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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

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

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"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.

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Enhanced biological phosphorus removal (EBPR) processes for wastewater have been widely used over the past several decades because of their low cost compared with chemical treatment methods. EBPR is characterized by the use of polyphosphate-accumulating organisms (PAOs) capable of accumulating polyphosphate as an intracellular storage compound using oxygen as an electron acceptor. However, EBPR processes combined with biological nitrogen removal processes have a drawback in that the organic substrate used is a limiting factor for the activities of both PAOs and denitrifying bacteria. Recently, the occurrence of DNPAOs capable of utilizing nitrite or nitrate instead of oxygen as an electron acceptor for phosphorus uptake has been reported (2, 10, 13). The use of DNPAOs in biological nutrient removal processes is advantageous because identical organic substrates such as acetate can be efficiently used as the energy source for both nitrogen and phosphorus removals. Other advantages associated with DNPAO activity include a reduction in surplus sludge production (14).

However, none of the DNPAOs and PAOs that have been isolated have been able to assimilate acetate and synthesize poly-hydroxy-alcanoates (PHA) anaerobically, concomitant with phosphorus release, which is different from EBPR sludge behavior (25). Thus, the biochemistry and genetics of PAOs remain to be elucidated. Recently, molecular techniques such as cloning and sequencing of rRNA genes have revealed that an organism related to the genus Rhodocyclus and provisionally named "Candidatus Accumulibacter phosphatis" (7) is abundant in acetate-fed laboratory-scale reactors under cyclic anaerobic/aerobic or anaerobic/anoxic conditions, particularly when efficient phosphorus removal is achieved (2, 31).

However, rRNA-targeted molecular analyses are unable to reveal the ecophysiological traits of "Ca. Accumulibacter phosphatis" as DNPAOs in an anaerobic/anoxic sequencing batch reactor (SBR). Therefore, metabolic analyses targeting "Ca. Accumulibacter phosphatis" were performed using fluorescence in situ hybridization (FISH) combined with microautoradiography (11, 12), polyphosphate staining, or PHA staining (5, 7, 15, 17, 31, 32). These studies revealed that "Ca. Accumulibacter phosphatis" could take up acetate, form PHA anaerobically, and accumulate polyphosphate under aerobic or anoxic condition.

However, it was not revealed by these metabolic analyses whether "Ca. Accumulibacter phosphatis" can denitrify or not. An attempt to obtain a better understanding of the population dynamics of denitrifying bacteria in anaerobic/aerobic SBR was made in our previous study (29), but the investigation of the denitrification ability of "Ca. Accumulibacter phosphatis" was not successful.

To clarify the denitrification ability of "Ca. Accumulibacter phosphatis," it is necessary to separate "Ca. Accumulibacter phosphatis" from microbial consortia. Several techniques such as flow cytometry (30) and density gradient centrifugation (8, 23) can be used to separate microbial cells. Recently, cells in complex microbial consortia stained by FISH with rRNA-targeted oligonucleotide probes have been successfully sorted by flow cytometry (24, 30).

On the other hand, PCR primer sets specific for functional genes involved in denitrification, namely, nirS and nirK, which encode cytochrome cd₁- and copper-containing nitrite reductases, respectively (4, 6), and nosZ, which encodes nitrous oxide reductase (21), have been developed.

In this study, bacteria hybridized with specific probes for "Ca. Accumulibacter phosphatis" were highly concentrated from sludge samples by flow cytometric sorting, and molecular analysis of the nitrite reductase gene was subsequently performed to obtain a better understanding of the ability of "Ca. Accumulibacter phosphatis" to denitrify.

