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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

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.

* 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.

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.



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