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One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson-Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution in vitro . To move AEGIS into living cells, metabolic pathways are next required to make AEGIS triphosphates economically from their nucleosides, eliminating the need to feed these expensive compounds in growth media. We report that "polyphosphate kinases" can be recruited for such pathways, working with natural diphosphate kinases and engineered nucleoside kinases. This pathway in vitro makes AEGIS triphosphates, including third-generation triphosphates having improved ability to survive in living bacterial cells. In α- 32 P-labeled forms, produced here for the first time, they were used to study DNA polymerases, finding cases where third-generation AEGIS triphosphates perform better with natural enzymes than second-generation AEGIS triphosphates.

Inorganic phosphate is stored as a polyphosphate (polyP) polymer inside every living cell. This polymer is synthesized by the polyP kinase (PPK) enzyme using the terminal phosphate of ATP as substrate and it performs important functions in the cell. In this study, effects of high levels of PPK on Bacillus thuringiensis subsp. israelensis were analyzed. Recombinant Bti ppk, a PPK overproducer, was found to uptake more phosphate into the cell and produce a consistently higher amount of endotoxin than the wild type under culture conditions including a range of temperatures (25 °C, 30 °C, and 37 °C), pH values (pH 5, 6, 8, and 9), and carbon sources (maltose, mannitol, sucrose, and starch). Moreover, this strain was found to overexpress sigB, which might cause a significant increase in the acid tolerance of this microorganism. Spores of Bti ppk were found to be smaller compared to wild-type spores; however, bioassay experiments with third-instar wild Culex pipiens larvae proved that high toxicity is not the result of small spore size. This hypertoxic recombinant Bti strain is very useful for industrial applications, not only because it produces more endotoxin than the wild type under different culture conditions, but also because it is more acid-tolerant under the conditions tested. [ABSTRACT FROM AUTHOR]

Acid stimulated accumulation of insoluble phosphorus within microbial cells is highly beneficial to wastewater treatment but remains largely unexplored. Using single cell analyses and next generation sequencing, the response of active polyphosphate accumulating microbial communities under conditions of enhanced phosphorus uptake under both acidic and aerobic conditions was characterised. Phosphorus accumulation activities were highest under acidic conditions (pH 5.5 > 8.5), where a significant positive effect on bioaccumulation was observed at pH 5.5 when compared to pH 8.5. In contrast to the Betaproteobacteria and Actinobacteria dominated enhanced biological phosphorus removal process, the functionally active polyP accumulators at pH 5.5 belonged to the Gammaproteobacteria , with key accumulators identified as members of the families Aeromonadaceae and Enterobacteriaceae . This study demonstrated a significant enrichment of key polyphosphate kinase and exopolyphosphatase genes within the community metagenome after acidification, concomitant with an increase in P accumulation kinetics. [ABSTRACT FROM AUTHOR]

Objective To construct the ppk mutant in Mycobacterium marinum (M. marinum) and to investigate its role in the physiology. Methods The ppk mutant in M.marinum was constructed by homologous recombination. Intracellular poly inorganic phosphate (poly-Pi) concentration was measured in cell suspensions by using a DAPI-based fluorescence approach. Then in vitro bacterial viability of ppk mutant type and the wild type strain was compared under different stress conditions. Furthermore, in vivo mycobacterial virulence was checked by infecting the adult zebrafish with mutant and wild type strain. And the intracellular growth ability was investigated by infection of the murine macrophage cell line RAW267.4. Results The intracellular poly-Pi concentration of M. marinum was significantly decreased after the deletion of ppk gene. In addition, the ppk mutant displayed attenuated phenotype in the zebrafish model. In mice-derived macrophage cell line, the cell proliferation of ppk mutant type declined significantly compared with wild type. Moreover, the ppk mutant type was more sensitive to nutrient starvation and antibiotic treatment, including rifampicin and levo-floxacin. Conclusions These results indicate that ppk is involved in stress response and required for mycobacterial virulence. [ABSTRACT FROM AUTHOR]

