Haloarcula salaria sp. nov. and Haloarcula tradensis sp. nov., isolated from salt in Thai fish sauce

The genus Haloarcula was firstly described by Torreblanca et al. (1986) with the type species Haloarcula vallismortis. To date, seven additional species have been described, Har. amylolytica, Har. argentinensis, Har. hispanica, Har. japonica, Har. marismortui, Har. mukohataei and Har. quadrata (Juez et al., 1986; Oren et al., 1990, 1999; Takashina et al., 1990, 1991; Ihara et al., 1997; Yang et al., 2007). Har. mukohataei has since been transferred to the genus Halomicrobium as Hmc. mukohataei (Oren et al., 2002). These extremely halophilic archaea are red-pigmented and short pleomorphic rods ranging from almost regular to irregular shapes. Fish sauce, which is a common fermented food in Thailand, is rich in various nutrients, particularly amino acids and peptides. It also contains a high concentration of NaCl, thus allowing halophilic bacteria and archaea to thrive (Lopetcharat et al., 2001). Previous studies of this product have found strains of halophilic archaea, Halobacterium salinarum, Halococcus thailandensis, Hcc. saccharolyticus and Natrinema gari (Thongthai et al., 1992; Namwong et al., 2007; Tapingkae et al., 2008; Tanasupawat et al., 2008–2009), and moderately halophilic bacteria, ‘Halobacillus thailandensis’, Lentibacillus salicampi, L. juripiscarius, L. halophilus and Chromohalobacter salexigens (Chaiyanan et al., 1999; Namwong et al., 2005; Tanasupawat et al., 2006, 2008–2009). This paper deals with the characterization of two novel extremely halophilic archaea from the salt in a sample of fish sauce based on their phenotypic and chemotaxonomic characteristics, DNA–DNA relatedness and 16S rRNA gene sequences.

The halophilic archaeal strains were isolated from salt from a sample of fish sauce collected in Trad province, Thailand. The collected salt was suspended in JCM medium No. 168 broth and the suspension was spread on agar plates of the same medium [composed of (l⁻¹): 200 g NaCl, 5 g Casamino acids (Difco), 5 g yeast extract (Difco), 2 g KCl, 3 g Na₃C₆H₅O₇, 1 g glutamic acid, 20 g MgSO₄ . 7H₂O, 0.36 g FeCl₂ . 4H₂O, 0.0036 g MnCl₂ . 4H₂O, 20 g agar, pH 7.2] and incubated at 37 °C for 1–2 weeks. Unless otherwise stated, the strains were grown in liquid or on agar media based on JCM medium No. 168.

Phenotypic characteristics were determined in accordance with the proposed minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997). Cell morphology and motility were examined by phase-contrast microscopy (BX50; Olympus). Gram staining was performed as described by Dussault (1955). Colonies were observed on JCM medium No. 168 agar plates after incubation at 37 °C for 7 days. Tests for catalase and oxidase activities, nitrate reduction, methyl red/Voges–Proskauer reactions, the formation of indole and the hydrolysis of gelatin, skimmed milk, Tween 80, tyrosine and xanthine were performed as described by Barrow & Feltham (1993). Carbon utilization from carbohydrates was determined in Leifson (1963) medium. Casitone was omitted for the carbon utilization test whereas Tris/HCl was omitted for the acid production test. Anaerobic growth was observed in the standard growth medium with 0.5 % nitrate using a Gaspak (BBL) anaerobic jar. Growth at various temperatures (5–60 °C) was examined using a temperature gradient incubator (model TN-3; Advantec). The effects of initial pH (5–9) for growth were determined in JCM medium No. 168. The effects of NaCl concentration on growth were tested in a medium containing 0–30 % (w/v) NaCl. At lower NaCl concentrations [0–2 % (w/v) NaCl], MgSO₄ . 7H₂O, KCl and Na₃C₆H₅O₇ . 2H₂O were omitted from the test medium. Likewise, the requirement for Mg²⁺ was tested in JCM medium No. 168 by omitting MgSO₂ . 7H₂O but supplementing with 2 % (w/v) Na₂SO₄ and 0–10 % (w/v) MgCl₂. Growth was determined by measuring the culture turbidity at 660 nm. The antibiotic susceptibility of the strains was tested as described by Stan-Lotter et al. (2002).

