Terribacillus aidingensis sp. nov., a moderately halophilic bacterium

Gram-positive, rod-shaped, spore-forming, moderately halophilic bacteria have been isolated from various saline or hypersaline environments (Ventosa et al., 1998; Kim et al., 2007). They were originally placed in the genus Bacillus (Claus & Berkeley, 1986). Subsequently, molecular and chemotaxonomic analyses have shown that they form several phylogenetically distinct lineages within the family Bacillaceae and many new genera and novel species of this kind of bacteria have been reported. To date, the spore-forming, moderately halophilic bacteria that have been described include Halobacillus (Spring et al., 1996; Chen et al., 2009a), Virgibacillus (Heyndrickx et al., 1998; Chen et al., 2009b; Niederberger et al., 2009), Gracilibacillus (Wainø et al., 1999; Tang et al., 2009), Oceanobacillus (Lu et al., 2001; Namwong et al., 2009), Filobacillus (Schlesner et al., 2001), Jeotgalibacillus (Yoon et al., 2001), Marinibacillus (Yoon et al., 2001), Lentibacillus (Yoon et al., 2002), Paraliobacillus (Ishikawa et al., 2002), Halolactibacillus (Ishikawa et al., 2005), Pontibacillus (Lim et al., 2005; Chen et al., 2009c), Thalassobacillus (García et al., 2005), Tenuibacillus (Ren & Zhou, 2005a), Salinibacillus (Ren & Zhou, 2005b), Salsuginibacillus (Carrasco et al., 2007a), Piscibacillus (Tanasupawat et al., 2007), Terribacillus (An et al., 2007), Sediminibacillus (Carrasco et al., 2008), Aquisalibacillus (Márquez et al., 2008) and Bacillus (Garabito et al., 1997; Arahal et al., 1999; Carrasco et al., 2007b; Xue et al., 2008).

At present, the genus Terribacillus includes three species. Terribacillus saccharophilus and Terribacillus halophilus were isolated from field soil in Japan (An et al., 2007), and Terribacillus goriensis DSM 18252^T was isolated from coastal water off the east coast of Korea (Kim et al., 2007; Krishnamurthi & Chakrabarti, 2008). The main phenotyptic characteristics of the genus Terribacillus are the formation of ellipsoidal endospores, gelatin liquefaction and the absence of H₂S and indole production and nitrate reduction. The DNA G+C content is 43.0–46.0 mol%, the major fatty acids are anteiso-C_15 : 0 and anteiso-C_17 : 0 and MK-7 is the major component of the quinone system (An et al. 2007; Krishnamurthi & Chakrabarti, 2008).

Three moderately halophilic bacterial strains, designated YI7-61^T, IA7 and DB2, were isolated from sediments of Aiding salt lake in Xinjiang, China (4 ° 32–49′ 10–13″ N 8 ° 10–54′ 32″ E). At the time of sampling, the sediments were 17 °C, 12.1–15.4 % (w/v) NaCl and pH 7.1–7.3. For isolation, the samples were suspended in sterilized water supplemented with 5 % NaCl, serially diluted and spread on plates of complex isolation and maintenance medium described previously (Liu et al., 2005). For cultivation of the isolates for characterization, the isolation medium was adjusted to pH 7.0 and incubation was at 35 °C, unless indicated otherwise. Phylogenetic analysis of the 16S rRNA gene sequences showed that the isolates belonged to the genus Terribacillus. The reference strains T. saccharophilus JCM 21759^T, T. halophilus JCM 21760^T and T. goriensis DSM 18252^T were grown according to conditions described by An et al. (2007) and Kim et al. (2007), respectively.

To characterize the isolates phenotypically, standard tests were performed according to the proposed minimal standards for the description of aerobic, endospore-forming bacteria (Logan et al., 2009). Cell morphology and motility were examined after cultivation for 16 h. Endospores were observed after 48 h using phase-contrast microscopy (Eclipse 50i; Nikon). Bacterial flagellation was observed using transmission electron microscopy (JEM-1230; JEOL). Gram reaction was determined according to the methods described by Doetsch (1981) and Gregersen (1978). Colony morphology was examined after 3 days at 35 °C on modified isolation medium (6 % NaCl, pH 7.0). Conditions for growth were determined in liquid isolation medium by monitoring the increase in optical density at 600 nm. Growth was tested with 0, 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 21, 22, 23, 25 or 30 % (w/v) NaCl, at pH 4, 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10 and 11, achieved using the appropriate biological buffers (pH 8.0, Na₂HPO₄/NaH₂PO₄; pH 9.0, Na₂CO₃/NaHCO₃; and pH 11.0, Na₂HPO₄/NaOH), as described by Gomori (1955), and at 4, 10, 15, 20, 25, 28, 30, 33, 35, 37, 40, 45, 50, 55 and 60 °C. The API 20E and API 50CHB systems (bioMérieux) were used to determine the physiological and biochemical characteristics, such as the Voges–Proskauer test, fermentation of 49 carbohydrates and hydrolysis of gelatin. All suspension media were supplemented with 5 % NaCl (w/v). Tests for catalase, urease and oxidase, hydrolysis of casein, starch, tyrosine, Tween 80 and aesculin, utilization of citrate, reduction of nitrate, Voges–Proskauer reaction, production of indole and H₂S and arginine dihydrolase, tryptophan deaminase, phenylalanine deaminase, lysine decarboxylase and ornithine decarboxylase activities were performed for the isolates and the reference strains according to the conventional methods described by Dong & Cai (2001). Cells of strains YI7-61^T, IA7 and DB2 were motile, Gram-positive and rod-shaped. Non-swollen ellipsoidal endospores were formed. Catalase and β-galactosidase were negative. Urease and oxidase were positive. Starch could be hydrolysed. H₂S and indole were not produced. Nitrate was not reduced to nitrite. Gelatin was liquefied. The Voges–Proskauer test was negative. The results for the reference strains in this study were the same as those reported before except for total salt concentration for growth (Table 1⇓). The morphological, physiological and biochemical characteristics are given in detail in Table 1⇓, Supplementary Fig. S1, available in IJSEM Online, and the species description.

