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Applied and Environmental Microbiology, August 2007, p. 5331-5337, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.00175-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Diversity of Nitrite Reductase Genes in "Candidatus Accumulibacter phosphatis"-Dominated Cultures Enriched by Flow-Cytometric Sorting{triangledown}

Ryuki Miyauchi, Kazuma Oki, Yoshiteru Aoi, and Satoshi Tsuneda*

Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan

Received 24 January 2007/ Accepted 11 May 2007

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.

* Corresponding author. Mailing address: Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan. Phone: 81 3 5286 3210. Fax: 81 3 3209 3680. E-mail: stsuneda{at}waseda.jp

{triangledown} Published ahead of print on 18 May 2007.

Applied and Environmental Microbiology, August 2007, p. 5331-5337, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.00175-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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