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

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JOURNAL OF BACTERIOLOGY, Sept. 2010, p. 4357–4366 0021-9193/10/$12.00 doi:10.1128/JB.00520-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 192, No. 17

Compartmentalized Glucose Metabolism in Pseudomonas putida Is Controlled by the PtxS Repressor䌤 Abdelali Daddaoua,1 Tino Krell,1 Carlos Alfonso,2 Bertrand Morel,3 and Juan-Luis Ramos1* Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, E-18008, Granada, Spain1; Department of Molecular Microbiology and the Biology of Infections, Centro de Investigacio ´nes Biolo ´gicas, Calle Ramiro de Maeztu 9, 28040 Madrid, Spain2; and Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, Campus de Fuentenueva, 18071 Granada, Spain3 Received 7 May 2010/Accepted 17 June 2010

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 KD (equilibrium dissociation constant) of 15 ␮M, 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°C and 57.6°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 ␮M. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5ⴕ-TGAAACCGGTTTCA-3ⴕ. This palindrome overlaps with the RNA polymerase binding site. phosphorylated to 6-phosphogluconate by gluconokinase, whereas two reactions mediated by KguK and KguD are needed to convert 2-ketogluconate into 6-phosphogluconate (Fig. 1). A third metabolic route present within P. putida, which operates in parallel with the above pathways (7, 8, 38), is the glucose-kinase pathway. This pathway takes place entirely in the cytoplasm and begins with glucokinase (Glk), which phosphorylates glucose to give glucose 6-phosphate (G6P). Next, the combined action of glucose 6-phosphate dehydrogenase (Zwf) and 6-phosphogluconolactonase (Pgl) converts G6P into 6-phosphogluconate (6PG). Subsequently, 6PG, produced by the three peripheral glucose catabolic enzymes, enters the Entner-Doudoroff route, where it is first converted into 2-keto-3-deoxy-6-phosphogluconate (KDPG) by the Edd enzyme (6-phosphogluconate dehydratase) and then hydrolyzed to produce glyceraldehyde-3-phosphate and pyruvate by action of the Eda enzyme (2-keto-3-deoxy-6-phosphogluconate aldolase). Glyceraldehyde-3-phosphate is further metabolized by the GAP-1 enzyme, whereas pyruvate is decarboxylated to acetyl-coenzyme A (CoA) and enters the Krebs cycle (6, 8, 20). The genes for the compartmentalized set of reactions that convert gluconate via 2-ketogluconate to 6-phosphogluconate are clustered in a region within the circular chromosome of P. putida KT2440 (20). The corresponding open reading frames (ORFs) are grouped into three transcriptional units, two of which are termed kgu and gad operon (Fig. 2) and encode the metabolic enzymes (see below), and a single transcriptional

The deciphering of the complete genomes of a number of strains of different species of the genus Pseudomonas has revealed that these microbes metabolize a limited number of sugars (3, 10, 13, 20, 21, 30, 38). However, glucose metabolism in the genus Pseudomonas is biochemically rich since up to three convergent pathways that transform this sugar into 6-phosphogluconate (6PG) have been described. Subsequently, 6PG is metabolized by the Entner-Doudoroff enzymes into central metabolites (6, 7, 8, 9, 11, 20, 34). A relevant feature of glucose metabolism is that the 2-ketogluconate (KG) pathway for glucose metabolism is compartmentalized. This pathway begins in the periplasm, where glucose is initially converted by glucose dehydrogenase into gluconate and then subsequently into 2-ketogluconate by gluconate dehydrogenase. Gluconate and 2-ketogluconate can be transported to the cytoplasm through energy-dependent processes mediated by the GnuK and KguP transporters, respectively. Flux studies in Pseudomonas fluorescens and Pseudomonas putida revealed that most gluconate produced from glucose (almost 90%) is transformed into 2-ketogluconate (8). The small fraction of gluconate that enters the cytoplasm is directly

* Corresponding author. Mailing address: Estacio ´n Experimental del Zaidín-CSIC, C/ Profesor Albareda 1, 18008 Granada, Spain. Phone: 34 958 181 608. Fax: 34 958 129 600. E-mail: juanluis.ramos 䌤 Published ahead of print on 25 June 2010. 4357




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-6phosphate 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.

