Diversification of polyphosphate end-labeling via bridging molecules.

Investigation of the biological roles of inorganic polyphosphate has been facilitated by our previous development of a carbodiimide-based method for covalently coupling primary amine-containing molecules to the terminal phosphates of polyphosphate. We now extend that approach by optimizing the reaction conditions and using readily available "bridging molecules" containing a primary amine and an additional reactive moiety, including another primary amine, a thiol or a click chemistry reagent such as dibenzocyclooctyne. This two-step labeling method is used to covalently attach commercially available derivatives of biotin, peptide epitope tags, and fluorescent dyes to the terminal phosphates of polyphosphate. Additionally, we report three facile methods for purifying conjugated polyphosphate from excess reactants.

Biogenic Polyphosphate Nanoparticles from a Marine Cyanobacterium Synechococcus sp. PCC 7002: Production, Characterization, and Anti-Inflammatory Properties In Vitro.

Probiotic-derived polyphosphates have attracted interest as potential therapeutic agents to improve intestinal health. The current study discovered the intracellular accumulation of polyphosphates in a marine cyanobacterium Synechococcus sp. PCC 7002 as nano-sized granules. The maximum accumulation of polyphosphates in Synechococcus sp. PCC 7002 was found at the late logarithmic growth phase when the medium contained 0.74 mM of KH2PO4, 11.76 mM of NaNO3, and 30.42 mM of Na2SO4. Biogenic polyphosphate nanoparticles (BPNPs) were obtained intact from the algae cells by hot water extraction, and were purified to remove the organic impurities by Sephadex G-100 gel filtration. By using 100 kDa ultrafiltration, BPNPs were fractionated into the larger and smaller populations with diameters ranging between 30⁻70 nm and 10⁻30 nm, respectively. 4',6-diamidino-2-phenylindole fluorescence and orthophosphate production revealed that a minor portion of BPNPs (about 14⁻18%) were degraded during simulated gastrointestinal digestion. In vitro studies using lipopolysaccharide-activated RAW264.7 cells showed that BPNPs inhibited cyclooxygenase-2, inducible nitric oxide (NO) synthase expression, and the production of proinflammatory mediators, including NO, tumor necrosis factor-α, interleukin-6, and interleukin-1ß through suppressing the Toll-like receptor 4/NF-κB signaling pathway. Overall, there is promise in the use of the marine cyanobacterium Synechococcus sp. PCC 7002 to produce BPNPs, an anti-inflammatory postbiotic.

Analysis of chitosan/tripolyphosphate micro- and nanogel yields is key to understanding their protein uptake performance.

Chitosan/tripolyphosphate (TPP) micro- and nanogels are widely explored as vehicles for protein drug and vaccine delivery. Yet, aside from the consensus that protein uptake into these particles is enhanced by stronger protein/particle binding, factors that control their uptake performance, such as differences in the chitosan, TPP and protein concentrations, remain poorly understood. Here, we show that many of the differences in the reported association efficiencies (AE-values) for protein uptake likely reflect the largely-ignored variability in the particle yield (XAgg), which is the fraction of the added chitosan that self-assembles into particles and (like the AE) varies with the chitosan, TPP and protein concentrations. Factors affecting XAgg are first systematically explored. The AE is then shown to scale almost linearly with the XAgg (which increases with the TPP and protein-to-chitosan ratios) until all chitosan aggregates into particles. Remarkably, the data collected at variable TPP and protein concentrations collapses onto a single AE∝XAgg curve for each protein type. Further analysis of protein/particle binding reveals this rise in AE with XAgg to reflect: (1) an increase in binding sites within the particles; and (2) a decrease in soluble (non-particulate) chitosan molecules, which form soluble protein/chitosan complexes and compete with the chitosan/TPP particles for the unassociated protein. These findings highlight the need to carefully analyze the effects of formulation parameters on chitosan/TPP particle yields and can likely be extended to other ionically crosslinked colloidal drug carriers.

Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli.

Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8-fold compared with non-targeted PPK1. Co-expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co-expression of PPX with non-targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal.

Performance of an Ultraviolet Mutagenetic Polyphosphate-Accumulating Bacterium PZ2 and Its Application for Wastewater Treatment in a Newly Designed Constructed Wetland.

