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Applied and Environmental Microbiology, May 1999, p. 2065-2071, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cloning and Characterization of Polyphosphate
Kinase and Exopolyphosphatase Genes from Pseudomonas
aeruginosa 8830
Anna
Zago,
Sudha
Chugani,
and
A. M.
Chakrabarty*
Department of Microbiology and Immunology,
College of Medicine, University of Illinois at Chicago, Chicago,
Illinois 60612
Received 6 November 1998/Accepted 12 February 1999
 |
ABSTRACT |
Pseudomonas aeruginosa accumulates polyphosphates in
response to nutrient limitations. To elucidate the function of
polyphosphate in this microorganism, we have investigated polyphosphate
metabolism by isolating from P. aeruginosa 8830 the genes
encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which
are involved in polyphosphate synthesis and degradation, respectively.
The 690- and 506-amino-acid polypeptides encoded by the two genes have
been expressed in Escherichia coli and purified, and their activities have been tested in vitro. Gene replacement was used to
construct a PPK-negative strain of P. aeruginosa 8830. Low residual PPK activity in the ppk mutant suggests a possible
alternative pathway of polyphosphate synthesis in this microorganism.
Primer extension analysis indicated that ppk is transcribed
from a
E-dependent promoter, which could be responsive
to environmental stresses. However, no coregulation between
ppk and ppx promoters has been demonstrated in
response to osmotic shock or oxidative stress.
 |
INTRODUCTION |
Inorganic polyphosphates (polyP) are
linear polymers in which inorganic orthophosphate (Pi)
residues are linked by energy-rich phosphoanhydride bonds
(21). Their chain lengths may vary from two to several
hundred Pi residues. PolyP are widely distributed in nature
and have been detected in bacteria, fungi, protozoa, plants, and
mammals (22, 24); however, uncertainty still remains regarding their exact physiological role.
Depending on the species, the cellular localization, and the
physiological conditions, polyP may have a variety of functions (19), including serving as an ATP substitute and energy
source, a phosphate reservoir, and a chelator for divalent cations
(16) and in the inhibition of RNA degradation (6)
and regulatory responses to stresses and nutritional deficiencies
(30). Moreover, it can be a structural element in DNA entry
and transformation (32).
Several polyP-metabolizing enzymes have been purified, and their genes
have been cloned. Accumulation of this polymer is known to occur in
microorganisms catalyzed by the enzyme polyphosphate kinase (PPK); PPK
catalyzes the reversible transfer of the terminal phosphate from ATP to
polyP (2, 13, 15, 36). PolyP degradation and utilization is
mediated by exopolyphosphatases (PPX) (3, 38),
endopolyphosphatases (23), and specific kinases
(37).
Extensive accumulation of polyP has been detected in the
Pseudomonas aeruginosa mucoid strain 8830, particularly
during the stationary phase (18) and in response to
phosphate and amino acid limitations (5), yet essentially
nothing is known about the genes and their regulation in the synthesis
of polyP. We report here the cloning and sequencing of the P. aeruginosa ppk and ppx genes and the characterization
of their promoters and gene products.
 |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Strains and
plasmids used in this work are described in Table
1. Escherichia coli and
P. aeruginosa were routinely grown at 37°C on
Luria-Bertani (LB) solid or liquid medium (Difco). For antibiotic
selection of E. coli, ampicillin was used at 50 or 100 µg/ml, tetracycline was used at 12.5 µg/ml, kanamycin was used at
50 µg/ml, and chloramphenicol was used at 34 µg/ml. For selection
of P. aeruginosa, carbenicillin, chloramphenicol, and tetracycline were used at 300 µg/ml in solid media and at 100 µg/ml
in liquid media. Triparental mating was performed by using pRK2013 as
the helper plasmid (12), and exconjugants were isolated on
Pseudomonas isolation agar (Difco) with appropriate
antibiotic selection. The broad-host-range plasmid pQF50 and its
constructs were introduced into P. aeruginosa via
electroporation (28).
DNA manipulations.
Steps involved in the cloning of the
P. aeruginosa 8830 ppk and ppx genes
are described in the Results section. Genomic DNA was prepared with a
GNome Kit (Bio 101, Inc.). Restriction endonucleases (Gibco BRL), T4
DNA ligase (Gibco BRL) and calf intestinal phosphatase (Pharmacia) were
used as specified by the manufacturers. Plasmid DNA was isolated on a
small scale as described by Zhou et al. (41) and on a large
scale by using the Qiagen Plasmid Kit (Qiagen).
DNA probes for Southern blotting were internally labeled with
[

-
32P]dCTP by using the Megaprime DNA labeling system
(Amersham) as
described by the manufacturer. Southern blotting was
performed
by capillary transfer of DNA fragments to positively charged
nylon
membranes (Hybond-N
+). Membranes were incubated in
Rapid-hyb buffer (Amersham) at
65°C and washed under high-stringency
conditions. PCR were performed
with
Pfu polymerase
(Stratagene); oligonucleotides (Table
2)
were purchased from Gibco BRL. DNA sequencing was done by the
Genetic
Engineering Laboratory of the University of Illinois at
Urbana-Champaign. Sequence analysis was performed with a Sequencer
3.0. Homology searches were performed by using BLASTP and BLASTX
(
4) provided by the National Center for Biotechnology
Information.
Amino acid alignments were performed with CLUSTAL X, an
updated
version of the general-purpose multiple alignment program
CLUSTAL
W (
35).
To isolate the
ppk gene, a 1.7-kb probe was obtained by PCR
by using the degenerate oligonucleotides PPK5R and PPK2F (Table
2). PCR
was carried out under low-stringency annealing conditions
(40°C) with
P. aeruginosa genomic DNA as a template. The 1.7-kb
PCR
product was cloned into PCR2.1 (pCR-F2R5) and
sequenced.
The tetracycline (Tc) cassette used to knock out the
ppk
gene was constructed by PCR amplification of the tetracycline
resistance
gene of pBR322 by using oligonucleotides Tc1 and Tc2. The
oligonucleotides
were designed to create
BglII sites at the
ends of the tetracycline
cassette. This cassette was inserted into the
unique
BamHI restriction
site of a 1.6-kb PCR product
amplified with the primers PPK5R
and PPK5F and with pCR-F2R5 as the
template. PPK5F has been designed
internally to the 1.7-kb PCR probe to
eliminate one of the two
BamHI sites (Table
2) and to
generate the shorter 1.6-kb PCR
product. The fragment was first cloned
into the PCR2.1 vector
and then ligated into the
HindIII
and
KpnI sites of pNOT19. To
complement the
ppk::Tc mutant, the
ppk gene and its
promoter were
amplified by using the oligonucleotides K1 and K2 and
pPPKB as
a template. The PCR product was ligated into pCR2.1 (pCR-5).
This
construct was digested with
HindIII and
EcoRV, and the 2.5-kb
fragment containing the
ppk
gene was cloned into pBBR1MCS (pBBR-K).
Cloning of ppk and ppx genes for
overexpression and protein purification.
The ppk gene
was amplified by using oligonucleotides PPK-N and PPK-C corresponding
to the 5' and 3' ends of the gene. The plasmid DNA extracted from PPKB
was used as template DNA. EcoRI and HindIII
sites were designed in the two oligonucleotides to allow the PCR
product to be cloned into the expression vector pET24a(+) (Novagen). In
PPK-C the stop codon at the 3' end of the ppk coding
sequence has been omitted to allow an in-frame fusion with six
histidine residues encoded in the vector.
The
ppx gene was amplified by using oligonucleotides PPX-N
and PPX-C. Plasmid DNA extracted from PPX5 was used as a template.
NdeI and
HindIII enzymes whose sites were
designed into the two
oligonucleotides were used to digest and clone
this PCR product
into pET28a(+) (Novagen). This strategy created an
N-terminal
fusion of PPX with a histidine tag encoded in the
vector.
