Molecular Microbiology (1998) 27(4), 717–725
Alginate, inorganic polyphosphate, GTP and ppGpp
synthesis co-regulated in Pseudomonas aeruginosa:
implications for stationary phase survival and
synthesis of RNA/DNA precursors
Hong-Yeoul Kim,1† D. Schlictman,2 Sandeep
Shankar,2 Zhidong Xie,2 A. M. Chakrabarty2 and A.
Kornberg1*
1
Department of Biochemistry, Beckman Center, Stanford
University School of Medicine, Stanford, CA 94305-5307,
USA.
2
College of Medicine, Department of Microbiology and
Immunology, University of Illinois, Chicago, IL 60612-
7344, USA.
Summary
The regulatory protein AlgR2 in Pseudomonas aeru-
ginosa positively regulates nucleoside diphosphate
kinase (Ndk) and succinyl-CoA synthetase, enzymes
critical in nucleoside triphosphate (NTP) formation.
AlgR2 positively regulates the production of alginate,
GTP, ppGpp and inorganic polyphosphate (poly P).
An algR2 mutant with low levels of these metabolites
has them restored by introducing and overexpressing
either the algR2 or the ndk gene into the algR2 mutant.
Thus, Ndk is involved in the formation of these com-
pounds and largely prevents the death of the algR2
mutant, which occurs early in the stationary phase.
We demonstrate that the 12 kDa Ndk–pyruvate kinase
(Pk) complex, previously shown to generate predomi-
nantly GTP instead of all the NTPs, has a low affinity
for the deoxynucleoside diphosphates and cannot gen-
erate the dNTPs needed for DNA replication and cell
division; this complex may thus be involved in regu-
lating the levels of both NTPs and dNTPs that modu-
late cell division and survival in the stationary phase.
Introduction
Many bacteria produce exopolysaccharides under starva-
tion conditions, particularly in response to prolonged nitro-
gen or phosphate limitation (Sutherland, 1990). Like many
Received 7 May, 1997; revised 24 October, 1997; accepted 2
December, 1997. †Present address: Department of Oral Micro-
biology, School of Dentistry, Kyung-hee University, 1 Hoeki-dong,
Dongdaemoon-Gu, Seoul 130-701, Korea. *For correspondence.
Tel. (415) 723 6167; Fax (415) 723 6783.
Q 1998 Blackwell Science Ltd
m
secondary metabolites, polysaccharide synthesis is usually
triggered at the onset of the stationary phase of growth
when nutrients in the growth media become limiting. An
example is alginate synthesis by Pseudomonas aerugi-
nosa , triggered in non-mucoid (non-alginate-producing)
cells during growth on poor phosphate or nitrogen sources.
With acetamide as a source of nitrogen, under phosphate
limitation in chemostats or in the presence of inhibitors of
energy metabolism that deplete the levels of nucleoside
triphosphates (NTPs), non-mucoid cells become heavily
mucoid because of secreted alginate (Speert et al ., 1990;
Woods et al., 1991; Terry et al ., 1992). Another example
of the transition of non-mucoid P . aeruginosa to mucoidy
is during pulmonary infections of cystic fibrosis (CF) patients
in whom mucoidy is believed to contribute to bacterial resis-
tance to phagocytosis, to the actions of neutrophils and to
allowing biofilm formation leading to resistance to antibio-
tics (May and Chakrabarty, 1994; Shankar et al ., 1995).
Another polymer produced in the stationary phase and
presumably in response to starvation is inorganic polyphos-
phate (poly P). Mutants of Escherichia coli lacking poly-
phosphate kinase (PPK), the enzyme that makes poly P,
fail to survive in stationary phase and lack resistance to
heat, oxidants and osmotic changes (Akiyama et al., 1992;
Crooke et al ., 1994; Rao and Kornberg, 1996). In Myxo-
coccus xanthus , the levels of poly P as well as poly P-
AMP phosphotransferase increase more than 10-fold after
the vegetative phase of growth (T. Shiba and A. Kornberg,
unpublished results). The increase in poly P level is pre-
ceded by an increase in the level of guanosine-38-di-58-
phosphate (ppGpp); mutants that fail to produce ppGpp
also fail to enhance this poly P level. Thus, ppGpp appears
to have a regulatory role in poly P accumulation (Kornberg,
1995). More recently, Kuroda et al . (1997) have demon-
strated in E . coli that high levels of ppGpp and guanosine
pentaphosphate (pppGpp) lead to massive accumulations
of poly P as a result of inhibition of the exopolyphospha-
tase (PPX) responsible for the hydrolytic breakdown of
poly P.
