Molecular Evidence for Novel Planctomycete Diversity in a Municipal Wastewater Treatment Plant

Abstract

We examined anoxic and aerobic basins and an anaerobic digestor of a municipal wastewater treatment plant for the presence of novel planctomycete-like diversity. Three 16S rRNA gene libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for Planctomycetes. Phylogenetic analysis of 234 16S rRNA gene sequences defined 110 operational taxonomic units. The majority of these sequences clustered with the four known genera, Pirellula (32%), Planctomyces (18.4%), Gemmata (3.8%), and Isosphaera (0.4%). More interestingly, 42.3% of the sequences appeared to define two distantly separated monophyletic groups. The first group, represented by 35.5% of the sequences, was related to the Planctomyces group and branched as a monophyletic cluster. It exhibited between 11.9 and 20.3% 16S rRNA gene sequence dissimilarity in comparisons with cultivated planctomycetes. The second group, represented by 6.8% of the sequences, was deeply rooted within the Planctomycetales tree. It was distantly related to the anammox sequences (level of dissimilarity, 20.3 to 24.4%) and was a monophyletic cluster. The retrieved sequences extended the intralineage phylogenetic depth of the Plantomycetales from 23 to 30.6%. The lineages described here may have a broad diversity of undiscovered biochemical and metabolic novelty. We developed a new 16S rRNA-targeted oligonucleotide probe and localized members of one of the phylogenetic groups using the fluorescent in situ hybridization technique. Our results indicate that activated sludge contains very diverse representatives of this group, which grow under aerobic and anoxic conditions and even under anaerobic conditions. The majority of species in this group remain poorly characterized.

Comparative sequence analysis of environmentally retrieved 16S rRNA gene sequences (16S rDNA) has become the “gold standard” for cultivation-independent assessment of bacterial diversity in natural and engineered systems (2). This method is based on identification of 16S rDNA genes in genomic DNA directly extracted from the environment, typically via PCR amplification, cloning, and sequencing (2, 26). To date, there are more than 62,000 16S rDNA sequences available from public databases. Thirty-seven division-level lineages have been detected (24), almost one-third of which are not represented by cultured microorganisms. This provides a high-resolution framework for assignment of novel sequences obtained in 16S rDNA libraries constructed from environmental diversity surveys.

The order Planctomycetales represents one of the main lines of descent of the domain Bacteria as defined on the basis of 16S rDNA sequence analysis (27, 32, 37, 43). Our knowledge of this group is limited because of the relatively few species that have been obtained in pure culture. All planctomycetes were originally isolated from aquatic habitats as diverse as acid bogs and sewage treatment plants. Four cultured genera have been described to date, Planctomyces, Pirellula, Gemmata, and Isosphaera. All of these organisms are aerobic chemoheterotrophs. Gemmata and Isosphaera were described on the basis of a single species each, Gemmata obscuriglobus and Isosphaera pallida (14, 17). Membership in the planctomycete group has been extended not only to chemoorganotrophs and obligate or facultative aerobes but also to obligate anaerobes, autotrophs, and phototrophs (15, 29). Members of two genera containing anaerobic ammonia-oxidizing autotrophs with Candidatus status, “Candidatus Brocadia anammoxidans” and “Candidatus Kuenenia stuttgartiensis” (34, 39), are examples of this extension. Considering the great phylogenetic depth of the order Planctomycetales (10), which is equivalent to the intralineage phylogenetic depth of other main lines of bacterial descent (e.g., the Proteobacteria), it is not surprising that the majority of the planctomycete strains have not been recovered yet from the environment, and greater metabolic versatility of planctomycetes has been assumed. In addition, in several studies workers have described clones of planctomycetes from 16S rDNA clone libraries prepared from different environments, such as marine organic aggregates, soil, anoxic bioreactors, and anoxic sediments (8, 18, 22, 27, 28, 44). Although several clone libraries from mainly lab-scale wastewater treatment reactors have been described (4, 6, 7, 28), only one full-scale municipal wastewater treatment plant (WWTP) has been analyzed (36). Low levels of planctomycetes, as well as some other bacterial groups, were detected in these studies for many reasons; for instance, the organisms might occur at lower levels, or their 16S rDNA might be subject to biased retrieval when universal primers are used.

In our molecular survey based on comparative sequence analysis of cloned 16S rDNA genes after amplification with Planctomycetales-specific primers, our goal was to obtain molecular evidence at a higher resolution for novel planctomycete diversity in a municipal WWTP. This work resulted in detection of novel molecular phylotypes related to the three known genera, Pirellula, Planctomyces, and Gemmata, found in aerobic, anoxic, and anaerobic digestors. More interestingly, we detected two major novel phylogenetic groups and several other minor groups for which pure-culture representatives are not known. We assert that the planctomycete group is not adequately represented by cultured species. This work enabled us, using a newly developed oligonucleotide hybridization probe and fluorescent in situ hybridization (FISH) technique, to localize in activated sludge flocs members of one of the two major groups, which represent a distinct and predominant group in our clone libraries.

MATERIALS AND METHODS

Description of the WWTP.

A large municipal WWTP in Evry, France, located 35 km south of Paris receives sewage from a 193-km sewer system. Pretreated wastewater is settled in two settling basins, and the average retention time is 2.2 h. Primary settled wastewater enters the biological basins in an anoxic zone (5,000 m³), where it is mixed with recycled biological sludge and mixed liquor. Nitrification occurs in the aerobic basin (19,000 m³). The average hydraulic retention time in the system is 17 h (21% in the anoxic zone), and the sludge retention time is 20 ± 7 days. Primary sludge and biological secondary sludge are mixed (56 and 44%, respectively) and anaerobically digested in a 7,000-m³ tank with a retention time of 39 ± 10 days.

Sludge sampling.

