Diversity of Nitrite Reductase Genes in “ Candidatus Accumulibacter phosphatis”-Dominated Cultures Enriched by Flow-Cytometric Sorting

Abstract

"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.

Analysis of the Fine-Scale Population Structure of “ Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge, Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting

“…Accumulibacter" lineages. Flow cytometric cell sorting has been used in environmental microbiology because it is possible to analyze complex microbial cell populations rapidly on a single-cell basis (35,38,41,46). However, our flow cytometric cell sorting of the four "Ca.…”

“…FISH and fluorescence-activated cell sorting (FACS) were carried out with modifications of methods described previously (35,42,43,46). Briefly, sludge samples collected from the SBR at the end of the anaerobic phase were fixed in the same way as described above for FISH experiments.…”

“…For example, Miyauchi et al (35) sorted PAOmix probe-labeled "Ca. Accumulibacter" cells from EBPR sludge and analyzed their nitrite reductase gene (nirS) diversity.…”

Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

“…In this analysis, we used a flow cytometry method to determine relative cellular polyP accumulation in different populations of human bone marrow mononuclear cells. Several authors have described similar approaches for detecting polyP in bacteria populations [34][35][36][37] but, to our knowledge, this is the first time that this technique has been applied in human cells to detect polyP.…”

What Flow Cytometry can Tell Us About Marine Micro-Organisms – Current Status and Future Applications

“…The combination of FCM and FISH has been successfully applied to describe microbial populations dispersed in a liquid suspension derived from different media (Lim et al, 1993;Joachimsthal et al, 2004;Rigottier-Gois et al, 2003;Barc et al, 2004;Lange et al, 1997;Wallner et al, 1993 and1995;Miyauchi et al, 2007).…”