JOURNAL OF BACTERIOLOGY, Dec. 2000, p. 6687-6693

Copyright 0 2000, American Society for Microbiology. All Rights Reserved.

002 1-9193/00/$04.00+0

Vol. 182, No. 23

Inorganic Polyphosphate in Vibrio cholerae: Genetic,
    Biochemical, and Physiologic Features
NOBUO OGAWA, CHI-MENG TZENG,P CRESSON D. FRALEY, AND ARTHUR KORNBERG"

Departmerit of Biochemistry, Stun ford University School iof Medicine, Stanfiird, Culifomia 94305-5307

Received 12 May 2000/Accepted 8 September 2000

Vibrio cholerue 01, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large
clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by theppk
gene, which has been cloned from V. cholerue, overexpressed, and knocked out by insertion-deletion mutagen-
esis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of
Escherichia coli, which it resembles biochemically. As in E. coli,ppk is part of an operon withppx, the gene that
encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts
of wild-type V. cholerue. The ppk null mutant of V. cholerue has diminished adaptation to high concentrations
of calcium in the medium as well as motility and abiotic surface attachment.

Inorganic polyphosphate poly P is a linear polymer of up to
hundreds of orthophosphate (P,) residues linked by high-en-
ergy phosphoanhydride bonds. Among known functions. poly
P can serve as a substitute for ATP in kinase reactions, a Pi
reservoir, and a chelator of divalent metals (9). Poly P is ubiq-
uitous in nature, having been found in all organisms examined
(15), yet little is known about its physiological roles (14).
Several poly P-metabolizing enzymes have been purified,
and the genes encoding them have been cloned (14, 28). The
enzyme primarily responsible for poly P synthesis in Esche-
n'chiu coli is poly P kinase (PPK), which catalyzes the polymer-
ization of the y phosphatc of ATP into a poly P chain (1). Poly
P can be hydrolyzed to Pi by an exopolyphosphatase (PPX) (3).
Jn E. coli, the encoding genes, ppk andppx, respectively, form
an operon. Thc inability to accumulate poly P upon deletion of
this operon or upon the overproduction of PPX has produced
several striking phenotypes in E. coli (6, 20, 15): decreased
long-term survival in stationary phase; increased sensitivity to
oxidative, osmotic, and thermal stresses; and defects in adap-
tive growth in minimal medium after a shift from rich medium.
Thcsc phcnotypcs arc likely due to the dccreascd cxpression of
the rpoS gene, which encodes the principal stationary-phase
sigma factor, us, or RpoS (25). These and related results (4)
suggest that poly P is an efector signal for rcsponses to acute
stringencies and adaptations in the stationary phase.
Recently available genome sequences have revealed that
PPK is highly conserved in many bactcrial spccics, including
some important pathogens (26). This also implies that PPK
and/or poly P has fundamental physiological roles in bacteria.
The ppk knockout mutant of Pseudomonas neiuginosa PA01
shows a dramatic deficiency in motility, both flagellar and pilus
mediated, an inability to form biofilms, and a loss of virulence
(22,23,24).ppk null mutants of scveral other pathogcns and of
E. coli also exhibit reduced motility and reduced abiotic sur-
face attachment (22, 24).
Vibrio spp. are among the most common microorganisms in
environmental surface waters, such as lakes and rivers. Vibrio

' Corresponding author. Mailing address: Department of Biochem-
istry, Beckman Center, Stanford University School of Medicine, Stan-
ford, CA 94305-5307. Phone: (650) 723-6167. Fax: (650) 723-6783.
E-mail: akornber@xmgm.stanford.edu.
t Present address: Institute of BioAgricultural Science, Academia
Sinica, Nankang, Taiwan.

cholerne 01 is an enteropathogenic gram-ncgative bacterium
that causes severe diarrheal disease. An rpuS mutant of V.
cholerae (29) revealed that RpoS is required for V. cholerae
persistence in a medium devised to simulate natural aquatic
habitats. A gene highly homologous to E. colippk was found in
the V. chokrae database of The Institute for Genomic Re-
search (TIGR) (26). Thc cssential role of poly P for the sta-
tionary-phase survival of E. coli and the possibility of a similar
role in V. cholerae promptcd us to study its PPK and to exam-
ine the phenotype of a ppk knockout mutant.

I/: chihw 01, biotype El Tor, accumulates much higher
lcvcls of poly P than E. coli undcr normal growth conditions.
High accumulations of poly P are stored as granules following
a shift from a defined medium lacking Pi to one with an excess
(20 mM). The ppk null mutant is dcfectivc in motility and
abiotic suriace attachment and fails to adapt to high concen-
trations of calcium.

MATERIALS AND METIIODS

Strains and plasmids. E. coli strains MG1655 (A- I-'-) and CM802 (MG1655
Appk Appx::Kan [17]) were the wild-type and niutant strains, respectively. Re-
combinant plasmids based on pBluescript II KS(+) and SK(+) (Stratagene, 1.a
Jolla, Calif.) were prepared from DHSn transformants. Suicide plasmids based
on pKNGl0l (11) were replicated inE. coli strain S17-l(hpir) (29); pFZYl (13)
was used as a low-copy-number vector. The 92A1552-RiP wild-type strain of V.
cholwue 0 I (El Tor, Inaba [2Y]) and the cnsmid lihrary of ib genomic DNA were
provided by F. Y ildiz, Department of Microhiology and Immunology, Stanfcrd

Media and growth conditions. A MOPS (morpholinepropanesulfoiiic add)-
hulfered minimal medium (21) was usrd to impose PrIirniting conditions for &he
growth of V. cliolernr. Mcdia were supplemented with ampicillin (IO0 ,&nl),
kanamycin (I50 pghil), or streptomycin (100 &mi) to select for antibiotic-
resistant transformants of V. cholerue.
Plasmid Construction. The V. cholerue PPK sequence was obtained by a
BLAST search of the TIGR genome sequence database using the E. coli se-
quence. To ohtain theppk region of K rholerue, two PCR primers were designed
VCPPKFOR I, CCTTCTACACAACrCTATGACACTAAAAC~CAC, and VC
PPKREVI, CCI'G?'CGACI'CTI'C;CCGATGAGAGAIMAGAC. VCPPKFORI
contains anXbuI site, and VCPPKREVl contains a Sal1 site, each located at the
5' end (underlined). These primers yielded a 1.95-kh PCR product using
genomic DNA prepared from the wild-type V. cholerue strain Y2A1552-RiF as a
template. This fragment begins at position -106 relative to the A in the start
codon at +I and ends at position +1820. After digestion with both Xbnl and
Sall, this PCR product was cloned into Xbnl- and Snll-digested pBluescript I1
KS( +); the resulting plasmid wits designated pVCK1.
The cosmid pVCK20 was obtained by colony hybridization from a V. cholerne
genome lihraiy using the 1.Y5-kb PCR fragment as a probe; pVCK2O contained
an insert of more than 40 kh in which theppk homologous region was limited to
:I 2.5-kh NcoI-Ug/ll fragment (pVCK35 [Fig. MI).

Plasmids containing the cloned V. choleme ppk gene were constructed as

University. .,.,

6687

6688 OGAWA ET AL.

J. BACTERIOL.

EcoRVz EcoRV3

A NcoI EcoRVl EcoRIi 1 BgnI I&oR;~~ Pit1

pho box

PCW
pVCKl
pVCK13 4-7

kan

Stem-loop

pVCK35

I I +I

CTCTTTAATGATGAAAACGTCAACTCAAAACGGCATAAGGTMGTmA

ppkORF-stan

c K A K G Q Q E T N D N S S Q ppkORF-slop

?AAGCAAAAGGGCAACAAGAAACGA&EACAACAGTAGTCAGTBB
+2062

ppxORF-sW M T T V V S N

D +p7 +3413 +3429

TBBGCCAGACTAGTCACTGCAATTCGATGACGCCCGCCCAACTC
ppx ORF-slop

AAGTTGGGCGTTTTTCATTTCATCACZTBGTCGA

~3432 +3?8

- uup (duwnstrm gene)

FIG. I. The ppk ppx operon in V. cholerae and recombinant plasmids. (A)
The large box represents the cloned and sequenced 4-kb Ncol-PslI chromosomal
fragment containing the operon. ORFs are indicated by arrows. The lines under
the box indicate the portions of the ppk ppx region inserted in the plasmids
indicated to the left. The nonunique EcoRl and EcoRV restriction sites in the
4-kb NcoI-Psfl fragment are denoted by subscripts. (B) Putative pho box se-
quence in the ppk promoter region. (C) Sequence overlap between the ppk and
pp ORFs. The center sequence is the nucleotide sequence of the ppkppx ORF
junction, and the top and bottom sequences are the deduced amino acid se-
quences for PPK and PPX, respectively. (D) Putative transcriptional terminator
sequence in the region downstredm of pp. The pair of sequences indicated by
the arrows have the potential to form a stem-loop structure. The underlined
nucleotides are the indicated start and stop codons.

follows. A 6.5-kb I~indIlI-f'stl fragment containingppk prepared from pVCK10
was inserted into similarly restricted pBluescript II SK( +). The resulting plas-
mid, pVCK27, was digested with Hirid111 and Ncol. and the 7-kh fragment was
isolated. After the ends were filled in, this fragment was self-ligated. The result-
ing plasmid, pVCK3I (Fig. IA), contains the 4-kb NcoI-At1 fragment in pBlue-
script 11 SK(+). A smaller plasmid, pVCK35 (Fig. In), was constructed by
digestion of pVCK31 with BgRl and BainIII, followed hy self-ligation of the
resulting longer fragment; pVCK35 harbors the 2.5-kb Ncol-8g/Il fragment in
pHhescript 11 SK(+).
A low-copy-number plasmid containing the V. cholerae ppk ppx loci, pVCK37,
was constructed as follows. pVCK31 was digested with BamlIl and Sall, and the
fragment with the 4-kb Ncol-Pstl ppk ppx region was isolated. The IlamHI-Sal1
fragmcnt of about 4 kb was inserted into a similarly restricted low-copy-number
vector, pFZY1, resulting in plasmid pVCK37.
Conslruction of the Y. choleraeppk knockout mutant. To make the deletion-
insertion mutation allele ofppk, a 1.2-kb EcoRV (site I)-EcoRI (site I) region of
pVCKl (Fig. 1A) was replaced with a 1.3-kb Hiricll-EcoN fragment containing
the kanamycin cassette from pVCK4, which was constructed hy insertion of a
1.3-kb Ifincll-Hincll fragment with the cassette from pUC4K (Amersham Phar-

   Biotech, Inc., Piscataway, N.J.) into the EcoKV site of pBluescript I1
KS(+). From the resulting plasmid, pVCK7, the 2.1-kb Xbal-Sal1 ppk::Kan
fragment was transferred into the suicide vector pKNG 101, and the resulting
plasmid was designated pVCK13 (Fig. IA).

pVCK13-integrated transformants (single-crossover recombinants) of the V.
cholerac wild-type strain 92A1552-RiP which exhibited both kanamycin and
streptomycin resistance phenotypes were obtained. After hvo successive over-
night cultivations of the integwnts in Ixria broth (1.B) supplemented with 5%
sucrose, the ppk knockout mutants (double-crossover recombinants) were ob-
tained as kanamycin-resistant but streptomyciii-sensitive colonies. "lie chromo-
soinill mutations were confirmed by Southern blot analysis, and one of the
resultant recombinants, KVC3, was selected as the ppk knockout mutant of
V. clicdcrac.

Biochemical assays. Cell extracts from V. cholerae were prepared as from E.
coli (IO), modified only by a 2-inin sonication after lysozyme treatment. PPK and
PPX activities were assayed as described previously (1, 17). Poly P levels were
determined by the nonradioactive method (4). The cloned DNA fragment was
sequenced by the PAN Facility.
Electron microscopy. Strains were cultivated as described in the legend to Fig.
3D. Cells were harvested at zero time and 2 h after the addition of Pi and were
washed three times with 0.9% sodium chloride (zero time) or phosphate-buff-
ered saline (2 h) solution. For negative staining, the samples were placed on a
carbon-coated grid and stained with 1%. uranyl acetate. Thin-section samples
were prepared by N. Ghori, Department crf Microhiology and Inimunology,
Stanford University.
Surface attachment assay. Cells (2 PI) cultured overnight in LB were inocu-
lated into 100 pl of 1.B in 96-well polyvinyl chloride plates (Falcon 391 I Mi-
crotester III flexible assay plate; Ikcton Dickinson, Oxnard, Calif.) and incu-
bated at 30C without shaking for 24 h. After the medium was removed. each well
was rinsed with sterile water, 100 pl of 1% cr).stal violet solution was added, and
the plate was incubated for 15 iiiin :it room temperature. The wells were thor-
oughly washed with water and air dricd. The adhcrent crystal violet was extracted
with 100 (*I of 95% ethanol. diluted in 95% ethanol, and measured at 595 nm in
a microplate spectrophotometer (model 550; Bio-Rad Laboratories, 1 lercules.
Calif.).
PI uptake assay. Cells were grown in a MOPS medium with 0.1 mM Pi
overnight, collccted, washed with a Pi-free MOPS medium, and resuspended in
50 ml of the Pi-free medium to an optical density at 6110 nm (OD,,,) of 0.1. The
cultures were shaken for 2 h at 37C arid were readjusted to an OD6,,,, of 0.1 by
dilution with the Pi-frec MOPS nicilium. K,HPO, (0.1 mM) with [32Pcj;]Pi
(1 pCiiml) was added to the shaking cultures. and samples (0.1 nil) were taken
at intervals and immediately filtered through an IA filter (3.5-nim diameter;
Millipore). The cells trapped on the filter were wished with 10 ml of Pi-free
MOPS medium without glucose, amino acids, or vitamins. The radioactivity on
the membrane filter was measured in a liquid scintillation counter; the values of
Pi uptake in the cells are indicated as counts per minute per milliliter per OD,
unit.
Purification of V. cholerae PPK E. coli strain CF5802 transformed with
pVCK31 was used as the starting material (4.2 X IO8 U in 610 mg of protein).
The purification was performed essentially as described previously (2). modified
only by the addition of a phenyl Scpharose column (Amersham Pharmncia
Biotech, Inc.) as the final step (yielding 4.6 X IO6 U in 0.12 mg of protein).
Nucleotide sequence accession number. The GenRank accession number for
thc sequence reported here is AFOX3(J28.