A process of l -theanine production by the recombinant γ-glutamylmethylamide synthetase (GMAS) coupled with an adenosine triphosphate (ATP) regeneration system that utilized inorganic polyphosphate (polyP) via polyphosphate kinase (PPK) was designed. The gmas gene from Methylovorus mays and ppk gene from Rhodobacter sphaeroides were cloned into pET-21a(+), then introduced into E. coli BL21(DE3) respectively. GMAS and PPK were both overexpressed in soluble forms. l -Theanine was efficiently produced from sodium glutamate, ethylamine hydrochloride and polyP with a very small amount of ATP. After 5 h at 37 °C and pH at 7.0, the 93% productivity of l -theanine was achieved at concentration of 200 mM sodium glutamate and 200 mM ethylamine hydrochloride with 5 mM ATP. Productivity of l -theanine decreased to 84% when using 400 mM sodium glutamate, and 26% when using 600 mM sodium glutamate. The productivity of l -theanine obtained at a very small amount of ATP and high substrate concentration provides the basis for the large-scale industrial production of l -theanine. [ABSTRACT FROM AUTHOR]

“ Candidatus Accumulibacter” are the dominant polyphosphate-accumulating organism (PAOs) to perform enhanced biological phosphate removal (EBPR) from wastewater. Free nitrous acid (FNA) as inhibitor is an intermediate of nitrification and denitrification commonly present in wastewater treatment plants (WWTPs) and lab-scale reactors. Impact of FNA on EBPR and community structures of Accumulibacter in domestic wastewater treatment was investigated. FNA complete inhibit concentration on aerobic P uptake at pH 6.5 was 6.85 × 10 −3 mg HNO 2 -N/L, higher than that at pH 7.0. Under complete inhibition, poly-β-hydroxyalkanoates degradation was used for nitrite reduction and detoxication rather than P-uptake. The P fraction existed in the forms of complex-P, interstitial-P, chemical participated-P and ortho-P at the end of aerobic phase, and complex-P proportion declined in comparison to non-inhibition. Dominant Accumulibacter clade was IIC, accounting for 70% of total Accumulibacter. Decline of IIF proportion indicated its sensitivity to FNA. Increase of IID percentage implied its strong tolerance to FNA. Under inhibition, Shannon index decreased from 3.45 of seed sludge to 1.92 at pH 7.0, indicating decline of biodiversity. Therefore, FNA inhibition weakened EBPR performance, and remarkably changed clade-level community structures of Accumulibacter. [ABSTRACT FROM AUTHOR]

5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli . ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160 min, ALA concentration was increased to 5.4 mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production. [ABSTRACT FROM AUTHOR]

Extraintestinal pathogenic Escherichia coli (ExPEC) causes a variety of acute infections in its hosts, and multidrug-resistant strains present significant challenges to public health and animal husbandry. Therefore, it is necessary to explore new drug targets to control E. coli epidemics. Previous studies have reported that ppk mutants of Burkholderia pseudomallei and Mycobacterium tuberculosis are more susceptible than the wild types (WTs) to stress. Therefore, we investigated the stress response to antibiotics mediated by polyphosphate kinase (PPK) in ExPEC strain PCN033. We observed that planktonic cells of a ppk knockout strain (Δppk) were more susceptible to antibiotics than was WT. However, biofilm-grown Δppk cells showed similar susceptibility to that of the WT and were more tolerant than the planktonic cells. During the planktonic lifestyle, the expression of genes involved in antibiotic tolerance (including resistance-conferring genes, and antibiotic influx, and efflux genes) did not change in the Δppk mutant without antibiotic treatment. However, the resistance-conferring gene bla and efflux genes were upregulated more in the WT than in the Δppk mutant by treatment with tazobactam. After treatment with gentamycin, the efflux genes and influx genes were upregulated and downregulated, respectively, more in the WT than in the Δppk mutant. The expression of genes involved in biofilm regulation also changed after treatment with tazobactam or gentamycin, and which is consistent with the results of the biofilm formation. Together, these observations indicate that PPK is important for the antibiotic stress response during the planktonic growth of ExPEC and might be a potential drug target in bacteria. [ABSTRACT FROM AUTHOR]

Proteus mirabilis ( P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis. [ABSTRACT FROM AUTHOR]