Total phosphorus (TP) removal performance and application for wastewater treatment of polyphosphate-accumulating bacteria (PAB) in constructed wetlands (CWs) were investigated. In this study, a novel isolated ultraviolet (UV) mutant PZ2 with phosphate-accumulating ability was screened from domestic wastewater and identified as Pseudomonas putida by 16S ribosomal DNA (rDNA) sequencing analysis. The TP removal performance of PZ2 in the synthetic wastewater reached the highest of 93.95 % within 45 h. Two vertical subsurface flow CWs planted with two aquatic macrophytes Canna indica and Acorus calamus were newly designed. After inoculating PZ2 into two CWs within 45 h, the average chemical oxygen demand (COD), TP, and ammonia-nitrogen (NH3-N) removal efficiencies reached 68.50, 60.22, and 66.81 %, respectively. Vegetation type and filter size significantly influenced the TP removal capability of PZ2 in CWs. Meanwhile, considerable qualitative differences were found in the pollutant removal efficiencies of PZ2 with and without CWs in synthetic wastewater. These results could also indicate potential applications of the UV mutagenesis in PAB isolation and the newly designed CWs in wastewater treatments.

PHO8 gene coding alkaline phosphatase of Saccharomyces cerevisiae is involved in polyphosphate metabolism.

It has been argued for a long time whether alkaline phosphatase (ALP) is involved in polyphosphate (polyP) metabolism in arbuscular mycorrhizal fungi. In the present study, we have analyzed the effects of disrupting the PHO8 gene, which encodes phosphate (Pi)-deficiency-inducible ALP, on the polyP contents of Saccharomyces cerevisiae. The polyP content of the Δpho8 mutant was higher than the wild type strain in the logarithmic phase under Pi-sufficient conditions. On the contrary, the chain length of polyP extracted from the Δpho8 mutant did not differ from the wild type strain. When cells in Pi-deficient conditions were supplemented with Pi, the increase of the polyP amounts in the Δpho8 mutant was similar to that in the wild type strain. These results suggest that ALP, which is encoded by PHO8, affects the polyP content, but not the chain length, and participates in polyP homeostasis in Pi-sufficient conditions.

Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

INTRODUCTION: Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. METHODS AND RESULTS: Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. CONCLUSIONS: Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

Recent advances in nutrient removal and recovery in biological and bioelectrochemical systems.

Nitrogen and phosphorous are key pollutants in wastewater to be removed and recovered for sustainable development. Traditionally, nitrogen removal is practiced through energy intensive biological nitrification and denitrification entailing a major cost in wastewater treatment. Recent innovations in nitrogen removal aim at reducing energy requirements and recovering ammonium nitrogen. Bioelectrochemical systems (BES) are promising for recovering ammonium nitrogen from nitrogen rich waste streams (urine, digester liquor, swine liquor, and landfill leachate) profitably. Phosphorus is removed from the wastewater in the form of polyphosphate granules by polyphosphate accumulating organisms. Alternatively, phosphorous is removed/recovered as Fe-P or struvite through chemical precipitation (iron or magnesium dosing). In this article, recent advances in nutrients removal from wastewater coupled to recovery are presented by applying a waste biorefinery concept. Potential capabilities of BES in recovering nitrogen and phosphorous are reviewed to spur future investigations towards development of nutrient recovery biotechnologies.

An efficient process for wastewater treatment to mitigate free nitrous acid generation and its inhibition on biological phosphorus removal.

Free nitrous acid (FNA), which is the protonated form of nitrite and inevitably produced during biological nitrogen removal, has been demonstrated to strongly inhibit the activity of polyphosphate accumulating organisms (PAOs). Herein we reported an efficient process for wastewater treatment, i.e., the oxic/anoxic/oxic/extended-idle process to mitigate the generation of FNA and its inhibition on PAOs. The results showed that this new process enriched more PAOs which thereby achieved higher phosphorus removal efficiency than the conventional four-step (i.e., anaerobic/oxic/anoxic/oxic) biological nutrient removal process (41 ± 7% versus 30 ± 5% in abundance of PAOs and 97 ± 0.73% versus 82 ± 1.2% in efficiency of phosphorus removal). It was found that this new process increased pH value but decreased nitrite accumulation, resulting in the decreased FNA generation. Further experiments showed that the new process could alleviate the inhibition of FNA on the metabolisms of PAOs even under the same FNA concentration.

Biological phosphorus removal from abattoir wastewater at very short sludge ages mediated by novel PAO clade Comamonadaceae.

Recent increases in global phosphorus costs, together with the need to remove phosphorus from wastewater to comply with water discharge regulations, make phosphorus recovery from wastewater economically and environmentally attractive. Biological phosphorus (Bio-P) removal process can effectively capture the phosphorus from wastewater and concentrate it in a form that is easily amendable for recovery in contrast to traditional (chemical) phosphorus removal processes. However, Bio-P removal processes have historically been operated at medium to long solids retention times (SRTs, 10-20 days typically), which inherently increases the energy consumption while reducing the recoverable carbon fraction and hence makes it incompatible with the drive towards energy self-sufficient wastewater treatment plants. In this study, a novel high-rate Bio-P removal process has been developed as an energy efficient alternative for phosphorus removal from wastewater through operation at an SRT of less than 4 days. The process was most effective at an SRT of 2-2.5 days, achieving >90% phosphate removal. Further reducing the SRT to 1.7 days resulted in a loss of Bio-P activity. 16S pyrotag sequencing showed the community changed considerably with changes in the SRT, but that Comamonadaceae was consistently abundant when the Bio-P activity was evident. FISH analysis combined with DAPI staining confirmed that bacterial cells of Comamonadaceae arranged in tetrads contained polyphosphate, identifying them as the key polyphosphate accumulating organisms at these low SRT conditions. Overall, this paper demonstrates a novel, high-rate phosphorus removal process that can be effectively integrated with short SRT, energy-efficient carbon removal and recovery processes.

The marine sponge-derived inorganic polymers, biosilica and polyphosphate, as morphogenetically active matrices/scaffolds for the differentiation of human multipotent stromal cells: potential application in 3D printing and distraction osteogenesis.

The two marine inorganic polymers, biosilica (BS), enzymatically synthesized from ortho-silicate, and polyphosphate (polyP), a likewise enzymatically synthesized polymer consisting of 10 to >100 phosphate residues linked by high-energy phosphoanhydride bonds, have previously been shown to display a morphogenetic effect on osteoblasts. In the present study, the effect of these polymers on the differential differentiation of human multipotent stromal cells (hMSC), mesenchymal stem cells, that had been encapsulated into beads of the biocompatible plant polymer alginate, was studied. The differentiation of the hMSCs in the alginate beads was directed either to the osteogenic cell lineage by exposure to an osteogenic medium (mineralization activation cocktail; differentiation into osteoblasts) or to the chondrogenic cell lineage by incubating in chondrocyte differentiation medium (triggering chondrocyte maturation). Both biosilica and polyP, applied as Ca²âº salts, were found to induce an increased mineralization in osteogenic cells; these inorganic polymers display also morphogenetic potential. The effects were substantiated by gene expression studies, which revealed that biosilica and polyP strongly and significantly increase the expression of bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase (ALP) in osteogenic cells, which was significantly more pronounced in osteogenic versus chondrogenic cells. A differential effect of the two polymers was seen on the expression of the two collagen types, I and II. While collagen Type I is highly expressed in osteogenic cells, but not in chondrogenic cells after exposure to biosilica or polyP, the upregulation of the steady-state level of collagen Type II transcripts in chondrogenic cells is comparably stronger than in osteogenic cells. It is concluded that the two polymers, biosilica and polyP, are morphogenetically active additives for the otherwise biologically inert alginate polymer. It is proposed that alginate, supplemented with polyP and/or biosilica, is a suitable biomaterial that promotes the growth and differentiation of hMSCs and might be beneficial for application in 3D tissue printing of hMSCs and for the delivery of hMSCs in fractures, surgically created during distraction osteogenesis.

Phosphate recovery as concentrated solution from treated wastewater by a PAO-enriched biofilm reactor.

Phosphorus recovery from wastewaters and its recycling are of importance for sustaining agricultural production. During the conventional enhanced biological phosphorus removal process, phosphorus is removed by withdrawing excess sludge from wastewater. However, excess sludge disposal is costly and energy intensive. A proposed novel process for phosphorus recovery from sewage treatment will result in no excess sludge if a polyphosphate accumulating organisms (PAOs) enrichment biofilm can be applied to effluents containing phosphate. This process allows the recovery of phosphate as phosphate-concentrated solutions by controlling PAOs to absorb and release phosphate. A reactor consisting of a modified trickling filter with a synthetic substrate (5 mg P L⁻¹) was operated to form a PAO-enriched biofilm. As a result of the enrichment, the concentration of phosphate of >100 mg P L⁻¹ was successfully achieved. During this experiment, no sludge withdrawal was carried out over the duration of the operation of 255 days. To highlight the new process, the principle of enriching PAOs on biofilm and concentrating phosphate from treated sewage is explained, and a discussion on phosphate recovery performance is given.

Fluorometric quantification of polyphosphate in environmental plankton samples: extraction protocols, matrix effects, and nucleic acid interference.

Polyphosphate (polyP) is a ubiquitous biochemical with many cellular functions and comprises an important environmental phosphorus pool. However, methodological challenges have hampered routine quantification of polyP in environmental samples. We tested 15 protocols to extract inorganic polyphosphate from natural marine samples and cultured cyanobacteria for fluorometric quantification with 4',6-diamidino-2-phenylindole (DAPI) without prior purification. A combination of brief boiling and digestion with proteinase K was superior to all other protocols, including other enzymatic digestions and neutral or alkaline leaches. However, three successive extractions were required to extract all polyP. Standard addition revealed matrix effects that differed between sample types, causing polyP to be over- or underestimated by up to 50% in the samples tested here. Although previous studies judged that the presence of DNA would not complicate fluorometric quantification of polyP with DAPI, we show that RNA can cause significant interference at the wavelengths used to measure polyP. Importantly, treating samples with DNase and RNase before proteinase K digestion reduced fluorescence by up to 57%. We measured particulate polyP along a North Pacific coastal-to-open ocean transect and show that particulate polyP concentrations increased toward the open ocean. While our final method is optimized for marine particulate matter, different environmental sample types may need to be assessed for matrix effects, extraction efficiency, and nucleic acid interference.

