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Partial nitrification (PN) and high glycogen accumulating metabolism (GAM) activity are the basis for efficient nitrogen (N) and phosphorus (P) removal in simultaneous nitrification endogenous denitrification and phosphorus removal (SNDPR) systems. However, achieving these processes in practical operations is challenging. This study proposes that light irradiation is a novel strategy to enhance the nutrient removal performance of the SNDPR system with low carbon to nitrogen ratios (C/N of 3.3-4.1) domestic wastewater. Light energy densities (Es) of 55-135 J/g VSS were found to promote the activity of ammonia-oxidizing bacteria (AOB) and GAM, while inhibiting the activity of nitrite-oxidizing bacteria (NOB) and polyphosphate accumulating metabolism (PAM). Long-term exposure to different light patterns at Es of 55-135 J/g VSS revealed that continuous light rapidly achieved PN by inhibiting NOB activity and promoted the growth of glycogen accumulating organisms (GAOs), allowing the removal of above 82 % N and below 80 % P. Intermittent light maintained stable PN by inhibiting the activity and growth of NOB and promoted the growth of polyphosphate accumulating organisms (PAOs) with high GAM activity (Accmulibacer IIC-ii and IIC-iii), allowing the removal of above 82 % N and 95 % P. Flow cytometry and enzyme activity assays showed that light promoted GAM-related enzyme activity and the metabolic activity of partial Accmulibacer II over other endogenous denitrifying bacteria, while inhibiting NOB translation activity. These findings provide a new approach for enhancing nutrient removal, especially for achieving PN and promoting GAM activity, in SNDPR systems treating low C/N ratio domestic wastewater using light irradiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Importance: Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic Mycobacterium species secrete polyP, and the polyP is necessary for the bacteria to prevent their killing after phagocytosis. Conversely, exogenous polyP prevents the killing of ingested bacteria that are normally killed after phagocytosis by human macrophages and the eukaryotic microbe Dictyostelium discoideum . This suggests the possibility that in these cells, a signal transduction pathway is used to sense polyP and prevent killing of ingested bacteria. In this report, we identify key components of the polyP signal transduction pathway in D. discoideum . In cells lacking these components, polyP is unable to inhibit killing of ingested bacteria. The pathway components have orthologs in human cells, and an exciting possibility is that pharmacologically blocking this pathway in human macrophages would cause them to kill ingested pathogens such as Mycobacterium tuberculosis ., Competing Interests: The authors declare no conflict of interest.

Tetrasphaera-related polyphosphate accumulating organisms (PAOs) are the key functional guilds for enhanced biological phosphorus removal (EBPR) systems. Their biomass enrichment can be enhanced by the nitrification inhibitor allylthiourea (ATU). However, the underlying assembly mechanism and the functional potential of the EBPR microbiome regulated by ATU are unclear. This study investigates the effect of ATU on microbiome assembly and functional potential by closely following the microbiota dynamics in an EBPR system enriched with Tetrasphaera-related PAOs for 288-days before, during and after ATU addition. The results showed that ATU addition increased microbiota structural similarity and compositional convergence, and enhanced determinism in the assembly of EBPR microbiome. During exposure to ATU, Tetrasphaera-related PAOs were governed by homogeneous selection and the dominant species revealed by 16S rRNA gene-based phylogenetic analysis shifted from clade III to clade I. Meanwhile, ATU supply promoted significant enrichment of functional genes involved in phosphate transport (pit) and polyphosphate synthesis and degradation (ppk1 and ppk2), whereas both Nitrosomonas and ammonia monooxygenase-encoding genes (amoA/B/C) assignable to this group of nitrifying bacteria decreased. Moreover, ATU addition relieved the significant abundance correlation between filamentous bacteria Ca. Promineofilum and denitrifying Brevundimonas (FDR-adjusted P < 0.01), damaging their potential synergic or cooperative interactions, thus weakening their competitiveness against Tetrasphaera-related PAOs. Notably, ATU withdrawn created opportunistic conditions for the unexpected explosive growth and predominance of Thiothrix filaments, leading to a serious bulking event. Our study provides new insights into the microbial ecology of Tetrasphaera-related PAOs in EBPR system, which could guide the establishment of an efficient microbiota for EBPR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Inorganic polyphosphates are evolutionarily conserved bioactive phosphate polymers found as various chain lengths in all living organisms. In mammals, polyphosphates play a vital role in the regulation of cellular metabolism, coagulation, and inflammation. Long-chain polyphosphates are found along with endotoxins in pathogenic gram-negative bacteria and can participate in bacterial virulence. We aimed to investigate whether exogenously administered polyphosphates modulate human leukocyte function in vitro by treating the cells with 3 different chain lengths of polyphosphates (P14, P100, and P700). The long-chain polyphosphates, P700, had a remarkable capacity to downregulate type I interferon signaling dose dependently in THP1-Dual cells while only a slight elevation could be observed in the NF-κB pathway with the highest dose of P700. P700 treatment decreased lipopolysaccharide-induced IFNβ transcription and secretion, reduced STAT1 phosphorylation, and downregulated subsequent interferon-stimulated gene expression in primary human peripheral blood mononuclear cells. P700 also augmented lipopolysaccharide-induced secretion of IL-1α, IL-1β, IL-4, IL-5, IL-10, and IFNγ. Furthermore, P700 has previously been reported to increase the phosphorylation of several intracellular signaling mediators, such as AKT, mTOR, ERK, p38, GSK3α/β, HSP27, and JNK pathway components, which was supported by our findings. Taken together, these observations demonstrate the extensive modulatory effects P700 has on cytokine signaling and the inhibitory effects specifically targeted to type I interferon signaling in human leukocytes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

We previously found that overexpression of phosphate starvation-responsive genes by disrupting PHO80 led to a shortened replicative lifespan in yeast. To identify lifespan-related genes, we screened upregulated genes in the pho80Δ mutant and focused on the VTC genes, which encode the vacuolar polyphosphate (polyP) polymerase complex. VTC1/VTC2/VTC4 deletion restored the lifespan and intracellular polyP levels in pho80Δ. In the wild type, overexpression of VTC5 or a combination of the other VTCs caused high polyP accumulation and shortened lifespan. Similar phenotypes were caused by the deletion of polyP phosphatase genes-vacuolar PPN1 and cytosolic PPX1. The polyP-accumulating strains exhibited stress sensitivities. Thus, we demonstrated that polyP metabolic enzymes participate in replicative lifespan, and extreme polyP accumulation shortens the lifespan., (© 2023 Federation of European Biochemical Societies.)

