polyP Synthesis Research Articles

Inorganic polyphosphate in host and microbe biology.

Inorganic polyphosphate (polyP) is produced by both bacteria and their eukaryotic hosts, and it appears to play multiple important roles in the interactions between those organisms. However, the detailed mechanisms of how polyP synthesis is regulated in bacteria, and how it influences both bacterial and host biology, remain largely unexplored. In this review, we examine recent developments in the understanding of how bacteria regulate the synthesis of polyP, what roles polyP plays in controlling virulence in pathogenic bacteria, and the effects of polyP on the mammalian immune system, as well as progress on developing drugs that may be able to target bacterial polyP synthesis as novel means of treating infectious disease.

Loss of two families of SPX domain-containing proteins required for vacuolar polyphosphate accumulation coincides with the transition to phosphate storage in green plants

Phosphorus is an essential nutrient for plants. It is stored as inorganic phosphate (Pi) in the vacuoles of land plants but as inorganic polyphosphate (polyP) in chlorophyte algae. Although it is recognized that the SPX-Major Facilitator Superfamily (MFS) and VPE proteins are responsible for Pi influx and efflux, respectively, across the tonoplast in land plants, the mechanisms that underlie polyP homeostasis and the transition of phosphorus storage forms during the evolution of green plants remain unclear. In this study, we showed that CrPTC1, encoding a protein with both SPX and SLC (permease solute carrier 13) domains for Pi transport, and CrVTC4, encoding a protein with both SPX and vacuolar transporter chaperone (VTC) domains for polyP synthesis, are required for vacuolar polyP accumulation in the chlorophyte Chlamydomonas reinhardtii. Phylogenetic analysis showed that the SPX-SLC, SPX-VTC, and SPX-MFS proteins were present in the common ancestor of green plants (Viridiplantae). The SPX-SLC and SPX-VTC proteins are conserved among species that store phosphorus as vacuolar polyP and absent from genomes of plants that store phosphorus as vacuolar Pi. By contrast, SPX-MFS genes are present in the genomes of streptophytes that store phosphorus as Pi in the vacuoles. These results suggest that loss of SPX-SLC and SPX-VTC genes and functional conservation of SPX-MFS proteins during the evolution of streptophytes accompanied the change from ancestral polyP storage to Pi storage.

The Multiple Roles of Polyphosphate in Ralstonia eutropha and Other Bacteria

An astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes. Besides being a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses such as exposure to heat, extreme pH, oxidative agents, high osmolarity, heavy metals and others have been ascribed to polyP. In this contribution, we will provide a historical overview on polyP, will then describe the key proteins of polyP synthesis, the polyP kinases, before we will critically assess of the underlying data on the multiple functions of polyP and provide evidence that – with the exception of a P-storage-function – most other functions of polyP are not relevant for survival of Ralstonia eutropha, a biotechnologically important beta-proteobacterial species.

Metabolic control of acclimation to nutrient deprivation dependent on polyphosphate synthesis.

Polyphosphate, an energy-rich polymer conserved in all kingdoms of life, is integral to many cellular stress responses, including nutrient deprivation, and yet, the mechanisms that underlie its biological roles are not well understood. In this work, we elucidate the physiological function of this polymer in the acclimation of the model alga Chlamydomonas reinhardtii to nutrient deprivation. Our data reveal that polyphosphate synthesis is vital to control cellular adenosine 5'-triphosphate homeostasis and maintain both respiratory and photosynthetic electron transport upon sulfur deprivation. Using both genetic and pharmacological approaches, we show that electron flow in the energy-generating organelles is essential to induce and sustain acclimation to sulfur deprivation at the transcriptional level. These previously unidentified links among polyphosphate synthesis, photosynthetic and respiratory electron flow, and the acclimation of cells to nutrient deprivation could unveil the mechanism by which polyphosphate helps organisms cope with a myriad of stress conditions in a fluctuating environment.

Polyphosphate: A Multifunctional Metabolite in Cyanobacteria and Algae.

