JOURNAL or BACT`EKIOLOGY, Feb. 1995, p. 491496

Copyright 0 1995, American Society for Microbiology

0021-9193/95/$04.00 +O

MINIREVIEW

Vol. 177, No. 3

Inorganic Polyphosphate: Toward Making a Forgotten
      Polymer Unforgettable

            ARTHUR KORNBERG
Depavtment of Biochemistry, Stanford University School of Medicine,
       Stanford, California 94305-5307

Inorganic polyphosphate (poly P) is a linear polymer of
many tens or hundreds of orthophosphate (P,) residues linked
by high-energy phosphoanhydride bonds (Fig. 1). Likely a
prominent precursor in prebiotic evolution, poly P is now
found in volcanic condensates, deep-oceanic steam vents, and
every living thing-bacteria, fungi, protozoa, plants, and mam-
mals (27). Yet, poly P has been ignored in textbooks and
dismissed as a "molecular fossil." This minireview intends to
make a case for poly P as a "molecule for many reasons" (24).
Numerous and varied biological functions are performed by
poly P, depending on the need and its location-species, cell,
or subcellular compartment. Among these functions are acting
as a reservoir of energy and phosphate, as a chelator of metals
(e.g., Mnz+ and Cazf), as a buffer against alkali, as a capsule
of bacteria, in competence for bacterial transformation, in
ecological disposal of pollutant phosphate, and, of great inter-
est, in physiologic adjustments to growth, development, stress,
and deprivation.

METACHROMATIC GRANULES ARE INORGANIC
          POLWHOSPHATE

Poly P was first seen as metachromatic granules in microor-
ganisms in the form of particles stained pink by basic blue dyes
and was called "volutin" early in this century (33). For some
time poly P granules were mistaken for nucleic acids. With the
advent of electron microscopy, these particles were seen to be
highly refractive and appeared to volatilize while viewed under
the electron beam; they were then identified as poly P (51).
Like other polyanions, poly P shifts the absorption of a bound
basic dye, such as toluidine blue, to a higher wavelength.
Historically, the poly P particle was recognized as a diagnos-
tic feature of medically important bacteria, such as Coiynebac-
teriiim diphtheriae. Decades later, poly P became of interest in
biochemistry in connection with the major biochemical riddle
of the 1940s, i.e., how Pi is fixed by an anhydride bond to ADP
in aerobic (oxidative) phosphorylation. Such studies led first to
the source of inorganic PP, (23) and then to a curiosity about
how the many more phosphoanhydride-linked residues in poly
P were assembled. Although Escherichia coli, a major source of
biochemical insights, lacks any visible content of poly P, it still
proved to be a rich source of an enzyme which makes poly P
(poly P kinase [PPK]) and catalyzes the more favored conver-
sion of poly P to ATP (25, 26) as follows:

nATP - poly P, + nADP

           ASSAY OF POLY P
Despite the prominence of poly P in many organisms, such
as in the vacuolar deposits in yeast cells that may represent 10

to 20% of cellular dry weight, this molecule has received rel-
atively scant attention. Studies by Kulaev, Harold, Wood, and
a few others (17, 29, 53) disclosed the ubiquity of poly P and
identified a few related enzyme activities. Yet, poly P has
remained a largely forgotten polymer. One of the reasons for
this is the lack of evidence for any essential metabolic role.
Another reason has been the inadequacy of methods to estab-
lish the authenticity and size of poly P and its abundance at
very low concentrations.
In addition to the staining and appearance of the granular
accumulations observed by light and electron microscopy, nu-
clear magnetic resonance (NMR) analysis has been used to
identify poly P in intact cells. However, NMR detection re-
quires high concentrations and fails to measure the poly P in
aggregates and in metal complexes. Otherwise, identification
of poly P rests on crude and cumbersome separations in cell
extracts from the known phosphate-containing polymers fol-
lowed by determination of the acid-lability characteristic of
phosphoanhydride bonds (Le., conversion to P, in 7 min at
100C in 1 M HC1). These assay methods are not sufficiently
quantitative to be conclusive, especially for low concentrations

Enzymology offers an attractive route to analysis as well as to
physiologic functions. Enzyme isolation has on many occasions
revealed a novel mechanism or sometimes an insight into a
metabolic or biosynthetic pathway. Now, the purified enzyme
has also opened the route of reverse genetics; its peptide se-
quence leads to its gene and thereby to the means to knock out
the gene or overexpress it. By manipulating expression of the
gene and the cellular levels of its product, phenotypes are
created which may provide clues to metabolic functions. More
immediate and decisive, as in the studies of poly P, the enzyme
can be a unique and invaluable reagent for analytic and pre-
parative work. Toward this end, several enzymes have been
purified and used for studies of poly P metabolism (1, 3, 54).
One enzyme, PPK, purified to homogeneity from E. coh,
catalyzes the readily reversible conversion of the terminal (y)
phosphate of ATP to poly P (1) (see equation above). The
enzyme, a tetramer of 80-kDa subunits bound to cell mem-
branes, is responsible for the processive synthesis of long poly
P chains (ca. 750 residues) in vivo; labeling with 32P in vitro
provides such chains for use as substrates and standards. With
ADP in excess, PPK converts near 90% of poly P to ATP,
identified by the use of either [14C]ADP or 32P-poly P as sub-
strates. A second E. coli enzyme, exopolyphosphatase (PPX),
encoded by a gene in the ppk operon (see below), hydrolyzes
the terminal residues of poly P to P, processively and nearly
completely with a strong preference for a long-chain substrate

A third enzyme, an exopolyphosphatase (sc PPX1) isolated

of poly P.

(3).

491

492 MINIREVIEW

J. BACI'ERIOL.

FIG. 1. Inorganic poly P as a substrate. Chains are attacked at their termini by AMP, ADP, glucose, or H,O, catalyzed respectively by poly P-AMP phospho-

transferase, poly P kinase, poly P glucohnase, and exopolyphosphatases and internally by endopolyphosphatases.

from Saccharomyces cerevisiue, is the most powerful of these
analytic reagents, releasing 30,000 P, residues per min per
enzyme molecule at 37C (54). It acts with about 40 times the
specific activity of the E. coli PPX, exhibits a far broader size
range among poly P chains (Le., 3 to 1000 residues), and
enables poly P to be measured accurately at a level of 0.5 pmol
when labeled with 32P. Cloning the gene for this polyphos-
phatasc enabled the enzyme to be overproduced in E. coli (55).
Application of this potent polyphosphatase to remove the poly
P that contaminates DNA preparations from yeast and other
poly P-rich organisms (42) may solve a problem that has be-
deviled the action of restriction nucleases and the use of shut-
tle vectors for expression of fungal gcncs in E. coli.
Two more enzymes available as reagents for analysis of poly
P are the glucokinase, which attacks the terminal residues of
the poly P chain with glucose (19) (Fig. l), and a phospho-
transferase, which attacks the termini with AMP (6).
With respect to the sources of poly P, it is imperative, as it
is with analysis of other cellular constituents, especially in
eukaryotes, to distinguish subcellular compartments: nucleus,
mitochondria, lysosomes (vacuoles), other vesicular entities,
and the cytosol. A dramatic example is the yeast vacuole, which
may contain more than 99% of the cellular poly P and mask the
not insignificant remainder in the mitochondria and the nu-
cleus.

BIOSYNTHESIS OF POLY P

The only pathway for the synthesis of poly P that has been
established is the polymerization of the terminal phosphate of
ATP through the action of PPK in E. coli (1). The gene en-
coding the kinase is part of an operon in which the gene for
PPX is located immediately downstream (2). Interruption of
the operon produces mutants which, for lack of long-chain poly
P, are defective in survival in the stationary phase (see below).
How the operon is regulated to balance two counteracting
enzymes that are roughly equal in activity has yet to be ex-
plained.

Although a PPK activity has been purified from other bac-
teria (41,47) and reported in yeast (29), such an enzyme action
has yet to be proven in animal systems. While PPK as the
device to produce long chains of poly P (ca. 750 residues) has
been validated in E. coli by genetic studies, mutants lacking this
enzyme can still make short poly P chains, about 60 residues
long, by an undefined pathway (9).
Several other plausible routes for the biosynthesis of poly P
need to be considered. These are from ADP by reversal of an
AMP phosphotransferase, from acetyl P, from 1,3-diphospho-
glycerate, from dolichyl pyrophosphate (43), and, of special
interest, by proton motive forces, known to fix Pi in inorganic
PP, (34) as well as in ATP.

FUNCTIONS OF POLY P

ATP substitute and energy source. PPK converts poly P to
ATP by catalyzing an ADP attack on the termini of the poly P
chain (Fig. 1). An aggrcgatc of poly P associated with this
membrane-bound enzyme could generate large amounts of
ATP at that very spot. Another source of ATP could come
from an AMP attack on poly P (Fig. 1) by AMP-phosphotrans-
ferase to produce ADP, which is readily converted to ATP by
coupling with PPK or with adenylate kinase:

poly P, + AMP -+ poly Pn.l + ADP

poly P,.l + ADP + poly Pn.2 + ATP

2 ADP - ATP + AMP

AMP-phosphotransfcrase has been purified from an Acmeto-
bacter strain (6) and has been identified in E. colr (21) and
Myxococcus xanthus (44); adenylate kinase is a potent and
ubiquitous enzyme.
Through the action of these enzymes, poly P is a potential
phosphagen in cells where and when its levels far exceed those
of ATP. Compared to the usual cellular ATP levels of 5 to 10
mM, the massive vacuolar deposits in yeast cells, expressed on

VOL. 177. 1995

MINIREVIEW 493

the basis of total cell volume, can exceed 200 mM; in Myxobac-
teriu cells in stationary phase the granular aggregates of poly P
can reach 50 mM (44, 49).
In view of its energy equivalence to ATP, poly P qualifies as
an ATP substitute in all of its kinase roles, involving a variety
of acceptors. In addition to the observed transfers to AMP and
ADP, poly P, as already noted, can replace ATP in the phos-
phorylation ol glucose in many bacteria. All of these glucoki-
nases use either ATP or poly P as donors; the more phyloge-
netically ancient species appear to show a preference for poly
P over ATP (19). One might expect to find poly P kinases for
other sugars, sugar derivatives (e.g., nucleosides and coenzyme
precursors), proteins, and carboxylic acids. Indeed, phosphor-
ylation of a 40-kDa protein in the ribosomal fraction of the
archaebacterium Sulfolobus acidocaldarius was observed with
poly P as the donor (46).
An energy recycling mechanism operating in the efflux of
organic end products (e.g., lactate in enteric bacteria) in sym-
port with protons can generate a proton motive force (48).
Such a mechanism may function in the utilization of poly P.
The efflux of a protonated metal chelate of P, released from
poly P creates a proton motive force that may be coupled to the
accumulation of amino acids from the medium or the synthesis
of ATP.
A reservoir for Pi. A stable level of Pi, essential for metab-
olism and growth, can be insured by a reservoir in which poly
P can be converted to P, by associated exopolyphosphatases.
The polymer, as an aggregate complexed with multivalent
counterions, enjoys a clear osmotic advantage over free P,.
Regulation of the ppk operon, which encodes both the poly P
kinase and exopolyphosphatase in E. coli (3), appears to be
responsive to thepho regulon that controls more than 20 genes
related to phosphate metabolism (39). Multiple exopolyphos-
phatases in E. coli (3,20) and in yeast (4,54,55) are potentially
available to produce Pi in various cellular locations.
Chelator nf metal inns. As expected of a phosphate poly-
anion, poly P is a strong chelator of metal ions. Lactobacillus
plantarurn, unusual in lacking a superoxide dismutase, a me-
talloenzyme that catalyzes the removal of the damaging super-
oxide radical, has an inorganic catalyst instead-an extraordi-
narily high, 30-mM level of Mn2' chelated to 60 mM poly P
(5). With regard to chelation of Ca2+, the regulation of cellular
Caz+ in yeast cells by vacuolar Ca2+ depends on its binding to
poly P; the poly P acts as a CaZ+ sink within the vacuole lumen
(12). Chelation of Ca2' and Mg2+, structurally essential in the
cell walls of gram-positive bacteria, is regarded as the basis for
the antibacterial action of poly P (31). Chelation of othcr
metals (e.g., Zn, Fe, Cu, and Cd) may either reduce their
toxicity or affect their functions.
Buffer against alkali ions. Algae, like yeasts, accumulate
poly P in their vacuoles. In the halotolerant green alga Du-
nuliellu salina, deposits of poly P reach levels near 1 M in Pi
equivalents. When stressed at alkaline pH, amines enter the
algal vacuoles and are neutralized by protons released by the
enzymatic hydrolysis of poly P (36). The specific polyphos-
phatase, presumably activated by the amines, produces tri-poly
P by mechanisms that have yet to be determined. Thus, poly P,
as a result of its hydrolysis, can provide a high-capacity buff-
ering system that sustains compartmentation of amines in
vacuoles and protects the cytoplasmic pH. This alga, cultivated
in large outdoor ponds, is an important commercial source of
p-carotene for health foods and for food coloring.
Channel for DNA entry. Transforming competent E. coli
with DNA for the cloning and expression of genes is currently
one of the world's favorite indoor sports. Despite the wide-
spread use of a Ca2+ recipe to induce competence, there is

little understanding of the mechanism whereby the highly charged
DNA molecule penetrates the lipid bilayer membranes sur-
rounding the cell. A significant advance was the discovery ol
polyhydroxybutyrate (PHB) complexed with Ca2+ and poly P
in the membranes of competent cells (40). In a proposed struc-
ture, Ca2+ is bonded by ion dipoles to the carbonyl ester
groups of PHB and by ionic interactions with poly P. This
complex produces profound physical changes in the compe-
tent-cell membranes-increased rigidity at ambient tempera-
tures and biphasic melting (9, 40). Whether and how these
alterations facilitate DNA entry remains unclear.
In current studies, the PHB-Ca2+-poly P complex has been
reconstituted in large, unilamellar vesicles by adding PHB,
Ca2+, and poly P to phospholipids (9). The capacity of these
vesicles for the uptake of small and large molecules, charged
and neutral, needs to be extended. Although mutants lacking
the long-chain poly P can attain competence, their membranes
still contain a short poly P chain of about 60 residues, synthe-
sized by a novel route during the development of competence

Regulator for stress and survival. Regulatory roles for poly
P, a phosphate polyanion with some resemblance to RNA and
DNA, seem reasonable. Poly P readily interacts with basic
proteins (e.g., histones) and with basic domains of proteins, as
in polymerases, and has been obscrvcd in association with
nonhistone nuclear proteins (35). Such roles could affect gene
functions in positive or negative ways. Inasmuch as poly P is
present in several sizes and complex forms, is located in the
nucleus and other cell compartments, and fluctuates in re-
sponse to nutritional and other parameters, it seems possible
that poly P might function in the network of responses to
stresses and the many signals that govern stages in the cell cycle
and development.
We were surprised to discover that a novel exopolyphos-
phatase, identified in E. coli mutants that lacked the exo-
polyphosphatase gene (ppx) encoded in the ppk operon, was
guanosine pentaphosphate (pppGpp) hydrolase (ZO), the en-
zyme that produces guanosine tetraphosphate (ppGpp; "magic
spot"), the powerful effector in the bacterial stringent response
(8). Upon deprivation of an amino acid, the RelA enzyme
generates pppGpp, which upon conversion to ppGpp represses
many genes and activates others. The significance of poly P and
of the polyPase activity of pppGpp hydrolase need to be stud-
ied. Also unclear is the part that poly P may play in other
stringencies, such as those of carbon, energy, or phosphate.
The possibility of poly P involvement in the stringent re-
sponse suggested that it might be among the multiple meta-
bolic adjustments in the stationary phase of the cell cycle. "Life
after log" in E. coli (45) is a dynamic interval in which many
genes are induced to cope with environmental stresses to en-
sure survival. Although theppk mutant lacking long-chain poly
P shows no phenotypic changes in the exponential phase of
growth, striking deficiencies are evident when it is examined in
the stationary phase (11, 38). The mutant survives less well, is
less resistant to heat, oxidants, and osmotic challenge, and
shifts to a small-colony phenotype, suggestive of an adaptive
genotypic change (18). Thus, poly P may enter in the cascade
of events that prepare cells for coping with "life in the slow
lane."
Regulator of development. Developmental changes in micro-
organisms-fruiting body and spore formation in Myxobucteria
species (e.g., M. xanthus), sporulation in bacteria (e.g., Bacillus
species), and fungi, and heterocyst formation in cyanobacteria
(e.g., Anabaena species)4ccur in response to starvation of
one or another nutrient. In view of the involvement of poly P

(9).

494 MINIREVIEW

J. BACTERIOL.

in the stationary stage of E. coli, poly P may well participate in
other instances of cellular adjustments to deprivation.
Upon the conclusion of vegetative growth in M. xunthus, the
levels of poly P and of poly P-AMP phosphotransferase activity
increase more than 10-fold (44). When present at concentra-
tions as high as 50 mM (49), poly P may be an energy source
for fruiting bodies and for deposition in spores. Inasmuch as an
increase in ppGpp precedes poly P formation and mutants that
fail to produce ppGpp also fail to increase their poly P levels
(44), it seems that ppGpp has a regulatory role in poly P
formation.
Cell capsule. Poly P is a component of the capsule (47)
which is loosely attached to the surface of Neisseria species and
represents about half of the cellular poly P. Whether the cap-
sule contributes to the pathogenesis of infections, and if so,
whether it is by combatting phagocytosis, by chelating metals
needed in complement fixation, or by some other way has yet
to be discovered.

POLY P IN ANIMAL CELLS AND TISSUES

Although the presence of poly P in fungi and algae has been
widely noted, the distribution and abundance of poly P in more
complex eukaryotic forms has remained uncertain. The very
low levels of poly P in animal cells (13) and subcellular com-
partments and the lack of definitive and sensitive methods
have left its metabolic and functional roles entirely obscure.
Our exploratory studies with improved enzymatic assay
methods have confirmed that poly P is present in a wide variety
of cell cultures and animal tissues. The concentrations of poly
P generally range from 10 to 100 pM (expressed in P, equiv-
alents) and in sizes of 100 to near 1,000 residues. Among the
subcellular organelles, poly P has been identified in lysosomes
(37) and in mitochondria (32) and is relatively enriched in
nuclei (30). In rat brain, poly P is present throughout the
course of embryonic and postnatal development (30). Uptake
of P, into poly P has been observed in cultured mammalian
cells (10, 30), lysosomes (37), mitochondria (32), and broken-
cell preparations (10). A strikingly rapid turnover of poly P was
seen in a confluent culture of PC 12 cells, a neuron-like cell
line derived from an adrenal pheochromocytoma. Although
these cells have a generation time of 48 to 72 h, the turnover
was nearly complete in 1 h (30). Studies of the dynamics of poly
P formation and utilization in a variety of cells should reveal
novel functions for the polymer in different stages of growth
and metabolism.

EVOLUTIONARY ROLE OF POLY P

RNA may have preceded DNA and proteins in prebiotic
evolution, but it seems likely that poly P appeared on earth
before any of these organic polymers. Poly P arises simply from
the dehydration and condensation of P, at elevated tempera-
tures and is evident in volcanic condensates (56) and oceanic
steam vents. The anhydride-bond energy and the phosphate of
poly P are plausible sources for nucleoside triphosphates, the
activated building blocks of RNA and DNA (28, 50); mixed
carboxylic-phosphate anhydrides provide a route to chemical
polypeptide synthesis starting with amino acids and poly P (15).
Among the species of poly P, mention should be made of the
very simplest, namely, inorganic pyrophosphate (PP,). Once
regarded solely as a metabolic product of biosynthetic reac-
tions (22) and as being fated to be hydrolyzed by potent and
ubiquitous inorganic pyrophosphatases to drive these path-
ways, PP, was revealed by later studies to be a substitute for
ATP (52). A role for PP, as well as for poly P in prebiotic

events leading to the evolution of ATP deserves attention.
Inasmuch as the synthesis of one ATP by extant de novo
pathways requires the input of at least eight ATPs, how was
ATP made in the first place?
Poly P accumulations, also prominent in archaebacteria,
may well be the substrates for enzymatic attack by nucleosides
to produce nucleoside mono-, di-, or triphosphates. A system-
atic search among these ancient organisms might uncover en-
zymes that carry out such salvage reactions in the biosynthesis
of nucleotides, coenzymes, and other factors.

INDUSTRIAL APPLICATIONS OF POLY P

Aspects of the chemical and physical properties of poly P
that emerge from its industrial uses are of general interest and
in some instances may impinge directly on its place and func-
tions in biological systems.
Depollution of Pi in the environment. Pi accumulation in
waste waters containing runoff of fertilizers and discharges of
industrial agents is a global problem that results in destructive
algal blooms in bays, lakes and waterways. The use of lime,
alum or ferric chloride to remove P, is expensive and ineffi-
cient. Currently, sanitary engineers employ a biological process
in which aerobically activated bacteria take up the P, and
convert it to poly P, which is then removed along with the
bacteria as a sludge. Because the microbial process is slow and
inadequate, genetically engineered improvements are needed
in Acinetobncter strains and other gram-negative organisms
that dominate the flora. A start has been made in this direction
by showing that theppk gene on a runaway plasmid introduced
into a selected coliform species vastly increases the rate and
extent of Pi removal from the medium (16). When coupled
with an improved pst uptake system for Pi, poly P accumula-
tions have reached 38 to 48% of the dry weight of the cell.
Antibacterial action. Poly P is a safe additive to meats,
enhancing water binding, emulsification, and color retention
while retarding oxidative rancidity. In its use in virtually all
processed meat, poultry, and fish products, poly P also serves
as an antibacterial agent (31).
Source of ATP. The cost of ATP for use as an enzymatic
phosphorylating agent on an industrial scale is prohibitive, as is
the cost of agents, such as creatine phosphate and phos-
phoenolpyruvate, that might be used in an enzymatic ATP-
regenerating system. In their place, poly P has been employed
to regenerate ATP using PPK immobilized on a column (7). In
this system, a commercial form of poly P costing 25$/lb can
provide ATP equivalents that would cost over $2,00O/Ib.
Insulating fibers. Phosphate fibers form bones and teeth.
Polyphosphates are added to cheese, meats, toothpaste, and
drinking water. A calcium polyphosphate fiber has been syn-
thesized with all the properties of asbestos (14) and could be a
safe substitute, but has been abandoned by its inventor, Mon-
santo Chemical Company, which cited its fear of litigation
from lawyers trained to bring suits for injuries from mineral
fibers. Unfortunately, the decision to abandon poly P fibers as
an insulator also extends to its use for nonflammable infant
sleepwear, hospital mattress covers, and fabric for aircraft in-
teriors.

SUMMARY

Pursuit of the enzymes that make and degrade poly P has
provided analytic reagents which confirm the ubiquity of poly P
in microbes and animals and provide reliable means for mea-
suring very low concentrations. Many distinctive functions ap-
pear likely for poly P, depending on its abundance, chain

VOL. 117, 1995

MINIREVIEW 495

length, biologic source, and subcellular location. These include
being an energy supply and ATP substitute, a reservoir for P,,
a chelator of metals, a buffer against alkali, a channel for DNA
entry, a cell capsule and, of major interest, a regulator of
responses to stresses and adjustments for survival in the sta-
tionary phase of culture growth and development. Whether
microbe or human, we depend on adaptations in the stationary
phase, which is really a dynamic phase of life. Much attention
has been focused on the early and reproductive phases of
organisms, which are rather brief intervals of rapid growth, but
more concern needs to be given to the extensive period of
maturity. Survival of microbial species depends on being able
to managc in the stationary phase. In view of the universality
and complexity of basic biochemical mechanisms, it would be
surprising if some of the variety of poly P functions observed in
microorganisms did not apply to aspects of human growth and
development, such as aging and the aberrations of disease.
Of theoretical interest regarding poly P is its antiquity in
prebiotic evolution, which along with its high energy and phos-
phate content make it a plausible precursor to RNA, DNA,
and proteins. Practical interest in poly P includes many indus-
trial applications, among which is its use in the microbial de-
pollution of P, in marine environments.

ACKNOWLEDGMENTS

I thank my colleagues Kyunghye Ahn, Masahiro Akiyama, LeRoy
Bertsch, Celina Castuma, Elliott Crooke, Jay Keasling, Anand
Kumhle, Sheng-jiang Liu, Narayana Rao, Toshikazu Shiba, and Hel-
mut Wurst for their courage in pursuing a largely forgotten polymer
and for the contributions that made it more memorable.
I am also grateful to the NIH and the Human Frontier Science
Program for their support of my research.

1.

2.

3.

4.

5.

6.

7.

8.

9.
10.

11.

12.

13.

14.
15.

REFERENCES

Ahn, IC, and A. Kornberg. 1990. Polyphosphate kinase from Escherichia coli.
J. Biol. Chem. 26511734-11739.
Akiyama, M., E. Crooke, and A. Kornberg. 1992. The polyphosphate kinase
gene of Escherichia coli. Isolation and sequence of theppk gene and mem-
brane location of the protein. J. Bid. Chem. 2672255622561.
Akiyama, M., E. Crooke, and A. Kornberg. 1993. An exopolyphosphatase of
Escherichia coli. The enzyme and itsppx gene in a polyphosphate operon. J.
Biol. Chem. 268633439.
Andreeva, N. A., and L. A. Okurukuv. 1993. Purification and characterization
of highly active and stablc polyphosphatase from Saccharomyces cerevuiue
cell envelope. Yeast 9127-139.
Archibald, F. S., and I. Fridovich. 1982. Investigations of the state of the
manganese in Lactobacillus pkantumm. Arch. Biochem. Biophys. 215589-
596.
Bunting, C. F. C., G. J. J. Kortstee, and A. J. B. Zehnder. 1991. Properties of
po1yphosphate:MP phosphotransferase of Acinetobacter strain 210A. J.
Bacteriol. 173:648&6488.
Butler, L. 1977. A suggested approach to ATP regeneration for enzyme
technology applications. Biotechnol. Bioeng. 19591-593.
Cashel, M., and K. E. Rudd. 1987. The stringent response, p. 1410-1438. In
F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter,
and H. E. Umbarger (ed.), Escherichiu coli and Sulmonella fyphrmunum:
cellular and molecular biology. American Society for Microbiology, Wash-
ington, D.C.
Castnma, C. E., and A. Kurnberg. Unpublished results.
Cowling, R. T., and II. C. Birnboim. 1994. Incorporation of ["PI orthophos-
phate into inorganic polyphosphates by human granulocytes and other hu-
man cell types. J. Bid. Chem. 269:9480-9485.
Crooke, E., M. Akiyama, N. N. Rao, and A. Kurnberg. 1994. Genetically
altcrcd lcvels of inorganic polyphosphate in Escherichiu coli. J. Biol. Chcm.
26962904295.
Dun, T., K. Gable, and T. Beeler. 1994. Regulation of cellular CaZf by yeast
vacuoles. J. Bid. Chem. 269:7273-7278.
Gabel, N. W., and V. Thomas. 1971. Evidence for the occurrence and dis-
tribution of inorganic polyphosphate in vertebrate tissues. J. Neurochem.
18: 1229-1242.
Griffith, E. J. 1992. In search of a safe mineral fiber. Chemtech 22:220-226.
Harada, K., and S. Fox. 1965. Thermal polycondensation of free amino acids
with polyphosphoric acid, p. 289-298. In S. Fox (cd.), Origins of prehiologi-
cal systems. Academic Press, Inc., New York.