Menaquinones were analysed by the reversed-phase HPLC method of Komagata & Suzuki (1987) and confirmed by MS running in EI mode and using a direct insertion probe (QPMS-QP5050A; Shimadzu). Polar lipids were extracted serially by using four different solvent solutions [first extraction with methanol : 0.3 % NaCl (100 : 1, v/v), followed by petroleum ether, chloroform : methanol : water (90 : 100 : 30, v/v) and finally chloroform : methanol : water (50 : 100 : 40, v/v)] and their extracts were separated by single dimensional silica gel TLC (Hezayen et al., 2002). Methanolysis of the polar lipids was conducted by mixing the dried cells with methanol (3 ml), toluene (3 ml) and concentrated H₂SO₄ (0.1 ml), followed by heating at 55 °C for 9 h. Core diether moieties were extracted with hexane and the hexane extracts were developed on TLC using the procedure described by Ross et al. (1981). Two-dimensional TLC of the total polar lipids was conducted according to the method of Minnikin et al. (1984).

Genomic DNA was isolated and purified according to the method of Saito & Miura (1963). The DNA G+C content was determined with the method of Tamaoka & Komagata (1984) using reversed-phase HPLC. DNA–DNA hybridization was conducted as reported previously (Ezaki et al., 1989) and detected by the colorimetric method reported by Tanasupawat et al. (2000). The almost complete 16S rRNA gene sequences of strains HST01-2R^T and HST03^T were PCR-amplified with primers 20F (5′-TCCGGTTGATCCTGCCG, position 8–24 according to the Escherichia coli numbering system) and 1540R (5′-GGAGGTGATCCAGCCG, position 1540–1525) (Enache et al., 2007). The amplified DNA fragments were separated by agarose gel electrophoresis and recovered by using a GenElute Minus EtBr Spin Column (Sigma). PCR products were ligated into the pT7blueT-vector (Novagen) using a Takara ligation kit version 2.1 according to the manufacturer's instructions and transformed into E. coli JM 109 by the heat-shock technique (Shao et al., 2004). Plasmids were extracted with a QIAprep Spin Miniprep kit (Qiagen) using the protocol supplied by the manufacturer. Inserts were amplified using the primers T7 (5′-TAATACGACTCACT ATAGGG-3′) and U19 (5′-GTTTTCCCAGTCACGACGT-3′) (Enache et al., 2007). PCR products were sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction kit (version 3.0; Applied Biosystems) in the ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The phylogenetic tree was constructed as described by Thompson et al. (1997), Saitou & Nei (1987), Kumar et al. (2001) and Felsenstein (1985).

Two strains of non-motile, red-pigmented and extremely halophilic archaea were isolated from the salt in the samples of fish sauce. Colonies on JCM medium No. 168 agar plates were small (0.8–2.3 mm in diameter), entire, translucent and convex. Cells from late-exponential to stationary phase cultures were pleomorphic, rod-shaped and approximately 0.6–1.2×1.0–2.5 μm (Fig. 1a and b⇓) and lysed when suspended in distilled water. The strains grew at salinities of 15–30 % (w/v) NaCl, showing optimal growth at 20–25 % (w/v) NaCl. They required at least 2 % (w/v) Mg⁺ and grew optimally when 4–6 % (w/v) Mg⁺ was added to the medium with 20 % (w/v) NaCl. The growth temperature range was 15–45 °C (optimum, 37 °C) and the pH range was 6–8 (optimum, pH 7). The physiological and biochemical properties of the novel strains are summarized in the species description and Table 1⇓.

