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Journal of Bacteriology, March 2000, p. 1333-1339, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Succinyl Coenzyme A Synthetase of Pseudomonas
aeruginosa with a Broad Specificity for Nucleoside Triphosphate
(NTP) Synthesis Modulates Specificity for NTP Synthesis by the
12-Kilodalton Form of Nucleoside Diphosphate Kinase
Vinayak
Kapatral,
Xiaowen
Bina,
and
A. M.
Chakrabarty*
Department of Microbiology and Immunology,
University of Illinois College of Medicine, Chicago, Illinois 60612
Received 14 September 1999/Accepted 7 December 1999
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ABSTRACT |
Pseudomonas aeruginosa secretes copious amounts of an
exopolysaccharide called alginate during infection in the lungs of
cystic fibrosis patients. A mutation in the algR2 gene of
mucoid P. aeruginosa is known to exhibit a nonmucoid
(nonalginate-producing) phenotype and showed reduced activities of
succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate
kinase (Ndk), implying coregulation of Ndk and Scs in alginate
synthesis. We have cloned and characterized the sucCD
operon encoding the 
and 
subunits of Scs from P. aeruginosa and have studied the role of Scs in generating GTP, an
important precursor in alginate synthesis. We demonstrate that, in the
presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and,
in the presence of ADP, Scs synthesizes ATP using GTP as a
phosphodonor. In the presence of inorganic orthophosphate,
succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes
essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP
synthesis can be an alternate source for the synthesis of alginate as
well as for the synthesis of other macromolecules requiring GTP such as
RNA and protein. Scs from P. aeruginosa is also shown to
exhibit a broad NDP kinase activity. In the presence of inorganic
orthophosphate (Pi), succinyl-CoA, and either GDP, ADP, UDP
or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown
to copurify with Ndk, presumably as a complex. In mucoid cells of
P. aeruginosa, Ndk is also known to exist in two forms, a
16-kDa cytoplasmic form predominant in the log phase and a 12-kDa
membrane-associated form predominant in the stationary phase. We have
observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells,
synthesizes all three of the nucleoside triphosphates from a mixture of
GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not
CTP. Such regulation may promote GTP synthesis in the stationary phase
when the bulk of alginate is synthesized by mucoid P. aeruginosa.
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INTRODUCTION |
Our laboratory is interested in
studying the regulation of biosynthesis of alginate in
Pseudomonas aeruginosa, an opportunistic pathogen. A number
of cystic fibrosis (CF) isolates have been studied for their enhanced
synthesis and secretion of alginate (16). Alginate is a
polymer of D-manuronic and its C-5 epimer L-guluronic acid. GDP-mannuronic acid, derived from
GDP-mannose, is the building block of alginate, which requires mannose
1-phosphate and GTP as precursors (17). Thus, alginate
synthesis demands a steady supply of GTP for every molecule of
mannuronic acid incorporated into a molecule of alginate. Recently,
Kamath et al. (9) have shown that the mucoid strains of
P. aeruginosa retain an active form of elastase in their
periplasm, and this periplasmic elastase acts on 16-kDa Ndk to generate
a truncated 12-kDa form that is membrane associated. It has been
demonstrated that the membrane-associated 12-kDa nucleoside diphosphate
(NDP) kinase (Ndk), in complex with pyruvate kinase or Pra (5,
24), specifically synthesizes GTP. An ndk knockout
mutation in P. aeruginosa is not lethal (27) because PK provides the nucleoside triphosphates (NTPs) required for
cell growth (24). However, the Ndk-deficient mutant still makes small amounts of alginate. Since an alginate-negative
algR2 mutant in P. aeruginosa is deficient in
both Ndk and Succinyl-coenzyme A (CoA) synthetase (Scs) levels
(23), an important question is whether Scs, like Ndk, plays
a role in providing the GTP precursors for alginate synthesis.