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SBR operation.
An SBR with a 2-liter working volume was operated using the anaerobic/aerobic cycle at room temperature. A separable flask (cylinder type with a flat bottom and 2,000-ml capacity; Sibata, Japan) capped with an acrylic cover was used as the reactor. Seed sludge was originally taken from a local municipal wastewater treatment plant (Ariake WWTP, Tokyo, Japan) where an anaerobic/anoxic/oxic process was adopted. Then, this seed sludge was cultivated in a laboratory-scale reactor under anaerobic/aerobic cycling for 1 year. The reactor was operated with an 8-h cycle that consisted of a 15-min filling time, a 90-min anaerobic condition, a 285-min aerobic condition, a 65-min settling time, and a 25-min withdrawing time. Since both influent and effluent volumes were 1 liter, 16 h of hydraulic retention was maintained. Once per day, 250 ml of mixed liquor was removed at the end of the aerobic condition so that the 8 days of sludge retention was maintained. The SBR was mixed constantly with a magnetic stirrer (300 rpm) in anaerobic and aerobic phases. Synthetic wastewater of the following composition was used as the feeding solution: 512 mg of CH₃COONa, 99.5 mg of KH₂PO₄, 90 mg of MgSO₄, 107 mg of NH₄Cl, 14 mg of CaCl₂·2H₂O, 1 mg of yeast extract, and 0.3 ml of nutrient solution (26) per liter. Soluble phosphate, nitrate/nitrite, and total organic carbon and pH were measured according to Tsuneda et al. (29). Acetate has been commonly used for enrichment of PAOs responsible for an enhanced biological phosphorus removal in wastewater treatment plants (25). An acetate-fed reactor containing phosphorus is basic and conventional to study enhanced biological phosphorus removal.

FISH and DNA staining.
After the reactor was operated for at least 6 months with more than 540 cycles, about 11 ml of sludge was withdrawn from the SBR and fixed in 4% paraformaldehyde-phosphate-buffered saline (PBS) solution (137 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄, pH 7.2) for 2 h at 4°C. The fixed sludge samples were washed twice in PBS, resuspended in PBS-ethanol solution (1:1, vol/vol), and then stored at –20°C. The following oligonucleotide probes specific for "Ca. Accumulibacter phosphatis" were used for hybridization: PAO462 (5'-CCGTCATCTACWCAGGGTATTAAC-3'), PAO651 (5'-CCCTCTGCCAAACTCCAG-3'), and PAO846 (5'-GTTAGCTACGGCACTAAAAGG-3') (5). These probes were labeled with 6-carboxyfluorescein at the 5' end. After centrifugation and discarding of the supernatant, approximately 10⁸ cells were resuspended by vortexing and sonication in a hybridization buffer containing 0.9 M NaCl, 20 mM Tris-HCl, 0.01% sodium dodecyl sulfate, 35% formamide (pH 7.2), and PAO462, PAO651, and PAO846 (2 ng/µl, each), and the suspension was incubated for 3 h at 46°C (30). Then, the cells were centrifuged and washed in a hybridization buffer for 15 min at 46°C. The supernatant was discarded, and 200 µl of PBS and 100 µl of DAPI (4',6'-diamidino-2-phenylindole; Molecular Probes, Eugene, OR) solution (50 µg/ml) was added to the cells. The cells were incubated for 10 min at room temperature to stain DNA. Afterwards, the cells were resuspended in PBS and stored at 4°C for subsequent flow cytometric sorting.

Dispersion of cells for single-cell sorting.
First, cells were dispersed by ultrasonic treatment (Sonifier II, model 150; Branson, Danbury, CT) and then filtered successively through a gauze filter (pore size, 35 µm; Falcon-type 2235 tube with strainer cap; Becton Dickinson, Franklin Lakes, NJ) and filter paper (pore size, 8 µm; Millipore, Billerica, MA) to remove large cell aggregates. The samples obtained were diluted and sonicated immediately before flow cytometric analysis and sorting.

Flow cytometric analysis and cell sorting.
Flow cytometric analysis and cell sorting were performed using a FACS Aria (Becton Dickinson) instrument equipped with 488-nm, 633-nm, and 407-nm lasers. The 488-nm laser was used for measuring forward scatter, side scatter (488-nm band-pass filter for detection), and probe-conferred fluorescence (530-nm band-pass filter). The 407-nm laser was used for measuring DAPI-DNA fluorescence (450-nm band-pass filter). Data analysis and instrument control were carried out using FACS DiVa software (Becton Dickinson). All analyses were performed at low-flow-rate settings (about 10 µl/min) so that cells would pass through the laser beam in a single-file stream. Data were collected from 10,000 cells per sample.