Inorganic polyphosphate (polyP) is a biopolymer composed of phosphoanhydride-linked orthophosphate molecules. PolyP is engaged in a variety of cellular functions, including mitochondrial metabolism. Here, we examined the effects of polyP on electron transport chain enzymes and F 1 F o ATP synthase in tick embryos during embryonic development. The study found that polyPs containing medium and long chains (polyP 15 and polyP 65 ) enhanced the activity of complex I, complex II, complex III, and F 1 F o ATP synthase, while short polyP chains (polyP 3 ) had no effect. The study also examined the activity of exopolyphosphatases (PPX) in various energy-demand situations. PPX activity was stimulated when ADP concentrations are high, characterizing a low-energy context. When complexes I-III and F 1 F o ATP synthase inhibitors were added in energized mitochondria, PPX activity decreased, whereas the mitochondrial uncoupler FCCP had no impact on PPX activity. Additionally, the study investigated the effect of polyP on mitochondrial swelling, finding that polyP causes mitochondrial swelling by increasing calcium effects on the mitochondrial permeability transition pore. The findings presented here to increase our understanding of the function of polyP in mitochondrial metabolism and its relationship to mitochondrial permeability transition pore opening in an arthropod model., (© 2023 Wiley Periodicals LLC.)

" Candidatus Accumulibacter" is the major polyphosphate-accumulating organism (PAO) in global wastewater treatment systems, and its phylogenetic and functional diversity have expanded in recent years. In addition to the widely recognized type I and II sublineages, we discovered a novel type enriched in laboratory bioreactors. Core gene and machine learning-based gene feature profiling supported the assertion that type III " Ca. Accumulibacter" is a potential PAO with the unique function of using dimethyl sulfoxide as an electron acceptor. Based on the correlation between ppk1 and genome similarity, the species-level richness of Accumulibacter was estimated to be over 100, suggesting that the currently recognized species are only the tip of the iceberg. Meanwhile, the interstrain transcriptional and morphological features of multiple " Ca. Accumulibacter" strains co-occurring in a bioreactor were investigated. Metatranscriptomics of seven co-occurring strains indicated that the expression level and interphasic dynamics of PAO phenotype-related genes had minimal correlation with their phylogeny. In particular, the expression of denitrifying and polyphosphate (poly-P) metabolism genes exhibited higher interstrain and interphasic divergence than expression of glycogen and polyhydroxyalkanoate metabolic genes. A strategy of cloning rRNA genes from different strains based on similar genomic synteny was successfully applied to differentiate their morphology via fluorescence in situ hybridization. Our study further expands the phylogenetic and functional diversity of " Ca. Accumulibacter" and proposes that deciphering the function and capability of certain " Ca. Accumulibacter" should be tailored to the environment and population in question. IMPORTANCE In the last 2 decades, " Ca. Accumulibacter" has garnered significant attention as the core functional but uncultured taxon for enhanced biological phosphorus removal due to its phylogenetic and functional diversity and intragenus niche differentiation. Since 2002, it has been widely known that this genus has two sublineages (type I and II). However, in this study, a metagenomic approach led to the discovery of a novel type (type III) with proposed novel functional features. By comparing the average nucleotide identity of " Ca. Accumulibacter" genomes and the similarity of ppk1 , a phylogenetic biomarker largely deposited in databases, the global species-level richness of " Ca. Accumulibacter" was estimated for the first time to be over 100. Furthermore, we observed the co-occurrence of multiple " Ca. Accumulibacter" strains in a single bioreactor and found the simultaneous transcriptional divergence of these strains intriguing with regard to their niche differentiation within a single community. Our results indicated a decoupling feature between transcriptional pattern and phylogeny for co-occurring strains., Competing Interests: The authors declare no conflict of interest.

Polyphosphate (PolyP) is a polymer comprised of linear phosphate units connected by phosphate anhydride bonds. PolyP exists in a diverse range of eukaryotes and prokaryotes with varied chain lengths ranging from six to thousands of phosphate units. Upon activation, human platelets and neutrophils release short-chain PolyP, along with other components, to initiate the coagulation pathway. Long-chain PolyP derived from cellular or bacterial organelles exhibits higher proinflammatory and procoagulant effects compared to short-chain PolyP. Notably, PolyP has been identified as a low-hemorrhagic antithrombotic target since neutralizing plasma PolyP suppresses the thrombotic process without impairing the hemostatic functions. As an inorganic polymer without uniform steric configuration, PolyP is typically targeted by cationic polymers or recombinant polyphosphatases rather than conventional antibodies, small-molecule compounds, or peptides. Additionally, because of its procoagulant property, PolyP has been incorporated in wound-dressing materials to facilitate blood hemostasis. This review summarizes current studies on PolyP as a low-hemorrhagic antithrombotic target and the development of hemostatic materials based on PolyP.

Phosphorus (P) is a growth-limiting nutrient for plants, which is taken up by root tissue from the environment as inorganic phosphate (Pi). To maintain an appropriate status of cellular Pi, plants have developed sophisticated strategies to sense the Pi level and modulate their root system architecture (RSA) under the ever-changing growth conditions. However, the molecular basis underlying the mechanism remains elusive. Inositol polyphosphate kinase (IPK2) is a key enzyme in the inositol phosphate metabolism pathway, which catalyzes the phosphorylation of IP3 into IP5 by consuming ATP. In this study, the functions of a rice inositol polyphosphate kinase gene (OsIPK2) in plant Pi homeostasis and thus physiological response to Pi signal were characterized. As a biosynthetic gene for phytic acid in rice, overexpression of OsIPK2 led to distinct changes in inositol polyphosphate profiles and an excessive accumulation of Pi levels in transgenic rice under Pi-sufficient conditions. The inhibitory effects of OsIPK2 on root growth were alleviated by Pi-deficient treatment compared with wild-type plants, suggesting the involvement of OsIPK2 in the Pi-regulated reconstruction of RSA. In OsIPK2-overexpressing plants, the altered acid phosphatase (APase) activities and misregulation of Pi-starvation-induced (PSI) genes were observed in roots under different Pi supply conditions. Notably, the expression of OsIPK2 also altered the Pi homeostasis and RSA in transgenic Arabidopsis. Taken together, our findings demonstrate that OsIPK2 plays an important role in Pi homeostasis and RSA adjustment in response to different environmental Pi levels in plants., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Polyphosphate (poly-P) biosynthesis in bacteria has been linked to many physiological processes and has been characterized as an interesting functional molecule involved in intestinal homeostasis. We determined the capacity for poly-P production of 18 probiotic strains mainly belonging to Bifidobacterium and former Lactobacillus genera, showing that poly-P synthesis varied widely between strains and is dependent on the availability of phosphate and the growth phase. Bifidobacteria were especially capable of poly-P synthesis and poly-P kinase (ppk) genes were identified in their genomes together with a repertoire of genes involved in phosphate transport and metabolism. In Bifidobacterium longum KABP042, the strain we found with highest poly-P production, variations in ppk expression were linked to growth conditions and presence of phosphate in the medium. Moreover, the strain produced poly-P in presence of breast milk and lacto-N-tetraose increased the amount of poly-P synthesized. Compared to KABP042 supernatants low in poly-P, exposure of Caco-2 cells to KABP042 supernatants rich in poly-P resulted in decreased epithelial permeability and increased barrier resistance, induction of epithelial protecting factors such as HSP27 and enhanced expression of tight junction protein genes. These results highlight the role of bifidobacteria-derived poly-P as a strain-dependent functional factor acting on epithelial integrity., (© 2023. The Author(s).)

