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

JOURNAL OF BACTERIOLOGY, Sept. 2010, p. 4357–4366 Vol. 192, No. 170021-9193/10/$12.00 doi:10.1128/JB.00520-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Compartmentalized Glucose Metabolism in Pseudomonas putida IsControlled by the PtxS Repressor�

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 Investigaciones Biologicas,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 thecells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by beingcompartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in theperiplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carriedout, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplas-mic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independentoperons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we showthat although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, invitro we found that only 2-ketogluconate binds to the regulator with an apparent KD (equilibrium dissociationconstant) 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 theeffector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized bymelting points of 48.1°C and 57.6°C and that, in the presence of 2-ketogluconate, the unfolding of the effectorbinding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate–PtxS inter-actions. 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 threepromoters and whose consensus sequence is 5�-TGAAACCGGTTTCA-3�. This palindrome overlaps with theRNA polymerase binding site.

The deciphering of the complete genomes of a number ofstrains of different species of the genus Pseudomonas has re-vealed that these microbes metabolize a limited number ofsugars (3, 10, 13, 20, 21, 30, 38). However, glucose metabolismin the genus Pseudomonas is biochemically rich since up tothree convergent pathways that transform this sugar into6-phosphogluconate (6PG) have been described. Subse-quently, 6PG is metabolized by the Entner-Doudoroff enzymesinto central metabolites (6, 7, 8, 9, 11, 20, 34).

A relevant feature of glucose metabolism is that the 2-keto-gluconate (KG) pathway for glucose metabolism is compart-mentalized. This pathway begins in the periplasm, where glu-cose is initially converted by glucose dehydrogenase intogluconate and then subsequently into 2-ketogluconate by glu-conate dehydrogenase. Gluconate and 2-ketogluconate can betransported to the cytoplasm through energy-dependent pro-cesses mediated by the GnuK and KguP transporters, respec-tively. Flux studies in Pseudomonas fluorescens and Pseudomo-nas putida revealed that most gluconate produced from glucose(almost 90%) is transformed into 2-ketogluconate (8). Thesmall fraction of gluconate that enters the cytoplasm is directly

phosphorylated to 6-phosphogluconate by gluconokinase,whereas two reactions mediated by KguK and KguD areneeded 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), isthe glucose-kinase pathway. This pathway takes place entirelyin the cytoplasm and begins with glucokinase (Glk), whichphosphorylates glucose to give glucose 6-phosphate (G6P).Next, the combined action of glucose 6-phosphate dehydroge-nase (Zwf) and 6-phosphogluconolactonase (Pgl) convertsG6P into 6-phosphogluconate (6PG). Subsequently, 6PG, pro-duced by the three peripheral glucose catabolic enzymes, en-ters the Entner-Doudoroff route, where it is first convertedinto 2-keto-3-deoxy-6-phosphogluconate (KDPG) by the Eddenzyme (6-phosphogluconate dehydratase) and then hydro-lyzed to produce glyceraldehyde-3-phosphate and pyruvate byaction of the Eda enzyme (2-keto-3-deoxy-6-phosphogluconatealdolase). Glyceraldehyde-3-phosphate is further metabolizedby the GAP-1 enzyme, whereas pyruvate is decarboxylated toacetyl-coenzyme A (CoA) and enters the Krebs cycle (6, 8, 20).

The genes for the compartmentalized set of reactions thatconvert gluconate via 2-ketogluconate to 6-phosphogluconateare 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 ofwhich are termed kgu and gad operon (Fig. 2) and encode themetabolic enzymes (see below), and a single transcriptional

* Corresponding author. Mailing address: Estacion Experimentaldel Zaidín-CSIC, C/ Profesor Albareda 1, 18008 Granada, Spain.Phone: 34 958 181 608. Fax: 34 958 129 600. E-mail: [email protected].

� Published ahead of print on 25 June 2010.

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unit, the ptxS gene, which encodes a regulator of the LacIfamily.

The operon kgu contains four ORFs predicted to encode theketogluconate reductase (kguD) and ketogluconate kinase(kguK), both of which are involved in the metabolism of glu-cose. The kguT gene encodes a major facilitator superfamily(MFS) transporter likely to be involved in ketogluconate up-take, whereas the kguE gene is predicted to encode an epime-rase. These four gene products share 56 to 83% sequenceidentity with their homologues in P. aeruginosa (32).

