Arthur Kornberg is Professor Emeritus and Cresson D. Fraley is Research Associate in the Department of Biochemistry, Stanford University School of Medicine, Stanford, Calif.
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- Inorganic Polyphosphate: a
Molecular Fossil Come to Life
This underappreciated polymer helps cells survive in stationary phase, can increase virulence, and plays other possibly critical roles
Arthur Kornberg and Cresson D. Fraley
Excitement about extremophiles on earth and possible extraterrestrial life tends to eclipse some of the extraordinary mysteries surrounding molecules found in commonplace biota. One such molecule is inorganic polyphosphate (poly P), a linear chain of hundreds of orthophosphate (Pi) residues linked by ATP-like, high-energy, phosphoanhydride bonds (Fig. 1).
Poly P is readily formed from Pi by dehydration at an elevated temperature and was likely involved as a catalyst and precursor in prebiotic evolution. Whatever its role, poly P is found in every type of cell that has been examined: bacterial, archaeal, fungal, protozoan, plant, and animal. For instance, metachromatic (volutin) granules, which were described a century ago in bacteria and yeast, are massive accumulations of poly P. Yet, for lack of any known function, most scientists dismiss poly P as a ``molecular fossil'' and so it is ignored in leading textbooks of microbiology, biochemistry, and chemistry.
However, recently compiled evidence indicates that poly P has numerous and varied biological functions depending on where it is--species, cell, or subcellular compartment--and when it is needed. These functions include substituting for ATP in kinase reactions, providing a reservoir of Pi, chelating metals (e.g., Mn2+, Mg2+, and Ca2+), buffering against alkali, providing part of the capsule of some bacteria, enabling competence for bacterial transformation, and participating in mRNA processing and its degradation, as well as in the microbial remediation of phosphate pollution. In addition, we recently found that poly P has a regulatory role in bacterial adaptations for stationary-phase survival and is essential for the motility and virulence of some major bacterial pathogens.
Sensitive Assays for Poly P Are Leading to New Insights
A major reason behind the delayed appreciation for poly P is that, until very recently, investigators lacked a facile, sensitive, and definitive means for measuring this molecule. However, we largely overcame this problem in the past few years once we could employ enzymes from Escherichia coli and yeast cells that are specific for synthesizing and metabolizing poly P.
Principal among these is poly P kinase (PPK), the enzyme that polymerizes the terminal phosphate of ATP into a poly P chain of about 700 residues; in reverse, PPK generates ATP from poly P and ADP:
nATP « poly Pn + nADP.
In cell extracts, PPK with an excess of added ADP readily converts poly P to ATP. In turn, that ATP is linked to the production and detection of light in a luciferin-luciferase system. This enzyme-catalyzed reaction is readily adapted to high-throughput assays. To eliminate any doubt that the light we are detecting derives from poly P, we can add the enzyme exopolyphosphatase (PPX), which degrades poly P and eliminates any signal. Still, we need to be mindful that the extraction procedures exclude short-chain poly P and also fail to detect poly P when it is complexed and unavailable to these enzymes, which act only on free ends of chains.
Stress-Induced Regulator, Other Adaptive Functions
Of the several known and potential functions for poly P, the regulation of gene expression now appears to be the most significant. Our experience in studying the roles played by this molecule in E. coli and Pseudomonas aeruginosa suggests that poly P serves as an ``alarmone'' to control other metabolic processes. For example, when Pi, an amino acid, or nitrogen is depleted or when cultured cells are downshifted into low-nutrient-growth media or subjected to the stress of high salt concentrations, we find alarmone-type responses. In some cases, the distressed cells promptly accumulate 100-fold or higher amounts of poly P, which subsequently decays in minutes or hours. However, we do not observe such responses when cells are depleted of carbon or are subjected to higher temperatures or abrupt shifts in pH.
Genetic experiments suggest that this capacity to accumulate poly P is vital for the survival of microbial cells in the stationary phase. For instance, mutants of E. coli that lack PPK and thus cannot produce poly P also fail to activate expression of the alternate RNA polymerase sigma factor, RpoS. This sigma factor controls the expression of more than 50 genes that are needed for resistance to stresses, stringencies, and survival in the stationary phase. Without it, these mutants fail to acquire resistances to a variety of stresses, including heat, oxidants, and UV damage that cells in the stationary phase typically encounter, and thus the mutants die within a few days.
Poly P as an Antimicrobial Drug Target among Pathogens
The recent dramatic increase in the number of microbial genome sequences that have been characterized reveals that the PPK amino acid sequence is highly conserved in more than 20 microbial species. Several of the major pathogens are included in this group, although some of the species appear to lack this particular enzyme (Table 1).
The need for PPK in E. coli for survival in the stationary phase--a stage during which some pathogens are known to produce their virulence factors--and the wide conservation of PPK suggest that the enzyme is needed for virulence. To explore this possibility, we prepared knockout mutants of ppk in six different bacterial pathogens, and we are using these mutants to test for dependence on PPK for virulence factors, growth, and long-term survival.
Several of our PPK null mutants are defective in flagellum-mediated motility, which can be restored by complementation with plasmids that bear ppk. The pathogens among these mutants include P. aeruginosa, Klebsiella pneumoniae, Vibrio cholerae, Salmonella enterica serovar Dublin SVA47, and Salmonella enterica serovar Typhimurium FIRN.
P. aeruginosa ppk mutants are defective in flagellum-mediated swimming and swarming (Fig. 2), type IV pilus-based twitching, and quorum sensing. The role of motility in pulmonary and burn infections in mice is well known and was confirmed in the ppk mutants in the burned-mouse model with regard to mortality, local persistence, and systemic spreading (Table 2). Phenotypic studies in progress of the ppk mutants of other pathogens include defects in growth, invasion of the cornea in guinea pigs (Sereny test, Shigella flexneri), and competition in vivo with the wild type for colonization of the pyloric mucosa in mice (Helicobacter pylori).
If PPK indeed proves necessary for virulence among these and other pathogens, then this enzyme would be an attractive target for antimicrobial drugs. It might be possible to identify specific PPK inhibitors with little toxicity, since homologs to this prokaryotic enzyme are not found in mammalian cells.
Multiple Roles for Poly P in Microbial Cells
Depending on the cell and circumstances, poly P may serve in a variety of roles. For example, in some cells where its levels far exceed those of ATP, poly P may act as a phosphate storage reserve and a source of high energy needed to drive biochemical reactions. Compared to the usual cellular ATP levels of 5 to 10 mM, the vacuolar deposits of poly P in yeast are massive, exceeding 200 mM (measured as Pi residues). In E. coli cells that are responding to stress, poly P levels reach as high as 50 mM, and in myxobacteria, during stationary phase, the granular aggregates of poly P can also reach 50 mM. In terms of driving metabolic reactions, poly P can serve as an ATP substitute in kinase reactions to several donor molecules, including AMP, ADP, and glucose, and may also be a donor of Pi to other sugars, sugar derivatives such as nucleosides and coenzyme precursors and, possibly, proteins as well.
Considering that poly P can serve as an ATP source and substitute and knowing that poly P levels can exceed those of ATP, two points need to be made regarding the role of poly P as an energy source. One is that the turnover of ATP molecules in cells is very rapid--less than a second in E. coli, even in stationary-phase cultures. Thus, even high levels of poly P would provide ATP equivalents for a few seconds at most. The other point involves the prominent regulatory role of poly P that enables E. coli to survive in the stationary phase. This regulatory function involves low poly P levels of about 0.1 mM, which is only 1% that of ATP. Hence, it seems unlikely that poly P serves cells as an energy source during stationary-phase growth.
A stable level of Pi in cells is essential for metabolism and growth. Poly P could serve as a reservoir for Pi, which PPXs could liberate from the polymer. This polymer, which forms complexes with multivalent counterions, confers a clear osmotic advantage to cells over free Pi. The PhoB component within the pho regulon, which controls more than 30 genes, including phosphatases involved in phosphate metabolism, apparently determines how much poly P will accumulate in a microbial cell. Both E. coli and yeast contain several PPXs that likely can be made available to produce Pi either in the cytoplasm or in the periplasm.
