Compartmentalized glucose metabolism in Pseudomonas putida is controlled by the PtxS repressor.
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.
Daddaoua
Abdelali
A
Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, Granada, Spain.
Krell
Tino
T
Alfonso
Carlos
C
Morel
Bertrand
B
Ramos
Juan-Luis
JL
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005942:Gluconates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D009876:Operon; D019897:Periplasm / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005942:Gluconates; D014157:Transcription Factors; C008229:2-ketogluconate; D005947:Glucose; C008884:6-phosphogluconic acid