A polyphosphate kinase (PPK2) widely conserved

in bacteria

Haiyu Zhang, Kazuya Ishige, and Arthur Kornberg*

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307

Contributed by Arthur Kornberg, October 29, 2002

Synthesis of inorganic polyphosphate (poly P) from the terminal
phosphate of ATP is catalyzed reversibly by poly P kinase (PPK,
now designated PPK1) initially isolated from Escherichia coli.
PPK1 is highly conserved in many bacteria, including some of the
major pathogens such as Pseudomonas aeruginosa. In a null
mutant of P. aeruginosa lacking ppk1, we have discovered a
previously uncharacterized PPK activity (designated PPK2) dis-
tinguished from PPK1 by the following: synthesis of poly P from
GTP or ATP, a preference for Mn2+ over Mg2*, and a stimulation
by poly P. The reverse reaction, a poly P-driven nucleoside
diphosphate kinase synthesis of GTP from GDP, is 75-fold greater
than the forward reaction, poly P synthesis from GTP. The gene
encoding PPK2 (ppk2) was identified from the amino acid se-
quence of the protein purified near 1,000-fold, to homogeneity.
The 5’-end is 177 bp upstream of the annotated genome se-
quence of a “conserved hypothetical protein’; ppk2 (1,074 bp)
encodes a protein of 357 aa with a molecular mass of 40.8 kDa.
Sequences homologous to PPK2 are present in two other pro-
teins in P. aeruginosa, in two Archaea, and in 32 other bacteria
(almost al! with PPK1 as well); these include rhizobia, cyanobac-
teria, Streptomyces, and several pathogenic species. Distinctive
features of the poly P-driven nucleoside diphosphate kinase
activity and structural aspects of PPK2 are among the subjects of
an accompanying report.

norganic polyphosphate (poly P) is a polymer of tens or

hundreds of phosphate residues found in all cells (bacterial,
fungal, plant, and animal) that have been examined (1-3).
Among its many functions, poly P is needed for bacterial survival
in the face of stress and stringencies and is required for the
virulence of some pathogens (4-12).

The gene ppk! encodes poly P kinase (PPK1), the enzyme that
converts ATP to poly P among other activities (13). Null mutants
of Pseudomonas aeruginosa PAO] lacking ppk/ are deficient in
motility, quorum sensing, biofilm formation, and virulence in
mouse models (9, 10, 12). Despite the lack of detectable PPK1
activity (<1% of wild type), these mutants still possess as much
as 20% of the wild-type levels of poly P (9).

The purpose of this study was to determine the source of poly
P in the ppkJ mutants of P. aeruginosa. No clue could be obtained
from extensive searches for homologies to PPK1 in the available
databases. Thus, the putative PPK2 would have to be revealed by
novel assays of poly P synthesis in cell-free extracts of the
mutants and the subsequent isolation of the responsible enzyme.

In this report, we describe the identification and novel features
of PPK2, its sequence in the P. aeruginosa genome, and the
conservation of this sequence among 35 microorganisms, nota-
bly rhizobia, cyanobacteria, Streptomyces, several pathogenic
species, and two Archaea.

Simultaneously with these studies, we pursued a very potent
activity in P. aeruginosa that uses poly P as a donor to convert
GDP to GTP (14). This activity, previously designated PNDK
(poly P-driven nucleoside diphosphate kinase; ref. 15), has now
been purified to homogeneity. Although seemingly different
from the activity that synthesizes poly P from GTP, both
activities reside in the very same protein, PPK2, encoded by

16678-16683 | PNAS | December 24,2002 | vol.99 | no. 26

ppk2. An account of this discovery is presented in the accom-
panying report (14).

Materials and Methods

Reagents. The sources of the reagents and related supplies used
in this study are as follows: creatine kinase, DNase I, and RNase
A were from Boehringer Mannheim; ATP, GTP, creatine phos-
phate, poly P (types P15 and P75), and BSA were from Sigma;
[y-?PJATP and [y-**P]GTP were from Amersham Pharmacia;
poly(vinylidene difluoride) membranes were from Bio-Rad.
Purified P. aeruginosa PPK1 was a gift from S. Lee ([COS
Corporation, Bothell, WA).

