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

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 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.

FIG. 1.

Glucose catabolism in P. putida as deduced from gene annotations. At the top are the events that occur in the outer membrane and the reactions that take place in the periplasmic space. Also shown are the transport of glucose, gluconate, and 2-ketogluconate into the cell. The set of catabolic reactions that take place in the cytoplasm is depicted. The genes encoding the enzymes involved are indicated for all of the steps. OM, outer membrane; PG, periplasmic space; IM, inner membrane.

FIG. 2.

In vivo carbon flux distribution in P. putida KT2440 obtained from METAFoR and net-flux analyses. All fluxes were normalized to the glucose uptake rate (6.31 nmol g [CDW]⁻¹ h⁻¹), and the thicknesses of the arrows are scaled to the relative percentages of flux. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; G3P, glyceraldeydehyde-3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; AcCoA, acetyl-CoA; ICT, isocitrate; KG, α-ketoglutarate; MAL, malate.

FIG. 3.

Organization of the peripheral glucose catabolic genes. The gene numbering and organization are derived from the annotation of the complete genome sequence of P. putida KT2440 in the TIGR database. (A) Set of 16 genes, most of which have been assigned a function based on enzymatic assays. (C) Set of nine genes, most of which have a specific role in glucose catabolism. (B and D) Physical organizations of the other genes involved in glucose catabolism in P. putida KT2440.

FIG. 4.

Transcriptional pattern of the gap-1, glk, and edd region involved in glucose catabolism. Lane 1, DNA size marker (Roche); lane 2, RT-PCR using primers based on the adjacent gap-1 and edd genes; lane 3, positive control for lane 2 with the same oligonucleotides but with DNA as a template; lane 4, negative control for lane 2 with the same oligonucleotides but without retrotranscriptase; lane 5, RT-PCR using primers complementary to RNA of the edd/glk genes; lane 6, positive control with the same primers as in lane 5 but with DNA as a template; lane 7, negative controls for the assay in lane 5 with the same primers but without retrotranscriptase; lane 8, RT-PCR using primers based on glk and gltR2; lane 9, positive control with the same oligonucleotides but with DNA as a template; lane 10, negative control for the assay in lane 8 but without retrotranscriptase.