As a deep subsurface biosphere is thought to be the biggest biosphere in the earth, it is very interesting to know the phylogenetic and functional... » More

As a deep subsurface biosphere is thought to be the biggest biosphere in the earth, it is very interesting to know the phylogenetic and functional diversity in such environments. However, there is a little biological information for them because it is very hard to recover whole microbial community by only culture-base methods. In that sense, metagenomics is one of major useful methods to elucidate the microbial flora in unknown biosphere, which seems to be constructed by mainly unculturable microbes. We used eight kinds of sediment core sample, which are 9 cm in diameter and 20 cm in length for metagenomic analysis from among all samples recovered by the ocean drilling to 365 meter below seafloor (mbsf) of the D/V Chikyu shakedown cruise in an offering of the Shimokita Peninsula on the northeast Honshu, Japan. DNA isolation directly from each sample was carried out by means of an improved beads beating method with a combination of beads beating and lytic enzymatic treatments. The amount of DNA isolated from the core samples decreased with an increasing of depth from seafloor and only several nanogram of DNA per 5g-core sample was isolated from the deepest layer (348 mbsf) although the total amount of DNA was enough for PCR amplification of 16S rRNA gene. However, since the amount of DNA was too small for construction of metagenomic libraries in some core samples, we attempted to amplify genomic DNA using GenomiPhi. Two thousands archaeal and 1000 bacterial 16S rDNA clones from each sample were sequenced and analyzed phylogenetically to figure out the vertical profile of prokaryotic community in the sediment from the seafloor to 348 mbsf. The patterns of archaeal and bacterial 16S rDNA varied depending on the layer depth and the majority of 16S rDNA were categorized into subsurface group 2 & 3 and a-proteobacteria in the deepest layer, respectively. We constructed the metagenomic shotgun libraries in 5 selected core samples ranging from 0.7 to 107 mbsf and have sequenced both ends of 40,000 clones of each library to investigate a feature of the functional genes in each layer depth.

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