Cell sorting was carried out in a single-cell mode to obtain the highest purity. For the sorting, autoclaved sodium chloride solution (0.1%) was used as the sheath fluid so as not to affect the subsequent microscopic and molecular analyses (30). Before the sorting, the internal sheath path was decontaminated with ethanol. Sorted cells were collected in sterile 1.5-ml tubes.

Cell counting.
The FISH/DAPI double-stained cells (before and after sorting) were sonicated (Sonifier II, model 150; Branson) and placed on microscopy slides. The relative abundance of hybridized cells in the sludge samples before and after sorting was estimated as the ratio of the number of hybridized cells to the number of DAPI-stained cells using epifluorescence microscopy (Zeiss Axioskop 2 plus; Hallbergmoos, Germany). For this cell counting, 10 images for unsorted cells (1,338 cells) and 24 images for sorted cells (1,086 cells) were arbitrarily selected, and cells were counted manually.

Detection of nirS and nirK genes.
DNAs were extracted from unsorted and sorted samples (designated as samples Ba and Bd, respectively) using Isoplant (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. For sample Bd, a coprecipitant (Ethachinmate; Nippon Gene) was used to recover DNA successfully during the ethanol precipitation step. The fragments of nirS genes (approximately 890 bp) were amplified using the primers nirS1F [5'-CCTA(C/T)TGGCCGCC(A/G)CA(A/G)T-3'] and nirS6R [5'-CGTTGAACTT(A/G)CCGGT-3'] (4). The fragments of nirK genes (approximately 514 bp) were amplified using the primers nirK1F [5'-GG(A/C)ATGGT(G/T)CC(C/G)TGGCA-3'] and nirK5R [5'-GCCTCGATCAG(A/G)TT(A/G)TGG-3'] (4). PCR amplification was conducted in an automated thermal cycler (iCycler; Bio-Rad Laboratories, Hercules, CA) using the following protocol: initial denaturation for 5 min at 94°C and 30 cycles (for nirS from sample Ba) or 35 cycles (for the other reactions) of denaturation for 1 min at 94°C, annealing for 1 min at 54°C, and extension for 1 min at 72°C, followed by a final extension for 7 min at 72°C. The PCR mixture had a final volume of 50 µl, which contained 5 µl of 10x PCR buffer (containing 20 mM Mg²⁺), 4 µl of deoxynucleoside triphosphate mixture (2.5 mM concentration of each), 1.25 U of TaKaRa Ex Taq polymerase (Takara Bio, Otsu, Japan), a 0.5 mM concentration of each primer, and 0.5 µl of purified DNA from sample Ba or 7 µl from sample Bd. PCR products were detected by agarose gel electrophoresis with ethidium bromide staining. Bands were visualized by UV excitation.

Cloning of PCR products.
nirS PCR products of the expected size (890 bp) were excised from a gel and purified using a Wizard SV gel and a PCR cleanup system (Promega, Madison, WI). Purified PCR products were cloned using a QIAGEN PCR Cloning Plus kit (Valencia, CA), and inserts were amplified using PCR mix (Insert Check-Ready; Toyobo, Osaka, Japan) according to Tsuneda et al. (29).

RFLP analysis of nirS clones.
Restriction fragment length polymorphism (RFLP) analysis was conducted to screen PCR products from clones. The products were digested in two separate reactions using the restriction enzymes HhaI and MspI at 37°C overnight. The digested products were electrophoresed on 3.2% (wt/vol) Metaphor agarose gels (Takara Bio) in freshly prepared, chilled 1x Tris-borate-EDTA buffer for approximately 90 min at 50 V. After electrophoresis, the gels were stained with SYBR Gold or SYBR Green I (Wako Pure Chemical Industries, Osaka, Japan) for 30 min and then visualized on a UV transilluminator. The RFLP patterns were compared visually, and clones showing identical RFLP patterns were grouped into operational taxonomic units (OTUs).

Sequencing of clones and phylogenetic analysis.
Representatives of each OTU were selected for sequencing. When an OTU was composed of two or more clones, at least two clones in each OTU were sequenced. DNA fragments were sequenced with nirS1F and nirS6R using a Big Dye Terminator (version 3.1) cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems) according to the manufacturer's instructions. The sequences and some nirS sequence fragments obtained from the DNA Data Bank of Japan (DDBJ) database were aligned using the CLUSTAL W program (28), and a phylogenetic tree was constructed by the neighbor-joining method (20).