In P . aeruginosa strain 8830, a mucoid CF isolate, the
algR2 gene positively regulates alginate synthesis and the
production of two key enzymes of energy metabolism,
nucleoside diphosphate kinase (Ndk), responsible for
718 H.-Y. Kim et al.

Fig. 1. Pathway of alginate biosynthesis in P . aeruginosa . Abbreviations are as follows: F6P, fructose 6-phosphate; M6P, mannose
6-phosphate; M1P, mannose 1-phosphate; GDP-Man, GDP-mannose; GDP-ManA, GDP-mannuronic acid. Enzyme names (PMI, PMM, etc.)
are shown below, while the corresponding genes are shown above the steps they catalyse. The four arrows depict polymerization, acetylation,
epimerization and export of the alginate polymer. PMI/GMP is a bifunctional enzyme with phosphomannose isomerase–GDP-mannose
pyrophosphorylase activity encoded by the algA gene. The union of GTP and M1P to produce GDP-mannose (and pyrophosphate) is
catalysed by the GMP domain of the algA gene product, PMI-GMP.

the generation of NTPs or deoxy-NTPs (dNTPs), and suc-
cinyl-CoA synthetase (Scs), an ATP-generating enzyme of
the tricarboxylic acid (TCA) cycle (Kavanaugh-Black et al .,
1994; Schlictman et al ., 1994). In this report, we demon-
strate that a null mutation in the algR2 gene leads to a
reduced level not only of alginate, but also of ppGpp and
poly P as well. Membrane fractions of this mutant have
been shown previously to allow reduced levels of GTP
synthesis (Sundin et al ., 1996a), and we demonstrate in
this report that the intracellular levels of poly P, GTP,
pppGpp and ppGpp are significantly reduced after growth
in a low-phosphate medium in the presence of serine
hydroxamate, an inducer of stringent response in E . coli
(Cashel and Rudd, 1987; Cashel, 1994). Introduction of
either the algR2 or the ndk gene and their hyperexpression
from the tac promoter restores the levels of alginate, poly
P, GTP and (p)ppGpp simultaneously, suggesting that
the reduced levels of these metabolites in the algR2
mutant are primarily caused by extremely low levels of
Ndk in this mutant. Additionally, we demonstrate that the
promoter of the algR2 gene is responsive to phosphate
starvation, so that an elevated level of Ndk under such star-
vation conditions enhances the efficiency of NTP and dNTP
generation, compensating for the low levels of intracellular
NTPs and dNTPs.
Results
Poly P and alginate production in P. aeruginosa is
co-regulated by algR2 and ndk genes
An alginate-negative (alg ) mutant of stably mucoid alginate-
producing (Alg þ ) P . aeruginosa strain 8830 is deficient in
Ndk and Scs formation (Schlictman et al ., 1994). A double
mutant, algR2 algH , has no detectable Ndk as measured
by autophosphorylation or Western blotting (Schlictman
et al ., 1995). Alginate synthesis requires phosphorylated
sugars, such as fructose 6-phosphate (F6P) or mannose
6-phosphate (M6P) as precursors of GDP-mannose (Fig.
1). Previous reports have suggested the presence of
poly P in pseudomonads (Miguez et al ., 1986; Takade et
al ., 1991), but there is little information on its relation to
other metabolic events. We, therefore, compared the levels
of poly P in the CF-isolate mucoid strain 8830 with those
of its spontaneous non-mucoid segregant 8822 and the
non-mucoid laboratory strain PAO1. During growth in L
broth, the bulk of poly P accumulation in strain 8830 takes
place at the onset of the stationary phase and continues
well into the late stationary phase (Fig. 2). The poly P
level in mucoid strain 8830 is much higher than is normally
found in other microorganisms, such as E . coli (30–
35 nmol mg¹1 protein during growth in low-phosphate
MOPS medium), Helicobacter pylori (180–190 nmol mg¹1
protein) and compares favourably with that found in yeasts
(850 nmol mg¹1 protein). It is noteworthy that poly P levels
are highly variable, depending upon media composition and
the state of growth of the microorganisms. Inasmuch as
poly P accumulation occurs predominantly under stringent
conditions in E . coli (Kornberg, 1995), poly P levels in P .
