Towards a Genome-Wide Mutant Library of Pseudomonas putida Strain KT2440

Duque, Estrella; Molina‐Henares, Antonio J.; Torre, J. de la; Molina‐Henares, María Antonia; Castillo, Teresa del; Lam, Jennifer; Ramos, Juan L.

doi:10.1007/978-1-4020-6097-7_8

Cited by 47 publications

(62 citation statements)

References 104 publications

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“…Mini-Tn5 transposon mutagenesis was carried out by performing triparental mating between P. putida KT2440R (Rif r ), E. coli CC118pir (pUT-Km), and the helper strain E. coli HB101(pRK600), as described by Duque et al (7). After 4 h of conjugation, the transconjugants were selected as Rif r Km r clones on M9 medium with citrate as a carbon source.…”

Section: Methodsmentioning

confidence: 99%

Convergent Peripheral Pathways Catalyze Initial Glucose Catabolism inPseudomonas putida: Genomic and Flux Analysis

et al. 2007

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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.

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Section: Methodsmentioning

confidence: 99%

Convergent Peripheral Pathways Catalyze Initial Glucose Catabolism inPseudomonas putida: Genomic and Flux Analysis

et al. 2007

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“…Mutants were generated by random mutagenesis of P. putida with mini-Tn5-Km as described (28). Further information on mutant generation and additional information of these 12 mcp genes in supplemental Table S1.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates

Lacal

Alfonso

Liu

et al. 2010

Journal of Biological Chemistry

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We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates butdoes not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T m ) and unfolding enthalpy (⌬H) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

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“…Pseudomonas putida KT2440 and a series of isogenic mutant strains with insertions in the gltR-2, hexR, ptxS, and gnuR genes are shown in Table 1. The GltR-2 and HexR mutant strains were identified by Duque et al (6) in a general screening after random mutagenesis of P. putida with mini-Tn5-Km. The ptxS and gnuR mutants were generated in this study and are described below.…”

Section: Methodsmentioning

confidence: 99%

“…Sequencing of its genome revealed that it possesses many genes encoding proteins involved in the degradation of plant-derived chemicals, including a variety of methoxylated aromatic acids, hydroxylated aromatic acids, and other compounds (6,12,23). The strain was also found to possess information for the degradation of fructose (39) and glucose (3,4,39), two of the most abundant sugars present in plant root exudates (14).…”

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A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate

2008

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Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism.

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Towards a Genome-Wide Mutant Library of Pseudomonas putida Strain KT2440

Cited by 47 publications

References 104 publications

Convergent Peripheral Pathways Catalyze Initial Glucose Catabolism inPseudomonas putida: Genomic and Flux Analysis

Convergent Peripheral Pathways Catalyze Initial Glucose Catabolism inPseudomonas putida: Genomic and Flux Analysis

Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates

A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate

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