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Characterization of the archaeal community in a minerotrophic fen and terminal restriction fragment length polymorphism-directed isolation of a novel hydrogenotrophic methanogen.

ABSTRACT: Minerotrophic fen peatlands are widely distributed in northern latitudes and, because of their rapid turnover of organic matter, are potentially larger sources of atmospheric methane than bog peatlands per unit area. However, studies of the archaeal community composition in fens are scarce particularly in minerotrophic sites. Several 16S rRNA-based primer sets were used to obtain a broad characterization of the archaeal community in a minerotrophic fen in central New York State. A wide archaeal diversity was observed in the site: 11 euryarchaeal and 2 crenarchaeal groups, most of which were uncultured. The E1 group, a novel cluster in the order Methanomicrobiales, and Methanosaetaceae were the codominant groups in all libraries and results of terminal restriction fragment length polymorphism (T-RFLP) analysis. Given its abundance and potential hydrogenotrophic methane contribution, the E1 group was targeted for culture attempts with a low-ionic-strength medium (PM1). Initial attempts yielded Methanospirillum-dominated cultures. However, by incorporating a T-RFLP analysis as a quick selection tool for treatments and replicates, we were able to select an enrichment dominated by E1. Further dilutions to 10(-9) and tracking with T-RFLP yielded a strain named E1-9c. E1-9c is a novel coccoid hydrogenotrophic, mesophilic, slightly acidophilic methanogen and is highly sensitive to Na(2)S concentrations (requires <0.12 mM for growth). We propose E1-9c as the first representative of a novel genus in the Methanomicrobiales order.

SUBMITTER: Cadillo-Quiroz H

PROVIDER: S-EPMC2292581 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Characterization of the archaeal community in a minerotrophic fen and terminal restriction fragment length polymorphism-directed isolation of a novel hydrogenotrophic methanogen.

Cadillo-Quiroz Hinsby H Yashiro Erica E Yavitt Joseph B JB Zinder Stephen H SH

Applied and environmental microbiology 20080215 7

Minerotrophic fen peatlands are widely distributed in northern latitudes and, because of their rapid turnover of organic matter, are potentially larger sources of atmospheric methane than bog peatlands per unit area. However, studies of the archaeal community composition in fens are scarce particularly in minerotrophic sites. Several 16S rRNA-based primer sets were used to obtain a broad characterization of the archaeal community in a minerotrophic fen in central New York State. A wide archaeal ...[more]

PMID: 18281434

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Project description:Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

Dataset Information

Characterization of the archaeal community in a minerotrophic fen and terminal restriction fragment length polymorphism-directed isolation of a novel hydrogenotrophic methanogen.

Publications

Characterization of the archaeal community in a minerotrophic fen and terminal restriction fragment length polymorphism-directed isolation of a novel hydrogenotrophic methanogen.

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