Published Ahead of Print 25 June 2010.

2010, 192(17):4357. DOI: 10.1128/JB.00520-10. J. Bacteriol.

Morel and Juan-Luis Ramos
Abdelali Daddaoua, Tino Krell, Carlos Alfonso, Bertrand

PtxS Repressor
Is Controlled by the Pseudomonas putida
Compartmentalized Glucose Metabolism in
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JOURNAL OF BACTERIOLOGY, Sept. 2010, p. 43574366 Vol. 192, No. 17
0021-9193/10/$12.00 doi:10.1128/JB.00520-10
Copyright 2010, American Society for Microbiology. All Rights Reserved.
Compartmentalized Glucose Metabolism in Pseudomonas putida Is
Controlled by the PtxS Repressor

Abdelali Daddaoua,
1
Tino Krell,
1
Carlos Alfonso,
2
Bertrand Morel,
3
and Juan-Luis Ramos
1
*
Department of Environmental Protection, Consejo Superior de Investigaciones Cientcas, Calle Profesor Albareda 1, E-18008,
Granada, Spain
1
; Department of Molecular Microbiology and the Biology of Infections, Centro de Investigaciones Biologicas,
Calle Ramiro de Maeztu 9, 28040 Madrid, Spain
2
; and Department of Physical Chemistry and Institute of Biotechnology,
Faculty of Sciences, University of Granada, Campus de Fuentenueva, 18071 Granada, Spain
3
Received 7 May 2010/Accepted 17 June 2010
Metabolic ux 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 periplas-
mic 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 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 48.1C and 57.6C 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-ketogluconatePtxS inter-
actions. Puried PtxS is a dimer that binds to the target promoters with afnities 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 5-TGAAACCGGTTTCA-3. This palindrome overlaps with the
RNA polymerase binding site.
The deciphering of the complete genomes of a number of
strains of different species of the genus Pseudomonas has re-
vealed 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. Subse-
quently, 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-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 into
gluconate and then subsequently into 2-ketogluconate by glu-
conate dehydrogenase. Gluconate and 2-ketogluconate can be
transported to the cytoplasm through energy-dependent pro-
cesses mediated by the GnuK and KguP transporters, respec-
tively. Flux studies in Pseudomonas uorescens and Pseudomo-
nas 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
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 dehydroge-
nase (Zwf) and 6-phosphogluconolactonase (Pgl) converts
G6P into 6-phosphogluconate (6PG). Subsequently, 6PG, pro-
duced by the three peripheral glucose catabolic enzymes, en-
ters the Entner-Doudoroff route, where it is rst converted
into 2-keto-3-deoxy-6-phosphogluconate (KDPG) by the Edd
enzyme (6-phosphogluconate dehydratase) and then hydro-
lyzed 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
* Corresponding author. Mailing address: Estacion Experimental
del Zaidn-CSIC, C/ Profesor Albareda 1, 18008 Granada, Spain.
Phone: 34 958 181 608. Fax: 34 958 129 600. E-mail: juanluis.ramos
@eez.csic.es.

Published ahead of print on 25 June 2010.

4357

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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 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% 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 ex-
pression of these operons.
We have concentrated our current efforts on understanding
the control of the genes whose expression is modulated by
PtxS. We have puried PtxS to homogeneity and have carried
out 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 are
listed in Table 1. Bacterial strains were grown in LB medium or in modied M9
minimal medium with a 5 mM concentration of glucose, gluconate, 2-ketoglu-
conate or citrate as the sole C source (15). When required, antibiotics were
added to the culture medium to reach a nal 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. 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 amplied 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. 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 rst 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.
4358 DADDAOUA ET AL. J. BACTERIOL.

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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 was
grown in 2-liter Erlenmeyer asks containing 250 ml of LB supplemented with 25
g/ml kanamycin. Cultures were incubated at 30C with shaking until a turbidity
at 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 P
lac
promoter. The cultures were then incubated at 18C overnight, and cells were
harvested by centrifugation (30 min at 20,000 g) and stored at 80C until
used for protein purication.
For PtxS purication, 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/in
2
. The cell suspension was then centrifuged at 20,000 g for 1 h. The pellet
was discarded, and the supernatant was ltered and loaded onto a 5-ml His-Trap
chelating 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 in
buffer A. Protein concentration was determined by the Bradford assay, and
protein purity was veried 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 80C.
Analytical gel ltration chromatography. In order to determine the oligomeric
state of PtxS in solution, we used analytical gel ltration chromatography using
an kta FLPC system (Amersham Biosciences). Puried 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 ow 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 K
D
(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 20C in an XL-I analytical ultracentrifuge (Beckman-
Coulter 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 coefcient (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 stan-
dard conditions (water, 20C, and innite 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
Strains
P. putida
KT2440 Wild type, prototroph; Cm
r
Rif
r
1
PSC303
b
ptxS::pCHESI-Km Rif
r
Km
r
6
E. coli
DH5F F hsdR17 recA1 gyrA 22
BL21(DE3) F