Nucleotide sequence accession numbers.
The sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers AB208081 to AB208105.

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EBPR reactor.
An anaerobic/aerobic reactor was operated over 6 months. Good phosphate removal was observed with rapid carbon consumption and phosphorus release under anaerobic conditions and phosphorus overaccumulation under the subsequent aerobic conditions (Fig. 1). The percentage of phosphorus weight in the sludge at the end of the aerobic phase was 21%. This sludge was taken for subsequent molecular analysis and flow cytometry.

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FIG. 1. Typical profiles of soluble P, N, and C concentrations and pH during a cycle of an anaerobic/aerobic SBR. , phosphate (mg P per liter); , nitrate (mg N per liter); •, nitrite (mg N per liter); , total organic carbon (TOC) (mg C per liter); , pH.

Cell sorting following FISH.
The sludge sample was hybridized with specific probes for "Ca. Accumulibacter phosphatis" and stained with DAPI. Probe-positive cells were 39% of DAPI-positive cells in the sample. Even after the successive filtration of stained cells using 35- and 8-µm-pore-size filters, the intensities of their fluorescence signals were almost the same as those before filtration. Thus, this sample was used for the flow sorting of "Ca. Accumulibacter phosphatis" cells with a high purity. Prior to the flow sorting, region 1 (Fig. 2A) in bivariate plots of side scatter versus forward scatter was set to separate cells from noise. Next, in bivariate plots of probe-conferred fluorescence versus DAPI-DNA fluorescence in which events of population 1 were only plotted, two groups were detected (Fig. 2B). Region 2 was set to distinguish probe-positive cells from other cells, and 3.4 x 10⁵ objects were sorted from region 2. The direct cell counting using epifluorescence microscopy revealed that the purity of sorted cells that hybridized with the probes was about 97% for DAPI-stained cells (Fig. 3). Among the sorted cells were some few relatively small, round cells that did not hybridize with the probes. This contamination was probably caused by their binding to hybridized cells in the droplets.

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FIG. 2. Flow cytometric analysis and sorting of bacteria hybridized with specific probes for "Ca. Accumulibacter phosphatis" from sludge taken from the SBR under anaerobic/aerobic conditions. (A) Region 1 was set to separate cells from noise. (B) Only the cells dotted within region 1 were plotted. Region 2 was defined in a bivariate dot plot of probe fluorescence versus DAPI-DNA fluorescence and was used for cell sorting.

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FIG. 3. Cells sorted from region 2 in Fig. 2B. Probe-conferred fluorescence (a) and DAPI-DNA fluorescence (b) are shown. Panel c shows a composite image of micrographs from panels a and b. Cells indicated by arrows did not hybridize with the probes.

PCR amplification of nirK and nirS genes.
nirS gene fragments were successfully amplified by PCR from both unsorted and sorted sludge samples (samples Ba and Bd, respectively). These results indicate that "Ca. Accumulibacter phosphatis" may have the nirS gene and an ability to reduce nitrite. nirK gene fragments were successfully amplified from sample Ba, though nonspecific bands were also detected by gel electrophoresis. On the other hand, specific nirK gene fragments were not detected from sample Bd, and only thin smears were observed (Fig. 4). From these results, it seems likely that "Ca. Accumulibacter phosphatis" lacks the nirK gene.

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FIG. 4. PCR amplification of nirS (890 bp) and nirK (514 bp) DNAs extracted from sorted cells. Lanes 1 and 4, 100-bp DNA ladder markers; lane 2, nirS PCR product; lane 5, nirK PCR product; lanes 3 and 6, negative controls.