The intricate dynamics of inorganic polyphosphate (polyP) in response to phosphorus (P) limitation and metal exposure typical of contaminated aquatic environments is poorly understood. Cyanobacteria are important primary producers in aquatic environments that are exposed to P stringency as well as metal contamination. There is a growing concern regarding migration of uranium, generated as a result of anthropogenic activities, into the aquatic environments owing to high mobility and solubility of stable aqueous complexes of uranyl ions. The polyP metabolism in cyanobacteria in context of uranium (U) exposure under P limitation has hardly been explored. In this study, we analyzed the polyP dynamics in a marine, filamentous cyanobacterium Anabaena torulosa under combination of variable phosphate concentrations (overplus and deficient) and uranyl exposure conditions typical of marine environments. Polyphosphate accumulation (polyP + ) or deficient (polyP - ) conditions were physiologically synthesized in the A. torulosa cultures and were ascertained by (a) toulidine blue staining followed by their visualization using bright field microscopy and (b) scanning electron microscopy in combination with energy dispersive X-ray spectroscopy (SEM/EDX). On exposure to 100 μM of uranyl carbonate at pH 7.8, it was observed that the growth of polyP + cells under phosphate limitation was hardly affected and these cells exhibited larger amounts of uranium binding as compared to polyP - cells of A. torulosa. In contrast, the polyP - cells displayed extensive lysis when exposed to similar U exposure. Our findings suggest that polyP accumulation played an important role in conferring uranium tolerance in the marine cyanobacterium, A. torulosa. The polyP-mediated uranium tolerance and binding could serve as a suitable strategy for remediation of uranium contamination in aquatic environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cells stabilize intracellular inorganic phosphate (P i ) to compromise between large biosynthetic needs and detrimental bioenergetic effects of P i . P i homeostasis in eukaryotes uses Syg1/Pho81/Xpr1 (SPX) domains, which are receptors for inositol pyrophosphates. We explored how polymerization and storage of P i in acidocalcisome-like vacuoles supports Saccharomyces cerevisiae metabolism and how these cells recognize P i scarcity. Whereas P i starvation affects numerous metabolic pathways, beginning P i scarcity affects few metabolites. These include inositol pyrophosphates and ATP, a low-affinity substrate for inositol pyrophosphate-synthesizing kinases. Declining ATP and inositol pyrophosphates may thus be indicators of impending P i limitation. Actual P i starvation triggers accumulation of the purine synthesis intermediate 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which activates P i -dependent transcription factors. Cells lacking inorganic polyphosphate show P i starvation features already under P i -replete conditions, suggesting that vacuolar polyphosphate supplies P i for metabolism even when P i is abundant. However, polyphosphate deficiency also generates unique metabolic changes that are not observed in starving wild-type cells. Polyphosphate in acidocalcisome-like vacuoles may hence be more than a global phosphate reserve and channel P i to preferred cellular processes. IMPORTANCE Cells must strike a delicate balance between the high demand of inorganic phosphate (P i ) for synthesizing nucleic acids and phospholipids and its detrimental bioenergetic effects by reducing the free energy of nucleotide hydrolysis. The latter may stall metabolism. Therefore, microorganisms manage the import and export of phosphate, its conversion into osmotically inactive inorganic polyphosphates, and their storage in dedicated organelles (acidocalcisomes). Here, we provide novel insights into metabolic changes that yeast cells may use to signal declining phosphate availability in the cytosol and differentiate it from actual phosphate starvation. We also analyze the role of acidocalcisome-like organelles in phosphate homeostasis. This study uncovers an unexpected role of the polyphosphate pool in these organelles under phosphate-rich conditions, indicating that its metabolic roles go beyond that of a phosphate reserve for surviving starvation., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to develop peptide antibiotic-polyphosphate nanoparticles that are able to overcome the enzymatic and mucus barriers providing a targeted drug release directly on the intestinal epithelium. Polymyxin B-polyphosphate nanoparticles (PMB-PP NPs) were formed via ionic gelation between the cationic peptide and the anionic polyphosphate (PP). The resulting NPs were characterized by particle size, polydispersity index (PDI), zeta potential, and cytotoxicity on Caco-2 cells. The protective effect of these NPs for incorporated PMB was evaluated via enzymatic degradation studies with lipase. Moreover, mucus diffusion of NPs was investigated with porcine intestinal mucus. Isolated intestinal alkaline phosphatase (IAP) was employed to trigger the degradation of NPs and consequent drug release. PMB-PP NPs exhibited an average size of 197.13 ± 14.13 nm, a PDI of 0.36, a zeta potential of -11.1 ± 3.4 mV and a concentration and time-dependent toxicity. They provided entire protection toward enzymatic degradation and exhibited significantly ( p < 0.05) higher mucus permeating properties than PMB. When incubated with isolated IAP for 4 h, monophosphate and PMB were constantly released from PMB-PP NPs and zeta potential raised up to -1.9 ± 0.61 mV. According to these findings, PMB-PP NPs are promising delivery systems to protect cationic peptide antibiotics against enzymatic degradation, to overcome the mucus barrier and to provide drug release directly at the epithelium.

Phosphorus (P) is the second most important macronutrient for crop growth and a limiting factor in food production. Choosing the right P fertilizer formulation is important for crop production systems because P is not mobile in soils, and placing phosphate fertilizers is a major management decision. In addition, root microorganisms play an important role in helping phosphorus fertilization management by regulating soil properties and fertility through different pathways. Our study evaluated the impact of two phosphorous formulations (polyphosphates and orthophosphates) on physiological traits of wheat related to yield (photosynthetic parameters, biomass, and root morphology) and its associated microbiota. A greenhouse experiment was conducted using agricultural soil deficient in P (1.49%). Phenotyping technologies were used at the tillering, stem elongation, heading, flowering, and grain-filling stages. The evaluation of wheat physiological traits revealed highly significant differences between treated and untreated plants but not between phosphorous fertilizers. High-throughput sequencing technologies were applied to analyse the wheat rhizosphere and rhizoplane microbiota at the tillering and the grain-filling growth stages. The alpha- and beta-diversity analyses of bacterial and fungal microbiota revealed differences between fertilized and non-fertilized wheat, rhizosphere, and rhizoplane, and the tillering and grain-filling growth stages. Our study provides new information on the composition of the wheat microbiota in the rhizosphere and rhizoplane during growth stages (Z39 and Z69) under polyphosphate and orthophosphate fertilization. Hence, a deeper understanding of this interaction could provide better insights into managing microbial communities to promote beneficial plant-microbiome interactions for P uptake.