unit, the ptxS gene, which encodes a regulator of the LacI family. The operon kgu contains four ORFs predicted to encode the ketogluconate reductase (kguD) and ketogluconate kinase (kguK), both of which are involved in the metabolism of glucose. The kguT gene encodes a major facilitator superfamily (MFS) transporter likely to be involved in ketogluconate uptake, whereas the kguE gene is predicted to encode an epimerase. These four gene products share 56 to 83% sequence identity with their homologues in P. aeruginosa (32). The expression of the two catabolic operons and the ptxS gene is induced in cells growing with glucose, gluconate, and 2-ketogluconate (8). Expression of these operons and ptxS is also high, regardless of the carbon source used for growth, in a mutant background lacking the PtxS protein (7), which was taken as evidence that PtxS is the local repressor of the expression of these operons. We have concentrated our current efforts on understanding the control of the genes whose expression is modulated by

PtxS. We have purified PtxS to homogeneity and have carried out studies that provide insight into the effectors of the pathway as well as insight into how PtxS binds to target promoters. MATERIALS AND METHODS Bacterial strains and plasmids used in this study. The genotype or the relevant characteristics of the bacterial strains and plasmids used in this study are listed in Table 1. Bacterial strains were grown in LB medium or in modified M9 minimal medium with a 5 mM concentration of glucose, gluconate, 2-ketogluconate or citrate as the sole C source (15). When required, antibiotics were added to the culture medium to reach a final concentration of 25 ␮g/ml kanamycin, 20 ␮g/ml rifampin, 50 ␮g/ml ampicillin, and 30 ␮g/ml chloramphenicol. Escherichia coli strain DH5␣ was used for plasmid construction, and E. coli BL21(DE3) was used for protein production. Recombinant expression of PtxS in E. coli. To produce polyhistidine-tagged PtxS, the ptxS gene was cloned into plasmid pET24b(⫹). To this end the ptxS gene was amplified from P. putida strain KT2440 chromosomal DNA using primers PtxS3.f and PtxS3.r (Table 2) which contain restriction sites for NdeI and BamHI, respectively. The fragment was then cloned into the pMBL vector to yield pMBL::PtxS (Table 1). The NdeI/BamHI fragment was then excised from this plasmid and cloned into NdeI-BamHI-restricted pET24b(⫹) to produce

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.

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TABLE 1. Strains and plasmids used in this study Strain or plasmid

Strains P. putida KT2440 PSC303b E. coli DH5␣F⬘ BL21(DE3) Plasmids pAD1 pAD2 pAD3 pGEM-T pET24b(⫹) pMBL::PtxS pET24b::PtxS pGEM-T:Pkgu pGEM-T:PptxS pGEM-T:Pgad a b

Genotype or relevant characteristic(s)a

Source or reference

Wild type, prototroph; Cmr Rifr ptxS::pCHESI⍀-Km Rifr Kmr

1 6

F⬘ hsdR17 recA1 gyrA F⫺ ompT hsdSB(rB⫺ mB⫺) gal dcm (DE3)

22 31

pMP220 bearing the promoter region of the kgu operon; Tcr pMP220 bearing the promoter region of the ptxs gene; Tcr pMP220 bearing the promoter region of the gad gene; Tcr Vector for cloning fragments; Apr Protein expression vector; Kmr ptxS gene in pMBL vector Derivative bearing the ptxS gene; Kmr pGEM-T containing the kgu promoter; Apr pGEM-T containing the ptxs promoter; Apr pGEM-T containing the gad promoter; Apr

This work This work This work Dominion Novagen This work This work This work This work This work

Cmr, Kmr, Rifr, and Apr stand for resistance to chloramphenicol, kanamycin, rifampin, and ampicillin, respectively. Our collection of KT2440 mutants from the group of degradation of toxic organic compounds.

pET24b::PtxS, which was used to produce PtxS containing a C-terminal hexahistidine tag. To this end E. coli BL21(DE3) transformed with pET24b::PtxS was grown in 2-liter Erlenmeyer flasks containing 250 ml of LB supplemented with 25 ␮g/ml kanamycin. Cultures were incubated at 30°C with shaking until a turbidity at 660 nm of 0.6 was reached, and then 1 mM isopropyl-␤-D-thiogalactopyranoside was added to induce the expression of the ptxS gene from the plasmid Plac promoter. The cultures were then incubated at 18°C overnight, and cells were harvested by centrifugation (30 min at 20,000 ⫻ g) and stored at ⫺80°C until used for protein purification. For PtxS purification, cells were resuspended in 25 ml of buffer A (50 mM Tris-HCl, pH 7.9, 300 mM NaCl, 1 mM dithiothreitol [DTT], 10 mM imidazole) supplemented with a tablet of complete EDTA-free protease inhibitor mixture. Cells were lysed by three passes through a French press at a pressure of 1,000 lb/in2. The cell suspension was then centrifuged at 20,000 ⫻ g for 1 h. The pellet was discarded, and the supernatant was filtered and loaded onto a 5-ml His-Trap chelating column (GE Healthcare, St. Gibes, United Kingdom) previously equilibrated with buffer A. The PtxS protein was eluted with a 10 to 500 mM gradient of imidazole in buffer A. Protein concentration was determined by the Bradford assay, and protein purity was verified by SDS-PAGE. The protein was dialyzed overnight against buffer B (50 mM HEPES, pH 7.9, 300 mM NaCl, 1 mM DDT, and 10% [vol/vol] glycerol), adjusted to 11 mg/ml and stored at ⫺80°C.

