Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal Journal Article uri icon



  • ABSTRACT; ; 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 β-; 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.;

publication date

  • October 1, 2002

has restriction

  • green

Date in CU Experts

  • May 12, 2014 9:42 AM

Full Author List

  • McMahon KD; Dojka MA; Pace NR; Jenkins D; Keasling JD

author count

  • 5

Other Profiles

International Standard Serial Number (ISSN)

  • 0099-2240

Electronic International Standard Serial Number (EISSN)

  • 1098-5336

Additional Document Info

start page

  • 4971

end page

  • 4978


  • 68


  • 10