RESULTS

Poly P accumulation. When E. coli was grown in rich mc-
dium (LB) under normal conditions (37C with aeration), poly
P levels did not exceed 1 nmol (in P, residues) per mg of total
cell protcin at any stage (21). Thc highest lcvels of transient
poly P accumulation, about 25 nmol/mg of protein, occur when
E. coli cells are exposed to specific stresses (4). However, V.
cholerue strain 92A1552-RiP, cvcn when grown in LB, accu-
mulated poly P to levels of more than 50 nmol/mg (Fig. 2A)
during logarithmic growth, while in stationary-phase cells $e
levels were low (-1 nmol/mg). In MOPS minimal salts me-
dium, V. cholerue accumulates more than 20 nmol ol poly P/mg
in stationary phase, far exceeding the <1-nmol/nig levels for E.
coli (21). Thus, K cholerue accumulates significantly higher
level\ of poly P than E. coli at all stages: during growth in rich
media and during stationary phase in minimal media.
To determine whether the high level of poly P accumulation
in V. cholerue relative to that in E. coli is due to a higher level
of PPK, wc measured PPK activity in V. cliolerue and found
that the membrane fraction activity (Table 1) was quite similar
to that of E. coli MGl6SS. PPK activity levels werc virtually the
samc in logarithmic- and stationary-phase cells (data not
shown). Thus, poly P accumulation levels do not reflect the
levels of PPK activity, which is also true in E. coli (21). In
contrast to E. coli, no dctcctablc activity of PPX was obsewcd
in V. cholerue (Table I). Since a measurable level of PPX
activity was detected in an E. coli Appk Appx strain bearing a
plasmid with thc K cholerue ppk ppx operon (sec bclow), V.
cholerue PPX should be expressed. The undetectable level of
native PPX activity may explain in part why K cliolcroe accu-
mulates largc amounts of poly P. Thc rapid degradation of poly

VOL. 182, 2000

POLYPHOSPHATE IN V. CHOLERAE 6689

W

0 2 4 6 8 1012
    Time (h)

O_ 1 2 3 4 5 6 7 8 9

4

C --

0 I2 3 4 5 6 7 8 9 10
    Time (h)

h

400

c

. .-

D2

   s
-300 &

rc

0
M

00-

ax

.-

0
a

0.1 0
0123456

Time (h)

FIG. 2. Poly P accumulations in V. cholerue. (A) Following overnight culture
in LB, the wild-type strain Y2A1552-RiP was subcultured into fresh LB at a 1:100
dilution and grown at 3PC with aeration. (B and C) Following overnight culture
in MOPS medium with 0.1 mM Pi, the wild-type strain was subcultured in MOPS
medium with 0.1 (squares), 0.5 (circles), or 2 (triangles) mM Pi. Solid symbols,
growth (A5.,,,); open symbols, poly P. (D) Following overnight culture in MOPS
with 0.1 mM Pi, the wild-type (squares) and KVC3 @pk mutant [circles]) strains
were harvested and washed with MOPS buffer. The cells were transferred to
Pi-free MOPS and cultivated for 1 hat 37C with aeration. Potassium phosphate
buffer (pH 7.4) was then added to a final concentration of 20 mM Pi at zero hour.
Soiid symbols, growth (AGw); open symbols, poly P.

TABLE 1. PPK and PPX activities and poly P
    levels in V. cholerae and E. coli"

PPK activity PPX Poly P

Strain Genotypeb (Um9 activity level

Soluble Membrane ("'%) (nmo"mg)

V. cholerae
92A1552-Rif WT
KVC3

1,120 17,300 <30 25.3

ppk::Kdn <30 <300 <30 c0.10

E. coli MG1655 WT 550 14,000 1,200

~~ ~~~

a PPK and PPX activities were determined using cell extracts prepared from
stationary-phase cultures in LB, and poly P levels were determined using cell
extracts prepared from stationary-phase cultures in 2 mM Pi MOPS medium.

WT, wild type.