Sulfate in wastewater can be reduced to sulfide and its impact on the stability of enhanced biological phosphorus removal (EBPR) is still unclear. In this study, the metabolic changes and subsequent recovery of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) were investigated at different sulfide concentrations. The results showed that the metabolic activity of PAOs and GAOs was mainly related to H 2 S concentration. Under anaerobic conditions, the catabolism of PAOs and GAOs was promoted at H 2 S concentrations below 79 mg/L S and 271 mg/L S, respectively, and inhibited above these concentrations; whereas anabolism was consistently inhibited in the presence of H 2 S. The phosphorus (P) release was also pH-dependent due to the intracellular free Mg 2+ efflux from PAOs. H 2 S was more destructive to the esterase activity and membrane permeability of PAOs than those of GAOs and prompted intracellular free Mg 2+ efflux of PAOs, resulting in worse aerobic metabolism and subsequent recovery of PAOs than GAOs. Additionally, sulfides facilitated the production of extracellular polymeric substances (EPS), especially tightly bound EPS. The amount of EPS in GAOs was significantly higher than that in PAOs. The above results indicated that sulfide had a stronger inhibition to PAOs than GAOs, and when sulfide was present, GAOs had a competitive advantage over PAOs in EBPR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Polyphosphate (polyP) is a conserved polymer of inorganic phosphate residues that can reach thousands of moieties in length. PolyP has been implicated in cellular functions ranging from energy and phosphate homeostasis to cell signalling in eukaryotes from yeast to humans. Despite the interest in the role of polyP as a signalling molecule, the spatiotemporal regulation of polyP itself remains poorly understood. This knowledge gap limits our ability to understand how polyP impacts the physiology of normal and diseased cells and how this might be exploited in a therapeutic context. Polyphosphatases, enzymes that degrade polyP to generate shorter chains and free inorganic phosphate are ideally positioned to mediate polyP dynamics. However, little is known about how the activities of these enzymes are linked to specific cellular functions and how they might be regulated. Here, we provide an in-depth overview of polyphosphatase enzymes in budding yeast, which has served as a workhorse for polyP research, and in mammalian cells where the enzymes that make and degrade polyP have remained elusive. We identify critical open questions in both systems and propose strategies to guide future work., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Lysosome-related organelles (LROs) are a class of heterogeneous organelles conserved in eukaryotes that primarily play a role in storage and secretion. An important function of LROs is to mediate metal homeostasis. Chlamydomonas reinhardtii is a model organism for studying metal ion metabolism; however, structural and functional analyses of LROs in C. reinhardtii are insufficient. Here, we optimized a method for purifying these organelles from 2 populations of cells: stationary phase or overloaded with iron. The morphology, elemental content, and lysosomal activities differed between the 2 preparations, even though both have phosphorus and metal ion storage functions. LROs in stationary phase cells had multiple non-membrane-bound polyphosphate granules to store phosphorus. Those in iron-overloaded cells were similar to acidocalcisomes (ACs), which have a boundary membrane and contain 1 or 2 large polyphosphate granules to store more phosphorus. We established a method for quantifying the capacity of LROs to sequester individual trace metals. Based on a comparative proteomic analysis of these 2 types of LROs, we present a comprehensive AC proteome and identified 113 putative AC proteins. The methods and protein inventories provide a framework for studying the biogenesis and modification of LROs and the mechanisms by which they participate in regulating metal ion metabolism., Competing Interests: Conflict of interest statement. The authors declare that the research was conducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular phosphate (P i ) homeostasis. To discover how the VTC complex performs its function, we determined a cryo-electron microscopy structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure reveals a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. Our findings, along with the functional data, allow us to propose a mechanism of polyP channel gating and VTC complex activation., (© 2023 The Authors.)

Macroautophagy/autophagy occurs basally under nutrient-rich conditions in most mammalian cells, contributing to protein and organelle quality control, and protection against aging and neurodegeneration. During autophagy, lysosomes are heavily utilized via their fusion with autophagosomes and must be repopulated to maintain autophagic degradative capacity. During starvation-induced autophagy, lysosomes are generated via de novo biogenesis under the control of TFEB (transcription factor EB), or by the recycling of autolysosome membranes via autophagic lysosome reformation (ALR). However, these lysosome repopulation processes do not operate under nutrient-rich conditions. In our recent study, we identify a sequential phosphoinositide conversion pathway that enables lysosome repopulation under nutrient-rich conditions to facilitate basal autophagy. Phosphatidylinositol-3,4-bisphosphate (PtdIns[3,4]P 2 ) signals generated downstream of phosphoinositide 3-kinase alpha (PI3Kα) during growth factor stimulation are converted to phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes by INPP4B (inositol polyphosphate-4-phosphatase type II B). We show that PtdIns3P is retained as endosomes mature into endolysosomes, and serves as a substrate for PIKFYVE (phosphoinositide kinase, FYVE-type zinc finger containing) to generate phosphatidylinositol-3,5-bisphosphate (PtdIns[3,5]P 2 ) to promote SNX2-dependent lysosome reformation, basal autophagic flux and protein aggregate degradation. Therefore, endosome maturation couples nutrient signaling to lysosome repopulation during basal autophagy by delivering PI3Kα-derived PtdIns3P to endolysosomes for PtdIns(3,5)P 2 -dependent lysosome reformation. Abbreviations: ALR: autophagic lysosome reformation; INPP4B: inositol polyphosphate-4-phosphatase type II B; PI3Kα: phosphoinositide 3-kinase alpha; PIKFYVE: phosphoinositide kinase FYVE-type zinc finger containing; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,4)P 2 : phosphatidylinositol-3,4-bisphosphate; PtdIns(3,5)P 2 phosphatidylinositol-3,5-bisphosphate; SNX2 sorting nexin 2; PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3.

