polyP Hydrolysis Research Articles

Prune regulates the metabolism of mammalian inorganic polyphosphates and bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved, ubiquitous polymer that is present in all studied organisms. It has shown a preferred localization within the mitochondria and it is composed of multiple orthophosphates (Pi) linked together by high energy phosphoanhydride bonds, identical to those found in ATP. The metabolism of polyP is well described in bacteria and yeast, but has yet to be elucidated in higher eukaryotes. However, recent evidence suggests that the mammalian F0F1 ATP synthase could be involved in both the synthesis and hydrolysis of polyP. The mitochondrial localized protein h-Prune, the human ortholog of Drosophila Prune, exhibits short chain exopolyphosphatase activity in vitro, in addition to its known cAMP phosphodiesterase activity. Here, we studied the role of prune orthologs in polyP metabolism in both mammalian HEK293 cells and Drosophila melanogaster. Our data shows that purified h-Prune is unable to hydrolyze polyP with chain lengths spanning from 13-33 Pi long. We then used interfering RNA to knock down the expression of prune orthologs and found that the polyP levels were significantly reduced in both HEK293 and Drosophila melanogaster. In HEK293 cells, ATP levels were also significantly decreased by h-Prune knockdown, although mitochondrial membrane potential was not affected. This suggests that h-Prune could either directly or indirectly influence the activity of the F0F1 ATP synthase. Concordantly, we found that h-Prune knockdown significantly decreased ATP synthase activity to levels comparable to inhibition by Oligomycin A. Our results collectively demonstrate the downstream ability of prune to regulate cellular polyP levels and bioenergetics, although further studies are required to elucidate the exact mechanism of this pathway.

Putative metabolism of Ca. Accumulibacter via the utilization of glucose

Ca. Accumulibacter was the predominant microorganism (relative FISH bio-abundance of 67 ± 5%) in a lab-scale sequential batch reactor that accomplished enhanced biological phosphorus removal (EBPR) while using glucose and acetate as the carbon sources (1:1 COD-based ratio). Both organic compounds were completely anaerobically consumed. The reactor's performance in terms of P/C ratio, phosphorous release and uptake, and overall kinetic and stoichiometric parameters were on the high end of the reported spectrum for EBPR systems (100:9.3 net mg phosphate removal per mg COD consumed when using glucose and acetate in a 1:1 ratio). The batch tests showed that, to the best of our knowledge, this is the first time a reactor enriched with Ca. Accumulibacter can putatively utilize glucose as the sole carbon source to biologically remove phosphate (COD:P (mg/mg) removal ratio of 100:6.3 when using only glucose). Thus, this research proposes that Ca. Accumulibacter directly anaerobically stored the fed glucose primarily as glycogen by utilizing the ATP provided via the hydrolysis of poly-P and secondarily as PHA by balancing its ATP utilization (glycogen generation) and formation (PHA storage). Alternative hypotheses are also discussed. The reported findings could challenge the conventional theories of glucose assimilation by Ca. Accumulibacter, and can be of significance for the biological removal of phosphorus from wastewaters with high contents of fermentable compounds or low VFAs.

Polyphosphate fertilizer use efficiency strongly relies on soil physicochemical properties and root-microbial activities

Limited phosphorus (P) bioavailability restricts global agriculture and food production. This element is considered the least plant-available nutrient, and it is highly susceptible to immobilization in the soil matrix. Among the mineral fertilizers used to increase soil P fertility, polyphosphates (PolyP) consist of polymers of OrthoP residues and have been shown to improve crop P uptake and resulting yields more than other forms of P amendments. PolyP fertilizers are also known for their progressive hydrolysis, improving P availability in the rhizosphere and plant P uptake throughout crop growth stages. However, PolyP behavior in soils is still understudied, including rhizosphere traits likely to be involved in PolyP use efficiency within the soil-root-microbe interface. To improve our knowledge of PolyP behavioral properties in the soil–plant continuum, this review is among the first studies to compile, discuss, and propose ideas regarding this research while focusing on the key soil biochemical factors responsible for PolyP hydrolysis and use by crop roots. A combination of exuded P-hydrolyzing enzymes and acidification of the rhizosphere can presumably mobilize available P from PolyP and thereby improve crop P acquisition. The importance of root-associated microbes (exhibiting high P-mobilization capacities) is also discussed as a promising rhizosphere trait that could contribute greatly to boost PolyP hydrolysis and thus increase PolyP agronomic efficiency.