P shown in Fig. IA, in the abscnce of significant PPX activity,
may be due to the reverse reaction of PPK (27), in which poly
P is converted to ATP. A Klebsiella aerogenesppx mutant strain
dcmotistrated profiles for poly P accumulation and degrada-
tion similar to those of the parental wild-type strain (A. Ku-
roda, personal communication).
Poly P overplus. The influcnce of Pi concentration on poly P
accumulation was determined in cells grown in a defined me-
dium (MOPS) (Fig. 2B and C). Poly P levels wcrc below thc
dctection limit (<OS nmol/mg) when cultivated with 0.1 mM Pi
overnight (Fig. 2C, 0 h). However, >100 nmol/mg accumulated
aftcr 4 11 in log phase with 0.1, 0.5, or 2 mM Pi. Cultures with
0.5 or 2 mM Pi maintained poly P levels of > 100 nmolfmg for
up to 9 h, it., after entering stationary phase. However, poly P
lcvcls in thc 0.1 mM Pi culture dccrcased gradually along with
the decrease in growth rate (Fig. ?B and C, 5 to 9 h). Given the
similarity in the growth curves for the 0.5 and 2 mM Pi cultures,
the slow growth of the 0.1 mM Pi culture is likely due to Pi
depletion. Thus, poly P levels in V. cholerae depend on the Pi
concentration in the medium.
When cclls werc shifted from a MOPS mcdium with 110 Pi
added to the same medium with 20 mM Pi, dramatic accumu-
lations of poly P occurred immediately after the upshift (Fig.
2D). Within 1 h, poly P accumulated to >300 nmol/mg andgto
near 400 nmol/mg after 2 h without any concomitant growth.
With the resumption of growth, the poly P level decreased to
250 nmolimg and rcinaincd there for at Icast fivc morc hours.
Similarly, large poly P accumulations have been reported in
AeroDacter nerogmes as the "poly P overplus" phenomenon (9).
These massive levels of poly P were observed by electron
microscopy as numerous bodies of relatively high electron den-
sity (Fig. 3). About 1 in 30 cells contained bodies 20 to 40 nm
in diameter (Fig. 3B), while the others had smaller bodies,
about 5 nni in diameter (Fig. 3A). Such granules were not
found in wild-type cells grown in Pi-free medium or in ppk
mutant cells (see below) grown in Pi-rich medium for 2 h (Fig.
3C and D). Inasmuch as these bodies were correlated with the
dmassivc accumulations of poly P, they arc prcsumed to bc
poly P. Similar granules observed in Myxococciis xzntliiu (9)
and Helicobacter pylori are somewhat localized at the flagellar
polc and in association with the inner mcmbranc as well as
being dispersed in the cytoplasm (5). As determined by thin-
section electron microscopy (Fig. 3E), the granules in K ckol-
erue arc distributcd in thc cytoplasm but not localizcd along thc
inner membrane.
The ppk gene. The region in the TIGR V. cholerue genome
database surrounding a scquence homologous to that of E. cdli
ppk was cloned from a V. cliolerar genome library by colBiiy
hybridization using a PCR fragment of the region as a probe
(see Materials and Methods). Thc E. coli Appk Appx knockout

6690 OGAWA ET AL.

J. BALIERIOI..

FIG. 3. Electron micrographs of V. rholerae strains. The bacteria were harvested at 0 and 2 11 ahcr the addition of Pi, as for Fig. 2D. Bars, 0.5 pm. Negatively stained
samples were prepared from the wild-type culture after 1) (C) and 2 (A and B) h of incubation and from KCV3 (theppk mutant) after 2 h of incubation (D). Thin-section
samples were prepared from the wild type (E) and KVCB (F) after 2 h of incubation. Magnifications, X28,OOO (A, C, E, and F). x35,OOO (D), and X45,OOO (B).

strain CF5802, transformed with pVCK35 containing the I/.
cholerae ppk homolog (Fig. 1 A), exhibited high levels of PPK
activity (38,000 U/nig), demonstrating that this region did in
fact contain the K cholerueppk gene; this was confirmed sub-
sequently by sequencing. `The deduced amino acid sequence of
K cholerae PPK is 701 amino acid residues long, with a calcu-
lated molecular mass of 81.6 kDa; it is 64% identical and 83%
similar (in conserved residues) to that of E. coli. Sequencing
also revealed a ppx homolog downstream ofppk in I/. cholerue,
as in E. coli, although with a two-cistron overlap. A transfor-
mant of the E. coli Appk Appx mutant strain CF5802 harboring
pVCK31, which contains both the ppk andppx homologs (Fig.
lA), gave low but significant levels of PPX activity (540 U/mg).
Its deduced amino acid sequence is 500 amino acid residues in
length, with a calculated molecular mass of 56.4 kDa, and is
51% identical and 70% similar (in conserved residues) to E.
coli PPX.
Upstream of the ppk open reading frame (ORF) (Fig. 1B)
lies a putative promoter region which contains a probablepho
box sequence with 15 of the 18 consensus base pairs at posi-
tions -84 to -67 from thc A in thcppk start codon (Fig. 1B).

In E. coli, pho boxes are the binding sites of the two-compo-
nent system regulator protein PhoB. Homologs of phoH and
phoR (the cognate sensor) are found in the I/. cholerue genome
database, suggesting that transcription of the ppk ppx operon
may be regulated by this system. Putative pho boxes are also
present in the ppk promoter regions of E. coli, K. aerogenes
(12), and Acinetohacfer sp. strain ADP1 (8). A pair of 17-bp
inverted-repeat sequences (from positions 24 to 40 and 43 to
59 from thc ppx stop codon) appear to constitutc a rho-inde-
pendent transcriptional terminator site downstream of the ppx
ORF (Fig. 1D). These features imply thatppk andppx form an
operon, as in E. coli and other gram-ncgativc bacteria. The
E. coli Appk Appx transformant with a high copy number of the
V. cholerue ppk ppx operon [CF5802(pVCK31)] expressed
690,000 U of PPK and 540 U of PPX activity per mg. We havc
shown previously that the E. cob wild type transformed with a
high copy number of the E. coli ppk ppx operon exhibited
630,000 U of PPK and 50,000 U of PPX activity pcr mg (2. 3).
Thus, I/. cholerue PPK was expressed in an E. colt host cell at
levels similar to those of E. coli PPK from the E. COIL operon,
but V. cholerue PPX was expresscd about 100-fold less than E.

