Search

Polyphosphate Accumulating Organisms (PAOs) microdiversity is a key factor to elucidate the mechanisms involved in the side-stream enhanced biological phosphorus removal (S2EBPR) systems, which has been shown to improve the process stability over conventional EBPR. However, fast, effective and cost-efficient methods to resolve PAO microdiversity in real-world activate sludge samples is still in absence. In this study, we applied oligotyping analysis following the regular 16S rRNA gene amplicon sequencing standard operation pipeline (SOP) to resolve subgenus-level PAO oligotypes, which cannot be achieved using traditional 16S rRNA sequencing SOP. The identified oligotype profiles of PAO-containing genera Ca. Accumulibacter, Tetrasphaera and Comamonas showed distinguished community-level differences across 12 water resource recovery facilities (WRRFs), which would not be revealed at the genus level. The WRRF-level differences were observed larger than the temporal differences in the same WRRF, indicating intrinsic sub-genus level microdiversity fingerprint between EBPR/S2EBPR systems. The identified oligotypes can be associated with known PAO clades phylogenetically, suggesting that oligotyping can suffice as a fast and cost-efficient approach for PAO microdiversity profiling. In addition, network analysis can be used to identify coexistence patterns between oligotypes with respect to EBPR/S2EBPR configurations and performance, enabling more detailed analysis between EBPR system performance and PAOs microdiversity. Correlation analyses between oligotype profiles and key EBPR performance parameters revealed potential different biological functional traits among these PAO species with P-removal performance implications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest related to this publication., (Copyright © 2024. Published by Elsevier Ltd.)

Acidocalcisomes of Trypanosoma brucei are membrane-bounded organelles characterized by their acidity and high content of polyphosphate and cations, like calcium and magnesium. They have important roles in cation and phosphorus storage, osmoregulation, autophagy initiation, calcium signaling, and virulence. Acidocalcisomes of T. brucei possess several membrane transporters, pumps, and channels, some of which were identified by proteomic and immunofluorescence analyses and validated as acidocalcisome proteins by their colocalization with the acidocalcisome marker vacuolar proton pyrophosphatase (VP1). Here, we report that a set of membrane transporters and enzymes, which were proposed to be present in acidocalcisomes by the morphological appearance of tagged proteins, colocalize with VP1, validating their character as acidocalcisome proteins., Importance: Acidocalcisomes are acidic organelles rich in polyphosphate and calcium present in a variety of eukaryotes and important for osmoregulation and calcium signaling. Several proteins were postulated to localize to acidocalcisomes based on their morphological characteristics. We provide validation of the localization of ten10 acidocalcisome proteins by their co-localization with enzymatic markers. These findings reveal the roles of acidocalcisomes in the storage of toxic metals, and the presence of enzymes involved in palmitoylation and polyphosphate metabolism., Competing Interests: The authors declare no conflict of interest.

Aberrant aggregation of α-Synuclein is the pathological hallmark of a set of neurodegenerative diseases termed synucleinopathies. Recent advances in cryo-electron microscopy have led to the structural determination of the first synucleinopathy-derived α-Synuclein fibrils, which contain a non-proteinaceous, "mystery density" at the core of the protofilaments, hypothesized to be highly negatively charged. Guided by previous studies that demonstrated that polyphosphate (polyP), a universally conserved polyanion, significantly accelerates α-Synuclein fibril formation, we conducted blind docking and molecular dynamics simulation experiments to model the polyP binding site in α-Synuclein fibrils. Here, we demonstrate that our models uniformly place polyP into the lysine-rich pocket, which coordinates the mystery density in patient-derived fibrils. Subsequent in vitro studies and experiments in cells revealed that substitution of the 2 critical lysine residues K43 and K45 with alanine residues leads to a loss of all previously reported effects of polyP binding on α-Synuclein, including stimulation of fibril formation, change in filament conformation and stability as well as alleviation of cytotoxicity. In summary, our study demonstrates that polyP fits the unknown electron density present in in vivo α-Synuclein fibrils and suggests that polyP exerts its functions by neutralizing charge repulsion between neighboring lysine residues., Competing Interests: UJ is a member of the PLOS Biology Editorial Board., (Copyright: © 2024 Huettemann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Due to their frequent coexistence in many polymicrobial infections, including in patients with cystic fibrosis or burn/chronic wounds, many studies have investigated the mechanistic details of the interaction between the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa rapidly outcompetes S. aureus under in vitro cocultivation conditions , which is mediated by several of P. aeruginosa 's virulence factors. Here, we report that polyphosphate (polyP), an efficient stress defense system and virulence factor in P. aeruginosa , plays a role in the pathogen's ability to inhibit and kill S. aureus in a contact-independent manner. We show that P. aeruginosa cells characterized by low polyP levels are less detrimental to S. aureus growth and survival while the Gram-positive pathogen is significantly more compromised by the presence of P. aeruginosa cells that produce high levels of polyP. The polyP-dependent phenotype of P. aeruginosa -mediated killing of S. aureus could at least in part be direct, as polyP was detected in the spent media and causes significant damage to the S. aureus cell envelope. However, more likely is that polyP's effects are indirect through modulating the production of one of P. aeruginosa's virulence factors, pyocyanin. We show that pyocyanin production in P. aeruginosa occurs polyP-dependently and harms S. aureus through membrane damage and potentially the generation of reactive oxygen species, resulting in the increased expression of antioxidant enzymes. In summary, our study adds a new component to the list of biomolecules that the Gram-negative pathogen P. aeruginosa generates to compete with S. aureus for resources.IMPORTANCEHow do interactions between microorganisms shape the course of polymicrobial infections? Previous studies have provided evidence that the two opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus generate molecules that modulate their interaction with potentially significant impact on disease outcomes. Our study identified the biopolymer polyphosphate (polyP) as a new effector molecule that impacts P. aeruginosa 's interaction with S. aureus . We show that P. aeruginosa kills S. aureus in a polyP-dependent manner, which occurs primarily through the polyP-dependent production of the P. aeruginosa virulence factor pyocyanin. Our findings add a new role for polyP to an already extensive list of functions. A more in-depth understanding of how polyP influences interspecies interactions is critical, as targeting polyP synthesis in bacteria such as P. aeruginosa may have a significant impact on other microorganisms and potentially result in dynamic changes in the microbial composition., Competing Interests: The authors declare no conflict of interest.

The inorganic biopolymer polyphosphate (polyP) occurs in all domains of life and affects myriad cellular processes. A longstanding observation is polyP's frequent proximity to chromatin, and, in many bacteria, its occurrence as magnesium (Mg 2+ )-enriched condensates embedded in the nucleoid region, particularly in response to stress. The physical basis of the interaction between polyP, DNA and Mg 2+ , and the resulting effects on the organization of the nucleoid and polyP condensates, remain poorly understood. Here, using a minimal system of polyP, Mg 2+ , and DNA, we find that DNA can form shells around polyP-Mg 2+ condensates. These shells show reentrant behavior, that is, they form within a window of Mg 2+ concentrations, representing a tunable architecture with potential relevance in other multicomponent condensates. This surface association tunes condensate size and DNA morphology in a manner dependent on DNA length and concentration, even at DNA concentrations orders of magnitude lower than found in the cell. Our work also highlights the remarkable capacity of two primordial inorganic species to organize DNA., (© 2024. The Author(s).)

