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

Abstract

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 (N 2 ) 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.

Environment-Dependent Distribution of the Sediment nifH -Harboring Microbiota in the Northern South China Sea

“…The "cluster IIIx" nifH genes of the ANME diazotrophic group (15) were only detected in the sampling stations of methane hydrate or its prospective areas (CF6, CF11, CF14, CF15, E407, and E422), with quite low abundance (3.88 ϫ 10 2 to 8.16 ϫ 10 3 copies g of sediment Ϫ1 ). Similarly, the nifD genes specific to the ANME diazotrophs (17) were also detected only in these sampling stations with low abundance (3.24 ϫ 10 2 to 5.84 ϫ 10 3 copies g of sediment Ϫ1 ). Previous studies indicate that the ANME diazotrophs are exclusively associated with the ANME-2c subgroup (15)(16)(17).…”

“…DNA concentrations were measured using PicoGreen (Molecular Probes, Eugene, OR) and a Modulus single-tube multimode reader fluorometer (Turner Biosystems, Sunnyvale, CA). Bacterial and archaeal nitrogenase reductase genes (nifH, including anfH and vnfH) were amplified with the primers nifHfw and nifHrv (12,15,17). To test the reproducibility of our experimental procedure and to identify any potential small-scale (ϳ20 cm) spatial variability of the sediment diazotrophic community, two separate nifH gene clone libraries (E407-I and E407-II) were constructed for sampling station E407, each from a distinct subcore DNA sample.…”

“…The abundances of sediment bacteria, archaea, nifH-harboring microorganisms, ANME diazotrophs, and methanogenic and methanotrophic archaea were quantified by using real-time fluorescent quantitative PCR (qPCR) methods with group-specific primers targeting, respectively, the 16S rRNA, nifH, nifD (encoding the ␣-subunit of nitrogenase molybdenum-iron protein), and mcrA (encoding the ␣-subunit of methyl-coenzyme M reductase) genes (see Table S1 in the supplemental material). All qPCR assays were carried out in triplicate with an ABI Prism 7500 sequence detection system (Applied Biosystems) using the SYBR green qPCR protocols (17,56). Reaction conditions of qPCR were optimized with reference plasmids carrying the respective target genes constructed in this and previous studies (15,51,56).…”

“…N 2 fixation, along with atmospheric N deposition, is the only source of new N that can lead to a net sequestration of atmospheric CO 2 in the deep ocean (31). In the marine benthic ecosystem, recent studies indicate that archaea, especially the anaerobic methanotrophic (ANME) archaeal functional guild (32), constitute a great portion of the in situ diazotrophic community and play an important role in supplying newly fixed nitrogen in deep-sea sediments rich in methane hydrates (14)(15)(16)(17). Due to the global distribution of methane hydrates in marginal sea sediments (4,33), the ANME diazotrophs' contribution to the marine and global N 2 fixation cannot be neglected.…”

“…Thus, diazotrophic microorganisms may be more widespread and play more important roles in marine environments than previously suggested. Although N 2 fixation (nitrogenase activity) is performed by numerous groups of microorganisms (9) and the nitrogenase reductase-encoding gene, nifH, has been frequently targeted as a functional biomarker for investigating the diazotrophic community in natural environments (10,11), the diversity and community structure of the nifH-harboring microorganisms are still poorly understood globally, and many important diazotrophs remain to be discovered (12)(13)(14)(15)(16)(17)(18).…”