Strains. The strains used in this study are P. aeruginosa PAOMS
[PAO1 Appki::tet (Tc‘)] (10) and Escherichia coli CF5802
[MG1655 Appk1-Appx::Kan (Kn‘)] (16).

Assays for PPK Activities. The PPK1 assay was performed as
described (13). For the PPK2 assay, the reaction mixture (25
pl) contained 50 mM Hepes (pH 7.2), 100 mM (NH4)2SQu, 0.1
mM poly P35 (in phosphate residues), 10 mM MnCl, and 2mM
[y-2P]GTP (5-50 cpm/pmol GTP). After incubation for 5 min
at 37°C, the reaction was stopped by adding 100 xl 2mM BSA
and 100 ul 7% percloride acid, then mixed well and put on ice.
[??P]poly P was measured in a liquid scintillation counter as in
the PPK1 assay by collecting on Whatman GF/C glass fiber
filters and washing four times with 20 ml of washing solution
(1M HCL and 0.1 M pyrophosphate) followed by ethanol. One
unit of enzyme is defined as the amount incorporating | pmol
of phosphate into acid-insoluble poly P per min at 37°C.

Plasmid Construction. To clone ppk2 from P. aeruginosa, PCR
primers PPK2-F (TTccatggGAGAGGTGTAAGGCTTTCCT
with an NcoI site near the 5’-end) and PPK2-R (TTggatccT-
GCCGTACAAGCAGATCGTGA with a BamHI site near the
5’-end) were used to amplify ppk2 from PAOMS genome DNA,
then cloned into the NcoI-BamHI site of pTrc99A (Amp’) (17)
to form pTrc-PPK2.

Other Methods. Protein mass spectrometry and amino terminal
sequence analysis were done by the Stanford Protein and Nucleic
Acid (PAN) facility. Protein on SDS/PAGE was digested by
LysC for matrix-assisted laser desorption/ionization-time of
flight (MALDI-TOF) mass spectrometry analysis. For amino-
terminal sequence analysis, proteins were blotted onto poly-
(vinylidene difluoride} membrane according to the guidelines of
the Stanford PAN facility. Sequencing was performed on an ABI
sequenator (Applied Biosystems; model Procise 494) based on
Edman degradation.

Abbreviations: poly P, inorganic polyphosphate; PPK, polyphosphate kinase; NDK, nucle-
oside diphosphate kinase.

Data deposition: The sequence reported in this paper (ppk2) has been deposited in the
GenBank database (accession no. AY 168003).

*To whom correspondence should be addressed. E-mail: akornber@cmgm.stanford.edu.

www.pnas.org/cgi/doi/10.1073/pnas.262655 199
Fractions

M A B
kDa 2.22 3.59 ug
200
150
100
i
50 ities
omnes il — AG kDa
37 atin, —«— -—- 42 kDa
—_ — 36 kDa
a a —3 kDa
25

ai:

Fig.1. SDS/PAGE analysis of purified PPK2. Samples were precipitated with
0.015% deoxycholate-5% trichloroacetic acid (18). Proteins were visualized by
Coomassie blue staining. Samples of two different purification operations
(A and B), both with nearly the same specific activity of 5.5 x 10° units/mg,
were applied as indicated. M, protein molecular mass markers (Precision
Protein Standards, Bio-Rad).