Polyphosphate (polyP) is a ubiquitous polyanion present throughout the tree of life. While polyP's widely varied functions have been interrogated in single-celled organisms, little is known about the cellular distribution and function of polyP in multicellular organisms. To study polyP in metazoans, we developed the nematode Caenorhabditis elegans as a model system. We designed a high-throughput, longitudinal-orientation cryosectioning method that allowed us to scrutinize the intracellular localization of polyP in fixed C. elegans using fluorescent polyP probes and co-immunostaining targeting appropriate marker proteins. We discovered that the vast majority of polyP is localized within the endo-lysosomal compartments of the intestinal cells and is highly sensitive toward the disruption of endo-lysosomal compartment generation and food availability. This study lays the groundwork for further mechanistic research of polyPs in multicellular organisms and provides a reliable method for immunostaining hundreds of fixed worms in a single experiment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polyphosphate (polyP) is an intriguing molecule that is found in almost any organism, covering a multitude of cellular functions. In industry, polyP is used due to its unique physiochemical properties, including pH buffering, water binding, and bacteriostatic activities. Despite the importance of polyP, its analytics is still challenging, with the gold standard being 31 P NMR. Here, we present a simple staining method using the fluorescent dye JC-D7 for the semi-quantitative polyP evaluation in yeast extracts. Notably, fluorescence response was affected by polyP concentration and polymer chain length in the 0.5-500 µg/mL polyP concentration range. Hence, for polyP samples of unknown chain compositions, JC-D7 cannot be used for absolute quantification. Fluorescence of JC-D7 was unaffected by inorganic phosphate up to 50 mM. Trace elements (FeSO 4  > CuSO 4  > CoCl 2  > ZnSO 4 ) and toxic mineral salts (PbNO 3 and HgCl 2 ) diminished polyP-induced JC-D7 fluorescence, affecting its applicability to samples containing polyP-metal complexes. The fluorescence was only marginally affected by other parameters, such as pH and temperature. After validation, this simple assay was used to elucidate the degree of polyP production by yeast strains carrying gene deletions in (poly)phosphate homeostasis. The results suggest that staining with JC-D7 provides a robust and sensitive method for detecting polyP in yeast extracts and likely in extracts of other microbes. The simplicity of the assay enables high-throughput screening of microbes to fully elucidate and potentially enhance biotechnological polyP production, ultimately contributing to a sustainable phosphorus utilization., (© 2024 The Author(s). Yeast published by John Wiley & Sons Ltd.)

The nucleos(t)ide analogs require phosphorylation to the pharmacologically active anabolites in cells. We investigated the hypothesis that single-nucleotide polymorphisms (SNPs) in genes that encode transporters and phosphodiesterase (PDE) enzymes involved in tenofovir (TFV), disoproxil fumarate (TDF), and lamivudine (3TC) disposition will be associated with concentrations of their phosphate anabolites and virologic response. Individuals with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) coinfection receiving TDF/3TC-containing antiretroviral therapy were enrolled. Steady-state TFV diphosphate (TFV-DP) and 3TC triphosphate (3TC-TP) concentrations in peripheral blood mononuclear cells (PBMCs) and dried blood spot samples were quantified. The relationship between genetic variants and TFV-DP and 3TC-TP concentrations as well as with virologic response were examined using multivariable linear regression. Of the 136 participants (median age 43 years; 63% females), 6.6% had HBV non-suppression, and 7.4% had HIV non-suppression. The multidrug resistance protein 2 (encoded by ABCC2 rs2273697) SNP was associated with 3TC-TP concentrations in PBMCs. The human organic anion transporter-1 (encoded by SLC28A2 ) rs11854484 SNP was associated with HIV non-suppression, and when evaluated together with SNPs with marginal associations ( ABCC2 rs717620 and PDE1C rs30561), participants with two or three variants compared to those with none of these variants had an adjusted odds ratio of 48.3 (confidence interval, 4.3-547.8) for HIV non-suppression. None of the SNPs were associated with HBV non-suppression. Our study identified ABCC2 SNP to be associated with 3TC-TP concentrations in PBMCs. Also, a combination of genetic variants of drug transporters and PDE was associated with HIV non-suppression., Competing Interests: The authors declare no conflict of interest.

Polyphosphate (polyP) mediates a plethora of biological functions. Understanding the polyP-protein interactome will help clarify the mechanisms underpinning these functions. Recent studies demonstrating a strong but noncovalent modification of lysine and histidine repeat proteins by polyP have provided new insights into polyP-protein biochemistry with implications for research and therapeutics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Phosphorus in wastewater influents is a global issue. Controlling eutrophic water is crucial. Biological phosphorus removal is an economically and environmentally sustainable method for removing phosphorus from wastewater. This study aims to isolate and improve the capacity of aerobic phosphorus-removing bacteria to reduce excessive phosphate concentrations in the environment. Only three out of fourteen bacterial isolates demonstrated the highest phosphate removal efficiency using Toluidine blue-O. Klebsiella pneumoniae 6A, Klebsiella quasipneumoniae 6R, and Enterobacter mori 8R were isolated from activated sludge and identified by 16srRNA. In a single-factor experiment, the effect of incubation periods, phosphate concentrations, carbon sources, sodium acetate concentrations, temperature, pH, and irradiation dosages were studied. Seventy-two hours of incubation, 55 mg/L PO 4 , sodium acetate as the carbon source, 30°C and pH 7 resulted in maximum phosphorus removal. After optimising the parameters, the removal efficiency of Klebsiella pneumoniae 6A, Klebsiella quasipneumoniae 6R, and Enterobacter mori 8R increased from 73.5% to 85.1%, 79.1% to 98.1%, and 80.6% to 91.9%, respectively. Gamma irradiation showed significant results only in Klebsiella pneumoniae 6A where 100 Gy increased the phosphorous removal efficiency from 85.1% to 100%. Immobilised mixed culture of the three strains adapted better to 100 mg/L Phosphorus than pure cells. Therefore, this technique holds great new promise for phosphorus-contaminated sites bioremediation.

The ability to generate polyphosphate (polyP) granules is important for survival for bacteria during resistance to diverse environmental stresses, however the genesis of polyP granules is poorly understood. Chlorobaculum tepidum (Cba tepidum) is a thermophilic green sulfur anoxygenic phototrophic bacterium which uses reduced sulfur compounds as electron donors. The presence of electron rich granules inside the Cba tepidum was reported, but no further information was provided. In this work we used cell thin sections at three different time points of cultivation to observe the biogenesis of the inclusions over time, and the in cell total phosphate concentration was monitored over time as well. Furthermore, the elemental analysis (EDS) of the electron rich inclusions showed the presence of phosphorus and oxygen. The existence of polyphosphate was demonstrated by 31 P NMR spectroscopy of cell lysates. Finally, we show that the biogenesis of the phosphorus granules correlates with an abundance of proteins that are closely related to polyphosphate metabolism., 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 © 2024 Elsevier B.V. All rights reserved.)