Solid-phase chemical synthesis of 5'-triphosphate DNA, RNA, and chemically modified oligonucleotides.

A chemical method for the solid-phase synthesis of 5'-triphosphate oligonucleotides is described. The full-length oligonucleotides are first constructed using standard solid-phase DNA/RNA synthesis, and then efficient implementation of a sequential 4-steps synthetic procedure, executed either manually or in a fully automated fashion, affords the corresponding solid-supported 5'-triphosphate oligonucleotides. Using this synthetic procedure, the full-length 5'-hydroxyl oligonucleotides are initially transformed into the corresponding 5'-H-phosphonate mono esters, subsequently oxidized in the presence of imidazole to the activated 5'-phosphorimidazolidates, and finally reacted with pyrophosphate on the solid support. The method uses safe, stable, and inexpensive reagents, and the process is scalable and readily applicable to automated synthesis compatible with the current commercially available DNA/RNA synthesizers. After cleavage from the solid support and deprotection, a range of DNA, RNA, and chemically modified 5'-triphosphate oligonucleotides are obtained in a convenient and efficient manner and isolated in good yields after HPLC purification.

High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i)) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i) but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i) in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i), which could be cytotoxic because of its high affinity for Ca(2+), thereby interfering with Ca(2+) signaling.

Process performance and polyphosphate-accumulating organism-glycogen-accumulating organism communities in an anaerobic/anoxic/oxic system operated with different carbon sources.

A pilot plant anaerobic/anoxic/oxic (A2/O) system fed with domestic wastewater was operated to examine the effect of varying different types of carbon source (acetic acid, propionic acid, and glucose), added as a complement to the wastewater, on the (1) process performance and (2) microbial population. The operational condition that lead to a significant removal of total nitrogen (82%) was achieved with acetic acid. When the complementary carbon source was propionic acid, an improved removal efficiency of orthophosphate (97%) was observed. Because this finding was concurrent with higher polyphosphate-accumulating organism (PAO) population fractions detected using fluorescent in situ hybridization analysis (41.9 +/- 3.0%), it suggests that members of PAO populations that were able to reduce nitrate gained importance over PAO members that could not, thus improving the denitrifying phosphorus removal.

Phosphorus release and uptake during start-up of a covered and non-aerated sequencing batch reactor with separate feeding of VFA and sulfate.

This study explored a sulfur cycle-associated biological phosphorus (P) removal process in a covered and non-aerated sequencing batch reactor (SBR) fed with volatile fatty acid (VFA) and sulfate separately. During the 60-day start-up, both phosphate release and uptake rates increased, while poly-phosphate cyclically increased and decreased accordingly. The P-release and P-uptake rates were associated with VFA uptake and sulfate reduction. The average ratio of potassium to phosphate during the P-uptake and P-release was also determined to be 0.29-0.31 mol K/mol P, which is close to a reported value (0.33) for biological phosphorus removal. All this evidence confirmed there was biological P removal in this reactor, in which metabolism could be different from conventional biological P removal.

[Biological characteristics of denitrifying polyphosphate-accumulating organisms].

A denitrifying phosphate-accumulating organisms (DNPAOs), which was called Q-hrb05, was isolated in the special medium from the anaerobic/aerobic/anoxic SBR reactor. Strain Q-hrb05 was identified by 16SrDNA gene analysis, and the accession number of 16SrDNA gene sequence of strain Q-hrb05 in GenBank was GU214826. Effects of the different pH values, temperature, carbon source of medium on nitrogen and phosphorus removal of strain Q-hrb05 were investigated. The result showed that strain Q-hrb05 belonged to Bacillus sp.. Meanwhile, extracellular exopolymers of strain Q-hrb05 was based on protein, about 120.6 mg x mL(-1), and it had 23.05 microg x mL(-1) nucleic acid, but little polysaccharide. There was no significant adsorption of phosphate. So phosphorus removal was mainly due to intracellular uptake. And when pH value was kept as 7, temperature was kept as 30 degrees C, and carbon source was kept sodium acetate, the highest nitrogen and phosphorus removal efficiency was achieved. Phosphorus uptake rate was averaged at 88%, and the denitrification rate reached 81%.

Direct quantification of inorganic polyphosphate in microbial cells using 4'-6-diamidino-2-phenylindole (DAPI).

Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.