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Applied and Environmental Microbiology, April 2005, p. 2145-2153, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2145-2153.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Novel Major Bacterial Candidate Division within a Municipal Anaerobic Sludge Digester

Rakia Chouari,1 Denis Le Paslier,1 Catherine Dauga,2 Patrick Daegelen,1 Jean Weissenbach,1 and Abdelghani Sghir1*

CNRS-UMR 8030, Genoscope and Université d'Evry Val d'Essonne, Evry,1 Génopole de l’Institut Pasteur, Paris, France2

Received 21 June 2004/ Accepted 26 October 2004


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ABSTRACT
 
In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.


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INTRODUCTION
 
The anaerobic sludge digester ecosystem has been partially described by classical methods (1), and several genera involved in the different steps of the degradation of organic matter to methane and carbon dioxide have been identified. However, to date there has been no comprehensive and exhaustive analysis of the microbial species composition in any anaerobic sludge digester. Despite the use of various media, generally microbiologists have found a remarkable difference in the number and morphology of organisms when comparing natural samples with those obtained from enrichment cultures and isolates from habitat-simulating or selective media. Application of modern molecular approaches, such as rRNA gene sequence analysis, has allowed the unambiguous assignment of cultivated and noncultivated organisms to their nearest phylogenetic neighbor and a better description of the complexity of the ecosystem. The number of bacterial groups has increased from 11 (25) to 50 to 55 main phylogenetic divisions; more than one-third are known only from detection of rRNA sequences and have no described cultivated representatives (18). These molecular inventories are based on the use of universal PCR primers described by Weisburg et al. (24) or Marshesi et al. (15). In many cases, DNA samples extracted from various sources were found to be poor templates for amplification of the 16S rRNA genes with certain so-called universal primers (15). However studies using bacterial division level-specific primers such as Planctomycete-Verrucomicrobia-specific primers revealed a huge hidden diversity at the subdivision level (3, 5). This diversity cannot be described based on the so-called bacterial universal primers, simply because most often these primers have many mismatches with their target in the 16S rRNA gene sequences of some important bacterial divisions.

In the present paper, using both qualitative and quantitative molecular approaches, we describe the occurrence of a novel high-order bacterial phylogenetic lineage never described before within an anaerobic sludge digester. Moreover we confirm the occurrence and extend the diversity of candidate divisions such as OD1, WS3, OP3, NBL-UPA2, and BRC1 previously described on the basis of a few phylotypes only (5, 11).

Anaerobic sludge samples were obtained from an anaerobic mesophilic digester at the Evry wastewater treatment plant (WWTP; 250,000 population equivalents), located about 35 km south of Paris, France. The digester temperature was 33°C, the pH was 7.2, and the digester had 37.5 days of retention time. Samples were centrifuged for 15 min at 20,000 x g. Sludge pellets were washed three times with phosphate-buffered saline and stored at –20°C. Genomic DNA extraction was performed as described by Chouari et al. (3). 16S rRNA genes were amplified from genomic DNA with a Planctomycetales-specific forward primer, Pla46F, and a universal reverse primer, 1390R (16). Molecular cloning and sequencing were done as described by Chouari et al. (3). The 16S rRNA gene sequences were treated as described by Ewing et al. (8) and compared with the complete EMBL nucleotide sequence databases. Sequences from EMBL with the best BLAST score were imported into the ARB data set when necessary (http://www.arb-home.de). Chimeric sequences were searched by the procedure described by Juretschko et al. (14) prior to phylogenetic analysis. All sequences with more than 1,200 nucleotides were imported into the ARB database and automatically aligned with the existing 16S rRNA gene sequences. The resulting alignments were manually checked and corrected when necessary, and 1,091 unambiguously aligned nucleotide positions were used for phylogenetic analyses. Phylogenetic placement was done in comparison with reference sequences representing the main lines of descent in the domain Bacteria, using the ARB program and database package. Tree topology was evaluated by applying neighbor-joining (NJ) analyses with Jukes and Cantor corrections and a 50% invariance criterion for inclusion of individual nucleotide sequence positions in the treeing analyses. We generated several trees which differed in the reference sequences, the set of alignment positions, and the outgroup sequence used. Maximum-parsimony (MP) and maximum-likelihood (ML) methods were also used. The statistical significance levels of interior nodes were determined by performing bootstrap analyses based on 100 resamplings by the NJ and MP methods. Overall, 16S rRNA gene sequence similarities were determined by using the distance matrix tool of the ARB program package (Fig. 1).