Scs has been widely studied in a variety of organisms (11,
20), although nothing is known of the Scs of P. aeruginosa. It is involved in the only step in the tricarboxylic
acid (TCA) cycle where a molecule of GTP or ATP is generated by
substrate level phosphorylation. In Escherichia coli, Scs
occurs predominantly in the A-form, where it exists as a heterotetramer
consisting of 2 and 2 subunits. During
aerobic growth, Scs catalyzes the hydrolysis of succinyl-CoA, using ADP
and inorganic orthophosphate (Pi), to form succinate and
ATP. However, Scs can freely interconvert ATP and succinate to form
succinyl-CoA, ADP, and inorganic Pi for anabolic reactions
as well, particularly during anaerobic growth (22). During
enzymatic catalysis, Scs undergoes an intermediate Scs- subunit
phosphorylation. In eukaryotes, the Scs occurs predominantly in the
G-form, where it is a heterodimer composed of the and subunits
(8, 25). It interconverts succinyl-CoA to succinate, resulting in the synthesis or hydrolysis of GTP. In this study, we show
that in P. aeruginosa Scs allows the synthesis of both ATP
and GTP from succinyl-CoA, Pi, and an equimolar mixture of ADP and GDP. P. aeruginosa Scs is capable of generating UTP
or CTP as well when exposed to succinyl-CoA, Pi, and UDP or
CDP, thus exhibiting Ndk activity, although the enzyme preferentially synthesizes ATP and/or GTP.
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MATERIALS AND METHODS |
Strains and plasmids.
The strains and plasmids used in this
study are listed in Table 1. Plasmids
were maintained in E. coli DH5 which were grown at 37°C
in Luria-Bertani broth containing ampicillin (100 µg/ml), kanamycin
(40 µg/ml), or chloramphenicol (20 µg/ml). Oligonucleotides used in
this study were synthesized by Gibco-BRL laboratories.
Cloning of the sucCD operon from P. aeruginosa PAO1.
A 20-kb EcoRI cosmid clone
(pSU6) from P. aeruginosa PAO1 genomic library that
hybridized to a 512-bp sucCD-specific probe (14)
was isolated. A 3.5-kb EcoRI-SalI fragment was
subcloned from pSU6 into pUC118 to generate pXL804. DNA sequencing was
performed on both the strands and was analyzed by using the TRANSLATE
and BLAST programs.
Overexpression and purification of the SCS and subunits.
To construct a plasmid for the overexpression of SucD
( subunit) from P. aeruginosa, PCR was performed using
pXL804 as a template and a combination of oligonucleotides 1 and 2 (Table 1). The PCR was carried out in a 50-µl reaction volume
containing 1× Pfu buffer, 2.5 mM concentrations of dNTPs, 2 ng of the template, and 2.5 U of Pfu polymerase
(Stratagene). The reaction was set to 30 cycles at 95°C for 2 min,
53°C for 2 min, and 72°C for 2 min per cycle. A PCR product of
about 864 bp was obtained, which was purified by using QiaQuick PCR
purification (Qiagen Corp.). The purified DNA was digested with
EcoRI and HindIII and was cloned into the
compatible sites of pET28a to generate pKV153. Similarly, with pXL804
as a template and a combination of oligonucleotides 3 and 4 (Table 1),
PCR was performed as described above to amplify a 1,164-bp DNA which
encodes sucC ( subunit). The PCR-amplified DNA was
purified, digested with EcoRI and HindIII,
and cloned into pET28a to generate pKV152.
Both the constructs pKV153 (encoding the Scs
subunit) and pKV152
(encoding the Scs
subunit) were introduced into
E. coli BL21(pLysS) for protein expression. Briefly,
E. coli
BL21(pLysS)
cells containing pKV152 or pKV153 were grown in 100 ml of
tryptone-yeast
extract (with 0.1% glucose) medium in a 500-ml flask to
an optical
density of 0.5 to 0.6 and were then induced with 1 mM IPTG
(isopropyl-
-
D-thiogalactopyranoside).
The cells were
further grown for 2 h and then harvested by centrifugation
at
12,000 rpm for 10 min. The cell pellet was resuspended in a
lysis
buffer, treated with lysozyme (1 mg/ml) on ice for 30 min,
and then
lysed by using a sonicator (Branso Sonifer model 450).
