Bacillus hwajinpoensis sp. nov. and an unnamed Bacillus genomospecies, novel members of Bacillus rRNA group 6 isolated from sea water of the East Sea and the Yellow Sea in Korea

Recent developments in genomic and chemical analyses have demonstrated the taxonomic heterogeneity of the genus Bacillus (Priest, 1981; Ash et al., 1991; Slepecky & Hemphill, 1991; Stackebrandt & Liesack, 1993; Rainey et al., 1994). In particular, from the results of 16S rDNA sequence analysis, Ash et al. (1991) revealed the presence of five phylogenetically distinct groups within the genus Bacillus. Subsequently, many Bacillus species that belong to these phylogenetic groups have been reclassified as members of novel genera or transferred to other genera (Wisotzkey et al., 1992; Ash et al., 1993; Shida et al., 1996; Spring et al., 1996; Wainø et al., 1999; Nazina et al., 2001; Yoon et al., 2001). Nielsen et al. (1994) established a new rRNA group, designated rRNA group 6, that contains some alkaliphilic and alkalitolerant bacilli. Bacillus species that belong to rRNA group 6 have been found to form a phylogenetic clade that is distinct from other related genera, as well as Bacillus rRNA group 1, which includes Bacillus subtilis, the type species of the genus Bacillus (Nielsen et al., 1994; Schlesner et al., 2001).

In this study, we describe two Gram-positive or -variable, endospore-forming, slightly halophilic rods, strains SW-72^T and SW-93, that were isolated from sea water of the East Sea and the Yellow Sea in Korea, respectively. The results of 16S rDNA sequence comparison indicated that these organisms are related phylogenetically to members of Bacillus rRNA group 6. Accordingly, the aim of the present work was to establish the exact taxonomic positions of strains SW-72^T and SW-93 by using a combination of phenotypic characterization, detailed phylogenetic analysis based on 16S rDNA sequences and genomic relatedness.

Strains SW-72^T and SW-93 were isolated by the dilution-plating technique on marine agar 2216 (MA) (Difco). Cell biomass of strains SW-72^T and SW-93 for cell-wall and isoprenoid quinone analyses and for DNA extraction was obtained from cultures in marine broth 2216 (MB) (Difco) at 30 °C. For fatty acid methyl ester analysis, cell mass of strains SW-72^T and SW-93 was obtained from agar plates after cultivation for 2 days at 30 °C on MA. Cell morphology was examined by light microscopy (using a Nikon E600 microscope) and transmission electron microscopy. Flagellation was examined by using transmission electron microscopy with cells from exponentially growing cultures. Gram-reaction was determined by using the bioMérieux Gram-stain kit according to the manufacturer's instructions. Catalase and oxidase activities, hydrolysis of casein and starch and phenylalanine deamination were determined as described by Cowan & Steel (1965). Aesculin hydrolysis and nitrate reduction were determined according to the method of Lanyi (1987), with the addition of 3 % (w/v) NaCl. Hydrolysis of gelatin and Tweens 20, 40, 60 and 80 was determined as described by Cowan & Steel (1965), with the modification that artificial sea water was used. Artificial sea water contained the following [(l distilled water)⁻¹]: 23·6 g NaCl, 0·64 g KCl, 4·53 g MgCl₂.6H₂O, 5·94 g MgSO₄.7H₂O and 1·3 g CaCl₂.2H₂O (Levring, 1946). Hydrolysis of hypoxanthine, tyrosine and xanthine was performed on MA, with substrate concentrations as described by Cowan & Steel (1965). Acid production from carbohydrates was determined as described by Leifson (1963). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with MA that had been prepared anaerobically by using nitrogen. Growth in the absence of NaCl was investigated in trypticase–soy broth that lacked NaCl. pH range for growth was determined in MB that was adjusted to pH values 4·5, 5·0, 5·5, 6·0, 6·5, 7·0, 7·5, 8·0, 8·5, 9·0, 9·5 and 10·0. Growth at various NaCl concentrations was investigated in MB. Growth at various temperatures (4–50 °C) was measured on MA.

The isomer type of diamino acid of the cell-wall peptidoglycan was determined by the method of Komagata & Suzuki (1987). Menaquinones were analysed as described previously (Komagata & Suzuki, 1987) by using reverse-phase HPLC. For quantitative analysis of cellular fatty acid compositions, a loop of cell mass was harvested and fatty acid methyl esters were prepared and identified by following the instructions of the Microbial Identification system (MIDI). Chromosomal DNA was isolated and purified according to a method described previously (Yoon et al., 1996), with the exception that ribonuclease T1 was used together with ribonuclease A. DNA G+C content was determined by the method of Tamaoka & Komagata (1984). DNA was hydrolysed and the resultant nucleotides were analysed by reverse-phase HPLC. 16S rDNA was amplified by PCR using two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rDNA and phylogenetic analysis were performed as described by Yoon et al. (2003). 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 by using five replicates for each sample; the highest and lowest values obtained for each sample were excluded and the remaining three values were used to calculate similarity values. The DNA–DNA relatedness values quoted are means of these three values.