VOL. 182, 2000

POLYPHOSPHATE IN V. CHOLERAE 6691

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0.5

A6001 0.3

0.2 j-/H

0 2 4 6 8 10 2022

Time (h)

FIG. 4. V. choleraeppk complements the adaptive growth defect in an E. coli
ppk mutant. E. coli strains MG1655(pFYZl) (squares), CF5802(pFYZl) (open
circles), and CF5802(pVCK37) (solid circles) were grown in 2XYT medium
supplemented with 50 Fg of ampicillin/ml to an A, of 0.5. The cells were
harvested and washed twice with MOPS medium devoid of nutrients and resus-
pended in MOPS medium with 2 mM P, and 0.4% glucose at zero hour.

coli PPX. Unlike in E. coli, there is a 20-bp overlap between
the ppk and ppx ORFs in the I.: cholerne operon (Fig. IC),
which may interfere with translation of the ppx ORF from the
ppk-pyx mRNA, accounting for the undctectable level of PPX
activity (Table 1).
Null mutant ofppk. In appk knockout mutant in V. cholerne
(KVC3) (see Materials and Methods), the levels of PPK activ-
ity and poly P accumulation were undetectable (Table I and
Fig. 2D), in contrast to the parental strain, 92A1.552-RiIT,
which accumulatcd more than 300 nniol of poly P per mg when
shifted from a Pi-free to a 20 mM Pi defined medium. We
tested KVC3 for the reported phenotypes of the E. coli ppk
mutant strain CAl0 (5, 18, 20). No phenotypes wcrc obscrvcd
for long-term survival in synthetic medium (for 30 days at
30"C), sensitivity to heat (at 45 and S5OC) and hydrogen per-
oxide (in 10, 3, and 1 mM). adaptivc growth following a shift
lrom rich medium to minimal medium, or long-term (IO days
at 30C) survival in artificial seawater (29).
With regard to adaptive growth in a nutrient downshift (Fig.
4), the E. coli wild-type strain MG1655 transformed with the
plasmid vector (pFZY1) grew in a minimal medium after a 2-11
lag following downshift from a rich medium (LB) whereas the
E. coli Appk Appx mutant CF.5802 harboring pFZYl was un-
able to grow even 22 h after the downshift. However, CF.5802
bearing the I< cholerue ppk plasmid pVCK37 grew in minimal
medium after the downshift with only a 4-h lag, after which the
growth rate and final OD wcre similar to that of the wild typc.
Thus, the V. choleme ppk complements the E. coli Appk mu-
tation in response to this stress condition.

A P. ueruginosa PA01 ppk mutant shows no defects in adap-
tive responses but is severely impaired in motility and surface
attachment (22, 23, 24), and the V. cholerue ppk mutant was
found to be deficient in these features as well (23). The swim
area of theppk mutant was 57% that of the wild type on 0.3%
agar plates (Table 2). The ppk mutant also exhibited a signif-
icantly lower ability for surface attachment (Table 2). Deficicn-
cies in motility and surface attachment have also been observcd
in ppk mutants of E. coli, K. pneurnoniue, and Sulmonellu spp.
(22).

TABLE 2. Swimming and surface attachment

Genotype Swim area Surface attachment

(anf)* (Adb

Strain

                                2.14 2 0.70
92A1552-Rif WT 1.52 2 0.07
KVC3 ppk::Kan 0.87 2 0.07 1.46 ? 0.30

a Data are from Rdshid et al. (23).

Average of 16 measurements; two-tail P value by f test with a 0.05 threshold

WT, wild type.

is 0.0012.

In view of the capacity of poly P to function as a chelator of
divalent metals (Y), the calcium sensitivity of the 1/. cholerue
ppk mutant was tested (Fig. 5). The growth lag time of the
wild-type strain (92A15.52-Rif) in LB containing 200 mM
CaCI, was 5 h, more than 4 h longer than in the absence of
CaCI, (Fig. 2A). The lag time of the ppk mutant KVC3 was
significantly longer, at 7 h. When complemented with the high-
copy-number plasmid pVCK31 harboring theppk gene, the lag
time was shortened to 3 h. As both the wild type and the pppk
mutant of 1/. cholerue had the same lag time when grown in LB
in the presence of 400 mM NaCl (data not shown), thcsc
results imply that theppk gene is involved in an adaptation to
excess levels of calcium but not chloride or osmolality.
Anothcr phcnotype of the ppk mutant was observed with
regard to Pi uptake (Fig. 6). After growth in a Pi-free medium
for 2 h, wild-type cells displayed a linear (nonsaturable) Pi
uptake for up to 2.5 min when incubated in 0.1 mM Pi (the
P,-limited condition) (Fig. 2B and C). The Appk mutant had
unique saturable profiles for Pi uptake. It showed a ratd"of
uptake similar to that of the wild type from 0 to 3 min, but the
uptake after 5 min was minimal and the rate was decreased
significantly. These data suggest that ppk is required for the
continual high-rate uptake of Pi under low-Pi conditions.
Characterization of PPK. PPK was purified from a transfor-
mant of the E. coli CF5802 strain (Appk Appx::Kan) bearing
the V. cliolerue ppk ppx operon on a high-copy-number vector,
pVCK31. Homogeneity of the purified protein was verified as