A modified University of Cape Town (MUCT) process was used to treat real municipal wastewater with low carbon to nitrogen ratio (C/N). To our knowledge, this is the first study where the influence of nitrite accumulation on “ Candidatus Accumulibacter” clade-level population structure was investigated during nitritation establishment and destruction. Real time quantitative PCR assays were conducted using the polyphosphate kinase 1 gene ( ppk 1) as a genetic marker. Abundances of total “ Candidatus Accumulibacter”, the relative distributions and population structure of the five “ Candidatus Accumulibacter” clades were characterized. Under complete nitrification, clade I using nitrate as electron acceptor was below 5% of total “ Candidatus Accumulibacter”. When the reactor was transformed into nitritation, clade I gradually disappeared. Clade IID using nitrite as electron acceptor for denitrifying phosphorus (P) removal was always the dominant “ Candidatus Accumulibacter” throughout the operational period. This clade was above 90% on average in total “ Candidatus Accumulibacter”, even up to nearly 100%, which was associated with good performance of denitrifying P removal via nitrite pathway. The nitrite concentrations affected the abundance of clade IID. The P removal was mainly completed by anoxic P uptake of about 88%. The P removal efficiency clearly had a positive correlation with the nitrite accumulation ratio. Under nitritation, the P removal efficiency was 30% higher than that under complete nitrification, suggesting that nitrite was appropriate as electron acceptor for denitrifying P removal when treating carbon-limited wastewater. [ABSTRACT FROM AUTHOR]

Inorganic polyphosphate (polyP) is composed of linear chains of phosphate groups linked by high-energy phosphoanhydride bonds. However, this simple, ubiquitous molecule remains poorly understood. The use of nonstandardized analytical methods has contributed to this lack of clarity. By using improved polyacrylamide gel electrophoresis we were able to visualize polyP extracted from Dictyostelium discoideum.We established that polyP is undetectable in cells lacking the polyphosphate kinase (DdPpk1). Generation of this ppk1 null strain revealed that polyP is important for the general fitness of the amoebae with the mutant strain displaying a substantial growth defect. We discovered an unprecedented accumulation of polyP during the developmental program, with polyP increasing more than 100-fold. The failure of ppk1 spores to accumulate polyP results in a germination defect. These phenotypes are underpinned by the ability of polyP to regulate basic energetic metabolism, demonstrated by a 2.5-fold decrease in the level of ATP in vegetative ppk1. Finally, the lack of polyP during the development of ppk1 mutant cells is partially offset by an increase of both ATP and inositol pyrophosphates, evidence for a model in which there is a functional interplay between inositol pyrophosphates, ATP, and polyP. [ABSTRACT FROM AUTHOR]

The most unique characteristic of Mycobacterium tuberculosis is persistence in the human host, and the biofilm formation is related to the persistance. Polyphosphate (polyP) kinase 1 (PPK1) is conserved in Mycobacteria and is responsible for polyP synthesis. polyP is a chain molecule linked by high-energy phosphate bonds, which is considered to play a very important role in bacterial persistence. However, the relationship of PPK1 and mycobacterial biofilm formation is still adequately unclear. In current study, ppk1 -deficient mutant (MT), ppk1 -complemented (CT) and wild-type strains of M. smegmatis mc 2 155 were used to investigate the formation, morphology and ultramicrostructure of the biofilm and to analyze the lipid levels and susceptibility to vancomycin antibiotic. And then WT, MT and CT strains were used to infect macrophages and to analyze the expression levels of various inflammatory factors, respectively. We found that PPK1 was required for M. smegmatis polyP production in vivo and polyP deficiency not only attenuated the biofilm formation, but also altered the phenotype and ultramicrostructure of the biofilm and reduced the cell lipid composition (except for C16.1 and C17.1, most of the fatty acid components from C8-C24). Moreover, the ppk1 -deficient mutant was also significantly more sensitive to vancomycin which targets the cell wall, and its ability to survive in macrophages was decreased, which was related to the change of the expression level of inflammatory factors in macrophage. This study demonstrates that the PPK1 can affect the biofilm structure through affecting the content of short chain fatty acid and promote intracellular survival of M. smegmatis by altering the expression of inflammatory factors. These findings establish a basis for investigating the role of PPK1 in the persistence of M. tuberculosis , and provide clues for treating latent infection of M. tuberculosis with PPK1 as a potential drug target., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors.)

The article reports on transcriptome analysis of Campylobacter jejuni polyphosphate kinase (ppk1 and ppk2) mutants. Topics discussed include Campylobacter jejuni being the cause of bacterial food-borne diarrhoea, role of PPK1 and PPK2 in the survival, adaptation, and virulence of bacterial pathogens, impact of deletion of ppk1 and ppk2 on the genes involved in C. jejuni, and the importance of poly-P metabolism in the survival and adaptation of C. jejuni.