Polyphosphate (polyP), a polymer of orthophosphate (PO4 3-) of varying lengths, has been identified in all kingdoms of life. It can serve as a source of chemical bond energy (phosphoanhydride bond) that may have been used by biological systems prior to the evolution of ATP. Intracellular polyP is mainly stored as granules in specific vacuoles called acidocalcisomes, and its synthesis and accumulation appear to impact a myriad of cellular functions. It serves as a reservoir for inorganic PO4 3- and an energy source for fueling cellular metabolism, participates in maintaining adenylate and metal cation homeostasis, functions as a scaffold for sequestering cations, exhibits chaperone function, covalently binds to proteins to modify their activity, and enables normal acclimation of cells to stress conditions. PolyP also appears to have a role in symbiotic and parasitic associations, and in higher eukaryotes, low polyP levels seem to impact cancerous proliferation, apoptosis, procoagulant and proinflammatory responses and cause defects in TOR signaling. In this review, we discuss the metabolism, storage, and function of polyP in photosynthetic microbes, which mostly includes research on green algae and cyanobacteria. We focus on factors that impact polyP synthesis, specific enzymes required for its synthesis and degradation, sequestration of polyP in acidocalcisomes, its role in cellular energetics, acclimation processes, and metal homeostasis, and then transition to its potential applications for bioremediation and medical purposes.

Interactions between DksA and Stress-Responsive Alternative Sigma Factors Control Inorganic Polyphosphate Accumulation in Escherichia coli.

Bacteria synthesize inorganic polyphosphate (polyP) in response to a variety of different stress conditions. polyP protects bacteria by acting as a protein-stabilizing chaperone, metal chelator, or regulator of protein function, among other mechanisms. However, little is known about how stress signals are transmitted in the cell to lead to increased polyP accumulation. Previous work in the model enterobacterium Escherichia coli has indicated that the RNA polymerase-binding regulatory protein DksA is required for polyP synthesis in response to nutrient limitation stress. In this work, I set out to characterize the role of DksA in polyP regulation in more detail. I found that overexpression of DksA increases cellular polyP content (explaining the long-mysterious phenotype of dksA overexpression rescuing growth of a dnaK mutant at high temperatures) and characterized the roles of known functional residues of DksA in this process, finding that binding to RNA polymerase is required but that none of the other functions of DksA appear to be necessary. Transcriptomics revealed genome-wide transcriptional changes upon nutrient limitation, many of which were affected by DksA, and follow-up experiments identified complex interactions between DksA and the stress-sensing alternative sigma factors FliA, RpoN, and RpoE that impact polyP production, indicating that regulation of polyP synthesis is deeply entwined in the multifactorial stress response network of E. coliIMPORTANCE Inorganic polyphosphate (polyP) is an evolutionarily ancient, widely conserved biopolymer required for stress resistance and pathogenesis in diverse bacteria, but we do not understand how its synthesis is regulated. In this work, I gained new insights into this process by characterizing the role of the transcriptional regulator DksA in polyP regulation in Escherichia coli and identifying previously unknown links between polyP synthesis and the stress-responsive alternative sigma factors FliA, RpoN, and RpoE.

Augmenting Colonoscopy using Extended and Directional CycleGAN for Lossy Image Translation.

Colorectal cancer screening modalities, such as optical colonoscopy (OC) and virtual colonoscopy (VC), are critical for diagnosing and ultimately removing polyps (precursors of colon cancer). The non-invasive VC is normally used to inspect a 3D reconstructed colon (from CT scans) for polyps and if found, the OC procedure is performed to physically traverse the colon via endoscope and remove these polyps. In this paper, we present a deep learning framework, Extended and Directional CycleGAN, for lossy unpaired image-to-image translation between OC and VC to augment OC video sequences with scale-consistent depth information from VC, and augment VC with patient-specific textures, color and specular highlights from OC (e.g, for realistic polyp synthesis). Both OC and VC contain structural information, but it is obscured in OC by additional patient-specific texture and specular highlights, hence making the translation from OC to VC lossy. The existing CycleGAN approaches do not handle lossy transformations. To address this shortcoming, we introduce an extended cycle consistency loss, which compares the geometric structures from OC in the VC domain. This loss removes the need for the CycleGAN to embed OC information in the VC domain. To handle a stronger removal of the textures and lighting, a Directional Discriminator is introduced to differentiate the direction of translation (by creating paired information for the discriminator), as opposed to the standard CycleGAN which is direction-agnostic. Combining the extended cycle consistency loss and the Directional Discriminator, we show state-of-the-art results on scale-consistent depth inference for phantom, textured VC and for real polyp and normal colon video sequences. We also present results for realistic pendunculated and flat polyp synthesis from bumps introduced in 3D VC models.

