Halostagnicola kamekurae sp. nov., an extremely halophilic archaeon from solar salt

The haloarchaea constitute a large group of extremely halophilic, aerobic archaea that are placed in the order Halobacteriales, family Halobacteriaceae (Grant et al., 2001). At the time of writing (December 2009), the family Halobacteriaceae contains 27 genera comprising 104 species (Savage et al., 2008; Oren et al., 2009; ; ) that display a wide variety of physiological characteristics, including ranges of salinity, temperature and pH for growth. The genus Halostagnicola, belonging to the family Halobacteriaceae, currently contains only one species and one strain, Halostagnicola larsenii XH-48^T, isolated from sediment of Lake Xilinhot, a saline lake in Inner Mongolia, China (Castillo et al., 2006). The strain is pleomorphic, neutrophilic and requires at least 15 % (w/v) NaCl. The highest 16S rRNA gene sequence similarities are obtained with Natrialba aegyptiaca 40^T and Natrialba asiatica 172P1^T. Polar lipids are phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, the latter derived from both C₂₀C₂₀ and C₂₀C₂₅ archaeol, and two unidentified glycolipids. We isolated a strain closely related to Hst. larsenii XH-48^T from an imported solar salt sample. In this report, we characterize strain 194-10^T and describe its identification. Hst. larsenii XH-48^T was obtained from the Japan Collection of Microorganisms as JCM 13463^T and used as a reference strain.

Strain 194-10^T was isolated from a sample of solar salt imported into Japan from the Philippines. The solar salt sample has been on the market from a Japanese firm as ‘Mangrove’ () as a 200 g package. To 1 g of the salt sample, 4 ml sterile distilled water was added gradually, and three drops of the solution were spread on agar plates of JCM medium no. 168 containing the following (l⁻¹): 5.0 g yeast extract, 5.0 g Casamino acids, 1.0 g sodium glutamate, 2.0 g KCl, 3.0 g sodium citrate, 20.0 g MgSO₄ . 7H₂O, 200.0 g NaCl, 36.0 mg FeCl₂ . 4H₂O and 0.36 mg MnCl₂ . 4H₂O. The medium was adjusted to pH 7.0 with 40 % (w/v) KOH. About 30 colonies appeared on an agar plate, and six colonies of different colours were selected and purified by repeated serial dilution; partial 16S rRNA gene sequences were then determined. Sequences of five of the strains were closely related (more than 99.2 % similarity) to those of haloarchaea of the genera Haloarcula and Halorubrum. A pink colony designated strain 194-10^T showed 98.5 % partial 16S rRNA gene sequence similarity to Hst. larsenii XH-48^T. Colony morphology was observed on agar medium under optimal growth conditions after incubation at 37 °C. Gram staining was performed according to Dussault (1955). Cell morphology and motility were examined using phase-contrast microscopy (Zeiss Axiovert 135). Colonies of strain 194-10^T on agar plates were circular, smooth, opaque and white and turned pink gradually after 2–3 weeks. The cells were motile and pleomorphic, exhibiting rod-shaped or teardrop-shaped morphology, and approximately 0.8–1.0×2.0–2.5 μm. Cells lysed in distilled water. There were no gas vesicles inside the cells.

Growth ranges and optima for NaCl and Mg²⁺ were determined by using the growth medium containing various concentrations of NaCl (0–30 %, w/v) and MgSO₄ . 7H₂O (0–100 mM), at intervals of 5 % (w/v) and 10 mM, respectively. The pH range for growth was assayed in liquid medium at pH 6.0–10.0 at intervals of 0.5 pH units. The temperature range for growth of strain 194-10^T was determined at 4, 10, 15, 20, 25, 30, 35, 37, 40, 45 and 50 °C at pH 7.0 with optimal NaCl and Mg²⁺ concentrations. Strain 194-10^T was capable of growing over a wide range of NaCl concentration [10–30 % (w/v)]. Mg²⁺ was not required for growth. The strain grew optimally in the presence of 15 % (w/v) NaCl and 60 mM MgSO₄ . 7H₂O. Strain 194-10^T grew at 20–45 °C (optimum, 30 °C) and pH 6.0–9.0 (optimum, pH 6.5–7.0).