aeruginosa were determined in a low-phosphate (0.4 mM)
MOPS medium containing serine hydroxamate, an inducer
of the amino acid stringent response (Cashel and Rudd,
1987). Strain 8830 grew slowly under such conditions
but accumulated large amounts of poly P up to 5 h, after
which the level declined (Fig. 3A). In contrast, the non-
mucoid PAO1 accumulated about 10% as much as the
mucoid strain under similar starvation conditions (Fig.
3A). As for the spontaneous segregant non-mucoid 8822
subjected to serine hydroxamate, poly P accumulation
was far less than in the mucoid strain (Fig. 3B), indicating
that the loss of alginate production is accompanied by a
lack of poly P accumulation. In each case, the level of
Fig. 2. Growth and poly P formation by P . aeruginosa strain 8830
in L broth (see Experimental procedures ).

Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 717–725

 G nucleotides, poly P and stationary phase regulation 719
Fig. 3. A. Poly P accumulation in mucoid strain 8830 and non-
mucoid strain PAO1. The cells were grown in a low-phosphate
(0.4 mM) MOPS medium and induced with serine hydroxamate at
an OD540 of 0.05, followed immediately by the addition of [32P]-Pi.
Time zero denotes the time of addition of [32P]-Pi in the low-
phosphate medium containing serine hydroxamate. B. Poly P
accumulation in the spontaneous segregant strain 8822, in the Alg¹
mutant algR2 and in an algR2 mutant complemented with algR2
(plasmid pJK662). Growth was in the low-phosphate MOPS
medium containing serine hydroxamate and incubated for the
indicated times after the addition of [32P]-Pi at an OD540 of about
0.1. Because of phosphate limitation and the presence of serine
hydroxamate, the strains, including the wild-type 8830, grew slowly
to a final OD540 of about 0.2 in 60 min. The measurement of
intracellular poly P levels is described in Experimental procedures .
poly P was much lower (10% or less) when serine hydrox-
amate was omitted or when the medium was supplemen-
ted with L broth, suggesting that conditions leading to poly
P accumulation in P . aeruginosa parallel those in E . coli .
Also, the levels were found to be variable in different experi-
ments, as shown by the results in Fig. 3A and B, presumably
because of differences in cell density used in individual
experiments and the physiological state of the cells.
Synthesis of both alginate and poly P requires NTPs, the
production of which is controlled by Ndk, which in turn is
positively regulated by algR2 . As with alginate synthesis,
the poly P level in the algR2 mutant is extremely low, but
is restored to a large extent in the presence of a functional
algR2 gene (pJK662) (Fig. 3B). This synthesis continued
until 4–5 h, as shown previously for the wild type, and
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 717–725
reached values approaching 65–70% of the wild-type
poly P level (data not shown). It is not clear why the poly
P level in the algR2 -complemented strain never reached
a value similar to that of the wild-type strain 8830. It is,
however, clear that both alginate synthesis and poly P
accumulation are regulated by the algR2 gene.
For lack of positive regulation of ndk by algR2 , the algR2
mutant has very low levels of Ndk. The loss of alginate in
the mutant can be restored to 60% of the wild-type level by
cloning ndk under the tac promoter and inducing enzyme
synthesis with IPTG (Sundin et al ., 1996a). Likewise, over-
expression of the ndk gene restored the poly P level to
about 60% of the wild-type level (Fig. 4), suggesting that
the level of NTPs generated by Ndk determines the accu-
mulation of both poly P and alginate.
