Haloarcula amylolytica sp. nov., an extremely halophilic archaeon isolated from Aibi salt lake in Xin-Jiang, China

The definition of the genus Haloarcula was based on the numerical taxonomy and polar lipid composition given by Torreblanca et al. (1986), adopting the generic name first suggested by Javor et al. (1982). At the time of writing, the genus Haloarcula comprises six recognized species (Fig. 1⇓): H. vallismortis (Gonzalez et al., 1978; Torreblanca et al., 1986), H. argentinensis (Ihara et al., 1997), H. hispanica (Juez et al., 1986), H. japonica (Takashina et al., 1990), H. marismortui (Elazari-Volcani, 1957; Oren et al., 1990) and H. quadrata (Oren et al., 1999). A common feature of representative strains of the genus Haloarcula is the presence of at least two heterogeneous 16S rRNA genes. During our surveys on halophilic archaeal diversity of the Aibi salt lake (82° 35′–83° 16′ E 44° 05′–45° 08′ N) in the Xin-Jiang region of China (Cui et al., 2006), a strain that harboured three heterogeneous 16S rRNA genes and that clustered tightly with members of the genus Haloarcula was obtained.

Strain BD-3^T was isolated from sediment of Aibi salt lake (pH 8.1, 112.4 g salt l⁻¹), the largest salt lake in the region. The standard growth medium used for strain BD-3^T and other Haloarcula strains contained (g l⁻¹): Casamino acids (Difco), 7.5; yeast extract (Difco), 10; trisodium citrate, 3.0; MgCl₂.6H₂O, 10.2; KCl, 20; FeSO₄.7H₂O, 0.05; NaCl, 180 (pH adjusted to 7.2 with 1 M NaOH solution). The methods used for isolation were as described by Xin et al. (2000).

Phenotypic tests were performed according to the proposed minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997). Cell motility and morphology were examined by phase-contrast microscopy of exponentially growing liquid cultures. Gram staining was carried out as described by Dussault (1955). Colony morphology was observed on salt-milk and the standard growth agar medium after incubation at 40 °C for 6–10 days. Anaerobic growth was tested in the presence of 5 g sodium nitrate, l-arginine or DMSO per litre in filled, stoppered tubes. To determine growth pH range (using increments of 0.5 pH units, from pH 5.0 to 10.0), 25 mM MES (pH 5.0–6.7), 25 mM PIPES (pH 6.1–7.5), 25 mM MOPS (pH 6.5–7.9), 25 mM HEPES (pH 6.8–8.2), 25 mM Tricine (pH 7.4–8.8) and 25 mM CHES (pH 8.6–10.0) were used. Tests for catalase and oxidase activities and hydrolysis of starch and of Tweens 20, 40, 60 and 80 were performed as described by Gonzalez et al. (1978). Nitrate reduction, H₂S formation, indole formation, and utilization of carbohydrates, sugar alcohols, amino acids and organic acids were examined as described by Oren et al. (1997).

Polar lipids were extracted with chloroform/methanol as described by Kamekura (1993). One- and two-dimensional TLC was performed by using silica gel plates (Kieselgel 60 F; Merck) (Kates, 1986).

DNA was extracted by using phenol/chloroform extraction followed by ethanol precipitation, as modified according to Ng et al. (1995). The gene encoding 16S rRNA was amplified by PCR with the forward primer 5′-TTCCGGTTGATCCTGCC-3′ (positions 7–23, according to the Escherichia coli numbering scheme) and the reverse primer 5′-AAGGAGGTGATCCAGCC-3′ (positions 1541–1525), modified according to Achenbach & Woese (1995). PCR was performed for 30 cycles with denaturation for 1 min at 95 °C, annealing for 1 min at 49 °C and polymerization for 90 s at 72 °C. The PCR product was cloned into T-vector, and sequenced by using an ABI BigDye3.1 sequencing kit (Applied Biosystems) and an automated DNA sequencer (model ABI3730; Applied Biosystems). Phylogenetic analyses were conducted using the neighbour-joining method (mega version 3.1; Kumar et al., 2004).

The G+C content of the DNA was determined by using the thermal denaturation method (Marmur & Doty, 1962), with a Beckman UV-Vis DU800 spectrophotometer at 260 nm. DNA–DNA hybridization was carried out by the thermal denaturation and renaturation method (De Ley et al., 1970) as modified by Huß et al. (1983).

Cells of strain BD-3^T were Gram-negative, motile rods, and were able to grow over a wide range of salinities (2.0–5.1 M NaCl; optimal growth at 2.9–3.2 M). Cells lysed in distilled water and 5 % brine. Colonies on salt-milk agar medium were red-pigmented. Phenotypic characteristics of strain BD-3^T are given in the species description below and Table 1⇓. As detailed in Table 1⇓, strain BD-3^T could be phenotypically differentiated from recognized Haloarcula species but had very similar phenotypic properties to H. hispanica ATCC 33960^T.

The polar lipid composition of strain BD-3^T was determined to be phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerosulfate, triglycosyl diether and an unknown diglycosyl diether (see Supplementary Fig. S1 in IJSEM Online). The genomic DNA G+C content of strain BD-3^T was 62.4 mol%.

Polymorphism of 16S rRNA genes of strain BD-3^T was observed. Three 16S rRNA genes were detected, designated rrnA (1472 bp), rrnB (1472 bp) and rrnC (1472 bp) (Fig. 1⇑). The rrnA gene sequence of BD-3^T showed high similarities to the rrnA gene sequence of H. hispanica (98.7 %), the 16S rRNA gene sequence of H. argentinensis (98.6 %), and the rrnB (98.6 %) and rrnC (98.6 %) gene sequences of H. marismortui. The rrnB gene sequence of BD-3^T showed highest similarity (99.5 %) to the H. marismortui rrnA gene sequence. The rrnC gene sequence of BD-3^T showed highest similarity (98.8 %) to the H. marismortui rrnA gene sequence and H. quadrata rrnB gene sequence. Levels of similarity between the three 16S rRNA genes of strain BD-3^T were 95.2 % (rrnA and rrnB), 94.6 % (rrnA and rrnC) and 99.0 % (rrnB and rrnC). DNA–DNA hybridization values between strain BD-3^T and H. japonica JCM 7785^T, H. vallismortis ATCC 29715^T, H. hispanica ATCC 33960^T, H. argentinensis JCM 9737^T, H. quadrata JCM 11048^T and H. marismortui ATCC 43049^T were 30, 30, 34, 38, 39 and 39 %, respectively.

Based on these results, it is concluded that strain BD-3^T represents a novel species of the genus Haloarcula, for which the name Haloarcula amylolytica sp. nov. is proposed.