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

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.     

Inorganic phosphate effect on alternate peripheral pathways of glucose catabolism in Pseudomonas cepacia

Abstract The effect of the inorganic phosphate concentration on the activity of the enzyme of alternate peripheral pathways of glucose catabolism was studied in Pseudomonas cepacia ATCC 17759. Growth with low glucose concentration (0.5% w/v) and 20 mM phosphate resulted in induced levels of the phosphorylative pathway enzymes when compared with the levels of these same enzymes in high glucose concentration (2% w/v). However, an expansion of the oxidative pathway was detected during growth with 0.5% (w/v) of glucose and high phosphate concentration (160 mM). Moreover, under high phosphate (160 mM) and high glucose (2% w/v) growth conditions, glucokinase activity was increased preferentially relative to levels of direct oxidative pathway enzymes.     

D- and L-isoleucine metabolism and regulation of their pathways in Pseudomonas putida

Pseudomonas putida oxidized isoleucine to acetyl-coenzyme A (CoA) and propionyl-CoA by a pathway which involved deamination of d-isoleucine by oxidation and l-isoleucine by transamination, oxidative decarboxylation, and beta oxidation at the ethyl side chain. At least three separate inductive events were required to form all of the enzymes of the pathway: d-amino acid dehydrogenase was induced during growth in the presence of d-isoleucine; branched-chain keto dehydrogenase was induced during growth on 2-keto-3-methylvalerate and enzymes specific for isoleucine metabolism; tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were induced by growth on isoleucine, 2-keto-3-methylvalerate, 2-methylbutyrate, or tiglate. Tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were purified simultaneously by several enzyme concentration procedures, but were separated by isoelectric focusing. Isoelectric points, pH optima, substrate specificity, and requirements for enzyme action were determined for both enzymes. Evidence was obtained that the dehydrogenase catalyzed the oxidation of 2-methyl-3-hydroxybutyryl-CoA to 2-methylacetoacetyl-CoA. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase catalyzed the oxidation of 3-hydroxybutyryl-CoA, but l-3-hydroxyacyl-CoA dehydrogenase from pig heart did not catalyze the oxidation of 2-methyl-3-hydroxybutyryl-CoA; therefore, they appeared to be different dehydrogenases. Furthermore, growth on tiglate resulted in the induction of tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase, but these two enzymes were not induced during growth on crotonate or 3-hydroxybutyrate.     

Multiple and interconnected pathways for L-lysine catabolism in Pseudomonas putida KT2440

L-lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates D-lysine, L-pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of L-lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either pathway failed to use L-lysine as the sole carbon and nitrogen source, although they still used L-lysine as the nitrogen source, albeit at reduced growth rates. New genes were identified in both pathways, including the davB and davA genes that encode the enzymes involved in the oxidation of L-lysine to delta-aminovaleramide and the hydrolysis of the latter to delta-aminovalerate, respectively. The amaA, dkpA, and amaB genes, in contrast, encode proteins involved in the transformation of Delta1-piperidine-2-carboxylate into aminoadipate. Based on L-[U-13C, U-15N]lysine experiments, we quantified the relative use of pathways in the wild type and its isogenic mutants. The fate of 13C label of L-lysine indicates that in addition to the existing connection between the D- and L-lysine pathways at the early steps of the catabolism of L-lysine mediated by a lysine racemase, there is yet another interconnection at the lower end of the pathways in which aminoadipate is channeled to yield glutarate. This study establishes an unequivocal relationship between gene and pathway enzymes in the metabolism of L-lysine, which is of crucial importance for the successful colonization of the rhizosphere of plants by this microorganism.     

Differential connectivity of splicing activators and repressors to the human spliceosome

BackgroundDuring spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions.ResultsHere, we investigate the protein connectivity of SR and hnRNP proteins to the core spliceosome using probabilistic network reconstruction based on the integration of interactome and gene expression data. We validate our model by immunoprecipitation and mass spectrometry of the prototypical splicing factors SRSF1 and hnRNPA1. Network analysis reveals that a factor’s properties as an activator or repressor can be predicted from its overall connectivity to the rest of the spliceosome. In addition, we discover and experimentally validate PPIs between the oncoprotein SRSF1 and members of the anti-tumor drug target SF3 complex. Our findings suggest that activators promote the formation of PPIs between spliceosomal sub-complexes, whereas repressors mostly operate through protein-RNA interactions.ConclusionsThis study demonstrates that combining in-silico modeling with biochemistry can significantly advance the understanding of structure and function relationships in the human spliceosome.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0682-5) contains supplementary material, which is available to authorized users.     

Production of toluene cis-glycol by Pseudomonas putida in glucose feb-batch culture

Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene Cis-Glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. Putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25h) of the process or the final concentration of TCG achieved. The rate of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. These changes were a consequence of TCG formation and a cooperative toxic effect was demonstrated for toluene and TCG. Adenylate energy charge values decreased from ca. 0.8 to 0.2 over the course of the biotransformation but were maintained above 0.5 in the absence of TCG. Similarly, cellular AMP levels increased dramatically during biotransformation, presumably as a consequence of RNA degradation, but were maintained at low levels in the absence of TCG. The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.     

Supra-operonic clustering of genes specifying glucose dissimilation in Pseudomonas putida.

Summary The linkage arrangements of genes governing glucolysis in Pseudomonas putida have been determined by transductional analysis. Five genes (gdh, kgtA, kgtB, edd and eda), comprising at least three operons, are cotransducible with each other, but not with ggu (glucose and gluconate uptake) nor with genes of a known supra-operonic cluster of genes specifying enzymes of other dissimilatory pathways, nor with a biochemically uncharacterized his marker. It thus appears that P. putida may have more than one chromosomal region in which genes with dissimilatory function are clustered in a supra-operonic fashion.     

Lignin catabolic pathways reveal unique characteristics of dye-decolorizing peroxidases in Pseudomonas putida

Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non-model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin-degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye-decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O₂-dependent ring cleavage strategy was utilized in central pathways to catabolize lignin-derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression-dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn²⁺ independent lignin depolymerization activity, while Mn²⁺ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 10¹² cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate-limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141-fold increase in DypB activity (56,565 U/l) was achieved, providing novel insights for lignin bioconversion.     

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real‐time RT‐PCR analysis revealed the intrinsic self‐regulation of GCD and GAD. Utilizing this self‐regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2‐ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm², 0.27 ± 0.02 mmol/g_CDW/hr and 0.25 ± 0.02 mmol/g_CDW/hr, which were 327%, 477%, and 644% of the values of wild‐type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.     

A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida

(S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids. The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction. In this study, we investigated the mechanism of this half-reaction in detail. At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction. This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin. The intermediate was not due to binding of anionic substrates or inhibitors. It was only observed with efficient substrates that have high k(cat) values. At higher temperatures, it was formed within the dead time of the stopped-flow instrument. The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect. This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton. This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065). Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway.     

Genetic control of the histidine dissimilatory pathway in Pseudomonas putida

Summary In P. putida the first four enzymes involved in the dissimilation of histidine are induced by the first intermediate of the pathway, urocanic acid. The genes specifying these enzymes, hutH, hutU, hutF and (probably) hutI appear to be clustered on the chromosome between pcaE and pcaA (genes involved in p-hydroxybenzoic acid catabolism). Two mutants which produce the histidine-dissimilitory enzymes constitutively have been isolated. They appear to carry mutations in a regulatory locus, which maps in the same region as the structural genes of the pathway.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.