Tetrasphaera-enhanced biological phosphorus removal (T-EBPR) was developed by augmenting conventional EBPR (C-EBPR) with Tetrasphaera to improve phosphorus removal from anaerobic digestate of swine wastewater. At influent total phosphorus (TP) concentrations of 45-55 mg/L, T-EBPR achieved effluent TP concentration of 4.17 ± 1.02 mg/L, 54 % lower than that in C-EBPR (8.98 ± 0.76 mg/L). The enhanced phosphorous removal was presumably due to the synergistic effect of Candidatus Accumulibacter and Tetrasphaera occupying different ecological niches. Bioaugmentation with Tetrasphaera promoted the polyphosphate accumulation metabolism depending more on the glycolysis pathway, as evidenced by an increase in intracellular storage compounds of glycogen and polyhydroxyalkanoates by 0.87 and 0.34 mmol C/L, respectively. The enhanced intracellular storage capacity was coincidentally linked to the increase in phosphorus release and uptake rates by 1.23 and 1.01 times, respectively. These results suggest bioaugmentation with Tetrasphaera could be an efficient way for improved phosphorus removal from high-strength wastewater., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity; this effect requires GrlD and Ppk1, but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, and to both decrease energy expenditures and retain nutrients.

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13 C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13 C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d -1 respectively) was higher than the expected value based on the solid removal rate (0.132 d -1 ), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d -1 . KEY POINTS: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations., (© 2023. The Author(s).)

Inorganic polyphosphate (polyP) is an ancient energy metabolite and phosphate store that occurs ubiquitously in all organisms. The vacuolar transporter chaperone (VTC) complex integrates cytosolic polyP synthesis from ATP and polyP membrane translocation into the vacuolar lumen. In yeast and in other eukaryotes, polyP synthesis is regulated by inositol pyrophosphate (PP-InsP) nutrient messengers, directly sensed by the VTC complex. Here, we report the cryo-electron microscopy structure of signal-activated VTC complex at 3.0 Å resolution. Baker's yeast VTC subunits Vtc1, Vtc3, and Vtc4 assemble into a 3:1:1 complex. Fifteen trans-membrane helices form a novel membrane channel enabling the transport of newly synthesized polyP into the vacuolar lumen. PP-InsP binding orients the catalytic polymerase domain at the entrance of the trans-membrane channel, both activating the enzyme and coupling polyP synthesis and membrane translocation. Together with biochemical and cellular studies, our work provides mechanistic insights into the biogenesis of an ancient energy metabolite., (© 2023. The Author(s).)

Phosphate (Pi) availability is a major factor limiting plant growth and development. The key transcription factor controlling Pi-starvation response (PSR) is PHOSPHATE STARVATION RESPONSE 1 (PHR1) whose transcript levels do not change with changes in Pi levels. However, how PHR1 stability is regulated at the post-translational level is relatively unexplored in Arabidopsis thaliana. Inositol polyphosphates (InsPn) are important signal molecules that promote the association of stand-alone SPX domain proteins with PHR1 to regulate PSR. Here, we show that NITROGEN LIMITATION ADAPTATION (NLA) E3 ligase can associate with PHR1 through its conserved SPX domain and polyubiquitinate PHR1 in vitro. The association with PHR1 and its ubiquitination is enhanced by InsP6 but not by InsP5. Analysis of InsPn-related mutants and an overexpression plant shows PHR1 levels are more stable in itpk4-1 and vih2-4/VIH1 amiRNA but less stable in ITPK4 overexpression plants. Under Pi-deficient conditions, nla seedlings contain high PHR1 levels, display long root hair and accumulate anthocyanin in shoots phenocopying PHR1 overexpression plants. By contrast, NLA overexpression plants phenocopy phr1 whose phenotypes are opposite to those of nla. Our results suggest NLA functions as a negative regulator of Pi response by modulating PHR1 stability and the NLA/PHR1 association depends on InsPn levels., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Excessive phosphorus can lead to eutrophication in marine and coastal ecosystems. Sulfur metabolism-associated microorganisms stimulate biological phosphorous removal. However, the integrating co-biotransformation mechanism of phosphorus and sulfur in subtropical marine mangrove ecosystems with Spartina alterniflora invasion is poorly understood. In this study, an ecological model of the coupling biotransformation of sulfur and phosphorus is constructed using metagenomic analysis and quantitative polymerase chain reaction strategies. Phylogenetic analysis profiling, a distinctive microbiome with high frequencies of Gammaproteobacteria and Deltaproteobacteria, appears to be an adaptive characteristic of microbial structures in subtropical mangrove ecosystems. Functional analysis reveals that the levels of sulfate reduction, sulfur oxidation, and poly-phosphate (Poly-P) aggregation decrease with increasing depth. However, at depths of 25-50 cm in the mangrove ecosystems with S. alterniflora invasion, the abundance of sulfate reduction genes, sulfur oxidation genes, and polyphosphate kinase (ppk) significantly increased. A strong positive correlation was found among ppk, sulfate reduction, sulfur oxidation, and sulfur metabolizing microorganisms, and the content of sulfide was significantly and positively correlated with the abundance of ppk. Further microbial identification suggested that Desulfobacterales, Anaerolineales, and Chromatiales potentially drove the coupling biotransformation of phosphorus and sulfur cycling. In particular, Desulfobacterales exhibited dominance in the microbial community structure. Our findings provided insights into the simultaneous co-biotransformation of phosphorus and sulfur bioconversions in subtropical marine mangrove ecosystems with S. alterniflora invasion., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vanillin is a potent growth-inhibiting factor in Saccharomyces cerevisiae during lignocellulose biorefineries. Here, a haploid gene-deletion library was screened to search for vanillin-tolerant mutants and explain the possible tolerance mechanisms. Twenty-two deletion mutants were identified. The deleted genes in these mutants were involved in phosphate and inositol polyphosphate metabolism and intracellular sterol transport. Activation of the phosphate signaling pathway is not conducive to yeast against the pressure of vanillin. Furthermore, the findings indicate the role of inositol polyphosphates in altering vanillin tolerance by regulating phosphate metabolism. Meanwhile, reducing the transport of sterols from the plasma membrane enhanced tolerance to vanillin. In the presence of vanillin, the representative yeast deletions, pho84Δ and lam3Δ, showed good growth performance and promoted rapid ethanol production. Overall, this study identifies robust yeast strain alternatives for ethanol fermentation of cellulose and provides guidance for further genomic reconstruction of yeast strains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Polyphosphate kinase 1 (Ppk1) generates polyphosphates (polyPs) by catalyzing phosphate transfer from ATP. In the presence of ATP, Myxococcus xanthus Ppk1 showed the highest activity with polyP60-70 but also showed high activity with orthophosphate and pyrophosphate. Ppk1 synthesizes long-chain polyPs with >1 000 phosphate residues from orthophosphate or pyrophosphate present in high concentrations, suggesting that in M. xanthus, Ppk1 uses intracellular ortho/pyrophosphate as an initial primer for polyP production. During M. xanthus starvation-induced development, the specific activity of Ppk1 peaked at 12 h (300-800 nmol/min/mg) and then gradually decreased. The polyP concentration was highest during mound formation (45 nmol phosphate/mg protein); then, the level of long-chain polyPs decreased and that of short-chain polyPs increased during fruiting body and spore formation. Myxococcus xanthus expresses two exopolyphosphatases, Ppx1 and Ppx2, which mainly degrade short- and long-chain polyPs, respectively, both of which were highest in vegetative cells and were detected during starvation, which may account for the degradation of polyPs. Thus, polyPs synthesized by Ppk1 early in starvation-induced development could be degraded by exopolyphosphatases and may also be used as substrates by polyP:AMP phosphotransferases and polyphosphate/ATP-NAD kinases to generate ADP and NADP+, respectively., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Phosphates and polyphosphates play ubiquitous roles in biology as integral structural components of cell membranes and bone, or as vehicles of energy storage via adenosine triphosphate and phosphocreatine. The solution phase space of phosphate species appears more complex than previously known. We present nuclear magnetic resonance (NMR) and cryogenic transmission electron microscopy (cryo-TEM) experiments that suggest phosphate species including orthophosphates, pyrophosphates, and adenosine phosphates associate into dynamic assemblies in dilute solutions that are spectroscopically "dark." Cryo-TEM provides visual evidence of the formation of spherical assemblies tens of nanometers in size, while NMR indicates that a majority population of phosphates remain as unassociated ions in exchange with spectroscopically invisible assemblies. The formation of these assemblies is reversibly and entropically driven by the partial dehydration of phosphate groups, as verified by diffusion-ordered spectroscopy (DOSY), indicating a thermodynamic state of assembly held together by multivalent interactions between the phosphates. Molecular dynamics simulations further corroborate that orthophosphates readily cluster in aqueous solutions. This study presents the surprising discovery that phosphate-containing molecules, ubiquitously present in the biological milieu, can readily form dynamic assemblies under a wide range of commonly used solution conditions, highlighting a hitherto unreported property of phosphate's native state in biological solutions.