GTP and ppGpp synthesis is co-regulated by algR2
and ndk genes
In order to understand how Ndk may promote both alginate
and poly P synthesis, we determined the levels of GTP
and (p)ppGpp in P . aeruginosa 8830. This is because
GTP is required for the synthesis of GDP-mannose, a pre-
cursor of alginate (Fig. 1) (May and Chakrabarty, 1994),
and GTP itself is a precursor of (p)ppGpp (Cashel, 1994),
the accumulation of which leads to poly P accumulation
(Kuroda et al ., 1997). Nucleoside diphosphate kinase is
known to be involved in preferential GTP synthesis in the
cell, both in P . aeruginosa and in other microorganisms,
such as Mycobacterium smegmatis , through complex for-
mation with specific proteins, such as pyruvate kinase, a
Ras-like protein Pra or the elongation factor Tu (Sundin
et al ., 1996a; Chopade et al ., 1997; Mukhopadhyay et
al ., 1997; Shankar et al ., 1997). Sundin et al . (1996a)
Fig. 4. Poly P accumulation in the algR2 mutant and in the algR2
mutant complemented with ndk (plasmid GWS95). Poly P
accumulation was induced in the low-phosphate MOPS medium in
the presence of serine hydroxamate and [32P]-Pi as described in
the legend to Fig. 3. Samples were removed after 1 h and 4 h of
growth, and poly P levels were determined as described in
Experimental procedures .
720 H.-Y. Kim et al.
Fig. 5. Cells (P . aeruginosa wild-type strain 8830, algR2 ::Cm and
algR2 ::Cm/pJK662) were grown in low-phosphate (0.4 mM) MOPS
medium containing serine hydroxamate at 100 mM to an initial
OD540 of 0.05. [32P]-H3PO4 was added to a final concentration of
10 mCi ml¹1 (3000 Ci mmol¹1 ) and the growth followed to an OD540
of 0.2. Aliquots (0.5 ml) of the cell cultures were mixed with 0.5 ml
of formic acid, frozen, thawed and the procedure repeated for a
total of four times. PEI-cellulose sheets were used for the
separation of the radioactive nucleotides (Cashel, 1994), and the
running solvent was 1 M LiCl–0.25 M formic acid. The reaction
products were visualized by autoradiography on Kodak XOMAT
film. Lanes represent 5 ml each of (A), P . aeruginosa 8830; (B),
algR2 ::Cm; (C), algR2 ::Cm/pJK662. Lane (D) represents 10 mCi of
[32P]-ATP, and lane (E) represents 10 mCi of [32P]-inorganic
phosphate (H3PO4).
previously reported that the membrane fractions of the
algR2 ::Cm null mutant generate only 10–15% of the
GTP generated by the wild type. To measure the level of
(p)ppGpp, we grew wild-type strain 8830, its algR2 ::Cm
null mutant and the complemented strains algR2 ::Cm with
either the ndk gene under the tac promoter (pGWS95) or
the algR2 gene under the tac promoter (pJK662) in a low-
phosphate (0.4 mM) MOPS medium containing [32P]-ortho-
phosphate (Pi) and induced with serine hydroxamate, as
described previously (Kuroda et al ., 1997). After 4 h of
growth, the cells were harvested at 1 h and 4 h, suspended
in cold 10 M formic acid, frozen in liquid nitrogen and
thawed, centrifuged, and the levels of GTP, (p)ppGpp and
poly P were determined in the supernatant after thin-layer
chromatography (TLC) separation on polyethyleneimine-
cellulose sheets. The results with poly P are shown in
Figs 3B and 4, while the results with GTP and (p)ppGpp
are shown in Fig. 5 and Table 1. The ppGpp levels, shown
in Table 1, were determined by autoradiography followed
by PhosphoImager scanning as described previously
(Kuroda and Kornberg, 1997). As can be seen from
Table 1, the ppGpp levels were significantly reduced in
the algR2 mutant, but were restored to near the wild-type
level by complementation with either the algR2 gene
(pJK662) or the ndk gene (pGWS95). Thus, the level of
ppGpp, like the alginate, poly P and GTP levels, is posi-
tively regulated by the algR2 or ndk gene, presumably
through enhanced synthesis of the precursor GTP.