The lysed cells
were pelleted at 15,000 rpm for 10 min at 4°C,
and the supernatant
was filtered by using a 0.22-µm (pore-size)
filter. The proteins were
purified by using a Ni-nitrilotriacetic
acid (Ni-NTA) purification
system according to the manufacturer's
instructions (Ni-NTA Spin-Kit;
Qiagen Corp.). The purified proteins
were dialyzed for 2 h with
two changes of buffer containing 50
mM Tris-HCl (pH 8.0) and 10 mM
MgCl
2 at 4°C.
Autophosphorylation and NDP kinase activities of the succinyl-CoA
synthetase.
The purified Scs and subunits were tested for
their autophosphorylation activity. Briefly, 3 µg of the purified
proteins (Scs and subunits), along with various amounts of ADP
or GDP (0, 5, 50 µM) and 1 µl (0.15 pmol) of
[
-32P]ATP (3,000 Ci/mM; Dupont NEN), was used for the
autophosphorylation reaction. The reaction was carried out in a final
20-µl volume in TMD buffer (50 mM Tris-HCl, pH 7.5; 10 mM
MgCl2; 25 mM KCl; 0.8 mM dithiothreitol) and was incubated
at room temperature for 10 min. The reaction was terminated by adding 4 µl of sodium dodecyl sulfate (SDS) loading buffer and was boiled for
10 min before electrophoresis. The samples were electrophoresed by
SDS-12% polyacrylamide gel electrophoresis (PAGE). The gel was
exposed to a PhosphorImager cassette and was analyzed by
using a STORM 860 PhosphorImager (Molecular Dynamics). Succinyl-CoA
synthetase activity was determined by the procedure described by
Kavanaugh-Black et al. (10).
Using the purified Scs
and
subunits (3 µg) and 0.15 pmol of
[
-
32P]ATP or 0.15 pmol of [
-
32P]GTP
(3,000 Ci/mM; Dupont NEN) as a phosphodonor, the effect
of various
concentrations of NDP mixtures such as NDPI (GDP-CDP-UDP)
or NDPII
(ADP-CDP-UDP) were determined, respectively. The reaction
was carried
out in 20 µl of TMD buffer with NDP concentrations
ranging from 0 to
1 mM. The nature of nucleotide synthesis by
the Scs complex was
determined by spotting 2 µl of the reaction
mixture onto a PEI-TLC
plate (Selecto Scientific, Norcross, Ga.)
as described by Sundin et al.
(
24). To the remaining reaction
mixture (18 µl), 4 µl of
loading buffer was added to terminate
the reaction. The samples were
boiled for 10 min and were run
on an SDS-12% PAGE gel. The gel was
exposed to a phosphorimager
cassette, and the amount of
32P
bound to the Scs
subunit was determined by using the PhosphorImager
(STORM 860; Molecular Dynamics). About 3 µg of the purified Scs
and
subunits was mixed with 100 µM succinyl-CoA (Sigma Chemicals,
St. Louis, Mo.) and inorganic [
32P]P
i (10 µCi) in the form of orthophosphoric acid (American Radiolabeled
Chemicals, Inc., St. Louis, Mo.), along with various concentrations
of
the NDPs in a 20-µl reaction volume. The reaction mixture was
incubated at room temperature for 10 min. About 1.5 µl of the
reaction was spotted onto a TLC-PEI plate and analyzed for the
triphosphate
synthesis.
The kinase activity of the Scs complex in the presence of Ndk was also
evaluated. Equal amounts (3 µg) of the purified Scs
and
subunits were incubated with equal amounts of either 12-
or 16-kDa Ndk
in 20 µl of TMD buffer. The nucleotide synthesis
assays were
performed as described above using 50 µM NDPI or 50
µM NDPII and
[
-
32P]ATP or [
-
32P]GTP as
phosphodonors,
respectively.
GenBank submission.
The DNA sequence encoding the
succinyl-CoA synthetase is given under GenBank accession number
AF128399.
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RESULTS |
Characterization of the sucCD operon from P. aeruginosa.
