Bacillus solisalsi sp. nov., a halotolerant, alkaliphilic bacterium isolated from soil around a salt lake

Many haloalkaliphilic and/or alkalitolerant Bacillus species that belong to rRNA group 6 (Ash et al., 1991; Nielsen et al., 1994; Yoon et al., 2004; Nogi et al., 2005; Lim et al., 2006; Borchert et al., 2007) are widely distributed in different saline and alkaline habitats with a high pH and a variable (up to saturation) salt concentration. This group of bacteria has attracted much interest because its members can secrete various potentially useful enzymes (Horikoshi, 2006). Recently, some haloalkaliphilic and alkalitolerant species have been isolated from saline soil samples (Olivera et al., 2005; Nowlan et al., 2006; Lee et al., 2006; Ghosh et al., 2007). This study describes the characterization and identification of a novel halotolerant and alkaliphilic species of the genus Bacillus that was isolated from a soil sample around Yuncheng Salt Lake (3 ° N 11 ° E), which is located in the South Shanxi Basin of Shanxi Province, China. Yuncheng Salt Lake is the third largest sodium sulfate inland lake in the world with an area of 132 km² and it was formed in the Quaternary Period of the Cenozoic Era. After the seas dried up and mountains formed, a large amount of saline mineral substances gathered and, after a long period of deposition and evaporation, a natural salt lake was formed.

Strain YC1^T was isolated using the dilution plating technique on Luria–Bertani (LB) medium which contained the following (g l⁻¹): yeast extract, 5; tryptone, 10; and NaCl, 10. The pH was adjusted to 7.0 with 1 M HCl. The organism was incubated at 30 °C and routinely cultivated on LB agar or in LB broth at the same temperature under aerobic conditions.

The primers used for PCR amplification of the 16S rRNA gene were 5′-AGAGTTTGATCCTGGCTCAG-3′ (forward) and 5′-AAGGAGGTGATCCAAGCCGCA-3′ (reverse), corresponding to positions 8–27 and 1521–1540, respectively, in the 16S rRNA gene sequence of Escherichia coli (Brosius et al., 1978). The 1424 bp sequence of strain YC1^T was compared with sequences retrieved from GenBank using the blast program () to determine an approximate phylogenetic affiliation. Phylogenetic analysis was performed using the phylip (Felsenstein, 1993) and mega version 3.1 (Kumar et al., 2001) software packages after multiple alignment of the data with clustal_x (Thompson et al., 1997). Distances (distance options according to the Kimura two-parameter model; Kimura, 1980, 1983) and clustering were based on the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) methods. Bootstrap analysis (1000 replications) was used to evaluate the topology of the neighbour-joining tree (Felsenstein, 1985). Comparison of the 16S rRNA gene sequences showed that strain YC1^T was clearly affiliated with the genus Bacillus and was most closely related to Bacillus macauensis JCM 13285^T (98.0 % similarity); sequence similarities with type strains of other recognized species of the genus Bacillus were less than 96.7 %. A phylogenetic tree based on the neighbour-joining method indicated that strain YC1^T formed a monophyletic clade adjacent to B. macauensis JCM 13285^T with a high bootstrap resampling value of 99 % (Fig. 1⇓).

Chromosomal DNA from strain YC1^T was extracted and purified according to the procedure of Yoon et al. (1996). The DNA G+C content, measured according to the method of Cashion et al. (1997), was 41.8 mol%. This value is slightly higher than that recorded for B. macauensis JCM 13285^T (40.8 mol%). DNA–DNA hybridization between strain YC1^T and B. macauensis JCM 13285^T was measured as described by De Ley et al. (1970) with the modifications described by Huß et al. (1983) and Escara & Hutton (1980). Renaturation rates were computed with the transfer.bas program described by Jahnke (1992). At the DNA–DNA level, there was 8.5 % genetic relatedness between strain YC1^T and B. macauensis JCM 13285^T, which provided decisive evidence that the two strains belong to different species when the recommended threshold value of 70 % DNA–DNA relatedness for the delineation of genomic species is considered (Stackebrandt et al., 2002).