Rationale: Tissue regeneration of skin and bone is an energy-intensive, ATP-consuming process that, if impaired, can lead to the development of chronic clinical pictures. ATP levels in the extracellular space including the exudate of wounds, especially chronic wounds, are low. This deficiency can be compensated by inorganic polyphosphate (polyP) supplied via the blood platelets to the regenerating site. Methods: The contribution of the different forms of energy derived from polyP (metabolic energy, mechanical energy and heat) to regeneration processes was dissected and studied both in vitro and in patients. ATP is generated metabolically during the enzymatic cleavage of the energy-rich anhydride bonds between the phosphate units of polyP, involving the two enzymes alkaline phosphatase (ALP) and adenylate kinase (ADK). Exogenous polyP was administered after incorporation into compressed collagen or hydrogel wound coverages to evaluate its regenerative activity for chronic wound healing. Results: In a proof-of-concept study, fast healing of chronic wounds was achieved with the embedded polyP, supporting the crucial regeneration-promoting activity of ATP. In the presence of Ca 2+ in the wound exudate, polyP undergoes a coacervation process leading to a conversion of fibroblasts into myofibroblasts, a crucial step supporting cell migration during regenerative tissue repair. During coacervation, a switch from an endothermic to an exothermic, heat-generating process occurs, reflecting a shift from an entropically- to an enthalpically-driven thermodynamic reaction. In addition, mechanical forces cause the appearance of turbulent flows and vortices during liquid-liquid phase separation. These mechanical forces orient the cellular and mineralic (hydroxyapatite crystallite) components, as shown using mineralizing SaOS-2 cells as a model. Conclusion: Here we introduce the energetic triad: metabolic energy (ATP), thermal energy and mechanical energy as a novel theranostic biomarker, which contributes essentially to a successful application of polyP for regeneration processes., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood. Our previous attempts to uncover PPK1 regulatory elements resulted in the discovery of PPK1* mutants, which accumulate more polyP in vivo, but do not produce more in vitro. In attempting to further characterize these mutant enzymes, we discovered that the most commonly-used PPK1 purification method - Ni-affinity chromatography using a C-terminal poly-histidine tag - altered intrinsic aspects of the PPK1 enzyme, including specific activity, oligomeric state, and kinetic values. We developed an alternative purification strategy using a C-terminal C-tag which did not have these effects. Using this strategy, we were able to demonstrate major differences in the in vitro response of PPK1 to 5-aminosalicylic acid, a known PPK1 inhibitor, and observed several key differences between the wild-type and PPK1* enzymes, including changes in oligomeric distribution, increased enzymatic activity, and increased resistance to both product (ADP) and substrate (ATP) inhibition, that help to explain their in vivo effects. Importantly, our results indicate that the C-terminal poly-histidine tag is inappropriate for purification of PPK1, and that any in vitro studies or inhibitor screens performed with such tags need to be reconsidered in that light., 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 © 2024 Elsevier Ltd. All rights reserved.)

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

Dissolved organic phosphorus (DOP) contains compounds with phosphoester, phosphoanhydride, and phosphorus-carbon bonds. While DOP holds significant nutritional value for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by Synechococcus strains from open and coastal oceans. Both strains exhibited comparable growth rates when provided phosphate, a phosphoanhydride [3-polyphosphate and 45-polyphosphate], or a DOP compound with both phosphoanhydride and phosphoester bonds (adenosine 5'-triphosphate). Growth rates on phosphoesters [glucose-6-phosphate, adenosine 5'-monophosphate, bis(4-methylumbelliferyl) phosphate] were variable, and neither strain grew on selected phosphorus-carbon compounds. Both strains hydrolyzed 3-polyphosphate, then adenosine 5'-triphosphate, and lastly adenosine 5'-monophosphate, exhibiting preferential enzymatic hydrolysis of phosphoanhydride bonds. The strains' exoproteomes contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5'-triphosphate under phosphate deficiency, suggests active mineralization of phosphoanhydride bonds by these exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity toward the phosphoanhydride 3-polyphosphate, with varying affinities between strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition and highlight varied growth and enzymatic responses to molecular diversity within DOP bond-classes, thereby expanding our understanding of microbially mediated DOP cycling in marine ecosystems., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Polyphosphate (polyP) is an evolutionary ancient inorganic molecule widespread in biology, exerting a broad range of biological activities. The intracellular polymer serves as an energy storage pool and phosphate/calcium ion reservoir with implications for basal cellular functions. Metabolisms of the polymer are well understood in procaryotes and unicellular eukaryotic cells. However, functions, regulation, and association with disease states of the polymer in higher eukaryotic species such as mammalians are just beginning to emerge. The review summarises our current understanding of polyP metabolism, the polymer's functions, and methods for polyP analysis. In-depth knowledge of the pathways that control polyP turnover will open future perspectives for selective targeting of the polymer.

Understanding the microbial community structure of sludge is crucial for improving the design, operation and optimisation of full-scale wastewater treatment plants (WWTPs). This study aimed to have a comprehensive comparison of microbial communities between aerobic granular sludge and flocculent sludge from two full-scale sequential batch reactors-based WWTPs with nutrient removal for the first time. To better understand key functional bacteria such as polyphosphate accumulating bacteria (PAOs), competitive bacteria such as glycogen accumulating bacteria (GAOs) and nitrifying bacteria for both nitrogen and phosphorus removal, another two full-scale WWTPs with only carbon (C) removal and C and nitrogen (N) removal were compared too. It was found that the richness and diversity of the microbial population in sludge increased with pollutant removal from only C, C and N, to C,N, P removal. For C, N P removal, granule structure led to a more diverse and rich microbial community structure than flocculent structure. Although more abundant nitrifying bacteria were enriched in granular sludge than flocculent sludge, the abundance of total putative PAOs was equivalent. However, the most typical putative PAOs such as Tetrasphaera and Candidatus Accumulibacter seemed to be more correlated with biological phosphorus removal performance, which might be more proper to be used as an indication for P removal potential. The higher abundance of GAOs in flocculent sludge with better phosphorus removal performance might suggest that further investigation is needed to understand the functions of GAOs. In addition, the equivalent abundances of PAOs in the WWTPs with only C removal and with C, N, and P removal, respectively, indicate that many newly reported putative PAOs might not contribute to P removal. This study provides insight into the microbial communities and functional bacteria in aerobic granular sludge and flocculent sludge in full-scale SBRs, which can provide microbes-informed optimisation of reactor operation for better nutrient removal., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Inorganic polyphosphate (polyP), one of the first high-energy compound on earth, defies its extreme compositional and structural simplicity with an astoundingly wide array of biological activities across all domains of life. However, the underlying mechanism of such functional pleiotropy remains largely elusive. In this review, we will summarize recent studies demonstrating that this simple polyanion stabilizes protein folding intermediates and scaffolds select native proteins. These functions allow polyP to act as molecular chaperone that protects cells against protein aggregation, as pro-amyloidogenic factor that accelerates both physiological and disease-associated amyloid formation, and as a modulator of liquid-liquid phase separation processes. These activities help to explain polyP's known roles in bacterial stress responses and pathogenicity, provide the mechanistic foundation for its potential role in human neurodegenerative diseases, and open a new direction regarding its influence on gene expression through condensate formation. We will highlight critical unanswered questions and point out potential directions that will help to further understand the pleiotropic functions of this ancient and ubiquitous biopolymer., 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 © 2024 Elsevier Ltd. All rights reserved.)