These constructs were amplified in DH5

and subsequently transformed
into
E. coli BL21(DE3)(pLys) (Novagen).
E. coli
transformants
freshly streaked on LB agar plates were inoculated into
500 ml
of LB broth (Difco) supplemented with appropriate antibiotics.
When this culture reached an optical density at 600 nm
(OD
600)
at 37°C of ca. 0.3, expression of the genes from
the T7 promoters
of the expression vectors was induced by the addition
of 1 mM
isopropyl-

-
D-thiogalactopyranoside (IPTG). Cells
were grown for
4 h before being harvested. Cells were resuspended
in 8 ml of
1× binding buffer of the His · Bind Buffer Kit
(Novagen) and sonicated
three times for 1 min with a Bronson Sonifier
450 at an output
of 3.5 (25 W) with a 50% duty cycle. Purification
under nondenaturing
conditions was performed by using Ni
2+
affinity chromatography with His · Bind resin (Novagen) as
described
by the manufacturer. Protein concentration was determined
with
Coomassie Plus protein assay reagent (Pierce). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins
was
conducted according to the methods described by Laemmli
(
26).
Protein bands were visualized by Coomassie blue
staining.
Assay for PPK activity.
PPK activity was measured in
membrane preparations as the production of acid-insoluble
32P-labeled polyP, as previously described (1).
Membranes were prepared by centrifugation of total cell extracts in a
Beckman ViTi65 rotor at 45,000 × g for 1 h after sonication.
The activity of purified PPK was tested in 20 µl of the same reaction
mixture. After incubation for 1 h at 37°C, the product
was
purified by using Glassmilk as described by Ault-Riché et
al.
(
5).
Assay for PPX activity.
The activity of purified PPX was
tested in 20 µl of 50 mM Tris-HCl buffer (pH 8)-1 mM
MgCl2-175 mM KCl-250 µM polyP. The synthesis and
purification of the [32P]polyP substrate was done as
described for the PPK assay. The PPX reaction was performed at 37°C.
Samples (2 µl) were taken periodically and loaded on a
polyethyleneimine plate. Polyphosphates remaining at the origin were
separated from orthophosphate by thin-layer chromatography (TLC) with
0.75 M KH2PO4 (pH 3.5). Dried plates were
visualized with a PhosphorImager (Molecular Dynamics).
Primer extension analysis.
Total RNA was isolated by using
TRIzol reagent (Gibco BRL) from P. aeruginosa 8830 culture
at an OD600 of 1.0. Samples of 30 µg of RNA were
hybridized overnight with 10 pmol of P1 primer labeled with the
MaxKinase kit (Ambion). After precipitation, RNA was resuspended in 10 µl of water, and the primer extension reaction was performed with the
First Strand cDNA synthesis kit (Pharmacia) at 37°C. Sequencing was
performed by use of the dideoxynucleotide chain termination reaction
method with T7 DNA polymerase (Sequenase 2.0 kit; USB Corp.).
-Galactosidase assay.
P. aeruginosa 8830 cells
harboring the promoter probe constructs were grown overnight in LB with
ampicillin (100 µg/ml). These overnight cultures were used to
inoculate fresh LB (1%). P. aeruginosa 8830 cells were
harvested in mid-log phase and stationary phase and then divided into
two parts. One part was treated with NaCl (added to a 2 M final
concentration) or H2O2, while the other was
used as a control. Samples (50 ml) were centrifuged, and the cell
pellets were resuspended in the assay buffer and sonicated. After
centrifugation to eliminate cell debris, the cell extract was assayed
for
-galactosidase activity. Quantitative determination of
-galactosidase activity was performed by the method of Miller (29). Each experiment was performed in triplicate.
Nucleotide sequence accession numbers.
The nucleotide
sequences of the P. aeruginosa 8830 ppk and
ppx genes have been deposited in the GenBank nucleotide
sequence database under accession numbers AF087931 and AF053463, respectively.
 |
RESULTS |
Cloning of P. aeruginosa 8830 ppk and
ppx genes.
On the basis of the homology alignment of
amino acid sequences of PPKs from Neisseria meningitidis,
Klebsiella aerogenes, and E. coli, we designed
degenerate primers that corresponded to highly conserved amino acid
sequences. These oligonucleotides were used to perform PCR as described
in Materials and Methods. The PCR product had the expected size of 1.7 kb. This product was sequenced, and the sequence homology to known
ppk genes suggested it could be the amplification of a
portion of the ppk gene.
A Southern blot of
P. aeruginosa 8830 DNA cleaved with
various restriction enzymes was probed with the 1.7-kb PCR product.
In
particular, this probe hybridized to a single 3.5-kb
SphI
fragment.
To isolate this fragment,
P. aeruginosa 8830
SphI DNA fragments
ranging in size from 2.5 to 5.5 kb were
purified by preparative
agarose gel electrophoresis. The purified DNA
was ligated into
the
SphI restriction site of pBR322 and
transformed into
E. coli DH5

. One hundred
tetracycline-sensitive (Tc
s) and ampicillin-resistant
(Amp
r) colonies were screened, and one positive clone
hybridizing with
the 1.7-kb probe was obtained. This clone had a 3.5-kb
insert
(pPPKB). The sequence of this fragment showed the presence of
an
open reading frame (ORF) encoding a 690-amino-acid protein.
The ORF
begins with a GTG start codon and has a putative ribosome
binding site,
CGGTGG, eight nucleotides upstream of the start
codon. The
translated amino acid sequence of this ORF shows 58%
identity with
Acinetobacter calcoaceticus PPK, 50% identity with
N. meningitidis PPK, 42% identity with
Synechocystis sp.
PPK,
36% identity with
Campylobacter coli PPK, 37%
identity with
Helicobacter pylori PPK, and 35% identity
with
E. coli PPK according to an
alignment performed with
BLASTP. A total of 155 nucleotides upstream
of the GTG start codon, the
3' end of the
hemB gene (X91820),
has been identified (Fig.
1A).

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FIG. 1.
(A) Organization of ppk and ppx
genes. (B) Alignment of P. aeruginosa 8830 PPX with E. coli PPX (accession number L06129) and E. coli
pppGpp-5'-phosphohydrolase (GppA) (accession number M83316) (CLUSTAL
X). Sequences were retrieved from GenBank. *, amino acid identity;
":" and ".", limited conserved substitutions.
|
|
Downstream of the
ppk gene, in the 3.5-kb
SphI
fragment, an incomplete ORF, which resembled the gene for
E. coli PPX, was
detected. To isolate the entire gene, a
SalI DNA fragment of ca.
3 kb was isolated that partially
overlapped the 3.5-kb
SphI DNA
fragment. A unique
SalI site was mapped in the
SphI DNA fragment,
and it was localized 927 bp upstream of the
ppk stop codon.
Southern
blot hybridization with the 1.7-kb PCR probe of
P. aeruginosa 8830 DNA digested with
SalI showed two bands
of ca. 3 and 2.5
kb. If the same probe was digested with
SalI and the purified
0.7-kb fragment at the 3' end was used
as a probe, a single band
of 3 kb was detected. To isolate this band,
SalI DNA fragments
of ca. 3 kb were cloned into pUC19 and
300 clones were screened
by using the 0.7-kb DNA probe. One positive
clone was detected
(PPX5). With an oligonucleotide designed near the 3'
end of the
ppk gene and by using sequencing by primer
walking on the DNA
insert, a complete ORF of 1,521 bp, coding for a
506-amino-acid
protein, was identified (Fig.
1A). The translated amino
acid sequence
of this ORF showed considerable identity with
E. coli PPX and
pppGpp-5'-phosphohydrolase (GppA) (
17)
(Fig.
1B). This ORF is
transcribed in the opposite orientation with
respect to
ppk, and
the two genes overlap with 14 nucleotides at their 3' ends. Located
183 bp upstream of the
ppx gene, in the opposite orientation,
was an ORF that
encodes a protein with 72% sequence identity to
E. coli thioredoxin.
Construction of ppk mutants with reduced PPK
activity.
