29 research outputs found
The Role of the Novel Exopolyphosphatase MT0516 in Mycobacterium tuberculosis Drug Tolerance and Persistence
Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions
Rapid accumulation of polyphosphate in extraradical hyphae of an arbulcular mycorrhizal fungus as revealed by histochemistry and a polyphosphate kinase/luciferase system
The rate of polyphosphate accumulation in extraradical hyphae of an arbuscular mycorrhizal fungus was investigated by conventional histochemistry and a new enzymatic method using a bacterial enzyme, polyphosphate kinase. Marigold (Tagetes patula cv. Bonanza Orange) was inoculated with Archaeospora leptoticha and grown under P-deficient conditions. Extraradical hyphae were harvested at 0, 1, 3 and 24 h after 1 mm P-application. PolyP levels were assessed by both metachromasy of Toluidine blue O and polyphosphate kinase which converted polyP to ATP followed by the ATP-luciferase assay. Percentage of hyphae with metachromatic granules was increased from 25 to 44% from 0 to 1 h, and a maximum of 50% was reach by 3 h. Polyphosphate content was doubled from 1 to 3 h after P-application (4.8–10.0 mol as Pi mg−1 protein) at a rate of 46.4 ± 15.1 nmol min−1 mg−1. The rate of polyphosphate accumulation in the hyphae was surprisingly rapid as those of polyphosphate-hyper accumulating microorganisms. The enzymatic method employed in the present study allows highly specific and sensitive assessment of polyphosphate in the mycorrhizal system.Ezawa, Tatsuhiro ; Cavagnaro, Timothy R. ; Smith, Sally E. ; Smith, F. Andrew ; Ohtomo, Ry
Effect of purine limitation caused by an amidophosphoribosyl transferase (purF) mutation on polyphosphate kinase 1 (ppk1) gene expression
Structure determination of selenomethionyl S-adenosylhomocysteine hydrolase using data at a single wavelength
Accumulation and enhanced cycling of polyphosphate by Sargasso Sea plankton in response to low phosphorus
Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography
A polyphosphate kinase 1 (ppk1) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects.
Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the beta-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions