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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

Lien, Stina Katrine; Niedenführ, Sebastian; Sletta, Håvard; Nöh, Katharina; Bruheim, Per
Journal article, Peer reviewed
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URI
http://hdl.handle.net/11250/283846
Date
2015
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  • Institutt for bioteknologi og matvitenskap [889]
  • Publikasjoner fra CRIStin - NTNU [19849]
Original version
BMC Systems Biology 2015, 9(6)   10.1186/s12918-015-0148-0
Abstract
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 13C-metabolic flux

analysis (13C-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 13C-label incorporation in primary metabolites by gas and liquid chromatography tandem mass

spectrometry.

R es u l t s : 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.

Keywords: Pseudomonas fluorescens, Anti-sigma factor MucA, Fluxome and fluxomics, Carbon labelling experiments,

GC-MS/MS, LC-MS/MS, 13C-metabolic flux analysis
Publisher
BioMed Central
Journal
BMC Systems Biology

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