Activated sludge samples were collected from different locations (aerobic basin, anoxic basin, and the digestor). After three washes with phosphate-buffered saline (PBS) and centrifugation at 20,000 × g for 15 min at 4°C, aliquots (approximately 200 mg) were immediately treated for genomic DNA extraction or stored at −20°C.

Genomic DNA extraction.

Given the peculiar nature of the planctomycete cell wall, which lacks peptidoglycan, we developed an enzymatic genomic DNA extraction protocol involving proteolytic enzymes to decrease biases introduced by the chemically based protocols. A 200-mg sludge sample was diluted in 200 μl of 100 mM Tris-10 mM EDTA (pH 8) and incubated at 95°C for 10 min. After cooling in ice, enzymes were added (20 μl of a solution containing 50 mg of lysozyme ml⁻¹, 20 μl of a solution containing 10 mg of pronase ml⁻¹, 8 μl of a solution containing 5,000 U of mutanolysin ml⁻¹, 20 μl of a solution containing 17.5 mg of lipase ml⁻¹, 4 μl of a solution containing 10 mg of RNase ml⁻¹), and samples were incubated at 37°C for 1 h. Two hundred microliters of extraction buffer (20 mM Tris-HCl, 100 mM EDTA [pH 8], 1% sodium dodecyl sulfate, 100 μg of proteinase K ml⁻¹) was added and incubated at 37°C for 1 h and then for an additional 30 min at 55°C. Genomic DNA was then extracted three times with phenol-chloroform-isoamyl alcohol and precipitated with ammonium acetate-ethanol.

PCR amplification of 16S rRNA genes, cloning, and sequencing.

16S rDNA genes were amplified from extracted DNA by using Planctomycetales-specific forward primer PLA-46F targeting the region corresponding to nucleotides 46 to 63 of Escherichia coli 16S rRNA genes (30) and universal reverse primer 1390R (31). The PCR thermal profile was as follows: initial denaturation at 94°C for 1 min and 30 cycles consisting of denaturation at 94°C for 1 min, primer annealing at 59°C for 1 min, and extension at 72°C for 1.5 min. The final elongation step was extended to 15 min. The 16S rDNA amplicons were cloned by using a TA cloning kit (pGEM-T Easy vector; Promega) in accordance with the manufacturer's instructions. 16S rDNA-containing clones were grown in Nunc microtiter plates containing 300 μl of Luria-Bertani medium supplemented with 5% glycerol and ampicillin (100 μg ml⁻¹). Plasmid extraction and 16S rDNA sequencing were performed as described by Artiguenave et al. (3).

Sequence analysis.

The 16S rDNA sequences were treated as described by Ewing et al. (11, 12). The 16S rDNA clone sequences recovered were compared with the complete EMBL nucleotide sequence databases. Sequences from the EMBL databases with the best BLAST scores were imported into the ARB data set (http://www.arb-home.de) when necessary. Chimeric sequences were searched by using the procedure described by Juretschko et al. (25) prior to phylogenetic analysis. Phylogenetic trees were constructed by using the ARB program and database package. All sequences having more than 1,200 nucleotides were imported into the ARB database and automatically aligned with the existing 16S rDNA sequences. The resulting alignments were manually checked and corrected when necessary.

Phylogenetic placement was done by comparison with reference sequences representing the main lines of descent in the domain Bacteria. Overall, 16S rDNA sequence similarities were determined by using the distance matrix tool of the ARB program package. Phylogenetic trees were constructed by neighbor joining (NJ) with the Jukes-Cantor correction and by the maximum-parsimony and the maximum-likelihood methods. The statistical significance levels of interior nodes were determined by performing bootstrap analysis by the NJ method.

Rarefaction analysis and OTU assignment.

Rarefaction curves were obtained by using the program ECOSIM 6.0 (20; http://homepages.together.net/∼gentsmin/ecosim.htm). Diversity coverage was calculated by Good's method (19), according to which the percentage of coverage was calculated with the formula [1 − (n/N)] × 100, where n is the number of molecular species represented by one clone (single-clone operational taxonomic units [OTUs]) and N is the total number of sequences analyzed. A 97% similarity threshold was used for OTU assignment (38).

Probe design and FISH.

The probe search function of the ARB program software package was used to design new probes. For FISH experiments, sludge samples were washed with PBS and fixed with a 4% paraformaldehyde solution in PBS (3:1) for 3 h at 4°C. The buffers and hybridization conditions used have been described previously (2, 25). Hybridization was performed for 1.5 h at 46°C. A stringent wash step was performed for 10 min at 48°C. The newly developed group-specific probe was 5′ end labeled with Cy3 and used simultaneously with fluorescein isothiocyanate-labeled EUB338 II (7) or Cy5-labeled PLA-46 (30). We tested the specificity of the new probe S-*-Plan-0322-a-A-18 targeting novel group I (see Table 4) using pure cultures of Planctomyces limnophilus and Pirellula staleyi as negative controls; no signal was obtained with these organisms, while they were positive when we used the EUB II probe and the Pla-46 probe specific for all Planctomycetales. Slides were visualized with a confocal laser scanning microscope (Zeiss) equipped with an argon ion laser (450 to 514 nm) and two helium-neon lasers (543 to 633 nm) used for recording optical sections.

TABLE 4.

Group-specific probe S-*-Plan-322-a-A-18 based on the 5′-3′ 16S rRNA sequence alignment^a

Probe, target, environmental clone, or taxon (accession no.)	Sequenceb
Probe S-*-Plan-0322-a-A-18	5′ GTTCCGGTCGTGGGGGCC 3′
Target	5′ GGCCCCCACGACCGGAAC 3′
032F07_P_BA_P3 (BX294886)	5′ ------------------ 3′
Uncultured sludge bacterium H28	5′ ------------------ 3′
(AF234749)
Escherichia coli O157 (AB035920)	5′ -A--AG--AC--U----- 3′
Planctomyces maris (AJ231184)	5′ ---------C--U--G-- 3′
Pirellula marina (X62912)	5′ -----GU-UC--U----- 3′
Isosphaera pallida (X64372)	5′ -----GG-AC--U----- 3′
“Candidatus Kuenenia stuttgartiensis”	5′ --U-GG--AC--U----- 3′
(AF375995)
“Candidatus Brocadia anammoxidans”	5′ --U-GG--AC--U----- 3′
(AF375994)
Parachlamydia sp. (AF366365)	5′ -A--G--AAC--U----- 3′
Verrucomicrobium spinosum (X90515)	5′ -A--AG--AC--U----- 3′
Spirochaeta halophila (M88722)	5′ -A--GG--AC--U----- 3′
Paracoccus denitrificans (Y16930)	5′ -AU-AG--AC--U----- 3′

^aThe probe designation is in accordance with the Oligonucleotide Probe Database nomenclature (1).