Classification and sequencing of nirS clones.
Since no specific nirK PCR products were detected, only nirS PCR products were cloned. Ninety-one and 65 clones were produced from samples Ba and Bd, respectively. These clones were classified by RFLP analysis and found to fall into 18 OTUs for sample Ba and 14 OTUs for sample Bd. Forty-three clones for sample Ba and 12 clones for sample Bd including representatives of all different RFLP patterns were sequenced. The sequences of nine clones for sample Ba and five clones for sample Bd could not be determined, probably because of the contamination of some other clones or the lack of nirS data in the DDBJ database using a BLAST search. We found that some OTUs had similar sequences (>98%), so they were treated as one OTU. As a result, OTUs in sample Ba decreased to seven consisting of 82 clones, and OTUs in sample Bd decreased to four consisting of 51 clones.

Figure 5 shows the distribution of RFLP patterns of the nirS clones obtained from each sample. In clone library Ba, clone Ba8 was most dominant (62% of the total number of clones). In clone library Bd, clone Bd69 was most dominant (59% of the total number of clones). The dendrogram of nucleotides in Fig. 6 shows the relationships between the cloned sequences and the nirS sequences in the DDBJ database. The nirN sequence (accession no. D84475) from Pseudomonas aeruginosa was used as an outgroup for the phylogenetic distance analysis of the nirS sequences. The sequence data and phylogenetic analysis revealed that representative of clones Ba120 (two clones) and Bd69 (five clones) had considerably high degrees of similarity with each other. The similarities determined by pairwise comparisons of these sequences were higher than 99%. Hence, these clones were considered to be identical. Clone Bd30 had 98% similarity to the nirS gene of Ralstonia eutropha. However, all the clones except clone Bd30 had less than 90% nucleotide sequence similarity to nirS genes of known denitrifying bacteria in the database.

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FIG. 5. Distribution of RFLP patterns of nirS clones obtained from DNAs in sludge sample before (a) and after (b) sorting. Clones Ba120 and Bd69 have the same RFLP patterns.

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FIG. 6. Neighbor-joining analysis of partial nucleotide sequences from cloned nirS PCR products. Evolutionary distance and parsimonious analyses were carried out using CLUSTAL W with 1,000 bootstrap resamplings. The scale indicates 0.1 nucleotide substitution per nucleotide position. The tree was rooted with the nirN sequence of P. aeruginosa as an outgroup. Clone Ba was obtained from the sludge sample before sorting. Clone Bd was obtained from the sorted cells. Clone 28-8 is from an anaerobic/aerobic SBR (29). Sequences of Sludge Oz, Jazz, and Phrap included in the tree were metagenomic sequences of nirS binned as "Ca. Accumulibacter phosphatis" (16). GOI, gene object identifier.

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In this study, the flow cytometric sorting of cells hybridized with specific probes for "Ca. Accumulibacter phosphatis" was performed, followed by molecular analysis of nitrite reductase genes (nirS and nirK), which are functional genes involved in denitrification. Such flow cytometric sorting following FISH and the subsequent molecular analysis have been reported previously (24, 30). In these studies, 16S rDNAs of sorted cells were analyzed, but functional genes were not analyzed.

In our study, FISH-positive cells were clearly discriminated from the other cells on dot plots of flow cytometric analysis (Fig. 2). This successful separation might be attributed to (i) the enhancement in the signal intensity of hybridized cells using three probes specific for "Ca. Accumulibacter phosphatis," (ii) the removal of aggregated cells by filtration, and (iii) the sonication of cells immediately before flow cytometric analysis. In previous studies (3, 22, 24), catalyzed reporter deposition-FISH, which could improve the sensitivity of FISH compared with probes with a single fluorochrome, was adopted for flow cytometric analysis of marine bacterioplanktons. In our study, however, the signal intensity was sufficiently strong to analyze with a flow cytometer without conducting catalyzed reporter deposition-FISH.

Since "Ca. Accumulibacter phosphatis" was present in samples Ba and Bd, nirS clones derived from "Ca. Accumulibacter phosphatis" should be present in both clone libraries Ba and Bd. Clones identical to clone Ba120 or Bd69 existed in both clone libraries Ba and Bd. However, all the other clones were included in only one of the libraries. Moreover, when the proportion of "Ca. Accumulibacter phosphatis" cells increased from 39% (sample Ba, before sorting) to 97% (sample Bd, after sorting), the proportion of clones identical to Ba120 or Bd69 to all nirS clones increased from 2% (sample Ba) to 59% (sample Bd). These observations indicate that the nirS sequences of these clones were that of "Ca. Accumulibacter phosphatis."