Biomass for chemotaxonomic analysis was harvested from cultures after incubation on complex medium at 35 °C for 18 h. The analysis of the cell-wall peptidoglycan was carried out using the method described by Schleifer & Kandler (1972) and Schleifer (1985). Cell-wall hydrolysates were separated by one-dimensional chromatography on micro-cellulose thin layers, using T. halophilus JCM 21760^T as a reference. Menaquinones were analysed as described by Collins (1985) using reversed-phase HPLC (HP-1050), using T. halophilus JCM 21760^T and Arthrobacter nicotinovorans CGMCC 1.1933^T as references. The fatty acid compositions of strain YI7-61^T, T. halophilus JCM 21760^T and T. saccharophilus JCM 21759^T were determined under the same laboratory conditions for cells grown in complex medium supplemented with 5 % (w/v) NaCl (pH 7.0) on a rotary shaker (200 r.p.m.) at 35 °C for 18 h, according to the manufacturer's instructions for the Microbial Identification System (MIDI). The diamino acid in the murein and the menaquinone of strain YI7-61^T were meso-diaminopimelic acid (m-DAP) and MK-7, respectively, which were the same as those of T. halophilus JCM 21760^T. The major cellular fatty acids of strains YI7-61^T, IA7 and DB2 were anteiso-C_15 : 0 (67.9, 58.8 and 48.6 %, respectively) and anteiso-C_17 : 0 (19.4, 21.4 and 34.8 %, respectively). Detailed cellular fatty acid compositions of strain YI7-61^T, T. halophilus JCM 21760^T, T. saccharophilus JCM 21759^T and T. goriensis DSM 18252^T are given in Supplementary Table S1.

Chromosomal DNA was extracted and purified according to standard methods (Marmur, 1961). The determination of DNA G+C content was carried out using the thermal denaturation method according to Marmur & Doty, (1962) with a BIO-20 UV spectrophotometer. For the calculation of DNA G+C content, the equation of Owen & Hill (1979) was used. The DNA G+C contents of strains YI7-61^T, IA7 and DB2 were 45.0, 44.6 and 44.9 mol%, respectively. These results are within the emended G+C content range (43–46 mol%) for the genus Terribacillus Krishnamurthi & Chakrabarti (2008).

The 16S rRNA gene sequence was amplified as described by Duckworth et al. (1996). PCR products were sequenced with a DNA sequencer (ABI 373A; Applied Biosystems) and the software provided by the manufacturer. The almost-complete 16S rRNA gene sequences of strains YI7-61^T, IA7 and DB2 (1472 bp, 1464 bp and 1485 bp) were compared with sequences from public databases using the blast program via the National Center for Biotechnology Information. Pairwise sequence similarities were calculated using the BioEdit software package (Hall, 1999). Phylogenetic trees were reconstructed using mega version 3.1 (Kumar et al., 2004). Distances were calculated by using distance options according to Kimura's two-parameter model (Kimura, 1980) and clustering was performed with the neighbour-joining, maximum-parsimony and minimum-evolution methods. The stability of the relationships was assessed by 1000 bootstrap analyses. A neighbour-joining phylogenetic tree is shown in Fig. 1⇓. The topologies of the trees generated with the minimum-evolution and maximum-parsimony methods were very similar to the neighbour-joining tree (data not shown). The phylogenetic analysis revealed that the three isolates belonged to the same phylogenetic lineage within the genus Terribacillus. 16S rRNA gene sequence similarities among the isolates were 98.5 % (YI7-61^T and IA7), 98.7 % (YI7-61^T and DB2) and 98.7 % (IA7 and DB2). Strains YI7-61^T, IA7 and DB2 showed 16S rRNA gene sequence similarities of 97.6, 96.8 and 96.9 % with T. halophilus 002-051^T, 97.2, 96.4 and 96.7 % with T. saccharophilus RB589 and 95.5, 95.4 and 95.4 % with T. goriensis CL-GR16^T, respectively. On the basis of 16S rRNA gene sequence analysis, it was evident that the novel isolates should be assigned to the genus Terribacillus.

To determine levels of DNA–DNA relatedness, extracted DNA was sheared by sonication (Braun Melsungen) at 50 W for three periods of 10 s, rehybridized in 2×SSC at 70 °C and analysed spectrophotometrically (De Ley et al., 1970; Huß et al., 1983). DNA–DNA relatedness among the isolates was 88 % (YI7-61^T and IA7), 90 % (YI7-61^T and DB2) and 92 % (IA7 and DB2). Strain YI7-61^T showed DNA–DNA relatedness of 24, 18 and 18 % with T. halophilus JCM 21760^T, T. saccharophilus JCM 21759^T and T. goriensis DSM 18252^T, respectively. Since <70 % DNA–DNA relatedness is a key marker for the delineation of a novel species (Wayne et al., 1987), these results showed that the three isolates belonged to the same species, which was different from the previously recognized members of the genus Terribacillus.

On the basis of morphological, physiological and chemotaxonomic characteristics, 16S rRNA gene sequence comparison and DNA–DNA relatedness, strains YI7-61^T, IA7 and DB2 should be treated as representatives of a novel species within the genus Terribacillus, for which the name Terribacillus aidingensis sp. nov. is proposed.