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 Lm Delta prfA and Lm Delta prfApPRFA* (showing no or enhanced expression, respectively, of the virulence factor PrfA) was determined by C-13 isotopologue perturbation. After growth of the bacteria in a defined medium containing a mixture of [U-C-13(6)]glucose and glucose with natural C-13 abundance (1:25, wt/wt), 14 amino acids were isolated and analyzed by NMR spectroscopy. Multiply C-13-labeled isotopologues were determined quantitatively by signal deconvolution. The C-13 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, lle, 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.