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  		FIG.1. Evolutionary distance dendrogram showing the affiliation of the environmental 16S rRNA gene sequences recovered from the anaerobic digester clone library to representative members of the divisions in the bacterial domain. The dendrogram was reconstructed by the NJ method in combination with a 50% consensus filter for the domain Bacteria with the ARB software package. The numbers at the nodes indicate the percentages of recovery of relevant branch points in 100 bootstrap resamplings (values above nodes by NJ, values below nodes by parsimony). The root was determined by using the archaeal 16S rRNA sequence of Methanobacterium formicicum (M36508) as the outgroup reference. EMBL, GenBank, and DDBJ accession numbers are given in parentheses. SR1, OP11, OD1, BRC1, OP3, and OP8 represent proposed candidate divisions. The scale bar represents the 10% estimated difference in nucleotide sequence positions.

To resolve the WWE1 position, a comprehensive set of spirochete 16S rRNA gene sequences was used in a separate analysis (Fig. 2). Aligned sequences were exported from the ARB database and analyzed with PAUP 4.0b10 (Sinauer Associates, Inc., Sunderland, Mass.). For MP analyses, we used a heuristic search strategy, the tree bisection reconnection (TBR) branch-swapping option. A strict consensus tree was drawn when multiple best trees were obtained. For the ML and NJ methods, the evolutionary model was evaluated with MODELTEST 3.0 (17). This test compared our sequence data matrix with various evolutionary models to identify models that best fit the data. We then performed phylogenetic reconstruction based on ML analyses, using the heuristic search strategy with TBR branch swapping. Distance analysis was performed with the same evolutionary model and the NJ algorithm (20). Statistical confidence levels for MP and NJ were evaluated by the nonparametric bootstrap method based on 100 resamplings. Bootstrap analyses for ML analysis were performed without TBR branch swapping to reduce computational time. Nodes represented in more than 90% of bootstrap replicates were considered strongly supported.



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  		FIG. 2. Evolutionary distance dendrogram constructed by the NJ method showing the affiliation of the environmental WWE1 16S rRNA gene sequences recovered from the anaerobic digester clone library to representative members of the closest divisions in the bacterial domain. A large data set of spirochete 16S rRNA gene sequences was used. Aligned sequences were analyzed by three methods (NJ, MP, and ML) provided by PAUP 4.0b10 as described in the text. The numbers at the nodes indicate the percentages of recovery of relevant branch points in 100 bootstrap resamplings (values above nodes by NJ, values below nodes by parsimony). The E. coli 16S rRNA gene sequence was used as the outgroup to define the root of the tree. The scale bar represent the 5% estimated difference in nucleotide sequence positions.

The probe search function of the ARB program software package was used to design new 16S rRNA probes, which were tested in silico with the "check probe" function program of RDP (http://rdp.cme.msu.edu/html/). For experimental validation of the WWE1-specific probe, since there is no cultivated microorganism representative of this novel division, 16S rRNA was generated by in vitro transcription of rRNA gene inserts of the WWE1 clones (Riboprobe in vitro transcription system; Promega). Washing temperature determination (Tw) of the new probe was achieved with washing buffer containing 3x SSC (1x SSC is 0.15 M NaCl plus 0.15 M sodium citrate) and 1% sodium dodecyl sulfate (SDS).

For dot blot hybridization (DBH) and quantification, total RNA was extracted from approximately 200 mg of six different digester samples (from April to December 2001) by bead beating, phenol extraction, and ethanol-sodium acetate precipitation as described previously (22, 23). The quality of the extracted RNA was checked by formaldehyde gel electrophoresis. Total rRNA was quantitatively normalized with a universal probe as described by Sghir et al. (22). Total rRNA of Escherichia coli (Boehringer, Mannheim, Germany) and a transcribed WWE1 rRNA gene were used as an rRNA standard. A total of 200 ng of RNA was blotted onto nylon membranes (Nytran Super Charge; Schleicher and Schuell) in triplicate and hybridized with 32P-labeled oligonucleotide probes (purchased from Eurogentec, Köln, Germany). A mixture of probes (EUB338, II, and III) was used to quantify specific bacterial rRNA as a percentage of the signal intensity obtained with a universal probe (Beta Imager; Packard Instruments). For the EUB probe mixture, we used 1% SDS and 1% SSC washing buffer under a Tw of 54°C. We determined the experimental Tw for the WWE1-specific probe, and we validated the probe using the same washing buffer composition. WWE1-specific rRNA was quantified and expressed as a percentage of total rRNA measured by EUB I, II, and III as described previously (22).

Fluorescent in situ hybridization (FISH) analyses of sludge samples were done as described by Chouari et al. (3). A stringent wash step was performed for 10 min at 48°C. The newly developed WWE1-specific probe was 5'-end labeled with Cy3 (MWG Biotech, Ebersberg, Germany) and used simultaneously with the fluorescein isothiocyanate-labeled EUB338 (a mixture of probes EUB I, II, and III). We have tested the specificity of the novel probe targeting the novel WWE1 lineage, using pure cultures of Bacillus firmus and Acinetobacter calcoaceticus as negative controls; no signal was obtained with these microorganisms, whereas they showed a positive signal when the mixture of EUB338 probes was used. The systematic use of unlabeled controls in our experiments confirmed that the bright cocci (Fig. 3A) corresponded with autofluorescence. Slides were visualized with a Zeiss axioplan epifluorescence microscope.



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  		FIG. 3. In situ identification of the WWE1 candidate division in an anaerobic municipal sludge digester, using Cy3-labeled WWE1-specific probe S-*-WWE1-1181-a-A-18. (A) Cells identified, marked with WWE1-specific Cy3-labeled probe. (B) EUB338, II, and III are green. (C) DAPI (4',6'-diamidino-2-phenylindole) staining. The bright cocci in panel A represent autofluorescence. Microscopic visualization was achieved with a Zeiss axioplan epifluorescence microscope.

Results obtained from previous work showed important planctomycete diversity in the aerobic and anoxic basins of a municipal WWTP, using a planctomycete-specific and universal primer set. However the 16S rRNA gene clone libraries generated from the anaerobic digester using the same primer set yielded a total of 396 rRNA gene sequences, of which only 4% of the clone libraries are affiliated with planctomycetes (3) (Table 1). The remaining 96% are affiliated with uncultured bacterial sequences and show a higher degree of diversity. We recovered 16S rRNA gene sequence types, which could not be assigned to the Planctomycetales but to two novel bacterial predominant groups we named WWE1 and WWE2 (for wastewater of Evry), represented by 85.9% (340 of 396 clones) and 4.5% (18 of 396 clones) of the clone library, respectively. Other 16S rRNA gene sequences are affiliated with the Verrucomicrobia, considered by Woese (25) as bacterial sister groups, and some other novel candidate divisions recently described by several authors, such as OD1 (11) or BRC1 and NBL-UPA2 (5).


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  		TABLE 1. Distribution of clone sequences and OTUs analyzed in the present studya

WWE1 and WWE2 are distantly related to 16S rRNA gene sequences of cultivated bacteria (Table 2 and Fig. 1). WWE2 exhibits a high-order diversity of rRNA gene sequences and is distantly affiliated with an unidentified 16S rRNA gene sequence (AB062771) retrieved from a termite gut (Coptotermus formosarus) with sequence homology ranging between 81.6 and 96.6%. This group of 18 16S rRNA gene sequences is monophyletic, encompassing a recently cultured bacterium, Victivallis vadensis, isolated from human feces (26). The authors placed this bacterium among a deeply branching group within the Verrucomicrobiales, but the present phylogenetic analysis using more closely related 16S rRNA gene sequences shows the WWE2 group branching independently from the Verrucomicrobia in all types of treeing analysis. The confidence node of WWE2 16S rRNA gene sequences is supported by a strong bootstrap value (≥97%). WWE2 seems to form a novel high-order phylogenetic cluster within the Bacteria domain comparable to Planctomycetes or Verrucomicrobia. They can be considered as a Planctomycetes, Verrucomicrobia, or Chlamydia sister group. Our results are corroborated by recent results of Cho et al. (2) showing that Victivallis vadensis could be rather placed within the new phylogenetic phylum of Lentisphaerae (Fig. 1). Thus, WWE2 16S rRNA gene sequences are strongly associated with this new phylum.


