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PubMed2005

Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus.

Inorganic polyphosphate (poly P) has been considered to be a translocatable form of phosphate (Pi) in arbuscular mycorrhizal fungi (AMF). Here we examined time-course changes in poly P content during the AMF colonization process. Onion (Allium cepa) plants were cultured with or without inoculation with Gigaspora margarita for 2-8 wk with periodic sampling. Poly P in the extracts, purified through gel filtration, was quantified by the reverse reaction of polyphosphate kinase. The length of poly P in mycorrhizal roots appeared to be shorter than in extraradical hyphae or in spores of the AMF, indicating that AMF depolymerize poly P before providing Pi to the host. The poly P content increased as colonization proceeded, and was highly correlated with the weight of the colonized roots. These results support the model that AMF supply Pi to the host through the poly P pool, and that the poly P content of a mycorrhizal root can be a good indicator of the Pi-supplying activity of AMF.

Mycorrhizae↗
PubMed1994

Iron-binding affinity of bacterial vaccine polysaccharides which contain phosphodiester linkages as part of the polymer chain and of other polyphosphates, including DNA.

The interaction of iron (II) with bacterial polysaccharides, possessing phosphodiester bonds as part of their polymer chain, has been studied by equilibrium binding dialysis using atomic absorption spectrophotometry. Ferrous ions were found to bind with a stoichiometry of one per two phosphates and with a binding constant of about 2.5 x 10(3) M-1. Similar results, but with larger (ca 1 x 10(4) M-1) binding constants were observed with DNA. This interaction helps explain the depolymerization of polyphosphates which has been observed in the presence of iron salts, and highlights the need to avoid iron contamination of vaccines (and other substances) which contain phosphodiester bonds. The interaction may also be a means of iron sequestration in bacteria which possess these cell-surface polyphosphates.

Bacterial Vaccines↗
PubMed1986

Factors affecting sensitivity of Staphylococcus aureus 196E to polyphosphates.

The effect of polyphosphates (eight compounds) on growth of Staphylococcus aureus 196E in brain heart infusion broth was studied. The organism was sensitive (in decreasing order) to chain polyphosphates with 21, 3, 13, and 15 PO4 groups, and bactericidal effects were observed with 0.5% of these compounds. No inhibition was effected by PPi or a metaphosphate. The inhibitory effects were pH dependent, and bacterial sensitivity was highest at pH greater than 7.4. Initial populations affected the number of survivors. No growth was observed after 24 h at 35 degrees C when the initial cell population was less than 10(4) CFU/ml, and a 100- to 1,000-fold decline in cell numbers occurred when initial populations were higher than 10(4) CFU/ml. Sodium tripolyphosphate produced less inhibition after heat sterilization (15 min, 121 degrees C) than after filter sterilization, whereas sodium hexametaphosphate (n = 21) retained most of its antimicrobial activity after heat sterilization. Supplementation of broth with Mg2+ was effective in overcoming inhibition by 0.5% sodium tripolyphosphate, and an addition of 0.25 to 1.0 mM cation restored most of the growth. Inhibition was partially eliminated by Ca2+ and Fe2+, but not by Zn2+ or Mn2+.

Culture Media↗
PubMed2000

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH.

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.

Candida↗
PubMed2004

Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants.

The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization. The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass. The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures. Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate. They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously. Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources. Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed. Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation. The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor. Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor. This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants.

In Situ Hybridization, Fluorescence↗
PubMed2002

Role of polyphosphate kinase in biofilm formation by Porphyromonas gingivalis.

In order to assess the role of polyphosphate kinase (PPK) in the physiology of Porphyromonas gingivalis, a ppk gene mutant, CW120, was constructed and characterized. P. gingivalis was demonstrated to synthesize short-chain polyphosphate (polyP) but not long-chain polyP. CW120 failed to survive in the stationary phase as well as the parental cell did, and it was attenuated in biofilm formation on polyvinylchloride and glass surfaces. Furthermore, the complementation by insertion of an intact copy of the ppk gene into the mutant CW120 restored its biofilm formation and stationary-phase survival. These results suggest that PPK may be important for incorporation of these organisms into subgingival plaque in the human oral cavity.

