Systems Biology Program, Centro Nacional de Biotecnología, Cantoblanco-Madrid, Spain.
mBio. 2012 Mar 20;3(2). doi: 10.1128/mBio.00028-12. Print 2012.
Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTS(Fru)), while the other (N-related PTS, or PTS(Ntr)) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTS(Fru) or PTS(Ntr) genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIA(Ntr)) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIA(Ntr) protein of the PTS(Ntr) thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology.
This study demonstrates that EIIA(Ntr) influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIA(Ntr) protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.
土壤细菌假单胞菌 KT2440 中有两个磷酸烯醇式丙酮酸磷酸转移酶系统（PTS）分支发挥作用。一个分支包含一套完整的果糖摄取酶（PTS（Fru）），而另一个（N 相关 PTS，或 PTS（Ntr））控制与碳水化合物运输无关的各种细胞功能。通过测量具有 PTS（Fru）或 PTS（Ntr）基因非极性突变的同基因菌株在果糖（PTS 底物）或葡萄糖上生长时的代谢通量，研究了这两个系统对调节中心碳分解代谢的潜力，葡萄糖在这种细菌中不受 PTS 控制。从每种糖流出的碳明显分为 Entner-Doudoroff、戊糖磷酸和 Embden-Meyerhof-Parnas 途径，在每个 PTS 突变体中都保持了这种比例。然而，缺乏 PtsN（EIIA（Ntr））的菌株在丙酮酸分流反应中显示出明显更高的通量，该分流反应绕过 TCA 循环中的苹果酸脱氢酶。这与相应 PTS 突变体中发现的苹果酸酶和丙酮酸羧化酶活性增加一致。遗传证据表明，PtsN 的这种代谢效应需要通过系统传递高能磷酸。因此，PTS（Ntr）的 EIIA（Ntr）蛋白有助于调整中心代谢通量，以满足整个细胞生理学的合成代谢和能量需求。
本研究表明，EIIA（Ntr）影响将碳从上、下中心代谢区域输送到假单胞菌消耗糖的生化反应。这些发现表明，EIIA（Ntr）蛋白是协调该细菌中各种生理目的地碳命运的关键因素。此外，这些结果强调了对现存酶复合物的翻译后调节对于增加相应代谢网络的稳健性的重要性。