Using a 512-bp specific probe for sucCD, a
20-kb cosmid clone was identified by screening the P. aeruginosa PAO cosmid library. Further, a 3.5-kb DNA fragment
containing the sucCD hybridizing fragment was subcloned and
sequenced. The organization of the sucCD operon from
P. aeruginosa is shown in Fig.
1. Translated DNA sequence analysis of
pXL804 (from the EcoRI end) revealed a sequence similarity
to the C-terminal region (52 amino acids) of dihyrolipoamide
dehydrogenase (lpd gene product) from P. fluorescens (2). The sucCD operon (2,025 bp)
is located 324 bp downstream from the lpd gene. The first
gene transcribed in this operon is sucC, which is 1,161 bp
and encodes the Scs subunit (387 amino acids). The Scs subunit
shows 72% amino acid sequence identity to the E. coli Scs
subunit. The ATG of the sucD gene (864 bp) overlaps with
the stop codon of the sucC gene. The sucD gene
encodes the Scs subunit (288 amino acids) and showed 89% amino
acid sequence identity to the E. coli Scs subunit. About
313 bp downstream of the sucCD operon is an open reading
frame that encodes the branched-chain amino acid carrier protein
(product of braB gene) in P. aeruginosa PAO1
(7).
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FIG. 1.
Genetic organization of the succinyl-CoA synthetase
operon in P. aeruginosa PAO1. sucC encodes the
subunit, while sucD encodes the subunit of the Scs
enzyme. The EcoRI-SalI fragment also contains the
3' region (129 bp) of the lpd gene.
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Succinyl-CoA synthetase activity assays.
The Scs subunit
was purified by use of the Ni-NTA spin column and was analyzed by
SDS-12% PAGE. A 30-kDa protein corresponding to the predicted
molecular size was obtained (Fig. 2A,
lanes 2 and 3). Similarly, the Scs subunit was purified by the
Ni-NTA spin column and was analyzed by SDS-12% PAGE. A 40-kDa protein corresponding to the predicted size was obtained (Fig. 2B, lanes 2 and
3). The purified proteins were tested for activity by performing the
phosphorylation of the Scs subunit by using
[-32P]ATP. Autophosphorylation was carried out in the
presence of various amounts of ADP or GDP and by using
[-32P]ATP as the phosphodonor. A 5 µM concentration
of GDP in the reaction mixture stimulated the phosphorylation of the
30-kDa Scs subunit, whereas 50 µM GDP in the reaction mixture
showed a significant decrease. Similarly, 5 µM ADP in the reaction
mixture weakly enhanced the phosphorylation, while 50 µM ADP did not
have any effect (data not shown).
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FIG. 2.
Induction and purification of the P. aeruginosa His-tagged Scs and subunits overexpressed in
E. coli. (A) Lane 1, E. coli BL21(pKV153)
(induced); lanes 2 and 3, partially purified Scs subunit obtained
from the Ni-NTA column after the first and second elutions. (B) Lane 1, E. coli BL21(pKV152) (induced); lanes 2 and 3, partially
purified Scs subunit.
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In order to test the activity of Scs
subunit, an Scs enzymatic
reaction was performed with succinate, CoA, and a mixture
of purified
Scs
and
subunits (3 µg each) in the presence of
[
-
32P]ATP. In this reaction the Scs enzyme converted
succinate, CoA,
and [
-
32P]ATP to succinyl-CoA, ADP,
and P
i. The amount of the phosphorylated
Scs
subunit
(in presence of [
-
32P]ATP) and in the presence of
equimolar amounts of succinate and
CoA was determined by counting the
32P-labeled Scs
subunit. A loss of phosphorylation of
the
subunit
is an indication of the binding of the nucleotide to
the catalytic
site of the
-subunit of Scs. A rapid decrease in the
phosphorylation
of the Scs
subunit in the presence of 20 µM
succinate and CoA
was observed, but succinate alone in the reaction
showed no decrease
in autophosphorylation (Fig.
3). Similarly, CoA alone in the reaction
had no effect on the Scs
subunit dephosphorylation (data not
shown). Taken together, the data indicated that both succinate
and CoA
must be present to allow the reaction to proceed to completion.