Dissolved organic phosphorus utilization by the marine bacterium Ruegeria pomeroyiDSS-3 reveals chain length-dependent polyphosphate degradation.

SummaryDissolved organic phosphorus (DOP) is a critical nutritional resource for marine microbial communities. However, the relative bioavailability of different types of DOP, such as phosphomonoesters (P‐O‐C) and phosphoanhydrides (P‐O‐P), is poorly understood. Here we assess the utilization of these P sources by a representative bacterial copiotroph, Ruegeria pomeroyi DSS‐3. All DOP sources supported equivalent growth by R. pomeroyi, and all DOP hydrolysis rates were upregulated under phosphorus depletion (−P). A long‐chain polyphosphate (45polyP) showed the lowest hydrolysis rate of all DOP substrates tested, including tripolyphosphate (3polyP). Yet the upregulation of 45polyP hydrolysis under −P was greater than any other substrate analyzed. Proteomics revealed three common P acquisition enzymes potentially involved in polyphosphate utilization, including two alkaline phosphatases, PhoD and PhoX, and one 5′‐nucleotidase (5′‐NT). Results from DOP substrate competition experiments show that these enzymes likely have broad substrate specificities, including chain length‐dependent reactivity toward polyphosphate. These results confirm that DOP, including polyP, are bioavailable nutritional P sources for R. pomeroyi, and possibly other marine heterotrophic bacteria. Furthermore, the chain‐length dependent mechanisms, rates and regulation of polyP hydrolysis suggest that these processes may influence the composition of DOP and the overall recycling of nutrients within marine dissolved organic matter.

Inorganic Polyphosphate and F0F1-ATP Synthase of Mammalian Mitochondria.

Inorganic polyphosphate is a polymer which plays multiple important roles in yeast and bacteria. In higher organisms the role of polyP has been intensively studied in last decades and involvements of this polymer in signal transduction, cell death mechanisms, energy production, and many other processes were demonstrated. In contrast to yeast and bacteria, where enzymes responsible for synthesis and hydrolysis of polyP were identified, in mammalian cells polyP clearly plays important role in physiology and pathology but enzymes responsible for synthesis of polyP or consumption of this polymer are still not identified. Here, we discuss the role of mitochondrial F0F1-ATP synthase in polyP synthesis with results, which confirm this proposal. We also discuss the role of other enzymes which may play important roles in polyP metabolism.

Simulating the Interplay between the Uptake of Inorganic Phosphate and the Cell Phosphate Metabolism under Phosphorus Feast and Famine Conditions in Chlorella vulgaris.

Using a mathematical simulation approach, we studied the dynamics of the green microalga Chlorella vulgaris phosphate metabolism response to shortage and subsequent replenishing of inorganic phosphate in the medium. A three-pool interaction model was used to describe the phosphate uptake from the medium, its incorporation into the cell organic compounds, its storage in the form of polyphosphates, and culture growth. The model comprises a system of ordinary differential equations. The distribution of phosphorous between cell pools was examined for three different stages of the experiment: growth in phosphate-rich medium, incubation in phosphate-free medium, and phosphate addition to the phosphorus-starving culture. Mathematical modeling offers two possible scenarios for the appearance of the peak of polyphosphates (PolyP). The first scenario explains the accumulation of PolyP by activation of the processes of its synthesis, and the decline in PolyP is due to its redistribution between dividing cells during growth. The second scenario includes a hysteretic mechanism for the regulation of PolyP hydrolysis, depending on the intracellular content of inorganic phosphate. The new model of the dynamics of P pools in the cell allows one to better understand the phenomena taking place during P starvation and re-feeding of the P-starved microalgal cultures with inorganic phosphate such as transient PolyP accumulation. Biotechnological implications of the observed dynamics of the polyphosphate pool of the microalgal cell are considered. An approach enhancing the microalgae-based wastewater treatment method based on these scenarios is proposed.

