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Akio Kuroda - One of the best experts on this subject based on the ideXlab platform.
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feasibility of thermophilic adenosine triphosphate regeneration system using thermus thermophilus Polyphosphate Kinase
Process Biochemistry, 2011Co-Authors: Elvi Restiawaty, Ryuichi Hirota, Akio Kuroda, Kohsuke Honda, Yoshihiro Iwasa, Shohei Maya, Takeshi Omasa, Hisao OhtakeAbstract:Abstract The gene encoding Polyphosphate Kinase from Thermus thermophilus (TtPPK) was expressed in Escherichia coli Rosetta2 (DE3) pLysS. The E. coli recombinant cells were heated at 70 °C to inactivate indigenous enzymes and used for regenerating adenosine triphosphate (ATP) from exogenous Polyphosphate (polyP) and adenosine diphosphate (ADP). The heat-treated cells having TtPPK were able to regenerate ATP at rates similar to those detected in cell-free extracts, suggesting that exogenous polyP and ADP could freely access TtPPK through the heat-damaged cell envelope. More than 80% of TtPPK activity was retained in the heated cells after incubation for at least 40 min at 70 °C. TtPPK in the heated cells could be easily recovered from the reaction mixture by centrifugation at 12,000 × g for 10 min. The gene encoding thermophilic ATP-dependent glycerol Kinase from Thermococcus kodakaraensis KOD1 (TkGK) was expressed in E. coli Rosetta2 (DE3) pLysS. Using the mixture of E. coli recombinants expressing TkGK and TtPPK, the production of glycerol 3-phosphate (G3P) from glycerol was examined at 70 °C as a model reaction. When polyP was added to the reaction mixture in a fed-batch mode, 100 mM glycerol was stoichiometrically converted to 80 mM G3P (a molar yield of 80%).
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Use of an Escherichia coli Recombinant Producing Thermostable Polyphosphate Kinase as an ATP Regenerator To Produce Fructose 1,6-Diphosphate
Applied and environmental microbiology, 2007Co-Authors: Seishi Iwamoto, Kei Motomura, Yasuharu Shinoda, Masaaki Urata, Junichi Kato, Noboru Takiguchi, Hisao Ohtake, Ryuichi Hirota, Akio KurodaAbstract:Heat-treated Escherichia coli producing Thermus Polyphosphate Kinase regenerated ATP by using exogenous Polyphosphate. This recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. In this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and Polyphosphate.
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Polyphosphate Kinase as a nucleoside diphosphate Kinase in Escherichia coli and Pseudomonas aeruginosa
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Akio Kuroda, Arthur KornbergAbstract:Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate Kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or overabundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of Polyphosphate Kinase (PPK) to use inorganic Polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E. coli; the activity was absent from the membrane fractions obtained from E. coli mutants lacking the ppk gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2–60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from Polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5′ tetraphosphate.
Arthur Kornberg - One of the best experts on this subject based on the ideXlab platform.
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Polyphosphate Kinase 1, a conserved bacterial enzyme, in a eukaryote, Dictyostelium discoideum, with a role in cytokinesis.
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Haiyu Zhang, María R. Gómez-garcía, Xiaobing Shi, Narayana N. Rao, Arthur KornbergAbstract:Polyphosphate Kinase 1 (PPK1), the principal enzyme responsible for reversible synthesis of Polyphosphate (poly P) from the terminal phosphate of ATP, is highly conserved in bacteria and archaea. Dictyostelium discoideum, a social slime mold, is one of a few eukaryotes known to possess a PPK1 homolog (DdPPK1). Compared with PPK1 of Escherichia coli, DdPPK1 contains the conserved residues for ATP binding and autophosphorylation, but has an N-terminal extension of 370 aa, lacking homology with any known protein. Polyphosphate or ATP promote oligomerization of the enzyme in vitro. The DdPPK1 products are heterogeneous in chain length and shorter than those of E. coli. The unique DdPPK1 N-terminal domain was shown to be necessary for its enzymatic activity, cellular localization, and physiological functions. Mutants of DdPPK1, as previously reported, are defective in development, sporulation, and predation, and as shown here, in late stages of cytokinesis and cell division.
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diverse phenotypes resulting from Polyphosphate Kinase gene ppk1 inactivation in different strains of helicobacter pylori
Journal of Bacteriology, 2005Co-Authors: Shumin Tan, Arthur Kornberg, Cresson D. Fraley, Maojun Zhang, Daiva Dailidiene, Douglas E BergAbstract:Connections among biochemical pathways should help buffer organisms against environmental stress and affect the pace and trajectory of genome evolution. To explore these ideas, we studied consequences of inactivating the gene for Polyphosphate Kinase 1 (ppk1) in strains of Helicobacter pylori, a genetically diverse gastric pathogen. The PPK1 enzyme catalyzes synthesis of inorganic Polyphosphate (poly P), a reservoir of high-energy phosphate bonds with multiple roles. Prior analyses in less-fastidious microbes had implicated poly P in stress resistance, motility, and virulence. In our studies, ppk1 inactivation caused the expected near-complete absence of poly P (>250-fold decrease) but had phenotypic effects that differed markedly among unrelated strains: (i) poor initial growth on standard brain heart infusion agar (five of six strains tested); (ii) weakened colonization of mice (4 of 5 strains); (iii) reduced growth on Ham's F-12 agar, a nutritionally limiting medium (8 of 11 strains); (iv) heightened susceptibility to metronidazole (6 of 17 strains); and (v) decreased motility in soft agar (1 of 13 strains). Complementation tests confirmed that the lack of growth of one Δppk1 strain on F-12 agar and the inability to colonize mice of another were each due to ppk1 inactivation. Thus, the importance of ppk1 to H. pylori differed among strains and the phenotypes monitored. We suggest that quantitative interactions, as seen here, are common among genes that affect metabolic pathways and that H. pylori's high genetic diversity makes it well suited for studies of such interactions, their underlying mechanisms, and their evolutionary consequences.