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FIG. 3.
Succinyl-CoA synthetase activity from P. aeruginosa, measured as the rate of dephosphorylation of the Scs
subunit. The purified Scs subunits were incubated with various
amounts of succinate and CoA ( ) or with succinate only ( ) using
[-32P]ATP as a phosphodonor.
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NDP kinase activities of the Scs with [-32P]ATP or
[-32P]GTP as phosphodonor.
In order to determine
if the purified Scs subunits exhibited kinase activity, we performed an
NDP kinase reaction by using 3 µg of the purified protein with
[-32P]ATP and various concentrations (0 to 1 mM) of
NDPI (GDP-CDP-UDP) in a 20-µl volume. Two microliters of the reaction
mixture was spotted onto a thin-layer chromatography plate, which was
analyzed for NTP synthesis. The remaining reaction was terminated with 4 µl of stop buffer and was analyzed on an SDS-12% PAGE for the phosphorylated Scs subunit. As shown in Fig. 4A and
B, the Scs complex transferred the
terminal phosphate from [-32P]ATP to GDP to form GTP;
higher concentrations (>100 µM) of the NDPI had an inhibitory effect
on GTP synthesis, and a significant amount of
[-32P]ATP remained unreacted (Fig. 4A). Very little
UTP or CTP formation was observed. The corresponding phosphorylated Scs
subunit separated on an SDS-12% PAGE showed a progressive
decrease in the level of phosphorylation with increasing NDP
concentration, suggesting substrate-induced binding at the catalytic
sites, leading to dephosphorylation and/or inhibition of
phosphorylation (Fig. 4C). When [-32P]GTP was used as
a phosphodonor and NDPII (ADP-CDP-UDP), ranging from 0 to 1,000 µM,
were used as substrates in the reaction mixture, predominantly ATP
synthesis was observed, although small amounts of UTP were also
detected (Fig. 4B). The generation of ATP and UTP was optimum at an NDP
concentration of 50 µM, beyond which there was progressive
inhibition. A corresponding decrease in the amount of phosphorylated
Scs subunit was observed under increasing NDP concentrations (Fig.
4D). It appears that the P. aeruginosa Scs enzyme can
generate either GTP or ATP and only traces of UTP, depending upon the
relative NDP concentration. At a high NDP concentration (>100 µM),
the kinase activity is significantly reduced with either ADP or GDP as
a substrate.
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FIG. 4.
(A) NDP kinase assays of the Scs and subunits
from P. aeruginosa in the presence of various concentrations
of NDPI (GDP, CDP, and UDP). Lane C, [-32P]ATP
control. (B) ATP (and small amounts of UTP) synthesis by the Scs and subunits when [-32P]GTP is used as a
phosphodonor in the presence of NDPII (ADP, CDP, and UDP). Lane C,
[-32P]GTP control. (C and D) Autophosphorylation of
the Scs subunit with [-32P]ATP in the presence of
NDPI (GDP, CDP, and UDP) shown in panel C or with
[-32P]GTP as a phosphodonor in the presence of NDPII
(ADP, CDP, and UDP) shown in panel D.
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NDP kinase activity of the Scs with inorganic Pi and
succinyl-CoA.
Using the purified Scs enzyme and inorganic
[32P]Pi in the form of orthophosphoric acid
and succinyl-CoA, we tested the succinyl-CoA synthetase enzyme activity
in the presence of GDP, ADP, UDP, or CDP either individually or in
combination. The reaction mixture was incubated at room temperature for
10 min, and 1.5 µl of the reaction mixture was analyzed for NTP
synthesis on a TLC-PEI plate. As shown in the Fig.
