07 N NaOH (pH 12.9); (2) 65 °C and 0.33 N NaOH; and (3) 94 °C and 0.07 N NaOH. Incubation time ranged from 30 to 90 min for the three other conditions. Using a universal primer set of Univ340F and Univ806R for prokaryotes, 16S rRNA gene sequences were amplified by PCR using LA Taq polymerase (TaKaRa Bio, Shiga, Japan). A reaction mixture was prepared in which concentration of each oligonucleotide primer was 0.1 μM and that of the DNA template was ca. 1.0 μL. Thermal cycling was performed as described previously (Takai & Horikoshi, 2000). A PCR product

with the expected size was confirmed Obeticholic Acid supplier by electrophoresis on TAE (40 mM Tris acetate, 1 mM EDTA, pH 8.3) agarose gels (1%), which was purified using an UltraClean PCR Clean-up Kit (MoBio Laboratories). Purified PCR product was cloned into vector pCR2.1 using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Plasmid DNA from the clones was sequenced with the ABI Big-Dye reaction mix using the vector M13 primers. Sequence similarity among all of the partial sequences was analyzed using the FASTA program equipped with DNAsis software (Hitachi Software, Tokyo, Japan). Partial 16S rRNA gene sequences with more than 97%

similarity were grouped in one 16S rRNA gene sequence type (phylotype), and the representative sequences were applied to sequence similarity analysis by fasta against DDBJ database (http://www.ddbj.nig.ac.jp/index-e.html). Fossariinae Classification of bacteria in this research was based on the NCBI taxonomy database (Sayers et al., 2009). It is well known that alkaline and heat treatments cause denaturation and fragmentation of DNA Staurosporine molecular weight (Ageno et al., 1969; Hill & Fangman, 1973). To determine the extent of DNA scission under different extraction conditions, cultured cells of P. stutzeri

were subjected to DNA extraction under various conditions described earlier. DNA fragmentation was evaluated by qPCR analysis using the primers that target 467 bp of the 16S rRNA gene (Univ340F and Univ806R). Additionally, fragmentation of the DNA extracts was also evaluated by electrophoresis. To visualize the size of the extracted DNA, 5 μL of extract solution was run on TAE agarose gels. Gel was stained for 30 min with 0.1 g mL−1 ethidium bromide, and gel images were captured using a Gel Doc EZ System (Bio-Rad Laboratories). Mineralogical composition of the sediment sample was determined by X-ray diffraction pattern analysis using an X-ray diffractometer (Rigaku RINT Ultima III). Powdered samples were mounted on glass holders, and diffraction patterns were obtained using a scan rate of 5° per min with monochromatized CuKa radiation at 30 kV and 15 mA. Relative abundance of opal-CT was calculated based on the ratio of peak heights of quartz and opal-CT. To dissolve the silica biomineral and cell membranes, the cell is incubated in 0.33 N NaOH at 94 °C for 5 min and at 65 °C for 1 h.