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  		TABLE 2. Affiliation of the bacterial 16S rRNA gene sequences analyzed in this study

WWE1 encompasses sequences affiliated with an unidentified clone 16S rRNA gene sequence of a clone retrieved from a methanogenic reactor (AY166854) (80.2 to 92.6% sequence similarity) (7) (Fig. 1 and Table 2). Phylogenetic analysis performed by Gu et al. (10) placed the latter clone sequence among a number of unidentified sequences deeply branching among a group of cultivated Spirochaetes 16S rRNA gene sequences, with ≤80% sequence homology. The authors' phylogenetic analysis was based on a small selection of 16S rRNA gene sequences. WWE1 also encompasses two 16S rRNA gene sequences (AJ390461 and AJ390485) retrieved from anoxic bulk soil of a flooded rice microcosm (5). In their phylogenetic analyses, Derakshani and coworkers showed that the two sequences cluster together in a monophyletic cluster they called SP lineage (with reference to spirochetes) showing distinct affiliation with the spirochetes with an overall level of 16S rRNA gene sequence dissimilarity ranging between 23 to 27%. If Leptospira spp., which form a deeply branching sublineage of spirochetes, are included in the phylogenetic analyses, the bootstrap support for assignment of SP lineage to the spirochete drops from 93% to 40 to 60%. In our study, we enriched this group of 16S rRNA gene sequences by 322 sequences. We used a comprehensive number of cultivated spirochete 16S rRNA gene sequences and included sequences from reports by Derakshani et al. (5) and Gu et al. (10): using the three treeing methods, phylogenetic analyses indicate that WWE1 and Spirochaetes are clearly making two monophyletic and distinct clusters (Fig. 2). We show that sequences from the reports by Derakshani or Gu et al., along with our WWE1 rRNA gene sequences, cluster in a monophyletic lineage with a bootstrap value of >93%. All treeing analyses using a number of different bacterial division sequence representatives show that WWE1 is a monophyletic lineage branching distinctly from Spirochaetes and other main lines of bacterial descent. Thus, WWE1 appears as a Spirochaetes division sister group, clearly distinguishable and forming a completely independent lineage (Fig. 2). 16S rRNA gene sequence dissimilarity between WWE1 and adjacent phyla (Lentisphaerae, Verrucomicrobia, and Planctomycetes; BRC1) is more than 20%. Sequence dissimilarity with the Spirochaetes phylum ranged between 20.9 and 28.7%. Hugenholtz et al. (12) suggested that 85% sequence identity be used as a cutoff for distinguishing new phyla; thus, by this criterion together with phylogenetic position, this clade was considered to be a novel phylum-level lineage in the domain Bacteria.

Comparison of 16S rRNA sequence signatures which distinguish among Bacteria, Archaea, and Eukarya was performed as described by Woese (25). Sequence signatures of WWE1 were clearly related to Bacteria and might be considered as Spirochaetes and Verrucomicrobia sister groups, respectively (data not shown). The percentage of intradivergence within WWE1 lineage is 29%, which is comparable to that of other well-established divisions (6).

Representative sequences of these lineages have also been retrieved from two other anaerobic digesters, such as the Corbeil (2 km upstream of Evry) and Creil (100 km downstream of Evry) WWTPs. They represented 14.7 and 19.2% of the generated clone libraries (data not shown). This means that microorganisms harboring such 16S rRNA gene sequences are environmentally widely distributed. They might be important components of the trophic chain of degradation of the organic matter under anaerobic conditions.

