Euzebya tangerina gen. nov., sp. nov., a deeply branching marine actinobacterium isolated from the sea cucumber Holothuria edulis, and proposal of Euzebyaceae fam. nov., Euzebyales ord. nov. and Nitriliruptoridae subclassis nov.

In the course of searching for novel lineages of bacteria from the intestine and epidermis of marine creatures, a novel bacterial strain, F10^T, was isolated, and this strain was shown to form a deep branch in the class Actinobacteria. As the field of marine actinobacterial research is still in its early stages (Bull et al., 2005), the isolation and taxonomic characterization of strain F10^T promises to increase our understanding of marine actinobacteria.

Stackebrandt et al. (1997) proposed a hierarchical classification system for the actinobacteria by conducting 16S rRNA gene sequence analyses and by distinguishing between patterns of signature nucleotides. As a result, the phylum Actinobacteria was classified into 5 subclasses within one class Actinobacteria Stackebrandt et al. 1997: Acidimicrobidae Stackebrandt et al. 1997, Rubrobacteridae Stackebrandt et al. 1997, Coriobacteridae Stackebrandt et al. 1997, Actinobacteridae Stackebrandt et al. 1997 and Sphaerobacteridae Stackebrandt et al. 1997. Sphaerobacter thermophilus was originally described by Demharter et al. (1989) and was subsequently placed in the subclass Sphaerobacteridae, which contains only the order Sphaerobacterales, the family Sphaerobacteraceae, the genus Sphaerobacter, and one species, S. thermophilus. More recently, Hugenholtz & Stackebrandt (2004) proposed the reclassification of S. thermophilus from the subclass Sphaerobacteridae in the phylum Actinobacteria to the class Thermomicrobia in the phylum Chloroflexi (green non-sulfur bacteria) on the basis of evolutionary distance and Bayesian inference. Additionally, Zhi et al. (2009) proposed an update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria. In a preliminary phylogenetic study, F10^T was classified to be a member of a novel order, that formed a distinct clade with the order Nitriliruptorales in the class Actinobacteria. The order Nitriliruptorales was recently described by Sorokin et al. (2009) within the class Actinobacteria, but the subclass to which the new order belonged was not defined.

Strain F10^T was isolated from the abdominal epidermis of a sea cucumber, Holothuria edulis, which had been collected off the coast of Aka Island, Okinawa prefecture, Japan, at a depth of 6 m. The novel marine actinobacterium, Iamia majanohamensis F12^T, was isolated from the same specimen (Kurahashi et al., 2009). Strain F10^T was isolated on SN medium agar plates as described by Kurahashi et al. (2009) after incubation at 25 °C for approximately 5 weeks. Subcultivation of the isolate was carried out on marine agar 2216 (MA; Becton Dickinson) at 20 °C. Stock cultures of the isolate in marine broth 2216 (Becton Dickinson) with 5 % DMSO were preserved at −80 °C.

Unless indicated otherwise, the phenotypic characteristics of the isolate were determined using MA at 25 °C. Cultures were incubated for 5 weeks to determine colony morphology, size and colour. To test the pH range for growth, 20 μl cell suspensions were transferred to test tubes containing 5 ml filter-sterilized TYSW (2 g tryptone and 1 g yeast extract dissolved in 1 l sterile artificial seawater) at pH 2–10, and incubated at 25 °C. Salt tolerance was tested with R2A agar (Becton Dickinson) suspended in seawater desalinated by means of an osmotic membrane. The medium was supplemented with 0–15 % NaCl and incubated at 25 °C for 5 weeks. The Gram reaction was carried out using a Gram Stain kit (Becton Dickinson), according to the manufacturer's instructions. The test for anaerobic growth was performed with a GasPak anaerobic jar system (Mitsubishi Gas Chemical). Cell motility and morphology were observed by using phase-contrast microscopy and transmission electron microscopy (TEM; H7600, Hitachi). Specimens for TEM were negatively stained with phosphotungstic acid. The biochemical characteristics of the isolate were examined by using a combination of standard approaches (Fukunaga et al., 2006), and by inoculating API 20E strips (bioMérieux) according to the manufacturer's instructions, with the exception that the inocula for the API 20E strips were prepared in sterile artificial seawater.