^bSequence for positions 305 to 322 (E. coli numbering [5]). Dashes indicate identity with the homologous nucleotides in the target sequence. There were at least three mismatches for all nontarget microorganisms.

Nucleotide sequences accession numbers.

Sequences determined in this study have been deposited in the EMBL, GenBank, and DDBJ databases under accession numbers BX294674 to BX294906.

RESULTS

Genomic DNA was extracted from sludge samples originating from the aerobic and anoxic basins and the anaerobic digestor of the municipal WWTP of Evry by using the procedures described in Material and Methods. 16S rRNA genes from each DNA preparation were separately amplified by PCR by using planctomycete-specific and universal primers and used for construction of three 16S rDNA clone libraries. Although clones with nonplanctomycete 16S rDNA, such as clones related to the orders Chlamydiales, Spirochetales, and Verrucomicrobiales, were observed, in this study we focused on the planctomycetes, the most abundant group of clones analyzed.

Determination and distribution of OTUs.

A total of 237 planctomycete-like 16S rDNA sequences were obtained; 140 of these 16S rDNA sequences were retrieved from the aerobic basin, 78 were retrieved from the anoxic basin, and only 16 were retrieved from the digestor (Tables 1 and 2). Three 16S rDNA clone sequences were identified as chimeric and thus excluded from further analysis. The remaining 234 clone sequences were grouped into 110 OTUs. The phylogenetic affiliations of the OTUs and their similarities to the most closely related 16S rRNA sequences available in public databases are shown in Table 1.

TABLE 1.

