Polyphosphate kinase from activated sludge performing enhanced biological phosphorus removal.
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.
D000693:Anaerobiosis; D020562:Betaproteobacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001673:Biodegradation, Environmental; D019149:Bioreactors; D017404:In Situ Hybridization, Fluorescence; D010710:Phosphates / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000302:isolation & purification* / Q000378:metabolism; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012722:Sewage / Q000382:microbiology*; D018505:Waste Management
D010710:Phosphates; D012336:RNA, Ribosomal, 16S; D012722:Sewage; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase