Development of 16S rRNA gene-targeted primers for detection of archaeal anaerobic methanotrophs (ANMEs)

Authors

  • Ai Miyashita,

    1. Subsurface Geobiology Advanced Research (SUGAR) Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Kanagawa, Japan
    2. Department of Environmental Systems Engineering, Nagaoka University of Technology, Niigata, Japan
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  • Hanako Mochimaru,

    1. Geomicrobiology Research Group, Institute for Geo-Resources and Environment, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan
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  • Hiromi Kazama,

    1. Subsurface Geobiology Advanced Research (SUGAR) Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Kanagawa, Japan
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  • Akiyoshi Ohashi,

    1. Department of Social and Environmental Engineering, Hiroshima University, Hiroshima, Japan
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  • Takashi Yamaguchi,

    1. Department of Environmental Systems Engineering, Nagaoka University of Technology, Niigata, Japan
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  • Takuro Nunoura,

    1. Subsurface Geobiology Advanced Research (SUGAR) Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Kanagawa, Japan
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  • Koki Horikoshi,

    1. Subsurface Geobiology Advanced Research (SUGAR) Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Kanagawa, Japan
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  • Ken Takai,

    1. Subsurface Geobiology Advanced Research (SUGAR) Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Kanagawa, Japan
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  • Hiroyuki Imachi

    1. Subsurface Geobiology Advanced Research (SUGAR) Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Kanagawa, Japan
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  • Editor: Mariet Hefting

Correspondence: Hiroyuki Imachi, Subsurface Geobiology Advanced Research (SUGAR) Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan. Tel.: +81 46 867 9709; fax: +81 46 867 9715; e-mail: imachi@jamstec.go.jp

Abstract

Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.

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