Oceanobacillus locisalsi sp. nov., isolated from a marine solar saltern

The genus Oceanobacillus was first proposed by Lu et al. (2001) with the description of a single species, Oceanobacillus iheyensis. Subsequently, four Oceanobacillus species with validly published names, Oceanobacillus oncorhynchi (Yumoto et al., 2005), Oceanobacillus chironomi (Raats & Halpern, 2007), Oceanobacillus profundus (Kim et al., 2007) and Oceanobacillus caeni (Nam et al., 2008), have been reported; a further species, Oceanobacillus picturae (Lee et al., 2006), was described by the reclassification of Virgibacillus picturae (Heyrman et al., 2003). In this study, the taxonomic characterization of an Oceanobacillus-like bacterium, strain CHL-21^T, which was isolated from a marine solar saltern, is reported.

Sediment samples were collected from a marine solar saltern (3 ° 41′ 59″ N 12 ° 34′ 33″ E) of the Yellow Sea, Korea, and used for the isolation of bacterial strains. Strain CHL-21^T was isolated by the dilution plating technique at 25 °C on a modified S-G medium (Sehgal & Gibbons, 1960) containing (l⁻¹ distilled water): 100 g NaCl; 20 g MgSO₄ . 7H₂O; 2 g KCl; 3 g trisodium citrate; 10 g yeast extract; and 7.5 g Casamino acids (pH 7.2). The type strains of the two subspecies of Oceanobacillus oncorhynchi and of two other species of the genus Oceanobacillus were used as reference strains for DNA–DNA hybridization and/or phenotypic characterization. Oceanobacillus oncorhynchi subsp. oncorhynchi JCM 12661^T was obtained from the Japan Collection of Microorganisms (JCM), Saitama, Japan. Oceanobacillus oncorhynchi subsp. incaldanensis DSM 16557^T, Oceanobacillus chironomi DSM 18262^T and Oceanobacillus iheyensis DSM 14371^T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. To investigate morphological, physiological and biochemical characteristics, strain CHL-21^T was routinely cultivated at 30 °C on another modified S-G medium (MSG), which contained (l⁻¹ distilled water): 50 g NaCl; 20 g MgSO₄ . 7H₂O; 2 g KCl; 3 g trisodium citrate; 10 g yeast extract; and 7.5 g Casamino acids (pH 7.2). Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. Flagellation was determined by using a Philips CM-20 transmission electron microscope with cells from exponentially growing cultures. For this purpose, cells were negatively stained with 1 % (w/v) phosphotungstic acid and grids were examined after being air-dried. The Gram reaction was performed by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth under anaerobic conditions was determined after incubation in a Forma anaerobic chamber on MSG agar [i.e. MSG medium containing 1.5 % (w/v) agar] and on MSG agar supplemented with 0.1 % (w/v) potassium nitrate, both of which had been prepared anaerobically under a nitrogen atmosphere. Growth at various temperatures (4–50 °C) was measured on MSG agar. The pH range for growth was determined in MSG broth adjusted to various pH (4.5–9.5, at intervals of 0.5) by the addition of HCl or Na₂CO₃. Growth at various NaCl concentrations [0–30 % (w/v), in increments of 1.0 %] was investigated by using liquid media prepared according to the formula of the MSG medium except that NaCl was excluded. Growth in the absence of NaCl was also investigated using trypticase soy broth, in the presence and absence of 0.45 % (w/v) MgCl₂ . 6H₂O, prepared according to the formula of the Difco medium except that NaCl was excluded. Catalase and oxidase activities were determined as described by Cowan & Steel (1965). Nitrate reduction and hydrolysis of aesculin, gelatin, urea and Tweens 20, 40, 60 and 80 were tested as described by Lányí (1987) by using artificial seawater (Bruns et al., 2001) for preparation of media. Hydrolysis of casein, hypoxanthine, starch and xanthine was tested on MSG agar using the substrate concentrations described by Cowan & Steel (1965). Acid production from carbohydrates was determined according to the method of Leifson (1963). Susceptibility to various antibiotics was investigated on MSG plates by using antibiotic discs of the following: polymyxin B (100 U), streptomycin (50 μg), penicillin G (20 U), ampicillin (10 μg), cephalothin (30 μg), gentamicin (30 μg), novobiocin (5 μg), chloramphenicol (100 μg), kanamycin (30 μg), lincomycin (15 μg), neomycin (30 μg), oleandomycin (15 μg), carbenicillin (100 μg), tetracycline (30 μg), rifampicin (5 μg) and erythromycin (22.5 μg). Enzyme activities were determined by using the API ZYM system (bioMérieux).