Total DNA was extracted by the method of Cline et al. (1989). The DNA G+C content was determined by the HPLC method of Tamaoka (1994). The DNA G+C content of strain 194-10^T was 59.8 mol%, which was similar to that reported for Hst. larsenii XH-48^T (61.0 mol%; Castillo et al. 2006). The 16S rRNA gene of strain 194-10^T was amplified by PCR with the forward and reverse primers 5′-ATTCCGGTTGATCCTGCCGG and 5′-AGGAGGTGATCCAGCCGCAG, as described previously (Fukushima et al., 2007). Amplification products were cloned into the pCR2.1 T vector (Invitrogen) and five clones were sequenced using the Big Dye sequencing kit version 3.1 (Applied Biosystems) using an ABI 310 DNA sequencer. 16S rRNA gene sequences retrieved from the DDBJ (Miyazaki et al., 2003) were aligned. A phylogenetic tree was reconstructed by the neighbour-joining method (Saitou & Nei, 1987) and was evaluated by bootstrap sampling with 1000 replicates (Felsenstein, 1985). Maximum-likelihood analysis was performed with RAxML 7.0.4 using the GTR+Γ model (Stamatakis et al., 2005), and support for the maximum-likelihood tree was obtained by bootstrapping (1000 replicates) using consense in phylip. The five 16S rRNA gene clones from strain 194-10^T gave exactly the same sequence, which was the closest to that of Hst. larsenii JCM 13463^T (DDBJ accession no. AB301489), with 98.5 % similarity; similarities to sequences of other species of the Halobacteriaceae with validly published names were less than 95.0 %, the highest being 94.9 % with Nab. hulunbeirensis AS 1.1986^T and 94.6 % with Nab. aegyptiaca 40^T. The sequence represented by DDBJ accession number AB301489 (1433 nt) was deposited by one of the authors of this paper (H. M.) in April 2007; this sequence was the same as that represented by AM117571 (1372 nt), deposited by A. Castillo, except for five nucleotides at positions 659–663 of AM117571. The neighbour-joining (Fig. 1⇓) and maximum-likelihood (Supplementary Fig. S1, available in IJSEM Online) trees also supported the conclusion that the strain 194-10^T was most closely related to Hst. larsenii XH-48^T. DNA–DNA hybridization was performed by the fluorometric method of Ezaki et al. (1989); the relatedness between strain 194-10^T and Hst. larsenii JCM 13463^T was 20.7 % (n=3). This value was well below the threshold value of 70 % generally accepted for the definition of distinct species (Wayne et al., 1987; Stackebrandt & Ebers, 2006).

Polar lipids were extracted with chloroform/methanol as described previously (Kamekura, 1993). TLC was performed using HPTLC silica gel 60 plates (20×10 cm; Merck) with the solvent system chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by vol.). Phospholipids were detected as blue spots by spraying with Dittmer–Lester reagent (Minnikin et al., 1984). Glycolipids were detected as purple spots by spraying with 0.5 % (w/v) α-naphthol in methanol/water (1 : 1) and then with sulfuric acid/ethanol (1 : 1), followed by brief heating at 160 °C. Polar lipids were detected as brown spots after prolonged heating. TLC of the polar lipids (Supplementary Fig. S2) suggested that strain 194-10^T contained phosphatidylglycerol and phosphatidylglycerolphosphate methyl ester (PGP-Me) derived from both C₂₀C₂₀ and C₂₀C₂₅ archaeol, as shown by the two spots for PGP-Me. Phosphatidylglycerol sulfate was not detected. Several unidentified glycolipid spots were detected. Two major spots detected above PGP-Me were not observed in Hst. larsenii JCM 13463^T in this study or by Castillo et al. (2006). Minor glycolipids similar to those of Hst. larsenii JCM 13463^T were also detected.

Phenotypic tests were performed according to the proposed minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997). All tests were done in our laboratory on both strain 194-10^T and Hst. larsenii JCM 13463^T. Tests for catalase and oxidase activities and for the hydrolysis of starch, gelatin, casein and Tween 80 were performed as described by Gonzalez et al. (1978). Reduction of nitrate was detected by using the sulfanilic acid and α-naphthylamine reagent (Smibert & Krieg, 1981). H₂S formation was determined by monitoring the production of a black sulfide precipitate in JCM medium no. 168 containing 0.5 % (w/v) sodium thiosulfate. Indole production from tryptophan and the utilization of sugars and organic acids were assessed as described by Oren et al. (1997). Strain 194-10^T was oxidase- and catalase-negative. Nitrate reduction and indole production from tryptophan were positive. Tween 80 was hydrolysed, but starch, gelatin and casein were not hydrolysed. Strain 194-10^T was urease-positive. Other results are included in the species description. Antibiotic sensitivity tests were performed by spreading a cell suspension on culture plates and applying discs impregnated with the following antibiotics (Becton Dickinson; amounts in mg unless indicated): ampicillin (10), bacitracin (10 U), chloramphenicol (30), erythromycin (15), gentamicin (120), kanamycin (30), nalidixic acid (30), neomycin (30), novobiocin (30), penicillin G (10 U), rifampicin (5), streptomycin (300), tetracycline (30) and vancomycin (30). Discs containing anisomycin (50 mg) and pravastatin (50 mg) were prepared in our laboratory and applied in the same way. The formation of acid from sugars and organic acids was assessed in a modified JCM medium no. 168 (Casamino acids, sodium glutamate and sodium citrate were omitted) with 0.5 g yeast extract l⁻¹, supplemented with 1 % (w/v) test substrate. After incubation for 2 weeks, acid formation was determined by using bromcresol green pH test paper. The results are included in the species description and differences between strain 194-10^T and Hst. larsenii JCM 13463^T are highlighted in Table 1⇓. There were no discrepancies in phenotypic test results for Hst. larsenii JCM 13463^T between those reported by Castillo et al. (2006) and those determined in our laboratory, showing that the differences between strain 194-10^T and Hst. larsenii JCM 13463^T highlighted in Table 1⇓ are reliable.

The phylogenetic and phenotypic characteristics indicated strongly that strain 194-10^T represents a novel species of the genus Halostagnicola, for which the name Halostagnicola kamekurae sp. nov. is proposed.