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Applied and Environmental Microbiology, October 2002, p. 4971-4978, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4971-4978.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal{dagger}

Katherine D. McMahon,1 Michael A. Dojka,2,{ddagger} Norman R. Pace,2,§ David Jenkins,1 and Jay D. Keasling3*

Departments of Civil and Environmental Engineering,1 Plant and Microbial Biology,2 Chemical Engineering, University of California at Berkeley, Berkeley, California 94720-14603

Received 24 April 2002/ Accepted 24 July 2002

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 ß-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 Mg2+, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.

* Corresponding author. Mailing address: Department of Chemical Engineering, University of California at Berkeley, Berkeley, CA 94720-1460. Phone: (510) 642-4862. Fax: (510) 643-1228. E-mail: keasling{at}socrates.berkeley.edu.

{dagger} This work is dedicated to the memory of our friend and colleague, Michael Anthony Dojka, Jr.

{ddagger} Deceased.

§ Present address: Department of Molecular Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347.

Applied and Environmental Microbiology, October 2002, p. 4971-4978, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4971-4978.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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