Affiliations of the 16S rDNA clones analyzed in this study

OTUa	Sequence representative (EMBL accession no.)	No. of clones in library			Microorganism or clone with highest 16S rDNA sequence similarity
		Aerobic basin	Anoxic basin	Digestor	Taxon	Accession no.	% Similarity
Planctomyces
1	026B03_P_BA_P3 (BX294719)	3	0	0	Planctomyces sp.	AJ231189	92.2-92.8
2	032B06_P_BA_P3 (BX294869)	1	0	0	Planctomyces sp.	AB015527	90.5
3	026D07_P_BA_P3 (BX294724)	3	3	0	Planctomyces sp.	X81953	90.0-90.4
4	028F09_P_BN_P5 (BX294807)	0	1	0	Planctomyces sp.	AJ2311	89-91.9
5	031B01_P_BA_P3 (BX294828)	1	0	0	Planctomyces sp.	AB015527	89.6
6	032B01_P_BA_P3 (BX294867)	1	0	0	Planctomyces sp.	X81953	89.3
7	025A02_P_BA_P3 (BX294675)	1	3	0	Planctomyces sp.	X81953	89.2-90
8	027C06_P_BN_P5 (BX294754)	0	1	0	Planctomyces sp.	AJ231189	88.8
9	026A07_P_BA_P3 (BX294717)	5	0	1	Planctomyces sp.	AB015527	88.7-90.4
10	025B04_P_BA_P3 (BX294685)	1	0	0	Planctomyces maris	AJ231184	88.7
11	031F07_P_BA_P3 (BX294849)	1	0	0	Planctomyces brasiliensis	X85247	88.7
12	028B12_P_BN_P5 (BX294787)	0	1	0	Planctomyces brasiliensis	X85247	88.6
13	032C05_P_BA_P3 (BX294872)	1	0	0	Planctomyces sp.	X81953	88.5
14	032A02_P_BA_P3 (BX294862)	1	0	0	Planctomyces brasiliensis	X85247	88.4
15	025C03_P_BA_P3 (BX294689)	1	0	0	Planctomyces brasiliensis	X85247	88.1
16	028F03_P_BN_P5 (BX294803)	0	1	0	Planctomyces brasiliensis	X85247	88.1
17	028D04_P_BN_P5 (BX294795)	1	1	0	Planctomyces brasiliensis	X85247	88-88.4
18	026D10_P_BA_P3 (BX294726)	1	0	0	Planctomyces maris	AJ231184	87.8
19	025H12_P_BA_P3 (BX294716)	1	0	0	Planctomyces sp.	X81953	87.7
20	027B06_P_BN_P5 (BX294748)	0	4	0	Planctomyces brasiliensis	X85247	87.4-88.6
21	028B05_P_BN_P5 (BX294783)	0	1	0	Planctomyces brasiliensis	X85247	86.8
22	028G10_P_BN_P5 (BX294812)	0	1	0	Planctomyces sp.	AJ231189	86.8
23	030H03_P_DI_P15 (BX294821)	0	0	1	Planctomyces brasiliensis	X85247	86.8
24	056E05_P_DI_P58 (BX294905)	0	0	1	Planctomyces maris	AJ231184	84.2
Total	24	23	17	3
Pirellula
25	031B03_P_BA_P3 (BX294830)	2	1	0	Pirellula sp.	X81947	97.5-98.4
26	025E05_P_BA_P3 (BX294698)	1	0	0	Uncultured planctomycete	AJ290171	97.5
27	028A09_P_BN_P5 (BX294778)	4	1	0	Uncultured bacterium PHOS-HE25	AF314429	97.3-99.5
28	055E08_P_DI_P58 (BX294901)	0	1	1	Uncultured planctomycete	AJ290171	97.0-97.3
29	056C02_P_DI_P58 (BX294903)	1	1	1	Uncultured planctomycete	AF271319	96.5-97.2
30	026G09_P_BA_P3 (BX294737)	4	0	0	Pirellula sp.	X81945	95.2-97.0
31	032B02_P_BA_P3 (BX294868)	3	0	0	Uncultured planctomycete	AJ290171	95.1-96.6
32	031C05_P_BA_P3 (BX294835)	2	0	0	Uncultured planctomycete	AF271331	95.0-95.5
33	027D05_P_BN_P5 (BX294759)	0	3	0	Uncultured planctomycete	AJ290171	94-95.3
34	027E08_P_BN_P5 (BX294767)	1	1	1	Uncultured sludge bacterium A17	AF234760	94.4-95.6
35	028H06_P_BN_P5 (BX294815)	1	2	2	Uncultured planctomycete	AF271331	94.1-96.2
36	055D08_P_DI_P58 (BX294899)	0	0	1	Uncultured sludge bacterium A17	AF234760	93.8
37	025G08_P_BA_P3 (BX294708)	1	0	0	Pirellula sp.	X81947	92.6
38	027A12_P_BN_P5 (BX294745)	8	1	0	Uncultured planctomycete	AJ290171	92.2-92.7
39	031F02_P_BA_P3 (BX294845)	1	0	0	Uncultured planctomycete	AF271319	92.2
40	028C11_P_BN_P5 (BX294792)	0	1	0	Uncultured sludge bacterium A4	AF234736	92.0
41	025H10_P_BA_P3 (BX294715)	1	0	0	Pirellula staleyi	X81946	92.0
42	025A12_P_BA_P3 (BX294682)	3	1	0	Pirellula staleyi	X81946	91.8-93.3
43	055C03_P_DI_P58 (BX294898)	4	1	1	Uncultured planctomycete	AJ290171	91.6-91.8
44	027E04_P_BN_P5 (BX294764)	0	1	0	Pirellula staleyi	X81946	90.7
45	031A04_P_BA_P3 (BX294823)	1	0	0	Planctomycete str292	P231182	90.2
46	028F08_P_BN_P5 (BX294806)	0	1	0	Uncultured planctomycete	AF271331	90.1
47	055H09_P_DI_P58 (BX294902)	0	0	1	Uncultured planctomycete	AF271331	90.0
48	027H04_P_BN_P5 (BX294772)	0	1	0	Pirellula staleyi	AJ231183	90.0
49	030F07_P_DI_P15 (BX294820)	1	0	1	Pirellula marina	X62912	89.6-90.8
50	025E11_P_BA_P3 (BX294700)	2	0	0	Pirellula sp.	X81945	89.4-89.6
51	025E01_P_BA_P3 (BX294696)	1	0	0	Uncultured planctomycete	AJ290171	89.4
52	025A04_P_BA_P3 (BX294677)	1	0	0	Pirellula staleyi	X81946	89.1
53	028D03_P_BN_P5 (BX294794)	0	1	0	Pirellula staleyi	AJ231183	88.9
54	026G04_P_BA_P3 (BX294733)	4	0	0	Uncultured Pirellula clone 55H12	AF029076	87.8-88.1
55	025F07_P_BA_P3 (BX294704)	1	0	0	Pirellula sp.	X81945	87.8
Total	31	9	17	9
Isosphaera
56	032F01_P_BA_P3 (BX294885)	1	0	0	Isosphaera sp.	X81960	87.7
Gemmata
57	025D04_P_BA_P3 (BX294693)	1	0	0	Uncultured planctomycete	AJ290195	99/PICK>
58	031A06_P_BA_P3 (BX294824)	3	0	0	Uncultured planctomycete	AJ290195	97.9-98.5
59	028F10_P_BN_P5 (BX294808)	0	1	0	Uncultured planctomycete	AJ290195	93.8
60	027A08_P_BN_P5 (BX294744)	0	2	0	Gemmata obscuriglobus	X81957	90.7
61	025D03_P_BA_P3 (BX294692)	1	0	0	Uncultured planctomycete	AF271341	89.9
62	028E08_P_BN_P5 (BX294799)	0	1	0	Gemmata obscuriglobus	X81957	87.4
Total	6	5	4	0
Group I
63	028D05_P_BN_P5 (BX294796)	1	1	0	Uncultured sludge bacterium H28	AF234749	96.7-99.0
64	027A05_P_BN_P5 (BX294742)	4	7	0	Uncultured sludge bacterium H28	AF234749	95.7-99.0
65	028E09_P_BN_P5 (BX294800)	1	1	0	Uncultured sludge bacterium H28	AF234749	95.3
66	026G07_P_BA_P3 (BX294735)	1	0	0	Uncultured sludge bacterium H28	AF234749	95.0
67	031G01_P_BA_P3 (BX294851)	1	0	2	Uncultured sludge bacterium H28	AF234749	94.4-94.9
68	027H07_P_BN_P5 (BX294774)	3	1	0	Uncultured sludge bacterium H28	AF234749	93.7-95.9
69	026F02_P_BA_P3 (BX294730)	3	1	0	Uncultured sludge bacterium H28	AF234749	93.9-94.6
70	028F02_P_BN_P5 (BX294802)	1	3	0	Uncultured sludge bacterium H28	AF234749	93.2-95.3
71	025A10_P_BA_P3 (BX294681)	3	5	0	Uncultured sludge bacterium H28	AF234749	92.9-96.4
72	028H01_P_BN_P5 (BX294813)	0	2	0	Uncultured sludge bacterium H28	AF234749	94.2-95.7
73	031E04_P_BA_P3 (BX294842)	1	0	0	Uncultured sludge bacterium H28	AF234749	94.2
74	028A10_P_BN_P5 (BX294779)	0	2	0	Uncultured sludge bacterium H28	AF234749	93.8-94.1
75	026B05_P_BA_P3 (BX294720)	7	1	0	Uncultured sludge bacterium H28	AF234749	93.7-96.8
76	025C06_P_BA_P3 (BX294690)	2	0	0	Uncultured sludge bacterium H28	AF234749	93.0-93.4
77	031H10_P_BA_P3 (BX294861)	6	0	0	Uncultured sludge bacterium H28	AF234749	93.7-94.1
78	025E06_P_BA_P3 (BX294699)	3	0	0	Uncultured sludge bacterium H28	AF234749	92.7-93.1
79	027E06_P_BN_P5 (BX294766)	0	1	0	Uncultured sludge bacterium H28	AF234749	92.2
80	028B10_P_BN_P5 (BX294786)	0	2	0	Uncultured sludge bacterium H28	AF234749	92.1-92.8
81	028H07_P_BN_P5 (BX294816)	0	1	0	Uncultured sludge bacterium H28	AF234749	91.8
82	032F07_P_BA_P3 (BX294886)	5	0	0	Uncultured sludge bacterium H28	AF234749	91.5-91.9
83	032G06_P_BA_P3 (BX294888)	1	0	0	Uncultured sludge bacterium H28	AF234749	91.5
84	026E07_P_BA_P3 (BX294727)	3	0	0	Uncultured sludge bacterium H28	AF234749	90.7-90.8
85	031G03_P_BA_P3 (BX294853)	1	0	0	Uncultured planctomycete	AJ290183	90.4
86	027D08_P_BN_P5 (BX294761)	0	2	0	Uncultured sludge bacterium H28	AF234749	89.9-90.7
87	025G09_P_BA_P3 (BX294709)	3	0	0	Uncultured sludge bacterium H28	AF234749	89.6-89.9
88	026A10_P_BA_P3 (BX294718)	1	0	0	Uncultured sludge bacterium H28	AF234749	89.3
Total	26	51	30	2
Group II
89	027A06_P_BN_P5 (BX294743)	0	1	0	Uncultured planctomycete	AJ290183	89.1
Group III
90	027B07_P_BN_P5 (BX294749)	0	1	0	Uncultured planctomycete	AJ290171	89.2
91	028C07_P_BN_P5 (BX294790)	0	1	0	Uncultured sludge bacterium H28	AF234749	88.5
92	032H11_P_BA_P3 (BX294894)	1	0	0	Planctomyces brasiliensis	X85247	85.3
Total	3	1	2	0
Group IV
93	055B12_P_DI_P58 (BX294897)	0	0	1	Pirellula sp.	X81945	84.9
94	029G09_P_DI_P15 (BX294818)	0	0	1	Uncultured planctomycete	X81945	83.9
Total	2	0	0	2
Group V
95	028H05_P_BN_P5 (BX294814)	0	1	0	Unidentified bacterium wb1_D18	AF317785	81.1
Total	1	0	1	0
Group VI
96	025H04_P_BA_P3 (BX294712)	1	0	0	Uncultured Crater Lake bacteria	AF316773	91.1
97	028G08_P_BN_P5 (BX294811)	0	1	0	Unidentified bacteria wb 1-D18	AF317785	90.4
98	031G08_P_BA_P3 (BX294856)	1	0	0	Uncultured Crater Lake bacteria	AF316773	90.3
99	031D04_P_BA_P3 (BX294837)	1	0	0	Uncultured Crater Lake bacteria	AF316773	90.2
100	025G12_P_BA_P3 (BX294710)	1	0	0	Uncultured Crater Lake bacteria	AF316773	90.0
101	032E06_P_BA_P3 (BX294883)	1	0	0	Uncultured Crater Lake bacteria	AF316773	89.4
102	025A05_P_BA_P3 (BX294678)	1	0	0	Uncultured Crater Lake bacteria	AF316773	88.6
103	027H11_P_BN_P5 (BX294775)	0	1	0	Unidentified bacteria wb1-D18	AF317785	84.1
104	028B09_P_BN_P5 (BX294785)	0	1	0	Unidentified bacteria wb1-D18	AF317785	83.3
105	025F10_P_BA_P3 (BX294705)	1	0	0	Unidentified bacteria wb1-D18	AF317785	83.2
106	032D01_P_BA_P3 (BX294875)	1	1	0	Unidentified bacteria wb1-D18	AF317785	83.0
107	032D06_P_BA_P3 (BX294877)	1	0	0	Unidentified bacteria wb1-D18	AF317785	82.5
108	028E10_P_BN_P5 (BX294801)	0	1	0	Unidentified bacteria wb1-D18	AF317785	82.4
109	031B05_P_BA_P3 (BX294831)	1	0	0	Unidentified bacteria wb1-D18	AF317785	81.9
110	027B03_P_BN_P5 (BX294746)	0	1	0	Uncultured Crater Lake bacteria	AF316773	88.1
Total	15	10	6	0
TOTAL	110	140	78	16