Biofilms↗
PubMed1980

Levels of acid-soluble polyphosphate in growing cultures of Saccharomyces cerevisiae.

Short-chain acid-soluble polyphosphates were extracted from growing cultures of Saccharomyces cerevisiae, and the changes in the levels of these compounds were determined. The production of acid-soluble polyphosphates correlated with the mitochondrial activities since it occurred in two bursts in respiration-competent yeast cells and in only one burst in respiration-deficient yeast cells. The possible role of these compounds is discussed.

Adenine↗
PubMed2000

Inorganic polyphosphate is required for motility of bacterial pathogens.

The ppk gene encodes polyphosphate kinase (PPK), the principal enzyme in many bacteria responsible for the synthesis of inorganic polyphosphate (polyP) from ATP. A null mutation in the ppk gene of six bacterial pathogens renders them greatly impaired in motility on semisolid agar plates; this defect can be corrected by the introduction of ppk gene in trans. In view of the fact that the motility of pathogens is essential to invade and establish systemic infections in host cells, this impairment in motility suggests a crucial and essential role of PPK or polyP in bacterial pathogenesis.

Bacteria↗
PubMed2005

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.

Animals↗
PubMed2006

Effect of temperature on synthesis of polyphosphates in Yersinia pseudotuberculosis and Listeria monocytogenes under starvation conditions.

It was found that at low temperature (6-8 degrees C) in the absence of nitrogen supply and at the presence of phosphate ions in the medium, Yersinia pseudotuberculosis and Listeria monocytogenes are able to actively synthesize reserve substances as polyphosphates. Most of the bacterial polyphosphates are alkali-soluble, especially at the preliminary stage of cell growth (lag-phase). This is proved by electron microscopic studies of ultrastructure of model microorganisms. During a long starvation period under conditions of carbon and energy source deficit, L. monocytogenes and Y. pseudotuberculosis consume this biopolymer for biosynthetic and bioenergetic processes.

Culture Media↗
PubMed1980

Influence of S-adenosylmethionine on DAPI-induced fluorescence of polyphosphate in the yeast vacuole.

Use of the fluorochrome 4',6-diamidino-2-phenylindole.2 HCl (DAPI) in ultraviolet microscopy revealed fluorescent objects in Brownian motion within the vacuoles of seven species of yeast. The abundance of these bodies increased when cells of Saccharomyces cerevisiae were transferred from growth medium to a glucose-phosphate solution, indicating that they contain polyphosphate. In addition, the effect on vacuolar fluorescence of supplementing a defined growth medium with amino acids provided evidence that they also contained S-adenosylmethionine. These deductions were supported by in vitro studies of the interaction and fluorescence of polyphosphate, S-adenosylmethionine, and DAPI. Vacuolar fluorescence of cells in suspension in flucose-phosphate solution was less after addition of exogenous arginine, lysine, or glutamine but not after addition of alanine, aspartic acid, or methionine. Mithramycin was superior to DAPI as a fluorochrome for ultraviolet demonstration of yeast nuclei since it stained the nuclei much more intensely and did not fluoresce with other material in the cells.

Amidines↗
PubMed1983

L-glutamine alteration of gene expression, not of polyphosphate and calcium metabolism, is a key event in arresting fungal sporulation.

