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Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA

BACKGROUND: The bacterium Pseudomonas fluorescens switches to an alginate-producing phenotype when the pleiotropic anti-sigma factor MucA is inactivated. The inactivation is accompanied by an increased biomass yield on carbon sources when grown under nitrogen-limited chemostat conditions. A previous...

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Autores principales: Lien, Stina K, Niedenführ, Sebastian, Sletta, Håvard, Nöh, Katharina, Bruheim, Per
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351692/
https://www.ncbi.nlm.nih.gov/pubmed/25889900
http://dx.doi.org/10.1186/s12918-015-0148-0
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author Lien, Stina K
Niedenführ, Sebastian
Sletta, Håvard
Nöh, Katharina
Bruheim, Per
author_facet Lien, Stina K
Niedenführ, Sebastian
Sletta, Håvard
Nöh, Katharina
Bruheim, Per
author_sort Lien, Stina K
collection PubMed
description BACKGROUND: The bacterium Pseudomonas fluorescens switches to an alginate-producing phenotype when the pleiotropic anti-sigma factor MucA is inactivated. The inactivation is accompanied by an increased biomass yield on carbon sources when grown under nitrogen-limited chemostat conditions. A previous metabolome study showed significant changes in the intracellular metabolite concentrations, especially of the nucleotides, in mucA deletion mutants compared to the wild-type. In this study, the P. fluorescens SBW25 wild-type and an alginate non-producing mucA- ΔalgC double-knockout mutant are investigated through model-based (13)C-metabolic flux analysis ((13)C-MFA) to explore the physiological consequences of MucA inactivation at the metabolic flux level. Intracellular metabolite extracts from three carbon labelling experiments using fructose as the sole carbon source are analysed for (13)C-label incorporation in primary metabolites by gas and liquid chromatography tandem mass spectrometry. RESULTS: From mass isotopomer distribution datasets, absolute intracellular metabolic reaction rates for the wild type and the mutant are determined, revealing extensive reorganisation of carbon flux through central metabolic pathways in response to MucA inactivation. The carbon flux through the Entner-Doudoroff pathway was reduced in the mucA- ΔalgC mutant, while flux through the pentose phosphate pathway was increased. Our findings also indicated flexibility of the anaplerotic reactions through down-regulation of the pyruvate shunt in the mucA- ΔalgC mutant and up-regulation of the glyoxylate shunt. CONCLUSIONS: Absolute metabolic fluxes and metabolite levels give detailed, integrated insight into the physiology of this industrially, medically and agriculturally important bacterial species and suggest that the most efficient way of using a mucA- mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12918-015-0148-0) contains supplementary material, which is available to authorized users.
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spelling pubmed-43516922015-03-07 Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA Lien, Stina K Niedenführ, Sebastian Sletta, Håvard Nöh, Katharina Bruheim, Per BMC Syst Biol Research Article BACKGROUND: The bacterium Pseudomonas fluorescens switches to an alginate-producing phenotype when the pleiotropic anti-sigma factor MucA is inactivated. The inactivation is accompanied by an increased biomass yield on carbon sources when grown under nitrogen-limited chemostat conditions. A previous metabolome study showed significant changes in the intracellular metabolite concentrations, especially of the nucleotides, in mucA deletion mutants compared to the wild-type. In this study, the P. fluorescens SBW25 wild-type and an alginate non-producing mucA- ΔalgC double-knockout mutant are investigated through model-based (13)C-metabolic flux analysis ((13)C-MFA) to explore the physiological consequences of MucA inactivation at the metabolic flux level. Intracellular metabolite extracts from three carbon labelling experiments using fructose as the sole carbon source are analysed for (13)C-label incorporation in primary metabolites by gas and liquid chromatography tandem mass spectrometry. RESULTS: From mass isotopomer distribution datasets, absolute intracellular metabolic reaction rates for the wild type and the mutant are determined, revealing extensive reorganisation of carbon flux through central metabolic pathways in response to MucA inactivation. The carbon flux through the Entner-Doudoroff pathway was reduced in the mucA- ΔalgC mutant, while flux through the pentose phosphate pathway was increased. Our findings also indicated flexibility of the anaplerotic reactions through down-regulation of the pyruvate shunt in the mucA- ΔalgC mutant and up-regulation of the glyoxylate shunt. CONCLUSIONS: Absolute metabolic fluxes and metabolite levels give detailed, integrated insight into the physiology of this industrially, medically and agriculturally important bacterial species and suggest that the most efficient way of using a mucA- mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12918-015-0148-0) contains supplementary material, which is available to authorized users. BioMed Central 2015-02-18 /pmc/articles/PMC4351692/ /pubmed/25889900 http://dx.doi.org/10.1186/s12918-015-0148-0 Text en © Lien et al.; licensee BioMed Central. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Lien, Stina K
Niedenführ, Sebastian
Sletta, Håvard
Nöh, Katharina
Bruheim, Per
Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA
title Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA
title_full Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA
title_fullStr Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA
title_full_unstemmed Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA
title_short Fluxome study of Pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA
title_sort fluxome study of pseudomonas fluorescens reveals major reorganisation of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor muca
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351692/
https://www.ncbi.nlm.nih.gov/pubmed/25889900
http://dx.doi.org/10.1186/s12918-015-0148-0
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