Cell biomass for DNA extraction and for analysis of cell-wall and isoprenoid quinones was obtained from cultures grown in MSG broth at 30 °C. Chromosomal DNA was isolated and purified as described by Yoon et al. (1996) with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed according to the method described by Yoon et al. (2003). The isomer type of the diamino acid in the cell-wall peptidoglycan was analysed using TLC according to the method described by Komagata & Suzuki (1987). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250×4.6 mm) column. For cellular fatty acid analysis, cell mass of strain CHL-21^T, Oceanobacillus chironomi DSM 18262^T, Oceanobacillus iheyensis DSM 14371^T, Oceanobacillus oncorhynchi subsp. incaldanensis DSM 16557^T and Oceanobacillus oncorhynchi subsp. oncorhynchi JCM 12661^T was harvested after incubation for 3 days at 30 °C on marine agar 2216 (Difco). Fatty acids were extracted and fatty acid methyl esters were prepared by the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined according to the procedure of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed using nuclease P1 (Sigma) and the resultant nucleotides were analysed by reversed-phase HPLC. DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded and the mean of the remaining three values was quoted as the DNA–DNA reassociation value.

Morphological, cultural, physiological and biochemical characteristics of strain CHL-21^T are given in the species description (see below) or are shown in Table 1⇓. The almost-complete 16S rRNA gene sequence of strain CHL-21^T determined in this study comprised 1520 nt. A comparative 16S rRNA gene sequence analysis and estimation of phylogenetic relationships showed that strain CHL-21^T was closely related to members of the genera Oceanobacillus, Ornithinibacillus and Paucisalibacillus (Fig. 1⇓). It exhibited 16S rRNA gene sequence similarity values of 97.1–97.2 % to the type strains of the two subspecies of Oceanobacillus oncorhynchi, 93.1–94.7 % to the type strains of the other species of the genus Oceanobacillus, and 92.0–93.3 % to the type strains of two species of the genus Ornithinibacillus and Paucisalibacillus globulus. Mean DNA–DNA reassociation values between strain CHL-21^T and the type strains of the subspecies of Oceanobacillus oncorhynchi were 19–21 % (19 % for O. oncorhynchi subsp. oncorhynchi JCM 12661^T; 21 % for O. oncorhynchi subsp. incaldanensis DSM 16557^T).

Strain CHL-21^T contained meso-diaminopimelic acid as diamino acid in the cell-wall peptidoglycan and MK-7 as the predominant menaquinone. The cellular fatty acid profile of strain CHL-21^T is shown in Table 2⇓, together with those of related species of the genus Oceanobacillus. Strain CHL-21^T had large amounts of branched fatty acids; the major components (>10 % of total fatty acids) were anteiso-C_15 : 0 and anteiso-C_17 : 0. The fatty acid profile of strain CHL-21^T was similar to those of other species of the genus Oceanobacillus in that anteiso-C_15 : 0 is the predominant fatty acid, although there were differences in the proportions of some fatty acids, particularly iso-C_15 : 0 and anteiso-C_17 : 0 (Table 2⇓). The DNA G+C content of strain CHL-21^T was 39.8 mol%.

Strain CHL-21^T was distinguishable from phylogenetically related species of the genus Oceanobacillus by differences in several phenotypic characteristics, as listed in Table 1⇑. The phylogenetic and genetic distinctiveness and differential phenotypic properties of strain CHL-21^T are sufficient to categorize it as a member of a species that is distinct from recognized species of the genus Oceanobacillus (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, based on the data presented, strain CHL-21^T is considered to represent a novel species of the genus Oceanobacillus, for which the name Oceanobacillus locisalsi sp. nov. is proposed.