Aim Polyphosphate kinase 1 ( PPK1) plays an important role in virulence, antibiotic resistance and survival under stress conditions and, therefore, is an attractive therapeutic target to control infections caused by multiple drug resistant Pseudomonas aeruginosa. This study explores the PPK1 inhibiting activity of ellagic acid derivatives ( EADs) from Terminalia chebula Retz. that could increase the susceptibility of Ps. aeruginosa to in vitro stress conditions. Methods and Results EADs reduced ppk1 gene expression by 93% ( P < 0·05) and completely inhibited its activity ( P < 0·01) at 0·5 mg ml−1. EADs-treated Ps. aeruginosa showed marked reduction in polyphosphate granules in cytosol. Expression of rpoS, the downstream master stress response regulator, was reduced by 94% ( P < 0·05) and the sensitivity of Ps. aeruginosa increased many fold to desiccation, oxidative (H2O2) and antibiotic (piperacillin) stresses. PPK-regulated swimming, swarming and twitching motilities and biofilm formation were also reduced significantly ( P ≤ 0·05) in MPAO1 and the clinical strains of Ps. aeruginosa. Conclusion EADs from T. chebula inhibited PPK1 expression and its activity and increased the sensitivity of Ps. aeruginosa to desiccation and oxidative stress while reducing tolerance to piperacillin. Significance and Impact of the Study The study underlines the potential of EADs as therapeutic agent against Ps. aeruginosa. [ABSTRACT FROM AUTHOR]

Lysinibacillus sphaericus produces parasporal crystal proteins (Bin toxins) that are specifically toxic to Culex and Anopheles mosquito larvae. It has long persistence in mosquito breeding sites due to the recycle of spores in mosquito cadaver and certain environments. This bacterium is unable to metabolize sugar (except N-acetylglucosamine) but can use proteinaceous materials. Previous studies have shown that inorganic polyphosphate (polyP) is essential for cell growth and responses to nutritional stringencies and environmental stresses and branched-chain amino acids (BCAAs) are useful intracellular signals for bacterial adaption to nutritional environments. However, relatively little is known about the role of polyP and the regulation link between polyP and BCAAs in nutritional limitation adaptation in a bacterium incapable of polysaccharide utilization. Here, a primary enzyme involved in polyP biosynthesis, polyphosphate kinase (PPK), was identified from L. sphaericus , and the polyP accumulation in different nutritional media was investigated. Furthermore, a ppk null mutant and the ppk -complemented strain were constructed and the effect of deletion on cell growth, polyP accumulation and expression of BCAAs biosynthetic genes ( ilvBHC ) was surveyed. The result showed that deletion of ppk resulted in a deficiency in polyP accumulation rather than cell growth in Luria-Bertani (LB) medium. Further studies demonstrated that the cell growth was evidently retarded and the ilvBHC expression was significantly reduced when ppk mutant was transferred from LB to limited-amino acid (LA) medium and that these phenotypes were significantly restored in the ppk -complemented strain. These results suggested that PPK plays a role in polyP accumulation and expression of genes involved in BCAAs biosynthesis, providing a hint for further understanding of the role of PPK in nutritional limitation in L. sphaericus . [ABSTRACT FROM AUTHOR]

Abnormalities in insulin-induced glucose incorporation in skeletal muscle were observed in Type 2 diabetes. Our previous studies revealed that the binding between skeletal muscle and kidney-enriched inositol polyphosphate phosphatase (SKIP) and p21-activated protein kinase (Pak1) at the plasma membrane is induced insulin-dependently and that this binding mediated a rapid and efficient termination of insulin signaling and a subsequent glucose uptake into skeletal muscle cells. Here, we identified 11-amino-acids peptide within kinase domain of Pak1, necessary and sufficient for SKIP binding. Expression of this region in C2C12 cells resulted in an increase in insulin signaling. Supplementation of a synthetic peptide of this sequence increased insulin signaling and insulin-induced glucose uptake into skeletal muscle cell lines. These findings suggest the physiological role of Pak1–SKIP binding in the regulation of insulin signaling in skeletal muscle. [ABSTRACT FROM AUTHOR]

Polyphosphate (polyP) is secreted by activated platelets and has been shown to contribute to thrombosis, suggesting that it could be a novel antithrombotic target. Previously reported polyP inhibitors based on polycationic substances, such as polyethylenimine, polyamidoamine dendrimers, and polymyxin B, although they attenuate thrombosis, all have significant toxicity in vivo, likely due to the presence of multiple primary amines responsible for their polyP binding ability. In this study, we examined a novel class of nontoxic polycationic compounds initially designed as universal heparin reversal agents (UHRAs) to determine their ability to block polyP procoagulant activity and also to determine their utility as antithrombotic treatments. Several UHRA compounds strongly inhibited polyP procoagulant activity in vitro, and 4 were selected for further examination in mouse models of thrombosis and hemostasis. Compounds UHRA-9 and UHRA-10 significantly reduced arterial thrombosis in mice. In mouse tail bleeding tests, administration of UHRA-9 or UHRA-10 was associated with significantly less bleeding compared with therapeutically equivalent doses of heparin. Thus, these compounds offer a new platform for developing novel antithrombotic agents that target procoagulant anionic polymers such as polyP with reduced toxicity and bleeding side effects. [ABSTRACT FROM AUTHOR]