The Histidine Ammonia Lyase of Trypanosoma cruzi Is Involved in Acidocalcisome Alkalinization and Is Essential for Survival under Starvation Conditions.

ABSTRACTTrypanosoma cruzi, the agent of Chagas disease, accumulates polyphosphate (polyP) and Ca2+ inside acidocalcisomes. The alkalinization of this organelle stimulates polyP hydrolysis and Ca2+ release. Here, we report that histidine ammonia lyase (HAL), an enzyme that catalyzes histidine deamination with production of ammonia (NH3) and urocanate, is responsible for acidocalcisome alkalinization. Histidine addition to live parasites expressing HAL fused to the pH-sensitive emission biosensor green fluorescent protein (GFP) variant pHluorin induced alkalinization of acidocalcisomes. PolyP decreased HAL activity of epimastigote lysates or the recombinant protein but did not cause its polyphosphorylation, as determined by the lack of HAL electrophoretic shift on NuPAGE gels using both in vitro and in vivo conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell fitness. This effect was enhanced by histidine and decreased when the lysine-rich C-terminal region was deleted. In conclusion, this study highlights a mechanism for stimulation of acidocalcisome alkalinization linked to amino acid metabolism.

Phenotypic dynamics in polyphosphate and glycogen accumulating organisms in response to varying influent C/P ratios in EBPR systems

This study employed molecular tools and single cell Raman micro-spectroscopy techniques to reveal the single cell- and population-level phenotypic dynamics and changes in functionally relevant organisms, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in response to influent loading readily biodegradable carbon to phosphorus ratio (C/P) changes in enhanced biological phosphorus removal (EBPR) systems. The results, for the first time, provided direct and cellular evidence confirming the adaptive anaerobic metabolic pathway shifts in PAOs in response to influent loading variations. Increase in influent readily biodegradable carbon to phosphorus (C/P) ratio from 20 to 50 led to nearly 50% decline in polyphosphate content and drastic rise of intracellular polyβhydroxybutyrate (PHB) to polyphosphate (polyP) ratio by nearly 6 times in PAOs, indicating corresponding diminishing reliance on polyP hydrolysis for energy as P becomes limiting. Influent carbon availability surge also impacted the intracellular carbon polymers in GAOs, with significant increase in the mean PHB content level but no observed changes in the intracellular glycogen level. Furthermore, the Raman-based quantification of differentiated intracellular polymer content associated with PAOs and GAOs, revealed new insights into the quantitative shift in intracellular carbon storage distribution between the two populations and their variations between the two carbon polymers (PHB, Glycogen). In summary, this investigation revealed high-resolution cellular level information regarding the metabolic flexibility in PAOs, phenotypic stoichiometry changes and carbon flux and distribution among PAOs and GAOs, in response to influent loading conditions. The new information will contribute to improvement in mechanistic EBPR modeling and design.

Comparison and optimization of extraction protocol for intracellular phosphorus and its polyphosphate in enhanced biological phosphorus removal (EBPR) sludge

Intracellular polyphosphate (poly-P) plays important roles in Enhanced biological phosphorus removal (EBPR) process, but an effective and reliable protocol for extracting intracellular P and its poly-P in EBPR sludge without hydrolysis of poly-P has not been setup yet. In the study, it was revealed that the severe hydrolysis of intracellular poly-P occurred during the different extraction processes, such as acid (i.e., HClO4, H2SO4 and HCl), basic (i.e., NaOH and KOH) and freezing-grind (under different solid-liquid ratios), but it did not occur during ultrasonic extraction process. The optimal extraction process of the ultrasonic protocol was 10 w/mL of ultrasonic power density and 15 min of ultrasonic time, when the extraction efficiency of intracellular P was 88.24 ± 1.56%. In addition, the extraction efficiency of intracellular P could be furtherly improved by that the 0.75 mol/L LiCl solution was used to resuspend the bacterial cell before ultrasonic extraction (i.e., LiCl-ultrasonic protocol). The ultrasonic protocol was more suitable to extract the intracellular P and its poly-P of EBPR sludge than the other 4 protocols (i.e., PCA-NaOH, EDTA-NaOH, freezing-grind and LiCl-ultrasonic), which had the technical characteristics of (i) with relatively high extraction efficiency of intracellular P, (ii) without hydrolysis of intracellular poly-P, (iii) with weak noise signal in 31P NMR spectrum and (iv) with simple extraction process and short extraction time. It was founded by the ultrasonic protocol that there was the high content (82.88%–89.79% of intracellular P content) of intracellular poly-P with long average chain length (376.4–383.2) in the EBPR sludges. Importantly, it was confirmed that the EBPR process was related to the combined action of extracellular and intracellular poly-P using a new fractionation method of P in EBPR sludge, which included the ultrasonic protocol at high power density for extracting the intracellular P and its poly-P.

The acidocalcisome inositol-1,4,5-trisphosphate receptor of Trypanosoma brucei is stimulated by luminal polyphosphate hydrolysis products

Acidocalcisomes are acidic calcium stores rich in polyphosphate (polyP) and are present in trypanosomes and also in a diverse range of other organisms. Ca2+ is released from these organelles through a channel, inositol 1,4,5-trisphosphate receptor (TbIP3R), which is essential for growth and infectivity of the parasite Trypanosoma brucei However, the mechanism by which TbIP3R controls Ca2+ release is unclear. In this work, we expressed TbIP3R in a chicken B lymphocyte cell line in which the genes for all three vertebrate IP3Rs were stably ablated (DT40-3KO). We show that IP3-mediated Ca2+ release depends on Ca2+ but not on ATP concentration and is inhibited by heparin, caffeine, and 2-aminomethoxydiphenyl borate (2-APB). Excised patch clamp recordings from nuclear membranes of DT40 cells expressing only TbIP3R disclosed that luminal inorganic orthophosphate (Pi) or pyrophosphate (PPi), and neutral or alkaline pH can stimulate IP3-generated currents. In contrast, polyP or acidic pH did not induce these currents, and nuclear membranes obtained from cells expressing rat IP3R were unresponsive to polyP or its hydrolysis products. Our results are consistent with the notion that polyP hydrolysis products within acidocalcisomes or alkalinization of their luminal pH activate TbIP3R and Ca2+ release. We conclude that TbIP3R is well-adapted to its role as the major Ca2+ release channel of acidocalcisomes in T. brucei.

Extraction and quantification of polyphosphates in activated sludge from waste water treatment plants by 31P NMR spectroscopy

Polyphosphate (poly-P) is a major constituent in activated sludge from wastewater treatment plants with enhanced biological phosphorus removal due to poly-P synthesis by poly-P accumulating organisms where it plays an important role for recovery of phosphorus from waste water. Our aim was to develop a reliable protocol for poly-P quantification by 31P NMR spectroscopy. This has so far been complicated by the risks of inefficient extraction and poly-P hydrolysis in the extracts. A protocol for complete extraction, identification and quantification of poly-P in activated sludge from a waste water treatment plant was identified based on test and evaluation of existing extraction protocols in combination with poly-P determination and quantification by solution and solid state 31P NMR spectroscopy. The total poly-P middle group content was quantified by solid state NMR for comparison with the poly-P middle groups quantified by solution NMR, which is novel. Three different extraction protocols previously used in literature were compared: 1) a single 0.25 M NaOH-0.05 M EDTA extraction, 2) a 0.05 M EDTA pre-extraction followed by a 0.25 M NaOH main extraction and 3) a 0.05 M EDTA pre-extraction followed by a 0.25 M NaOH-0.05 M EDTA main extraction. The results showed that the extraction protocol 2 was optimal for fresh activated sludge, extracting 10.8 ± 0.4 to 11.4 ± 1.2 mgP/gDW poly-P. Extraction protocols 1 and 3 extracted less than 9.4 ± 0.5 mgP/gDW poly-P. A comparison of the quantification of poly-P by 31P solution NMR and by 31P solid state NMR spectroscopy of lyophilised activated sludge showed 86 ± 9% extraction efficiency of poly-P, which confirms that the extraction protocol recovered most of the poly-P from the samples without pronounced poly-P degradation.