algR2 expression responds to phosphate starvation
To determine whether algR2 is regulated by phosphate, an
algR2 – lacZ transcriptional fusion (in which the lacZ expres-
sion depends on the algR2 promoter) proved to be respon-
sive to phosphate starvation (Fig. 6). During growth in a
basal synthetic medium with low (0.1 mM) phosphate,
the algR2 promoter was activated and remained active
throughout the stationary phase of growth. This activation
was absent in a phoB mutant (which is unresponsive to
low Pi) or in the presence of high (2 mM) phosphate. It is
notable that the increased activity of the algR2 promoter
is not merely the result of cessation of growth, because
then an increase would also be seen in the phoB mutant,
which is not the case. Furthermore, we have tested
algR2 – lacZ activity under conditions of carbon and nitro-
gen starvation, in which only a small increase in activity
is seen. Thus, under prolonged phosphate starvation,
which results in low NTP levels, the algR2 promoter is acti-
vated to generate higher levels of AlgR2, which in turn sti-
mulates Ndk and Scs formation; elevated levels of Ndk
and Scs then raise NTP levels to overcome the effects of
starvation. AlgR2, therefore, appears to play an additional
role in restoring the energy balance during periods of
phosphate starvation. A similar induction of ppGpp forma-
tion by phosphate starvation in E . coli has also been
reported (Spira et al ., 1995). It is interesting to note that
the algR2 promoter activation under phosphate starvation
is abolished in a phoB mutant. Visual examination does
Table 1. Level of ppGpp in 8830 and its algR2 mutant complemented
by ndk or algR2 .
Strain ppGpp (nmol mg¹1 of protein)
8830 4.2
algR2 ::Cm 0.05
algR2 ::Cm þ pGWS95 (ndk ) 3.7
algR2 ::Cm /pJK662 (algR2 ) 3.5
Cells were grown in low-phosphate medium and induced with IPTG
for 60 min in the presence of serine hydroxamate before being
extracted for ppGpp measurement, as described by Cashel (1994)
and under Experimental procedures . PGWS95 is a plasmid with
the ndk gene and pJK662 with the algR2 gene, both under the tac
promoter.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 717–725
 G nucleotides, poly P and stationary phase regulation 721

Fig. 6. Regulation of algR2 expression by

phosphate starvation. Wild-type PAO1 or its
phoB mutant strains containing an algR2–lacZ
transcriptional fusion were grown in complete
MOPS minimal medium (2 mM PO4) or with
limiting phosphate (0.1 mM). Samples were
taken at various time intervals, and the levels
of b-galactosidase were measured. Time zero
represents the time of inoculation of the
cultures in the respective high (2.0 mM) or low
(0.1 mM)-phosphate MOPS media.

not reveal a Pho box in the algR2 promoter region, sug- inhibited by anti-Ndk antibody (Fig. 8). Anti-Pk antibody
gesting that the effect of PhoB on algR2 expression is changes the specificity from GTP to all three NTPs. With
likely to be indirect. Given the regulation of algR2 , a key regard to the synthesis of dNTPs, the membrane fraction
regulator of alginate synthesis, by PhoB, it would be of has very little activity to begin with and is unaffected by
interest to construct a phoB mutant of the mucoid strain anti-Ndk or anti-Pk antibodies (Fig. 8). The reconstituted
8830 to identify directly any role of PhoB in alginate synthesis. complex between purified 12 kDa Ndk and Pk resembles
activities found in the membrane fraction (Fig. 9). Pk and
the 16 kDa Ndk can each generate NTPs and dNTPs
algR2 and ndk contribute to stationary phase survival
from NDPs, dNDPs and [g-32P]-ATP; however, Pk requires
Inasmuch as algR2 promotes the expression of ndk , it may PEP and Mn2þ for this activity (Sundin et al ., 1996a).
upregulate the levels of GTP and ppGpp and, hence, poly Because the 16 kDa Ndk fails to form a complex with Pk,
P. Furthermore, poly P accumulation is defective in the the addition of Pk had no effect (Fig. 9). However, with
algR2 mutant, and this defect can be overcome by com- the 12 kDa form and adequate levels of Pk, the resulting
plementation by ndk (Fig. 4). The fact that poly P is essen-
tial for the survival of E . coli in the stationary phase (Rao
and Kornberg, 1996) prompted the study of the viability of
the algR2 mutant. As in E . coli , survival of the mutant in
stationary phase is greatly diminished, a defect that in
this case can be overcome by plasmids bearing either
the algR2 or ndk genes (Fig. 7). Thus, the levels of GTP,
ppGpp or poly P may singularly or collectively promote
the survival of P . aeruginosa in the stationary phase.

