Compartmentalized glucose metabolism in Pseudomonas putida is controlled by the PtxS repressor

Abstract

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.

FIG. 1.

Summary of glucose metabolism in P. putida KT2440, as deduced from gene annotations and functional analysis in the wild-type strain and a series of mutants. OM, outer membrane; PS, periplasmic space; IM, inner membrane; Gcd, glucose dehydrogenase; Gad, gluconate dehydrogenase; KguD, 2-ketogluconate reductase; Glk, glucokinase; GnuK, gluconokinase; KguK, 2-ketogluconate kinase; Zwf-1, glucose-6-phosphate 1-dehydrogenase; Pgl, 6-phosphoglucose lactonase; Edd, phosphogluconate dehydratase; Eda, 2-keto-3-deoxy gluconate aldolase; GntP, gluconate permease; KguT, 2-ketogluconate transporter; PYR, pyruvate. Proteins highlighted in bold are those whose transcription is controlled by PtxS.

FIG. 2.

Genetic organization of open reading frames that are under the control of PtxS. Gene order was first established by Nelson et al. (20) when the genome of KT2440 was described. The operon structures of gadCBA and kguEKTD were established previously by our group (8). PP3381 is predicted to be a transposase, and PP3385 is an outer transmembrane protein.

FIG. 3.

Determination of the oligomeric state of PtxS. (A) Gel filtration elution profile of PtxS. (B) Calibration curve of the gel filtration column using the following marker proteins: carbonic anhydrase (A; molecular weight of 29,000), albumin from chicken egg white (B; 45,000), albumin from bovine serum monomer (C; 66,000) and dimer (D; 132,000), and urease (E; 545,000). The elution volume determined for PtxS is indicated. AU, arbitrary units.

FIG. 4.

Analytical ultracentrifugation analysis. (a) Sedimentation coefficient distributions, c(s), corresponding to the sedimentation speed (48,000 rpm at 20°C) of 70 μM PtxS alone (solid line) and in the presence of 1 mM 2-ketogluconate (dotted line). (b) Sedimentation equilibrium analysis of the association state of PtxS sedimentation equilibrium absorbance gradients (10,000 rpm at 20°C) of PtxS at 70 μM (circles), 30 mM (squares), and 10 μM (triangles). The solid lines show the corresponding best-fit gradients for a single sedimenting species at sedimentation equilibrium. The residuals (difference between the experimental data and the fitted data for each point) are shown at the bottom of this panel (see Materials and Methods for details). OD₂₉₀, optical density at 290 nm.

FIG. 5.

Differential scanning calorimetry of homogeneous PtxS. Calorimetry profiles obtained from DSC experiments with PtxS (30 μM) in the absence and presence of different concentrations of 2-ketogluconate. The concentration (in mM) of 2-ketogluconate is indicated alongside each thermogram. For the sake of clarity, the thermograms were displaced along the vertical axis.

FIG. 6.

Microcalorimetric titration of PtxS with 2-ketogluconate. (Top) Raw data for the injection of 4.8-μl aliquots of 1 mM 2-ketogluconate into 20 μM PtxS. (Bottom) Integrated, dilution-corrected and protein concentration-normalized peak areas of the raw data. Data were fitted with the one-binding-site model of the MicroCal version of ORIGIN.

FIG. 7.

Interaction of PtxS with promoters P_kgu, P_ptxS, and P_gad. (Left) DNase I footprint experiment using a DNA sequence of the promoter of P_gad and PtxS. The protected region is highlighted, and the corresponding sequence is indicated. (Right) Electrophoretic mobility shift assays for the binding of PtxS to different regions: P_kgu (A), P_ptxS (B), and P_gad (C). Experiments were carried out with PtxS concentrations ranging between 0.1 to 3 μM. Images were analyzed densitometrically to determine the fraction of bound DNA which was plotted against the logarithm of the concentration of PtxS and fitted with ORIGIN to determine affinities. (D) EMSA of P_gad in the presence of 6 μM PtxS and a range of 2-ketogluconate concentrations.

FIG. 8.

Analysis of the P_kgu, P_ptxS, and P_gad promoters. (A) Determination of the transcription start point of kguE, gadC, and ptxS using primer extension analysis. Details are in Materials and Methods. (B) Sequences of the three promoters. The transcriptional start site and the start codon are indicated in bold. Arrows indicate the palindromic PtxS binding site, and the −10 and −35 binding sites for the RNA polymerase are marked.