Results

Assay of PPK2. The standard assay of PPK1 in lysates of the ppk/
null mutant in P. aeruginosa PAOMS showed no activity
(<1%) using the measure of conversion of [y-*?P]ATP to
acid-insoluble poly P. By improving the separation of poly P,
the sensitivity was increased so that as few as 200 cpm of 2 x
10® cpm [y-*P]GTP added could be detected as poly P
(expressed as 10 units/mg protein). By modifying cultural
conditions and components of the assay mixture, such as
replacing Mg** with Mn?* and addition of poly P, a novel
PPK2 activity in the lysate was increased 1,000-fold to 10,000

Table 1. P. aeruginosa PPK1 and PPK2 in the synthesis and
utilization of poly P

 

PPK1* PPK2
Synthesis (NTP—>poly P)
ATP or GTP ATP only ATP or GTP
Metal ion Mg?* Mn2+
Poly P stimulation No Yes
Vmax, units/mg 2.1 x 108 6.7 x 10°
Poly P product, residues 500-800 200-800
Processivity . Yes Yes
Utilization (Poly P->NTP)
ADP/GDP 34 0.9
Poly P size preference Long Short
Metal ion Mg?2+ Mg?*
Vmax UNits/mg 5.1 x 105 5.0 x 108
Processivity Yes Yes
Synthesis/utilization
Vmax ratio 4.1 0.013

 

*His-tagged P. aeruginosa PPK? was a gift from S. Lee (ICOS Corporation).

Zhang et al.

0.5 1 2 5 10 2030 min
7 BB — PolyP750

  

we GTP

Fig.2. Processivity of PPK2 poly P synthesis. A 25-yl assay mixture contained
56 ng of PPK2. After incubation at 37°C for the indicated times, the products
were separated by PAGE (20% gel) containing 7 M urea and visualized by
Phosphorlmager (Molecular Dynamics); the [y-22P]GTP and poly P759 migrated
on the gel as indicated.

units/mg protein, a level comparable to that of PPK1 in
wild-type lysates.

Purification. PPK2 was purified near 1,000-fold to apparent
homogeneity from the P. aeruginosa PAOMS soluble lysate by
fractionation on heparin-Sepharose, GTP agarose, Mono Q,
and Mono S columns. Yet, the purified PPK2 fractions with a
specific activity of 5.5 < 10° units/mg protein showed on some
occasions several bands on SDS/PAGE with molecular masses
of ~46, 42, 36, and 32 kDa (Fig. 1). By mass spectroscopy, all four

PPK2 _PPK1_
Pa. Pa. Ec.

'”

 

Fig.3. Chain lengths of poly P synthesized by PPKs. PPK2 of P. aeruginosa (Pa;
140 ng); PPK1 of P. aeruginosa (Pa; 120 ng); PPK1 of E. coli (Ec; 29 ng). After
45 min at 37°C, the products were separated by PAGE (20% gel) containing 7
M urea and visualized by Phosphorlmager (Molecular Dynamics); the positions
to which [y-32P]ATP and [y-32P]GTP migrated on the gel are indicated.

PNAS [| December 24,2002 | vol.99 [| no.26 | 16679

BIOCHEMISTRY
181

24)