Chemotaxonomic analyses of the major respiratory quinones, and the DNA G+C content as well as determination of the whole-cell fatty acid, amino acid and sugar compositions, were performed as described by Kurahashi & Yokota (2004). The chemotaxonomic characteristics were determined using MA at 25 °C, unless otherwise indicated. The cellular fatty acids in cell mass grown on half-strength MA for 5 weeks at 25 °C were analysed according to the standardized procedures of the Microbial Identification System (MIDI). Whole-cell or cell-wall hydrolysates were analysed for their amino acid and sugar compositions by TLC (Hasegawa et al., 1983) and HPLC; the amino acid phenylthiocarbamoyl derivatives were identified according to the manufacturer's instructions (Wako Pure Chemical Industries, 1989). After the cells had been cultured for 5 weeks, purified cell walls were obtained by using the method of Schleifer & Kandler (1972). Polar lipids were extracted and purified by the method of Minnikin et al. (1984), and were identified by two-dimensional TLC followed by spraying with the detection reagents (Komagata & Suzuki, 1987). The N-acyl type of muramic acid was determined by the colorimetric method of Uchida & Aida (1977).

The phylogenetic relationships of strain F10^T were examined by 16S rRNA gene sequencing, as described by Kurahashi & Yokota (2004). To reveal the exact taxonomic affiliation of strain F10^T in the domain Bacteria, the F10^T sequence was aligned against an arb dataset using the arb program package (Ludwig et al., 2004).

The sequence was compared to reference 16S rRNA gene sequences using the fasta program (Pearson, 1994). Alignments of 16S rRNA gene sequences of the isolate and related strains were carried out with clustal x software (version 1.8) (Thompson et al., 1994). The evolutionary distance matrix was calculated using the Kimura two-parameter method (Kimura, 1980). Distances and clustering with the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) algorithms were determined by using bootstrap values based on 1000 sample replications according to the method of Felsenstein (1993).

After 5 weeks at 25 °C, the colonies grown on MA were circular and pulvinate along the entire edge, tangerine in colour, nearly opaque, and the texture was hard. Strain F10^T formed rod-shaped cells ranging from 1.5 to 6.0 μm in length and 0.6 to 0.8 μm in width (see Supplementary Fig. S1, available in IJSEM Online). Cells of strain F10^T showed Gram-positive staining and were non-spore-forming and non-motile. The optimal growth temperature was between 20 and 28 °C. No growth occurred at 10 or 40 °C. Growth occurred in NaCl concentrations of 0.5–12 %, whereas no growth occurred in 0 or 15 % NaCl. The pH range for growth was pH 7–9, while no growth was observed at either pH 6 or pH 10. Cells liquid dried (Sakane & Kuroshima, 1997) in preservation medium SM 1 [per litre of 0.1 M phosphate buffer solution (pH 7.0): 30 g sodium glutamate, 15 g ribitol and 0.5 g l-cysteine monohydrochloride] lost viability upon long-term preservation (one month at 25 °C). The physiological and biochemical characteristics of the isolate are summarized in the genus and species descriptions below.