Inorganic polyphosphate (polyP) is a ubiquitous polymer that controls fundamental processes. To overcome the absence of a genetically tractable mammalian model, we developed an inducible mammalian cell line expressing Escherichia coli polyphosphate kinase 1 (EcPPK1). Inducing EcPPK1 expression prompted polyP synthesis, enabling validation of polyP analytical methods. Virtually all newly synthesized polyP accumulates within the nucleus, mainly in the nucleolus. The channeled polyP within the nucleolus results in the redistribution of its markers, leading to altered rRNA processing. Ultrastructural analysis reveals electron-dense polyP structures associated with a hyper-condensed nucleolus resulting from an exacerbation of the liquid-liquid phase separation (LLPS) phenomena controlling this membraneless organelle. The selective accumulation of polyP in the nucleoli could be interpreted as an amplification of polyP channeling to where its physiological function takes place. Indeed, quantitative analysis of several mammalian cell lines confirms that endogenous polyP accumulates within the nucleolus., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H 2 O 2 -supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H 2 O 2 -supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Living organisms ranging from bacteria to animals have developed their own ways to accumulate and store phosphate during evolution, in particular as the polyphosphate (polyP) granules in bacteria. Degradation of polyP into phosphate is involved in phosphorus cycling, and exopolyphosphatase (PPX) is the key enzyme for polyP degradation in bacteria. Thus, understanding the structure basis of PPX is crucial to reveal the polyP degradation mechanism. Here, it is found that PPX structure varies in the length of ɑ-helical interdomain linker (ɑ-linker) across various bacteria, which is negatively correlated with their enzymatic activity and thermostability - those with shorter ɑ-linkers demonstrate higher polyP degradation ability. Moreover, the artificial DrPPX mutants with shorter ɑ-linker tend to have more compact pockets for polyP binding and stronger subunit interactions, as well as higher enzymatic efficiency (k cat /K m ) than that of DrPPX wild type. In Deinococcus-Thermus, the PPXs from thermophilic species possess a shorter ɑ-linker and retain their catalytic ability at high temperatures (70 °C), which may facilitate the thermophilic species to utilize polyP in high-temperature environments. These findings provide insights into the interdomain linker length-dependent evolution of PPXs, which shed light on enzymatic adaption for phosphorus cycling during natural evolution and rational design of enzyme., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM. For complete details on the use and execution of this protocol, please refer to Neville et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ammonium polyphosphate (APP), a pivotal constituent within environmentally friendly flame retardants, exhibits notable decomposition susceptibility and potentially engenders ecological peril. Consequently, monitoring the APP concentration to ensure product integrity and facilitate the efficacious management of wastewater from production processes is of great significance. A fluorescent assay was devised to swiftly discern APP utilizing 4',6'-diamino-2-phenylindole (DAPI). With increasing APP concentrations, DAPI undergoes intercalation within its structure, emitting pronounced fluorescence. Notably, the flame retardant JLS-PNA220-A, predominantly comprising APP, was employed as the test substrate. Establishing a linear relationship between fluorescence intensity (F-F0) and JLS-PNA220-A concentration yielded the equation y = 76.08x + 463.2 (R 2 = 0.9992), with a LOD determined to be 0.853 mg/L. The method was used to assess the degradation capacity of APP-degrading bacteria. Strain D-3 was isolated, and subsequent analysis of its 16S DNA sequence classified it as belonging to the Acinetobacter genus. Acinetobacter nosocomialis D-3 demonstrated superior APP degradation capabilities under pH 7 at 37 °C, with degradation rates exceeding 85% over a four-day cultivation period. It underscores the sensitivity and efficacy of the proposed method for APP detection. Furthermore, Acinetobacter nosocomialis D-3 exhibits promising potential for remediation of residual APP through environmental biodegradation processes.

Polyphosphates (polyP) are ubiquitous biomolecules that play a multitude of physiological roles in many cells. We have studied the presence and role of polyP in a unicellular alga, the freshwater diatom Achnanthidium minutissimum. This diatom stores up to 2.0 pg·cell -1 of polyP, with chain lengths ranging from 130 to 500 inorganic phosphate units (P i ). We applied energy dispersive X-ray spectroscopy, Raman/fluorescence microscopy, and biochemical assays to localize and characterize the intracellular polyP granules that were present in large apical vacuoles. We investigated the fate of polyP in axenic A. minutissimum cells grown under phosphorus (P), replete (P (+) ), or P deplete (P (-) ) cultivation conditions and observed that in the absence of exogenous P, A. minutissimum rapidly utilizes their internal polyP reserves, maintaining their intrinsic growth rates for up to 8 days. PolyP-depleted A. minutissimum cells rapidly took up exogenous P a few hours after P i resupply and generated polyP three times faster than cells that were not initially subjected to P limitation. Accordingly, we propose that A. minutissimum deploys a succession of acclimation strategies regarding polyP dynamics where the production or consumption of polyP plays a central role in the homeostasis of the diatom., (© 2024 The Authors. Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Polyphosphates, chains of polymerized phosphate subunits, are used as food additives for various applications such as conservation, water retention, and pH buffering. Currently, the value chain of phosphates is linear, based on mining fossil phosphate rock, which is anticipated to be depleted in a few hundred years. With no replacement available, a transition to a circular phosphate economy, to which biological systems can contribute, is required. Baker's yeast can hyperaccumulate phosphate from various phosphate-rich waste streams and form polyphosphates, which can be used directly or as polyphosphate-rich yeast extract with enhanced properties in the food industry. By maturing the technology to an industrial level and allowing upcycled waste streams for food applications, substantial contributions to a sustainable phosphate economy can be achieved., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest. LMB filed two patent applications: “Zusammensetzung, enthaltend getrocknetes Polyphosphat und Verfahren zur Gewinnung von Polyphosphat aus polyphosphat-haltigen Hefezellen dazu” (DE 10 2019 131 561.1) and “Polyphosphatreiche Hefeextrakte und Herstellverfahren dazu” (DE 10 2018 130 081.6, PCT/EP2019/082709)., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The linear polymer polyphosphate (poly-P) is present across all three domains of life and serves diverse physiological functions. The enzyme polyphosphate kinase (Ppk) is responsible for poly-P synthesis, whereas poly-P degradation is carried out by the enzyme exopolyphosphatase (Ppx). In many Lactobacillaceae , the Ppk-encoding gene ( ppk ) is found clustered together with two genes encoding putative exopolyphosphatases ( ppx1 and ppx2 ) each having different domain compositions, with the gene order ppx1-ppk-ppx2 . However, the specific function of these ppx genes remains unexplored. An in-frame deletion of ppx1 in Lacticaseibacillus paracasei BL23 resulted in bacteria unable to accumulate poly-P, whereas the disruption of ppx2 did not affect poly-P synthesis. The expression of ppk was not altered in the Δ ppx1 strain, and poly-P synthesis in this strain was only restored by expressing ppx1 in trans . Moreover, no poly-P synthesis was observed when ppk was expressed from a plasmid in the Δ ppx1 strain. Purified Ppx2 exhibited in vitro exopolyphosphatase activity, whereas no in vitro enzymatic activity could be demonstrated for Ppx1. This observation corresponds with the absence in Ppx1 of conserved motifs essential for catalysis found in characterized exopolyphosphatases. Furthermore, assays with purified Ppk and Ppx1 evidenced that Ppx1 enhanced Ppk activity. These results demonstrate that Ppx1 is essential for poly-P synthesis in Lc. paracasei and have unveiled, for the first time, an unexpected role of Ppx1 exopolyphosphatase in poly-P synthesis.IMPORTANCEPoly-P is a pivotal molecular player in bacteria, participating in a diverse array of processes ranging from stress resilience to pathogenesis while also serving as a functional component in probiotic bacteria. The synthesis of poly-P is tightly regulated, but the underlying mechanisms remain incompletely elucidated. Our study sheds light on the distinctive role played by the two exopolyphosphatases (Ppx) found in the Lactobacillaceae bacterial group, of relevance in food and health. This particular group is noteworthy for possessing two Ppx enzymes, supposedly involved in poly-P degradation. Remarkably, our investigation uncovers an unprecedented function of Ppx1 in Lacticaseibacillus paracasei , where its absence leads to the total cessation of poly-P synthesis, paralleling the impact observed upon eliminating the poly-P forming enzyme, poly-P kinase. Unlike the anticipated role as a conventional exopolyphosphatase, Ppx1 demonstrates an unexpected function. Our results added a layer of complexity to our understanding of poly-P dynamics in bacteria., Competing Interests: The authors declare no conflict of interest.

Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo et al. described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31 P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo et al. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo et al. (2024), published in this issue., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria., (© 2024. The Author(s).)

Glutamine synthetase (GS) is vital in maintaining ammonia and glutamate (Glu) homeostasis in living organisms. However, the natural enzyme relies on adenosine triphosphate (ATP) to activate Glu, resulting in impaired GS function during ATP-deficient neurotoxic events. To date, no reports demonstrate using artificial nanostructures to mimic GS function. In this study, we synthesize aggregation-induced emission active polyP-Mn nanosheets (STPE-PMNSs) based on end-labeled polyphosphate (polyP), exhibiting remarkable GS-like activity independent of ATP presence. Further investigation reveals polyP in STPE-PMNSs serves as phosphate source to activate Glu at low ATP levels. This self-feeding mechanism offers a significant advantage in regulating Glu homeostasis at reduced ATP levels in nerve cells during excitotoxic conditions. STPE-PMNSs can effectively promote the conversion of Glu to glutamine (Gln) in excitatory neurotoxic human neuroblastoma cells (SH-SY5Y) and alleviate Glu-induced neurotoxicity. Additionally, the fluorescence signal of nanosheets enables precise monitoring of the subcellular distribution of STPE-PMNSs. More importantly, the intracellular fluorescence signal is enhanced in a conversion-responsive manner, allowing real-time tracking of reaction progression. This study presents a self-sustaining strategy to address GS functional impairment caused by ATP deficiency in nerve cells during neurotoxic events. Furthermore, it offers a fresh perspective on the potential biological applications of polyP-based nanostructures., (© 2024. The Author(s).)

The maintenance of phosphate homeostasis serves as a foundation for energy metabolism and signal transduction processes in all living organisms. Inositol pyrophosphates (PP-InsPs), composed of an inositol ring decorated with monophosphate and diphosphate moieties, and inorganic polyphosphate (polyP), chains of orthophosphate residues linked by phosphoanhydride bonds, are energy-rich biomolecules that play critical roles in phosphate homeostasis. There is a complex interplay between these two phosphate-rich molecules, and they share an interdependent relationship with cellular adenosine triphosphate (ATP) and inorganic phosphate (Pi). In eukaryotes, the enzymes involved in PP-InsP synthesis show some degree of conservation across species, whereas distinct enzymology exists for polyP synthesis among different organisms. In fact, the mechanism of polyP synthesis in metazoans, including mammals, is still unclear. Early studies on PP-InsP and polyP synthesis were conducted in the slime mould Dictyostelium discoideum, but it is in the budding yeast Saccharomyces cerevisiae that a clear understanding of the interplay between polyP, PP-InsPs, and Pi homeostasis has now been established. Recent research has shed more light on the influence of PP-InsPs on polyP in mammals, and the regulation of both these molecules by cellular ATP and Pi levels. In this review we will discuss the cross-talk between PP-InsPs, polyP, ATP, and Pi in the context of budding yeast, slime mould, and mammals. We will also highlight the similarities and differences in the relationship between these phosphate-rich biomolecules among this group of organisms., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Inorganic polyphosphate (polyP) is widely recognized for playing important roles and processes involved in energy and phosphate storage, regulation of gene expression, and calcium signaling. The less well-known role of polyP is as a direct mediator of ion transport across biological membranes. Here, we will briefly summarize current knowledge of the molecular mechanisms of how polyP can be involved in membrane ion transport. We discuss three types of mechanisms that might involve polyP: (1) formation of non-protein channel complex that includes calcium, polyP, and polyhydroxybutyrate (PHB); (2) modulation of the channel activity of PHBlated protein channels; and (3) direct effects of polyP on the function of the voltage-gated ion channels in the process that do not involve PHB., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa . Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Baijal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