5A to D, either ADP or GDP could act as a substrate at the lowest concentration tested (50 µM), while UDP and
CDP were active only at concentrations above 100 µM. When all of the
NDPs (ADP, GDP, UDP, and CDP) were used in the reaction, GTP and ATP
were the prominent products with traces of UTP as well (Fig. 5E). Based
on these results, we conclude that the Scs enzyme exhibits NDP kinase
activity with GDP = ADP > UDP > CDP with respect to
its relative affinities for the NDPs. To further delineate the relative
affinity of ADP and GDP as substrates, a substrate competition
experiment was conducted at various concentrations in the presence of
inorganic [32P]Pi, succinyl-CoA, and GDP or
ADP. In one experiment, 50 µM ADP was added in the reaction mixture,
along with various amounts of GDP. As shown in Fig.
6A, GTP synthesis was observed in the presence of ADP even when the concentration of GDP was 5 µM (Fig. 6A,
lane 2). At equal concentrations, both GTP and ATP were synthesized in
essentially equal amounts. Conversely, when GDP concentration was fixed
at 50 µM and various concentrations of ADP were used, ATP synthesis
was observed with as little as 5 µM ADP (Fig. 6B, lane 2), while at
50:50 concentrations, both ATP and GTP were synthesized in essentially
identical amounts.
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FIG. 5.
NDP kinase activity of the purified Scs enzyme with
inorganic Pi, succinyl-CoA, and various concentrations of
individual NDPs. (A to D) A, ADP; B, GDP; C, UDP; D, CDP. Lane 1, 0;
lane 2, 50 µM; lane 3, 100 µM; lane 4, 500 µM; lane 5, 1 mM; lane
6, 2.5 mM. (E) NDP kinase activity of the purified Scs enzyme of
P. aeruginosa with inorganic Pi, Succinyl-CoA
and various concentrations of NDPs (ADP, GDP, CDP, and UDP). Lane 1, 0;
lane 2, 50 µM; lane 3, 100 µM; lane 4, 500 µM; lane 5, 1 mM; lane
6, 2.5 mM.
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FIG. 6.
Succinyl-CoA synthetase from P. aeruginosa
utilizes GDP or ADP and synthesizes GTP or ATP, respectively, with
radiolabeled Pi as a phosphodonor. (A) GTP synthesis by the
Scs enzyme in the presence of 50 µM ADP in each lane with various
micromolar amounts of GDP in the reaction mixture as indicated. (B) ATP
synthesis by the Scs enzyme in the presence of 50 µM GDP in each lane
with various micromolar amounts of ADP in the reaction mixture as
indicated.
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Modulation of 12-kDa Ndk activity by the and subunits of
Scs.
The Scs protein was shown to copurify with Ndk in P. aeruginosa (10). Ndk exists in two forms in mucoid
P. aeruginosa, a cytoplasmic 16-kDa form capable of
generating all of the NTPs and a 12-kDa membrane-associated form which
forms complexes with pyruvate kinase, Pra, or EF-Tu, and these
complexes predominantly use GDP as a substrate, generating GTP (5,
12, 18). Since Scs copurifies with Ndk, it was of interest to see
if Scs modulates Ndk activity in any way. We therefore evaluated NTP
synthesis by the 16- and 12-kDa forms of Ndk in the presence of the and subunits of Scs. As shown in Fig.
7, the Scs subunit alone (lane 3) or
a combination and subunits (lane 7) generated GTP in presence
of GDP, CDP, and UDP. Either the 16-kDa Ndk or the 12-kDa Ndk generated
all the three NTPS (GTP, CTP, and UTP) in the presence of the NDPs
(lanes 2 and 9). The presence of Scs , , or both in association
with the 16-kDa Ndk did not lead to any alteration of NTP synthesis
(Fig. 7, lanes 4, 6, and 8). When the and subunits of Scs were
separately present with 12-kDa Ndk, no appreciable change in the nature
of NTP was observed (Fig. 7, lanes 9, 10, and 11). However, when both
the and subunits of Scs were added with 12-kDa Ndk, very little
CTP (<10%) was generated, suggesting that a complex of 12-kDa Ndk and
Scs may modulate Ndk activity to primarily generate NTPs other than CTP
at the NDP concentrations used.
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FIG. 7.