361

422

481

601

661

F2i

781

841

901

961

102%

CGGCAGGGGCATAGAGTGGGGGACTGCATAGCTGAAAACGTAGAACGGAAGGAGTCCCGC

ATGAGCGAAGAACCCACTGTCAGTCCCCCCTCCCCCGAGCAACCCGCCGCGCAGCCGGCC
M S EF EB PTV SS PPS P BQ PA AKA QP A

AAGCCGGCCCGGCCAGCCECCCGCCECGCCCCECGCAAGCCGGCGACCCGCCGCCCGCGA
K P A R P A AR R A PR K P AT R R PR

GTGGCCAGCCCGGCGCAGAAGGCCCGCGAGGAGATCCAGGCAATCAGCCAGAAGCCGGTG
VA S P A @ K A R BE E IQ Ad S$ @ K P Y¥

GCCCTGCAGGTCGCCAGTGCGCCCCACGGCAGCAGCGAGGACAGCACCTCGGCGAGCCTG
A L @ VA S A P H GCG & &§ EB D § T S AS EL

CCGGCGAACTATCCCTATCACACGCGGATGCGCCGCAACGAGTACGAGAAGGCCAAGCAC
P A N ¥ P ¥ H T R M R R N E ¥ E K A K EF

GACCTGCAGATCGAACTGCT CAAGGT GCAGAGCTGGGTGAAGGAGACCGGCCAGCGCGTG
DL@ tit ELL K VQ § W VY K FE T G QR V

GTGCTCCTGTTCGAAGGCCGCGACGCCGCCGGCAAGGGCGGCACCATCAAGCGCTTCATG
VV b FE @ RUDUALALGUELGUGIP I KOR OF OM
GAACACCTGAACCCGCGCGGCGCGCGGATCGTAGCCTTGGAGAAACCGTCCTCCCAGGAG
BE H L N P R G A R IV A L E K P §& §&S§ Q@ E

CAGGGCCAGTGGTATTT CCAGCGCTACATCCAACATCTGCCCACCSCCOGCGAGATGSTC
Q GoW Y F 9 R ¥ I @ H L BP T A G EM V¥

TTICTTCGACCGCTCCTGGTACAACCGCGCCGGCGTCGAGCGGGTCATGGGCTTCTGTTCG
F F D R S W ¥Y N R AG V ER VM G@ F C S

COGCTGCAATACCTGGAGTTCATGCGCCAGGCGCCGGAGCTGGAGCGCATGCTCACCAAC
P LQ ¥ L E F M R Q A P E L E R M EL T WN

AGCGGCATCCTGCTGTTCAAGTACTGGTTCTCGGTGAGCCGCGAGGAACAACTGCGGCGC
8 G TT L Lb F Kk ¥ W F S&S V S R E E Q L RR

TPCATCTCGCGCCGCGACGATCCGCTCAAGCACTGGAAGCTGTCGCCCATCGACATCAAG
FIs R R Dp PP} LK H WK LS PID IK

TCTCTGGACAAGTGGGACGACTACACCGCCGCCAAGCAGGCGATGTTCTTCCATACCGAC
S$ L DB K WDD YY T A A K Q A M F F H T D

ACCGCCGACGCGCCGTGGACGGTCATCAAGTCCGACGACAAGAAGCGCGCGCGACTCAAC
T A DA PW TV § KF S DD K K R A R LN

TGCATCCGCCACTTCCTGCACTCGCTGGACTACCCGGACAAGGACCGGCGCATCGCCCAT
c I R KH F LH S LoD Y p DK DR RIA H

GAGCCCGACCCGT TGCTGGTGGGGCCGGCCTCGCGGGTGATCGAGGAGGACGAGAAGGTC

BE Pp p PP] Lb uv G PAS RV IEE DEK V

TACGCCGAGGCGGCCGCCGCGCCGGGCCACGCGAACCTGGATATCCCGGCCTGA

Y A E A A A A P G H A N L D FT PA *

Fig. 4. Nucleotide (ppk2) and the predicted amino acid (PPK2) sequences of P. aeruginosa. The nucleotide sequence (5’ to 3’) of the noncoding strand starts
at nucleotide 1 and terminates at 1074. A possible Shine~-Delgarno sequence (AGGAGT) for translation initiation starting at —11 bp is marked by a wavy line.
The amino-terminal amino acid sequence determined from purified PPK2 (underlined) agrees with the deduced amino acid sequence from the start codon. The
incorrect original annotation of the start codon at nucleotide 178 (GTG) is shaded. The P-loop sequence is marked by a dotted line, and the conserved amino
acids are shaded. Three aspartyl-prolyl (D-P) bonds are identified in boxes.

bands referred to the same protein (see below), all with the same
N-terminal sequence. The three bands with lower molecular
mass were likely artifacts of acid-catalyzed cleavage of aspartyl-
prolyl (D-P) bonds (19, 20).

As described in detail in the accompanying report (14), a parallel
purification of PPK2 to homogeneity was achieved by assay of the
reverse reaction: poly P as a donor to [7H]GDP to form GTP with

a 1,270-fold purification and a remarkable yield of 72% (14).

