13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA
13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA
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DOI：
10.1073/pnas.0507580103
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发表时间：
2006-02-14
影响因子：
11.1
通讯作者：
Bacher, A
中科院分区：
文献类型：
--
作者：
Eisenreich, W;Slaghuis, J;Bacher, A
通过C-13同位素扰动法测定了单核细胞增生李斯特菌（Lm）EGD和两种同基因突变株Lm Delta prfA和Lm Delta prfApPRFA*（分别显示毒力因子PrfA不表达或表达增强）的碳代谢。细菌在含有[U-C-13（6）]葡萄糖和具有天然C-13丰度的葡萄糖的混合物（1：25，wt/wt）的确定成分培养基中生长后，分离14种氨基酸并通过NMR光谱分析。多重C-13-标记的同位素物通过信号去卷积定量测定。C-13富集和同位素模式允许大多数氨基酸生物合成途径的重建，并说明过量产生的PrfA可能强烈影响某些氨基酸的合成，特别是支链氨基酸（瓦尔、Ile和Leu）的合成。同位素组成的逆生物合成分析表明，葡萄糖的降解发生在很大程度上通过戊糖磷酸途径和柠檬酸循环是不完整的，因为没有2-酮戊二酸脱氢酶活性。草酰乙酸的重建标记模式表明其形成的丙酮酸羧化。该代谢反应似乎对在限定基本培养基中的生长要求具有强烈影响。生物信息学稳态网络分析和通量分布预测证实了实验数据和预测代谢物通量通过酶的途径正在研究中。
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.