Analysis of isoprenoid quinones was performed according to Komagata & Suzuki (1987) on cells harvested from an LB broth culture incubated at 30 °C for 2 days. Peptidoglycan diamino acids were analysed in cell-wall preparations as described by Bousfield et al. (1985). The presence of meso-diaminopimelic acid as the diagnostic diamino acid of the cell-wall peptidoglycan and MK-7 as the predominant menaquinone were features that were consistent with those of numerous Bacillus species, including B. macauensis JCM 13285^T (Zhang et al., 2006) and Bacillus barbaricus DSM 14730^T (Täubel et al., 2003).

Polar lipids were extracted, examined by two-dimensional TLC and identified by using published procedures (Minnikin et al., 1984). Fatty acid methyl esters were prepared and identified following the instructions of the Microbial Identification system (MIDI), as described by Sasser (1990), after cells were grown on tryptic soy agar (Merck) for 2 days at 30 °C. The type strain of the closely related species B. macauensis (JCM 13285^T) was also cultured under the same nutrient and temperature conditions and characterized concurrently with strain YC1^T. The polar lipids present in strain YC1^T and B. macauensis were essentially similar, showing only slight quantitative differences; the polar lipid patterns for strain YC1^T and B. macauensis JCM 13285^T are available as Supplementary Fig. S1 in IJSEM Online. The major polar lipids present in both strains were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) and minor components were an unknown aminolipid (AL) and an unknown aminophospholipid (APL); glycolipids were not detected. The polar lipids DPG, PG, PE, AL and APL have also been detected in B. barbaricus DSM 14730^T. The absence of glycolipids in strain YC1^T confirmed its close phylogenetic relatedness to B. macauensis and B. barbaricus, neither of which have glycolipids in their polar lipid profile, and its distant phylogenetic relatedness to Bacillus subtilis, which has been reported to contain glycolipids (Brundish et al., 1965; Bishop et al., 1967; Kämpfer et al., 2006).

The major cellular fatty acids of strain YC1^T were iso-C_15 : 0 and anteiso-C_15 : 0, comprising about 38.6 and 35.9 %, respectively, of the total fatty acid content. As shown by Kämpfer (1994), the predominance of 14- to 17-carbon iso- and anteiso-branched fatty acids is typical of members of the genus Bacillus. The fatty acid profile of strain YC1^T was similar to those of its close relatives, B. macauensis JCM 13285^T, B. barbaricus DSM 14730^T and Bacillus gelatini LMG 21880^T, but the fatty acid compositions were somewhat different (Table 1⇓).

Cell morphology and motility were examined using light microscopy (model BH2; Olympus) and transmission electron microscopy (model H-7650; Hitachi) after 48 h growth on LB agar at 30 °C. For electron microscopic observation, cells were air-dried and negatively stained with 1 % (w/v) phosphotungstic acid (see Supplementary Fig. S2 available in IJSEM Online). Gram-staining and analysis of endospore-forming features were performed according to the method described by Smibert & Krieg (1994). Growth at different temperatures and pH was investigated as described by Xu et al. (2005) using LB as the basal medium. NaCl tolerance was tested at concentrations between 0 and 20 % (w/v) in combination with LB medium. Growth was tested on nutrient agar, Simmons' citrate agar, cetrimide agar and MacConkey agar. Oxidase, catalase and phenylalanine deaminase activities and hydrolysis of casein, gelatin, Tweens 80 and 20, carboxylmethylcellulose, aesculin, tyrosine, DNA and starch were investigated as described by Smibert & Krieg (1994). Acid production from carbohydrates was determined using the medium and method described by Yamaguchi & Yokoe (2000). Single carbon source utilization studies were carried out as described by Liu et al. (2008). Sensitivity to antibiotics was tested using the diffusion method as described by Park et al. (2007) on LB agar at 30 °C with antibiotic filter-paper discs (8 mm diameter; Sanofi Pasteur). All tests were performed at 30 °C and properties were recorded after 2 days. Additional biochemical features were tested using API 20E kits (bioMérieux) according to the instructions of the manufacturer. Phenotypic characteristics of strain YC1^T are shown in the species description and in Table 2⇓. As shown in Table 2⇓, the novel isolate can be readily differentiated from its closest phylogenetic relatives in the genus Bacillus by several phenotypic properties.

On the basis of the above evidence, it is proposed that strain YC1^T should be classified in the genus Bacillus as the type strain of a novel species, for which the name Bacillus solisalsi sp. nov. is proposed.