Derived Postbiotics of a Multi‐strain Probiotic Formula Clinically Validated for the Treatment of Irritable Bowel Syndrome

IntroductionThe pathophysiology of Irritable Bowel Syndrome (IBS) involves increased intestinal permeability, micro‐inflammation and gut dysbiosis with associated visceral hypersensitivity. The probiotic mix of Lactobacillus plantarum strains CECT7484 (KABP022) and CECT7485 (KABP023) and Pediococcus acidilactici CECT7483 (KABP021) has shown positive clinical outcomes in IBS. However, their exact mechanisms of action have not been elucidated. Postbiotics are probiotic‐derived metabolites or cell components that can improve host health. In this work we sought to characterize said strains about the production of specific postbiotics known to influence gut permeability and inflammation, as well as their inhibitory activity against bacteria known to be increased in IBS.MethodsWe measured the capacity to biosynthesize acetate from 3 carbon sources (glucose, xylose and arabinose) by colorimetric assay, acetylcholine (ACh) by HPLC‐ESI‐MS/MS and polyphosphate (polyP) granules by fluorescence, using Lactobacillus rhamnosus ATCC53103 as control. Inhibitory activity was tested on 6 IBS‐associated bacteria (2 enterobacteria, 2 Actinomyces spp. and 2 Streptococcus spp. strains). Supernatants from co‐cultures of probiotic and target bacteria were obtained and adjusted to pH 6 to only detect organic‐acid independent inhibition. Target bacteria were cultured on the supernatants for 16 h. Genomes were sequenced by Hiseq 2500–3000 and key genes involved in the biosynthesis of these postbiotics were searched. Additionally, susceptibility of the 3 strains to 8 antibiotics were determined according to ISO10932.ResultsResults are summarized in Table 1. Production of acetate was dependent on carbon source. The production from glucose was low (< 1 mM). The fermentation of xylose increased the acetate production by all the strains (3 mM approx.) while arabinose yielded the greatest concentrations (up to 13.5 mM for L. plantarum CECT7484). L. plantarum strains synthesized the largest amounts of ACh (12.5 mg/L for CECT7484 and 13.8 mg/L for CECT7485) whereas P. acidilactici CECT7483 produced 1.9 mg/L and control strain produced none. After 6 h of growth, accumulation of PolyP granules was observed for the L. plantarum and control strains but not for Pediococcus. The 3 strains produced antimicrobials against all 6 target bacteria. Consistent with phenotypic analysis, in silico study confirmed the presence of phosphofructokinase (pfk) gene (responsible for acetate synthesis) in the 3 genomes and polyphosphate kinase (ppk) gene (polyP synthesis) in the 2 L. plantarum genomes. Bacteriocin (antimicrobial compound) genes were found in the genomes of L. plantarum CECT7484 and CECT7485. Moreover, the 3 strains were susceptible to all the antibiotics tested.ConclusionThe probiotic strains were proven to produce postbiotic molecules including acetate, polyP, ACh and antimicrobial compounds (potentially bacteriocins) against IBS‐associated microorganisms. This unique combination of postbiotics could explain the clinically beneficial effects of the probiotic mix in IBS subjects.Support or Funding InformationAB‐Biotics ‐KANEKA Postbiotics produced by the 3 probiotic strains and the control strain. Acetate production was tested from three carbon sources. Production of antimicrobials was determined by testing the inhibition of growth of 6 IBS‐related bacteria. ACh, acetylcholine; PolyP, polyphosphate (−/+, traces; +, production). Strains Acetate ACh PolyP No. bacteria inhibited Glucose Xylose Arabinose L. plantarum CECT7484 (KABP022) −/+ ++ ++++ ++++ +++ 6 out of 6 L. plantarum CECT7485 (KABP023) −/+ ++ +++ ++++ ++ 6 out of 6 P. acidilactici CECT7483 (KABP021) −/+ ++ +++ + − 6 out of 6 L. rhamnosus ATCC53103 (Control) −/+ ++ + − + NA

The PolS-PolR Two-Component System Regulates Genes Involved in Poly-P Metabolism and Phosphate Transport in Microlunatus phosphovorus.