To isolate a knockout mutation in the ppk
gene, the tetracycline cassette was inserted in the unique
BamHI site of the 1.6-kb PCR fragment as described in
Materials and Methods, and pMOB2 was inserted in the NotI
site (33). The resulting plasmid (pNotK::Tc) was
conjugated into P. aeruginosa 8830 by triparental mating, and several tetracycline-resistant but carbenicillin-sensitive colonies
were obtained. Southern blotting of chromosomal DNA from one of these
transformants digested with SphI and by using the 1.7-kb PCR
probe showed two bands of ca. 2.3 and 3 kb, instead of a single 3.5-kb
band. This finding was consistent with the insertion of the 1,720-bp Tc
cassette which had one SphI restriction site and no
KpnI site. Moreover, DNA digested with KpnI
showed an increase in fragment size from 8 to ca. 9.7 kb (Fig.
2).

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FIG. 2.
Southern blot analysis of ppk knockout mutant
genomic DNA, determined by using the 1.7-kb PCR probe. Lanes: 1, P. aeruginosa 8830 chromosomal DNA digested with
SphI; 2, ppk::Tc genomic DNA digested
with SphI; 3, P. aeruginosa 8830 chromosomal DNA
digested with KpnI; 4, ppk::Tc genomic
DNA digested with KpnI. The different band patterns for the
parental and mutant strains are consistent with the insertion of the Tc
cassette of 1,720 bp having one SphI restriction site and no
KpnI sites.
|
|
PPK activity was tested in the membrane preparations of mid-log phase
cultures of the
ppk::Tc mutant and of the parental
strain
P. aeruginosa 8830. A residual PPK activity was
detected in the
mutant (430 ± 1 U mg
1), but it was
23 times lower than in the parental strain (10,000
± 10 U
mg
1). When the mutant was complemented with the
ppk gene as part
of a high-copy-number vector (pBBR-K), the
activity (35,700 ±
3,400 U mg
1) was 3.5 times
higher than in the wild type. The activity measured
in the strain
containing the vector pBBR1MCS alone was 130 ± 5
U
mg
1.
Purification and activity of recombinant PPK.
The His-tagged
protein was purified by metal-chelate affinity chromatography on an
Ni-iminodiacetic acid column. Eluted protein (3.5 µg) was loaded onto
an SDS-PAGE gel and stained with Coomassie blue R250. Protein
purification was homogeneous since a single 75-kDa band was detected.
The purified protein was tested for its ability to synthesize polyP in
vitro. For this purpose PPK was incubated with
[
-32P]ATP, and the product of the in vitro reaction
was purified with Glassmilk and loaded onto a TLC plate. After TLC
analysis, the product of the in vitro reaction was retained at the
origin (Fig. 3, lane 2), but after
digestion for 1 h with an excess of recombinant yeast PPX (rPPX1),
as described by Wurst and Kornberg (38), it was completely
degraded to free Pi (Fig. 3, lane 3). Similar results were
observed when purified E. coli PPK (Fig. 3, lanes 4 and 5)
was used. When the same product was incubated with an ATPase
purified in our laboratory from Mycobacterium bovis
(40), no release of free Pi was detected (Fig.
3, lanes 7 and 8). However, the ATPase activity of this enzyme was
confirmed by digestion of ATP to free Pi in the PPK
reaction mixture (Fig. 3, lane 6).

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FIG. 3.
TLC analysis of PPK enzymatic activity product. Lanes:
1, P. aeruginosa PPK reaction mixture; 2, polyP purified
with Glassmilk from the reaction mixture shown in lane 1; 3, P. aeruginosa polyP digested with rPPX1; 4, purified polyP
synthesized by E. coli PPK; 5, E. coli polyP
digested with rPPX1; 6, reaction mixture in lane 1 digested with an
M. bovis ATPase; 7, E. coli polyP digested with
M. bovis ATPase; 8, P. aeruginosa polyP digested
with M. bovis ATPase.
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Purification of recombinant PPX and polyP degradation.