The expression of the two catabolic operons and the ptxSgene is induced in cells growing with glucose, gluconate, and2-ketogluconate (8). Expression of these operons and ptxS isalso high, regardless of the carbon source used for growth, ina mutant background lacking the PtxS protein (7), which wastaken as evidence that PtxS is the local repressor of the ex-pression of these operons.

We have concentrated our current efforts on understandingthe control of the genes whose expression is modulated by

PtxS. We have purified PtxS to homogeneity and have carriedout studies that provide insight into the effectors of the path-way 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 rele-vant characteristics of the bacterial strains and plasmids used in this study arelisted in Table 1. Bacterial strains were grown in LB medium or in modified M9minimal medium with a 5 mM concentration of glucose, gluconate, 2-ketoglu-conate or citrate as the sole C source (15). When required, antibiotics wereadded to the culture medium to reach a final concentration of 25 �g/ml kana-mycin, 20 �g/ml rifampin, 50 �g/ml ampicillin, and 30 �g/ml chloramphenicol.Escherichia coli strain DH5� was used for plasmid construction, and E. coliBL21(DE3) was used for protein production.

Recombinant expression of PtxS in E. coli. To produce polyhistidine-taggedPtxS, the ptxS gene was cloned into plasmid pET24b(�). To this end the ptxSgene was amplified from P. putida strain KT2440 chromosomal DNA usingprimers PtxS3.f and PtxS3.r (Table 2) which contain restriction sites for NdeI andBamHI, respectively. The fragment was then cloned into the pMBL vector toyield pMBL::PtxS (Table 1). The NdeI/BamHI fragment was then excised fromthis plasmid and cloned into NdeI-BamHI-restricted pET24b(�) to produce

FIG. 1. Summary of glucose metabolism in P. putida KT2440, as deduced from gene annotations and functional analysis in the wild-type strainand a series of mutants. OM, outer membrane; PS, periplasmic space; IM, inner membrane; Gcd, glucose dehydrogenase; Gad, gluconatedehydrogenase; 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 controlledby 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). PP3381is predicted to be a transposase, and PP3385 is an outer transmembrane protein.

4358 DADDAOUA ET AL. J. BACTERIOL.

pET24b::PtxS, which was used to produce PtxS containing a C-terminal hexa-histidine tag. To this end E. coli BL21(DE3) transformed with pET24b::PtxS wasgrown 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 turbidityat 660 nm of 0.6 was reached, and then 1 mM isopropyl-�-D-thiogalactopyrano-side 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 wereharvested by centrifugation (30 min at 20,000 � g) and stored at �80°C untilused for protein purification.

For PtxS purification, cells were resuspended in 25 ml of buffer A (50 mMTris-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 1,000lb/in2. The cell suspension was then centrifuged at 20,000 � g for 1 h. The pelletwas discarded, and the supernatant was filtered and loaded onto a 5-ml His-Trapchelating column (GE Healthcare, St. Gibes, United Kingdom) previously equil-ibrated with buffer A.

The PtxS protein was eluted with a 10 to 500 mM gradient of imidazole inbuffer A. Protein concentration was determined by the Bradford assay, andprotein purity was verified by SDS-PAGE. The protein was dialyzed overnightagainst 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 oligomericstate of PtxS in solution, we used analytical gel filtration chromatography usingan Åkta FLPC system (Amersham Biosciences). Purified PtxS (27 �M) wasloaded onto a Superdex-200 10/300GL column (Amersham Biosciences) that wasequilibrated in buffer B; it was eluted at a constant flow rate of 0.7 ml/min, andthe absorbance of the eluate was monitored at 280 nm. The molecular mass ofPtxS was estimated from a plot of the elution volume against the logarithm of themolecular masses of the following protein standards: carbonic anhydrase (29kDa), 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 PtxSwas performed at several protein concentrations (in the range of 10 to 100 �M).Effector concentration corresponded to 50 times its KD (equilibrium dissociationconstant) for PtxS (1 mM for 2-ketogluconate), which was determined before byisothermal titration calorimetry (ITC). Sedimentation velocity runs were carriedout at 48,000 rpm and 20°C in an XL-I analytical ultracentrifuge (Beckman-Coulter Inc.) with a UV-visible light optics detection system, using an An50Tirotor and 12-mm double-sector centerpieces. Absorbance scans were run at 290nm. Sedimentation coefficient (S) distributions were calculated by least-squaresboundary modeling of sedimentation velocity data using the c(s) method (25) asimplemented in the SEDFIT program. These S values were corrected to stan-dard conditions (water, 20°C, and infinite dilution [35]) using the SEDNTERP

