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1 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
2 Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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ABSTRACT |
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-type meso-diaminopimelic acid; major fatty acids, iso-C15 : 0 and anteiso-C15 : 0) also supported the affiliation of the isolate to the genus Pontibacillus. Although the 16S rRNA gene sequence similarity between strain BH030004T and Pontibacillus chungwhensis DSM 16287T was relatively high (99·1 %), physiological properties and DNA–DNA hybridization (about 7 % DNA–DNA relatedness) allowed genotypic and phenotypic differentiation of strain BH030004T from the type strain of P. chungwhensis. Therefore, strain BH030004T represents a novel species of the genus Pontibacillus, for which the name Pontibacillus marinus sp. nov. is proposed. The type strain is BH030004T (=KCTC 3917T=DSM 16465T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BH030004T is AY603977.
Fatty acid profiles of strain BH30004T and P. chungwhensis DSM 16287T are available as supplementary material in IJSEM Online.
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; Stackebrandt & Liesack, 1993; Nielsen et al., 1994). Some of these distinct lineages have been described as the novel genera Halobacillus (Spring et al., 1996; Yoon et al., 2003), Gracilibacillus (Wainø et al., 1999), Virgibacillus (Heyndrickx et al., 1998; Heyrman et al., 2003), Filobacillus (Schlesner et al., 2001) and Lentibacillus (Yoon et al., 2002). Recently, Lim et al. (2005) reported the species Pontibacillus chungwhensis, which is the only species described in the genus Pontibacillus to date. In this study, another novel Gram-positive bacterium belonging to the genus Pontibacillus was isolated from a solar saltern in Korea and its taxonomic status and phenotypic properties are described.
Strain BH030004T was isolated from a solar saltern of the Yellow Sea in Korea, using marine agar 2216 (MA) (Difco) supplemented with 5 % (w/v) NaCl and 3 days incubation at 35 °C. Requirements for, and tolerance of, NaCl were determined in nutrient broth (Difco) supplemented with modified artificial sea water [containing (l–1) 0–30 % (w/v) NaCl, 5·94 g MgSO4.7H2O, 4·53 g MgCl2.6H2O, 0·64 g KCl and 1·3 g CaCl2]. The isolates were routinely grown aerobically on MA for 2 days at 30 °C unless otherwise indicated. Anaerobic growth was determined by incubation in an anaerobic chamber for 5 days at 30 °C on MA. Growth was tested at different temperatures (4–55 °C) on MA and at different pH values (5·0–10·0) in nutrient broth supplemented with artificial sea water containing 2·36 % (w/v) NaCl. The cellular morphology of strain BH030004T was examined using light microscopy and transmission electron microscopy. Motility was observed at 12 and 36 h in wet mounts by means of a light microscope (E600 apparatus; Nikon). For visualization of flagella, cells were mounted on Formvar-coated copper grids (Electron Microscopy Science), negatively stained with 2 % (w/v) uranyl acetate for 15 s and then subjected to transmission electron microscopy (JEM-1010 apparatus; JEOL). Gram staining was determined using the bioMérieux Gram stain kit, according to the manufacturer's instructions. Oxidase activity was tested using a Bactident Oxidase strip (Merck) and catalase activity was determined by bubble production in 3 % (v/v) hydrogen peroxide solution. Nitrate reduction and hydrolysis of aesculin, casein, starch, Tween 80, urea, hypoxanthine, tyrosine, gelatin and xanthine were determined on MA as described by Lanyi (1987). Acid production from carbohydrates was tested as described by Leifson (1963); all suspension media were supplemented with artificial sea water containing 2·36 % (w/v) NaCl.
