Caldisericum exile gen. nov., sp. nov., an anaerobic, thermophilic, filamentous bacterium of a novel bacterial phylum, Caldiserica phyl. nov., originally called the candidate phylum OP5, and description of Caldisericaceae fam. nov., Caldisericales ord. nov. and Caldisericia classis nov.

A novel anaerobic, thermophilic, thiosulfate-reducing bacterium, strain AZM16c01^T, was isolated from a hot spring in Otari, Nagano Prefecture, Japan (Mori et al., 2008). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain AZM16c01^T was not closely related to any of the recognized phyla and was reproducibly placed in a candidate phylum called OP5. The candidate phylum OP5 was represented originally by environmental clone sequences retrieved from the Obsidian Pool in Yellowstone National Park (Hugenholtz et al., 1998a) and indicated an independent phylum-level lineage in the domain Bacteria (Hugenholtz et al., 1998a, b; Mori et al., 2008; Rappé & Giovannoni, 2003). Since then, environmental clone sequences belonging to this phylum have been detected in various environments (Dojka et al., 1998; Humayoun et al., 2003; Inagaki et al., 2006; Kaksonen et al., 2004; Ley et al., 2006; Skirnisdottir et al., 2000; Teske et al., 2002). Strain AZM16c01^T was the first cultivable isolate accommodated in OP5. In the present study, we have characterized strain AZM16c01^T in detail and propose the name Caldisericum exile gen. nov., sp. nov. for this strain. In addition, the higher taxa of the genus Caldisericum are proposed in this paper.

Cells of strain AZM16c01^T were multicellular filaments (0.3 μm in width and 1.6–8.3 μm in length) with a single polar flagellum and formed complicated aggregates (Fig. 1 a–c⇓). Observation of negative-staining with a transmission electron microscope (model H-7600; Hitachi) revealed that cells of strain AZM16c01^T possessed an envelope outside of the cell membrane (Fig. 1c⇓). Although this envelope was sometimes observed in ultrathin sections, it was composed of an electron-lucent material and often could not be observed (Fig. 1d–h⇓). The cell-wall structure of strain AZM16c01^T was shown to be Gram-negative, by using both conventional Gram-staining and Gram test with 3 % (w/v) KOH (Powers, 1995). Stalk structures (Fig. 1e and g⇓) and intracellular vacuole-like structures (Fig. 1e and h⇓) were often observed. Motility and spore formation were not observed. Strain AZM16c01^T was negative for catalase and oxidase activities.

Cellular fatty acids, respiratory quinones and the genomic DNA G+C content were analysed using methods described previously by Mori & Suzuki (2008). Strain AZM16c01^T possessed iso-C_17 : 0 (49 % of the total fatty acids) as the predominant fatty acid; iso-C_15 : 0 (23 %), iso-C_17 : 0 3-OH (16 %), C_16 : 0 (8 %) and anteiso-C_17 : 0 (4 %) were also present. Strain AZM16c01^T possessed the respiratory quinones menaquinone-8(H₆), menaquinone-8(H₈) and menaquinone-8(H₁₀) (39, 40 and 21 % of the total isoprenoid quinones, respectively). The genomic DNA G+C content of strain AZM16c01^T was 34.6 mol%.

For a detailed study of the cell envelope, we attempted to extract the cell wall to analyse its peptidoglycan content by using the method employed for Gram-positive bacteria (Schleifer & Kandler, 1972; Yamaguchi, 1965); however, our attempt was unsuccessful. To investigate whether the component of the cell wall was peptidoglycan or protein S-layer, we observed negative-stained cells using an electron microscope after enzyme treatments with lysozyme [Nacalai Tesque; 2 mg l⁻¹ in 10 mM Tris buffer (pH 8) containing 1 M urea], proteinase K [Nacalai Tesque; 2 mg l⁻¹ in 10 mM Tris buffer (pH 8) containing 1 mM Na₂EDTA and 1 M urea], proteinase mix (Pronase, Roche; 2 mg l⁻¹ in 10 mM Tris buffer containing 1 mM Na₂EDTA and 1 M urea) or trypsin (Nacalai Tesque; 2 mg l⁻¹ in a solution comprising 1 mM Na₂EDTA, 72 mM NaCl, 2.6 mM KCl, 0.2 mM NaH₂PO₄ . 2H₂O and 1 M urea, adjusted to pH 8 with 8 % NaHCO₃) at 37 °C for overnight. Compared with untreated cells in 10 mM Tris buffer containing 1 M urea, the cells treated with lysozyme, proteinase K and proteinase mix were shrunk in the envelope, but these digestions could not remove the envelope (data not shown). Because of these results, we could not come to a conclusion on the outer envelope structure. However, the detection of 3-hydroxy fatty acid in the cellular fatty acid analysis suggested that strain AZM16c01^T possessed an outer membrane identical to that of Gram-negative bacteria.

