Abstract
The Roseobacter clade of aerobic marine proteobacteria, which compose 10–25% of the total marine bacterial community, has been reported to fix CO2, although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO2 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 B12 (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 13C-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 CO2; (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.
Highlights
Two of the most important sources of carbon sinks known in nature are absorption of CO2 by the oceans and photosynthesis by photosynthetic organisms
CO oxidation was confirmed experimentally for the non-phototrophic Roseobacter clade bacterium Silicibacter pomeroyi [5] and other Roseobacter clade [6], and CO2 fixation was suggested in several marine Aerobic Anoxygenic Phototrophs (AAPs) [7], while bioinformatic analysis in Roseobacter clade with completed genome sequence indicated that the genes encoding ribulose bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoribulokinase required in the Calvin cycle for carbon fixation, as well as genes for other autotrophic CO2 fixation pathways, are missing in these bacteria [5,8,9,10]
CO2 fixation in R. denitrificans and other AAPs In this paper, we reported that R. denitrificans uses the anaplerotic pathways to fix 10–15% of protein carbon from CO2
Summary
Two of the most important sources of carbon sinks known in nature are absorption of CO2 by the oceans and photosynthesis by photosynthetic organisms. Some members of the Roseobacter clade belong to a group known as Aerobic Anoxygenic Phototrophs (AAPs), the only known organisms performing photosynthesis requiring oxygen but not producing oxygen, while other members are non-phototrophic. CO oxidation was confirmed experimentally for the non-phototrophic Roseobacter clade bacterium Silicibacter pomeroyi [5] and other Roseobacter clade [6], and CO2 fixation was suggested in several marine AAPs [7], while bioinformatic analysis in Roseobacter clade with completed genome sequence indicated that the genes encoding ribulose bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoribulokinase required in the Calvin cycle for carbon fixation, as well as genes for other autotrophic CO2 fixation pathways, are missing in these bacteria [5,8,9,10]. Given that organisms in the Roseobacter clade are known to require organic carbon sources for growth [12,13], understanding how Roseobacter clade bacteria utilize organic carbon and assimilate CO2 will help us understand the bio-energy metabolism, production of bioactive metabolites, and roles of global carbon cycle in these wide-spread marine bacteria
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