Quantitative DBH and FISH techniques are providing quantitative information on the abundance of a microorganism or a group of microorganisms, their shape and their spatial localization within the original environmental setting. We developed a WWE1-specific probe, S-*-WWE1-1181-a-A-18 (Table 3). The washing temperatures for the WWE1 probe was determined to be 50°C, using a washing buffer containing 1x SSC and 1% SDS as described by Sghir et al. (21, 22). Application of the probe using DBH on six different total RNA extracts from anaerobic digester samples showed that WWE1 rRNA represents 11.9% ± 3.1% of the total bacterial rRNA, which is comparable to the relative rRNA index of a bacterial phylum like Proteobacteria, represented by 14% of total rRNA (unpublished data). The same probe was Cy3-5'-end labeled and used to track representatives of WWE1 within the digester samples by the FISH technique. Results show that the probe binds to rods and filamentous shaped microorganisms but not spiral shaped organisms, which is a characteristic of the Spirochaetes phylum (Fig. 3). Similar results were obtained by Rossetti et al. (19) using the SPL998 16S rRNA probe, which targets a set of sequences affiliated with the WWE1 lineage (data not shown). This provides further support for considering this major lineage as a candidate division, which may be recovered from methanogenic environments. The novel probe described in this study will enable us to determine the distribution and population dynamics of the WWE1 lineage in environmental samples such as sludge digesters and termite, animal, or human gut.


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  		TABLE 3. Novel candidate division-specific probe S-*-WWE1-1181-a-A-18 based on the 5'->3' 16S rRNA sequence alignment

Sequences affiliated with other bacterial divisions such as the Verrucomicrobiales, OD1, BRC1, and NBL-UPA2 were also found in the clone library (Table 1 and Fig. 1). For the Verrucomicrobia, the phylogenetic tree depicted in Fig. 1 shows that besides the five Verrucomicrobia subdivisions defined by Hugenholtz et al. (13), a novel subdivision might be defined. Sequence similarity of all of those Verrucomicrobia sequences to the most closely affiliated 16S rRNA gene sequence ranges between 80.7 and 94.9%. The intradivergence of 20% known for this division increased with the newly characterized sequences to 22%. The novel sequences also show at least three mismatches with probe EUB338 III, which was designed for Verrucomicrobiales (4).

Twelve 16S rRNA gene sequences were grouped within a recently described candidate division, OD1 (11). Sequence similarity of OD1 rRNA gene sequences to known environmental sequences ranges between 77.4 and 79.6%. The BRC1 candidate division proposed on the basis of 11 sequences by Derakshani et al. (5) is represented by 12 sequences defining four operational taxonomic units (OTUs), showing between 77.9 and 85.6% similarity with the most closely affiliated uncultured soil bacterium AF390481 (Fig. 1). Thirteen more BRC1 sequences were recovered from aerobic and anoxic basins (data not shown). Other sequences were found to be affiliated with the candidate division NBL-UPA2 (5), represented by three sequences defining one OTU. Five more sequences were retrieved from the aerobic and anoxic basins (data not shown); they cluster together and show 90.6% similarity to the uncultured soil bacterium AJ390463. They extend this recently described candidate division and thus confirm the existence of this novel bacterial lineage (Fig. 1).

Due to the limitation imposed by the so-called universal primers, we explored a novel fruitful strategy based on the use of a specific division-level primer set for assessment of the diversity of quantitatively important but less known bacterial divisions. Many unique lineages were sampled within the digester ecosystem by using a specific primer set targeting the Planctomycetales division. These groups may constitute novel divisions or subphyla, greatly expanding the known higher-order diversity of Bacteria. The discovery of division or subphylum phylogenetic lineages is a step toward exploring the relationship between bacterial diversity and biogeochemical function within the digester ecosystems. However, further elucidation of their role in such complex ecosystem may require new culturing approaches or metagenomic studies that allow the linkage of functional and rRNA genes.


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Nucleotide sequence accession number.
 
Sequences reported in this study have been submitted to the EMBL, GenBank, and DDBJ databases under accession numbers CR933019 to CR933097.


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ACKNOWLEDGMENTS
 
This study was partly supported by a grant from the European Union for Research project WIRES (EVK1-CT2000-00050).

We are very grateful to S. Cure for reading the manuscript, the excellent technical assistance of the Genoscope sequencing team, and M. Trouvé and D. Dehon for providing samples from the WWTP.


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FOOTNOTES
 
* Corresponding author. Mailing address: CNRS-UMR 8030, Genoscope, 2, rue Gaston Crémieux, 91057 Evry, France. Phone: (33) 1 60 87 25 31. Fax: (33) 1 60 87 25 14. E-mail: sghir{at}genoscope.cns.fr. Back


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Applied and Environmental Microbiology, April 2005, p. 2145-2153, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2145-2153.2005
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