10 ,

wt[vector]

ppk-[vector]

A600

0.5

::~&. , . , . . ,I

0.05

0 2 4 6 8 10 12 14 22 24

Time (h)

FIG. 5. Growth adaptation to an excess amount of calcium. V. cholerae
strains Y2A1552-Rif(pBluescript II) (squares), KVC3(pBluescript 11) (open cir-
cles), and KVC3(pVCK31) (solid circles) were grown in LB medium supple-
mented with 10 mM KH,PO, and 50 kg of ampicillin/ml overnight. The cells
were inoculated in LB supplemented with 200 mM CaCI, and shaken at 37C.

6692 OGAWA ET AL.

J. BACTERIOL.

0 5 10 15 20 25 30
       Time (min)

FIG. 6. Pi uptake of V. cholerae. Pi uptake activities of 92A1552-RiP
(squares) and KVC3 (circles) were tested as described in Materials and Methods.

a single band on a Cooniassie-stained sodium dodecyl sulfatc-
polyacrylamide gel (data not shown) with a molecular mass
estimated at 87 kDa, compared to the calculated mass of 81.6
kDa. Comparison of the PPK retention time to those of ref-
erence proteins in a high-performance liquid chromatography
gel filtration column showed a molecular mas of 310 kDa
(data not shown), indicating that V. cholerue PPK is a homotet-
ramer like E. coli PPK (1). The final fraction has a specific
activity of 40 X lo6 U/mg, slightly higher than that of E. colt
PPK (29 X 106 U/mg) (1).
The optimal reaction conditions for V. cholerue PPK are
almost the same as those for E. coli PPK, e.g., a pH optimum
of 7.2 in HEPES buffer and 4 mM MgZf, and 40 mM ammo-
nium sulfate increases activity twofold. Autophosphorylated
PPK was observed in a reaction with [y-32P]Ai'P. Like the E.
coli cnzymc (16), the 1/. cholerue cnzymc can catalyzc the
synthesis of ATP from ADP and poly P and can catalya the
5ynthcsis of GTP and ppppG (linear guanosine 5'-tetraphos-
phate) from GDP and poly P. Significant differences in the
hinetic parameters for the PPKs of V. cholerue and E. coli are
shown by the 40-fold decrease in the I.: cholerue K,,, value for
ATP for the forward (poly P synthesis) reaction (Tablc 3). Thc
k,,/K, ratio for the ATP synthesis reaction of I/. cholerue PPK
is more than 10 times higher than that for E. coli. On the other
hand. the k,,JK,,,s ratios for the GTP and ppppG synthcsis
reactions of V. cholerue PPK are 5 and 20 times lower, respec-
tively, than those for E. coli. These data suggest that the K
clzolerue PPK is niorc specific for the generation of ATP than
GTP or ppppG.

DISCUSSION

Accumulations of poly P in V. cliolerue arc rcmarkablc for
being so great and sustained compared to those in E. coli. The
levels in excess of 50 nmol/mg of protein during exponential
growth in a rich mcdium (Fig. 2A) and 150 nmol/nig in sta-
tionary phase in a defined medium (Fig. 2C) are roughly 100
times those in E. coli. Yet the PPK activities in extracts arc
ncarly the same (Table 1). Some of the reason may lie in the
undetectable levels of PPX activity in extracts of V. cliolerue
(Tablc 1). In that organism, as in E. coli (3), an operon con-
tains thc ppk as wcll as thc pp.r gcne (Fig. 1). Unlikc in E. coli,