The cells of Saccharomyces cerevisiae are capable for phosphate surplus: the increased uptake of phosphate (Pi) and accumulation of inorganic polyphosphate (polyP) occur when the cells after Pi limitation were cultivated in a medium supplemented with Pi. We demonstrated that single knockout mutations in the PHO84, PHO87, and PHO89 genes encoding plasma membrane phosphate transporters suppressed the Pi uptake and polyP accumulation under phosphate surplus at nitrogen starvation. The knockout strains in the PHM6 and PHM7 genes encoding unannotated PHO-proteins showed decreased polyP accumulation under Pi surplus both at nitrogen starvation and in complete YPD medium. This is due to the suppression of Pi uptake in the cells of these mutant strains. We speculate that Pi transporters of plasma membrane, and Phm6 and Phm7 proteins function in concert providing increased Pi uptake at phosphate surplus conditions., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Inorganic polyphosphates (polyP), according to literature data, are involved in the regulatory processes of molecular complex of the Saccharomyces cerevisiae cell wall (CW). The aim of the work was to reveal relationship between polyP, acid phosphatase Pho3p, and the major CW protein, glucanosyltransglycosylase Bgl2p, which is the main glucan-remodelling enzyme with amyloid properties. It has been shown that the yeast cells with deletion of the PHO3 gene contain more high molecular alkali-soluble polyP and are also more resistant to exposure to alkali and manganese ions compared to the wild type strain. This suggests that Pho3p is responsible for hydrolysis of the high molecular polyP on the surface of yeast cells, and these polyP belong to the stress resistance factors. The S. cerevisiae strain with deletion of the BGL2 gene is similar to the Δpho3 strain both in the level of high molecular alkali-soluble polyP and in the increased resistance to alkali and manganese. Comparative analysis of the CW proteins demonstrated correlation between the extractability of the acid phosphatase and Bgl2p, and also revealed a change in the mode of Bgl2p attachment to the CW of the strain lacking Pho3p. It has been suggested that Bgl2p and Pho3p are able to form a metabolon or its parts that connects biogenesis of the main structural polymer of the CW, glucan, and catabolism of an important regulatory polymer, polyphosphates.

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study, we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate in vitro using polyP as phosphate donor. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency. The predicted masses and identities of the mono- and oligo-phosphorylated thiamine metabolites were identified by high-performance liquid chromatography tandem mass spectrometry. Moreover, the biological activity of ThP2, that was synthesized by phosphorylation of ThP1 with polyP and PPK, as a cofactor of ThP2-dependent enzymes (here transketolase TktA from Escherichia coli) was confirmed in a coupled enzyme assay. Our study shows that PPKs are promiscuous enzymes in vitro that could be involved in the formation of a variety of phosphorylated metabolites in vivo., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro . In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S . siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg 2+ ), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S . siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.

The microalgal species Chlorella vulgaris was cultivated in batch conditions to identify the optimum set of initial conditions for the best biomass growth rate, phosphate removal, polyphosphate accumulation, and protein productivity. To study the effect of phosphorus deficiency caused stress, the microalgal biomass was exposed to phosphorus deficiency conditions for periods varying between 1 and 10 days and inoculated at different initial biomass and phosphate concentrations. A 10-day period of phosphate deficiency, supported by low initial biomass concentration (∼0.25 mg DW L -1 ), increased the phosphate removal by 62-175% when compared to the reference conditions. A 10-day period of biomass P-deficiency also boosted the polyphosphate accumulation and protein productivity, increasing them up to 40 and 46.8 times, respectively, if compared to reference conditions. At the same time, optimization algorithm model results suggested one-day biomass P-starvation with low initial biomass concentration as the optimum combination to achieve the highest performance while the initial phosphate concentration had less impact. The initial conditions suggested by the optimization model were validated in a sequencing batch photobioreactor, giving 101.7 and 138.0% more phosphate removal and polyphosphate accumulation, compared to the reference conditions. The obtained results present microalgae exposure to phosphorus stress as a supplementary tool for wastewater post-treatment targeted on rapid phosphorus removal., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Linda Mezule reports financial support was provided by Latvian Council of Sciences., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Glycerol is abundantly present in wastewater from industries such as biodiesel production facilities. Glycerol is also a potential carbon source for microbes that are involved in wastewater nutrient removal processes. The conversion of glycerol in biological phosphorus removal of aerobic granular sludge processes has not been explored to date. The current study describes glycerol utilization by aerobic granular sludge and enhanced biological phosphorus removal (EBPR). Robust granules with good phosphorus removal capabilities were formed in an aerobic granular sludge sequencing batch reactor fed with glycerol. The interaction between the fermentative conversion of glycerol and product uptake by polyphosphate accumulating organisms (PAO) was studied using stoichiometric and microbial community analysis. Metagenomic, metaproteomic and microscopic analysis identified a community dominated by Actinobacteria (Tessaracoccus and Micropruina) and a typical PAO known as Ca. Accumulibacter. Glycerol uptake facilitator (glpF) and glycerol kinase (glpK), two proteins involved in the transport of glycerol into the cellular metabolism, were only observed in the genome of the Actinobacteria. The anaerobic conversion appeared to be a combination of a substrate fermentation and product uptake-type reaction. Initially, glycerol fermentation led mainly to the production of 1,3-propanediol (1,3-PDO) which was not taken up under anaerobic conditions. Despite the aerobic conversion of 1,3-PDO stable granulation was observed. Over time, 1,3-PDO production decreased and complete anaerobic COD uptake was observed. The results demonstrate that glycerol-containing wastewater can effectively be treated by the aerobic granular sludge process and that fermentative and polyphosphate accumulating organisms can form a food chain in glycerol-based EBPR processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