PPX
with an oligohistidine domain at the N terminus was purified to
homogeneity as visualized by Coomassie blue R250 staining of 6 µg of
elution fraction run on an SDS-PAGE gel. The molecular mass of the
denatured histidine-tagged protein was determined by SDS-PAGE to be 60 kDa, with a linear regression curve of the standard low-molecular-mass
protein (14,400 to 97,400 Da) (Bio-Rad). The theoretical molecular mass
calculated from the amino acid sequence encoded by the ppx
gene, including six histidine terminal residues, was 58.7 kDa. The
hydrolytic activity of this enzyme toward polyP was demonstrated in
vitro by using polyP as a substrate (Fig.
4). Its specific activity, measured with
E. coli polyP as a substrate, was 7 × 106
pmol of Pi produced min
1 mg of
protein
1.

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FIG. 4.
TLC analysis of P. aeruginosa PPX enzymatic
activity. [32P]polyP (267 µM) was incubated for various
time periods as specified with 200 ng of PPX protein in 20 µl of
total reaction mixture. Samples (1 µl) were taken periodically and
spotted onto a polyethyleneimine plate.
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|
ppk and ppx promoter investigation.
The region upstream of the ppk gene was investigated for its
promoter activity through construction of a promoter-reporter gene
fusion. For this purpose, a 376-bp XhoI fragment including 251 bp upstream of the GTG translational start site was cloned into the
SalI site of the promoterless lacZ gene of the
pQF50 plasmid in both orientations. These constructs were
electroporated into P. aeruginosa 8830, and the
-galactosidase activity was tested at various stages of growth. When
the fragment was cloned in the inverted orientation, the activity was
8.7 times lower (37.27 ± 1.4 Miller units) than in the direct
orientation (327.4 ± 12.87 Miller units) (pQF-376K). This
suggested that the promoter could be located upstream of the
ppk coding sequence. Primer extension analysis identified
three putative transcriptional start sites (Fig.
5A) differing by one or two bases. The
start site farthest upstream is a C residue that produces the most
intense transcript band. This start site is accompanied by
35 and
10 promoter sequences, which resemble the consensus promoter sequence
for
E-dependent promoters as exemplified by E. coli htrA and rpoH or P. aeruginosa algT,
algR1, or algD (10, 14) (Fig. 5B). A
649-bp SphI fragment upstream of the ppx gene was
cloned in the direct orientation into pQF50 upstream of the
promoterless lacZ gene (pQF-649X). When two strains of
P. aeruginosa 8830, which contained the promoter probe
construct (pQF-649X) and the control plasmid (pQF50), respectively,
were grown to log phase (OD600 = 1.50) and the
-galactosidase activity was measured, it was 7.7 times greater in
pQF-649X (93 ± 0.4 Miller units) than in the control (12 ± 0.2 Miller units). To investigate a putative coregulation at the
transcriptional level between ppx and ppk, the
-galactosidase activity was measured in P. aeruginosa
8830 strains containing pQF-376K or pQF-649X in response to stress
conditions such as a high concentration of NaCl or oxidative stress,
which are known to influence polyP synthesis in E. coli
(30).
-Galactosidase levels were measured after 30 min of
exposure to 2 M NaCl. Compared to the activity measured in each
control, a modest increase was seen for both ppk and
ppx promoters (Table 3). The
increase became more significant after 2 h, but the response of
the ppk promoter to such a stress was greater (3.2 times) in
the stationary phase than in the log phase (Table 3). In contrast,
ppx promoter activation was stronger in the log phase. The
response to oxidative stress was evaluated by using 53 mM
H2O2 (final concentration) and measuring the
-galactosidase activity after 10 min. Stress-induced ppx promoter activity decreased slightly during both the log and the stationary phases, while a small increase of ppk promoter
activity was measured during the stationary phase.

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FIG. 5.
(A) Primer extension analysis of the transcription start
sites of the ppk gene. P. aeruginosa 8830 total
RNA was annealed to oligonucleotide P1 and extended with murine reverse
transcriptase. Lanes G, A, T, and C represent the dideoxy sequencing
ladder carried out with the same primer. Lane 1, primer extension
products. (B) Comparison of 35 and 10 motifs identified upstream of
the transcription start site (the C residue that gave the most intense
band) with E consensus sequences.