16. Harduyu, K. Yamada, H. Shinjo, J. Kato, and H. Ohtake. 1994. Production
and release of polyphosphate by a genetically engincered strain of Esche-
nchiu coli. Appl. Environ. Microbiol. 603485-3490.

17. Harold, F. M. 1967. Inorganic polyphosphates in biology: structure, metab-
  olism and function. Bacteriol. Rev. 30:772-794.
18. Harris, R. S., S. Longerich, and S. M. Rosenberg. 1994. Recombination in
adaptive mutation. Science 264258-260.

19. Hsieh, P.-C., B. C. Sheuuy, J. E. Jentoft, and N. F. B. Phillips. 1993. Puri-
fication of polyphosphate and ATP glucose phosphotransferase from Myco-
bacfenum fuberculosis I13,Ra: Evidence that poly (P) and ATP glucokinase
activities are catalyzed by the same enzyme. Protein Exp. Purif. 476-84.
20. Keasling, J. D., L. Bertsch, and A. Kornberg. 1993. Guanosine pentaphos-
phate phosphohydrolase of Escherichia coli is a long-chain exopolyphos-
phatase. Proc. Natl. Acad. Sci. USA 90:7029-7033.

21. Kim, H.-Y., and A. Kornberg. Unpublished observations.
22. Kornberg, A. 1957. Pyrophosphorylases and phosphorylases in biosynthetic
reactions. Adv. Enzymol. 18:191-240.
23. Kornberg, A. 1993. Recollections. ATP and inorganic pyro- and polyphos-
phate. Protein Sci. 2131-132.

24. Kornberg, A. 1994. Inorganic polyphosphate: a molecular fossil come to life,
p. 204-208. In A. Torriani-Gorini, S. Silver and E. Yagil (ed.), Phosphate in
microorganisms: cellular and molecular biology. American Society for Mi-
crobiology, Washington, D.C.
25. Kornberg, A., S. R. Kornberg, and E. S. Simms. 1956. Metaphosphate
synthcsis by an enzyme from Escherichia cnh. Biochim. Biophys. Acta 20:
  215-227.

26. Kornberg, S. R. 1957. Adenosine triphosphate synthesis from polyphosphate
by an enzyme from Escherichia coli. Biochim. Biophys. Acta 26294-300.
27. Kulaev, I. S. 1979. The biochemistry of inorganic polyphosphates. John

Wiley & Sons, Inc., New York.

28. Kulaev, I. S., and K. G. Skryabin. 1974. Reactions of nonenzymic transphos-
phorylation performed by high-polymeric polyphosphates and their role in
abiogcncsis. Zh. Evol. Biokhim. Fiziol. 10533.
29 Kulaev, I. S., V. M. Vagabov, and Y. A. Shabalin. 1987. New data on
biosynthesis of polyphosphates in yeast, p. 233-238. In A. Torriani-Gorini.
F. G. Rothman, S. Silver, k Wright, and E. Yagil (ed.), Phosphate metab-
olism and cellular regulation in microorganisms. American Society for Mi-
crobiology, Washington, D.C.

30. Knmble, K., and A. Kurnberk. Unpublished observations.

31. Lee, R. M., P. A. Hartman, H. M. Stahr, D. G. Olson, and F. D. Williams.
1994. Antibacterial mechanism of long-chain polyphosphate in S~upl~ylococ-
  cus aurew. J. Food Prot. 57:289-294.