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Polyphosphate Kinase is essential for biofilm development quorum sensing and virulence of pseudomonas aeruginosa
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Harunur M Rashid, Kendra P Rumbaugh, Luciano Passador, David G Davies, Abdul N Hamood, Barbara H Iglewski, Arthur KornbergAbstract:‡§ ¶ The human opportunistic pathogen Pseudomonas aeruginosa causes a variety of infections in immunocompromised hosts and in individuals with cystic fibrosis. A knockout mutation in the Polyphosphate Kinase (ppk) gene, encoding PPK responsible for the synthesis of inorganic Polyphosphate from ATP, renders P. aeruginosa cells unable to form a thick and differentiated biofilm. The mutant is aberrant in quorum sensing and responses in that production of the quorum-sensing controlled virulence factors elastase and rhamnolipid are severely reduced. In a burned-mouse pathogenesis model, the virulence of the mutant is greatly reduced with severe defects in the colonization of mouse tissues. The conservation of PPK among many bacterial pathogens and its absence in eukaryotes suggest that PPK might be an attractive target for antimicrobial drugs.
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Polyphosphate Kinase as a nucleoside diphosphate Kinase in Escherichia coli and Pseudomonas aeruginosa
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Akio Kuroda, Arthur KornbergAbstract:Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate Kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or overabundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of Polyphosphate Kinase (PPK) to use inorganic Polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E. coli; the activity was absent from the membrane fractions obtained from E. coli mutants lacking the ppk gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2–60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from Polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5′ tetraphosphate.
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Phosphohistidyl active sites in Polyphosphate Kinase of Escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Krishnanand D. Kumble, Kyunghye Ahn, Arthur KornbergAbstract:In the synthesis of inorganic Polyphosphate (polyP) from ATP by Polyphosphate Kinase (PPK; EC 2.7.4.1) of Escherichia coli, an N-P-linked phosphoenzyme was previously identified as the intermediate. The phosphate is presumed to be linked to N3 of the histidine residue because of its chemical stabilities and its resemblance to other enzymes known to contain N3-phosphohistidine. Tryptic digests of [32P]PPK contain a predominant 32P-labeled peptide that includes His-441. Of the 16 histidine residues in PPK of E. coli, 4 are conserved among several bacterial species. Mutagenesis of these 4 histidines shows that two (His-430 and His-598) are unaffected in function when mutated to glutamine, whereas two others (His-441 and His-460) mutated to glutamine or alanine fail to be phosphorylated, show no enzymatic activities, and fail to support polyP accumulation in cells bearing these mutant enzymes.
Cesar Torres - One of the best experts on this subject based on the ideXlab platform.
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Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and Polyphosphate Kinase
BMC biotechnology, 2011Co-Authors: Oscar N. Ruiz, Derry Alvarez, Gloriene Gonzalez-ruiz, Cesar TorresAbstract:Background The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and Polyphosphate Kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation.
Jay D. Keasling - One of the best experts on this subject based on the ideXlab platform.
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Polyphosphate Kinase genes from full-scale activated sludge plants
Applied Microbiology and Biotechnology, 2007Co-Authors: Katherine D Mcmahon, Daniel L Gall, Suzan Yilmaz, David Jenkins, Jay D. KeaslingAbstract:The performance of enhanced biological phosphorus removal (EBPR) wastewater treatment processes depends on the presence of bacteria that accumulate large quantities of Polyphosphate. One such group of bacteria has been identified and named Candidatus Accumulibacter phosphatis. Accumulibacter-like bacteria are abundant in many EBPR plants, but not much is known about their community or population ecology. In this study, we used the Polyphosphate Kinase gene ( ppk1 ) as a high-resolution genetic marker to study population structure in activated sludge. Ppk1 genes were amplified from samples collected from full-scale wastewater treatment plants of different configurations. Clone libraries were constructed using primers targeting highly conserved regions of ppk1 , to retrieve these genes from activated sludge plants that did, and did not, perform EBPR. Comparative sequence analysis revealed that ppk1 fragments were retrieved from organisms affiliated with the Accumulibacter cluster from EBPR plants but not from a plant that did not perform EBPR. A new set of more specific primers was designed and validated to amplify a 1,100 bp ppk1 fragment from Accumulibacter-like bacteria. Our results suggest that the Accumulibacter cluster has finer-scale architecture than previously revealed by 16S ribosomal RNA-based analyses.