The predominant menaquinone in the novel isolate was MK-9(H₄) (98.8 %). This differs from that of the closely related genera Iamia, Acidimicrobium and Frankia (Table 1⇓). The genomic DNA G+C content of strain F10^T was found to be 68.3 mol%, which is lower than that of I. majanohamensis (74.4 mol%). The cellular fatty acid composition of strain F10^T is given in Table 2⇓; the predominant fatty acids identified were C_16 : 1ω7c, C_16 : 0 and C_17 : 1ω8c. This profile also differs from that of the phylogenetically closely related N. alkaliphilus; however, discrepant findings were reported by Sorokin et al. (2009), which may reflect the use of different media and temperatures for the growth of the biomass. The cell-wall peptidoglycan of strain F10^T contained glutamic acid, alanine and meso-diaminopimelic acid in a molar ratio of 1 : 2 : 1; this finding corresponds to peptidoglycan type A1γ, according to Schleifer & Kandler (1972). The acyl type of the muramic acid in the peptidoglycan was acetyl. However, the cell-wall muramic acid of the phylogenetically closely related genus Iamia was of the glycolyl type. Unfortunately, in the genera Nitriliruptor and Acidimicrobium, the type of muramic acid has not been defined. The cell-wall sugars of strain F10^T were rhamnose and galactose. However, the type strain of the genus Iamia also contains mannose, arabinose and xylose, in addition to rhamnose and galactose. The phospholipids included phosphatidylglycerol as well as several unidentified glycolipids and glycophospholipids.

The 16S rRNA gene analysis placed strain F10^T within the class Actinobacteria. Similarity values calculated for isolate F10^T indicated a remote relationship (less than 88 % similarity) with members of the class Actinobacteria. Sequence-similarity calculations indicated that strain F10^T showed the greatest degree of similarity to N. alkaliphilus EF422408 (87.4 %), Acidothermus cellulolyticus CP000481 (86.1 %), I. majanohamensis AB360448 (84.7 %) and Acidimicrobium ferrooxidans U75647 (84.3 %). Phylogenetic trees obtained by using the neighbour-joining (Fig. 1⇓) and maximum-likelihood (data not shown) methods revealed clear affiliations of the novel isolate with the uncultured isolates Dolo_10 (AB257636, 95.2 % similarity), TX1A_108 (FJ152660, 91.2 %), TX1A_82 (FJ152634, 91.0 %), TX1A_83 (FJ152635, 91.2 %) and AT425_EubY10 (AY053479, 91.1 %), and placed the novel strain on a separate branch within the class Actinobacteria. This phylogenetic position of strain F10^T was also supported by a tree generated using the arb program. Hugenholtz & Stackebrandt (2004) argued for the reclassification of S. thermophilus from the subclass Sphaerobacteridae in the phylum Actinobacteria to the class Thermomicrobia in the phylum Chloroflexi (green non-sulfur bacteria) based on the lack of suitable outgroups in the analysis. Our phylogenetic trees generated using Escherichia coli (X80725) and Bacillus subtilis (D26185) as outgroups, as described by Hugenholtz & Stackebrandt (2004), both indicated that the phylogenetic position of strain F10^T is within the phylum Actinobacteria (data not shown).

The characteristics that differentiate strain F10^T from members of phylogenetically related genera are shown in Table 1⇑. The data obtained from the phenotypic characterization support a separate position for strain F10^T in the 16S rRNA gene sequence-based phylogenetic tree for the class Actinobacteria. A proposed novel subclass, composed of Nitriliruptor alkaliphilus and strain F10^T, could be distinguished from the subclass Acidimicrobidae on the basis of evolutionary distance, the low degree of similarity (82.4–85.4 %) of the 16S rRNA gene sequence, and the low bootstrap value (less than 50 %) between the subclass Acidimicrobidae clade and the isolate clade. The characteristic signature oligonucleotides for the examined taxa were defined (see the family description below). Phenotypic and phylogenetic characteristics distinguish the proposed novel subclass, composed of N. alkaliphilus and strain F10^T, from the related taxa at the subclass level.

Based on the results described above, we concluded that strain F10^T represents a novel species in a new genus, Euzebya tangerina gen. nov., sp. nov. We also propose the name Nitriliruptoridae subclassis nov., with a new order Euzebyales ord. nov. and a new family Euzebyaceae fam. nov., based on the distinct phylogenetic position of E. tangerina gen. nov., sp. nov. within the class Actinobacteria.