16680 | www.pnas.org/cgi/doi/10.1073/pnas.262655199

Zhang et al.
Table 2. PPK2 or PPK1 homologs among microorganisms

PPK2

PPK2 and PPK1
Agrobacterium tumefaciens
Brucella melitensis
Burkholderia fungorum (2)*
Campylobacter jejuni
Caulobacter crescentus
Chlorobium tepidum
Chloroflexus aurantiacus
Deinococcus radiodurans
Magnetospirillum magnetotacticum (2)
Mesorhizobium loti (2)
Methanosarcina acetivorans (2)*
Methanosarcina mazei (2)*
Mycobacterium tuberculosis
Myxococcus xanthus
Nostoc punctiforme (2)
Nostoc sp. PCC7120 (2)
Prochlorococcus marinus
P. aeruginosa (3)
Pseudomonas fluorescens (2)
Ralstonia metallidurans (5)
Ralstonia solanacearum
Rhodobacter sphaeroides (2)
Rhodopseudomonas palustris
Rhodospirillum rubrum
Sinorhizobium meliloti (3)
Streptomyces coelicolor
Synechococcus sp. WH 8102
Synechocystis sp. PCC6803
Thermosynechococcus elongatus
Vibrio cholerae
Xanthomonas axonopodis
Xanthomonas campestris

PPK2 only
Corynebacterium glutamicum (2)
Magnetococcus MC-1 (2)
Plectonema boryanum

PPK? only

Genome complete
Bacillus anthracis
Bacillus halodurans
Clostridium acetobutylicum
E. coli
Helicobacter pylori
Mycobacterium leprae
Neisseria meningitidis
Salmonella typhi
Salmonella typhimurium
Xylella fastidiosa
Yersinia pestis

Genome incomplete
Acidithiobacillus ferrooxidans
Acinetobacter baumannii
Acinetobacter calcoaceticus
Acinetobacter sp. ADP1
Aphanizomenon sp. TR183
Aphanizomenon baltica
Arthrobacter sp. KM
Bordetella pertussis
Burkholderia cepacia
Campylobacter coli
Cytophaga hutchinsonii
Dictyostelium discoideum*
Geobacter sulfurreducens
Haloferax volcanii*
Klebsiella aerogenes
Leuconostoc mesenteroides
Microbulbifer degradans
Mycobacterium marinum
Mycobacterium ulcerans
Neisseria gonorrhoea
Nitrosomonas europaea
Nodularia spumigena
Oenococcus oeni
Porphyromonas gingivalis
Propionibacterium shermanii
Rhodocyclus tenuis
Salmonella dublin
Serratia marcescens
Shigella flexneri
Streptomyces griseus
Streptomyces lividans
Thermobifida fusca

Neither PPK1 nor PPK2

Aeropyrum pernix*
Aquifex aeolicus
Archaeoglobus fulgidus*
Bacillus subtilis
Borrelia burgdorferi
Buchnera aphidicola str. Sg
Buchnera sp. APS
Chlamydia muridarum
Chlamydia trachomatis
Chlamydophila pneumoniae
Clostridium perfringens
Fusobacterium nucleatum
Haemophilus influenzae Rd
Halobacterium sp. NRC-1*
Lactococcus lactis
Listeria innocua
Listeria monocytogenes
Methanococcus jannaschi*
Methanopyrus kandleri*
Methanothermobacter
thermautotrophicus*
Mycoplasma genitalium
Mycoplasma pneumoniae
Mycoplasma pulmonis
Pasteurella multocida
Pyobaculum aerophilum*
Pyrococcus abyssi*
Pyrococcus furiosus*
Pyrococcus horikoshii*
Rickettsia conorii
Rickettsia prowazekii
Saccharomyces cerevisiae*
Schizosaccharomyces pombe?
Staphylococcus aureus
Streptococcus pneumoniae
Streptococcus pyogenes
Sulfolobus solfataricus*
Sulfolobus tokodaii*
Thermoanaerobacter tengcongensis
Thermoplasma acidophilum*
Thermoplasma volcanium*
Thermotoga maritima
Treponema pallidum
Ureaplasma urealyticum

 

*Archaea.

tThe number in parentheses indicates the number of homologs of PPK2 in the organism.