Analytical gel filtration chromatography. In order to determine the oligomeric state of PtxS in solution, we used analytical gel filtration chromatography using an Åkta FLPC system (Amersham Biosciences). Purified PtxS (27 ␮M) was loaded onto a Superdex-200 10/300GL column (Amersham Biosciences) that was equilibrated in buffer B; it was eluted at a constant flow rate of 0.7 ml/min, and the absorbance of the eluate was monitored at 280 nm. The molecular mass of PtxS was estimated from a plot of the elution volume against the logarithm of the molecular masses of the following protein standards: carbonic anhydrase (29 kDa), albumin from chicken egg white (45 kDa), albumin from bovine serum (monomer, 66 kDa, and dimer, 132 kDa) and urease (545 kDa) (Sigma). Analytical ultracentrifugation. Analytical ultracentrifugation analysis of PtxS was performed at several protein concentrations (in the range of 10 to 100 ␮M). Effector concentration corresponded to 50 times its KD (equilibrium dissociation constant) for PtxS (1 mM for 2-ketogluconate), which was determined before by isothermal titration calorimetry (ITC). Sedimentation velocity runs were carried out at 48,000 rpm and 20°C in an XL-I analytical ultracentrifuge (BeckmanCoulter Inc.) with a UV-visible light optics detection system, using an An50Ti rotor and 12-mm double-sector centerpieces. Absorbance scans were run at 290 nm. Sedimentation coefficient (S) distributions were calculated by least-squares boundary modeling of sedimentation velocity data using the c(s) method (25) as implemented in the SEDFIT program. These S values were corrected to standard conditions (water, 20°C, and infinite dilution [35]) using the SEDNTERP

TABLE 2. The sequences of primers used in this study Primer



Kgu 1.f Kgu 1.r Kgu 2.f Kgu 2.r PtxS 1.f PtxS 1.r PtxS 2.f PtxS 2.r PtxS 3.f PtxS 3.R Gad 1.f Gad1.r Gad 2.f Gad 2.r


Fusion to ⬘lacZ Fusion to ⬘lacZ EMSA, footprinting, and primer extension EMSA and footprinting EMSA, footprinting, and primer extension EMSA and footprinting Fusion to ⬘lacZ Fusion to ⬘lacZ PtxS purification PtxS purification Fusion to lacZ Fusion to lacZ EMSA, footprinting, and primer extension EMSA and footprinting



