Halostagnicola alkaliphila sp. nov., an alkaliphilic haloarchaeon from commercial rock salt

The genus Halostagnicola belonging to the family Halobacteriaceae was first described by Castillo et al. (2006) and, at the time of writing, the genus comprised two species, Halostagnicola larsenii (Castillo et al., 2006) and Halostagnicola kamekurae (Nagaoka et al., 2010). Cells of the type strains of both species are pleomorphic, neutrophilic and strictly aerobic. In this report, we describe an alkaliphilic haloarchaeon, strain 167-74^T, isolated from commercial rock salt, that belongs to the genus Halostagnicola.

Strain 167-74^T was isolated from a sample of rock salt produced in Hubei Province, China. The rock salt is labelled ‘BEST ancient rock salt’ and is sold in Japan by Hanamasa Co., Ltd. The salt sample (1.0 g) was dissolved in 5 ml sterile 5 % NaCl solution and spread on JCM no. 167 medium agar plates. JCM no. 167 medium contains (l⁻¹) 200 g NaCl, 5.0 g Na₂CO₃, 0.24 g MgSO₄ . 7H₂O, 1.0 g KCl, 1.0 g NH₄Cl, 5.0 g yeast extract (Difco), 5.0 g Casamino acids (Difco), 1.0 g sodium glutamate, 1.0 g KH₂PO₄, 0.17 g CaSO₄ . 2H₂O and 1.0 ml trace metal solution. The trace metal solution contained (l⁻¹) 0.1 g ZnSO₄ . 7H₂O, 0.03 g MnCl₂ . 4H₂O, 0.3 g H₃BO₃, 0.2 g CoCl₂ . 6H₂O, 0.01 g CuCl₂ . 2H₂O, 0.02 g NiCl₂ . 6H₂O and 0.03 g Na₂MoO₄ . 2H₂O (adjusted to pH 3.6 with HCl). All components, except Na₂CO₃, were dissolved in distilled water and made to 900 ml. The medium was adjusted to pH 6.5 with 40 % KOH, and autoclaved. Sodium carbonate was dissolved in 100 ml distilled water and autoclaved separately. After autoclaving, the two solutions were mixed aseptically. The final pH was 9.0. After incubation of agar plates at 37 °C for 2–4 weeks, various coloured colonies developed. Four colonies were transferred to fresh agar plates, and pure cultures were obtained by plating serial dilutions and repeated transfers on agar plates. Partial 16S rRNA gene sequences of the four isolates were almost the same, and the most alkaliphilic strain, 167-74^T, was chosen as a representative for further experiments.

Hst. larsenii JCM 13463^T, obtained from the Japan Collection of Microorganisms, and Hst. kamekurae 194-10^T, isolated in our previous study (Nagaoka et al., 2010), were used as reference strains.

Colony morphology was observed on agar medium after incubation for 2–3 weeks at 37 °C. Gram-staining was performed according to Dussault (1955). Cell morphology and motility were examined using phase-contrast microscopy (Axiovert 135; Zeiss). Total DNA was extracted by the method of Cline et al. (1989). The 16S rRNA gene was analysed as described previously (Nagaoka et al., 2010). 16S rRNA gene sequences retrieved from the DDBJ (Miyazaki et al., 2003; Pearson & Lipman, 1988) were aligned using clustal_x version 2.0.12 (Larkin et al., 2007). A phylogenetic tree was reconstructed by the neighbour-joining method (Saitou & Nei, 1987) and evaluated by bootstrap sampling, from 1000 replicates (Felsenstein, 1985). Maximum-likelihood analysis was performed with RAxML 7.0.4 using the GTR+Γ model (Stamatakis et al., 2005), and confidence values for the maximum-likelihood tree were obtained by bootstrapping (1000 replicates) using consense in phylip (Felsenstein, 2002). The 16S rRNA gene sequence of strain 167-74^T was most similar to those of Hst. larsenii JCM 13463^T (98.5 %) and Hst. kamekurae 194-10^T (97.2 %). Lower similarities (<94.6 %) were found with sequences from other species of the family Halobacteriaceae with validly published names. The neighbour-joining (Fig. 1) and maximum-likelihood (Supplementary Fig. S1, available in IJSEM Online) trees also supported the conclusion that strain 167-74^T was most closely related to members of the genus Halostagnicola.

