Inorganic polyphosphate: essential for growth and survival.

Inorganic polyphosphate (Poly P) is a polymer of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds as in ATP. Found in abundance in all cells in nature, it is unique in its likely role in the origin and survival of species. Here, we present extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth. Beyond that, Poly P has proved in a variety of ways to be essential for growth of cells, their responses to stresses and stringencies, and the virulence of pathogens. In this review, we pay particular attention to the enzyme, polyphosphate kinase 1 (Poly P kinase 1 or PPK1), responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens. Mutants lacking PPK1 are defective in motility, quorum sensing, biofilm formation, and virulence. Structural studies are cited that reveal the conserved ATP-binding site of PPK1 at atomic resolution and reveal that the site can be blocked with minute concentrations of designed inhibitors. Another widely conserved enzyme is PPK2, which has distinctive kinetic properties and is also implicated in the virulence of some pathogens. Thus, these enzymes, absent in yeast and animals, are novel attractive targets for treatment of many microbial diseases. Still another enzyme featured in this review is one discovered in Dictyostelium discoideum that becomes an actin-like fiber concurrent with the synthesis, step by step, of a Poly P chain made from ATP. The Poly P-actin fiber complex, localized in the cell, lengthens and recedes in response to metabolic signals. Homologs of DdPPK2 are found in pathogenic protozoa and in the alga Chlamydomonas. Beyond the immediate relevance of Poly P as a target for anti-infective drugs, a large variety of cellular operations that rely on Poly P will be considered.

The long and short of it - polyphosphate, PPK and bacterial survival.

Inorganic polyphosphate (poly P) is present in all species tested to date, from each of the three kingdoms of life. Studied mainly in prokaryotes, poly P and its associated enzymes are important in diverse basic metabolism, in at least some structural functions and, notably, in stress responses. These numerous and unrelated roles for poly P are probably the consequence of its presence in life-forms from early in evolution. The genomes of many bacterial species, including pathogens, encode a homologue of a major poly P synthetic enzyme, poly P kinase 1 (PPK1). Loss of PPK1 results in reduced poly P levels, and deletion of the ppk1 gene in pathogens also results in a loss of virulence towards protozoa and animals. Thus far, no PPK1 homologue has been identified in higher-order eukaryotes and, therefore, PPK1 exhibits potential as a novel target for chemotherapy.

Polyphosphate kinase 1, a conserved bacterial enzyme, in a eukaryote, Dictyostelium discoideum, with a role in cytokinesis.

Polyphosphate kinase 1 (PPK1), the principal enzyme responsible for reversible synthesis of polyphosphate (poly P) from the terminal phosphate of ATP, is highly conserved in bacteria and archaea. Dictyostelium discoideum, a social slime mold, is one of a few eukaryotes known to possess a PPK1 homolog (DdPPK1). Compared with PPK1 of Escherichia coli, DdPPK1 contains the conserved residues for ATP binding and autophosphorylation, but has an N-terminal extension of 370 aa, lacking homology with any known protein. Polyphosphate or ATP promote oligomerization of the enzyme in vitro. The DdPPK1 products are heterogeneous in chain length and shorter than those of E. coli. The unique DdPPK1 N-terminal domain was shown to be necessary for its enzymatic activity, cellular localization, and physiological functions. Mutants of DdPPK1, as previously reported, are defective in development, sporulation, and predation, and as shown here, in late stages of cytokinesis and cell division.

A polyphosphate kinase 1 (ppk1) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects.

Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the beta-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions.

Inorganic polyphosphate essential for lytic growth of phages P1 and fd.

Transduction frequency with phage P1 had been observed to be very low in Escherichia coli K-12 mutants lacking the operon (ppk1-ppx) responsible for the synthesis of inorganic polyphosphate (poly P). We now find that these mutants, for lack of poly P, are lysogenic for P1 and when infected with phage P1 produce only approximately 1% the number of infective centers compared with the WT host. Both phage adsorption and release were unaffected. The host-encoded P1 late-gene transcriptional activator, SspA, failed to show the transcriptional increase in the mutant, observed in the WT. UV induction of a P1-infected mutant resulted in a 200-fold increase in the production of infectious phage particles. The lysogenized P1 (P1mut) and P1 progeny from the mutant host (Deltappk1-ppx) produced plaques of differing morphologies, whereas P1 progeny from the WT yielded only small, clear plaques. Two discernable variants, one producing small and clear plaques (P1small) and the other large plaques with turbid rims (P1large), had broader host range and produced larger burst sizes in WT compared with P1. Transmission electron microscopy showed P1mut had contractile sheath defects. Thus, the lack of poly P/PPK1 in the mutant host resulted in the formation of defective P1 particles during intracellular growth. A filamentous phage, fd, also failed to produce plaques on a mutant lawn. Although fd adsorbed to the F-pilus, its DNA failed to enter the mutant host.

Diverse phenotypes resulting from polyphosphate kinase gene (ppk1) inactivation in different strains of Helicobacter pylori.