Modulation of NDP kinase (Ndk) activity by the and
subunits of Scs. Two forms of Ndk, a 16-kDa form and a truncated
12-kDa form obtained by treatment with purified elastase (9)
were used. Lane 1, [-32P]ATP control; lane 2, 16-kDa
Ndk; lane 3, Scs ; lane 4, Scs plus 16-kDa Ndk; lane 5, Scs ;
lane 6, Scs plus 16-kDa Ndk; lane 7, Scs plus Scs ; lane 8, Scs plus Scs plus 16-kDa Ndk; lane 9, 12-kDa Ndk; lane 10, Scs
plus 12-kDa Ndk; lane 11, Scs plus 12-kDa Ndk; lane 12, Scs plus Scs plus 12-kDa Ndk. A 50 µM concentration of NDPI
(GDP-CDP-UDP) was used in all of the reactions in addition to
[-32P]ATP as described previously (24).
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DISCUSSION |
GTP plays a central role in cellular metabolism (21).
Apart from its role in the synthesis of RNA, GTP is involved in a variety of cellular processes, including cellular signal transduction and protein synthesis, that require GTP-binding (G) proteins (4, 6, 18). GTP is also important in polysaccharide synthesis since
GDP-mannose, which is derived from mannose 1-phosphate and GTP
(17), is often an intermediate in the synthesis of mannose (or its uronic acid mannuronate)-containing polysaccharides. A pertinent example is the synthesis of the polysaccharide alginate by
P. aeruginosa cells isolated from the lungs of CF patients (16). Such strains produce copious amounts of alginate. A
molecule of alginate contains hundreds of mannuronate residues, the
incorporation of each of which requires consumption of a molecule of
GTP. Thus, synthesis of each molecule of alginate by mucoid P. aeruginosa requires the consumption of hundreds of molecules of
GTP (4, 9). The transition of nonmucoid P. aeruginosa to mucoidy (alginate production) in the CF lung
therefore requires that P. aeruginosa be able to
specifically generate large amounts of GTP to allow production of large
amounts of alginate. We previously reported that Ndk, which is normally
responsible for all NTP formation in the cell, is important for
alginate synthesis (24), since it forms complexes with a
number of cellular proteins in P. aeruginosa and since such
complexes generate predominantly GTP (4, 5). It should be
noted, however, that GTP-generating complexes of Ndk with proteins such
as pyruvate kinase or Pra (4, 5, 24) involves the truncated
12-kDa form of Ndk, which is generated uniquely in the mucoid cells by
the periplasmic retention of elastase (9, 12). An
ndk knockout mutant (27), while deficient in alginate synthesis, nevertheless, makes small amounts of alginate. Thus, we were interested in knowing what other enzymes may provide an
alternative source of GTP. Besides Ndk, other kinases, such as pyruvate
kinase (24), adenylate kinase (15), or
polyphosphate kinase (13), can substitute for Ndk,
generating NTPs, including GTP. Since Scs was previously shown to
copurify with Ndk, presumably as a tight complex (10) and
since Scs is known to generate either ATP or GTP (8, 19,
20), it was of interest to us to find out the nature of the Scs
enzyme in P. aeruginosa and its putative role in GTP
synthesis, with or without complexation with Ndk. Since very little is
known about the Scs enzyme or its genetic organization in pseudomonads,
our studies additionally provide important information about this TCA
cycle enzyme.
The substrate level phosphorylation catalyzed by Scs is known to
involve adenine nucleotides in plants as well as in prokaryotes such as
E. coli generating ATP during the operation of the TCA cycle
(19, 20). In contrast, Scs from eukaryotes such as pig heart
(8) or Dictyostelium discoidium (1, 25,
26) is known to generate GTP. However, both ATP- and GTP-specific
Scs isoforms have been characterized from multicellular eukaryotes (8) and, in general, the nucleotide preference varies widely within organisms (11, 20). It is interesting to note that the purified Scs and subunits of P. aeruginosa
generate predominantly GTP from [-32P]ATP and a
mixture of GDP, CDP, and UDP (Fig. 4A). It is, however, capable of
transferring the terminal phosphate from GTP to ADP (Fig. 4B). Thus, in
the presence of succinyl-CoA, Pi, and an equimolar mixture
of ADP and GDP, P. aeruginosa Scs can efficiently synthesize almost an equimolar mixture of ATP and GTP. The Scs subunit can be
efficiently autophosphorylated either in presence of ATP or in presence
of GTP, and small quantities of GDP or ADP can enhance such
phosphorylation, while larger concentrations inhibit it, suggesting
that intracellular concentrations of ADP and GDP modulate the
interconversion of ATP and GTP by Scs. The stimulatory effect of low
concentrations of GDP and ADP in the autophosphorylation of the subunit of Scs may be due to binding to a high-affinity allosteric site
(3, 26) of Scs. The stimulation of Scs phosphorylation by
GDP is well documented in the case of eukaryotes (8, 25), but it also occurs in P. aeruginosa.
