Dataset Information

Polyphosphate:AMP phosphotransferase as a polyphosphate-dependent nucleoside monophosphate kinase in Acinetobacter johnsonii 210A.

ABSTRACT: We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.

SUBMITTER: Shiba T

PROVIDER: S-EPMC1063994 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Polyphosphate:AMP phosphotransferase as a polyphosphate-dependent nucleoside monophosphate kinase in Acinetobacter johnsonii 210A.

Shiba Toshikazu T Itoh Hiromichi H Kameda Atsushi A Kobayashi Keiju K Kawazoe Yumi Y Noguchi Toshitada T

Journal of bacteriology 20050301 5

We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produc ...[more]

PMID: 15716459

Similar Datasets

Project description:Acinetobacter baumannii, one of the most successful bacteria causing severe nosocomial infection, was identified as a top-priority pathogen by the WHO. Thus, genetic manipulations to clarify the potential targets for fighting A. baumannii resistance and virulence are vital. Polyphosphate (polyP) kinase (PPK) is conserved in nearly all bacteria and is responsible for polyP formation, which is associated with bacterial pathogenicity and antibiotic resistance. In this study, ppk1-deficient (Δppk1::Apr), ppk1-complemented (Δppk1::Apr/PJL02-ppk1), and wild-type strains of A. baumannii ATCC 17978 were used to determine the influence of PPK1 on A. baumannii virulence and persistence mainly by polyP quantification, surface motility, biofilm formation, and bacterial persistence assays. Our work found that PPK1 is indispensable for polyP formation in vivo and that the motility of the PPK1-deficient strain was significantly impaired due to the lack of a pilus-like structure typically present compared with the complemented and wild-type strains. The deficiency of PPK1 also inhibited the biofilm formation of A. baumannii and decreased bacterial persistence under stimuli of high-concentration ampicillin (Amp) treatment, H2O2 stress, heat shock, and starvation stress. Furthermore, ppk1-deficient bacterium-infected mice showed a significantly reduced bacterial load and a decreased inflammatory response. However, complementation with PPK1 effectively rescued the impaired virulence and persistence of ppk1-deficient A. baumannii. In addition, metabonomic analysis revealed that PPK1 was associated with glycerophospholipid metabolism and fatty acid biosynthesis. Taken together, our results suggest that targeting PPK1 to control A. baumannii pathogenicity and persistence is a feasible strategy to fight this pathogen. IMPORTANCE A. baumannii was identified as a top-priority pathogen by the WHO due to its antibiotic resistance. Meanwhile, the pathogenicity of A. baumannii mediated by several vital virulence factors also cannot be ignored. Here, the role of PPK1 in A. baumannii was also explored. We found that the motility ability and biofilm formation of a PPK1-deficient strain were significantly impaired. Furthermore, PPK1 was essential for its persistence maintenance to resist stimuli of high-concentration Amp treatment, H2O2 stress, heat shock, and starvation stress. Metabonomic analysis revealed that PPK1 was associated with glycerophospholipid metabolism and fatty acid biosynthesis. In addition, ppk1-deficient bacterium-infected mice showed significantly reduced bacterial loads and a decreased inflammatory responses in vivo. Together, our results suggest that PPK1 is vital for A. baumannii pathogenicity and persistence.

Project description:Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism. Abbreviations: AXIN1: axin 1; 3-MA: 3-methyladenine; 5-FU: 5-fluorouracil; AA mutant: double alanine mutant (S192A, T284A) of PRKAG1; ACACA: acetyl-CoA carboxylase alpha; AICAR: 5-Aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ATM: ataxia telangiectasia mutated; ATR: ATM serine/threonine kinase; AV: autophagic vacuole; AVd: degradative autophagic vacuole; AVi: initial autophagic vacuole; BECN1: beclin 1; BSA: bovine serum albumin; CBS: cystathionine beta-synthase; CDK7: cyclin dependent kinase 7; CDKN1A: cyclin dependent kinase inhibitor 1A; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S transferase; H2AX/H2AFX: H2A.X variant histone; HBSS: Hanks balanced salt solution; IP: immunopurification; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K9: mitogen-activated protein kinase kinase kinase 9; mRFP: monomeric red fluorescent protein; mCh: mCherry; MCM7: minichromosome maintenance complex component 7; MTORC1: mechanistic target of rapamycin kinase complex 1; NHEJ: non-homologous end joining; NRBP2: nuclear receptor binding protein 2; NTC: non-targeting control; NUAK1: NUAK family kinase 1; PBS: phosphate-buffered saline; PIK3AP1: phosphoinositide-3-kinase adaptor protein 1; PIK3CA: phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha; PIKK: phosphatidylinositol 3-kinase-related kinase; PRKAA: protein kinase AMP-activated catalytic subunit alpha; PRKAB: protein kinase AMP-activated non-catalytic subunit beta; PRKAG: protein kinase AMP-activated non-catalytic subunit gamma; PRKDC: protein kinase, DNA-activated, catalytic subunit; RLuc: Renilla luciferase; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TP53: tumor protein p53; TSKS: testis specific serine kinase substrate; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

Dataset Information

Polyphosphate:AMP phosphotransferase as a polyphosphate-dependent nucleoside monophosphate kinase in Acinetobacter johnsonii 210A.

Publications

Polyphosphate:AMP phosphotransferase as a polyphosphate-dependent nucleoside monophosphate kinase in Acinetobacter johnsonii 210A.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets