scholarly journals Simultaneous Catabolite Repression between Glucose and Toluene Metabolism in Pseudomonas putida Is Channeled through Different Signaling Pathways

2007 ◽  
Vol 189 (18) ◽  
pp. 6602-6610 ◽  
Author(s):  
Teresa del Castillo ◽  
Juan L. Ramos
Keyword(s):  
Glucose Metabolism ◽  
Pseudomonas Putida ◽  
Catabolite Repression ◽  
Carbon Sources ◽  
Flux Analysis ◽  
Glucose Determination ◽  
Net Flux ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Metabolism Gene

ABSTRACT Pseudomonas putida KT2440(pWW0) can use toluene via the TOL plasmid-encoded catabolic pathways and can use glucose via a series of three peripheral chromosome-encoded routes that convert glucose into 6-phosphogluconate (6PG), namely, the glucokinase pathway, in which glucose is transformed to 6PG through the action of glucokinase and glucose-6-phosphate dehydrogenase. Alternatively, glucose can be oxidized to gluconate, which can be phosphorylated by gluconokinase to 6PG or oxidized to 2-ketogluconate, which, in turn, is converted into 6PG. Our results show that KT2440 metabolizes glucose and toluene simultaneously, as revealed by net flux analysis of [13C]glucose. Determination of glucokinase and gluconokinase activities in glucose metabolism, gene expression assays using a fusion of the promoter of the Pu TOL upper pathway to ′lacZ, and global transcriptomic assays revealed simultaneous catabolite repression in the use of these two carbon sources. The effect of toluene on glucose metabolism was directed to the glucokinase branch and did not affect gluconate metabolism. Catabolite repression of the glucokinase pathway and the TOL pathway was triggered by two different catabolite repression systems. Expression from Pu was repressed mainly via PtsN in response to high levels of 2-dehydro-3-deoxygluconate-6-phosphate, whereas repression of the glucokinase pathway was channeled through Crc.

scholarly journals Crc Is Involved in Catabolite Repression Control of the bkd Operons of Pseudomonas putida andPseudomonas aeruginosa

2000 ◽  
Vol 182 (4) ◽  
pp. 1144-1149 ◽  
Author(s):  
Kathryn L. Hester ◽  
Jodi Lehman ◽  
Fares Najar ◽  
Lin Song ◽  
Bruce A. Roe ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Catabolite Repression ◽  
Carbon Sources ◽  
Keto Acid ◽  
Carbon Regulation ◽  
Acid Dehydrogenase ◽  
Transposon Mutants ◽  
Branched Chain ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of thebkd operon, were isolated and identified as crcand vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.

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

2008 ◽  
Vol 190 (7) ◽  
pp. 2331-2339 ◽  
Author(s):  
Teresa del Castillo ◽  
Estrella Duque ◽  
Juan L. Ramos
Keyword(s):  
Glucose Metabolism ◽  
Pseudomonas Putida ◽  
Transcriptional Regulator ◽  
Transport Systems ◽  
Transcriptional Repressors ◽  
Periplasmic Space ◽  
Host Chromosome ◽  
Catabolic Enzymes ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Transcriptional Fusions

ABSTRACT 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.

scholarly journals Carbon Catabolite Repression in Bacillus subtilis: Quantitative Analysis of Repression Exerted by Different Carbon Sources

2008 ◽  
Vol 190 (21) ◽  
pp. 7275-7284 ◽  
Author(s):  
Kalpana D. Singh ◽  
Matthias H. Schmalisch ◽  
Jörg Stülke ◽  
Boris Görke
Keyword(s):  
Bacillus Subtilis ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Carbon Sources ◽  
Transport Activity ◽  
Phosphotransferase System ◽  
Phosphorylation State ◽  
Mechanism Operative

ABSTRACT In many bacteria glucose is the preferred carbon source and represses the utilization of secondary substrates. In Bacillus subtilis, this carbon catabolite repression (CCR) is achieved by the global transcription regulator CcpA, whose activity is triggered by the availability of its phosphorylated cofactors, HPr(Ser46-P) and Crh(Ser46-P). Phosphorylation of these proteins is catalyzed by the metabolite-controlled kinase HPrK/P. Recent studies have focused on glucose as a repressing substrate. Here, we show that many carbohydrates cause CCR. The substrates form a hierarchy in their ability to exert repression via the CcpA-mediated CCR pathway. Of the two cofactors, HPr is sufficient for complete CCR. In contrast, Crh cannot substitute for HPr on substrates that cause a strong repression. Determination of the phosphorylation state of HPr in vivo revealed a correlation between the strength of repression and the degree of phosphorylation of HPr at Ser46. Sugars transported by the phosphotransferase system (PTS) cause the strongest repression. However, the phosphorylation state of HPr at its His15 residue and PTS transport activity have no impact on the global CCR mechanism, which is a major difference compared to the mechanism operative in Escherichia coli. Our data suggest that the hierarchy in CCR exerted by the different substrates is exclusively determined by the activity of HPrK/P.