Microlunatus phosphovorus NM-1 is a polyphosphate (poly-P)-accumulating bacterium that accumulates poly-P under aerobic conditions and degrades poly-P under anaerobic conditions. In this study, the two-component system (TCS) PolS-PolR was identified in NM-1, and the response regulator PolR was found to directly bind to the promoters of genes related to phosphate transport (MLP_RS00235, MLP_RS23035, and MLP_RS24590); poly-P catabolism (MLP_RS12905) and poly-P synthesis (MLP_RS23025). RT-qPCR assays showed that ppgk (MLP_RS12905), ppk (MLP_RS23025), pstS (MLP_RS23035), and pit (MLP_RS24590) were down-regulated during the aerobic-anaerobic shift. The sequence GTTCACnnnnnGTTCaC was identified as a recognition sequence for PolR by MEME analysis and DNase I footprinting. EMSAs and ChIP-qPCR assays indicated that PolR binds to the promoters of pit (MLP_RS00235), ppgk (MLP_RS12905), ppk (MLP_RS23025), pstS (MLP_RS23035) and pit (MLP_RS24590), and ChIP-qPCR further suggested that the binding affinity of PolR was lower under anaerobic conditions than under aerobic conditions in vivo. These findings indicate that the PolS-PolR TCS in M. phosphovorus may be involved in the regulation of poly-P metabolism in response to levels of dissolved oxygen in the environment, and our results provide insights into new approaches for understanding the mechanisms of phosphorus accumulation and release.

Proteins required for vacuolar function are targets of lysine polyphosphorylation in yeast.

Polyphosphates (polyP) are long chains of inorganic phosphates that can be attached to lysine residues of target proteins as a nonenzymatic post-translational modification. This modification, termed polyphosphorylation, may be particularly prevalent in bacterial and fungal species that synthesize large quantities of polyP. In this study, we evaluated the polyphosphorylation status of over 200 candidate targets in Saccharomycescerevisiae. We report eight new polyphosphorylated proteins that interact genetically and physically with previous targets implicated in ribosome biogenesis. The expanded target network includes vacuolar proteins Prb1 and Apl5, whose modification with polyP suggests a model for feedback regulation of polyP synthesis, while raising questions regarding the location of polyphosphorylation invivo.

Fluorescence enzymatic assay for bacterial polyphosphate kinase 1 (PPK1) as a platform for screening antivirulence molecules.

Inorganic polyphosphate (polyP) and its metabolic enzymes are important in several cellular processes related with virulence and antibiotic susceptibility. Accordingly, bacterial polyP synthesis has been proposed as a good target for designing novel antivirulence molecules as alternative to conventional antibiotics. In most pathogenic bacteria, polyphosphate kinase 1 (PPK1), in charge of polyP synthesis from ATP, is widely conserved. Current colorimetric and radioactive polyP synthesis enzymatic assays are not suitable for high-throughput screening of PPK1 inhibitors. Given the ability of polyP to modify the excitation-emission spectra of DAPI (4ʹ-6-diamidino-2-phenylindole), a fluorescence assay was previously developed by using a purified recombinant PPK1 enzyme from Escherichia coli. In this work we have developed a suitable methodology for high-throughput measurement of E. coli PPK1 activity. This platform can be used for the screening putative antimicrobial molecules for related enteropathogenic bacteria.

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

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

Cadmium accumulation and toxicity affect the extracytoplasmic polyphosphate level in Chlamydomonas reinhardtii