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TABLE 3.
Transcriptional response of ppk and
ppx promoters to osmotic stress (2 M NaCl) after 30 min and
after 2 h
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|
 |
DISCUSSION |
Accumulation of polyP has been detected in P. aeruginosa in response to nutrient limitation (5, 18),
but the implications of this physiological response to such stress
conditions remain unclear. The pathway of synthesis and degradation of
polyP in P. aeruginosa has never been investigated. We
isolated the P. aeruginosa genes encoding the major enzymes
involved in polyP metabolism, PPK and PPX. In contrast to E. coli, where the ppk and ppx genes are
organized in an operon, in P. aeruginosa the two genes are
transcribed divergently (Fig. 1A). Investigation of the two promoters
did not show any coregulation between the expression of these two
genes, at least under the conditions we analyzed, such as low or high
Pi levels (data not shown), osmotic shock, or oxidative
stress. This observation is in agreement with the observation that
polyP accumulation is regulated at the enzymatic level through PPX
inhibition by the stress response nucleotides ppGpp and pppGpp
(25) without any modulation of the transcription rate of
these two genes.
After ppk gene inactivation, the ppk knockout
mutant does not show a growth defect compared to the parental strain.
However, a residual low PPK activity, measured as polyP synthesis, in
membrane preparations has been detected in the mutant. This may
represent a separate mechanism of polyP synthesis, as suggested also by the observations that a small amount of polyP remains in the cells of
E. coli (8) and N. meningitidis
(36) where the ppk gene has been inactivated.
Downstream of the ppk gene in P. aeruginosa is an
ORF encoding a putative thioredoxin. The ppx gene product
has been shown to be able to hydrolyze polyP to Pi with a
specific activity comparable to that of E. coli PPX
(3). Moreover, this enzyme exhibits significant sequence
similarity with E. coli PPX and E. coli GppA. Both of these enzymes belong to a large superfamily that includes sugar
kinases, actin, and hsp70 proteins (31). Since we have now
cloned both the genes with their respective promoters, we should be
able to define various environmental parameters that might modulate the
expression of either or both of the genes that allow the cells to
accumulate polyP.
An interesting observation from our studies is that the ppk
gene of P. aeruginosa appears to be expressed from a
E-responsive promoter (Fig. 5B). The
E-responsive promoters have been reported to regulate
alginate synthesis through activation of critical alginate biosynthetic genes such as algR1, algT, algD, and
algC (34). It is interesting to note that both
polyP accumulation and alginate synthesis are coregulated in P. aeruginosa such that alginate-positive mucoid cells accumulate
much more polyP than their nonmucoid counterparts (18).
Indeed, a mutation in the algR2 gene that results in reduced alginate production also results in reduced polyP accumulation (18). Whether a common regulator controls the expression of key
E-responsive promoters such as algT/algR1
and ppk is not known at present. Further investigations
looking for a common link in the genetic expression of two
stress-inducible polymers, such as polyP and alginate, presumably
through
E-responsive promoters, may provide important
insights into the mode of regulation of the biosynthesis of these two polymers.
 |
ACKNOWLEDGMENTS |
We are grateful to A. Kornberg for the kind gift of yeast rPPX
and E. coli PPK. We are also very grateful to H. P. Schweizer for providing pMOB2.
This research was supported by Public Health Service grant AI16790-18
from the National Institutes of Health.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology (M/C 790), College of Medicine, University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612. Phone: (312) 996-4586. Fax: (312) 996-6415. E-mail:
Ananda.Chakrabarty{at}uic.edu.
Present address: Howard Hughes Medical Institute, University of
Chicago, Chicago, IL 60637.
 |
REFERENCES |
| 1.
|
Ahn, K., and A. Kornberg.
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Applied and Environmental Microbiology, May 1999, p. 2065-2071, Vol. 65, No. 5
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