The applicability of the enhanced biological phosphorus removal (EBPR) process for the removal of phosphorus in warm climates is uncertain due to frequent reports of EBPR deterioration at temperature higher than 25 °C. Nevertheless, a recent report on a stable and efficient EBPR process at 28 °C has inspired the present study to examine the performance of EBPR at 24 °C–32 °C, as well as the PAOs and GAOs involved, in greater detail. Two sequencing batch reactors (SBRs) were operated for EBPR in parallel at different temperatures, i.e., SBR-1 at 28 °C and SBR-2 first at 24 °C and subsequently at 32 °C. Both SBRs exhibited high phosphorus removal efficiencies at all three temperatures and produced effluents with phosphorus concentrations less than 1.0 mg/L during the steady state of reactor operation. Real-time quantitative polymerase chain reaction (qPCR) revealed Accumulibacter-PAOs comprised 64% of the total bacterial population at 24 °C, 43% at 28 °C and 19% at 32 °C. Based on fluorescent in situ hybridisation (FISH), the abundance of Competibacter-GAOs at both 24 °C and 28 °C was rather low (<10%), while it accounted for 40% of the total bacterial population at 32 °C. However, the smaller Accumulibacter population and larger population of Competibacter at 32 °C did not deteriorate the phosphorus removal performance. A polyphosphate kinase 1 ( ppk1 )-based qPCR analysis on all studied EBPR processes detected only Accumulibacter clade IIF. The Accumulibacter population shown by 16S rRNA and ppk1 was not significantly different. This finding confirmed the existence of single clade IIF in the processes and the specificity of the clade IIF primer sets designed in this study. Habitat filtering related to temperature could have contributed to the presence of a unique clade. The clade IIF was hypothesised to be able to perform the EBPR activity at high temperatures. The clade's robustness most likely helps it to fit the high-temperature EBPR sludge best and allows it not only to outcompete other Accumulibacter clades but coexist with GAOs without compromising EBPR activity. [ABSTRACT FROM AUTHOR]

Polyphosphate kinases 2 (PPK2) are key enzymes for polyphosphate utilisation in bacteria. The genome of Ruegeria pomeroyi, a marine α-proteobacterium, includes three Pseudomonas aeruginosa PPK2 homologs. We expressed these homologs in Escherichia coli as soluble proteins, purified the protein products and compared their metal, pH and nucleotide preferences. The optimal pH was 8.0 for SPO1727 and 9.0 for SPO1256. The SPO0224 gene product had two pH optima at eight and ten. The SPO0224 protein showed little dependence on metal presence, while SPO1256 required Mg. SPO1727 required Mg but accepted other ions as well. [ABSTRACT FROM AUTHOR]

Biofilms increase C. jejuni's resilience to detergents, antibiotics, and environmental stressors. In these investigations, we studied the modulation of biofilm in response to phosphate related stressors. We found that the deletion of ppk1, phoX, and ppk2 (polyphosphate associated [poly P] genes) in C. jejuni modulated different stages of biofilm formation such as attached microcolonies, air-liquid biofilms, and biofilm shedding. Additionally, inorganic phosphate also modulated attached microcolonies, air-liquid biofilms, and biofilm shedding both independently of and additively in the poly P associated mutants. Furthermore, we observed that these different biofilm stages were affected by biofilm age: for example, the adherent microcolonies were maximum on day 2, while biofilm growth at the air-liquid interface and shedding was highest on day 3. Also, we observed altered calcofluor white reactive polysaccharides in poly P-associated mutants, as well as increased secretion of autoinducer-2 (AI-2) quorum sensing molecules in the Δppk2 mutant. Further, the polysaccharide and flagellar biosynthesis genes, that are associated with biofilm formation, were altered in these poly P-associated mutants. We conclude that the phosphate limiting condition modulates C. jejuni biofilm formation. [ABSTRACT FROM AUTHOR]

