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.
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 (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.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, N-7491, Trondheim, Norway. stina.lien@ntnu.no.
authors.forename: Stina K
authors.initials: SK
authors.lastname: Lien
authors.affiliation: Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425, Jülich, Germany. s.niedenfuehr@fz-juelich.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Niedenführ
authors.affiliation: Department of Bioprocess technology, SINTEF Materials and Chemistry, Sem Sælands vei 2a, N-7465, Trondheim, Norway. havard.sletta@sintef.no.
authors.forename: Håvard
authors.initials: H
authors.lastname: Sletta
authors.affiliation: Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425, Jülich, Germany. K.Noeh@fz-juelich.de.
authors.forename: Katharina
authors.initials: K
authors.lastname: Nöh
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, N-7491, Trondheim, Norway. Per.Bruheim@ntnu.no.
authors.forename: Per
authors.initials: P
authors.lastname: Bruheim
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002244:Carbon / Q000378:metabolism*; D055786:Gene Knockout Techniques; D064688:Metabolic Flux Analysis; D053858:Metabolic Networks and Pathways; D009154:Mutation; D009243:NAD / Q000096:biosynthesis; D009249:NADP / Q000096:biosynthesis / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C107895:MucA protein, Pseudomonas; D009243:NAD; D009249:NADP; D002244:Carbon