The chemotaxonomic features of the two strains, i.e. the presence of C₂₀C₂₀ glycerol diether derivatives of phosphatidylglycerol sulfate (PGS) and triglycosyl diether (TGD-2), and MK-8 and MK-8(H₂) as the major menaquinone components, conformed to the previously published descriptions of the genus Haloarcula (Torreblanca et al., 1986; Juez et al., 1986; Grant & Larsen, 1990; Oren et al., 1990, 1999, 2009; Takashina et al., 1990; Ihara et al., 1997; Yang et al., 2007). Thin layer chromatograms of the polar lipid fractions of strains HST01-2R^T and HST03^T revealed C₂₀C₂₀ glycerol diether derivatives of phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), PGS, TGD-2, diglycosyl diether (DGD) and two unknown phospholipids (see Supplementary Fig. S1 in IJSEM Online). The TLC of the methanolysis samples from strains HST01-2R^T and HST03^T, as well as those from Har. amylolytica JCM 13557^T, Har. argentinensis JCM 9737^T, Har. hispanica JCM 8911^T and Hcc. thailandensis JCM 13552^T revealed that the two novel strains contained only C₂₀C₂₀, core lipids (see Supplementary Fig. S2). The two-dimensional TLC of the total polar lipids of strains HST01-2R^T and HST03^T is presented in Supplementary Fig. S3. The novel strains contained MK-8 (91.8–91.9 %) and MK-8(H₂) (8.2–8.8 %) as the menaquinone components. The genomic DNA G+C contents of the strains were 61.6–62.2 mol% (Table 2⇓).

It has been demonstrated that the recognized members of the genus Haloarcula examined so far harbour at least two different 16S rRNA gene copies, which have been categorized into two 16S rRNA gene types (type I and II) showing 4.8–5.6 % divergence from each other (Gemmell et al., 1998; Arahal et al., 1996; Ihara et al., 1997; Oren et al., 1999; Yang et al., 2007; Baliga et al., 2004; Cui et al., 2009). The two novel isolates also revealed this 16S rRNA gene polymorphism: strain HST01-2R^T contained at least two 16S rRNA gene copies, rrnA (1479 bp) and rrnB (1484 bp), and strain HST03^T contained three 16S rRNA gene copies, rrnA (1489 bp), rrnB (1487 bp) and rrnC (1487 bp), as shown in Fig. 2⇓. All of the 16S rRNA gene copies belonged to type II. On the phylogenetic tree (Fig. 2⇓), the three gene copies of strain HST03^T seemed to be separated from the hitherto reported type II genes, and only one sequence from Haloarcula hispanica ATCC 33960^T (GenBank accession no. U68541) was closely related to the rrnC gene (97.91 % similarity). On the other hand, the rrnA and rrnB gene sequences of strain HST01-2R^T were included in the radiation of the type II genes. The DNA–DNA hybridization study revealed that strains HST01-2R^T and HST03^T had low DNA–DNA relatedness (<46.9 %) to each other and to the type strains of recognized species of the genus Haloarcula, i.e. Har. argentinensis JCM 9737^T, Har. amylolytica JCM 13557^T, Har. hispanica JCM 8911^T, Har. vallismortis JCM 8877^T, Har. marismortui JCM 8966^T, Har. japonica JCM 7785^T and Har. quadrata JCM 11048^T (Wayne et al., 1987), as shown Table 2⇑. In addition, several phenotypic characteristics of strains HST01-2R^T and HST03^T, i.e. acid production from raffinose, utilization of sugars and amino acids as energy sources, the NaCl and Mg²⁺ ranges for growth and the hydrolysis of tyrosine, clearly distinguished these strains from each other and from recognized species of the genus Haloarcula. Therefore, strains HST01-2R^T and HST03^T represent two novel species in the genus Haloarcula, for which the names Haloarcula salaria sp. nov. and Haloarcula tradensis sp. nov. are proposed, respectively.