The effects of salinity on highly enriched polyphosphate- or glycogen-accumulating organisms (PAOs or GAOs) have been revealed, which is meaningful but idealized. In this study, three salinity levels (0.5%, 1.0%, and 0.75%) were sequentially adopted in a PAOs and GAOs coexisted biological phosphorus removal (BPR) reactor within 150 days. Compared to a slight decrease of phosphorus removal efficiency (PRE) under 0.5% salinity (from 96.09% to 73.68%), doubled salinity (1.0%) resulted in a lengthy recovery period and a sharp PRE decline (13.89%), and the PRE was merely kept at 27.39% even through salinity was decreased to 0.75% hereafter. Salinity was also found to stimulate more extracellular protein secretion, resulting in sludge volume index reduction (<32.87 mL/g) and particle size enlargement (222.78 μm on average). Hyphomicrobium (0.96%-1.76%) and unclassified&#95;f&#95;Rhodobacteraceae (4.72%-13.33%) could resist certain salinity and conduct BPR, but better salt-tolerant Candidatus&#95;Competibacter eventually became the predominant genus (>40%)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOG 1 (ASK1), and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Understanding and characterizing the molecular background of the maintenance of genomic integrity might be a major factor in comprehending the exceptional ability of the malaria parasite, Plasmodium falciparum to adapt at a fast pace to antimalarials. A balanced nucleotide pool is an essential factor for high-fidelity replication. The lack of detailed studies on deoxynucleotide-triphosphate (dNTP) pools in various intraerythrocytic stages of Plasmodium falciparum motivated our present study. Here, we focused on the building blocks of DNA and utilized an EvaGreen-based dNTP incorporation assay to successfully measure the temporal dynamics of dNTPs in every intraerythrocytic stage and in drug-treated trophozoites. Our findings show that the ratio of dNTPs in the ring-stage parasites significantly differs from the more mature trophozoite and schizont stages. We were also able to detect dGTP levels that have never been shown before and found it to be the least abundant dNTP in all stages. Treatment with WR99210, a TS-DHFR inhibitor drug, affected not only dTTP, but also dGTP levels, despite its presumed selective action on pyrimidine biosynthesis. Results from our studies might assist in a better understanding of genome integrity mechanisms and may potentially lead to novel drug related aspects involving purine and pyrimidine metabolic targets., (© 2022. The Author(s).)

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF is to bind to and protect the blood circulating Factor VIII (FVIII). Therefore, here, we study the role of polyP in the VWF-FVIII complex in vitro and suggest its biological significance. Surface plasmon resonance and electrophoretic mobility assays indicated that polyP binds dynamically to VWF only in the absence of FVIII. Using the VWF Ristocetin Cofactor assay, the most accepted method for studying VWF in platelet adhesion, we found that polyP activates this role of VWF only at low levels of FVIII, such as in plasmas with chemically depleted FVIII and plasmas from severe hemophilia A patients. Moreover, we demonstrated that FVIII competes with polyP in the activation of VWF. Finally, polyP also increases the binding of VWF to platelets in samples from patients with type 2 and type 3 von Willebrand disease. We propose that polyP may be used in designing new therapies to activate VWF when FVIII cannot be used.

Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G + polymorphonuclear neutrophils, depletion of CD11c + SiglecF + alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1 -/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Roewe, Walachowski, Sharma, Berthiaume, Reinhardt and Bosmann.)

Obesity increases the risk of insulin resistance and type 2 diabetes through increased inflammation at cellular and tissue levels. Therefore, study of the molecular elements involved in obesity-related inflammation may contribute to preventing and controlling it. Inorganic polyphosphate is a natural phosphate polymer that has recently been attracting more attention for its role in inflammation and hemostasis processes. Polyphosphates are one of the main constituents of human platelets, which are secreted after platelet activation. Among other roles, they interact with multiple proteins of the coagulation cascade, trigger bradykinin release, and inhibit the complement system. Despite its importance, determinations of polyphosphate levels in blood plasma had been elusive until recently, when we developed a method to detect these levels precisely. Here, we perform cross sectional studies to evaluate plasma polyphosphate in: 25 children, most of them with obesity and overweight, and 20 adults, half of them with severe type 2 diabetes. Our results show that polyphosphate increases, in a significant manner, in children with insulin resistance and in type 2 diabetes patients. As we demonstrated before that polyphosphate decreases in healthy overweight individuals, these results suggest that this polymer could be an inflammation biomarker in the metabolic disease onset before diabetes.