Total lipids were extracted with chloroform/methanol as described previously (Kamekura, 1993). TLC of polar lipids (Supplementary Fig. S2) suggested that strain 167-74^T and the type strains of the other two species of the genus Halostagnicola contained C₂₀C₂₀ and C₂₀C₂₅ archaeol derivatives of phosphatidylglycerol (PG) and phosphatidylglycerol phosphate methyl ester (PGP-Me), as shown by the double spot for PGP-Me. Phospholipids were the same as for the two type strains of the genus Halostagnicola, but no glycolipid spot was detected in strain 167-74^T.

Results of physiological characterization are given in the species description, with methods mentioned in the proposed minimal standards for the descriptions of new taxa in the order Halobacteriales (Oren et al., 1997), as described previously (Castillo et al., 2006; Nagaoka et al., 2010). The range and optimal NaCl concentration for growth were determined by using the growth medium containing various concentrations of NaCl (0–30 %, w/v, at intervals of 5 %, w/v). The pH range for growth was assayed from pH 7.0 to 11.0 at intervals of 0.5 pH units in liquid medium with various buffers (glycyl glycine/NaOH, pH 7.0–8.0; glycine/NaOH, pH 8.5–11.0), each at 25 mM. The temperature for growth was determined at 4, 10, 15, 20, 25, 30, 35, 37, 40, 45, 50, 55 and 60 °C in a medium at pH 8.0 with optimal NaCl concentration. Strain 167-74^T was capable of growth in the presence of 20–30 % (w/v) NaCl, at pH 8.0–10.0 and at 20–55 °C. Optimal growth of strain 167-74^T occurred in 25 % (w/v) NaCl, at pH 9.0 and at 37 °C. Phenotypic testing of the strain used the above optimal growth conditions.

Tests for catalase and oxidase activities and for hydrolysis of starch, gelatin, casein and Tween 80 were performed as described by Gonzalez et al. (1978). Reduction of nitrate was detected by using sulfanilic acid/α-naphthylamine reagent (Smibert & Krieg, 1994). H₂S formation was determined by formation of a black sulfide precipitate in medium containing 0.5 % (w/v) sodium subsulfite. Indole production from tryptophan and the utilization of sugars and organic acids were assessed as described by Oren et al. (1997). Antibiotic sensitivity tests were performed by spreading cell suspensions on culture plates and then placing discs impregnated with antibiotics on top (Becton Dickinson). Utilization of single or complex carbon sources was assessed in a modified JCM no. 167 medium (Casamino acids and sodium glutamate omitted; tested under optimal growth conditions) with 0.5 g yeast extract l⁻¹, supplemented with 1.0 % (w/v) test sugar or organic acid.

The G+C content of the total DNA of strain 167-74^T, determined by the HPLC method (Tamaoka & Komagata, 1984), was 60.7 mol%. DNA–DNA hybridization between strain 167-74^T, Hst. larsenii JCM 13463^T and Hst. kamekurae 194-10^T was assessed by using the fluorometric method of Ezaki et al. (1989). The relatedness of strain 167-74^T to Hst. larsenii JCM 13463^T and Hst. kamekurae 194-10^T was 19.5 and 18.8 %, respectively. These values are well below the threshold value of 70 % DNA–DNA relatedness generally accepted for the definition of a novel species (Wayne et al., 1987; Stackebrandt & Ebers, 2006).

Detailed results are included in the species description and differences between strain 167-74^T, Hst. larsenii JCM 13463^T and Hst. kamekurae 194-10^T are highlighted in Table 1. On the basis of the data presented, we conclude that strain 167-74^T represents a novel species of the genus Halostagnicola, for which we propose the name Halostagnicola alkaliphila sp. nov.