program (17) to obtain the corresponding standard S values (S20,w). Sedimentation equilibrium studies were conducted to determine the state of association of PtxS. The sedimentation equilibrium runs were carried out at multiple speeds (10,000, 12,000, and 15,000 rpm) and wavelengths (280, 290, and 296 nm) with a short column (85 ␮l), using the same experimental conditions and instruments as for the sedimentation velocity experiments. After the equilibrium scans, a highspeed centrifugation run (43,000 rpm) was done to estimate the corresponding baseline offsets. The weight-average buoyant molecular weight of PtxS was determined by fitting data to the single species model using either the MATLAB program (kindly provided by Allen Minton, NIH), based on the conservationof-signal algorithm, or the HeteroAnalysis program (retrieved from the FTP site of the Analytical Ultracentrifugation Facility of the University of Connecticut, Storrs, CT). Both analyses gave similar results. The molecular weight of the protein was determined from the experimental buoyant masses using 0.735 as the partial specific volume of PtxS (calculated from the amino acid composition using the SEDNTERP program [17]). Isothermal titration calorimetry. Microcalorimetric experiments were carried out at 25°C using a VP-microcalorimeter (Microcal, Amherst, MA). Protein and substrates were dialyzed against 50 mM HEPES, pH 7.9, 300 mM NaCl, 1 mM DTT, and 10% (vol/vol) glycerol. Typically, 4.8-␮l aliquots of 1 mM effector solution were injected into 20 ␮M PtxS. All data were corrected using the heat changes arising from injection of the effector into buffer. Data were analyzed using the one-binding-site model of the MicroCal version of ORIGIN. Titration curves were fitted by a nonlinear least-squares method to a function for the binding of one molecule of substrate to one molecule of target protein. The parameters ⌬H (reaction enthalpy) and KA (binding constant; KA ⫽ 1/KD) were determined from the curve fit. The change in free energy (⌬G) and in entropy (⌬S) were calculated from the values of KA and ⌬H using the following equation: ⌬G ⫽ ⫺RT ln KA ⫽ ⌬H ⫺ T⌬S, where R is the universal molar gas constant and T is the absolute temperature. Differential scanning calorimetry. The assays were carried out at a scan rate of 60°C/h in a Valerian-Plotnikov differential scanning calorimeter (VP-DSC) capillary cell from MicroCal (Northampton, MA). Protein concentration was kept constant at 30 ␮M. Calorimetric cells (operating volume, 0.137 ml) were kept under an excess pressure of 60 lb/in2 to avoid any possible degassing on heating. DSC experiments were carried out in 50 mM HEPES, 100 mM NaCl, and 0.5 mM tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), pH 7.9. Reversibility of the transitions was checked by reheating the solution in the DSC cell after it was cooled from the first run. Since thermal transitions were always found to be irreversible, the reheating thermograms were used as instrumental baselines and were subtracted from the original experimental thermograms to obtain apparent specific heat (Cp) profiles. In addition the thermograms were dynamically corrected using the determined time constant of the calorimeter. The enthalpy changes upon unfolding were estimated from the area under each transition peak in the DSC curve. Transcriptional fusions to ⴕlacZ. To obtain a transcriptional fusion of the promoters of the kgu, ptxS, and gad genes to the ⬘lacZ reporter, the corresponding regions were amplified using P. putida strain KT2440 chromosomal DNA as a template, and primers introduced the appropriate restriction sites at their ends (Table 2). PstI-BglII restriction sites were incorporated into the amplified kgu and gad promoter regions, whereas PstI-BamHI sites were introduced into the ptxS intergenic region. Upon digestion, the DNA fragments were cloned into the pGEM-T plasmid cut with the appropriate restriction enzymes (Table 1). Cloned DNA was then sequenced to verify the absence of mutations. The PstI-BglII or PstI-BamHI fragments were subsequently excised from the pGEM-T derivative and cloned into the pMP220 promoter probe vector using the same restriction sites. Resulting plasmids were then introduced into wild-type P. putida KT2440 or its ptxS isogenic mutant. ␤-Galactosidase assays. Wild-type P. putida KT2440 and its isogenic ptxS mutant were grown in minimal medium with citrate as the sole C source and 10 ␮g/ml tetracycline. Overnight cultures were diluted to a turbidity of 0.05 in the same minimal medium, and cells were grown until they reached a turbidity of 0.6; then the inducer molecules were added at a concentration of 5 mM. Growth was continued at 30°C, and after another 6 h aliquots were taken, and ß-galactosidase activity was determined in permeabilized whole cells (19) by using o-nitrophenylß-D-galactoside as a substrate. At least three independent assays were performed, and activity was expressed in Miller units. RNA extraction and primer extension. RNA was extracted from P. putida KT2440 cells growing on medium supplemented with 5 mM 2-ketogluconate using TRI reagent (Ambion). RNA concentration was determined spectrophotometrically at 260 nm, and RNA integrity was assessed by agarose gel electrophoresis. Primer extension reactions were performed as described by Marque´s et al. (18) with the set of primers indicated in Table 2.

J. BACTERIOL. Electrophoresis mobility shift assays (EMSAs). The Pkgu, PptxS, and Pgad promoter regions were amplified by PCR using pGEM-T:Pkgu, pGEM-T:PptxS, and pGEM-T:Pgad, respectively, as templates and the set of primer pairs indicated in Table 2. Amplified fragments were isolated from agarose gels and end labeled with [␥-32P]dATP using the T4 polynucleotide kinase. A 10-␮l sample containing about 2 nM labeled DNA (1.5 ⫻ 104 cpm) was incubated with increasing concentrations of purified PtxS for 1 h in 10 ␮l of binding buffer (50 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5 M magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 5% [vol/vol] glycerol) containing 20 ␮g/ml of poly(dI-dC) and 200 ␮g/ml bovine serum albumin. The DNA-protein complexes were resolved by electrophoresis in 4% (wt/vol) nondenaturing polyacrylamide gels in 1⫻ Trisborate-EDTA (TBE) buffer using Bio-Rad electrophoresis equipment, as previously described (23, 24). DNase I footprinting. The DNA fragment containing Pgad was amplified as outlined above. DNA was labeled with [␥-32P]dATP. Ten-microliter samples containing 2 nM probe were mixed with different amounts of PtxS (5 to 20 ␮M) in binding buffer for the formation of the DNA-PtxS complex. Samples were incubated at 30°C for 1 h, which was followed by the addition of DNase I (0.4 U; Roche Biochemicals). After the mixture was incubated for 2 min, the reaction was stopped by the addition of 2 ␮l of 500 mM EDTA. DNA was extracted with phenol-chloroform, ethanol precipitated, and dissolved in 10 ␮l of sequence loading buffer. After incubation at 95°C for 5 min, DNA was loaded onto a 6.5% (wt/vol) DNA sequencing gel (22). Appropriate sequencing reaction mixtures were loaded onto the gels along with the footprinting samples and used as a size ladder for identification of the sequences of protected sites.