ompT hsdS
B
(r
B

m
B

) gal dcm (DE3) 31

Plasmids
pAD1 pMP220 bearing the promoter region of the kgu operon; Tc
r
This work
pAD2 pMP220 bearing the promoter region of the ptxs gene; Tc
r
This work
pAD3 pMP220 bearing the promoter region of the gad gene; Tc
r
This work
pGEM-T Vector for cloning fragments; Ap
r
Dominion
pET24b() Protein expression vector; Km
r
Novagen
pMBL::PtxS ptxS gene in pMBL vector This work
pET24b::PtxS Derivative bearing the ptxS gene; Km
r
This work
pGEM-T:P
kgu
pGEM-T containing the kgu promoter; Ap
r
This work
pGEM-T:P
ptxS
pGEM-T containing the ptxs promoter; Ap
r
This work
pGEM-T:P
gad
pGEM-T containing the gad promoter; Ap
r
This work
a
Cm
r
, Km
r
, Rif
r
, and Ap
r
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 lacZ
Kgu 1.r 5-AGATCTGCCAACCTGATCATCCGC-3 Fusion to lacZ
Kgu 2.f 5-GCACAAAGTCGGCGCCGTAGC-3 EMSA, footprinting, and primer extension
Kgu 2.r 5-GCCTGCTCGGTCGCTTGCG-3 EMSA and footprinting
PtxS 1.f 5-TGGTGTGCTGCTTTGCTCCCG-3 EMSA, footprinting, and primer extension
PtxS 1.r 5-ATGGGCAGGCGCGTCGGT-3 EMSA and footprinting
PtxS 2.f 5-CTGCAGCGTTCGCGGGTATGG-3 Fusion to lacZ
PtxS 2.r 5-GGATCCGGGGTATCAACTGGTGGCC-3 Fusion to lacZ
PtxS 3.f 5-ATGACCGACGCGCCTGCCCA-3 PtxS purication
PtxS 3.R 5-CTGGGGTTGGGTTGAACCGC-3 PtxS purication
Gad 1.f 5-CTGCAGGGGATCAGGGTCAAGGT-3 Fusion to lacZ
Gad1.r 5-AGATCTTGCGGTCGGACTCTTTGG-3 Fusion to lacZ
Gad 2.f 5-CCTCATCGGCTGTGGGGCG-3 EMSA, footprinting, and primer extension
Gad 2.r 5-TGCGGTCGGACTCTTTGGGC-3 EMSA and footprinting
VOL. 192, 2010 SUGAR CATABOLISM CONTROL IN BACTERIA 4359