scholarly journals Convergent Peripheral Pathways Catalyze Initial Glucose Catabolism in Pseudomonas putida: Genomic and Flux Analysis

2007 ◽  
Vol 189 (14) ◽  
pp. 5142-5152 ◽  
Author(s):  
Teresa del Castillo ◽  
Juan L. Ramos ◽  
José J. Rodríguez-Herva ◽  
Tobias Fuhrer ◽  
Uwe Sauer ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Krebs Cycle ◽  
Open Reading Frames ◽  
Transport Systems ◽  
Flux Analysis ◽  
Uptake System ◽  
Direct Oxidation ◽  
Gene Encoding ◽  
Glucose Catabolism ◽  
Glucose 6 Phosphate Dehydrogenase

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 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 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.

scholarly journals Multicopy FZF1 (SUL1) Suppresses the Sulfite Sensitivity but Not the Glucose Derepression or Aberrant Cell Morphology of a grr1 Mutant of Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 511-521 ◽  
Author(s):  
Dorina Avram ◽  
Alan T Bakalinsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Glucose Metabolism ◽  
Cell Morphology ◽  
Glucose Repression ◽  
Carbon Sources ◽  
Single Copy ◽  
Aberrant Cell ◽  
Growth Defects ◽  
Putative Transcription Factor

Abstract An ssu2 mutation in Sacccharomyces cermisiae, previously shown to cause sulfite sensitivity, was found to be allelic to GRR1, a gene previously implicated in glucose repression. The suppressor rgt1, which suppresses the growth defects of grr1 strains on glucose, did not fully suppress the sensitivity on glucose or nonglucose carbon sources, indicating that it is not strictly linked to a defect in glucose metabolism. Because the Cln1 protein was previously shown to be elevated in grr1 mutants, the effect of CLN1 overexpression on sulfite sensitivity was investigated. Overexpression in GRR1 cells resulted in sulfite sensitivity, suggesting a connection between CLN1 and sulfite metabolism. Multicopy FZF1, a putative transcription factor, was found to suppress the sulfite sensitive phenotype of grr1 strains, but not the glucose derepression or aberrant cell morphology. Multicopy FZF1 was also found to suppress the sensitivity of a number of other unrelated sulfite-sensitive mutants, but not that of ssu1 or met20, implying that FZF1 may act through Ssulp and Met20p. Disruption of FZF1 resulted in sulfite sensitivity when the construct was introduced in single copy at the FZF1 locus in a GRR1 strain, providing evidence that FZF1 is involved in sulfite metabolism.

Bicarbonate and CO2 transport across the skin of the leopard frog, Rana pipiens: effect of PGF2alpha

1997 ◽  
Vol 272 (2) ◽  
pp. R640-R647 ◽  
Author(s):  
O. A. Candia ◽  
T. Yorio
Keyword(s):  
Methodological Approach ◽  
Transport Processes ◽  
Leopard Frog ◽  
Bicarbonate Transport ◽  
Amphibian Skin ◽  
Acid Base ◽  
Reliable Determination ◽  
Net Flux ◽  
Unidirectional Fluxes

The amphibian skin represents an important organ for osmoregulation and, like the mammalian kidney, maintains acid-base balance by secreting protons or base. However, the lack of a reliable and accurate method to measure the contribution of unidirectional fluxes of HCO3- ions to this mechanism has been an obstacle for the determination of the role of bicarbonate in epithelial acid-base homeostasis. Recently, one of us developed a method that allows for the reliable determination of transepithelial fluxes of bicarbonate, and this method was applied to determine unidirectional fluxes of (14)CO2 and H(14)CO3 under a variety of conditions. We report that the combined CO2 and HCO3- mucosal-to-serosal flux under 5% CO2 was 40% larger than the opposing flux, giving a net flux in the mucosal-to-serosal direction. This net flux was inhibited by acetazolamide. In CO2-free conditions, there was no detectable net flux; however, acetazolamide and PGF(2alpha) attenuated the mucosal-to-serosal flux and established an apparent secretion of HCO3-. A model is presented that depicts twelve vectors or components to the CO2 plus HCO3- fluxes in the frog skin. This model can accurately reproduce the experimental values measured from unidirectional fluxes of CO2 and HCO3- under a variety of conditions and can explain the effects of PGF(2alpha) on unidirectional 14C-labeled fluxes as a consequence of inhibition of H+ secretion to the apical bath, similar to what was previously suggested by our laboratory using a different methodological approach. The present method, utilizing radiolabeled HCO3-, may be useful as a means to evaluate the mechanism of action of hormones and drugs that may regulate acid-base homeostasis by altering proton and bicarbonate transport processes.

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