Limits...
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.

Show MeSH
Proposed metabolic flux for carbon fixation by R. denitrificans.Bold curves represent proposed stronger metabolic fluxes.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2749216&req=5

pone-0007233-g005: Proposed metabolic flux for carbon fixation by R. denitrificans.Bold curves represent proposed stronger metabolic fluxes.

Mentions: To investigate metabolic fluxes through the anaplerotic pathways, [3-13C]pyruvate was used and the 13C-isotopomer abundance in aspartate (from OAA), serine (from PEP, 3-PGA) and alanine (from pyruvate) was analyzed (Table S1h). Both QRT-PCR and 13C-labeling pattern suggest that the flux of pyruvate to PEP is weak (different isotopomer distribution between alanine and serine), consistent with low gene expression of ppdK (RD1_1948, pyruvate phosphate dikinase, EC 2.7.9.1, pyruvate→PEP) and that the pathway from OAA to PEP is likely to be active and pyruvate to OAA is not as strong. This is in agreement with the gene expression profile of pckA (RD1_1376) and pyc (RD1_3376). Hence, most of the 13C-labeling in OAA likely comes from the TCA cycle flux (from malate), in which the pyruvate to malate pathway (via malic enzyme) should be rather active, compatible with higher expression of tme (RD1_0421) than pyc (RD1_3376). The proposed anaplerotic flux is summarized in Figure 5.


Carbohydrate metabolism and carbon fixation in Roseobacter denitrificans OCh114.

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

Proposed metabolic flux for carbon fixation by R. denitrificans.Bold curves represent proposed stronger metabolic fluxes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007233-g005: Proposed metabolic flux for carbon fixation by R. denitrificans.Bold curves represent proposed stronger metabolic fluxes.
Mentions: To investigate metabolic fluxes through the anaplerotic pathways, [3-13C]pyruvate was used and the 13C-isotopomer abundance in aspartate (from OAA), serine (from PEP, 3-PGA) and alanine (from pyruvate) was analyzed (Table S1h). Both QRT-PCR and 13C-labeling pattern suggest that the flux of pyruvate to PEP is weak (different isotopomer distribution between alanine and serine), consistent with low gene expression of ppdK (RD1_1948, pyruvate phosphate dikinase, EC 2.7.9.1, pyruvate→PEP) and that the pathway from OAA to PEP is likely to be active and pyruvate to OAA is not as strong. This is in agreement with the gene expression profile of pckA (RD1_1376) and pyc (RD1_3376). Hence, most of the 13C-labeling in OAA likely comes from the TCA cycle flux (from malate), in which the pyruvate to malate pathway (via malic enzyme) should be rather active, compatible with higher expression of tme (RD1_0421) than pyc (RD1_3376). The proposed anaplerotic flux is summarized in Figure 5.

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.

Show MeSH