Strain SW-72^T had no flagella, whereas strain SW-93 was motile by means of peritrichous flagella (see Supplementary Figure A, available in IJSEM Online). Maximum growth temperatures of strains SW-72^T and SW-93 were 40 and 42 °C, respectively. Strain SW-72^T grew at pH values in the approximate range 5·0–9·5, with an optimum at pH 6·0–7·0. Strain SW-93 grew better at pH 8·5–9·5 than at neutral pH. Strain SW-93 grew at pH 5·0, but not at pH 4·5. Neither strain showed any growth in the absence of NaCl. Strains SW-72^T and SW-93 did not grow in the presence of >20 and >21 % (w/v) NaCl, respectively. Phenotypic properties of strains SW-72^T and SW-93 are shown in Table 1⇓ or are given in the species description (see below).

Strains SW-72^T and SW-93 contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The predominant isoprenoid quinone in strains SW-72^T and SW-93 was unsaturated menaquinone with seven isoprene units (MK-7). Both strains had cellular fatty acid profiles that contained large amounts of straight-chain, branched and unsaturated fatty acids (Table 2⇓), and both contained anteiso-C_15 : 0 as the major fatty acid (Table 2⇓). The DNA G+C contents of strains SW-72^T and SW-93 were 40·9 and 41·0 mol%, respectively.

The 16S rDNA sequences of strains SW-72^T and SW-93 determined in this study comprised 1507 and 1508 nt, respectively, representing approximately 96 % of the Escherichia coli 16S rDNA sequence. 16S rDNA sequence similarity between strains SW-72^T and SW-93 was 99·7 %. In the phylogenetic tree based on the neighbour-joining algorithm, strains SW-72^T and SW-93 fall within the radiation of the cluster that comprises Bacillus species, particularly Bacillus species that have been designated as belonging to rRNA group 6 (Fig. 1⇓). In trees generated by using the maximum-likelihood and maximum-parsimony algorithms, the cluster that includes strains SW-72^T and SW-93 and Bacillus rRNA group 6 is also separate from the clade that includes Bacillus rRNA group 1 and other related genera, although there are some differences in tree topology (data not shown). 16S rDNA similarity levels between strains SW-72^T and SW-93 and the type strains of Bacillus species that are assigned to rRNA group 6 were in the range 93·1–95·2 %. Strains SW-72^T and SW-93 exhibited 16S rDNA sequence similarity levels of <94·0 and 94·3 %, respectively, to Bacillus species of rRNA group 1 and members of related genera that were used for phylogenetic analysis. DNA–DNA hybridization was performed to determine genomic relatedness between strains SW-72^T and SW-93. These strains exhibited two independent levels of DNA–DNA relatedness (23·2 and 19·7 %) when their DNA was used reciprocally as the labelled DNA probe.

Phylogenetic analysis based on 16S rDNA sequences, together with phenotypic properties, reveals that strains SW-72^T and SW-93 are members of the genus Bacillus, and particularly Bacillus rRNA group 6 (Fig. 1⇑). The results obtained from chemotaxonomic analyses are consistent with those obtained from 16S rDNA sequence analysis. Strains SW-72^T and SW-93 are characterized chemotaxonomically by the presence of meso-diaminopimelic acid in the peptidoglycan and MK-7 as the predominant menaquinone. Cellular fatty acid patterns of strains SW-72^T and SW-93 are similar to a pattern that is characteristic of other Bacillus species (Table 2⇑). From the results of phylogenetic inference, it is apparent that the bacilli of rRNA group 6 may have to be reclassified as one or more novel genera (Nielsen et al., 1994). Nevertheless, there are a few clear-cut differences in phenotypic characteristics, particularly chemotaxonomic characteristics, making it possible to differentiate this group from members of Bacillus rRNA group 1, which includes B. subtilis, the type species of the genus. A decision as to whether rRNA group 6 bacilli exist as members of the genus Bacillus that are separate from the phylogenetic radiation that comprises rRNA group 1 bacilli or should be included in a novel genus (or genera) should await additional phenotypic data and detailed phylogenetic analysis. As no proposal concerning taxonomic re-evaluation of rRNA group 6 bacilli has yet been put forward, strains SW-72^T and SW-93 are still described as members of the genus Bacillus.

Analysis of 16S rDNA sequences reveals that strains SW-72^T and SW-93 have a high degree of relatedness, there being only five 16S rDNA nucleotide sequence differences (99·7 % similarity) (Fig. 1⇑). However, the DNA–DNA relatedness value for strains SW-72^T and SW-93 is much lower than 70 %, a value that has been recognized as the threshold for species delineation (Wayne et al., 1987). The same result was obtained from repeated tests, as well as from cross-hybridization between DNA extracted from the two strains. The possibility of contamination of DNA from strains SW-72^T and SW-93 was investigated by sequencing 16S rDNA that was amplified from DNA of each strain. However, there was no contamination of the DNA of the two strains. Strains SW-72^T and SW-93 are similar in their phenotypic characteristics, although there are minor differences in some features. Therefore, it is better that these two are considered as members of genomic species that cannot be differentiated phenotypically from each other (Wayne et al., 1987). The taxonomic positions of strains SW-72^T and SW-93 within the genus Bacillus, particularly Bacillus rRNA group 6, become distinct from the results of 16S rDNA sequence comparison (Fig. 1⇑). 16S rDNA sequence similarity values obtained for strains SW-72^T and SW-93 and the type strains of Bacillus species are low enough to exclude the possibility of assigning strains SW-72^T and SW-93 to any existing Bacillus species (Stackebrandt & Goebel, 1994).

On the basis of these data, we propose that strain SW-72^T should be placed within the genus Bacillus as the type strain of a novel species, Bacillus hwajinpoensis sp. nov., and that strain SW-93 should be placed within the genus Bacillus as an unnamed Bacillus genomospecies.