The whole-cell fatty acid profile of strain BH030004T was determined by using GC/MS according to the instructions of the Microbial Identification System (MIDI; Microbial ID) after cultivation of the micro-organism for 2 days at 30 °C on either MA or MA supplemented with 3 % (w/v) NaCl. The peptidoglycan and isoprenoid quinones of the strain were analysed as described by Komagata & Suzuki (1987). The DNA G+C content of strain BH030004T was determined by reverse-phase HPLC using the method of Tamaoka & Komagata (1984). DNA–DNA hybridization was carried out to evaluate the genomic DNA relatedness between strain BH030004T and P. chungwhensis KCTC 3890T. Genomic DNA from the isolate and from P. chungwhensis KCTC 3890T was isolated and purified according to the method described by Yoon et al. (1996). The isolated DNA samples were serially diluted from 200 to 0·5 ng and then denatured by boiling for 10 min. The series of dilutions was applied to positively charged nylon membranes with a slot device (Schleicher & Schuell) under a slight vacuum. Random-primed DNA labelling with digoxigenin-dUTP and hybridization were performed using a DIG High Prime DNA labelling kit and hybridization solutions (Roche Applied Science) according to the manufacturer's instructions. Detection of hybrids on the nylon membrane was performed using an enzyme immunoassay detection kit (Roche Applied Science). The membranes were incubated in 5 ml freshly prepared Coloursubstrate solution (Roche Applied Science) for 16 h in the dark. The membranes were washed with distilled water and then the colour intensities of the series of dilutions were quantified using Bio-Rad GelDoc scanning software. The signals produced by self-hybridization were inferred as 100 %, and relatedness values (percentages) were calculated from duplicate samples.
The 16S rRNA gene sequence of strain BH030004T was analysed as described previously (DeLong, 1992). The 16S rRNA gene sequence was aligned together with those of representative members of selected genera by using the CLUSTAL W program (Thompson et al., 1994). Sequence similarity values were computed using Similarity Matrix version 1.1 (Ribosomal Database Project II; http://rdp.cme.msu.edu/index.jsp; Cole et al., 2003). Gaps at the 5' and 3' ends of the alignment were omitted for further analyses. Phylogenetic trees were constructed using three different methods, the neighbour-joining, maximum-likelihood and maximum-parsimony algorithms, available in PHYLIP software, version 3.6 (Felsenstein, 2002). Evolutionary distance matrices were calculated according to the algorithm of the Kimura two-parameter model for the neighbour-joining method. A bootstrap analysis (1000 replications) was performed, using the neighbour-joining method in the PHYLIP package, to evaluate the stability of the phylogenetic tree.
On MA, strain BH030004T formed creamy, smooth and circular/slightly irregular colonies. It grew optimally on media containing 2–5 % (w/v) NaCl, but did not grow without NaCl or in the presence of more than 10 % (w/v) NaCl. The organism grew in nutrient agar (Difco) supplemented with artificial sea water, but not in nutrient agar with just NaCl, i.e. the strain requires other salts in addition to NaCl for growth. Anaerobic growth was not observed in an anaerobic chamber. Growth occurred from pH 6·0 to 9·0 (optimum, pH 7·0–7·5) and at temperatures between 15 and 40 °C (optimum, 30 °C). Strain BH030004T showed obvious Gram-, catalase- and oxidase-positive reactions and reduced nitrate to nitrite. The cells were rods, 0·4–0·9 µm wide and 3·3–4·0 µm long, and were motile by means of peritrichous flagella after 2 days incubation at 35 °C on MA. Spherical endospores were formed in a terminal position in swollen sporangia. Analysis of the cell-wall peptidoglycan of the isolate showed that the diagnostic diamino acid was meso-diaminopimelic acid (A1-type). The major isoprenoid quinone was MK-7. The genomic DNA G+C content was about 42·0 mol%.