Differential synthesis of GTP, NTPs and dNTPs by

the 12 kDa form of Ndk
In P . aeruginosa 8830, the 16 kDa cytoplasmic Ndk pro-
duces all the NTPs and dNTPs during the exponential
phase of growth, while the 12 kDa membrane form pro-
duces predominantly GTP at the onset of the stationary
phase (Shankar et al ., 1996). Both RNA and DNA synth-
eses slow down at the onset of stationary phase, caused
perhaps in part by reduced levels of NTPs and dNTPs.
Fig. 7. Growth and survival of 8830 (wild-type strain), the algR2
To determine whether the truncated 12 kDa form of Ndk, mutant and the algR2 mutant harbouring either the algR2 gene
complexed with Pk, has an altered capacity to synthesize (pJK662) or the ndk gene (pGWS95) under the tac promoter. Cells
were grown in a complete MOPS medium and sampled for colony-
dNTPs, the specificities of the complex were compared
forming units (cfus) at various times indicated by dilution and
with those of the membrane fraction (Fig. 8). The mem- plating on L agar plates. The vector plasmid used to clone the
brane fraction makes predominantly GTP, an activity algR2 or the ndk gene under the tac promoter was pMMB66EH.

Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 717–725

722 H.-Y. Kim et al.
Fig. 8. Specificity of NTP and dNTP synthesis by membrane
fractions of P . aeruginosa 8830. Cells were grown for 15 h up to the
stationary phase, lysed by sonication and centrifuged at 10 000 × g
for 15 min. The supernatant was centrifuged at 50 000 × g
(Beckman Model L350) for 1 h, and the pellet represented the
membrane fraction (Schlictman et al ., 1995). For assay of NTP or
dNTP synthesis, 10 mg of membrane fraction protein was incubated
with or without antibodies (diluted 1:1000) for 5 min at 48C followed
by the addition of 0.1 mM each of GDP, CDP, UDP or their dNDP
derivatives. The reaction mixture was treated with 1 mCi of
[g-32P]-ATP and incubated at room temperature for 15 s, followed
by the addition of 5 ml of 4 × SDS stop buffer. The reaction mixture
(1 ml) was spotted on a polyethyleneimine thin-layered plate
(Aldrich) and developed in 0.75 M KH 2PO4 at pH 3.75. The plate
was then dried, covered with Saran wrap and autoradiographed at
room temperature.
complex was ineffective in the formation of dNTPs (Fig. 9);
the complex has reduced specificity for dNDPs compared
with either the 12 kDa Ndk or Pk separately.
Discussion
AlgR2 positively regulates nucleoside diphosphate kinase
(NdK) synthesis and, as a result, may modulate the levels
of GTP, ppGpp, alginate and poly P. Alginate synthesis
requires a supply of GTP, as does (p)ppGpp; the latter pro-
motes the accumulation of poly P (Kuroda et al ., 1997).
The conversion of the 16 kDa cytoplasmic form of Ndk to
the 12 kDa membrane-associated form at the onset of sta-
tionary phase (Shankar et al ., 1996) promotes the syn-
thesis of GTP and, in turn, ppGpp (Cashel, 1994). The
latter induces expression of the stationary phase sigma fac-
tor, sS, in E . coli (Gentry et al ., 1993), which controls over
40 genes essential for stationary phase resistance and
survival (Loewen and Hengge-Aronis, 1994). However,
the effect of ppGpp on the level of RpoS in P . aeruginosa
is unknown at present. As the membrane-associated
12 kDa Ndk appears at the onset of the stationary phase
(Shankar et al ., 1996; Chopade et al ., 1997) and makes
GTP preferentially and very little dNTPs (Figs 8 and 9),
the precursor pools of RNA and DNA are probably reduced
with a consequent slowdown in RNA and DNA synthesis.