While the adsorption/bio-oxidation (A/B) process has been widely studied for carbon capture and shortcut nitrogen (N) removal, its integration with enhanced biological phosphorus (P) removal (EBPR) has been considered challenging and thus unexplored. Here, full-scale pilot testing with an integrated system combining A-stage high-rate activated sludge with B-stage partial (de)nitrification/anammox and side-stream EBPR (HRAS-P(D)N/A-S2EBPR) was conducted treating real municipal wastewater. The results demonstrated that, despite the relatively low influent carbon load, the B-stage P(D)N-S2EBPR system could achieve effective P removal performance, with the carbon supplement and redirection of the A-stage sludge fermentate to the S2EBPR. The novel process configuration design enabled a system shift in carbon flux and distribution for efficient EBPR, and provided unique selective factors for ecological niche partitioning among different key functionally relevant microorganisms including polyphosphate accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). The combined nitrite from B-stage to S2EBPR and aerobic-anoxic conditions in our HRAS-P(D)N/A-S2EBPR system promoted DPAOs for simultaneous internal carbon-driven denitrification via nitrite and P removal. 16S rRNA gene-based oligotyping analysis revealed high phylogenetic microdiversity within the Accumulibacter population and discovered coexistence of certain oligotypes of Accumulibacter and Competibacter correlated with efficient P removal. Single-cell Raman micro-spectroscopy-based phenotypic profiling showed high phenotypic microdiversity in the active PAO community and the involvement of unidentified PAOs and internal carbon-accumulating organisms that potentially played an important role in system performance. This is the first pilot study to demonstrate that the P(D)N-S2EBPR system could achieve shortcut N removal and influent carbon-independent EBPR simultaneously, and the results provided insights into the effects of incorporating S2EBPR into A/B process on metabolic activities, microbial ecology, and resulted system performance., 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.)

We piloted the incorporation of side-stream enhanced biological phosphorus removal (S2EBPR) with A/B stage short-cut nitrogen removal processes to enable simultaneous carbon-energy-efficient nutrients removal. This unique configuration and system conditions exerted selective force on microbial populations distinct from those in conventional EBPR. Interestingly, effective P removal was achieved with the predominance of Acinetobacter (21.5 ± 0.1 %) with nearly negligible level of known conical PAOs (Ca. Accumulibacter and Tetrasphaera were 0.04 ± 0.10 % and 0.47 ± 0.32 %, respectively). Using a combination of techniques, such as fluorescence in situ hybridization (FISH) coupled with single cell Raman spectroscopy (SCRS), the metabolic tracing of Acinetobacter-like cells exerted PAO-like phenotypic profiling. In addition, comparative metagenomics analysis of the closely related Acinetobacter spp. revealed the EBPR relevant metabolic pathways. Further oligotyping analysis of 16s rRNA V4 region revealed sub-clusters (microdiversity) of the Acinetobacter and revealed that the sub-group (oligo type 1, identical (100 % alignment identity) hits from Acinetobacter_midas_s_49494, and Acinetobacter_midas_s_55652) correlated with EBPR activities parameters, provided strong evidence that the identified Acinetobacter most likely contributed to the overall P removal in our A/B-shortcut N-S2EBPR system. To the best of our knowledge, this is the first study to confirm the in situ EBPR activity of Acinetobacter using combined genomics and SCRS Raman techniques. Further research is needed to identify the specific taxon, and phenotype of the Acinetobacter that are responsible for the P-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 © 2023. Published by Elsevier Ltd.)

Phosphorus is an essential but non-renewable nutrient resource critical for agriculture. Luxury phosphorus uptake allows microalgae to synthesize polyphosphate and accumulate phosphorus, but, depending on the strain of algae, polyphosphate may be degraded within 4 hours of accumulation. We studied the recovery of phosphorus from wastewater through luxury uptake by an engineered strain of Synechocystis sp. with inhibited polyphosphate degradation and the effect of this engineered Synechocystis biomass on lettuce growth. First, a strain (Δ phoU ) lacking the phoU gene, which encodes a negative regulator of environmental phosphate concentrations, was generated to inhibit polyphosphate degradation in cells. Polyphosphate concentrations in the phoU knock-out strain were maintained for 24 h and then decreased slowly. In contrast, polyphosphate concentrations in the wild-type strain increased up to 4 h and then decreased rapidly. In addition, polyphosphate concentration in the phoU knockout strain cultured in semi-permeable membrane bioreactors with artificial wastewater medium was 2.5 times higher than that in the wild type and decreased to only 16% after 48 h. The biomass of lettuce treated with the phoU knockout strain (0.157 mg P/m 2 ) was 38% higher than that of the lettuce treated with the control group. These results indicate that treating lettuce with this microalgal biomass can be beneficial to crop growth. These results suggest that the use of polyphosphate-accumulating microalgae as biofertilizers may alleviate the effects of a diminishing phosphorous supply. These findings can be used as a basis for additional genetic engineering to increase intracellular polyphosphate levels.

Enhanced biological phosphorus removal (EBPR) is an effective process for phosphorus removal from wastewater. In this study, two lab-scale sequencing batch reactors (SBR) were used to perform EBPR process, in which genus Propioniciclava was unexpectedly accumulated and its relative abundance was over 70 %. A series of tests were conducted to explore the role of Propioniciclava in the two EBPR systems. The two systems performed steadily throughout the study, and the phosphorus removal efficiencies were 96.6 % and 93.5 % for SBR1 and SBR2, respectively. The stoichiometric analysis related to polyphosphate accumulating organisms (PAOs) indicated that polyphosphate accumulating metabolism (PAM) was achieved in the anaerobic phase. It appeared that the Propioniciclava-dominated systems could not perform denitrifying phosphorus removal. Instead, phosphorus was released under anoxic conditions without carbon sources. According to the genomic information from Integrated Microbial Genomes (IMG) database, Propioniciclava owns ppk1, ppk2 and ppx genes that are associated with phosphorus release and uptake functions. By phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2) analysis, the abundance of genes related to phosphorus metabolism was much higher than that of genes related to denitrification. Therefore, Propioniciclava was presumed to be a potential PAO without denitrifying phosphorus uptake function. In addition to Propioniciclava, Tessaracoccus and Thiothrix were also enriched in both systems. Overall, this study proposes a novel potential PAO and broadens the understanding of EBPR microbial communities., 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.)