Unexpectedly, the proportions of this nirS clone group to all clones were much lower than the proportions of "Ca. Accumulibacter phosphatis" cells determined by microscopic analysis in samples Ba and Bd. This phenomenon was also reported by Sekar et al. (24).

The first possible explanation of this discrepancy is the effect of bias caused by PCR (9, 27). Possibly, the primers used in the present study were not perfectly suitable for the PCR amplification of the nirS gene of "Ca. Accumulibacter phosphatis," and they tended to amplify some other nirS genes. In sample Bd (after sorting), few microorganisms other than "Ca. Accumulibacter phosphatis" were included (3%). There might have existed some nirS clones derived from these microorganisms if they were easily amplified under the PCR conditions used in this study. Moreover, there is a possibility that none of the observed clones is actually from "Ca. Accumulibacter phosphatis" but that all are from the 3% contaminants in the sorted sample. To confirm that obtained clones include nirS sequences of "Ca. Accumulibacter phosphatis," additional experiments were performed. Samples were artificially prepared. One consisted of 97% nirS-positive cells (Marinobacter) and 3% nirS-negative cells (Alcaligenes xylosoxidans); the other consisted of 3% nirS-positive and 97% nirS-negative cells. The total number of cells of each sample was adjusted to be identical, and then DNA extraction and PCR amplification were conducted under the same conditions as we used for sorted cells. As a result, PCR products from 97% nirS-positive cells were detected, but PCR products from 3% nirS-positive cells were not detected (Fig. 7). These results support the finding that DNAs extracted from "Ca. Accumulibacter phosphatis" included in sorted cells were actually amplified and cloned.

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FIG. 7. PCR amplification of nirS (890 bp) DNAs. Lane 1, PCR product from the sample consisting of 97% nirS-positive cells and 3% nirS-negative cells; lane 2, PCR product from the sample consisting of 3% nirS-positive cells and 97% nirS-negative cells; lane 3, negative control; lane 4, 200-bp DNA ladder markers. Marinobacter was used as nirS-positive cells, and A. xylosoxidans was used for nirS-negative cells.

The second possible explanation may be the existence of another strain of "Ca. Accumulibacter phosphatis." In clone library Bd, the second dominant clone represents 27% of the total clone population. It can be expected that this clone is derived from a "Ca. Accumulibacter phosphatis" whose strain is different from the strain having the sequence of the most dominant clone. The total of the most dominant and the second-most dominant clones in the clone library Bd is 86%. This ratio is comparable to the ratio determined by microscopic analysis. Recently, metagenomic analysis of "Ca. Accumulibacter phosphatis"-dominated sludge communities was performed (16). According to our search of the metagenomic data, two types of nirS genes classified as belonging to "Ca. Accumulibacter phosphatis" were found. One shows high similarity to clone Bd69 (85%) which is the most dominant in the clone library of sorted cells; the other shows high similarity to clone Bd86 (86%), which is second-most dominant clone in the clone library of sorted cells. These observations may support the second explanation.

In our previous study (29), the cloning of the nirS gene fragment with the same primer set used in the present study was also performed for similar sludge samples without flow cytometric sorting. As a result, one of the nirS clones occupied approximately 70% of all nirS clones. As shown in Fig. 6, this clone (28-8; accession number AB185906) is identical to clone Ba8 which accounted for about 62% of clone library Ba. However, the "Ca. Accumulibacter phosphatis" putative clone was not found in the clone library in our previous study (29). This result is not in conflict with the present study because the "Ca. Accumulibacter phosphatis" putative clone represented only 2% of clone library Ba.

The combination of flow sorting and molecular analysis described in this study could be adapted to other uncultured bacteria. In addition, single-cell amplification of total DNA through multiple displacement amplification is a powerful tool for analyzing genes in uncultured bacterial cells especially from small-scale samples (1, 19). Thus, the combined use of multiple displacement amplification will advance our methods and contribute to revealing characteristics of uncultured bacteria in natural environments.

 
* 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

Published ahead of print on 18 May 2007.

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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.

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