Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach - PubMed (original) (raw)

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

Sonda Guermazi et al. Environ Microbiol. 2008 Aug.

Abstract

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA-DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.

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Figures

Fig. 1

Fig. 1

Maximum-likelihood phylogenetic tree showing the relationship of the environmental WWE3 sequences to representatives of the OP11, WS6, OD1 and TM7 divisions. Sequences were aligned with the ARB database and software package. Aligned sequences were analysed by three methods (BioNJ, maximum likelihood and maximum parsimony) provided by PAUP 4.0b10 as described in the text. A total of 1176 homologous positions was used for tree construction. The numbers at the nodes indicate the percentage of recovery of relevant branch points in 100 bootstrap re-samplings. The Anabaena circinalis 16S rDNA sequence was used as the outgroup to define the root of the tree. The scale bar represents the 10% estimated difference in nucleotide sequence positions.

Fig. 2

Fig. 2

WWE3 DIGA11YD11 16S rDNA secondary structure. This planar structure was determined using the 16S rRNA secondary structure from E. coli rrsA as a reference. Coloured spots indicate nucleotides not conserved between the two secondary structures: yellow, supplementary nucleotides; pink, both nucleotides of a base pair are different; green, only one nucleotide of the base pair is different; blue, loop nucleotide variants. The H17 stacked helix from E. coli rrsA is represented in grey. The first two nucleotides U437 and U438 and the last G497 of H17 (represented and circled in pale red) could correspond to nucleotides U410, U411 and G412 of DIGA11YD11, while the nucleotides C408 and G414 in DIGA11YD11 (represented and circled in red) probably correspond to nucleotides C436 and A498 in E. coli rrsA, in 5′ and 3′ of the H17 helix respectively. Tertiary interactions supported by strong comparative data (RDPII) are not represented except for the H1/H2 pseudoknot.

Fig. 3

Fig. 3

Epifluorescence micrographs of WWE3 bacteria in sludge samples from the anaerobic digester of Evry. A and D. Cy3-labelled DIGA11YD11-21-specific probe (red). B. FITC-labelled Eub338 probe mix (green). C. Colour combination of (A) and (B), WWE3 bacteria were not labelled by the Eub338 mix probe and then did not appear yellow. E. SYTO 9 staining (green). F. Colour combination of (D) and (E), WWE3 bacteria appear yellow.

References

    1. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995;59:143–169. - PMC - PubMed
    1. Baker GC, Smith JJ, Cowan DA. Review and re-analysis of domain-specific 16S primers. J Microbiol Methods. 2003;55:541–555. - PubMed
    1. Baker BJ, Tyson GW, Webb RI, Flanagan J, Hugenholtz P, Allen EE, Banfield JF. Lineages of acidophilic archaea revealed by community genomic analysis. Science. 2006;314:1933–1935. - PubMed
    1. Barbe V, Vallenet D, Fonknechten N, Kreimeyer A, Oztas S, Labarre L, et al. Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res. 2004;32:5766–5779. - PMC - PubMed
    1. Behrens S, Rühland C, Inácio J, Huber H, Fonseca A, Spencer-Martins I, et al. In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl Environ Microbiol. 2003;69:1748–1758. - PMC - PubMed

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