Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida:: Genomic and flux analysis
Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida:: Genomic and flux analysis
复制标题
DOI：
10.1128/jb.00203-07
复制
发表时间：
2007-07-01
影响因子：
3.2
通讯作者：
Duque, Estrella
中科院分区：
文献类型：
--
作者：
del Castillo, Teresa;Ramos, Juan L.;Duque, Estrella
在这项研究中，我们表明，葡萄糖catenase在pudda假单胞菌发生通过同时操作的三个途径，收敛在6-磷酸葡萄糖酸，这是代谢的Edd和Eda Entner/Doudoroff酶的中央代谢产物的水平。当葡萄糖通过特异性OprB孔蛋白进入周质空间时，它可以被内化到细胞质中或被氧化成葡萄糖酸盐。葡萄糖在由开放阅读框PP 1015至PP 1018编码的ABC摄取系统介导的过程中转运至细胞质，然后被葡糖激酶（由glk基因编码）磷酸化并被葡萄糖-6-磷酸脱氢酶（由zwf基因编码）转化为6-磷酸葡萄糖酸盐。周质中的葡萄糖酸盐可以被转运到细胞质中，随后被葡糖激酶磷酸化为6-磷酸葡萄糖酸盐或氧化为2-酮葡萄糖酸盐，其被转运到细胞质中，随后被磷酸化并还原为6-磷酸葡萄糖酸盐。在野生型菌株中，葡萄糖以约6 mmol g（-1）h（-1）的速率消耗，这允许0.58 h（-1）的生长速率和0.44 g/g所用碳的生物质产量。C-13标记的葡萄糖的通量分析表明，在克雷布斯循环中，大部分的丙酮酸盐的乙酸部分是由丙酮酸分流，而不是由苹果酸脱氢酶直接氧化苹果酸。酶和微阵列分析表明，酶，调节剂，和运输系统的三个外周葡萄糖途径诱导响应于葡萄糖在外部介质。我们在所有三种途径的一个或多个步骤中产生了一系列同基因突变体，并发现，尽管所有三种途径同时起作用，但葡萄糖激酶途径和2-酮葡萄糖酸环在数量上比葡萄糖酸的直接磷酸化更重要。从物理学的角度来看，葡萄糖催化剂基因被组织成一系列的簇，沿着染色体分散。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途径的酶是物理连接的，并且同时被诱导。因此，可以得出结论，葡萄糖激酶途径是P. pudda与葡萄糖一起生长的必要条件。
In this study, we show that glucose catabolism in Pseudomonas pudda 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 C-13-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. pudda to grow with glucose.