del Castillo Teresa, Ramos Juan L, Rodríguez-Herva José J, Fuhrer Tobias, Sauer Uwe, Duque Estrella
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, E-18008 Granada, Spain.
J Bacteriol. 2007 Jul;189(14):5142-52. doi: 10.1128/JB.00203-07. Epub 2007 May 4.
In this study, we show that glucose catabolism in Pseudomonas putida occurs through the simultaneous operation of three pathways that converge at the level of 6-phosphogluconate, which is metabolized by the Edd and Eda Entner/Doudoroff enzymes to central metabolites. When glucose enters the periplasmic space through specific OprB porins, it can either be internalized into the cytoplasm or be oxidized to gluconate. Glucose is transported to the cytoplasm in a process mediated by an ABC uptake system encoded by open reading frames PP1015 to PP1018 and is then phosphorylated by glucokinase (encoded by the glk gene) and converted by glucose-6-phosphate dehydrogenase (encoded by the zwf genes) to 6-phosphogluconate. Gluconate in the periplasm can be transported into the cytoplasm and subsequently phosphorylated by gluconokinase to 6-phosphogluconate or oxidized to 2-ketogluconate, which is transported to the cytoplasm, and subsequently phosphorylated and reduced to 6-phosphogluconate. In the wild-type strain, glucose was consumed at a rate of around 6 mmol g(-1) h(-1), which allowed a growth rate of 0.58 h(-1) and a biomass yield of 0.44 g/g carbon used. Flux analysis of (13)C-labeled glucose revealed that, in the Krebs cycle, most of the oxalacetate fraction was produced by the pyruvate shunt rather than by the direct oxidation of malate by malate dehydrogenase. Enzymatic and microarray assays revealed that the enzymes, regulators, and transport systems of the three peripheral glucose pathways were induced in response to glucose in the outer medium. We generated a series of isogenic mutants in one or more of the steps of all three pathways and found that, although all three functioned simultaneously, the glucokinase pathway and the 2-ketogluconate loop were quantitatively more important than the direct phosphorylation of gluconate. In physical terms, glucose catabolism genes were organized in a series of clusters scattered along the chromosome. Within each of the clusters, genes encoding porins, transporters, enzymes, and regulators formed operons, suggesting that genes in each cluster coevolved. The glk gene encoding glucokinase was located in an operon with the edd gene, whereas the zwf-1 gene, encoding glucose-6-phosphate dehydrogenase, formed an operon with the eda gene. Therefore, the enzymes of the glucokinase pathway and those of the Entner-Doudoroff pathway are physically linked and induced simultaneously. It can therefore be concluded that the glucokinase pathway is a sine qua non condition for P. putida to grow with glucose.
在本研究中，我们表明恶臭假单胞菌中的葡萄糖分解代谢通过三条途径同时运行，这些途径在6-磷酸葡萄糖酸水平汇聚，6-磷酸葡萄糖酸由Edd和Eda Entner/Doudoroff酶代谢为中心代谢物。当葡萄糖通过特定的OprB孔蛋白进入周质空间时，它既可以内化到细胞质中，也可以氧化为葡萄糖酸。葡萄糖在由开放阅读框PP1015至PP1018编码的ABC摄取系统介导的过程中转运至细胞质，然后由葡萄糖激酶（由glk基因编码）磷酸化，并由葡萄糖-6-磷酸脱氢酶（由zwf基因编码）转化为6-磷酸葡萄糖酸。周质中的葡萄糖酸可以转运到细胞质中，随后由葡萄糖酸激酶磷酸化为6-磷酸葡萄糖酸，或者氧化为2-酮葡萄糖酸，2-酮葡萄糖酸转运到细胞质中，随后磷酸化并还原为6-磷酸葡萄糖酸。在野生型菌株中，葡萄糖的消耗速率约为6 mmol g(-1) h(-1)，这使得生长速率为0.58 h(-1)，生物量产量为0.44 g/g所利用的碳。对(13)C标记的葡萄糖进行通量分析表明，在三羧酸循环中，大多数草酰乙酸部分是由丙酮酸分流产生的，而不是由苹果酸脱氢酶直接氧化苹果酸产生的。酶促和微阵列分析表明，三条外周葡萄糖途径的酶、调节因子和转运系统是响应外部培养基中的葡萄糖而被诱导的。我们在所有三条途径的一个或多个步骤中产生了一系列同基因突变体，发现尽管所有三条途径同时发挥作用，但葡萄糖激酶途径和2-酮葡萄糖酸循环在数量上比葡萄糖酸的直接磷酸化更重要。从物理角度来看，葡萄糖分解代谢基因沿着染色体分散排列成一系列簇。在每个簇中，编码孔蛋白、转运蛋白、酶和调节因子的基因形成操纵子，这表明每个簇中的基因共同进化。编码葡萄糖激酶的glk基因与edd基因位于一个操纵子中，而编码葡萄糖-6-磷酸脱氢酶的zwf-1基因与eda基因形成一个操纵子。因此，葡萄糖激酶途径的酶和Entner-Doudoroff途径的酶在物理上是相连的，并且同时被诱导。因此可以得出结论，葡萄糖激酶途径是恶臭假单胞菌利用葡萄糖生长的必要条件。