where thepyx gene is separated by 7 bp from the upstreamppk
gene, thc amino terminus-cncoding region of ppx in V. cholerue
overlaps the carboxy terminus-encoding region ofppk by 20 bp
(Fig. 1C). The E. coli transformant with the V. cholerueppx
gene on a high-copy-number plasmid did cxprcss PPX activity.
How these genes and possibly others are regulated pre- and
posttranscriptionally remains to be determined.
When cells arc switchcd from a Pi starvation nicdium to one
with adequate Pi, there is a massive accumulation ol poly P
(Fig. 2D). as observed in A. izerogenes (9) and dcsignated the
poly P overplus phenomenon. The accumulation of poly P is
evident as electron-dense granules up to 40 nm in diameter
with an ordered matrix structure as in crystal complexes (Fig.
3B). The granules appcar to be largely cytoplasmic (Fig. 3E),
unlike some of the granules in H. pylori, which have polar and
membrane-oriented locations (5). The dynamic accumulation
and removal of poly P and its mobilization at a molecular level,
as well as the nature of the granules and their cellular loca-
tions, nccd to be clarificd.
K cholerue PPK resembles that of E. coli in size and in its
multiple activities: processive poly P synthesis from ATP, nu-
clcoside diphosphate kinase action on ADP and GDP by donor
poly P, pyrophosphoral transfer to GDP to form ppppG, and
autophosphorylation by ATP. The most notable differences are
in thc kinetic parametcrs (Tablc 3), cg., a K, for ATP of 0.2
mM for V. cliolerue PPK compared to 2.0 mM for E. coli PPK.
Also, kinetic parameters for ATP, GTP, and ppppG synthesis
rcactions indicate that K cholcrue PPK is more specific for thc
generation of ATP than for GTP or ppppG cornpared to E. coli
PPK. These enzyme characteristics are similar to those of H.
pylori PPK purified as a rccombinant protcin (27; C.-M. Tzeng
and A. Kornberg, unpublished data).
The strong PPK sequence homologies of 20 or more bacte-
rial specics includc a number of the major pathogcns (26), V.
cholerue among them. In view of the striking dependence of E.
coli on PPK for a variety of adaptive responses in the stationary
phase and the cxprcssion of virulcncc factors in the stationary
phases of some pathogens (7), the phenotypes of null mutants
of ppk in these pathogens have been sought. Furthermorc,
there is the attractive possibility that PPK might prove a novel
target for an antimicrobial drug with a broad spectrum and
minimal side effects, inasmuch as PPK has not been found in
animal species.
Among the pathogenic features of the PPK null mutants, a
decrease in motility (coinnionly associated with a loss of viru-
lence [191) and weakened attachment to abiotic surfaces (a

vi.

rL

     TABLE 3. Kinetic parameters of PPK

Keactioii and K,,, vmar hat Lt&

SOLI~C~ (mM) (pmol mg-' min-') (s-') (s-l mM-')

Poly P bynthesis
v. clrolerue
1.1 coli"

ATP synthesis
V. choleruc.
E. colic
GTP synthesis
L'. clrolerue
E. coli'

ppppG synthesis
V. choleruc
E. coli

0.05
2

0.12b

0.25

2.5'
0.63

2.3'
0.46

1.6 X 10"
51 X lob

95 x 106
3.7 x loh

1.5 x 104
5.7 x 104

1.1 x 103

9.0 X lo3

8.2

59


65.3
10.5

0.076
0.088


0.006
0.027

164

29.5

544
42

0.03
0.14

0.002
0.04

"E. coli data for poly P synthesis arc from Ahn and Kornherg (1).

Values for ADP or GDP.
E. coli data for ATP and GTP synthesis are from Kuroda and Kornhcrg (16).

VOL. 182. 2000

POLYPHOSPHATE IN V. CHOLERAE 6693

frequent correlate of poor biofilm formation) have been ob-
scrvcd in several pathogens. Particularly striking arc mutants
of P. aemginosu (22, 23, 24) which are defective in quorum
sensing and have also lost their virulence in mouse models. The
V. cholerue ppk mutant also had diminished attachment to an
abiotic surface (Table 2), with the activity decreased by 68%
compared to that of the wild type. Although this was not a
dramatic reduction as with P. aeruginosn (22, 24), it could be
    ed by using the rugose colony variant of V. cholerae
01 (30), which prefcrs to form biofilm, compared to thc
smooth colony variant which was used in this study.

The failure to adapt to stress and the lack of survival in
stationary phase observed in the E. coli ppk mutant (6) have
not been apparent in the I/. cholerue mutant. In addition to
decreased motility and decreased attachment to abiotic sur-
faces (Table 2), other dcfects have been observed. One is ii
delayed adaptation to high calcium levels in the medium (Fig.
5); complementation of the mutant with the ppk gene more
than corrects for the cxtcndcd lag in growth. It is plausible that
a large amount of poly P can trap excess calcium in the cell and
maintain the calcium level at a low enough level to permit
growth. Another defect of the ppk mutant is an abnormal ratc
of uptake of Pi from the medium (Fig. 6). Whereas the initial
rate resembled that of the wild type, the subsequent rate was
considcrably rcduccd. Not cnough is known about Pi uptake in
V. cholerue to identify which system might be affected by the
lack of PPK function. Interestingly, budding-yeast mutants de-
ficicnt in poly P accumulation also showed a similar saturable
curve for Pi uptake (18a).
The phenotypic tests of the V. choleme ppk mutant were
pattcrncd on thosc pcrformcd on bactcrial species which diffcr
sharply from V. choleme in their physiologic features, commen-
sal intcractions, and host invasiveness (10). In view of the
unique aspects of the aquatic ecology of I.: cholernc, much
needs to be studied to evaluate what effect the lack of PPK and
poly P might have on its survival and pathogenesis.

ACKNOWLEDGMENTS

We thank F. N. Yildiz and G. Schoolnik in thc Departmcnt of
Micr(>t)iciI(igy and Immunology for providing strains, plasmids, the
gene library, and technical advice, N. Ghori in the same department
for electron microscopy, S. 1I;indy in the Department of Chemistry for
high-performancc liquid chromatography analyses, N. Rao in our lab-
oratory for performing surface attachment assays, and L. Bertsch for
help with the manuscript.
This work was supportcd by a grant from the National Institutc of
General Medical Sciences.

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