Connections among biochemical pathways should help buffer organisms against environmental stress and affect the pace and trajectory of genome evolution. To explore these ideas, we studied consequences of inactivating the gene for polyphosphate kinase 1 (ppk1) in strains of Helicobacter pylori, a genetically diverse gastric pathogen. The PPK1 enzyme catalyzes synthesis of inorganic polyphosphate (poly P), a reservoir of high-energy phosphate bonds with multiple roles. Prior analyses in less-fastidious microbes had implicated poly P in stress resistance, motility, and virulence. In our studies, ppk1 inactivation caused the expected near-complete absence of poly P (>250-fold decrease) but had phenotypic effects that differed markedly among unrelated strains: (i) poor initial growth on standard brain heart infusion agar (five of six strains tested); (ii) weakened colonization of mice (4 of 5 strains); (iii) reduced growth on Ham's F-12 agar, a nutritionally limiting medium (8 of 11 strains); (iv) heightened susceptibility to metronidazole (6 of 17 strains); and (v) decreased motility in soft agar (1 of 13 strains). Complementation tests confirmed that the lack of growth of one Deltappk1 strain on F-12 agar and the inability to colonize mice of another were each due to ppk1 inactivation. Thus, the importance of ppk1 to H. pylori differed among strains and the phenotypes monitored. We suggest that quantitative interactions, as seen here, are common among genes that affect metabolic pathways and that H. pylori's high genetic diversity makes it well suited for studies of such interactions, their underlying mechanisms, and their evolutionary consequences.

Inorganic polyphosphate in the social life of Myxococcus xanthus: motility, development, and predation.

Inorganic polyphosphate (poly P), a polymer of tens or hundreds of phosphate residues linked by high-energy, ATP-like bonds, is found in all organisms and performs a wide variety of functions. Myxococcus xanthus, a social bacterium that feeds on other bacteria and forms fruiting bodies and spores, depends on poly P for motility, development, and nutritional predation. Two poly P metabolizing enzymes were studied in M. xanthus: poly P kinase 1, which synthesizes poly P reversibly from ATP, and poly P:AMP phosphotransferase, which uses poly P as a donor to also reversibly convert AMP to ADP. The null mutant of ppk1 is defective in social motility, overproduces pilin protein on the cell surface, is delayed in fruiting body formation, produces fewer spores, is delayed in germination, and forms far smaller plaques on a lawn of Klebsiella aerogenes. The pap mutant is also impaired in social motility, but shows only slightly reduced abilities in development and predation.

Endopolyphosphatase in Saccharomyces cerevisiae undergoes post-translational activations to produce short-chain polyphosphates.

Endopolyphosphatase (Ppn), responsible for cleavage of long chain inorganic polyphosphate (poly P) of several hundred residues to generate progressively shorter chains, has been identified in mammalian cells and purified from Saccharomyces cerevisiae. Disruption of the encoding gene, PHM5, in S. cerevisiae resulted in a mutant that showed limited growth and failure to survive in a minimal medium. The limited digestion products of the yeast enzyme Ppn1 judged to be P(3) and P(60) have now, with the homogeneous enzyme and improved separation methods, been demonstrated to be P(i) and P(3). Ppn1, a homotetramer of a 35-kDa subunit, is of vacuolar origin and requires protease activation of a 78 kDa (674-aa) precursor polypeptide (prePpn1). The protease-processed Ppn1 has been purified 3800-fold to homogeneity and the protease cleavage sites determined. Both termini of prePpn1 and the post-translational modification of N-glycosylations are essential for the protease-mediated maturation of Ppn1.

Inorganic polyphosphate in Dictyostelium discoideum: influence on development, sporulation, and predation.

Dictyostelium discoideum, a social slime mold that forms fruiting bodies with spores, depends on inorganic polyphosphate (poly P) for its cycles of development and for nutritional predation on bacteria. The synthesis of poly P, a polymer of tens or hundreds of phosphate residues linked by high energy, ATP-like bonds, is catalyzed in most bacteria by poly P kinase (PPK1). The eukaryote D. discoideum possesses a homolog of PPK1. We report here that mutants of D. discoideum PPK1 (DdPPK1) have reduced levels of poly P and are deficient in development. Fruiting bodies are smaller and produce fewer spores, which appear to germinate like the wild type (WT). The DdPPK1 mutant formed smaller plaques on bacterial lawns compared with those of the WT. Predation by D. discoideum, assessed by uptake and digestion of Klebsiella aerogenes, showed that fewer bacteria were taken up by the DdPPK1 mutant compared with the WT and were killed less rapidly, indicating a role of poly P and/or DdPPK1 in phagocytosis. On Pseudomonas aeruginosa lawns, cleared plaques were observed with the bacterial PPK1 mutant but not with the WT P. aeruginosa. Thus, poly P is important in predation both for the predator and prey.

Inorganic polyphosphate in Bacillus cereus: motility, biofilm formation, and sporulation.

Chains of inorganic polyphosphate (poly-P) with hundreds of P(i) residues linked by phosphoanhydride bonds, as in ATP, are found in every bacterial, fungal, plant, and animal cell, in which they perform various functions. In the spore-forming Bacillus cereus, we have identified three principal enzymes and genes involved in the metabolism of poly-P, namely, (i) poly-P kinase (PPK), which synthesizes poly-P reversibly from ATP, (ii) exopolyphosphatase (PPX), which hydrolyzes poly-P to P(i), and (iii) poly-P/AMP phosphotransferase (PAP), which uses poly-P as a donor to convert AMP to ADP, reversibly. In the null mutant of ppk, poly-P levels are reduced to <5% of the WT; in the ppx mutant, the PPK activity is elevated 10-fold, and the accumulation of poly-P is elevated approximately 1,000-fold. All of the null mutants of ppk, ppx, and pap showed defects in motility and biofilm formation, but sporulation efficiency was impaired only in the ppx mutant. These enzymes and genes in B. cereus are nearly identical to those in the very closely related pathogen Bacillus anthracis, and, thus, they may provide attractive targets for the treatment of anthrax.

Inorganic polyphosphate in the origin and survival of species.

Inorganic polyphosphate (poly P), in chains of tens to hundreds of phosphate residues, linked by high-energy bonds, is environmentally ubiquitous and abundant. In prebiotic evolution it could have provided a flexible, polyanionic scaffold to assemble macromolecules. It has been conserved in every cell in nature. In prokaryotes, a major poly P synthetic enzyme is poly P kinase 1 (PPK1), which is found in 100 bacterial genomes, including numerous pathogens. Null mutants of PPK1, with low poly P levels, are defective in survival: namely, they show defective responses to physical/chemical stresses and predation. Pathogens with a PPK1 deletion are defective in biofilm formation, quorum sensing, general stress and stringent responses, motility, and other virulence properties. With the exception of Dictyostelium, PPK1 is absent in eukaryotes and provides a novel target for chemotherapy that would affect both virulence and susceptibility to antibacterial compounds. Remarkably, another PPK in Dictyostelium discoideum (PPK2) is an actin-related protein (Arp) complex that is polymerized into an actin-like filament, concurrent with its reversible synthesis of a poly P chain from ATP.

Formation of an actin-like filament concurrent with the enzymatic synthesis of inorganic polyphosphate.

Inorganic polyphosphate (poly P), a chain of hundreds of phosphate residues linked by ATP-like bonds, is found in every cell in nature and is commonly produced from ATP by poly P kinases (e.g., PPK1). Dictyostelium discoideum, the social slime mold, possesses a PPK activity (DdPPK1) with sequence similarity to bacterial PPKs. We find here a previously unrecognized PPK (DdPPK2) in D. discoideum with the sequences and properties of actin-related proteins (Arps) that are similar to muscle actins in size, properties, and globular-filamentous structural transitions. Significantly, the unique actin inhibitors, phalloidin and DNase I, also inhibit synthesis of poly P by DdPPK2. Thus, this particular Arp complex is an enzyme that can polymerize into an actin-like filament concurrent with its synthesis of a poly P chain in a fully reversible reaction.

Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells.

Inorganic polyphosphate (poly P), chains of hundreds of phosphate residues linked by "high-energy" bonds as in ATP, has been conserved from prebiotic times in all cells. Poly P is essential for a wide variety of functions in bacteria, including virulence in pathogens. In this study, we observe the unique and many-fold stimulation by poly P in vitro of the protein kinase mTOR (mammalian target of rapamycin). To explore the role of poly P in mammalian cells, a yeast polyphosphatase, PPX1, was inserted into the chromosomes of MCF-7 mammary cancer cells. The transfected cells are markedly deficient in their response to mitogens, such as insulin and amino acids, as seen in their failure to activate mTOR to phosphorylate one of its substrates, PHAS-I (the initiation factor 4E-binding protein). In addition, the transfected cells are severely reduced in their growth in a serum-free medium. On the basis of these findings, we suggest that poly P (and/or PPX1) serves as a regulatory factor in the activation of mTOR in the proliferative signaling pathways of animal cells.

Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP.

An enzyme that uses inorganic polyphosphate (poly P) as a donor to convert GDP to GTP has been purified 1,300-fold to homogeneity from lysates of Pseudomonas aeruginosa PAOM5. Poly P chains of 30-50 residues are optimal; those of 15-700 residues can also serve. GDP is preferred over ADP among nucleoside diphosphate acceptors. This nucleoside diphosphate kinase (NDK) activity resides in the same protein isolated for its synthesis of poly P from GTP and designated PPK2 in an accompanying report. The reaction that synthesizes poly P and the reaction that utilizes poly P differ in their kinetic features. Especially notable is the catalytic potency of the NDK activity, which is 75-fold greater than that of poly P synthesis. PPK2 appears in the stationary phase of growth and reaches NDK levels of 5-10% that of the classic NDK; both kinase activities may figure in the generation of the guanosine precursors in the synthesis of alginate, an exopolysaccharide essential for the virulence of P. aeruginosa.

A polyphosphate kinase (PPK2) widely conserved in bacteria.

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) distinguished 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 sequence of the protein purified near 1,000-fold, to homogeneity. The 5'-end is 177 bp upstream of the annotated genome sequence 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 proteins in P. aeruginosa, in two Archaea, and in 32 other bacteria (almost all with PPK1 as well); these include rhizobia, cyanobacteria, 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.