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Genotype or relevant characteristic(s)a Source or reference

StrainsP. putida

KT2440 Wild type, prototroph; Cmr Rifr 1PSC303b ptxS::pCHESI�-Km Rifr Kmr 6

E. coliDH5�F� F� hsdR17 recA1 gyrA 22BL21(DE3) F� ompT hsdSB(rB

� mB�) gal dcm (DE3) 31

PlasmidspAD1 pMP220 bearing the promoter region of the kgu operon; Tcr This workpAD2 pMP220 bearing the promoter region of the ptxs gene; Tcr This workpAD3 pMP220 bearing the promoter region of the gad gene; Tcr This workpGEM-T Vector for cloning fragments; Apr DominionpET24b(�) Protein expression vector; Kmr NovagenpMBL::PtxS ptxS gene in pMBL vector This workpET24b::PtxS Derivative bearing the ptxS gene; Kmr This workpGEM-T:Pkgu pGEM-T containing the kgu promoter; Apr This workpGEM-T:PptxS pGEM-T containing the ptxs promoter; Apr This workpGEM-T:Pgad pGEM-T containing the gad promoter; Apr This work

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

TABLE 2. The sequences of primers used in this study

Primer Sequence Use

Kgu 1.f 5�-CTGCAGGACTGATGGAAACGGGG-3� Fusion to �lacZKgu 1.r 5�-AGATCTGCCAACCTGATCATCCGC-3� Fusion to �lacZKgu 2.f 5�-GCACAAAGTCGGCGCCGTAGC-3� EMSA, footprinting, and primer extensionKgu 2.r 5�-GCCTGCTCGGTCGCTTGCG-3� EMSA and footprintingPtxS 1.f 5�-TGGTGTGCTGCTTTGCTCCCG-3� EMSA, footprinting, and primer extensionPtxS 1.r 5�-ATGGGCAGGCGCGTCGGT-3� EMSA and footprintingPtxS 2.f 5�-CTGCAGCGTTCGCGGGTATGG-3� Fusion to �lacZPtxS 2.r 5�-GGATCCGGGGTATCAACTGGTGGCC-3� Fusion to �lacZPtxS 3.f 5�-ATGACCGACGCGCCTGCCCA-3� PtxS purificationPtxS 3.R 5�-CTGGGGTTGGGTTGAACCGC-3� PtxS purificationGad 1.f 5�-CTGCAGGGGATCAGGGTCAAGGT-3� Fusion to lacZGad1.r 5�-AGATCTTGCGGTCGGACTCTTTGG-3� Fusion to lacZGad 2.f 5�-CCTCATCGGCTGTGGGGCG-3� EMSA, footprinting, and primer extensionGad 2.r 5�-TGCGGTCGGACTCTTTGGGC-3� EMSA and footprinting

VOL. 192, 2010 SUGAR CATABOLISM CONTROL IN BACTERIA 4359

program (17) to obtain the corresponding standard S values (S20,w). Sedimenta-tion equilibrium studies were conducted to determine the state of association ofPtxS. 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 ashort column (85 �l), using the same experimental conditions and instruments asfor the sedimentation velocity experiments. After the equilibrium scans, a high-speed centrifugation run (43,000 rpm) was done to estimate the correspondingbaseline offsets. The weight-average buoyant molecular weight of PtxS was de-termined by fitting data to the single species model using either the MATLABprogram (kindly provided by Allen Minton, NIH), based on the conservation-of-signal algorithm, or the HeteroAnalysis program (retrieved from the FTP siteof the Analytical Ultracentrifugation Facility of the University of Connecticut,Storrs, CT). Both analyses gave similar results. The molecular weight of theprotein was determined from the experimental buoyant masses using 0.735 as thepartial specific volume of PtxS (calculated from the amino acid compositionusing the SEDNTERP program [17]).