An almost-complete 16S rRNA gene sequence of strain BH030004T was obtained and used for initial BLAST searches (http://www.ncbi.nlm.nih.gov/blast/) in GenBank and the phylogenetic analyses. The tree constructed by the neighbour-joining method showed that strain BH030004T formed a phyletic line (with a 100 % bootstrap value) distinct from the most closely related species, P. chungwhensis (Fig. 1). The topologies of phylogenetic trees constructed using the maximum-likelihood and maximum-parsimony algorithms were similar to that of the tree constructed using neighbour-joining analysis (data not shown). The strain was most closely related to P. chungwhensis KCTC 3890T (99·1 % 16S rRNA gene sequence similarity) and it was confirmed that differences between the two sequences were mainly localized in the second variable region of the 16S RNA molecules. Levels of 16S rRNA gene sequence similarity between strain BH030004T and other related taxa were less than 95·1 %. The predominant cellular fatty acids of strain BH030004T were iso-C15 : 0 (
65·1 %), anteiso-C15 : 0 (10·2 %), C16 : 1
7c alcohol (8·8 %) and iso-C14 : 0 (4·0 %) on MA (details available in a supplementary table in IJSEM Online). No significant differences in fatty acid profiles were found between cells grown on MA and those grown on MA supplemented with 3 % (w/v) NaCl. Two major cellular fatty acids, iso-C15 : 0 and anteiso-C15 : 0, of the test strain composed about 65 and 10 % of the total fatty acid content, respectively, whereas the corresponding fatty acids in P. chungwhensis KCTC 3890T accounted for about 28 and 31 % of the total fatty acid content, respectively. Although strain BH030004T and P. chungwhensis DSM 16287T had different fatty acid ratios, the profiles were similar. Therefore, comparative 16S rRNA gene sequence analysis clearly indicated that the novel strain was a member of the genus Pontibacillus. In addition to the results of phylogenetic analysis, many chemotaxonomic properties also supported the affiliation of the isolate to the genus Pontibacillus (Table 1).
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). Phenotypic properties of strain BH030004T, including pigmentation, NaCl range, oxidase, nitrate reduction, hydrolyses and acid production, also supported its distinction from closely related taxa (Table 1
). On the basis of the phenotypic and phylogenetic properties, it is proposed that strain BH030004T represents a novel species of the genus Pontibacillus, for which the name Pontibacillus marinus sp. nov. is proposed.
Description of Pontibacillus marinus sp. nov.
Pontibacillus marinus (ma'rin.us. L. masc. adj. marinus of the sea).
Cells are rods, approximately 0·4–0·9 µm wide and 3·3–4·0 µm long, motile by means of peritrichous flagella. Spherical endospores are formed terminally in swollen sporangia. Strictly aerobic. Catalase- and oxidase-positive. Nitrate is reduced to nitrite. Colonies are cream in colour, flat, smooth and circular/slightly irregular on MA. Growth occurs at 15–40 °C (optimum, 30 °C), pH 6·0–9·0 (optimum, pH 7·0–7·5) and 1–9 % (w/v) NaCl (optimum, 2–5 %). Tween 80 and aesculin are hydrolysed. Hydrolysis of casein, starch, gelatin, L-tyrosine, hypoxanthine, xanthine and urea is not observed. Acids are produced from sucrose, D-melibiose, D-trehalose, D-raffinose, D-fructose, D-ribose and maltose, but not from D-glucose, glycerol, D-xylose, L-arabinose, L-rhamnose, 
-D-lactose, adonitol, D-mannitol, inositol or D-mannose. Major cellular fatty acids on MA are iso-C15 : 0 (65·12 %), anteiso-C15 : 0 (10·15 %), C16 : 17c alcohol (8·82 %) and iso-C14 : 0 (4·0 %). DNA G+C content is 42·0 mol% (HPLC).
The type strain is BH030004T (=KCTC 3917T=DSM 16465T), isolated from a solar saltern of the Yellow Sea in Korea.
Emended description of the genus Pontibacillus Lim et al. 2005
The description is as given by Lim et al. (2005) with the following amendments. DNA G+C content is 40·8–42·0 mol% (HPLC). On MA, predominant cellular fatty acids are anteiso-C15 : 0 and iso-C15 : 0.
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ACKNOWLEDGEMENTS |
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