Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal

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 ␤-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.

Phosphorus sequestration in the form of polyphosphate by microbial symbionts in marine sponges

“…RT of RNA was performed using RevertAid Reverse Transcriptase (Thermo Scientific) with random primers and following the manufacturer's protocol. The ppk genes were amplified from genomic DNA and cDNA using primers designed by McMahon et al (39), with the following conditions: 94°C for 5 min; 35 cycles of 94°C for 1 min, 50°C for 45 s, and 72°C for 2 min; and a final extension at 72°C for 12 min. RNA samples without the RT step were included as a PCR template to check for residual DNA in the RNA samples.…”

Emticicia oligotrophica gen. nov., sp. nov., a new member of the family ‘Flexibacteraceae’, phylum Bacteroidetes

“…T is a member of the phylum Bacteroidetes showing closest similarity (94?0 %) with an uncultured bacterial clone, HP1A92, which was a representative sequence of a sludge microbial community (McMahon et al, 2002). Among the culturable bacteria, it showed closest similarity to Leadbetterella byssophila 4M15 T (87?8 %).…”

Comparative genomics of two ‘Candidatus Accumulibacter’ clades performing biological phosphorus removal

“…In comparison, the Accumulibacter Clade IIA UW-1 chromosome and three plasmids totaled 5.3 Mbp of sequence and had 4792 predicted genes. We also identified a ppk1 homolog in the largest scaffold (Scaffold01135) sharing 98% nucleotide identity with a Clade IA ppk1 identified previously (GenBank AF502200.1; McMahon et al, 2002). As this locus was originally used to define the Accumulibacter clade phylogeny, the presence of a Clade IA ppk1 homolog within this putative Clade IA scaffold provides confidence that the scaffolds were assembled and binned properly.…”

“…An archetypal EBPR organism is readily enriched with acetate as a primary carbon source in laboratory-scale sequencing batch reactors McMahon et al, 2010), and is named 'Candidatus Accumulibacter phosphatis' (Hesselmann et al, 1999) (henceforth referred to as Accumulibacter). The Accumulibacter lineage is phylogenetically subdivided into two types (Types I and II) based on comparative sequence analysis of the gene encoding polyphosphate kinase (ppk1) (McMahon et al, 2002). Each type is comprised of several coherent clades exhibiting unique distribution patterns in wastewater treatment systems (He et al, 2007;He et al, 2010a) and natural aquatic environments .…”