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.

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.

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.

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.

General enzymatic screens identify three new nucleotidases in Escherichia coli. Biochemical characterization of SurE, YfbR, and YjjG.

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.

Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp.

The importance of inorganic polyphosphate (poly P) and poly P kinase (PPK), the enzyme principally responsible for its synthesis, has been established previously for stationary-phase survival of Escherichia coli and virulence in Pseudomonas aeruginosa. The gene (ppk) that encodes PPK is highly conserved among many bacterial pathogens, including Shigella and Salmonella spp. In view of the phylogenetic similarity of the enteropathogens and the frequency with which virulence factors are expressed in stationary phase, the ppk gene of pathogenic Shigella flexneri, Salmonella enterica serovar Dublin, and Salmonella enterica serovar typhimurium have been cloned and deleted. In some of these mutants lacking ppk, the phenotypes included features indicative of decreased virulence such as: (i) growth defects, (ii) defective responses to stress and starvation, (iii) loss of viability, (iv) polymyxin sensitivity, (v) intolerance to acid and heat, and (vi) diminished invasiveness in epithelial cells. Thus PPK may prove, as it has with P. aeruginosa, to be an attractive target for antibiotics, with low toxicity because PPK is not found in higher eukaryotes.