In this study we also demonstrate that P. aeruginosa Scs
enzyme exhibits NDP kinase activity and can synthesize GTP, ATP, UTP,
and CTP by using inorganic Pi and the corresponding
diphosphates. Both GDP or ADP were equally preferred at lower
concentrations; however, at higher concentrations CDP and UDP could
also be used. Since the intracellular level of NDPs may reach
millimolar concentrations, it is conceivable that P. aeruginosa Scs may generate all of the NTPs under certain
conditions. We also considered the possibility that the broad NDP
substrate range of P. aeruginosa Scs is due to trace
contamination with Ndk, since Ndk is known to copurify with Scs
(10). However, Ndk uses only NTPs such as ATP or GTP as a
phophodonor, not Pi. When we tested the ability of purified P. aeruginosa Ndk to generate any NTP from a mixture of
Pi, succinyl-CoA, and ADP-GDP-CDP-UDP, no NTP formation was
detected (data not shown). Thus, the formation of UTP or CTP by Scs is
not due to contamination with Ndk.
The loss of CTP synthesis by Scs, when complexed with the 12-kDa form
of Ndk, is interesting. Unlike pyruvate kinase or Pra, which generates
predominantly GTP in complexation with the 12-kDa form of Ndk (5,
24), Scs-12-kDa-Ndk complexes generated both UTP and GTP. It is
unclear if such a complex plays any role in generating the GTP involved
in alginate synthesis or if Scs is involved in residual alginate
synthesis by the ndk knockout mutant cells. The availability
of the scs genes will now allow us to make double knockout
mutations in both ndk and scs genes to examine the extent of alginate synthesis by such a double mutant if it is viable.
Finally, nothing is known about the regulation of Scs in P. aeruginosa. In E. coli, the sucCD operon
encoding the and subunits of Scs is part of sucABCD
operon, where sucAB encodes -ketoglutarate dehydrogenase,
forming a single transcript. The oxygen and carbon control of
sucABCD gene expression has been shown to occur by
transcriptional regulation of the upstream sdhC promoter
(22). A weak sucABCD promoter upstream also
allows a low constitutive level of this operon expression, whereas the negative regulation seen under anaerobic conditions is mediated by
arcA and fnr gene products (22). Since
the organization of the sucCD operon in P. aeruginosa differs from that of E. coli and the
P. aeruginosa Scs has a different specificity for NTP production, it is likely that the regulation of Scs in P. aeruginosa would be markedly different from that of E. coli. Such studies are currently under way in our laboratory.
| |
ACKNOWLEDGMENTS |
We thank Bob Hancock for providing the sucCD probe and
the P. aeruginosa PAO1 library. We thank T. K. Misra
for technical help on protein purification and Page Goodlove for DNA
sequencing at the DNA Sequencing Facility of the University of Illinois
at Urbana.
This work was funded by Public Health Service grant AI-16790-18 from
The National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology (M/C790), University of Illinois College of
Medicine, 835 S. Wolcott Ave., Chicago, IL 60612. Phone: (312) 996-4586. Fax: (312) 996-6415. E-mail:
Ananda.Chakrabarty{at}uic.edu.
Present address: Department of Microbiology and Immunology, Tufts
University, Boston, MA 02111.
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Journal of Bacteriology, March 2000, p. 1333-1339, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
