A New Member of the Order Sulfolobales, Sulfurisphaera ohwakuensis gen. nov., sp. nov., and Its Family B DNA Polymerase
Norio KUROSAWA*a, Yuko H. ITOHa, Toshie IWAIa, Akihiko SUGAIb, Ikuko UDAb, Haruko HIROSEc, Naohiro KIMURAa, Tadao HORIUCHIa, and Toshihiro ITOHb
a Department of Bioengineering, Faculty of Engineering, Soka University 1-236 Tangi-cho, Hachioji, Tokyo 192, Japan
b Division of Chemistry, Center of Liberal Arts and Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan
c Material Analysis Research Center, Teijin Limited, 4-3-2 Asahigaoka, Hino, Tokyo 191, Japan
In the sulfur-dependent thermoacidophilic archaea, four genera; Sulfolobus, Metallosphaera, Acidianus, and Stygiolobus have been assigned to the order Sulfolobales. All members of the order Sulfolobales are acidophilic cocci. Sulfolobus spp. and Metallosphaera spp. is an obligate aerobe. In contrast, Acidianus spp. are facultative anaerobes. Stygiolobus sp. is an obligate anaerobe. During recent attempts to isolate additional thermoacidophilic archaeal species from hot springs in Hakone, Japan, three spherical thermoacidophilic archaea (strains TA-1, TA-13, and TA-14) were obtained. The partial 16S rRNA gene sequence (about 650 bp) from these strains possessed over 99% sequence similarities with each other. All the isolates are facultatively anaerobic, and grew optimally at around 85℃, pH 2.0.
Thin sections of cells from isolate TA-1 revealed an S-layer about 24 nm wide covering the cell membrane. Cells grow at temperatures ranging from 63℃ to 92℃, with optimal growth occurring at 84℃. The pH suitable for growth ranges from 1.0 to 5.0, with optimal growth around pH 2.0. The doubling time was 5.9 h under optimal conditions. Cells contain caldarchaeol and calditoglycerocaldarchaeol as their core lipids. The G+C content of DNA from TA-1 is 33 mol%. Although these properties resemble those of the genus Acidianus, the sequence of the 16S rRNA gene from strain TA-1 was more similar to the sequences of species of Stygiolobus than Acidianus. DNA-DNA hybridization experiments also indicated that strain TA-1 is clearly distinguished phylogenetically from the members of Acidianus, Sulfolobus, and Metallosphaera. On the basis of the distinct physiological and molecular properties, we describe the new strains as members of the new genus Sulfurisphaera. The type species and type strain is Sulfurisphaera ohwakuensis, TA-1 (IFO 15161).
We cloned and sequenced the gene encoding the DNA polymerase from isolate TA-1. The gene was found to encode a 872 residues polypeptide chain with a deduced molecular mass of 101 kDa. By comparison with other archaeal genes, a putative transcription promoter sequences were identified in the 5'-flanking region. The conserved motifs characteristic of family B DNA polymerases which are assumed to polymerization and exonuclease domains have been found in the deduced primary structure of this enzyme. Expression in E. coli, purification and characterization of this enzyme are currently in progress.