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program (17) to obtain the corresponding standard S values (S
20,w
). Sedimenta-
tion 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 high-
speed centrifugation run (43,000 rpm) was done to estimate the corresponding
baseline offsets. The weight-average buoyant molecular weight of PtxS was de-
termined by tting data to the single species model using either the MATLAB
program (kindly provided by Allen Minton, NIH), based on the conservation-
of-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 specic volume of PtxS (calculated from the amino acid composition
using the SEDNTERP program [17]).
Isothermal titration calorimetry. Microcalorimetric experiments were carried
out at 25C 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 tted 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 K
A
(binding constant; K
A
1/K
D
) were
determined from the curve t. The change in free energy (G) and in entropy
(S) were calculated from the values of K
A
and H using the following equation:
G RT ln K
A
H TS, 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
60C/h in a Valerian-Plotnikov differential scanning calorimeter (VP-DSC) cap-
illary 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/in
2
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 rst 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
specic heat (Cp) proles. In addition the thermograms were dynamically cor-
rected 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 lacZ. To obtain a transcriptional fusion of the
promoters of the kgu, ptxS, and gad genes to the lacZ reporter, the correspond-
ing regions were amplied 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 amplied 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 30C, and after another 6 h aliquots were taken, and -galactosidase
activity was determined in permeabilized whole cells (19) by using o-nitrophenyl-
-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. putida
KT2440 cells growing on medium supplemented with 5 mM 2-ketogluconate
using 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 Marque s et
al. (18) with the set of primers indicated in Table 2.
Electrophoresis mobility shift assays (EMSAs). The P
kgu
, P
ptxS
, and P
gad
promoter regions were amplied by PCR using pGEM-T:P
kgu
, pGEM-T:P
ptxS
,
and pGEM-T:P
gad,
respectively, as templates and the set of primer pairs indi-
cated in Table 2. Amplied fragments were isolated from agarose gels and end
labeled with [-
32
P]dATP using the T4 polynucleotide kinase. A 10-l sample
containing about 2 nM labeled DNA (1.5 10
4
cpm) was incubated with
increasing concentrations of puried 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 Tris-
borate-EDTA (TBE) buffer using Bio-Rad electrophoresis equipment, as previ-
ously described (23, 24).
DNase I footprinting. The DNA fragment containing P
gad
was amplied as
outlined above. DNA was labeled with [-
32
P]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 30C 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 95C 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 identication 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 puried by afnity chromatography from the soluble frac-
tion of the E. coli lysate. We obtained an average yield of 40 mg
of pure protein per liter of E. coli culture. Gel ltration exper-
iments 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 puried protein. Eluted protein was
submitted to SDS-PAGE, which conrmed that it was full-
length protein. When this elution volume was plotted against
the ln of the molecular mass of the proteins used in the cali-
bration 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 pro-
vides a mass of the monomer of 36.8 kDa). Homogenous PtxS
protein was subjected to analytical ultracentrifugation analy-
ses. Figure 4a shows sedimentation velocity data for a 70 M
PtxS solution in the absence and presence of 1 mM 2-ketoglu-
conate. The major species (95%) sedimented with a standard
S
20,w
value of 3.8 (0.1), and 2-ketogluconate had no effect on
the association state of PtxS. To conrm 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-ketoglu-
conate) tted 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 identied the two domains of this set of proteins, with
the helix-turn-helix (HTH) DNA binding domain from resi-
dues 12 to 67 at the N terminus of the protein and the effector
domain located at the C-terminal end. We generated a homol-
ogy model of the DNA binding domain of PtxS using the
structure of the transcriptional regulator CcpA of Bacillus sub-
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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). To
gain insight into the independent folding of the domains, pu-
ried PtxS protein was submitted to thermal unfolding as de-
scribed in Materials and Methods. Thermal unfolding of PtxS
was characterized by two events centered at 48.1 and 57.6C
(Fig. 5), with respective enthalpy changes of about 860 and 130
kJ/mol. According to Pfam protein families database, the ef-
fector binding domain consists of 262 amino acid residues
while the HTH DNA binding region is composed of 46 resi-
dues. 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 rst and the second event, respectively, were
found be similar, which is consistent with the notion that the
rst 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 accor-
dance with the proposed homology model of the PtxS protein,
it consists of two domains that unfold independently.
PtxS specically recognizes 2-ketogluconate. The three pro-
moter regions shown above to be regulated by PtxS, namely,
P
ptxS
, P
kgu
, and P
gad
, were fused to the lacZ gene, and gene
expression studies in the wild-type and in the ptxS-decient
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 P
kgu
, P
ptxS
, and P
gad
, 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 conrmed that PtxS represses gene expression in the
wild-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 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 signicant 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 calo-
rimetry 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, signif-
icant 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
specically 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 that
binding is driven by favorable enthalpy changes (H
14.0 0.1 kcal/mol) and counterbalanced by unfavorable
entropy changes (TS 7.4 0.2 kcal/mol). The corre-
sponding change in free energy,G, of 6.6 0.1 kcal/mol
corresponds to a K
D
of 14.5 0.4 M. The mathematical
algorithm also makes it possible to estimate the binding stoi-
chiometry, 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 (T
m
) (33). To evaluate the inuence of 2-ketoglu-
conate 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 T
m
shift of the rst
FIG. 3. Determination of the oligomeric state of PtxS. (A) Gel ltration elution prole of PtxS. (B) Calibration curve of the gel ltration
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.
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unfolding event was observed (Fig. 5), whereas no signicant
impact on the unfolding characteristics of the second event was
noticed.
This is consistent with the notion that 2-ketogluconate bind-
ing stabilizes the effector binding domain and has no signicant
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 rst 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 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 PtxS
resulted in retardation of the target DNA. In a series of com-
plementary EMSAs, the effect of 2-ketogluconate on binding
of PtxS to its target DNA was tested. First, we used a concen-
tration 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 2-
ketogluconate 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 frac-
tion of bound DNA was plotted against the logarithm of pro-
tein concentration and then tted using the sigmoid tting 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 promot-
ers P
kgu
, P
ptxS
, and P
gad
, respectively.
To identify the binding site of PtxS within the target se-
quences with respect to the RNA polymerase binding site, we
rst 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 transcrip-
tional units were transcribed from a main transcription start
point (Fig. 8). The leader sequence to the proposed rst ATG
was 3 nucleotides for kguE, 35 nucleotides for ptxS, and 84
FIG. 4. Analytical ultracentrifugation analysis. (a) Sedimentation
coefcient distributions, c(s), corresponding to the sedimentation
speed (48,000 rpm at 20C) 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 20C) of PtxS at 70
M (circles), 30 mM (squares), and 10 M (triangles). The solid lines
show the corresponding best-t gradients for a single sedimenting
species at sedimentation equilibrium. The residuals (difference be-
tween the experimental data and the tted data for each point) are
shown at the bottom of this panel (see Materials and Methods for
details). OD
290
, optical density at 290 nm.
FIG. 5. Differential scanning calorimetry of homogeneous PtxS.
Calorimetry proles 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.
4362 DADDAOUA ET AL. J. BACTERIOL.