RESULTS PtxS is dimeric in solution. To characterize in detail the PtxS protein, it was overproduced as a His tag fusion in E. coli and purified by affinity chromatography from the soluble fraction of the E. coli lysate. We obtained an average yield of 40 mg of pure protein per liter of E. coli culture. Gel filtration experiments were carried out to determine the oligomeric state of the protein in solution (Fig. 3). An elution volume of 19.5 ml was determined for the purified protein. Eluted protein was submitted to SDS-PAGE, which confirmed that it was fulllength protein. When this elution volume was plotted against the ln of the molecular mass of the proteins used in the calibration curve, an apparent molecular mass of 72 kDa was determined, which indicated that PtxS is more likely dimeric in solution (primary sequence deduced from DNA sequence provides a mass of the monomer of 36.8 kDa). Homogenous PtxS protein was subjected to analytical ultracentrifugation analyses. Figure 4a shows sedimentation velocity data for a 70 ␮M PtxS solution in the absence and presence of 1 mM 2-ketogluconate. The major species (⬎95%) sedimented with a standard S20,w value of 3.8 (⫾ 0.1), and 2-ketogluconate had no effect on the association state of PtxS. To confirm the size of the protein, sedimentation equilibrium assays were carried out as described in Materials and Methods (Fig. 4b), and the sedimentation equilibrium gradient of PtxS (without and with 2-ketogluconate) fitted best with a single species with a molecular weight of 72,000 ⫾ 2,000. All sets of data are compatible with PtxS being a dimer in solution. PtxS belongs to the LacI family, and its members are often involved in sugar catabolism control in proteobacteria (29, 37, 39). Multialignment of PtxS with other members of the LacI family identified the two domains of this set of proteins, with the helix-turn-helix (HTH) DNA binding domain from residues 12 to 67 at the N terminus of the protein and the effector domain located at the C-terminal end. We generated a homology model of the DNA binding domain of PtxS using the structure of the transcriptional regulator CcpA of Bacillus sub-

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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.

tilis as a template (25% sequence identity) (27). Frequently, the thermal unfolding of proteins which consist of two individual domains is characterized by two unfolding events (33). To gain insight into the independent folding of the domains, purified PtxS protein was submitted to thermal unfolding as described in Materials and Methods. Thermal unfolding of PtxS was characterized by two events centered at 48.1 and 57.6°C (Fig. 5), with respective enthalpy changes of about 860 and 130 kJ/mol. According to Pfam protein families database, the effector binding domain consists of 262 amino acid residues while the HTH DNA binding region is composed of 46 residues. In general, the enthalpy changes normalized for the number of amino acids per unfolding unit are in the same range. Based on the estimated number of amino acids present in each of the PtxS domains, the enthalpy changes per amino acid have been calculated. Both values, 3.28 kJ/mol and 2.82 kJ/mol for the first and the second event, respectively, were found be similar, which is consistent with the notion that the first event represented the unfolding of the effector binding domain (3.28 kJ/mol per amino acid), whereas the second event could be due to the unfolding of the DNA binding domain (2.82 kJ/mol per amino acid). Therefore, in accordance with the proposed homology model of the PtxS protein, it consists of two domains that unfold independently. PtxS specifically recognizes 2-ketogluconate. The three promoter regions shown above to be regulated by PtxS, namely, PptxS, Pkgu, and Pgad, were fused to the ⬘lacZ gene, and gene expression studies in the wild-type and in the ptxS-deficient backgrounds were carried out. In the parental background and in the absence of effectors, large differences in the basal levels were detected, such as 20, 1,000, and 100 Miller units for promoters Pkgu, PptxS, and Pgad, respectively (Table 3). In all three cases the mutation of the ptxS gene gave rise to an increase in gene expression by a factor of 3 to 10 (Table 3), which confirmed that PtxS represses gene expression in the wild-type strain, in accordance with previous global transcriptional assays (8). Subsequent experiments were aimed at evaluating the effect