In addition to Pk, a Ras-like protein, Pra, also appears in
the membrane at the stationary phase and forms a com-
plex with 12 kDa Ndk, directing its specificity towards GTP
synthesis (Chopade et al ., 1997). Apart from 12 kDa Ndk,
Pra can also form a complex with Pk, modulating its
specificity from NTP synthesis to GTP (Chopade et al .,
1997). In the presence of 12 kDa Ndk, however, Pk has
a higher affinity for 12 kDa Ndk, so that the Pk–Pra
complex dissociates. In the algR2 mutant, which has
very little Ndk, it is Pk that allows NTP synthesis, and it
Fig. 9. Effect of pyruvate kinase (Pk) on
dNTP synthesis by the 16 kDa and 12 kDa
forms of Ndk. Ndk (10 mg) was incubated with
2–10 mg of Pk for 15 min at room temperature
followed by the addition of 0.1 mM each of
d(CDP/GDP/TDP), [g-32P]-ATP, 25 mM
phosphoenolpyruvate (PEP) and 10 mM Mn2þ.
Reaction mixtures were then treated as in
Fig. 8. Numbers next to Pk represent the
amounts of Pk in mg. The number 10 on the
extreme left after Pk refers to 10 mg of Pk
without any Ndk addition, showing that Pk
generates all three dNTPs in the absence of
Ndk, similar to 16 kDa or 12 kDa Ndk by
themselves.
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 G nucleotides, poly P and stationary phase regulation 723
is likely that the Pk–Pra complex generates the GTP
and very little dNTP for transition to stationary phase
(Chopade et al ., 1997).
The co-regulation of two polymers, alginate and poly P,
by a common regulator, such as AlgR2, is interesting. The
mucoid Alg þ cells are rich in poly P, while the non-mucoid
segregant or the non-mucoid laboratory isolate produces
much less poly P. Thus, the genetic switch that turns on
alginate synthesis in P . aeruginosa also turns on poly P
synthesis, implying a common mode of genetic regulation.
As hyperexpression of the ndk gene largely restores algi-
nate, GTP, ppGpp and poly P synthesis in the algR2
mutant (Table 1, Figs 3 and 4), Ndk plays a critical role
in the synthesis of these two polymers. As AlgR2 positively
regulates Ndk and Scs formation and as hyperexpression
of the ndk gene can compensate for AlgR2 deficiency, it is
likely that the phenotypes demonstrated by the algR2
mutant are largely caused by the reduced formation of
Ndk. Although P . aeruginosa is normally non-mucoid, it
is converted to a mucoid form under several conditions:
in the lungs of CF patients (Shankar et al ., 1995), during
growth under starvation conditions and in the presence
of inhibitors of energy metabolism (Speert et al ., 1990;
Woods et al., 1991; Terry et al ., 1992). Starvation condi-
tions, particularly for phosphate, allow an activation of
the algR2 promoter, leading to enhanced Ndk synthesis
and, presumably, an enhanced formation of GTP, pppGpp
and ppGpp. High concentrations of pppGpp or ppGpp are
known to lead to the accumulation of poly P (Kuroda et al .,
1997). Indeed, the poly P accumulation in P . aeruginosa is
maximal in the stationary phase (Fig. 2) when the mem-
brane-associated complexes of Pk and Pra with the
12 kDa Ndk generate large amounts of GTP. Alginate is
also known to accumulate predominantly in the late expo-
nential–early stationary phase cells (Tatnell et al ., 1993;
Hassett, 1996). As alginate synthesis requires both phos-
phorylated sugars and GTP (Fig. 1) and as poly P may
substitute for ATP (Kornberg, 1995), it is possible that
poly P can contribute to alginate synthesis. Recent efforts
in our laboratory to isolate polyphosphate kinase-negative
(ppk ) mutants of stably mucoid P . aeruginosa strain 8830
have produced presumptive mutants, many of which are
non-mucoid with little PPK activity (A. Zago, unpublished
observations). When the cloned ppk gene of P . aerugi-
nosa becomes available, its capacity to restore alginate
synthesis in the presumptive ppk non-mucoid mutants
can be determined. Given the ubiquitous nature of poly
P and its accumulation in the stationary phase (Kornberg,
1995), it can be imagined that poly P, among its many
functions, can be used by bacteria to produce capsular
polysaccharides for their protection in stressful situations,
as for P . aeruginosa in the CF lung. Similarly, isolation of a
relA mutant, incapable of converting GTP to pppGpp or
ppGpp, in the mucoid strain 8830 may provide insights
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 717–725
into the role of these regulatory nucleotides in alginate
synthesis.