32. Liu, S. J., and A. Kornberg. Unpublished observations.
33. Meyer, A. 1904. Orientierende Untersuchungen ueber Verbreitung, Mor-
phologie, und Chemie des Volutins. Bot. Zeit. 62(1):113-152.

34. Nyren, P., B. F. Nore, and A. Strid. 1991. Proton-pumping NN'-dicyclohex-
ylcarbodiimide-sensitive inorganic pyrophosphate synthase from Khodospi-
rilluni rubrum: purification, characterization, and reconstitution. Biochemis-
try 30:2883-2887.
35. Offenhacher, S., and E. S. Kline. 1984. Evidence for polyphosphate in phos-
phorylated nonhistone nuclear proteins. Arch. Biochem. Biophys. 231:114-
  123.
36. Pick, U., and M. Weiss. 1991. Polyphosphate hydrolysis within acidic vacu-
oles in response to amine-induced alkaline stress in the halotolerant alga
Dunuliello salina. Plant Physiol. 97:1234-1240.
37. Pisoni, R. L., and E. R. Lindley. 1991. Incorporation of ["PI orthophosphate
into long chains of inorganic polyphosphate within lysosomes of human
  fibroblasts. J. Biol. Chem. 2673626-3631.

38. Rao, N. N., and A. Kornberg. Unpublished observations,
39. Rao, N. N., and A. Torriani. 1990. Molecular aspects of phosphate transport
  in Escherichiu coli. Mol. Microbiol. 41083-1090.

40. Reusch, R. N., and H. L. Sadoff. 1988. Putative structure and functions of a
poly-P-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma
  membranes. Proc. Natl. Acad. Sci. USA 8541764180.
41. Robinson, N. A., J. E. Clark, and H. G. Wood. 1987. Polyphosphate kinase
from Prnpinnrharcterium shermunii. J. Biol. Chem. 262:5216-5222.
42. Rodriguez, R. J. 1993. Polyphosphate present in DNA preparations from
filamentous fungal species of Cnllefotnchum inhibits restriction endonucle-
ases and other enzymes. Anal. Biochem. 209:291-297.
43. Shabalin, Y. A., and I. S. Kulaev. 1989. Soluhilization and properties of yeast
dolichylpyrophosphate: polyphosphate phosphotransferase. Biokhimiya 54:
68-75. (In Russian.)

44. Shiba, T., and A. Kornberg. Unpublished observations.
4.5. Siegele, D. A., and R. Kolter. 1992. Life after log. J. Bacteriol. 174345-348.
46. Skorko, R. 1989. Polyphosphate as a source of phosphoryl group in protein

modification in the archaebacterium Suvolohus acidocaldarius. Biochimie
71:1089-1093.

47. Tinsley, C. R., B. N. Manjula, and E. C. Gotschlich. 1993. Purification and
characterization of polyphosphate kinase from Neisseriu meningitidis. Infect.
  Immun. 61:3703-3710.
48. Van Veen, H. W., T. Abee, A. W. F. Kleefsman, B. Melgers, G. J. J. Kurtstee,
W. N. Konings, and A. J. II. Zehnder. Energetics of alanine, lysine and

496 MINIREVIEW

J. BACTERIOL.

proline transport in cytoplasmic membranes of the polyphosphdte-accumu-
lating Acinetohncter johnvonii strain 210A. J. Bacteriol. 1762670-2676.

49. Voelz, H., U. Voelz, and R. 0. Ortigoza. 1966. The "polyphosphatc overplus"
phenomenon in Mvxococcus urnthus and its influence on the architecture of
  the cell. Arch. Mikrobiol. 53371-388.

SO. Waehneldt, T. V., and S. Fox. 1967. Phosphorylation of nucleosides with

polyphosphoric acid. Biochim. Biophys. Acta 1349-16.
51. Wiame, J.-M. 1947. Yeast metaphosphate. Fed. Proc. 6:302.
52. Wood, H. G. 1985. Inorganic pyrophosphate and polyphosphates as sources

of energy. Curr. Top. Cell. Reg. 263355-369.

phosphates. Annu. Rev. Biochem. 57:235-260.

churonzyces cerevisiue. J. Biol. Chem. 26910996-11001.

53. Wood, H. G., and J. E. Clark. 1988. Biological aspects of inorganic poly-

54. Wurst, H., and k Kornberg. 1994. A soluble exopolyphosphatase of Suc-

55. Wurst, H., and k Kornberg. Unpuhlished ohservations.

56. Yamagata, Y., H. Watanabe, M. Saitoh, and T. Namba. 1991. Volcanic
production of polyphosphates and its relevance to prebiotic evolution. Na-
ture (London) 352:516-519.