Isothermal titration calorimetry. Microcalorimetric experiments were carriedout at 25°C using a VP-microcalorimeter (Microcal, Amherst, MA). Protein andsubstrates were dialyzed against 50 mM HEPES, pH 7.9, 300 mM NaCl, 1 mMDTT, and 10% (vol/vol) glycerol. Typically, 4.8-�l aliquots of 1 mM effectorsolution were injected into 20 �M PtxS. All data were corrected using the heatchanges arising from injection of the effector into buffer. Data were analyzedusing the one-binding-site model of the MicroCal version of ORIGIN. Titrationcurves were fitted by a nonlinear least-squares method to a function for thebinding of one molecule of substrate to one molecule of target protein. Theparameters H (reaction enthalpy) and KA (binding constant; KA 1/KD) weredetermined 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 � TS, where R is the universal molar gas constant andT is the absolute temperature.

Differential scanning calorimetry. The assays were carried out at a scan rate of60°C/h in a Valerian-Plotnikov differential scanning calorimeter (VP-DSC) cap-illary cell from MicroCal (Northampton, MA). Protein concentration was keptconstant at 30 �M. Calorimetric cells (operating volume, 0.137 ml) were keptunder 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 0.5mM tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), pH 7.9. Reversibilityof the transitions was checked by reheating the solution in the DSC cell after itwas cooled from the first run. Since thermal transitions were always found to beirreversible, the reheating thermograms were used as instrumental baselines andwere subtracted from the original experimental thermograms to obtain apparentspecific heat (Cp) profiles. In addition the thermograms were dynamically cor-rected using the determined time constant of the calorimeter. The enthalpychanges upon unfolding were estimated from the area under each transition peakin the DSC curve.

Transcriptional fusions to �lacZ. To obtain a transcriptional fusion of thepromoters of the kgu, ptxS, and gad genes to the �lacZ reporter, the correspond-ing regions were amplified using P. putida strain KT2440 chromosomal DNA asa template, and primers introduced the appropriate restriction sites at their ends(Table 2). PstI-BglII restriction sites were incorporated into the amplified kguand gad promoter regions, whereas PstI-BamHI sites were introduced into theptxS intergenic region. Upon digestion, the DNA fragments were cloned into thepGEM-T plasmid cut with the appropriate restriction enzymes (Table 1). ClonedDNA was then sequenced to verify the absence of mutations. The PstI-BglII orPstI-BamHI fragments were subsequently excised from the pGEM-T derivativeand cloned into the pMP220 promoter probe vector using the same restrictionsites. Resulting plasmids were then introduced into wild-type P. putida KT2440or its ptxS isogenic mutant.

�-Galactosidase assays. Wild-type P. putida KT2440 and its isogenic ptxSmutant 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 thesame 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 wascontinued at 30°C, and after another 6 h aliquots were taken, and ß-galactosidaseactivity was determined in permeabilized whole cells (19) by using o-nitrophenyl-ß-D-galactoside as a substrate. At least three independent assays were per-formed, and activity was expressed in Miller units.

RNA extraction and primer extension. RNA was extracted from P. putidaKT2440 cells growing on medium supplemented with 5 mM 2-ketogluconateusing TRI reagent (Ambion). RNA concentration was determined spectropho-tometrically at 260 nm, and RNA integrity was assessed by agarose gel electro-phoresis. Primer extension reactions were performed as described by Marques etal. (18) with the set of primers indicated in Table 2.

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 indi-cated in Table 2. Amplified fragments were isolated from agarose gels and endlabeled with [�-32P]dATP using the T4 polynucleotide kinase. A 10-�l samplecontaining about 2 nM labeled DNA (1.5 � 104 cpm) was incubated withincreasing concentrations of purified PtxS for 1 h in 10 �l of binding buffer (50mM 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 byelectrophoresis in 4% (wt/vol) nondenaturing polyacrylamide gels in 1� Tris-borate-EDTA (TBE) buffer using Bio-Rad electrophoresis equipment, as previ-ously described (23, 24).

DNase I footprinting. The DNA fragment containing Pgad was amplified asoutlined above. DNA was labeled with [�-32P]dATP. Ten-microliter samplescontaining 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 wereincubated 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 reactionwas stopped by the addition of 2 �l of 500 mM EDTA. DNA was extracted withphenol-chloroform, ethanol precipitated, and dissolved in 10 �l of sequenceloading 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 mixtureswere loaded onto the gels along with the footprinting samples and used as a sizeladder for identification of the sequences of protected sites.