Dissolved Organic Phosphorus Utilization by Phytoplankton Reveals Preferential Degradation of Polyphosphates Over Phosphomonoesters

The nutritionally available pool of dissolved organic phosphorus (DOP) supports marine primary productivity in a range of ocean ecosystems but remains poorly resolved. Here, the relative lability of model phosphorous (P) compounds representing the major P(V) bond classes of marine DOP – phosphomonoesters (P-O-C) and phosphoanhydrides (P-O-P) – was assessed in diatom cultures of the genus Thalassiosira, as well as coastal field sites of the western North Atlantic. In diatom samples, maximum enzymatic hydrolysis rates revealed that the P-anhydride bonds of inorganic tripolyphosphate (3poly-P), followed by the P-anhydride bonds of adenosine 5’-triphosphate (ATP), were preferentially degraded relative to the P-monoesters adenosine 5’-monophosphate (AMP) and 4-methylumbelliferone phosphate (MUF-P). Consistent with these rate measurements, targeted proteomics analysis demonstrated that the underlying phosphatase diversity present in diatom samples was dominated by P-anhydride degrading enzymes (inorganic pyrophosphatases and nucleoside triphosphatases). Furthermore, biomass-normalized rates of ATP degradation were always suppressed under P-replete conditions in diatom cultures, but the effect of overall P availability on 3poly-P degradation was inconsistent among diatom strains, suggesting that inorganic polyphosphate (poly-P) degradation may persist irrespective of prevailing P levels in the marine environment. Indeed, the majority of field sites examined in the P-replete coastal western North Atlantic exhibited significantly higher maximum rates of inorganic poly-P hydrolysis relative to P-monoester hydrolysis, which was largely driven by phytoplankton dynamics. Based on these results, the possibility that P-anhydride utilization may contribute comparably or even more substantially than P-esters to community-level P demand, phytoplankton growth, and primary productivity should be considered.

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Enzymatic quantification and length determination of polyphosphate down to a chain length of two

Polyacrylamide gel electrophoresis, being the current method of choice for length determination of inorganic polyphosphate (polyP), requires a sequencing apparatus, relies on commercially not available polyP length standards and yields only a chain length distribution. State of the art polyP quantification involves enzymatic hydrolysis of polyP to orthophosphate with the Saccharomyces cerevisiae exopolyphosphatase 1 (scPpx1p) and subsequent colorimetric orthophosphate detection. Because scPpx1p leaves one pyrophosphate per polyP, short chain polyPs are only partially detected. To overcome this analytical limitation, a method involving both the scPpx1p and the S. cerevisiae inorganic pyrophosphatase (scIpp1p) is proposed. Differential enzymatic hydrolysis of polyP with scPpx1p, and a combination of scIpp1p and scPpx1p allows not only for comprehensive quantification of polyP (excluding cyclic polyP) down to a chain length of two, but also absolute average chain length determination in the range of two to approximately 80. An optimized one-reagent method for rapid (2 min) orthophosphate quantification is part of the assay. Biological phosphorous containing molecules at equimolar phosphorous concentrations regarding polyP do not interfere. The method requires 1.5 μg polyP and calls only for a plate reader. This is the first enzymatic method for simultaneous average polyP chain length determination as well as comprehensive quantification.

