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Glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H: a combined fluxomics and transcriptomics analysis

ABSTRACT: In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by 13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For 13C-MFA, cells were cultivated with specifically 13C-labeled glucose and intracellular metabolites were analyzed for their labeling pattern by LC-MS. In growth phase I, 90% of the glucose was oxidized periplasmatically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. Since G. oxydans lacks phosphofructokinase, glucose 6-phosphate can only be metabolized via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP). 13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phase I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II. The transcriptome comparisons of G. oxydans growth phase II vs. growth phase I were repeated independently three times in biological replicates resulting in 3 hybridizations as termed by sample 1 to 3.

ORGANISM(S): Gluconobacter oxydans 621H

SUBMITTER: Tino Polen

PROVIDER: E-GEOD-42223 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Combined fluxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H.

Hanke Tanja T Nöh Katharina K Noack Stephan S Polen Tino T Bringer Stephanie S Sahm Hermann H Wiechert Wolfgang W Bott Michael M

Applied and environmental microbiology 20130201 7

In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by (13)C-based metabolic flux analysis ((13)C-MFA) in combination with transcriptomics and enzyme assays. For (13)C-MFA, cells were cultivated with specifically (13)C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxi ...[more]

PMID: 23377928

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Project description:The obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H oxidizes sugars and sugar alcohols primarily in the periplasm and only a small fraction is metabolized in the cytoplasm. The latter can occur either via the Entner-Doudoroff pathway (EDP) or via the pentose phosphate pathway (PPP). The Embden-Meyerhof pathway is non-functional and a cyclic operation of the tricarboxylic acid cycle is prevented by the absence of succinate dehydrogenase. In this work, the cytoplasmic catabolism of fructose formed by oxidation of mannitol was analyzed with a M-NM-^Tgnd mutant lacking the oxidative PPP and a M-NM-^Tedd-eda mutant devoid of the EDP. The growth characteristics of the two mutants under controlled conditions with mannitol as carbon source and enzyme activities showed that the PPP is the main route for cytoplasmic fructose catabolism, whereas the EDP is dispensable and even unfavorable. The M-NM-^Tedd-eda mutant (lacking 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) formed 24% more cell mass than the reference strain. In contrast, deletion of gnd (6-phosphogluconate dehydrogenase) severely inhibited growth and caused a strong selection pressure for secondary mutations inactivating glucose-6-phosphate dehydrogenase, thus preventing fructose catabolism via the EDP, too. These M-NM-^Tgnd zwf* mutants were almost totally disabled in fructose catabolism, but still produced about 14% of the carbon dioxide of the reference strain, possibly by catabolizing substrates from the yeast extract. Overexpression of gnd in the reference strain improved biomass formation in a similar manner as deletion of edd-eda, further confirming the importance of the PPP for cytoplasmic fructose catabolism. The three transcriptome comparisons of M-NM-^Tupp M-NM-^Tgnd zwf* vs. M-NM-^Tupp and of M-NM-^Tupp M-NM-^Tedd-eda vs. M-NM-^Tupp, and of M-NM-^Tupp M-NM-^Tgnd versus M-NM-^Tupp, were repeated independently three times in biological replicates resulting in 3 x 3 hybridizations as termed by sample 1 to 9.

Project description:The genome-wide transcriptional responses of the strictly aerobic -proteobacterium Gluconobacter oxydans 621H to oxygen limitation, to the absence of the cytochrome bc1 complex, and to low pH were studied using DNA microarray analyses. Oxygen limitation caused expression changes of 486 genes, representing 20% of the chromosomal genes. Genes with an increased mRNA level included those for terminal oxidases, the cytochrome bc1 complex, transhydrogenase, two alcohol dehydrogenases, heme biosynthesis, PTS proteins, proteins involved in cyclic diGMP synthesis and degradation, two sigma factors, flagella and chemotaxis proteins, several stress proteins, and a putative exporter protein. The downregulated genes comprised those for respiratory dehydrogenases, enzymes of central metabolism, PQQ biosynthesis, outer membrane receptors, Sec proteins, and proteins involved in transcription and translation. A M-NM-^TqrcABC mutant of G. oxydans showed a growth defect during cultivation on mannitol at pH 4 under oxygen saturation. Comparison of the transcriptomes of this mutant versus the wild type under these conditions revealed 51 differentially expressed genes. Interestingly, almost all of the 45 genes with increased expression in the M-NM-^TqrcABC mutant at pH 4 were also upregulated in the wild type grown at pH 6 under oxygen limitation. These results support an active role of the cytochrome bc1 complex in G. oxydans respiration. The transcriptome comparison of G. oxydans wild type at pH 4 versus pH 6 in mannitol medium under oxygen-saturated conditions uncovered only 72 differentially expressed genes. The 35 upregulated genes included those for cytochrome bd oxidase, major polyol dehydrogenase, iron storage and oxidative stress proteins. Among the 37 downregulated genes were some encoding enzymes dealing with carbon dioxide, such as biotin carboxylase, biotin carboxyl carrier protein, and carboanhydrase. These results give first insights into global transcriptional responses of G. oxydans. DNA microarray experiments were repeated independently three times for G. oxydans M-NM-^TqrcABC versus wild type and G. oxydans grown at pH 4 versus pH 6 and four times for G. oxydans grown at oxygen limitation versus pH oxygen saturation in biological replicates.

Dataset Information

Glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H: a combined fluxomics and transcriptomics analysis

Publications

Combined fluxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H.

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