2-ketogluconate and 6-phosphogluconic-acid

In the human pathogen Pseudomonas aeruginosa, the GltR regulator is required for glucose transport, whereas GtrS is a sensor kinase that plays a key role in mediating bacteria-host interaction and pathogen dissemination in the host. We show that GtrS and GltR form a two-component system that regulates the expression from the promoters Pedd/gap-1, PoprB and Pglk, which control the expression of genes involved in glucose metabolism and transport. In addition, the GtrS/GltR pair regulates the expression of toxA that encodes exotoxin A, the primary virulence factor. Microcalorimetry-based ligand screening of the recombinant GtrS ligand-binding domain revealed specific binding of 2-ketogluconate (2-KG) (KD=5 μM) and 6-phosphogluconate (KD=98 μM). These effectors accelerate GtrS autophosphorylation, with concomitant transphosphorylation of GltR leading to a three-fold increase in transcription. Surprisingly, in vivo a similar increase in expression from the above promoters was observed for the mutant deficient in GltR regardless of the presence of effectors. The GltR operator site was found to contain the consensus sequence 5'-tgGTTTTTc-3'. We propose that 2-KG is a key metabolite in the stringent transcriptional control of genes involved in virulence and glucose metabolism. We show that GltR is a transcriptional repressor that is released from DNA upon phosphorylation.

Topics: ADP Ribose Transferases; Bacterial Proteins; Bacterial Toxins; Binding Sites; Exotoxins; Gene Expression Regulation, Bacterial; Gluconates; Glucose; Monosaccharide Transport Proteins; Promoter Regions, Genetic; Protein Kinases; Protein Structure, Tertiary; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Repressor Proteins; Transcriptional Activation; Virulence Factors

Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent K(D) (equilibrium dissociation constant) of 15 muM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1 degrees C and 57.6 degrees C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 muM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5'-TGAAACCGGTTTCA-3'. This palindrome overlaps with the RNA polymerase binding site.

Topics: Bacterial Proteins; Calorimetry, Differential Scanning; Cytoplasm; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Gluconates; Glucose; Operon; Periplasm; Promoter Regions, Genetic; Protein Binding; Pseudomonas putida; Transcription Factors

Glucose and other C sources exert an atypical form of catabolic repression on the sigma54-dependent promoter Pu, which drives transcription of an operon for m-xylene degradation encoded by the TOL plasmid pWW0 in Pseudomonas putida. We have used a genetic approach to identify the catabolite(s) shared by all known repressive C sources that appears to act as the intracellular signal that triggers downregulation of Pu. To this end, we reconstructed from genomic data the pathways for metabolism of repressor (glucose, gluconate) and nonrepressor (fructose) C sources. Since P. putida lacks fructose-6-phosphate kinase, glucose and gluconate appear to be metabolized exclusively by the Entner-Doudoroff (ED) pathway, while fructose can be channeled through the Embden-Meyerhof (EM) route. An insertion in the gene fda (encoding fructose-1,6-bisphosphatase) that forces fructose metabolism to be routed exclusively to the ED pathway makes this sugar inhibitory for Pu. On the contrary, a crc mutation known to stimulate expression of the ED enzymes causes the promoter to be less sensitive to glucose. Interrupting the ED pathway by knocking out eda (encoding 2-dehydro-3-deoxyphosphogluconate aldolase) exacerbates the inhibitory effect of glucose in Pu. These observations pinpoint the key catabolites of the ED route, 6-phosphogluconate and/or 2-dehydro-3-deoxyphosphogluconate, as the intermediates that signal Pu repression. This notion is strengthened by the observation that 2-ketogluconate, which enters the ED pathway by conversion into these compounds, is a strong repressor of the Pu promoter.

Topics: Bacterial Proteins; Culture Media; DNA-Binding Proteins; DNA-Directed RNA Polymerases; Fructose; Gene Expression Regulation, Bacterial; Gluconates; Glucose; Lac Operon; Phosphorylation; Promoter Regions, Genetic; Pseudomonas putida; RNA Polymerase Sigma 54; Sigma Factor