Re-visiting the Phostrip process to recover phosphorus from municipal wastewater

This study examined an innovative approach to make use of the Phostrip process to recover phosphorus (P) from municipal wastewater. Returned activated sludge (RAS) from a municipal wastewater treatment plant was systematically studied to examine P release kinetics of RAS in a recovery stream that contained high concentrations of phosphate (PO43−-P). Findings suggested that the specific P release rate in RAS declined with increasing concentration of PO43−-P in the recovery stream. However, there was a strong positive linear correlation between acetate consumed and P released by the RAS (Pearson product-moment correlation coefficient [r = 0.98, n = 45, p < 0.005]). The data also suggest that acetate concentration in the recovery stream was not a factor in the observed reduction of specific P release rate with increasing PO43−-P in the recovery stream. When P release rates (poly-P hydrolysis rate) at different initial P concentrations were modelled using a modified Michaelis-Menten equation, a good fit was achieved between the experimental and the modelled data. According to the model, the maximum specific P release rate (18 mg-P/g-MLSS.h) halved when PO43−-P concentration in the recovery stream reached approximately 83 mg-P/L. Additionally, the RAS demonstrated a Prelease/Cacetate uptake molar ratio of approximately 0.5. An application of the derived P release kinetics into an innovative side stream process configuration showed that a Phostrip tank with a small footprint (9 m3) is sufficient to facilitate P recovery from a wastewater treatment plant that receives 61 ML/d of influent.

Highly effective wastewater phosphorus removal by phosphorus accumulating organism combined with magnetic sorbent MFC@La(OH)3

To enhance the efficiency of phosphate removal by phosphorus accumulating organisms (PAOs), a magnetic Fe3O4 core-shell (MFC) sorbent was synthesized to remove the intracellular phosphorus of PAOs. The MFC was prepared by the hydrothermal method, and then functionalized with La(OH)3 by depositing on the MFC surface. The sorbent (MFC@La(OH)3) was easy to separate from wastewater and had a high phosphate adsorption capacity of 45.45 mg/g at 30 °C. The batch experiments were conducted to probe the influence of adsorbent on PAOs (Pseudomonas putida). The results showed that the adsorbent could inhibit the growth of microorganism by competing for the phosphate in solution, and further improving the hydrolysis of polyphosphates (polyP) from microorganism. The microorganism after the hydrolysis of polyP could accumulate phosphate more rapidly when it was added into phosphorus-rich medium. The optimized condition for polyP hydrolysis from microorganism was 0.1 g/100 mL of sorbent dosage and 1 h of contacting time. The sorbent exhibited stable phosphate adsorption behavior after 5 adsorption-desorption cycles. Phosphate could be effectively recovered through the regeneration of phosphate loaded MFC@La(OH)3 under alkaline condition. The present findings indicate that PAOs combined with MFC@La(OH)3 is a novel and highly effective method for removing phosphate in wastewater and recovering phosphorus resource.

Temperature Effects on Zn(II) Toxicity to Metabolisms of Polyphosphate Accumulating Organisms in Aerobic and Anaerobic Conditions

This study explores the influence of temperature on the tolerance of polyphosphate accumulating organisms (PAOs) to Zn(II) in enhanced biological phosphorus removal. The results show anaerobic and aerobic metabolisms of PAOs decreased with increasing Zn(II) concentration varying between 0-2 mg L-1 and temperature indeed affected inhibitive degrees. Furthermore, Zn(II) is more toxic to anaerobic poly-P hydrolysis and glycogen degradation at 10°C than at 20 and 30°C. For anaerobic polyhydroxyalkanoate (PHA) synthesis, Zn(II) had a highly inhibitive effect at 10°C too. The inhibitions of PAOs aerobically taking up phosphorus, degrading PHA and replenishing glycogen in the presence of Zn(II) were amplified at 10°C. The metabolism of aerobic PHA degradation at 10°C was completely terminated and the aerobic replenishment of glycogen at 10° C was also completely terminated at 0.1 mg L-1 of Zn(II) due to the complete inhibition of aerobic PHA metabolism, which provides the required reducing power for synthesizing glycogen.

Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner (Lepidoptera: Noctuidae): A role for inorganic polyphosphate towards yolk mobilization

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.

Potentiation of the Cytotoxic Activity of Copper by Polyphosphate on Biofilm-Producing Bacteria: A Bioinspired Approach

Adhesion and accumulation of organic molecules represent an ecologically and economically massive problem. Adhesion of organic molecules is followed by microorganisms, unicellular organisms and plants together with their secreted soluble and structure-associated byproducts, which damage unprotected surfaces of submerged marine structures, including ship hulls and heat exchangers of power plants. This is termed biofouling. The search for less toxic anti-biofilm strategies has intensified since the ban of efficient and cost-effective anti-fouling paints, enriched with the organotin compound tributyltin, not least because of our finding of the ubiquitous toxic/pro-apoptotic effects displayed by this compound [1]. Our proposed bio-inspired approach for controlling, suppressing and interfluencing the dynamic biofouling complex uses copper as one component in an alternative anti-fouling system. In order to avoid and overcome the potential resistance against copper acquired by microorganisms we are using the biopolymer polyphosphate (polyP) as a further component. Prior to being functionally active, polyP has to be hydrolyzed to ortho-phosphate which in turn can bind to copper and export the toxic compound out of the cell. It is shown here that inhibition of the hydrolysis of polyP by the bisphosphonate DMDP strongly increases the toxic effect of copper towards the biofilm-producing Streptococcus mutans in a synergistic manner. This bisphosphonate not only increases the copper-caused inhibition of cell growth but also of biofilm production by the bacteria. The defensin-related ASABF, a marine toxin produced by the sponge Suberites domuncula, caused only an additive inhibitory effect in combination with copper. We conclude that the new strategy, described here, has a superior anti-biofilm potential and can be considered as a novel principle for developing bio-inspired antifouling compounds, or cocktails of different compounds, in the future.

Metabolic shift of polyphosphate-accumulating organisms with different levels of polyphosphate storage

Previous studies have shown that polyphosphate-accumulating organisms (PAOs) are able to behave as glycogen-accumulating organisms (GAOs) under different conditions. In this study we investigated the behavior of a culture enriched with Accumulibacter at different levels of polyphosphate (poly-P) storage. The results of stoichiometric ratios Glydegraded/HAcuptake, PHBsynthesized/HAcuptake, PHVsynthesized/HAcuptake and Prelease/HAcuptake confirmed a metabolic shift from PAO metabolism to GAO metabolism: PAOs with high poly-P content used the poly-P to obtain adenosine tri-phosphate (ATP), and glycogen (Gly) to obtain nicotinamide adenine dinucleotide (NADH) and some ATP. In a test where poly-P depletion was imposed on the culture, all the acetate (HAc) added in each cycle was transformed into polyhydroxyalkanoate (PHA) despite the decrease of poly-P inside the cells. This led to an increase of the Glydegraded/HAcuptake ratio that resulted from a shift towards the glycolytic pathway in order to compensate for the lack of ATP formed from poly-P hydrolysis. The shift from PAO to GAO metabolism was also reflected in the change in the PHA composition as the poly-P availability decreased, suggesting that polyhydroxyvalerate (PHV) is obtained due to the consumption of excess reducing equivalents to balance the internal NADH, similarly to GAO metabolism. Fluorescence in situ hybridization analysis showed a significant PAO population change from Type I to Type II Accumulibacter as the poly-P availability decreased in short term experiments. This work suggests that poly-P storage levels and GAO-like metabolism are important factors affecting the competition between different PAO Types in enhanced biological phosphorus removal systems.