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nucleotides for P
gad
. 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 P
ptxS
pro-
moter, the PtxS binding site overlaps the transcriptional start
point, whereas for promoters P
kgu
and P
gad
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 poly-
merase binding, which is exemplied in the case of P
gad
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 becomes
dissociated from its target promoter. To conrm this hypoth-
esis, we carried out EMSAs with a saturating concentration of
PtxS in the absence and in the presence of 3 mM 2-ketoglu-
coante. 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 exo-
toxin 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 eluci-
dated. In P. aeruginosa and P. putida, the PtxS protein is in-
volved in the control of the compartmentalized metabolism of
glucose 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 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 ux of carbon from
glucose 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 using
the -galactosidase reporter, we showed that PtxS binds to the
three promoters with an afnity 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
TABLE 3. Expression from P
kgu
, P
ptxS
, and P
gad
promoters in the wild-type and a ptxS mutant
Host and
promoter
Activity (Miller units)
a
Without substrate With gluconate With glucose With 2-ketogluconate
Wild-type
P
kgu
::lacZ 20 3 170 2 130 2 275 20
P
ptxS
::lacZ 950 10 4,495 103 5,560 129 11,545 50
P
gad
::lacZ 100 12 1,090 10 1,060 25 6,430 9
ptxS strain
P
kgu
::lacZ 180 6 175 3 220 10 360 30
P
ptxS
::lacZ 3,470 100 5,930 150 6,740 50 12,010 150
P
gad
::lacZ 1,380 2 1,270 20 1,500 10 7,005 20
a
The three promoter regions were cloned into pMP220 derivative (Tc
r
) 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.
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-corrected
and protein concentration-normalized peak areas of the raw data.
Data were tted with the one-binding-site model of the MicroCal
version of ORIGIN.
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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 P
gad
, between 12 and 26 in P
kgu
, and between 5
and 9 in P
ptxS
; therefore, these regions overlap with the RNA
polymerase binding sites, suggesting that regulation of tran-
scription from these promoters involves impairment of RNA
polymerase binding. Azotobacter vinelandii and P. uorescens
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 se-
quences 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. uorescens, and in the
nitrogen-xing 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.
PtxSa repressor of the LacI family. PtxS belong to the
LacI family of transcriptional regulators and exhibits strong
sequence similarity indicative of structural relationships. Ge-
netic 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 bind-
ing domain of transporters and in transcriptional regulators of
the LacI family. While lactose, fructose, and rafnose repres-
sors exist as tetramers, all other members of the LacI family
appear to be dimers, as happens with PtxS. Structural infor-
mation on the effector binding domain revealed that it binds
sugars 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 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 M. Images were analyzed densitometrically to determine the fraction of bound DNA which was plotted against the logarithm
of the concentration of PtxS and tted with ORIGIN to determine afnities. (D) EMSA of P
gad
in the presence of 6 M PtxS and a range of
2-ketogluconate concentrations.
4364 DADDAOUA ET AL. J. BACTERIOL.

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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 of
PtxS, 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 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 pro-
teins 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. aerugi-
nosa (32, 36) and P. putida (8) PtxS binds to its target opera-
tors, 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 bind-
ing, a conformational change occurs within PtxS that likely
decreases its afnity 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 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.
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FIG. 8. Analysis of the P
kgu
, P
ptxS
, and P
gad
promoters. (A) Deter-
mination 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 palin-
dromic PtxS binding site, and the 10 and 35 binding sites for the
RNA polymerase are marked.
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