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

      Proceedings of the National Academy of Sciences of the United States of America
      Amino Acids, Branched-Chain, biosynthesis, Carbon, metabolism, Carbon Isotopes, analysis, Computational Biology, Glucose, Listeria monocytogenes, chemistry, genetics, Magnetic Resonance Spectroscopy, Mutation, Oxaloacetic Acid, Peptide Termination Factors, Pyruvic Acid, Virulence

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          Abstract

          The carbon metabolism of Listeria monocytogenes (Lm) EGD and the two isogenic mutant strains LmDeltaprfA and LmDeltaprfApPRFA* (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.

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            Comparative genomics of Listeria species.

            Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.
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              Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering.

              Rational metabolic engineering requires powerful theoretical methods such as pathway analysis, in which the topology of metabolic networks is considered. All metabolic capabilities in steady states are composed of elementary flux modes, which are minimal sets of enzymes that can each generate valid steady states. The modes of the fructose-2,6-bisphosphate cycle, the combined tricarboxylic-acid-glyoxylate-shunt system and tryptophan synthesis are used here for illustration. This approach can be used for many biotechnological applications such as increasing the yield of a product, channelling a product into desired pathways and in functional reconstruction from genomic data.
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