As expected of a phosphate polyanion, poly P is a strong chelator of metal ions and thus can play yet another important role in microbial cells. For instance, cells of Lactobacillus plantarum can withstand superoxide toxicity by employing a 30 mM Mn2+-poly P complex, which serves as an inorganic superoxide dismutase. Meanwhile, yeast can chelate Ca2+ within a vacuole, thereby regulating cytosol levels of this cation.
Because both Ca2+ and Mg2+ are essential for the structural integrity of the cell walls of gram-positive bacteria, chelating them provides the basis for at least one form of poly P antibacterial action. Its ability to chelate other metals, including zinc, iron, copper, and cadmium may explain how cells manage to reduce the toxicity of some of those metals and alter or control the functions of others.
Poly P also plays a role in introducing DNA molecules to transform E. coli that are ``competent'' to accept such molecules. Despite the widespread use of Ca2+ in recipes that are used to induce competence in recipient cells, little is understood about how highly charged DNA molecules penetrate the bilayer lipid membranes surrounding such cells. A likely mechanism is through a complex of b -polyhydroxybutyrate with Ca2+-poly P in the membranes of competent cells. In a proposed structure, Ca2+ is bonded by ion dipoles to the carbonyl ester groups of b -polyhydroxybutyrate and by ionic interactions with poly P. This complex produces profound physical changes in the competent-cell membranes--including increased rigidity at ambient temperatures and biphasic melting--and might facilitate DNA entry.
In Neisseria meningitidis, poly P is a component of an extracellular capsule that is loosely attached to the surface and contains about half of the total cellular poly P. Whether the capsule contributes to the pathogenesis of infections and, if so, chelates metals needed to combat phagocytosis during complement fixation is not known. We do know that ppk-defective mutants lack the resistance to human serum displayed by wild-type cells and that this resistance is restored in the mutants by complementation with ppk-bearing plasmids.
Mammalian Cells and Tissues
The newer enzyme-based assay methods now enable us reliably to measure the concentration of poly P in rodent or other mammalian cells such as brain, heart, kidneys, liver, and lungs and in subcellular fractions, including nuclei, mitochondria, plasma membranes, and microsomes. Typically, poly P levels within such tissues and subcellular fractions range from 25 to 120 m M (in terms of Pi residues) in chains containing 50 to 800 residues. In brain tissue poly P occurs predominantly in the very large molecules, most of which are nearly 800 residues long and are present at similar levels pre- and postnatally.
Diverse cell lines, including fibroblasts, T cells, and kidney and adrenal cells convert Pi into poly P at rates in excess of 10 pmol/mg of cell protein per h. In a confluent culture of PC 12 cells, which are neuron-like cells derived from an adrenal pheochromocytoma, the turnover of poly P is strikingly rapid. Although these cells have a generation time of 48 to 72 h, the turnover is nearly complete within 1 h. By contrast, cultured fibroblast cells show little turnover of poly P within 4-h periods.
The ubiquity of poly P and variations in its size, location, and metabolism in a variety of cells suggest it plays several functions in mammalian systems. Thus far, however, no synthetic activity for poly P has been observed in any cell-free system of any mammalian cell culture or tissue. Because the synthesis of poly P from Pi in the medium bypasses intracellular Pi and ATP pools, energy sources in cell membranes or the channeling of Pi through a sequestered ATP compartment may be involved in synthesizing poly P in such cells.
Summary and Perspective
Although many biologists speculate that RNA preceded DNA and proteins in prebiotic evolution, it seems even more likely that poly P appeared on earth before any of these organic polymers. Poly P can arise simply and without cells or enzymes from the dehydration and condensation of Pi at elevated temperatures. Thus, it can be found in volcanic condensates and along oceanic steam vents.
Because poly P is so readily formed abiotically, its anhydride bond energy and store of phosphate residues are plausible sources for nucleoside triphosphates, which in cells are the essential building blocks of RNA and DNA. Moreover, the cyclic trimetaphosphate that is readily split from poly P can form the 5¢ ribo- and deoxyribonucleoside triphosphates directly from nucleosides, and polypeptides can be synthesized from mixed carboxylic-phosphate anhydrides, starting with amino acids and poly P.
Scientists often follow fashionable trends within their specific fields, sometimes ignoring classes of molecules because they seem either obscure or unglamorous. Yet those same molecules later may become the centers of scientific attention. In the past half-century, nucleic acids, histones, kinases, and phosphatases were once snubbed by scientific fashion and yet subsequently have emerged as the focus of intensive research efforts.
Surely, a molecule such as inorganic polyphosphate (poly P)--conserved from prebiotic time, found in every cell in nature, and containing high-energy bonds and other structural features resembling the nucleic acids--performs essential and perhaps even glamorous functions in living cells. Based on its known capacity as an ATP substitute, as an obvious reservoir of phosphate, and as a powerful chelator of divalent metal ions, poly P is clearly a molecule with many functions.
In E. coli, we believe that a most significant function of poly P is its regulatory role in gene expression. Cells that are deficient in poly P fail to activate many genes expressed by wild-type cells in stationary phase, cannot adapt to nutrient deficiencies and other environmental stresses, and thus die off rapidly. Because so many studies focus on cells that are growing rapidly (exponential phase), little attention has been given to the profound metabolic changes that occur during stationary growth. Our work on poly P has underscored the importance of ``life in the slow lane.'' Changes occurring during stationary growth may be essential for the survival of a microbial species within a host or free-living in an open environment.
Information from the accelerating accumulation of microbial genome sequences indicates that PPK, the enzyme responsible for synthesizing poly P, is highly conserved among many major pathogens. Because some virulence factors are expressed during stationary-phase growth of pathogens, they may also depend on poly P as has been observed for such gene expression in E. coli. In fact, null mutants of several pathogens lacking PPK are defective in growth, motility, quorum sensing, biofilm formation, and virulence in animal hosts. Antimicrobial drugs targeted to PPK may display a broad spectrum of activity and possibly low toxicity, inasmuch as the enzyme has not been observed in mammalian cells.
Among the many current questions regarding poly P, the most fundamental concerns the molecular basis of its regulatory and other metabolic roles. Genetic and physiologic studies will further clarify some of the metabolic aspects of poly P accumulation and utilization, but what will remain essential is the resolution and complete characterization of these poly P functions and their reconstitution at the molecular level.
SUGGESTED READING
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deKievit, T. R., and B. H. Iglewski. The role of quorum sensing in Pseudomonas aeruginosa virulence gene expression in bacterial pathogenesis, in press. ASM Press, Washington, D.C.
Kolter, R., D. A. Siegele, and A. Tormo. 1993. The stationary phase of the bacterial life cycle. Annu. Rev. Microbiol. 47:855-874.
Kornberg, A. 1995. Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J. Bacteriol. 177:491-496.
Kornberg, A., N. N. Rao, and D. Ault-Riche. 1999. Inorganic polyphosphate: a molecule of many functions. Annu. Rev. Biochem. 68:89-125.
Kulaev, I. S. 1979. The biochemistry of inorganic polyphosphates. Wiley, New York, N.Y.
Rashid, M. H., and A. Kornberg. Inorganic polyphosphate is needed for swimming, swarming and twitching motilities of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA, in press.
Rashid, M. H., N. N. Rao, and A. Kornberg. 2000. Inorganic polyphosphate is required for motility of bacterial pathogens. J. Bacteriol. 182:225-227.
Reusch, R. N., and H. L. Sadoff. 1988. Putative structure and functions of a poly-b -hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes. Proc. Natl. Acad. Sci. USA 85:4176-4180.
Tseng, C.-M., and A. Kornberg. 1998. Polyphosphate kinase is highly conserved in many bacterial pathogens. Mol. Microbiol. 29:381-382.
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