Vegetatively growing cells of the coenocytic freshwater mould Achlya developed asexual sporangia and sporulated within 6 h of postransfer to a nutrient-free (starvation) medium. Sporangial development was arrested by the addition of L-glutamine to starving cells. During starvation (minus glutamine), three polyphosphate substances accumulated intracellularly, ATP was rapidly depleted, and a protein of molecular weight 42 000 (presumed to be actin) was actively synthesized, whereas synthesis of the most abundant detergent-soluble protein of molecular weight 83 000 (p83) ceased. In the presence of glutamine, starving cells used up the polyphosphates faster than they were formed. ATP depletion was delayed, cell calcium (Ca) exited rapidly, and synthesis of actin diminished while p83 synthesis continued unabated. Several pyrimidine analogues, including 5-diazouracil (which inhibited pyridimide nucleotide biosynthesis), and inorganic phosphate prevented Ca exit from glutamine-supplemented starving cells. The pyrimidine analogues delayed but did not inhibit sporangial development; however, they did not overcome glutamine suppression of sporangial development. Vegetatively growing and starving cells displayed significantly different protein synthesis patterns (monitored by polyacrylamide gel electrophoresis) but, when glutamine was added, it changed the protein synthesis pattern of starving cells to a form typical of vegetatively growing cells. Glutamine withdrawal reversed the effect and the cells differentiated. Pyrimidine analogues and inorganic phosphate did not alter the protein synthesis patterns of starving cells in the presence and absence of glutamine. The conclusion is that glutamine inhibition of sporangial development may be linked to its ability to subvert starving cell metabolism by making it vegetative like.

Calcium↗
PubMed2006

Accumulation of mercury in transgenic tobacco expressing bacterial polyphosphate.

The feasibility of transgenic tobacco, engineered to express bacterial polyphosphate (polyP), for phytoremediation of mercury pollution was evaluated. T3 progeny of the transgenic tobacco produced a large amount of polyP in leaves and showed a relatively high resistant phenotype to Hg2+ than its wild-type progenitors. These results suggest that the integrated ppk gene, encoding polyphosphate kinase (PPK), a key enzyme for polyP biosynthesis, is stably conserved in tobacco genome, and translated to active PPK which catalyzed biosynthesis of polyP, and suggest that polyP is capable of reducing the cytotoxicity of Hg2+, probably via chelation formation with polyP. The transgenic tobacco expressing polyP accumulated significantly more mercury than its wild-type progenitors from Hg2+-containing agar medium and simulated soils without taxing the tobacco plants suggesting that the transported Hg2+ was accumulated as a less toxic Hg-polyP complex in the tobacco tissues. Based on the results obtained in the present study, the polyP-mediated accumulation of mercury from mercurial-contaminated soils may provide an ecologically compatible approach for phytoremediation of mercury pollution.

Escherichia coli↗
PubMed2005

Characterization of denitrifying polyphosphate-accumulating organisms in activated sludge based on nitrite reductase gene.

Nitrite reductase gene (nirS) fragments in the activated sludge obtained from a sequencing batch reactor (SBR) under anaerobic-aerobic condition were cloned and classified by restriction fragment length polymorphism (RFLP) analysis, and representative fragments were sequenced. One of the nirS clones was approximately 70% of all nirS clones in anaerobic/aerobic (existing oxygen and nitrate) cycle operation in which a large amount of anoxic phosphate uptake was observed. Although the activated sludge samples analyzed might contain bacteria that did not accumulate polyphosphate, it was likely that this nirS fragment sequence was that from denitrifying polyphosphate-accumulating organisms (DNPAOs) which can utilize both oxygen and nitrate as electron acceptors. The sequence was similar to the nirS sequences of Thauera mechernichensis (83% similarity) and Azoarcus tolulyticus (83% similarity) both of which belong to the Rhodocyclus group.

Genes, Bacterial↗
PubMed1998

Use of Escherichia coli polyphosphate kinase for oligosaccharide synthesis.

The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.

Adenine Nucleotides↗
PubMed2006

Evaluation of sludge reduction and phosphorus recovery efficiencies in a new advanced wastewater treatment system using denitrifying polyphosphate accumulating organisms.