of glucose, gluconate, and 2-ketogluconate on in vivo gene expression. To this end, cells were precultured in M9 minimal medium with citrate, and when the culture reached a turbidity of about 0.6, a 5 mM concentration of the test compound was added, and ␤-galactosidase activity was determined 6 h later. In the wild-type background, for all three promoters each of these three compounds caused a significant increase (5- to 60-fold) in expression, of which the increase observed with 2-ketogluconate was most pronounced. To study the molecular recognition of effector molecules by PtxS, the protein was submitted to isothermal titration calorimetry studies (15) using glucose, gluconate, 2-ketogluconate, pyruvate, and 6-phosphogluconate. The results showed heats indistinguishable from the dilution heats in buffer with glucose, gluconate, pyruvate, and 6-phosphogluconate, indicating that these molecules do not directly bind to PtxS. However, significant heat changes were obtained using 2-ketogluconate. The titration of 20 ␮M PtxS with 1 mM 2-ketogluconate revealed large exothermic heat changes (Fig. 6), indicating that PtxS specifically recognizes 2-ketogluconate. Fitting of the integrated and dilution-corrected raw data with the one-bindingsite model of the ORIGIN software (MicroCal) revealed that binding is driven by favorable enthalpy changes (⌬H ⫽ ⫺14.0 ⫾ 0.1 kcal/mol) and counterbalanced by unfavorable entropy changes (T⌬S ⫽ ⫺7.4 ⫾ 0.2 kcal/mol). The corresponding change in free energy,⌬G, of ⫺6.6 ⫾ 0.1 kcal/mol corresponds to a KD of 14.5 ⫾ 0.4 ␮M. The mathematical algorithm also makes it possible to estimate the binding stoichiometry, which was two molecules of 2-ketogluconate per PtxS dimer. It is known that, typically, small ligand binding to protein results in an increase in the thermal denaturation midpoint temperature (Tm) (33). To evaluate the influence of 2-ketogluconate binding on the thermal unfolding characteristics of PtxS, the DSC analysis was repeated in the presence of 100 ␮M 2-ketogluconate. For this reason DSC assays of PtxS were also carried out in the presence of 100 ␮M 2-ketogluconate. We found that in the presence of its effector, a Tm shift of the first




FIG. 5. Differential scanning calorimetry of homogeneous PtxS. Calorimetry profiles obtained from DSC experiments with PtxS (30 ␮M) in the absence and presence of different concentrations of 2ketogluconate. 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. 4. Analytical ultracentrifugation analysis. (a) Sedimentation coefficient distributions, c(s), corresponding to the sedimentation speed (48,000 rpm at 20°C) of 70 ␮M 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 ␮M (circles), 30 mM (squares), and 10 ␮M (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.

unfolding event was observed (Fig. 5), whereas no significant impact on the unfolding characteristics of the second event was noticed. This is consistent with the notion that 2-ketogluconate binding stabilizes the effector binding domain and has no significant

impact on the unfolding of the DNA binding domain. This lends support to the hypothesis derived from the analysis of the unfolding enthalpy per amino acid. This set of data provides further support for the proposal that the first unfolding event corresponds to the effector binding domain, as proposed from the enthalpic changes discussed above. PtxS targets DNA complexes. In order to study the binding of PtxS with target sequences, EMSAs using the three promoters in the presence of increasing PtxS concentrations (0.1 to 3 ␮M) were carried out (Fig. 7). In all cases the addition of PtxS resulted in retardation of the target DNA. In a series of complementary EMSAs, the effect of 2-ketogluconate on binding of PtxS to its target DNA was tested. First, we used a concentration of PtxS (5 ␮M) that retarded more than 50% of the target DNA, and the complex was incubated with increasing concentrations of 2-ketogluconate. It was observed that 2ketogluconate freed PtxS from its target and that, at saturating concentrations of the effector, almost no PtxS was retained bound to DNA. Gels were analyzed densitometrically; the fraction of bound DNA was plotted against the logarithm of protein concentration and then fitted using the sigmoid fitting tool of ORIGIN (data not shown). Dissociation constants of 2.3 ⫾ 0.5, 1.3 ⫾ 0.1, and 3.1 ⫾ 0.5 ␮M were determined for promoters Pkgu, PptxS, and Pgad, respectively. To identify the binding site of PtxS within the target sequences with respect to the RNA polymerase binding site, we first determined the transcription start point (TSP) of the three promoters and then carried out footprint analysis with the promoter regions and homogenous PtxS protein. To determine the TSP, we cultured cells on minimal medium with glucose as the sole carbon source and prepared total RNA as described in Materials and Methods. We found that the three transcriptional units were transcribed from a main transcription start point (Fig. 8). The leader sequence to the proposed first ATG was 3 nucleotides for kguE, 35 nucleotides for ptxS, and 84

