Twenty-eight strains were selected on the basis of cell or colony morphology among the Mariana isolates for 16S rDNA analysis. Each strain was cultivated on an agar plate under appropriate growth conditions as shown in Table 2. One loop of cell pellet was suspended in 50 μl of 25 mM Tris-HCl, 50 mM glucose, 10 mM EDTA and 1 mg ml−1 of lysozyme. The cell suspension was incubated at 37°C for 20 min and 5 ml of 10% SDS solution was added to the cell suspension to extract chromosomal DNA. The DNA was purified by phenol/chloroform treatment and then used as template DNA for PCR amplification of 16S rDNA. The following prokaryote-specific primers were used for the gene amplification and sequencing: EU10F (5′-AGAGTTTGATCCTGGCTCAG-3′), EU1500R (5′-GGTTACCTTGTTACGACTT-3′), EU500F (5′-GTGCCAGCAGCCGCGG-3′), EU500R (5′-GTATTACCGCGGCTGCTG-3′), EU1100F (5′-AAGTCCCGCAACGAGCGCA-3′), and EU1100R (5′-TTGCGCTCGTTGCGGGACT-3′). The amplified fragments were purified with Suprec™-02 (Takara Shuzo Co., Ltd., Japan) and sequenced using LI-COR DNA sequencer Model 4000L (LI-COR, Inc., Nebraska, USA).
16S rDNA sequences were aligned using the Clustal multiple-alignment program (Clustal W). Sites involving gaps were excluded from all analysis. A phylogenetic tree was inferred by the neighbor-joining method. The sequences determined in this study were aligned with previously deposited sequences in the GenBank and Ribosomal Database of the University of Illinois at Urbana-Champaign. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL and GenBank nucleotide sequence databases under the accession numbers AB002633 for HTA333, AB002634 for HTA474, AB002635 for HTA275, AB002636 for HTA218, AB002637 for HTA064, AB002638 for HTA459, AB002639 for HTA212, AB002640 for HTA484, AB002641 for HTA563, AB002642 for HTA437, AB002643 for HTA506, AB002644 for HTA454, AB002645 for HTA426, AB002646 for HTA462, AB002647 for HTA1418, AB002648 for HTA1422, AB002649 for HTA1417, AB002650 for HTA1420, AB002651 for HTA1416, AB002652 for HTA1415, AB002653 for HTA473, AB002654 for HTA456, AB002655 for HTA557, AB002656 for HTA554, AB002657 for HTA527, AB002658 for HTA608, AB002659 for HTA580, AB002660 for HTA208, and AB002661 for alkaliphilic Bacillus sp. C-125.