Cadmium is a non-essential metal and highly toxic for biological functions. Depending on the dose of Cd2+, the biochemical response will differ. In this study, we investigated the level of extracytoplasmic polyphosphate (polyP) when Chlamydomonas reinhardtii was exposed to the effect of CdCl2. When compared to control cells, Cd2+-treated cells to 400–600 µM showed a decrease in the growth rate from 0.78 to 0.38 d−1 for the strain CC-125, and a decrease from 0.81 to 0.32–0.35 d−1 for CC-503. This indicated a strong toxicity effect on the population growth of cells during 72 h. In addition, the results demonstrated the decrease in the synthesis and/or the degradation of polyP that was correlated with the accumulation of Cd2+ in both algal strains. Furthermore, the level of polyP decreased in relation to the decrease of FV/FM value. The toxicity of Cd2+ induced a high level of cell necrosis for CC-503, and the level of polyP could not be recovered at 72 h. In response to the toxic effects of Cd2+, the observed formation of palmelloid colonies by CC-125 cells was correlated with the recovery of the polyP level. Nevertheless, both algal strains were able to accumulate significant amount of Cd2+ in their biomass. Therefore, our study demonstrated a distinct impact of Cd2+ on the metabolism of polyP (synthesis and/or degradation), which was dependent on the concentration of CdCl2 and the Chlamydomonas strain. Based on this study, the level of polyP can be used as a biomarker of Cd2+ toxicity at 24–48 h, even with the cell wall-deficient strain CC-503.

Polyphosphate in Lactobacillus and Its Link to Stress Tolerance and Probiotic Properties.

The synthesis of the inorganic polymer polyphosphate (poly-P) in bacteria has been linked to stress survival and to the capacity of some strains to sequester heavy metals. In addition, synthesis of poly-P by certain strains of probiotic lactobacilli has been evidenced as a probiotic mechanism due to the homeostatic properties of this compound at the intestinal epithelium. We analyzed the link between poly-P synthesis, stress response, and mercury toxicity/accumulation by comparing wild-type strains of Lactobacillus and their corresponding mutants devoid of poly-P synthesis capacity (defective in the poly-P kinase, ppk, gene). Results showed that resistance to salt (NaCl) and acidic (pH 4) stresses upon ppk mutation was affected in Lactobacillus casei, while no effect was observed in two different Lactobacillus plantarum strains. Inorganic [Hg(II)] and organic (CH3Hg) mercury toxicity was generally increased upon ppk mutation, but no influence was seen on the capacity to retain both mercurial forms by the bacteria. Notwithstanding, the culture supernatants of ppk-defective L. plantarum strains possessed a diminished capacity to induce HSP27 expression, a marker for cell protection, in cultured Caco-2 cells compared to wild-type strains. In summary, our results illustrate that the role of poly-P in stress tolerance can vary between strains and they reinforce the idea of probiotic-derived poly-P as a molecule that modulates host-signaling pathways. They also question the relevance of this polymer to the capacity to retain mercury of probiotics.

Importance of polyphosphate in the Leishmania life cycle.

Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these poly-phosphate (polyP) chains serve as an energy source and phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 (VTC4) is responsible for polyP synthesis in Leishmania parasites. During Leishmania in vitro culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient (vtc4-) Leishmania proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In in vivo mouse infections, L. major VTC4 knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of VTC4 did not alter the course of L. guyanensis infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures, Leishmania promastigotes highly consumed polyP, and both knockdown or deletion of VTC4 diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans.

Screening a Protein Array with Synthetic Biotinylated Inorganic Polyphosphate To Define the Human PolyP-ome.

Phenotypes are established by tight regulation on protein functions. This regulation can be mediated allosterically, through protein binding, and covalently, through post-translational modification (PTM). The integration of an ever-increasing number of PTMs into regulatory networks enables and defines the proteome complexity. Protein PTMs can occur enzymatically and nonenzymatically. Polyphosphorylation, which is a recently discovered PTM that belongs to the latter category, is the covalent attachment of the linear ortho-phosphate polymer called inorganic polyphosphate (polyP) to lysine residues. PolyP, which is ubiquitously present in nature, is also known to allosterically control protein function. To date, lack of reagents has prevented the systematic analysis of proteins covalently and/or allosterically associated with polyP. Here, we report on the chemical synthesis of biotin-modified monodisperse short-chain polyP (bio-polyP8-bio) and its subsequent use to screen a human proteome array to identify proteins that associate with polyP, thereby starting to define the human polyP-ome.

Discovery of potent polyphosphate kinase 1 (PPK1) inhibitors using structure-based exploration of PPK1Pharmacophoric space coupled with docking analyses.