Inorganic polyphosphate (polyP) plays a vital role in cellular energy metabolism and signaling, owing to its structure and high-energy phosphate bonds. Intracellular ATP functions both as a cellular energy source and a key factor in cell death, and ATP dynamics in tumor cells are crucial for advancing cancer therapy. In this study, we explored the interplay between polyP and ATP in cellular energy metabolism. Treatment with polyP did not affect cell proliferation of human non-small cell lung cancer H1299 and human glioblastoma T98G cell lines as compared to their respective control cells until 72 h post-treatment. The mitochondrial membrane potential (MMP) in polyP-treated cells was low, contrasting with the time-dependent increase observed in control cells. While the ATP content increased over time in untreated and Na-phosphate-treated control cells, it remained unchanged in polyP-treated cells. Furthermore, the addition of cyclosporine A, a mitochondrial permeability transition pore (mPTP) inhibitor, failed to restore ATP levels in polyP-treated cells. We performed lactate assays and western blot analysis to evaluate the effect of polyP on glucose metabolism and found no significant differences in lactate secretion or glucose-6-phosphate dehydrogenase (G6PD) activity between polyP-treated and control cells. Additional pyruvate restored MMP but had no effect on the cellular ATP content in polyP-treated cells. We observed no correlation between the Warburg effect and glucose metabolism during ATP depletion in polyP-treated cells. Further investigation is warranted to explore the roles of polyP and ATP in cancer cell energy metabolism, which might offer potential avenues for therapeutic interventions., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The yeast Snf1/AMP-activated kinase (AMPK) maintains energy homeostasis, controlling metabolic processes and glucose derepression in response to nutrient levels and environmental cues. Under conditions of nitrogen or glucose limitation, Snf1 regulates pseudohyphal growth, a morphological transition characterized by the formation of extended multicellular filaments. During pseudohyphal growth, Snf1 is required for wild-type levels of inositol polyphosphate (InsP), soluble phosphorylated species of the six-carbon cyclitol inositol that function as conserved metabolic second messengers. InsP levels are established through the activity of a family of inositol kinases, including the yeast inositol polyphosphate kinase Kcs1, which principally generates pyrophosphorylated InsP 7 . Here, we report that Snf1 regulates Kcs1, affecting Kcs1 phosphorylation and inositol kinase activity. A snf1 kinase-defective mutant exhibits decreased Kcs1 phosphorylation, and Kcs1 is phosphorylated in vivo at Ser residues 537 and 646 during pseudohyphal growth. By in vitro analysis, Snf1 directly phosphorylates Kcs1, predominantly at amino acids 537 and 646. A yeast strain carrying kcs1 encoding Ser-to-Ala point mutations at these residues (kcs1-S537A,S646A) shows elevated levels of pyrophosphorylated InsP 7 , comparable to InsP 7 levels observed upon deletion of SNF1. The kcs1-S537A,S646A mutant exhibits decreased pseudohyphal growth, invasive growth, and cell elongation. Transcriptional profiling indicates extensive perturbation of metabolic pathways in kcs1-S537A,S646A. Growth of kcs1-S537A,S646A is affected on medium containing sucrose and antimycin A, consistent with decreased Snf1p signaling. This work identifies Snf1 phosphorylation of Kcs1, collectively highlighting the interconnectedness of AMPK activity and InsP signaling in coordinating nutrient availability, energy homoeostasis, and cell growth., Competing Interests: Conflict of 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous researches have recognized the vital role of Tetrasphaera elongata in enhanced biological phosphorus removal systems, but the underlying mechanisms remain under-investigated. To address this issue, this study investigated the metabolic characteristics of Tetrasphaera elongata when utilizing glucose as the sole carbon source. Results showed under aerobic conditions, Tetrasphaera elongata exhibited a glucose uptake rate of 136.6 mg/(L·h) and a corresponding phosphorus removal rate of 8.6 mg P/(L·h). Upregulations of genes associated with the glycolytic pathway and oxidative phosphorylation were observed. Noteworthily, the genes encoding the two-component sensor histidine kinase and response regulator transcription factor exhibited a remarkable 28.3 and 27.4-fold increase compared with the group without glucose. Since these genes play a pivotal role in phosphate-specific transport systems, collectively, these findings shed light on a potential mechanism for simultaneous decarbonization and phosphorus removal by Tetrasphaera elongata under aerobic conditions, providing fresh insights into phosphorus removal from wastewaters., 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.)

Inorganic polyphosphate (polyP) is primarily synthesized by Polyphosphate Kinase-1 (PPK-1) and regulates numerous cellular processes, including energy metabolism, stress adaptation, drug tolerance, and microbial pathogenesis. Here, we report that polyP interacts with acyl CoA carboxylases, enzymes involved in lipid biosynthesis in Mycobacterium tuberculosis . We show that deletion of ppk-1 in M. tuberculosis results in transcriptional and metabolic reprogramming. In comparison to the parental strain, the Δ ppk-1 mutant strain had reduced levels of virulence-associated lipids such as PDIMs and TDM. We also observed that polyP deficiency in M. tuberculosis is associated with enhanced phagosome-lysosome fusion in infected macrophages and attenuated growth in mice. Host RNA-seq analysis revealed decreased levels of transcripts encoding for proteins involved in either type I interferon signaling or formation of foamy macrophages in the lungs of Δ ppk-1 mutant-infected mice relative to parental strain-infected animals. Using target-based screening and molecular docking, we have identified raloxifene hydrochloride as a broad-spectrum PPK-1 inhibitor. We show that raloxifene hydrochloride significantly enhanced the activity of isoniazid, bedaquiline, and pretomanid against M. tuberculosis in macrophages. Additionally, raloxifene inhibited the growth of M. tuberculosis in mice. This is an in-depth study that provides mechanistic insights into the regulation of mycobacterial pathogenesis by polyP deficiency., Competing Interests: Competing interests statement:The authors declare no competing interest.

