13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA.
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.
Eisenreich
Wolfgang
W
Lehrstuhl für Organische Chemie und Biochemie, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Slaghuis
Jörg
J
Laupitz
Ralf
R
Bussemer
Johanna
J
Stritzker
Jochen
J
Schwarz
Christine
C
Schwarz
Roland
R
Dandekar
Thomas
T
Goebel
Werner
W
Bacher
Adelbert
A
D000597:Amino Acids, Branched-Chain / Q000096:biosynthesis*; D002244:Carbon / Q000378:metabolism*; D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D019295:Computational Biology; D005947:Glucose / Q000378:metabolism; D008089:Listeria monocytogenes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D062907:Oxaloacetic Acid / Q000378:metabolism; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D014774:Virulence
D000597:Amino Acids, Branched-Chain; D002247:Carbon Isotopes; D010454:Peptide Termination Factors; D062907:Oxaloacetic Acid; D002244:Carbon; D019289:Pyruvic Acid; D005947:Glucose