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J. Bacteriol. doi:10.1128/JB.00203-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida: Genomic and flux analysis

Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Prof. Albareda, 1, E-18008 Granada, Spain; Institute for Molecular Systems Biology, ETH, CH-8093 Zurich, Switzerland

^* To whom correspondence should be addressed. Email: jlramos{at}eez.csic.es.

	Abstract

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 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 ¹³C-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 system 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 co-evolved. The glk gene encoding glucokinase was located in an operon with the edd gene; whereas the zwf-1gene, 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 Pseudomonas putida to grow with glucose.

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