Parts of the Mn crusts and sediments were transferred to a DNA/RNA-free plastic tube and stored at −80 °C until DNA extraction. One liter of the seawater sample was filtered with a 0.2-μm-pore-size Rapamycin price polycarbonate membrane to trap the suspended particles (Advantec, Tokyo, Japan) on board and then the filter was stored in a DNA/RNA-free plastic tube at −80 °C until DNA extraction. Analysis of the 16S rRNA genes present in the collected

solid and liquid samples was performed as described previously (Kato et al., 2009c, 2010). In brief, genomic DNA was extracted from the samples using a Fast DNA kit for soil (Qbiogene, Carlsbad, CA). Partial 16S rRNA genes were amplified by PCR with the prokaryote-universal primer set, Uni515F and Uni1406R. The PCR products were cloned using a TOPO TA cloning kit (Invitrogen, CA). The nucleotide sequences of randomly selected clones were determined using M13 forward and reverse primers (Invitrogen) on an ABI PRISM 3130xl Genetic analyser (Applied Biosystems, CA). Nucleotide sequences were aligned

and distance matrices were generated from alignment data sets from each clone library using arb (Ludwig et al., 2004). Clones having 97% sequence similarity or higher were treated as the same phylotype using dotur (Schloss & Handelsman, 2005). Maximum-likelihood trees were constructed using phyml (Guindon & Gascuel, 2003) with non-gap homologous positions in the alignment dataset. Bootstrap values were estimated using 100 replicates. Rarefaction analysis, the Poziotinib manufacturer Shannon diversity index and Chao1 richness estimators were estimated using dotur based on the distance matrices generated from the alignment data sets of the clones from each clone library. Chao1 species richness estimates of shared phylotypes were calculated using sons (Schloss & Handelsman, 2006). The phylogenetic (P)-test

and the UniFrac significance test were performed using UniFrac (Lozupone et al., 2006). Bacterial and archaeal rRNA gene copy numbers in DNA extracts from each sample were determined by Q-PCR as described previously (Kato et al., 2009b). For bacterial rRNA genes, the bacterial-specific PCR primers, Bac1369F (5′-CGGTGAATACGTTCYCGG-3′) and Prok1492R (5′-GGWTACCTTGTTACGACTT-3′), and the TaqMan probe, TM1389F (5′-CTTGTACACACCGCCCGTC-3′), were used. For archaeal rRNA genes, the archaeal ADAM7 PCR primers, Arc349F (5′-CCTACGGGRBGCASCAG-3′) and Arc806R (5′-GGACTACNNGGGTATCTAAT-3′), and a TaqMan probe, Arc516F (5′-TGYCAGCMGCCGCGGTAAHACVNRS-3′), were used. The purified PCR products from the 16S rRNA gene of Escherichia coli and environmental archaeal clones belonging to Marine group I (MGI) were used as the standard DNA for bacterial and archaeal analyses, respectively. All assays were performed in triplicate. Regression coefficient (r2) values of the standard curve were 0.994 and 0.999 for bacterial and archaeal analyses, respectively.