Previous Article | Next Article 
Applied and Environmental Microbiology, November 2009, p. 7153-7162, Vol. 75, No. 22
0099-2240/09/$08.00+0 doi:10.1128/AEM.01184-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Molecular Characterization of Potential Nitrogen Fixation by Anaerobic Methane-Oxidizing Archaea in the Methane Seep Sediments at the Number 8 Kumano Knoll in the Kumano Basin, Offshore of Japan
,
Junichi Miyazaki,1*
Ryosaku Higa,2
Tomohiro Toki,2
Juichiro Ashi,3
Urumu Tsunogai,4
Takuro Nunoura,1
Hiroyuki Imachi,1 and
Ken Takai1
Subground Geobiology and Advanced Research Project, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan,1 Department of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan,2 Department of Ocean Floor Geoscience, Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakanoku, Tokyo 164-8639, Japan,3 Earth and Planetary System Science, Faculty of Science, Hokkaido University, N10 W8, Kita-ku, Sapporo 060-0810, Japan4
Received 22 May 2009/ Accepted 18 September 2009
The potential for microbial nitrogen fixation in the anoxic methane seep sediments in a mud volcano, the number 8 Kumano Knoll, was characterized by molecular phylogenetic analyses. A total of 111 of the nifH (a gene coding a nitrogen fixation enzyme, Fe protein) clones were obtained from different depths of the core sediments, and the phylogenetic analysis of the clones indicated the genetic diversity of nifH genes. The predominant group detected (methane seep group 2), representing 74% of clonal abundance, was phylogenetically related to the nifH sequences obtained from the Methanosarcina species but was most closely related to the nifH sequences potentially derived from the anoxic methanotrophic archaea (ANME-2 archaea). The recovery of the nif gene clusters including the nifH sequences of the methane seep group 2 and the subsequent reverse transcription-PCR detection of the nifD and nifH genes strongly suggested that the genetic components of the gene clusters would be operative for the in situ assimilation of molecular nitrogen (N2) by the host microorganisms. DNA-based quantitative PCR of the archaeal 16S rRNA gene, the group-specific mcrA (a gene encoding the methyl-coenzyme M reductase 
subunit) gene, and the nifD and nifH genes demonstrated the similar distribution patterns of the archaeal 16S rRNA gene, the mcrA groups c-d and e, and the nifD and nifH genes through the core sediments. These results supported the idea that the anoxic methanotrophic archaea ANME-2c could be the microorganisms hosting the nif gene clusters and could play an important role in not only the in situ carbon (methane) cycle but also the nitrogen cycle in subseafloor sediments.
* Corresponding author. Mailing address: Subground Geobiology and Advanced Research Project, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan. Phone: 81 46 867 9692. Fax: 81 46 867 9715. E-mail: miyazaki11{at}jamstec.go.jp
Published ahead of print on 25 September 2009.
Supplemental material for this article may be found at http://aem.asm.org/.
Applied and Environmental Microbiology, November 2009, p. 7153-7162, Vol. 75, No. 22
0099-2240/09/$08.00+0 doi:10.1128/AEM.01184-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»