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Polyphosphate Kinase genes from activated sludge carrying out enhanced biological phosphorus removal.
Water science and technology : a journal of the International Association on Water Pollution Research, 2002Co-Authors: Katherine D Mcmahon, David Jenkins, Jay D. KeaslingAbstract:The community structure and metabolic function of activated sludge carrying out enhanced biological phosphorus removal have been investigated. Laboratory-scale sequencing batch reactors were operated at several influent COD/P ratios to obtain sludges with a range of phosphorus contents. Molecular microbiological techniques based on small subunit ribosomal RNA were used to characterize the community structure of these sludges. The dominant Polyphosphate accumulating organism was a close relative of Rhodocyclus tenuis , a member of the β subclass of the Proteobacteria. Fragments of genes coding for Polyphosphate Kinase (PPK), thought to be responsible for Polyphosphate accumulation, were retrieved from one of the sludges. The relative abundance of PPK gene copies in genomic DNA extracted from sludges was determined to confirm that at least one of the PPK gene sequences was derived from the dominant Polyphosphate accumulating organism.
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Polyphosphate Kinase from activated sludge performing enhanced biological phosphorus removal.
Applied and environmental microbiology, 2002Co-Authors: Katherine D Mcmahon, David Jenkins, Michael A. Dojka, Norman R. Pace, Jay D. KeaslingAbstract:A novel Polyphosphate Kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like beta-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg(2+), and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.
Katherine D Mcmahon - One of the best experts on this subject based on the ideXlab platform.
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Polyphosphate Kinase genes from full-scale activated sludge plants
Applied Microbiology and Biotechnology, 2007Co-Authors: Katherine D Mcmahon, Daniel L Gall, Suzan Yilmaz, David Jenkins, Jay D. KeaslingAbstract:The performance of enhanced biological phosphorus removal (EBPR) wastewater treatment processes depends on the presence of bacteria that accumulate large quantities of Polyphosphate. One such group of bacteria has been identified and named Candidatus Accumulibacter phosphatis. Accumulibacter-like bacteria are abundant in many EBPR plants, but not much is known about their community or population ecology. In this study, we used the Polyphosphate Kinase gene ( ppk1 ) as a high-resolution genetic marker to study population structure in activated sludge. Ppk1 genes were amplified from samples collected from full-scale wastewater treatment plants of different configurations. Clone libraries were constructed using primers targeting highly conserved regions of ppk1 , to retrieve these genes from activated sludge plants that did, and did not, perform EBPR. Comparative sequence analysis revealed that ppk1 fragments were retrieved from organisms affiliated with the Accumulibacter cluster from EBPR plants but not from a plant that did not perform EBPR. A new set of more specific primers was designed and validated to amplify a 1,100 bp ppk1 fragment from Accumulibacter-like bacteria. Our results suggest that the Accumulibacter cluster has finer-scale architecture than previously revealed by 16S ribosomal RNA-based analyses.
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candidatus accumulibacter population structure in enhanced biological phosphorus removal sludges as revealed by Polyphosphate Kinase genes
Applied and Environmental Microbiology, 2007Co-Authors: Shaomei He, Daniel L Gall, Katherine D McmahonAbstract:We investigated the fine-scale population structure of the “Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the Polyphosphate Kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of “Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “Candidatus Accumulibacter” lineage. Sequences from at least five clades of “Candidatus Accumulibacter” were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “Candidatus Accumulibacter”-specific 16S rRNA and “Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “Candidatus Accumulibacter” clades. The relative distributions of “Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.
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Polyphosphate Kinase genes from activated sludge carrying out enhanced biological phosphorus removal.
Water science and technology : a journal of the International Association on Water Pollution Research, 2002Co-Authors: Katherine D Mcmahon, David Jenkins, Jay D. KeaslingAbstract:The community structure and metabolic function of activated sludge carrying out enhanced biological phosphorus removal have been investigated. Laboratory-scale sequencing batch reactors were operated at several influent COD/P ratios to obtain sludges with a range of phosphorus contents. Molecular microbiological techniques based on small subunit ribosomal RNA were used to characterize the community structure of these sludges. The dominant Polyphosphate accumulating organism was a close relative of Rhodocyclus tenuis , a member of the β subclass of the Proteobacteria. Fragments of genes coding for Polyphosphate Kinase (PPK), thought to be responsible for Polyphosphate accumulation, were retrieved from one of the sludges. The relative abundance of PPK gene copies in genomic DNA extracted from sludges was determined to confirm that at least one of the PPK gene sequences was derived from the dominant Polyphosphate accumulating organism.
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Polyphosphate Kinase from activated sludge performing enhanced biological phosphorus removal.
Applied and environmental microbiology, 2002Co-Authors: Katherine D Mcmahon, David Jenkins, Michael A. Dojka, Norman R. Pace, Jay D. KeaslingAbstract:A novel Polyphosphate Kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like beta-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg(2+), and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.