^aClones exhibiting levels of 16S rDNA sequence similarity of ≥97% were grouped in the same OTU.

TABLE 2.

Distribution of plantomycete OTUs in known and novel groups in the three libraries from the WWTP

Source	No. of clones	No. of OTUs	No. of OTUs represented by a single clone	No. of Planctomyces OTUs	No. of Pirellula OTUs	No. of Isosphaera OTUs	No. of Gemmata OTUs	No. of group I OTUs	No. of group VI OTUs	% Coverage
Clone library
Aerobic basin	140	49	36	11	13	1	3	11	9	74.3
Anoxic basin	78	31	24	7	6	0	3	6	5	69.2
Digestor	16	6	6	2	2	0	0	0	0
Shared OTUs
Aerobic and anoxic basins		16		3	4	0	0	8	1
Aerobic basin and digestor		3		1	1	0	0	1	0
Anoxic basin and digestor		1		0	1	0	0	0	0
Total	234	110	66	24	31	1	6	26	15	71.8

All related clone sequences found in databases were retrieved mainly from polluted environments, such as sequencing batch reactors (4, 6) and industrial treatment plants (25), or from river biofilm samples (13) and marine environments (40). Only two OTUs, represented by seven sequences, exhibited more than 97% similarity to a cultivated species (Pirellula), while six OTUs (7.3%) were related to uncultivated planctomycetes with more than 97% similarity. These sequences were distributed in three groups, Pirellula, Gemmata, and group I (Table 1). The remaining 102 OTUs (92.7%) generated in this study are related to either cultivated or noncultivated microorganisms with less than 97% similarity. They clearly were derived from undescribed microorganisms and probably represent novel phylotypes.

