalyzed by 6-phosphogluconate dehydrogenase. Predicted 13C-labeling distributions using D-[1-13C]glucose and D-[6-13C]glucose in the oxidative PP and non-oxidative PP pathways are shown. Experimentally identified 13C-labeling patterns are reported in the context.
Mentions: The ED pathway also leads to different predicted labeling in the aromatic amino acids. Phosphoenolpyruvate (PEP), synthesized from 3-PGA, and erythrose-4-phosphate (E4P), the intermediate in the non-oxidative PP pathway, are the precursors of phenylalanine, tyrosine and tryptophan. Labeled E4P in the non-oxidative PP pathway (Figure S2) leads to lower M+0 value in the aromatic amino acids (0.50±0.03) compared to serine (0.74) using D-[1-13C]glucose. The ED and PP pathways can explain much higher M+0 value in phenylalanine (0.48) and tyrosine (0.53) using D-[1-13C]glucose than the value in these amino acids (0.04) using D-[6-13C]glucose, and more labeled carbon can be incorporated into PEP and E4P using D-[6-13C]glucose (Figure 2a and 4).

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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