Experimental procedures
Bacterial strains and growth
P . aeruginosa 8830 is a stable alginate-producing strain. The
organism was routinely propagated in L broth at 378C. The
algR2 ::Cm , which is a null mutant in the algR2 gene because
of insertion of a chloramphenicol cassette, was propagated in
L broth or in MOPS media for studies of poly P accumulation.
Assay for guanosine tri- and tetraphosphates
GTP and ppGpp production was analysed by one-dimensional
chromatography (PEI-cellulose; E. Merck) as described by
Cashel (1994). Non-labelled E . coli ppGpp and pppGpp
were used as standards and detected by UV light. The wild-
type strain 8830, the algR2 null mutant and the algR2 mutant
complemented by either the algR2 or the ndk gene under the
tac promoter were grown from an OD540 of 0.05 to an OD of
0.25 in a low-phosphate (0.4 mM) MOPS medium containing
[32P]-orthophosphate. Aliquots were transferred to cold 10 M
formic acid, frozen in liquid nitrogen, and the thawed lysates
were examined for GTP and (p)ppGpp levels, as described
by Cashel (1994) and shown in Fig. 5. The authenticity of
the (p)ppGpp was determined by its susceptibility to purified
yeast exopolyphosphatase (PPX), which converts it to GTP
and Pi, all of which can be separated on polyethyleneimine
TLC plates as described earlier (Kuroda and Kornberg,
1997).
Quantitative measurement of poly P
Assays according to S. Liu and A. Kornberg (unpublished)
used overnight cultures grown with [32P]-Pi in a synthetic
medium. Cells were harvested and lysed in a FUSE buffer
(3 M formic acid, 2 M urea, 20 mM EDTA, 1% SDS, 1 mg ml¹1
polyphosphate 65) and sonicated briefly. Poly P in the lysate
was adsorbed on a DE81 filter disc, washed with a TKP buffer
(10 mM Tris-HCl, pH 8, 100 mM KCl, 5 mM K3PO4) and eluted
with TKP buffer containing 500 mM KCl. Poly P in the eluant
was further enriched by removal of 32P contaminants with
Norit (activated charcoal), then readsorbed to a DE81 filter
disc, washed and treated with yeast PPX (Kuroda and Korn-
berg, 1997) in 40 mM Tris-HCl, pH 7.4, 100 mM ammonium
acetate/4 mM MgCl2 , 10 mg ml¹1 BSA and 5 mg ml¹1 PPX.
The amount of poly P was determined by the loss of 32P
from the filter. This loss was subsequently correlated with the
appearance of [32P]-Pi in the supernatant, and the level of
poly P was also occasionally determined by averaging the
loss of 32P from the filter and the appearance of [32P]-Pi in
the eluant.
algR2–lacZ assays
Plasmid pDS25 (algR2 – lacz ) was introduced into strain PAO1
and its phoB mutant (Shortridge et al ., 1992). The strains
724 H.-Y. Kim et al.
were grown in MOPS minimal media (Neidhardt et al ., 1974),
containing either 2 mM or 0.1 mM Pi. Strains were grown in 1 l
of media with 100 mg ml¹1 carbenicillin in 2 l flasks at 378C
with shaking at 250 r.p.m. b-Galactosidase activity was deter-
mined according to Miller (1972). Protein was determined
using the Bio-Rad Protein Assay kit with BSA as a standard.
Assay for NTP and dNTP synthesis by membrane
fractions or by combinations of Ndk and Pk
Isolation of membrane fractions, purified Ndk (16 kDa and
12 kDa forms) and Pk and the separation of NTPs (and
dNTPs) were as described previously (Shankar et al ., 1996;
Sundin et al ., 1996a,b).
Acknowledgements
We thank Dr George Sundin for gifts of strains and valuable
advice. This work was supported by NIH grants AI-31546-4
and AI-16790-17 to A.M.C. and grant GM-07581-33 to A.K.
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