DNA damage-induced autophagy is a new type of autophagy that differs from traditional macroautophagy; however, this type of autophagy has not been identified in the pathogenic fungus Candida albicans. Inositol polyphosphates are involved in the regulation of DNA damage repair and macroautophagy; however, whether inositol polyphosphates are involved in the regulation of DNA damage-induced autophagy remains unclear. In this study, we identified DNA damage-induced autophagy in C. albicans and systematically investigated the mechanisms of inositol polyphosphate pathway regulation. We found that the core machinery of macro autophagy is also essential for DNA damage-induced autophagy, and that inositol polyphosphate synthetases Kcs1, Ipk1, and Vip1 play a critical role in autophagy. In this study, we focused on Kcs1 and Vip1, which are responsible for the synthesis of inositol pyrophosphate. The kcs1Δ/Δ and vip1Δ/Δ strains exhibited reduced number of phagophore assembly sites (PAS) and autophagic bodies. The recruitment of autophagy-related gene 1 (Atg1) to PAS was significantly affected in the kcs1Δ/Δ and vip1Δ/Δ strains. Target of rapamycin complex 1 kinase activity was elevated in kcs1Δ/Δ and vip1Δ/Δ strains, which significantly inhibited the initiation of autophagy. Atg18 Localization was altered in these mutants. The absence of Kcs1 or Vip1 caused the downregulation of RAD53, a key gene in the DNA damage response. These data provide further understanding of the mechanism of autophagy regulation in C. albicans., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Inorganic polyphosphate (polyP) is an ancient polymer that is well-conserved throughout evolution. It is formed by multiple subunits of orthophosphates linked together by phosphoanhydride bonds. The presence of these bonds, which are structurally similar to those found in ATP, and the high abundance of polyP in mammalian mitochondria, suggest that polyP could be involved in the regulation of the physiology of the organelle, especially in the energy metabolism. In fact, the scientific literature shows an unequivocal role for polyP not only in directly regulating oxidative a phosphorylation; but also in the regulation of reactive oxygen species metabolism, mitochondrial free calcium homeostasis, and the formation and opening of mitochondrial permeability transitions pore. All these processes are closely interconnected with the status of mitochondrial bioenergetics and therefore play a crucial role in maintaining mitochondrial and cell physiology. In this invited review, we discuss the main scientific literature regarding the regulatory role of polyP in mammalian mitochondrial physiology, placing a particular emphasis on its impact on energy metabolism. Although the effects of polyP on the physiology of the organelle are evident; numerous aspects, particularly within mammalian cells, remain unclear and require further investigation. These aspects encompass, for example, advancing the development of more precise analytical methods, unraveling the mechanism responsible for sensing polyP levels, and understanding the exact molecular mechanism that underlies the effects of polyP on mitochondrial physiology. By increasing our understanding of the biology of this ancient and understudied polymer, we could unravel new pharmacological targets in diseases where mitochondrial dysfunction, including energy metabolism dysregulation, has been broadly described., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Enhanced biological phosphate removal and aerobic sludge granulation are commonly studied with fatty acids as substrate. Fermentative substrates such as glucose have received limited attention. In this work, glucose conversion by aerobic granular sludge and its impact on phosphate removal was studied. Long-term stable phosphate removal and successful granulation were achieved. Glucose was rapidly taken up (273 mg/gVSS/h) at the start of the anaerobic phase, while phosphate was released during the full anaerobic phase. Some lactate was produced during glucose consumption, which was anaerobically consumed once glucose was depleted. The phosphate release appeared to be directly proportional to the uptake of lactate. The ratio of phosphorus released to glucose carbon taken up over the full anaerobic phase was 0.25 Pmol/Cmol. Along with glucose and lactate uptake in the anaerobic phase, poly‑hydroxy-alkanoates and glycogen storage were observed. There was a linear correlation between glucose consumption and lactate formation. While lactate accounted for approximately 89 % of the observed products in the bulk liquid, minor quantities of formate (5 %), propionate (4 %), and acetate (3 %) were also detected (mass fraction). Formate was not consumed anaerobically. Quantitative fluorescence in-situ hybridization (qFISH) revealed that polyphosphate accumulating organisms (PAO) accounted for 61 ± 15 % of the total biovolume. Metagenome evaluation of the biomass indicated a high abundance of Micropruina and Ca. Accumulibacter in the system, which was in accordance with the microscopic observations and the protein mass fraction from metaproteome analysis. Anaerobic conversions were evaluated based on theoretical ATP balances to provide the substrate distribution amongst the dominant genera. This research shows that aerobic granular sludge technology can be applied to glucose-containing effluents and that glucose is a suitable substrate for achieving phosphate removal. The results also show that for fermentable substrates a microbial community consisting of fermentative organisms and PAO develop., 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Phosphate (Pi) is a macronutrient, and Pi homeostasis is essential for life. Pi homeostasis has been intensively studied; however, many questions remain, even at the cellular level. Using Schizosaccharomyces pombe, we sought to better understand cellular Pi homeostasis and showed that three Pi regulators with SPX domains, Xpr1/Spx2, Pqr1, and the VTC complex synergistically contribute to Pi homeostasis to support cell proliferation and survival. SPX domains bind to inositol pyrophosphate and modulate activities of Pi-related proteins. Xpr1 is a plasma membrane protein and its Pi-exporting activity has been demonstrated in metazoan orthologs, but not in fungi. We first found that S. pombe Xpr1 is a Pi exporter, activity of which is regulated and accelerated in the mutants of Pqr1 and the VTC complex. Pqr1 is the ubiquitin ligase downregulating the Pi importers, Pho84 and Pho842. The VTC complex synthesizes polyphosphate in vacuoles. Triple deletion of Xpr1, Pqr1, and Vtc4, the catalytic core of the VTC complex, was nearly lethal in normal medium but survivable at lower [Pi]. All double-deletion mutants of the three genes were viable at normal Pi, but Δpqr1Δxpr1 showed severe viability loss at high [Pi], accompanied by hyper-elevation of cellular total Pi and free Pi. This study suggests that the three cellular processes, restriction of Pi uptake, Pi export, and polyP synthesis, contribute synergistically to cell proliferation through maintenance of Pi homeostasis, leading to the hypothesis that cooperation between Pqr1, Xpr1, and the VTC complex protects the cytoplasm and/or the nucleus from lethal elevation of free Pi., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Adenosine triphosphate (ATP) regeneration systems are essential for efficient biocatalytic phosphoryl transfer reactions. Polyphosphate kinase (PPK) is a versatile enzyme that can transfer phosphate groups among adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, and polyphosphate (Poly P). Utilization of PPK is an attractive solution to address the problem of ATP regeneration due to its ability to use a variety of inexpensive and stable Poly P salts as phosphate group donors. This review comprehensively summarizes the structural characteristics and catalytic mechanisms of different types of PPKs, as well as the variations in enzyme activity, catalytic efficiency, stability, and coenzyme preference observed in PPKs from different sources. Moreover, recent advances in PPK-mediated ATP regeneration systems and protein engineering of wild-type PPK are summarized.

Previous research suggested that two major groups of polyphosphate-accumulating organisms (PAOs), i.e., Ca. Accumulibacter and Tetrasphaera, play cooperative roles in enhanced biological phosphorus removal (EBPR). The fermentation of complex organic compounds by Tetrasphaera provides carbon sources for Ca. Accumulibacter. However, the viability of the fermentation products (e.g., lactate, succinate, alanine) as carbon sources for Ca. Accumulibacter and their potential effects on the metabolism of Ca. Accumulibacter were largely unknown. This work for the first time investigated the capability and metabolic details of Ca. Accumulibacter cognatus clade IIC strain SCUT-2 (enriched in a lab-scale reactor with a relative abundance of 42.8%) in using these fermentation products for EBPR. The enrichment culture was able to assimilate lactate and succinate with the anaerobic P release to carbon uptake ratios of 0.28 and 0.36 P mol/C mol, respectively. In the co-presence of acetate, the uptake of lactate was strongly inhibited, since two substrates shared the same transporter as suggested by the carbon uptake bioenergetic analysis. When acetate and succinate were fed at the same time, Ca. Accumulibacter assimilated two carbon sources simultaneously. Proton motive force (PMF) was the key driving force (up to 90%) for the uptake of lactate and succinate by Ca. Accumulibacter. Apart from the efflux of proton in symport with phosphate via the inorganic phosphate transport system, translocation of proton via the activity of fumarate reductase contributed to the generation of PMF, which agreed with the fact that PHV was a major component of PHA when lactate and succinate were used as carbon sources, involving the succinate-propionate pathway. Metabolic models for the usage of lactate and succinate by Ca. Accumulibacter for EBPR were built based on the combined physiological, biochemical, metagenomic, and metatranscriptomic analyses. Alanine was shown as an invalid carbon source for Ca. Accumulibacter. Instead, it significantly and adversely affected Ca. Accumulibacter-mediated EBPR. Phosphate release was observed without alanine uptake. Significant inhibitions on the aerobic phosphate uptake was also evident. Overall, this study suggested that there might not be a simply synergic relationship between Ca. Accumulibacter and Tetrasphaera. Their interactions would largely be determined by the kind of fermentation products released by the latter., 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.)