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

doi:10.1073/pnas.0507580103

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

^†Lehrstuhl für Organische Chemie und Biochemie, Lichtenbergstrasse 4, D-85747 Garching, Germany; and Lehrstühle
^§für Mikrobiologie und
^¶Bioinformatik, Biozentrum der Universität Würzburg, D-97074 Würzburg, Germany

Edited by Rowena G. Matthews, University of Michigan, Ann Arbor, MI, and approved December 26, 2005
↵ ^‡W.E. and J.S. contributed equally to this work. (received for review September 6, 2005)

Abstract

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

Footnotes

^∥To whom correspondence should be addressed. E-mail: goebel{at}biozentrum.uni-wuerzburg.de
Author contributions: W.E., J. Slaghuis, J. Stritzker, R.S., T.D., W.G., and A.B. designed research; W.E., J. Slaghuis, R.L., J.B., J. Stritzker, C.S., and R.S. performed research; W.E., J. Slaghuis, R.L., J.B., C.S., R.S., T.D., W.G., and A.B. analyzed data; and W.E., J. Slaghuis, W.G., and A.B. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

EM,

elementary mode;

KEGG,

Kyoto Encyclopedia of Genes and Genomes;

Lm,

Listeria monocytogenes;

PPP,

pentose phosphate pathway.