*Eukaryote.

Requirements of PPK2. Requirements of pure PPK2 for the
synthesis of poly P (forward reaction) differed markedly from
those for the utilization of poly P to make GTP from GDP
(reverse reaction) (Table 1). Mn2* (10 mM) was preferred over
Mg?~ in poly P synthesis, but the opposite was true of poly P
as a donor in GTP synthesis (14). GTP (Km, 0.68 mM; Vinax,
6.7 X 10° units/mg protein) and ATP (Ki, 0.5 mM; Vinax, 7.6 X
10° units/mg protein) were equally active in poly P synthesis,
as were GDP and ADP in GTP and ATP synthesis (14). Poly
P stimulated poly P synthesis in the lysate 10-fold, but the
effect was muted with the pure enzyme to which poly Pjs5 had
been added (1.5 «M in Pj residues when diluted 1,000-fold in
the assay mixture). Poly P chains of 15-700 were all effective,
but poly P; was not.

Zhang et ai.

Processivity and Product Size. The poly P-synthesizing reaction
seems to be processive, because a maximal chain length of
~ 700-800 residues appeared from the very beginning of the
reaction (Fig. 2). Compared with E. coli PPK1, which produces
a homogeneous product of 700-800 residues, the P. aeruginosa
PPK1 and PPK2 products have a broader range: ~500-800 for
PPK1 and 200-800 for PPK2 (Fig. 3).

Identification of the ppk2 Sequence. Mass spectroscopic analysis
of the purified PPK2 bands revealed a perfect match with
a “conserved hypothetical protein” (GenBank accession no.
NP_248831) of P. aeruginosa (21), but the amino-terminal se-
quence “SEEPTVSPP” detected in our isolated protein was
absent in the annotated protein. Also absent from the annotated

PNAS | December 24, 2002 | voi.99 | no. 26 | 16681

BIOCHEMISTRY
 

Fig.5. Alignment of PPK2 and two homologs in P. aeruginosa. The identical
amino acids are shaded in solid black. Hom1 is homolog 1 (hypothetical
protein PA2428, GenBank accession no. NP_251118); Hom2 is homolog 2
(hypothetical protein PA3455, GenBank accession no. NP.252145).

gene was a Shine—Delgarno (SD) sequence and the common
ATG start codon. However, at 177 bp upstream in the same
reading frame, we identified an ATG start codon with a strong
SD sequence (AGGAGT) 11 bp upstream of it; translation of
this alternative ORF gives exactly the same NHb2-terminal se-
quence as that found in purified PPK2. Thus, PPK2 contains 357
aa with a molecular mass of 40.8 kDa encoded by a ppk2 of 1,074

bp (Fig. 4).

Cloning of ppk2 and the Activity of the Gene Product. The ppk2 was
cloned into pTrc99A and transformed into £. coli CF5802. Both
PPK2 poly P synthesis and utilization activities were detected in
the lysate of isopropy! B-p-thiogalactoside (IPTG)-induced E.
coli CF5802 (pTrc-PPK2) with a specific activity of 1.7 x 104
units/mg protein for synthesis and 1.3 x 10° units/mg protein for
utilization, each about two to four times of the PPK2 activity in
lysates of P. aeruginosa PAOMS; E. coli CF5802 (pTrc99A)
containing only the vector, had no PPK2 activity [<0.01% of that
of E. coli CF5802 (pTrce-PPK2)].

