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13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA

Item Type:Article
Title:13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA
Creators Name:Eisenreich, W. and Slaghuis, J. and Laupitz, R. and Bussemer, J. and Stritzker, J. and Schwarz, C. and Schwarz, R. and Dandekar, T. and Goebel, W. and Bacher, A.
Abstract:The carbon metabolism of Listeria monocytogenes (Lm) EGD and the two isogenic mutant strains Lm{delta}prfA and Lm{delta}prfApPRFA* (showing no or enhanced expression, respectively, of the virulence factor PrfA) was determined by 13C isotopologue perturbation. After growth of the bacteria in a defined medium containing a mixture of [U-13C6]glucose and glucose with natural 13C abundance (1:25, wt/wt), 14 amino acids were isolated and analyzed by NMR spectroscopy. Multiply 13C-labeled isotopologues were determined quantitatively by signal deconvolution. The 13C enrichments and isotopologue patterns allowed the reconstruction of most amino acid biosynthesis pathways and illustrated that overproduced PrfA may strongly influence the synthesis of some amino acids, notably that of the branched amino acids (Val, Ile, and Leu). Retrobiosynthetic analysis of the isotopologue compositions showed that degradation of glucose occurs to a large extent via the pentose phosphate pathway and that the citrate cycle is incomplete because of the absence of 2-oxoglutarate dehydrogenase activity. The reconstructed labeling pattern of oxaloacetate indicated its formation by carboxylation of pyruvate. This metabolic reaction seems to have a strong impact on the growth requirement in defined minimal medium. Bioinformatical steady-state network analyses and flux distribution predictions confirmed the experimental data and predicted metabolite fluxes through the enzymes of the pathways under study.
Keywords:NMR Analysis, Intracellular Bacteria, Metabolic Flux, Extracellular Metabolom
Source:Proceedings of the National Academy of Sciences of the United States of America
ISSN:0027-8424
Publisher:National Academy of Sciences (U.S.A.)
Volume:103
Number:7
Page Range:2040-2045
Date:14 February 2006
Official Publication:https://doi.org/10.1073/pnas.0507580103
PubMed:View item in PubMed

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