As indicated in a previous report (Mori et al., 2008), strain AZM16c01^T could grow on yeast extract but not on any other substrate in the presence of sulfur compounds (thiosulfate, sulfite and elemental sulfur) as electron acceptors. Optimum conditions for growth with 10 mM thiosulfate and 10 g Bacto yeast extract l⁻¹ (Difco) were determined by assessing the time-course of optical density with a spectrophotometer (model U-2800; Hitachi) and the decrease in thiosulfate concentration by using HPLC (Mori & Suzuki, 2008). The results are shown in Supplementary Fig. S1 (available in IJSEM Online). Strain AZM16c01^T could grow between 55 and 70 °C, the optimum temperature being 65 °C. The pH range for growth was 5.5–7.5 and the optimum pH was 6.5. Strain AZM16c01^T was very sensitive to the NaCl concentration. Optimum growth was observed when no NaCl was added to the medium. Although growth was observed in 0.5 and 1 % (w/v) NaCl, the cell concentration increased only scarcely and the amount of thiosulfate reduction was very slight. Strain AZM16c01^T had a doubling time of 10.6 h under the optimum growth conditions (65 °C, pH 6.5 and in 0 % NaCl).

Phylogenetic analysis based on the 16S rRNA gene sequence (Mori et al., 2008) indicated that strain AZM16c01^T belonged to the OP5 lineage (Fig. 2⇓). This lineage was independent at the phylum level from other bacteria, and the branching point of the OP5 lineage was strongly supported with 93 and 97 % bootstrap values by using the neighbour-joining and maximum-likelihood methods, respectively. The neighbour-joining tree of the OP5 phylum is shown in Fig. 3⇓. This phylum consisted mainly of clone sequences obtained from anaerobic environments, and the sequence divergence for the phylum was 27.2 %.

The aim of this study was to characterize in detail strain AZM16c01^T, isolated from the Otari hot spring. The phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain AZM16c01^T belonged in the OP5 lineage described by Hugenholtz et al. (1998a) and Rappé & Giovannoni (2003). In addition, strain AZM16c01^T was not closely related to any of the known bacteria in recognized phyla, with less than 85 % sequence similarity (Mori et al., 2008), and was sufficiently distinct to represent a new bacterial phylum (Hugenholtz et al., 1998a). The low DNA G+C content of strain AZM16c01^T and the presence of the highly saturated menaquinone-8 in strain AZM16c01^T were not common in members of the domain Bacteria (Collins, 1994). Also, the fatty acid profile was typical of thermophiles and resembled particularly that of the phylum ‘Deinococcus-Thermus’ (Garrity & Holt, 2001). Considering the morphological observations, although the occurrence of 3-hydroxy fatty acid strongly suggested the presence of an outer membrane containing lipopolysaccharide as observed for Gram-negative bacteria, it would be interesting to analyse the cell envelope of this organism in detail in a further study. In conclusion, the phylogenetic and physiological findings indicated that strain AZM16c01^T represents a new genus and novel species within the candidate phylum OP5. Thus, we propose the name Caldisericum exile gen. nov., sp. nov. for this strain. In addition, corresponding to the candidate phylum OP5, we propose the phylum name Caldiserica phyl. nov., after C. exile, the first and sole representative of the phylum.