Phosphorus-accumulating organisms (PAOs), which harbor metabolic mechanisms for phosphorus removal, are widely applied in wastewater treatment. Recently, novel PAOs and phosphorus removal metabolic pathways have been identified and studied. Specifically, Dechloromonas and Tetrasphaera can remove phosphorus via the denitrifying phosphorus removal and fermentation phosphorus removal pathways, respectively. As the main PAOs in biological phosphorus removal systems, the conventional PAO Candidatus Accumulibacter and the novel PAOs Dechloromonas and Tetrasphaera are thoroughly discussed in this paper, with a specific focus on their phosphorus removal metabolic mechanisms, process applications, community abundance and influencing factors. Dechloromonas can achieve simultaneous nitrogen and phosphorus removal in an anoxic environment through the denitrifying phosphorus removal metabolic pathway, which can further reduce carbon source requirements and aeration energy consumption. The metabolic pathways of Tetrasphaera are diverse, with phosphorus removal occurring in conjunction with macromolecular organics degradation through anaerobic fermentation. A collaborative oxic phosphorus removal pathway between Tetrasphaera and Ca. Accumulibacter, or a collaborative anoxic denitrifying phosphorus removal pathway between Tetrasphaera and Dechloromonas are future development directions for biological phosphorus removal technologies, which can further reduce carbon source and energy consumption while achieving enhanced phosphorus removal., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Bacterial antibiotic resistance is a major threat to human health. A combination of antibiotics with metals is among the proposed alternative treatments. Only one such combination is successfully used in clinics; it associates antibiotics with the metal bismuth to treat infections by Helicobacter pylori. This bacterial pathogen colonizes the human stomach and is associated with gastric cancer, killing 800,000 individuals yearly. The effect of bismuth in H. pylori treatment is not well understood in particular for sublethal doses such as those measured in the plasma of treated patients. We addressed this question and observed that bismuth induces the formation of homogeneously sized membrane vesicles (MVs) with unique protein cargo content enriched in bismuth-binding proteins, as shown by quantitative proteomics. Purified MVs of bismuth-exposed bacteria were strongly enriched in bismuth as measured by inductively coupled plasma optical emission spectrometry (ICP-OES), unlike bacterial cells from which they originate. Thus, our results revealed a novel function of MVs in bismuth detoxification, where secreted MVs act as tool to discard bismuth from the bacteria. Bismuth also induces the formation of intracellular polyphosphate granules that are associated with changes in nucleoid structure. Nucleoid compaction in response to bismuth was established by immunogold electron microscopy and refined by the first chromosome conformation capture (Hi-C) analysis of H. pylori. Our results reveal that even low doses of bismuth induce profound changes in H. pylori physiology and highlight a novel defense mechanism that involves MV-mediated bismuth extrusion from the bacteria and a probable local DNA protective response where polyphosphate granules are associated with nucleoid compaction. IMPORTANCE Bacterial resistance to antibiotics is a major threat to human health. Treatments combining antibiotics with metals were proposed to circumvent this hurdle. Only one such combination is successfully used in clinics associating antibiotics with the metal bismuth to treat infections by the human pathogen Helicobacter pylori. H. pylori causes 800,000 deaths by gastric cancer yearly. How bismuth impacts H. pylori and its response to this toxic metal were ill defined. We discovered that upon bismuth exposure, H. pylori secretes membrane vesicles that are enriched in bismuth. Bismuth also induces the formation of intracellular polyphosphate granules associated with compaction of the chromosome. Upon bismuth exposure, H. pylori displays both defense and protection mechanisms, with bismuth extrusion by vesicles and shielding of the chromosome.

Acinetobacter baumannii is an opportunistic pathogen known for high morbidity and mortality. It causes life-threatening infections, such as ventilator-associated pneumonia (VAP), bacteremia, meningitis, wound and urinary tract infections (UTI). Increase in carbapenem resistance exhibited by A. baumannii has accentuated the need for novel targets for effective treatment. Despite the pronounced relevance of PPK2 as a pathogenicity determinant in several pathogens, it has not been explored as a drug target in A. baumannii. The present study was piloted to investigate the substrate binding by A. baumannii PPK2 (AbPPK2), a two-domain Class II polyphosphate kinase 2. A homology model of AbPPK2 was developed and validated for molecular docking of ATP and ADP in the predicted binding pocket. Further analysis of AbPPK2 revealed a set of common residues in the catalytic cleft interacting with ATP and ADP which would be useful for the screening of inhibitors against A. baumannii., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, financial or otherwise., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tetrasphaera are polyphosphate accumulating organisms (PAOs) that play an important role in enhanced biological phosphorus removal (EBPR) from wastewater. The effect of a wide range of temperature changes (1-30 °C) on phosphorus removal, metabolism and clade-level community structure of Tetrasphaera-dominated PAOs was investigated. At 10 °C, the bioactivities of Tetrasphaera-dominated communities were obviously inhibited and the EBPR efficiency was only 73 %. Yet at 20-30 °C, EBPR efficiency reached 99 % and the relative abundance of Tetrasphaera was up to 90 %. The temperature variation changed the community distribution of Tetrasphaera clades, which was possibly a main reason for EBPR performance. Amino acids and PHA with different contents were intracellular metabolite of Tetrasphaera-dominated communities during phosphorus release and uptake at different temperatures. Moreover, Tetrasphaera fermented protein and amino acids and released VFAs. The outcomes suggested the great potential of Tetrasphaera-PAOs in the treatment of wastewater with varying temperatures and limited carbon sources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Excess phosphate (Pi) is stored into the vacuole through Pi transporters so that cytoplasmic Pi levels remain stable in plant cells. We hypothesized that the vacuolar Pi transporters may harbor a Pi-sensing mechanism so that they are activated to deliver Pi into the vacuole only when cytosolic Pi reaches a threshold high level. We tested this hypothesis using Vacuolar Phosphate Transporter 1 (VPT1), a SPX domain-containing vacuolar Pi transporter, as a model. Recent studies have defined SPX as a Pi-sensing module that binds inositol polyphosphate signaling molecules (InsPs) produced at high cellular Pi status. We showed here that Pi-deficient conditions or mutation of the SPX domain severely impaired the transport activity of VPT1. We further identified an auto-inhibitory domain in VPT1 that suppresses its transport activity. Taking together the results from detailed structure-function analyses, our study suggests that VPT1 is in the auto-inhibitory state when Pi status is low, whereas at high cellular Pi status InsPs are produced and bind SPX domain to switch on VPT1 activity to deliver Pi into the vacuole. This thus provides an auto-regulatory mechanism for VPT1-mediated Pi sensing and homeostasis in plant cells., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4',6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes.

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually outgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to outgrow their food supply and starve, causing the D. discoideum cells to inhibit their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases plasma membrane recycling and apparent cell membrane fluidity, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1 and the inositol hexakisphosphate kinase I6kA. PolyP alters protein contents in detergent-insoluble crude cytoskeletons, but does not significantly affect random cell motility, cell speed or F-actin levels. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

This study assessed the impact of the operating conditions of the biofilm sequencing batch reactor (BSBR) on the community structure and the growth/metabolic pathways of its polyphosphate-accumulating organisms (PAOs). There are significant difference with reference to the enhanced biological phosphorus removal (EBPR) process. The leading PAOs in BSBR generally are capable of high affinity acetate metabolism, gluconeogenesis, and low affinity phosphate transport, and have various carbon source supplementation pathways to ensure the efficient circulation of energy and reducing power. A new model of the metabolic mechanism of PAOs in the BSBR was formulated, which features low glycogen metabolism with simultaneous gluconeogenesis and glycogenolysis and differs significantly from the classic mechanism based on Candidatus&#95;Accumulibacter and Tetrasphaera. The findings will assist the efficient recovery of low concentration phosphate in municipal wastewater., (Copyright © 2022 Elsevier Ltd. All rights reserved.)