A new biological nutrient removal process, anaerobic-oxic-anoxic (A/O/A) system using denitrifying polyphosphate-accumulating organisms (DNPAOs), was proposed. To attain excess sludge reduction and phosphorus recovery, the A/O/A system equipped with ozonation tank and phosphorus adsorption column was operated for 92 days, and water quality of the effluent, sludge reduction efficiency, and phosphorus recovery efficiency were evaluated. As a result, TOC, T-N and T-P removal efficiency were 85%, 70% and 85%, respectively, throughout the operating period. These slightly lower removal efficiencies than conventional anaerobic-anoxic-oxic (A/A/O) processes were due to the unexpected microbial population in this system where DNPAOs were not the dominant group but normal polyphosphate-accumulating organisms (PAOs) that could not utilize nitrate and nitrite as electron acceptor became dominant. However, it was successfully demonstrated that 34-127% of sludge reduction and around 80% of phosphorus recovery were attained. In conclusion, the A/O/A system equipped with ozonation and phosphorus adsorption systems is useful as a new advanced wastewater treatment plant (WWTP) to resolve the problems of increasing excess sludge and depleted phosphorus.

Adsorption↗
PubMed2006

Competition between polyphosphate- and glycogen-accumulating organisms in enhanced-biological-phosphorus-removal systems: effect of temperature and sludge age.

Temperature and sludge age were found to be important factors in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-polyphosphate organisms (GAOs) and the resultant stability of enhanced-biological-phosphorus removal (EBPR). At 20 degrees C and a 10-day sludge age, PAOs were dominant in an anaerobic/aerobic (A/O) sequencing-batch reactor (SBR), as a result of their higher anaerobic-acetate-uptake rate and aerobic-biomass yield than GAOs. However, at 30 degrees C and a 10-day sludge age, GAOs were able to outcompete PAOs in the A/O SBR because of their higher anaerobic-acetate-uptake rate than PAOs. At 30 degrees C and a 5-day sludge age, GAOs coexisted with PAOs in the A/O SBR, resulting in unstable EBPR performance. At 30 degrees C, reducing the sludge age from 5 to 3 days improved the EBPR efficiency drastically, and the EBPR performance was stable. The maximum specific-anaerobic-acetate-uptake rates of GAO-enriched sludge were affected by temperature with the Arrhenius temperature coefficient theta of 0.042 (degrees C(-1) between 10 and 30 degrees C. The effect of sludge age (5 and 10 days) on the maximum specific-anaerobic-acetate-uptake rates of GAO-enriched activated sludge, however, was not significant. For the PAO-enriched activated sludge, the maximum specific-anaerobic-acetate-uptake rate did not change significantly between 20 and 30 degrees C, but significantly increased from 0.38 to 0.52 mmol-C/ mmol-C/h as the sludge age decreased from 10 to 3 days at 30 degrees C.

Bacteria, Aerobic↗
PubMed2002

Polyphasic approaches to the identification of predominant polyphosphate-accumulating organisms in a laboratory-scale anaerobic/aerobic activated sludge system.

By combination of denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA (PCR-DGGE), quinone profiling, and 16S rRNA-targeted fluorescence in situ hybridization (FISH), a polyphosphate-accumulating organism (PAO) responsible for phosphate (P)-removal was identified in activated sludge with high P-removal ability from a laboratory-scale anaerobic/aerobic continuous flow reactor. The DNA fragment from the most dense band on the DGGE gel was closely related to that of 'Candidatus Accumulibacter phosphatis' (beta-Proteobacteria). Quinone profiling also suggested the predominance of beta-Proteobacteria. FISH with a specific oligonucleotide probe designed for the sequence showed that the targeted bacterium was dominant in the activated sludge, and the accumulation and consumption of polyphosphate were observed by dual staining with 4',6-diamidino-2-phenylindole. The bacterium was concluded to be the responsible PAO in the reactor. However, when the P-removal ability per cell slightly decreased, the dominance of the PAO greatly diminished in the activated sludge. Such sludge might be dominated by other types of PAOs.

Base Sequence↗