RESULTS

PtxS is dimeric in solution. To characterize in detail thePtxS protein, it was overproduced as a His tag fusion in E. coliand purified by affinity chromatography from the soluble frac-tion of the E. coli lysate. We obtained an average yield of 40 mgof pure protein per liter of E. coli culture. Gel filtration exper-iments were carried out to determine the oligomeric state ofthe protein in solution (Fig. 3). An elution volume of 19.5 mlwas determined for the purified protein. Eluted protein wassubmitted to SDS-PAGE, which confirmed that it was full-length protein. When this elution volume was plotted againstthe ln of the molecular mass of the proteins used in the cali-bration curve, an apparent molecular mass of 72 kDa wasdetermined, which indicated that PtxS is more likely dimeric insolution (primary sequence deduced from DNA sequence pro-vides a mass of the monomer of 36.8 kDa). Homogenous PtxSprotein was subjected to analytical ultracentrifugation analy-ses. Figure 4a shows sedimentation velocity data for a 70 �MPtxS solution in the absence and presence of 1 mM 2-ketoglu-conate. The major species (�95%) sedimented with a standardS20,w value of 3.8 ( 0.1), and 2-ketogluconate had no effect onthe association state of PtxS. To confirm the size of the protein,sedimentation equilibrium assays were carried out as describedin Materials and Methods (Fig. 4b), and the sedimentationequilibrium gradient of PtxS (without and with 2-ketoglu-conate) fitted best with a single species with a molecular weightof 72,000 2,000. All sets of data are compatible with PtxSbeing a dimer in solution.

PtxS belongs to the LacI family, and its members are ofteninvolved in sugar catabolism control in proteobacteria (29, 37,39). Multialignment of PtxS with other members of the LacIfamily identified the two domains of this set of proteins, withthe helix-turn-helix (HTH) DNA binding domain from resi-dues 12 to 67 at the N terminus of the protein and the effectordomain located at the C-terminal end. We generated a homol-ogy model of the DNA binding domain of PtxS using thestructure of the transcriptional regulator CcpA of Bacillus sub-


tilis as a template (25% sequence identity) (27). Frequently,the thermal unfolding of proteins which consist of two individ-ual domains is characterized by two unfolding events (33). Togain insight into the independent folding of the domains, pu-rified PtxS protein was submitted to thermal unfolding as de-scribed in Materials and Methods. Thermal unfolding of PtxSwas characterized by two events centered at 48.1 and 57.6°C(Fig. 5), with respective enthalpy changes of about 860 and 130kJ/mol. According to Pfam protein families database, the ef-fector binding domain consists of 262 amino acid residueswhile the HTH DNA binding region is composed of 46 resi-dues. In general, the enthalpy changes normalized for thenumber of amino acids per unfolding unit are in the samerange. Based on the estimated number of amino acids presentin each of the PtxS domains, the enthalpy changes per aminoacid have been calculated. Both values, 3.28 kJ/mol and 2.82kJ/mol for the first and the second event, respectively, werefound be similar, which is consistent with the notion that thefirst event represented the unfolding of the effector bindingdomain (3.28 kJ/mol per amino acid), whereas the secondevent could be due to the unfolding of the DNA bindingdomain (2.82 kJ/mol per amino acid). Therefore, in accor-dance with the proposed homology model of the PtxS protein,it consists of two domains that unfold independently.

PtxS specifically recognizes 2-ketogluconate. The three pro-moter regions shown above to be regulated by PtxS, namely,PptxS, Pkgu, and Pgad, were fused to the �lacZ gene, and geneexpression studies in the wild-type and in the ptxS-deficientbackgrounds were carried out. In the parental background andin the absence of effectors, large differences in the basal levelswere detected, such as 20, 1,000, and 100 Miller units forpromoters Pkgu, PptxS, and Pgad, respectively (Table 3). In allthree cases the mutation of the ptxS gene gave rise to anincrease in gene expression by a factor of 3 to 10 (Table 3),which confirmed that PtxS represses gene expression in thewild-type strain, in accordance with previous global transcrip-tional assays (8).

Subsequent experiments were aimed at evaluating the effect

of glucose, gluconate, and 2-ketogluconate on in vivo geneexpression. To this end, cells were precultured in M9 minimalmedium with citrate, and when the culture reached a turbidityof about 0.6, a 5 mM concentration of the test compound wasadded, and �-galactosidase activity was determined 6 h later.In the wild-type background, for all three promoters each ofthese three compounds caused a significant increase (5- to60-fold) in expression, of which the increase observed with2-ketogluconate was most pronounced.