Conservation of the PPK2 Sequence. The P. aeruginosa PPK2
sequence was used in BLAST searches of the NCBI, TIGR,
EMBL, and DDBJ peptide sequence databases for homologs
with an E value <1 x 10-' (Table 2). In the P. aeruginosa
genome (Fig. 5), two homologs are present: one of similar size

16682 | www.pnas.org/cgi/doi/10.1073/pnas.262655199

(305 aa) and 51.5% identity and the other much larger (497 aa)
with identity only in the carboxyl half of the sequence. Among
other microorganisms, 51 homologs have been identified in 34
species; two of them are Archaea (M. acetivorans and M. mazei)
each with two PPK2 homologs. Homologs are found in both
Gram-positive and Gram-negative bacteria and, notably, in-
clude rhizobia, cyanobacteria, Streptomyces, and many patho-
genic species. Most of the original annotations of these
proteins are “conserved hypothetical protein” or “hypothet-
ical protein,” many with a phosphate binding motif (P-loop)
sequence, GXXXXGK (Fig. 4) commonly found in ATP- and
GTP-binding proteins (22, 23).

The E. coli PPK1 sequence was also used for the same BLAST
searches. Homologs were identified in 32 species that con-
tained both PPK1 and PPK2; only three species with PPK2 lack
PPK1i. Among 43 species that contain only PPK1 are the slime
mold D. discoideum and one Archae, H. volcanii. Lacking both
PPK1 and PPK2 sequences in completely sequenced genomes
are 14 of 17 Archael species, 27 of 55 bacterial species, and
S. cerevisiae (Table 2).

Discussion

Certain mutants of P. aeruginosa lacked the highly conserved
PPK1 that catalyzes the synthesis of poly P from ATP (10). The
persistence of significant levels of poly P in these mutants
sparked our search for the responsible enzyme activity. For lack
of any clues from the PPK1 sequence in the available genomic
databases, we resorted to classical biochemical approaches. We
developed more sensitive activity assays to detect poly P, un-
covered novel requirements for this activity, purified it to
homogeneity and identified its gene. This activity in mutants and
wild-type strains that synthesizes poly P from GTP or ATP is now
designated PPK2 and the encoding gene is ppk2.

Concurrent with these studies, another very potent activity
in P. aeruginosa was pursued in our laboratory, an activity that
utilizes poly P as a donor to convert GDP to GTP (14). Some
kinetic features made it seem that the poly P synthetic enzyme
was separate from the poly P-driven NDK activity. Yet, on
purification of each of these two activities to homogeneity,
both proved to reside in the same protein (PPK2) and to derive
from the same gene, ppk2. We recount here the properties of
PPK2 as a poly P synthetase and the sequence of ppk2 and
discuss the significance of its distribution in a wide variety of
microorganisms. In the accompanying report (14), we focus on
the remarkably strong capacity of PPK2 to use poly P in
the synthesis of GTP and discuss its likely physiological
importance.

Several characteristics distinguish the poly P synthetic activity
of PPK2 from those of PPK1 in P. aeruginosa (Table 1); these
distinctions also apply to the PPK1s of £. coli, V. cholerae,
and H. pylori (11). PPK2 can use either GTP or ATP in the
synthesis of poly P, but the PPK1s use only ATP. In cell lysates,
PPK2 activity is stimulated 10-fold by the addition of poly P,
but no such effect was observed with PPK1. Inasmuch as
the poly P added to PPK2 was not extended (data not shown),
its stimulatory effect may be to promote oligomerization and
stability (14).

Mn?" is preferred over Mg?” by PPK2 for poly P synthesis
activity, whereas the reverse is true of PPK1is. The optimal
level of Mn** for PPK2 is reached at 10 mM, but levels of even
25 mM are tolerated. Whether Mn?* serves by binding the
nucleoside triphosphate or the active site of the enzyme or
both are unknown. A requirement for Mn?* is shared by many
enzymes, including the NDK activity of pyruvate kinase, which
is sustained by Mn?* at the very low level of 0.05 mM (24).
Mn?" can also perform as an inorganic superoxide dismutase
as in Lactobacillus plantarum where, as a complex of 30 mM

Zhang et al.
Mn?~ with 60 mM poly P, it serves the cell in disposing of
superoxide (25).

The processivity of E. coli PPK1 is extraordinary. The uniform
product is 700-800 residues in average length; no shorter lengths
are detectable in the course of the reaction. The P. aeruginosa
PPK 1 is also processive although the distribution of chain lengths
(500-800) is somewhat broader. PPK2 is processive as well (Fig.
3) both in the synthesis of poly P and its utilization (14).