Most clone sequences were affiliated with the Pirellula group (31 OTUs), the Planctomyces group (24 OTUs), group I (26 OTUs), group VI (15 OTUs), and the genus Gemmata (6 OTUs). The Isosphaera group was represented by only one 16S rDNA sequence (Table 2). Sixty-six OTUs (60% of the OTUs recovered from the three libraries) were represented by a single 16S rDNA sequence.

Phylogenetic analysis.

Phylogenetic analysis revealed 10 phylogenetic groups of distantly related microorganisms. Most of the organisms were assigned to cultivated members of the Planctomycetales, including the genera Planctomyces, Pirellula, Isosphaera, and Gemmata. Phylogenetic groups V and VI seem to branch deeply within the planctomycetes. Except for groups III (three sequences) and V (one sequence), whose positions changed according to the taxonomic sampling method used, the same branching order was obtained by using the data set and the maximum-likelihood and maximum-parsimony tree construction methods.

Known phylogenetic groups within the planctomycetes.

The Planctomyces group has a common node with groups I and II. It is divided into four main subclusters encompassing sequences retrieved from all three clone libraries. The intralineage phylogenetic depth which represents the maximum difference in the sequence set within the Planctomyces group is 23.8%.

The Pirellula group is represented by two main subgroups with many subclusters, and its intralineage phylogenetic depth is 20.5%.

Fewer sequences related to Isosphaera and Gemmata are represented in the clone libraries. The intralineage phylogenetic depth within the Gemmata group is 21.9%. Only a single sequence, found in the aerobic basin, is related to the Isosphaera group.

Deeply branching phylogenetic groups.

The phylogenetic relationships of the novel groups within the planctomycete group were analyzed by using the same NJ trees. No representatives of group I (Table 1) are related to any cultured microorganism with ≥97% similarity. Like the Planctomyces and Pirellula groups, group I was retrieved from all three clone libraries. As shown in the phylogenetic tree in Fig. 1A, group I and group II have a common node with the Planctomyces group and branch as Planctomyces sister groups. Signature nucleotide analysis showed that group I and group II are closely related to the genus Planctomyces (Table 3); group I has two transversions at positions 948 and 1100, whereas group II has the same two transversions and two additional transversions at positions 680 and 710. The intralineage phylogenetic depth within group I is 16.6%. Group II consists of a single sequence and OTU retrieved from the anoxic basin. This sequence has a common node with group I and may represent a new phylogenetic group.

FIG. 1.

Evolutionary distance dendrograms constructed by the NJ method and showing the affiliations of the environmental 16S rDNA sequences recovered from the aerobic basin, from the anoxic basin, and from the digestor to representative members of the Planctomycetales. (A) General tree, showing the affiliations of different groups. The numbers in the boxes or on the branches indicate the numbers of OTUs. (B) Isosphaera, Gemmata, and group II, III, IV, V, and VI clones. The trees were constructed by using the ARB software package, as described in Materials and Methods. The outgroup used in the analysis was Methanobacterium formicicum. The numbers in parentheses are the numbers of sequences. Scale bar = 10% estimated difference in nucleotide sequence positions.

TABLE 3.

Comparison of Pirellula- and Planctomyces-specific 16S rRNA signature nucleotides for Pirellula-like and Planctomyces-like clones^a

Taxon	Nucleotide at positionb:
	114	115	312	313	680	710	948	1100	1233
Planctomyces	G	U	A	C	C	G	C	G, A	G
Group I	G	U	A	C	C	G	G	U	C
Group II	G	U	A	C	A	U	G	U	C
Pirellula	A	G	C	U	A	U	G	U	C
Group III	A	G (C)	C (G)	U	A (C)	G (U)	G	U	C
Group IV	A	U	A	U	G	C	G	U	C

^aSignature nucleotides were obtained from reference 16. Nucleotide differences are underlined. Bases present in a minority of the clones are in parentheses.

^bE. coli numbering (5).

(i) Group III.

Group III (Fig. 1A and Table 1) is represented by only three sequences representing three OTUs that branch deeply within the Pirellula group. In fact, compared to the group I and II sequences, the group III nucleotide signature seems to be more closely related to the Pirellula group, and there are only occasional changes and a transversion at position 710 (Table 3).

(ii) Group IV.

The two OTUs of group IV (Fig. 1A) share five of nine nucleotide signatures with their closest relative, Pirellula, and have two transversions at positions 312 and 710 and two transitions at positions 115 and 680 (Table 3). They branch deeply from the Pirellula group and are an independent monophyletic group.

(iii) Group V.

Group V (Fig. 1B) is represented by a single sequence that branches deeply in the Planctomycetales tree and diverges after and independent of group VI. Its signature nucleotides have two nucleotide transversions at the division level at positions 340 and 570 (data not shown)

(iv) Group VI.

Group VI (Fig. 1B) is the most deeply branching monophyletic lineage in the Planctomycetales phylogenetic tree. The levels of similarity of group VI to cultivated planctomycetes are low, ranging from 78.6 to 81.1%. This group can be divided into four monophyletic subgroups that have a common node. One of the subgroups encompasses the recently described anaerobic ammonium-oxidizing bacteria that are culturable only in a mixed microbial community (41). Representatives of this subgroup exhibit between 78.1 and 79.3% similarity to the anaerobic ammonia-oxidizing bacteria “Candidatus Brocadia anammoxidans” and “Candidatus Kuenenia stuttgartiensis.” The other subgroups include unidentified aquatic environmental planctomycetes representing the closest relatives of our sequences, and the levels of similarity range from 81.9 to 91.1%. Examination of the 16S rDNA sequences of group VI revealed an insertion consisting of approximately 20 variable nucleotides located between helices 10 and 11. This insertion seems to be different from that described for the anammox sequences (35) located in helix 9. This feature may explain in part the divergence of this group of sequences from the sequences of the other groups.

No sequence that belonged to this novel lineage was retrieved from the anaerobic digestor clone library. The intralineage phylogenetic depth of this group is 24.4%. Nucleotide signature analysis of group VI at the phylum level showed that only three of seven nucleotides are unchanged (data not shown).

Most groups described in this study have the same signature nucleotides at the phylum level; the exceptions are groups V and VI, which have many differences (data not shown).

With the present sequences the phylogenetic depth of the Planctomycetales ranges from 23 to 30.6%, which exceeds the phylogenetic depths of well-known divisions of bacteria, such as the Proteobacteria (23%) (9). As determined by using sequence representatives of candidate division OP3 or the Chlamydiales as an outgroup, group VI is deeply branching within the Planctomycetales but has a low bootstrap value.

Diversity estimates.

To figure out whether the clone sequences analyzed represent a sufficient sample size, we determined coverage estimates for the aerobic and anoxic basins and the digestor. We found that there was 74.3 and 69.2% coverage for the aerobic and anoxic basins, respectively. These values provide estimates of how well the clones analyzed accounted for the biodiversity of planctomycetes within the clone library when the present methodology was used. Another estimate was obtained by plotting the cumulative number of planctomycete OTUs as a function of the number of planctomycete clone sequences. Diversity curves for the 16S rDNA clone libraries generated from genomic DNA from the aerobic and anoxic basins sludge samples are shown in Fig. 2. These curves show that more clones have to be sequenced and analyzed in order to approach saturation.

FIG. 2.

Rarefaction curves for the planctomycete phylotypes analyzed. All sequences with more than 3% dissimilarity were considered different. The cumulative number of OTUs is expressed as a function of the number of clones sequenced; 95% confidence intervals (95% conf.) are indicated. Squares, aerobic basin; triangles, anoxic basin.

Simultaneous occurrence of planctomycete OTUs in the three locations.

We also analyzed the planctomycete-like sequences in order to determine whether they were simultaneously present in the three locations (Table 2). Overall, 72, 52, and 14 OTUs were recovered from the aerobic and anoxic basins and the digestor, respectively, and these OTUs represented a total of 110 different OTUs. Twenty-four OTUs (20.9%) were simultaneously present at least in two locations. A detailed breakout for all three libraries analyzed is shown in Table 2 for the known lineages, as well as for novel groups I and VI.

Visualization of group I sequences by FISH.

Using the probe design function of the ARB software, we developed a group-specific probe, S-*-Plan-0322-a-A-18 targeting group I. The nucleotide sequence and the target region used for probe design are shown in Table 4. Among the sequences available in ARB and the Ribosomal Database Project the closest sequence representing a nontarget 16S rDNA (Planctomyces maris) had three mismatches with this new probe.

Group I may represent the third phylogenetic group after Pirellula and Planctomyces in terms of the number of OTUs retrieved (Table 2). Using the Pla-46 probe in conjunction with the new group-specific probe S-*-Plan-0322-a-A-18 labeled with different dyes, we were able to visualize and localize representatives of the new group in activated sludge samples (Fig. 3). Only a fraction of the microorganisms labeled with the general planctomycete probe Pla-46 were labeled with the newly developed probe S-*-Plan-0322-a-A-18. The newly localized microorganisms were organized in microcolonies or were present as single cells, but they were always localized inside the flocs, which suggests that there were interactions between the diverse microorganisms constituting the flocs.

FIG. 3.

In situ identification of group I in the activated sludge of an aerobic basin by using probe probe S-*-Plancto-0322-a-A-18. Two images were recorded as a single optical section by using a confocal laser scanning microscope. Cells labeled with the general planctomycete probe Pla-46 are blue, cells labeled with both the new specific probe S-*-Plancto-0322-a-A-18 and the Pla-46R probe are pink, and autofluorescence is green.

DISCUSSION

Extensive phylogenic diversity among Planctomycetales.

In this study we performed a detailed analysis of the molecular diversity of the Planctomycetales in a municipal WWTP, which had not been done at this level previously. This group is among the least-known groups in the bacterial domain. Currently, it includes four known genera (Planctomyces, Isosphaera, Pirellula, and Gemmata) and only seven described species represented by pure cultures isolated from various aquatic ecosystems (14, 15, 17, 21, 33, 42).

Our survey indicated that the representatives of the order Planctomycetales in activated sludge are very diverse. A total of 110 Planctomycetes-related OTUs were detected among 234 Planctomycetes sequences harvested from the three 16S rDNA clone libraries, and 101 of these OTUs were novel OTUs that had never been detected before. We found only two OTUs (seven rDNA sequences) corresponding to cultured species belonging to the genus Pirellula, and no OTU corresponding to any cultivated species belonging to the genus Planctomyces, Gemmata, or Isosphaera. The remaining OTUs are related either to cultivated species (but with <97% similarity) or to environmental clone sequences retrieved in other environmental surveys with levels of similarity ranging from 81.1 to 99.5%. Thus, 98% of the described OTUs seem to represent putative planctomycete species that have not been cultivated yet.

In addition to the phylogenetic groups Pirellula, Planctomyces, and Gemmata, other monophyletic groups (groups I and VI) were detected. These five groups together account for 92.7% of the total OTUs recovered from the three libraries. The genus Isosphaera and other minor deeply divergent groups accounted for only 7.3% of the OTUs retrieved.

In this study we doubled the number of available 16S rDNA sequences of members of the bacterial order Planctomycetales from 281 to 515 (Ribosomal Database Project data, January 2003). Members of the planctomycete group have been detected very rarely or at low levels in numerous previous environmental surveys of WWTPs, reactors, soil, and rice roots and have even been associated with aquatic invertebrates, but they have never been detected at the level of diversity that we describe in this paper. In diversity surveys of WWTPs and reactors, an average of six planctomycete OTUs were detected in various separate studies (for details see reference 41), compared to the 110 OTUs detected in the present study. Primer sequences used in these surveys were considered to be universal for 16S rDNA of the domain Bacteria. Mismatches in the target sites of the 16S rDNA primers are often encountered (7) and probably result in inefficient PCR amplification. Our results show clearly that the use of specific primers is a powerful tool for a thorough survey of a particular group in an ecosystem.

It remains to be seen whether the primers used in this study for 16S PCR amplification cover the Planctomycetales exhaustively. In addition to the use of Planctomycetales-specific primers (30), the use of a new enzymatic combination for genomic DNA extraction that takes into account the cell wall chemical composition of Planctomycetales may also contribute to the observation of extensive diversity within a WWTP. The Planctomycetales diversity observed at the Evry municipal WWTP is not yet exhaustive, as suggested by the partial coverage rates of 74.3% for the aerobic basin clone library and 69.2% for the anoxic basin clone library, and additional novel phylogenetic groups remain undetected. Moreover, some novel groups in the phylogenetic tree (Fig. 1A) are represented by only a few rDNA sequences when our set of primers and PCR conditions are used.

The plantomycete diversity detected in the anoxic and aerobic basins is clearly different and quantitatively greater than that detected in the digestor (Table 2). Sequences related to Planctomyces and Pirellula were retrieved from the aerobic basin, anoxic basin, and digestor clone libraries, suggesting that representatives of these groups have adapted to both aerobic and anaerobic conditions, whereas no sequence related to Gemmata or Isosphaera was found in the anaerobic digestor.

Novel deeply branching group within the Planctomycetales.

During this survey we identified a monophyletic group, group VI, that is deeply rooted in the Planctomycetales tree and comprises 15 novel OTUs and some other sequences distantly related to the Planctomycetales. The distantly related sequences include the recently described anaerobic ammonia oxidizers “Candidatus Brocadia anammoxidans” (39) and “Candidatus Kuenenia stuttgartiensis” (34). As shown in the phylogenetic tree in Fig. 1B, this group has long branches. However, using different methods for tree construction (NJ, parsimony, maximum likelihood), we obtained the same tree topology.

This group of sequences, which exhibited less than 90.1% similarity to previously reported sequences, may be regarded as a candidate division (23) very distantly related to the Planctomycetales for the following reasons: (i) the low level of conservation of Planctomycetales nucleotide signatures at the phylum level; (ii) the substantial sequence dissimilarity to the most closely related cultivated planctomycete (≥20%) (the levels of similarity to cultured planctomycetes ranged from 78.6 to 81.1%); and (iii) the metabolic specificity of some members of this group (anammox metabolism). However, the statistical support for this lineage (34%) is low, and more sequences, preferably from other ecosystems, need to be retrieved to determine its phylogenetic position.

With the present data set, phylogenetic analyses performed with representatives of the main bacterial divisions do not affect the deeply branching characteristics of this group within the Planctomycetales. If this remains true, because of the intralineage phylogenetic depth of the whole order, we can say that the Planctomycetales displays at present the second largest extent of rDNA sequence divergence in the domain Bacteria (30.6%) after the OP11 candidate division, which shows 33% sequence divergence (9).

This group, which has paramount ecological importance, is being investigated in the aerobic and anoxic basins of other WWTPs by molecular cloning and sequencing. The preliminary results of molecular analyses of two other digestors confirmed the absence of group VI rRNA gene sequences in the anaerobic and mesophilic digestors. A set of six oligonucleotide probes targeting rRNA gene sequences of representatives of group VI were designed; they are now being tested and evaluated by using both FISH and dot blot hybridization techniques. This should help in assessing the population structure of group VI, as well as its dynamics and its overall activity within the ecosystem.

The sequences generated should facilitate design of new species- and group-specific probes and subsequent isolation of corresponding organisms, providing powerful tools for studying sludge ecology. The novel major phylogenetic groups, retrieved from sludge, will be quantified by dot blotting and FISH to assess their contributions to the total sludge microflora and their population dynamics without the bias resulting from PCR amplification.

In summary, we conducted a molecular analysis of rDNA amplicons generated directly from activated sludge samples taken from three distinct metabolic environments. This study provided the first large-scale and high-resolution insight into the composition of the planctomycete community present in a municipal WWTP. The results show that 92.7% of the Planctomycetales OTUs (102 of 110 OTUs) could be assigned to three major cultivated and two uncultivated phylogenetic groups (Pirellula, Planctomyces, Gemmata, group I, and group VI); 37.3% of the OTUs (41 OTUs) were assigned to the novel major phylogenetic groups I and VI. As expected, 108 of 110 OTUs (99%) correspond to uncultivated planctomycetes or loosely related species and are awaiting cultivation and further characterization; the only exceptions are two OTUs represented by Pirellula-like sequences.

The use of simple methods for isolation of planctomycetes should allow more extensive studies of the distribution, physiology, and ecology of this division of the domain Bacteria with unique cell organization and cell walls, large evolutionary distances, and few cultivated representatives. This division may harbor microorganisms representing functional groups, which may play a much more significant role in the environment than first imagined with regard to the cycling of organic and inorganic matter. Increased effort for development of suitable cultivation strategies for these bacteria is badly needed. In parallel, the use of techniques referred to as environmental genomics should allow investigation of the genome composition of these bacteria without a requirement for cultivation.