To study the molecular recognition of effector molecules byPtxS, the protein was submitted to isothermal titration calo-rimetry studies (15) using glucose, gluconate, 2-ketogluconate,pyruvate, and 6-phosphogluconate. The results showed heatsindistinguishable from the dilution heats in buffer with glucose,gluconate, pyruvate, and 6-phosphogluconate, indicating thatthese molecules do not directly bind to PtxS. However, signif-icant heat changes were obtained using 2-ketogluconate. Thetitration of 20 �M PtxS with 1 mM 2-ketogluconate revealedlarge exothermic heat changes (Fig. 6), indicating that PtxSspecifically recognizes 2-ketogluconate. Fitting of the inte-grated and dilution-corrected raw data with the one-binding-site model of the ORIGIN software (MicroCal) revealed thatbinding is driven by favorable enthalpy changes (H �14.0 0.1 kcal/mol) and counterbalanced by unfavorableentropy changes (TS �7.4 0.2 kcal/mol). The corre-sponding change in free energy,G, of �6.6 0.1 kcal/molcorresponds to a KD of 14.5 0.4 �M. The mathematicalalgorithm also makes it possible to estimate the binding stoi-chiometry, which was two molecules of 2-ketogluconate perPtxS dimer.

It is known that, typically, small ligand binding to proteinresults in an increase in the thermal denaturation midpointtemperature (Tm) (33). To evaluate the influence of 2-ketoglu-conate binding on the thermal unfolding characteristics ofPtxS, the DSC analysis was repeated in the presence of 100 �M2-ketogluconate. For this reason DSC assays of PtxS were alsocarried out in the presence of 100 �M 2-ketogluconate. Wefound that in the presence of its effector, a Tm shift of the first

FIG. 3. Determination of the oligomeric state of PtxS. (A) Gel filtration elution profile of PtxS. (B) Calibration curve of the gel filtrationcolumn 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 isindicated. AU, arbitrary units.


unfolding event was observed (Fig. 5), whereas no significantimpact on the unfolding characteristics of the second event wasnoticed.

This is consistent with the notion that 2-ketogluconate bind-ing stabilizes the effector binding domain and has no significant

impact on the unfolding of the DNA binding domain. Thislends support to the hypothesis derived from the analysis of theunfolding enthalpy per amino acid. This set of data providesfurther support for the proposal that the first unfolding eventcorresponds to the effector binding domain, as proposed fromthe enthalpic changes discussed above.

PtxS targets DNA complexes. In order to study the bindingof PtxS with target sequences, EMSAs using the three promot-ers in the presence of increasing PtxS concentrations (0.1 to 3�M) were carried out (Fig. 7). In all cases the addition of PtxSresulted in retardation of the target DNA. In a series of com-plementary EMSAs, the effect of 2-ketogluconate on bindingof PtxS to its target DNA was tested. First, we used a concen-tration of PtxS (5 �M) that retarded more than 50% of thetarget DNA, and the complex was incubated with increasingconcentrations of 2-ketogluconate. It was observed that 2-ketogluconate freed PtxS from its target and that, at saturatingconcentrations of the effector, almost no PtxS was retainedbound to DNA. Gels were analyzed densitometrically; the frac-tion of bound DNA was plotted against the logarithm of pro-tein concentration and then fitted using the sigmoid fitting toolof ORIGIN (data not shown). Dissociation constants of 2.3 0.5, 1.3 0.1, and 3.1 0.5 �M were determined for promot-ers Pkgu, PptxS, and Pgad, respectively.

To identify the binding site of PtxS within the target se-quences with respect to the RNA polymerase binding site, wefirst determined the transcription start point (TSP) of the threepromoters and then carried out footprint analysis with thepromoter regions and homogenous PtxS protein. To determinethe TSP, we cultured cells on minimal medium with glucose asthe sole carbon source and prepared total RNA as described inMaterials and Methods. We found that the three transcrip-tional units were transcribed from a main transcription startpoint (Fig. 8). The leader sequence to the proposed first ATGwas 3 nucleotides for kguE, 35 nucleotides for ptxS, and 84

FIG. 4. Analytical ultracentrifugation analysis. (a) Sedimentationcoefficient distributions, c(s), corresponding to the sedimentationspeed (48,000 rpm at 20°C) of 70 �M PtxS alone (solid line) and in thepresence of 1 mM 2-ketogluconate (dotted line). (b) Sedimentationequilibrium analysis of the association state of PtxS sedimentationequilibrium absorbance gradients (10,000 rpm at 20°C) of PtxS at 70�M (circles), 30 mM (squares), and 10 �M (triangles). The solid linesshow the corresponding best-fit gradients for a single sedimentingspecies at sedimentation equilibrium. The residuals (difference be-tween the experimental data and the fitted data for each point) areshown at the bottom of this panel (see Materials and Methods fordetails). OD290, optical density at 290 nm.

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 2-ketogluconate. The concentration (in mM) of 2-ketogluconate is indi-cated alongside each thermogram. For the sake of clarity, the thermo-grams were displaced along the vertical axis.


nucleotides for Pgad. Upstream from �1, canonical sequencesat �10 to �35 were found for the three transcriptional units,which supports that the hypothesis that these promoters aretranscribed by RNA polymerase with sigma-70.

Footprint analysis revealed that PtxS protects a single regionwithin each of the promoters (Fig. 7). These binding sites werefound to correspond with a perfect palindromic sequence, 5�-TGAAACCGGTTTCA-3�. Interestingly, in the PptxS pro-moter, the PtxS binding site overlaps the transcriptional startpoint, whereas for promoters Pkgu and Pgad the PtxS bindingsites were shown to overlap, respectively, the �10 and �35binding sites of the RNA polymerase (Fig. 8). This is consistentwith the idea that PtxS binding interferes with the RNA poly-merase binding, which is exemplified in the case of Pgad in Fig.7. These results support the notion that the mechanism of PtxS

repression is that of competing with RNA polymerase for bind-ing and that in the presence of 2-ketogluconate PtxS becomesdissociated from its target promoter. To confirm this hypoth-esis, we carried out EMSAs with a saturating concentration ofPtxS in the absence and in the presence of 3 mM 2-ketoglu-coante. We found that in the presence of 2-ketogluconate, PtxSwas in part released from DNA.

DISCUSSION

PtxS has common and different functions in P. aeruginosaand P. putida. The initial description of PtxS in P. aeruginosademonstrated that this protein reduces the expression of thePtxR regulator. This regulator controls the production of exo-toxin A, which, in turn, is considered to be the most toxicvirulence factor of this pathogen (4, 5, 12). In P. aeruginosa theptxS and ptxR genes are transcribed divergently, but PtxS wasnot found to directly regulate ptxR expression (5). Instead, anindirect mechanism seems to exist and remains to be eluci-dated. In P. aeruginosa and P. putida, the PtxS protein is in-volved in the control of the compartmentalized metabolism ofglucose via 2-ketogluconate.

In vivo, gluconate dehydrogenase and the enzymes respon-sible for the conversion of 2-ketogluconate into 6-phosphoglu-conate 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 effec-tive signal molecule that triggers the pathway. Gluconate de-hydrogenase is present in the periplasm (Fig. 1) and convertsgluconate into 2-ketogluconate. Since the gene products of thekgu operon are involved in the transport and in the metabolismof ketogluconate, the concerted and coordinated regulation ofthe gad and kgu operon favors the steady flux of carbon fromglucose to 6-phosphogluconate via the 2-ketogluconate path-way, 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 usingthe �-galactosidase reporter, we showed that PtxS binds to thethree promoters with an affinity of around 2 �M. The PtxSproteins from P. aeruginosa and P. putida share 72% overallsequence identity and almost 100% identity at their DNAbinding motif. The conservation of the HTH motif in the twoproteins is probably the reason for the absolute conservation of

TABLE 3. Expression from Pkgu, PptxS, and Pgad promoters in the wild-type and a ptxS mutant

Host andpromoter

Activity (Miller units)a

Without substrate With gluconate With glucose With 2-ketogluconate

Wild-typePkgu::lacZ 20 3 170 2 130 2 275 20PptxS::lacZ 950 10 4,495 103 5,560 129 11,545 50Pgad::lacZ 100 12 1,090 10 1,060 25 6,430 9

ptxS strainPkgu::lacZ 180 6 175 3 220 10 360 30PptxS::lacZ 3,470 100 5,930 150 6,740 50 12,010 150Pgad::lacZ 1,380 2 1,270 20 1,500 10 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 mediumwith 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 donein duplicate.

FIG. 6. Microcalorimetric titration of PtxS with 2-ketogluconate.(Top) Raw data for the injection of 4.8-�l aliquots of 1 mM 2-keto-gluconate into 20 �M PtxS. (Bottom) Integrated, dilution-correctedand protein concentration-normalized peak areas of the raw data.Data were fitted with the one-binding-site model of the MicroCalversion of ORIGIN.


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 revealedthat P. putida PtxS recognized this target between �36 and�50 in Pgad, between �12 and �26 in Pkgu, and between �5and �9 in PptxS; therefore, these regions overlap with the RNApolymerase binding sites, suggesting that regulation of tran-scription from these promoters involves impairment of RNApolymerase binding. Azotobacter vinelandii and P. fluorescenshave PtxS homologues that share 72% and 68% sequenceidentity, respectively, with the P. putida sequence. We searchedfor the presence of the PtxS recognition palindrome in thegenomes of these bacteria. We found PtxS recognition se-quences in both genomes, which, in analogy to P. putida and P.aeruginosa, are located just upstream of the ptxS gene and thekgu and gad operons. This is consistent with the idea that PtxSmodulates the expression of the kgu and gad operons, as well asits own expression, in P. aeruginosa, P. fluorescens, and in thenitrogen-fixing bacterium A. vinelandii. This also suggests that

information derived from studies in P. putida KT2440 can berelevant to understanding the regulation of 2-ketogluconatemetabolism in other species of the genus Pseudomonas.

PtxS—a repressor of the LacI family. PtxS belong to theLacI family of transcriptional regulators and exhibits strongsequence similarity indicative of structural relationships. Ge-netic and biochemical studies have shown that the proteins ofthis 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 bind-ing domain of transporters and in transcriptional regulators ofthe LacI family. While lactose, fructose, and raffinose repres-sors exist as tetramers, all other members of the LacI familyappear to be dimers, as happens with PtxS. Structural infor-mation on the effector binding domain revealed that it bindssugars primarily, as is the case for the LacI or CcpA transcrip-tional regulators, or purine derivatives (e.g., hypoxanthine),

FIG. 7. Interaction of PtxS with promoters Pkgu, PptxS, and Pgad. (Left) DNase I footprint experiment using a DNA sequence of the promoterof Pgad and PtxS. The protected region is highlighted, and the corresponding sequence is indicated. (Right) Electrophoretic mobility shift assaysfor the binding of PtxS to different regions: Pkgu (A), PptxS (B), and Pgad (C). Experiments were carried out with PtxS concentrations rangingbetween 0.1 to 3 �M. Images were analyzed densitometrically to determine the fraction of bound DNA which was plotted against the logarithmof 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 of2-ketogluconate concentrations.


and also the PurR transcriptional regulator (26, 28). The clos-est PtxS homologue with a resolved three-dimensional struc-ture is the B. subtilis transcriptional regulator CcpA. The struc-ture of CcpA (27) was used to generate a homology model ofPtxS, which supported the hypothesis that PtxS has two do-mains that may fold independently. A number of transcrip-tional regulators consisting of an effector binding domain anda DNA binding domain have been described previously, i.e.,TetR (2) and the NmrA regulator (16), to cite some, and havebeen analyzed by DSC. These studies showed that these pro-teins unfold in a single event, pointing toward the cooperativeunfolding of both domains (14, 16). In contrast, PtxS domainsseem to unfold independently, as suggested by our DSC studiesshown in Fig. 5. Furthermore, we have shown that the bindingof 2-ketogluconate to PtxS increased thermal stability of theeffector binding site, whereas the stability of the DNA bindingdomain remained unchanged.

In summary, numerous studies have shown that in P. aerugi-nosa (32, 36) and P. putida (8) PtxS binds to its target opera-tors, which overlap with the RNA polymerase binding site atthe corresponding promoter. Our in vitro and in vivo assaysindicated that 2-ketogluconate leads to the release of PtxSfrom its target DNA, and we suggest that upon effector bind-ing, a conformational change occurs within PtxS that likelydecreases its affinity for the target site and eases the entry of

RNA polymerase to transcribe target operons for the catabo-lism of glucose via the 2-ketogluconate loop.

ACKNOWLEDGMENTS

This study was supported by Fondos FEDER via grant BIO-2006-05668 from the Ministry of Science and Innovation and by grantsCVI-344 and CVI-3010 of the Junta de Andalucia.

We thank M. M. Fandila and C. Lorente for secretarial assistanceand B. Pakuts for checking the English in the manuscript. We thankGerman Rivas at CIB-CSIC for help with analytical ultracentrifugationassays.

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