A remarkable feature of PPK2 is that the catalytic potency
(Vinax and ear) in the poly P-driven synthesis of GTP from
GDP is far greater than the synthesis of poly P. The ratio of
poly P utilization to poly P synthesis is 75 for PPK2, but only
0.07 for PPK1 of E. coli. Thus, the ratio with PPK2 differs from
that with PPK1 >1,000-fold. Another distinctive characteristic
is the selectivity for GDP vs. ADP as the substrate for the poly
P-driven NDK activity. The ratio of GDP over ADP for PPK2
at Vinax is 1.1; by contrast, the PPK1s in their NDK actions
strongly favor ADP over GDP with GDP/ADP ratios ranging
from 0.015 for E. coli PPK1 to 0.00016 for V. cholerae PPK1.

A search of the available databases disclosed the wide
conservation of the PPK2 sequence (Table 2). Of immediate
interest are the two homologs in the P. aeruginosa genome.
One is similar in size to PPK2, whereas the other is twice the
size and homologous only in the carboxyl half of the molecule.
It remains to be determined whether these homologs have PPK
activity. Among bacterial species, 33 have PPK2 homologs as
do two archaeal species; most possess PPK1 as well. Among
those listed are some bacteria with developmental cycles (e.g.,
cyanobacteria, caulobacter, rhizobia) and several pathogens.
Also listed are microorganisms that contain only PPK1 (un-
completed genome sequences may have a PPK2 homolog); also
listed are microorganisms that have neither PPK1 nor PPK2.
There are instances of closely related species in which only one
possesses a PPK. As examples are the following: B. subtilis
lacks any PPK but B. anthracis (with two virulence plasmids)
has PPK1; M. tuberculosis has PPK1 and PPK2 but M. leprae
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Porn

a

an

Zhang et al.

Further studies are now contemplated to prepare null
mutants of PPK2 and its several homologs in conjunction with
available ppk1, ndk mutants. The phenotypic features of these
new constructs will be of considerable interest along with
comparisons with clinical isolates of P. aeruginosa (e.g., strain
8830) as well as studies of the other microorganisms that
possess PPK2.

At this stage in our studies, it seems likely that PPKi in P.
aeruginosa, and in other organisms, functions in the synthesis of
poly P from ATP and that PPK2 functions by using poly P to
generate GTP from GDP. In keeping with this proposed func-
tion for PPK2, it is significant that the appearance of the enzyme
at the late-log stage (14) is at the very time when GTP is needed
for the massive synthesis of alginate, the exopolysaccharide that
covers the organism in a mucoid envelope (26, 27). PPK2 may
also have other functions because some organisms (e.g., Strep-
tomyces, etc.) that have PPK2 homologs do not have exopolysac-
charide coats like alginate.

The fact that P. aeruginosa and many other organisms (Table
2) have multiple PPKs suggests that they attach importance to
maintaining poly P levels and its use in a variety of functions,
That PPK can serve as a target for discovery of antibiotics has
already been demonstrated for PPK1 in P. aeruginosa (S. Lee,
personal communication); PPK2 now invites a similar effort.
Among considerations that favor PPK as a target for antibiotics
is its distribution in a wide spectrum of pathogens, the loss of
virulence in ppk mutants (9, 10, 12, 28, 29), and the expected lack
of host toxicity because the PPK sequence seems to be absent in
the sequences of yeast and animal cells.

To Leroy Bertsch we are indebted for advice and assistance in prepa-
ration of this paper. We thank Dr. Anand Sethuraman, who found a PPK
activity in a ppk/ mutant of Klebsiella pneumoniae that used GTP and
that alerted us to GTP as a possible donor for poly P synthesis. We thank
the National Institute of General Medical Science of the National
Institutes of Health for the support of our research and the Yamasa
Corporation for fellowship support of K_I.

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