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TABLE 3. Expression from Pkgu, PptxS, and Pgad promoters in the wild-type and a ptxS mutant Activity (Miller units)a

Host and promoter

Without substrate

With gluconate

With glucose

With 2-ketogluconate

Wild-type Pkgu::lacZ PptxS::lacZ Pgad::lacZ

20 ⫾ 3 950 ⫾ 10 100 ⫾ 12

170 ⫾ 2 4,495 ⫾ 103 1,090 ⫾ 10

130 ⫾ 2 5,560 ⫾ 129 1,060 ⫾ 25

275 ⫾ 20 11,545 ⫾ 50 6,430 ⫾ 9

⌬ptxS strain Pkgu::lacZ PptxS::lacZ Pgad::lacZ

180 ⫾ 6 3,470 ⫾ 100 1,380 ⫾ 2

175 ⫾ 3 5,930 ⫾ 150 1,270 ⫾ 20

220 ⫾ 10 6,740 ⫾ 50 1,500 ⫾ 10

360 ⫾ 30 12,010 ⫾ 150 7,005 ⫾ 20

a The three promoter regions were cloned into pMP220 derivative (Tcr) bearing the indicated fusion to ⬘lacZ. P. putida cells were grown on M9 minimal medium with citrate (15 mM), and overnight cultures were diluted 50-fold in the same medium in the absence or in the presence of gluconate, glucose, and 2-ketogluconate (5 mM), and ␤-galactosidase activity was determined when culture cells had reached a cell density of about 0.7. Data are the average of three independent assays done in duplicate.

nucleotides for Pgad. Upstream from ⫹1, canonical sequences at ⫺10 to ⫺35 were found for the three transcriptional units, which supports that the hypothesis that these promoters are transcribed by RNA polymerase with sigma-70. Footprint analysis revealed that PtxS protects a single region within each of the promoters (Fig. 7). These binding sites were found to correspond with a perfect palindromic sequence, 5⬘TGAAACCGGTTTCA-3⬘. Interestingly, in the PptxS promoter, the PtxS binding site overlaps the transcriptional start point, whereas for promoters Pkgu and Pgad the PtxS binding sites were shown to overlap, respectively, the ⫺10 and ⫺35 binding sites of the RNA polymerase (Fig. 8). This is consistent with the idea that PtxS binding interferes with the RNA polymerase binding, which is exemplified in the case of Pgad in Fig. 7. These results support the notion that the mechanism of PtxS

FIG. 6. Microcalorimetric titration of PtxS with 2-ketogluconate. (Top) Raw data for the injection of 4.8-␮l aliquots of 1 mM 2-ketogluconate into 20 ␮M 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.

repression is that of competing with RNA polymerase for binding and that in the presence of 2-ketogluconate PtxS becomes dissociated from its target promoter. To confirm this hypothesis, we carried out EMSAs with a saturating concentration of PtxS in the absence and in the presence of 3 mM 2-ketoglucoante. We found that in the presence of 2-ketogluconate, PtxS was in part released from DNA. DISCUSSION PtxS has common and different functions in P. aeruginosa and P. putida. The initial description of PtxS in P. aeruginosa demonstrated that this protein reduces the expression of the PtxR regulator. This regulator controls the production of exotoxin A, which, in turn, is considered to be the most toxic virulence factor of this pathogen (4, 5, 12). In P. aeruginosa the ptxS and ptxR genes are transcribed divergently, but PtxS was not found to directly regulate ptxR expression (5). Instead, an indirect mechanism seems to exist and remains to be elucidated. In P. aeruginosa and P. putida, the PtxS protein is involved in the control of the compartmentalized metabolism of glucose via 2-ketogluconate. In vivo, gluconate dehydrogenase and the enzymes responsible for the conversion of 2-ketogluconate into 6-phosphogluconate are induced by glucose, gluconate, or 2-ketogluconate; however, in vitro only 2-ketogluconate is able to bind to PtxS, which indicates that 2-ketogluconate is the functionally effective signal molecule that triggers the pathway. Gluconate dehydrogenase is present in the periplasm (Fig. 1) and converts gluconate into 2-ketogluconate. Since the gene products of the kgu operon are involved in the transport and in the metabolism of ketogluconate, the concerted and coordinated regulation of the gad and kgu operon favors the steady flux of carbon from glucose to 6-phosphogluconate via the 2-ketogluconate pathway, as described before by del Castillo et al. (8). With the aid of techniques such as footprinting, EMSA, primer extension analysis, and gene expression studies using the ␤-galactosidase reporter, we showed that PtxS binds to the three promoters with an affinity of around 2 ␮M. The PtxS proteins from P. aeruginosa and P. putida share 72% overall sequence identity and almost 100% identity at their DNA binding motif. The conservation of the HTH motif in the two proteins is probably the reason for the absolute conservation of