Inorganic polyphosphate (polyP) is present in all living forms of life. Studied mainly in prokaryotes, polyP and its associated enzymes are vital in diverse metabolic activities, in some structural functions, and most importantly in stress responses. Bacterial species, including many pathogens, encode a homolog of a major polyP synthesis enzyme, Poly Phosphate Kinase (PPK) with 2 different genes coding for PPK1 and PPK2. Genetic deletion of the ppk1 gene leads to reduced polyP levels and the consequent loss of virulence and stress adaptation responses. This far, no PPK1 homolog has been identified in higher-order eukaryotes, and, therefore, PPK1 represents a novel target for chemotherapy. The aim of the current study is to investigate PPK1 from Escherichia coli with comprehensive understanding of the enzyme's structure and binding sites, which were used to design pharmacophores and screen a library of compounds for potential discovery of selective PPK1 inhibitors. Verification of the resultant inhibitors activities was conducted using a combination of mutagenic and chemical biological approaches. The metabolic phenotypic maps of the wild type E. coli (WT) and ppk1 knockout mutant were generated and compared with the metabolic map of the chemically inhibited WT. In addition, biofilm formation ability was measured in WT, ppk1 knockout mutant, and the chemically inhibited WT. The results demonstrated that chemical inhibition of PPK1, with the designed inhibitors, was equivalent to gene deletion in altering specific metabolic pathways, changing the metabolic fingerprint, and suppressing the ability of E. coli to form a biofilm.

A screen for novel targets casts polyphosphorylation of lysine as a common post‐translational modification

Polyphosphates (polyP) are long chains of linked inorganic phosphates ranging from 3–1000s of residues in length. They are found in all organisms and play critical roles in a range of functions including blood clotting and bacterial virulence. Given its diverse roles, polyP is an attractive target for novel therapies. Recently, it was shown that polyP can be non‐enzymatically added to protein targets within poly‐acidic, serine, and lysine rich (PASK) motifs as a lysine post‐translational modification termed polyphosphorylation. This modification was described for two yeast proteins, Nsr1 and Top1. In yeast, polyP is initially synthesized by a polyP synthetase (VTC4) and subsequently added as a PTM non‐enzymatic. Our lab has developed a screen to assess polyphosphorylation of yeast substrates wherein we uncovered 25 novel substrates including a conserved network of proteins functioning in ribosome biogenesis. Disruption of this polyP synthesis through vtc4D deletion results in translation defects as measured by polysome profiling. Finally, we found that expression of E. coli polyP synthetase (EcPPK1) in vtc4D mutant yeast results in extreme sensitivity to rapamycin and cycloheximide, despite its ability to restore polyphosphorylation of multiple yeast targets. We propose a model wherein polyphosphorylation of unique targets regulates multiple aspects of cell growth. Moreover, regulation of polyphosphorylation in space and time is critical to preserve essential cell functions, such as ribosome function.Support or Funding InformationThe funding agencies that support this research are: University of Ottawa and CIHRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation invivo.

Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels.

Bacteria synthesize inorganic polyphosphate (polyP) in response to a wide variety of stresses, and production of polyP is essential for stress response and survival in many important pathogens and bacteria used in biotechnological processes. However, surprisingly little is known about the molecular mechanisms that control polyP synthesis. We have therefore developed a novel genetic screen that specifically links growth of Escherichia coli to polyP synthesis, allowing us to isolate mutations leading to enhanced polyP production. Using this system, we have identified mutations in the polyP-synthesizing enzyme polyP kinase (PPK) that lead to dramatic increases in in vivo polyP synthesis but do not substantially affect the rate of polyP synthesis by PPK in vitro These mutations are distant from the PPK active site and found in interfaces between monomers of the PPK tetramer. We have also shown that high levels of polyP lead to intracellular magnesium starvation. Our results provide new insights into the control of bacterial polyP accumulation and suggest a simple, novel strategy for engineering bacteria with increased polyP contents.IMPORTANCE PolyP is an ancient, universally conserved biomolecule and is important for stress response, energy metabolism, and virulence in a remarkably broad range of microorganisms. PolyP accumulation by bacteria is also important in biotechnology applications. For example, it is critical to enhanced biological phosphate removal (EBPR) from wastewater. Understanding how bacteria control polyP synthesis is therefore of broad importance in both the fields of bacterial pathogenesis and biological engineering. Using Escherichia coli as a model organism, we have identified the first known mutations in polyP kinase that lead to increases in cellular polyP content.