Highlights: [•] A new configuration of SBR was designed to save reactor volume in the AEI regime. [•] The new configuration of SBR showed higher BPR than the A/O-SBR and AEI-SBR. [•] The biochemical metabolisms in the new configuration of SBR were followed by those in the AEI-SBR. [•] The activities of PPX and PPK between the A/O and AEI regimes were compared for the first time. [ABSTRACT FROM AUTHOR]

Phytoplankton alter their biochemical composition according to nutrient availability, such that their bulk elemental composition varies across oceanic provinces. However, the links between plankton biochemical composition and variation in biogeochemical cycling of nutrients remain largely unknown. In a survey of phytoplankton phosphorus stress in the western North Atlantic, we found that phytoplankton in the phosphorus-depleted subtropical Sargasso Sea were enriched in the biochemical polyphosphate (polyP) compared with nutrient-rich temperate waters, contradicting the canonical oceanographic view of polyP as a luxury phosphorus storage molecule. The enrichment in polyP coincided with enhanced alkaline phosphatase activity and substitution of sulfolipids for phospholipids, which are both indicators of phosphorus stress. Further, polyP appeared to be liberated preferentially over bulk phosphorus from sinking particles in the Sargasso Sea, thereby retaining phosphorus in shallow waters. Thus, polyP cycling may form a feedback loop that attenuates the export of phosphorus when it becomes scarce, contributes bioavailable P for primary production, and supports the export of carbon and nitrogen via sinking particles. [ABSTRACT FROM AUTHOR]

Abstract: Solids reduction in activated sludge processes (ASP) at source using process manipulation has been researched widely over the last two-decades. However, the absence of nutrient removal component, lack of understanding on the organic carbon, and limited information on key microbial community in solids minimizing ASP preclude the widespread acceptance of sludge minimizing processes. In this manuscript, we report simultaneous solids reduction through anaerobiosis along with nitrogen and phosphorus removals. The manuscript also reports carbon mass balance using stable isotope of carbon, microbial ecology of nitrifiers and polyphosphate accumulating organisms (PAOs). Two laboratory scale reactors were operated in anaerobic-aerobic-anoxic (A2O) mode. One reactor was run in the standard mode (hereafter called the control-SBR) simulating conventional A2O type of activated sludge process and the second reactor was run in the sludge minimizing mode (called the modified-SBR). Unlike other research efforts where the sludge minimizing reactor was maintained at nearly infinite solids retention time (SRT). To sustain the efficient nutrient removal, the modified-SBR in this research was operated at a very small solids yield rather than at infinite SRT. Both reactors showed consistent NH3–N, phosphorus and COD removals over a period of 263 days. Both reactors also showed active denitrification during the anoxic phase even if there was no organic carbon source available during this phase, suggesting the presence of denitrifying PAOs (DNPAOs). The observed solids yield in the modified-SBR was 60% less than the observed solids yield in the control-SBR. Specific oxygen uptake rate (SOUR) for the modified-SBR was almost 44% more than the control-SBR under identical feeding conditions, but was nearly the same for both reactors under fasting conditions. The modified-SBR showed greater diversity of ammonia oxidizing bacteria and PAOs compared to the control-SBR. The diversity of PAOs in the modified-SBR was even more interesting in which case novel clades of Candidatus Accumulibacter phosphatis (CAP), an uncultured but widely found PAOs, were found. [Copyright &y& Elsevier]

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by "high-energy" phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities. [ABSTRACT FROM AUTHOR]

Diphospho- myo-inositol polyphosphates have many roles to play, including roles in apoptosis, vesicle trafficking, the response of cells to stress, the regulation of telomere length and DNA damage repair, and inhibition of the cyclin-dependent kinase Pho85 system that monitors phosphate levels. This review focuses on the three classes of enzymes involved in the metabolism of these compounds: inositol hexakisphosphate kinases, inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinases and diphosphoinositol polyphosphate phosphohydrolases. However, these enzymes have roles beyond being mere catalysts, and their interactions with other proteins have cellular consequences. Through their interactions, the three inositol hexakisphosphate kinases have roles in exocytosis, diabetes, the response to infection, and apoptosis. The two inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinases influence the cellular response to phosphatidylinositol (3,4,5)-trisphosphate and the migration of pleckstrin homology domain-containing proteins to the plasma membrane. The five diphosphoinositol polyphosphate phosphohydrolases interact with ribosomal proteins and transcription factors, as well as proteins involved in membrane trafficking, exocytosis, ubiquitination and the proteasomal degradation of target proteins. Possible directions for future research aiming to determine the roles of these enzymes are highlighted. [ABSTRACT FROM AUTHOR]