FIG. 7. Interaction of PtxS with promoters Pkgu, PptxS, and Pgad. (Left) DNase I footprint experiment using a DNA sequence of the promoter of Pgad 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: Pkgu (A), PptxS (B), and Pgad (C). Experiments were carried out with PtxS concentrations ranging between 0.1 to 3 ␮M. 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 Pgad in the presence of 6 ␮M PtxS and a range of 2-ketogluconate concentrations.

the PtxS DNA recognition sequence in P. aeruginosa and P. putida, which corresponds to the 5⬘-TGAAACCGGTTTCA-3⬘ palindrome in both species (32). Footprint analysis revealed that P. putida PtxS recognized this target between ⫺36 and ⫺50 in Pgad, between ⫺12 and ⫺26 in Pkgu, and between ⫹5 and ⫺9 in PptxS; therefore, these regions overlap with the RNA polymerase binding sites, suggesting that regulation of transcription from these promoters involves impairment of RNA polymerase binding. Azotobacter vinelandii and P. fluorescens have PtxS homologues that share 72% and 68% sequence identity, respectively, with the P. putida sequence. We searched for the presence of the PtxS recognition palindrome in the genomes of these bacteria. We found PtxS recognition sequences in both genomes, which, in analogy to P. putida and P. aeruginosa, are located just upstream of the ptxS gene and the kgu and gad operons. This is consistent with the idea that PtxS modulates the expression of the kgu and gad operons, as well as its own expression, in P. aeruginosa, P. fluorescens, and in the nitrogen-fixing bacterium A. vinelandii. This also suggests that

information derived from studies in P. putida KT2440 can be relevant to understanding the regulation of 2-ketogluconate metabolism in other species of the genus Pseudomonas. PtxS—a repressor of the LacI family. PtxS belong to the LacI family of transcriptional regulators and exhibits strong sequence similarity indicative of structural relationships. Genetic and biochemical studies have shown that the proteins of this family contain two domains. The DNA binding domain (InterPro signature IPR000843) is located at the N terminus (amino acids 11 to 82) and contains a helix-turn-helix motif. This is followed in sequence by an effector binding domain (IPR001761) that has been found both in the periplasmic binding domain of transporters and in transcriptional regulators of the LacI family. While lactose, fructose, and raffinose repressors exist as tetramers, all other members of the LacI family appear to be dimers, as happens with PtxS. Structural information on the effector binding domain revealed that it binds sugars primarily, as is the case for the LacI or CcpA transcriptional regulators, or purine derivatives (e.g., hypoxanthine),

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RNA polymerase to transcribe target operons for the catabolism of glucose via the 2-ketogluconate loop. ACKNOWLEDGMENTS This study was supported by Fondos FEDER via grant BIO-200605668 from the Ministry of Science and Innovation and by grants CVI-344 and CVI-3010 of the Junta de Andalucia. We thank M. M. Fandila and C. Lorente for secretarial assistance and B. Pakuts for checking the English in the manuscript. We thank Germa´n Rivas at CIB-CSIC for help with analytical ultracentrifugation assays. REFERENCES

FIG. 8. Analysis of the Pkgu, PptxS, and Pgad 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.

and also the PurR transcriptional regulator (26, 28). The closest PtxS homologue with a resolved three-dimensional structure is the B. subtilis transcriptional regulator CcpA. The structure of CcpA (27) was used to generate a homology model of PtxS, which supported the hypothesis that PtxS has two domains that may fold independently. A number of transcriptional regulators consisting of an effector binding domain and a DNA binding domain have been described previously, i.e., TetR (2) and the NmrA regulator (16), to cite some, and have been analyzed by DSC. These studies showed that these proteins unfold in a single event, pointing toward the cooperative unfolding of both domains (14, 16). In contrast, PtxS domains seem to unfold independently, as suggested by our DSC studies shown in Fig. 5. Furthermore, we have shown that the binding of 2-ketogluconate to PtxS increased thermal stability of the effector binding site, whereas the stability of the DNA binding domain remained unchanged. In summary, numerous studies have shown that in P. aeruginosa (32, 36) and P. putida (8) PtxS binds to its target operators, which overlap with the RNA polymerase binding site at the corresponding promoter. Our in vitro and in vivo assays indicated that 2-ketogluconate leads to the release of PtxS from its target DNA, and we suggest that upon effector binding, a conformational change occurs within PtxS that likely decreases its affinity for the target site and eases the entry of

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