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Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Oeundong, Yusong, Daejon 305-333, Republic of Korea
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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ABSTRACT |
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-meso-diaminopimelic acid as the diagnostic diamino acid. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. DNA G+C content was about 41 mol% and the major isoprenoid quinone was MK-7. On the basis of their physiological and molecular properties, the isolates represent a new genus, Pontibacillus gen. nov., and novel species, Pontibacillus chungwhensis sp. nov. The type strain is BH030062T (=KCTC 3890T=DSM 16287T).
Published online ahead of print on 30 July 2004 as DOI 10.1099/ijs.0.63315-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains BH030062T, BH030049 and BH030080 are AY553296, AY603361 and AY603362, respectively.
A transmission electron micrograph showing the general morphology of strain BH030062T is available as supplementary material in IJSEM Online.
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). Taxonomically, they include both Gram-positive and Gram-negative micro-organisms. Aerobic spore-forming moderately halophilic Gram-positive rods are also taxonomically diverse and have been isolated from marine environments and related habitats. They were originally assigned to the genus Bacillus, but molecular and chemical analyses have shown that they form several phylogenetically distinct lineages within the group classically defined as the genus Bacillus (Ash et al., 1991; Stackebrandt & Liesack, 1993; Nielsen et al., 1994). The distinct lineages have been described as the genera Halobacillus (Spring et al., 1996), Gracilibacillus (Wainø et al., 1999), Virgibacillus (Heyndrickx et al., 1998), Filobacillus (Schlesner et al., 2001) and Lentibacillus (Yoon et al., 2002). Recently, some members of the genera Salibacillus and Bacillus have been transferred to the genera Virgibacillus and Gracilibacillus (Wainø et al., 1999; Heyrman et al., 2003). Some members of the genera Filobacillus and Halobacillus have shown Gram-negative or Gram-variable reactions, although phylogenetically they belong to the Gram-positive bacteria (Schlesner et al., 2001; Yoon et al., 2003). In this study, three moderately halophilic Gram-positive bacteria were isolated from the Chungwha solar saltern in Korea. Using a polyphasic approach, the strains were classified in a new genus, for which the name Pontibacillus gen. nov. is proposed. The three strains (BH030062T, BH030049 and BH030080) were isolated from a solar saltern of the Tae-An area on the Yellow Sea in Korea. The strains were isolated from soil samples that were diluted serially, spread on marine agar 2216 (MA; Difco) with the addition of 5 % (w/v) NaCl [final concentration: 6·94 % (w/v) NaCl] and incubated for 2 days at 35 °C. Requirement of and tolerance to NaCl were determined in nutrient broth (NB; Difco; 3·0 g beef extract l–1 and 5·0 g peptone l–1) supplemented with modified artificial sea water (ASW) 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 35 °C, except where otherwise indicated. Anaerobic growth was determined by incubation in an anaerobic chamber at 35 °C on MA. Growth was tested at different temperatures (4–55 °C) on MA and at different pH values (pH 5·0–10·0) in NB supplemented with ASW containing 2·36 % (w/v) NaCl.
Cell morphology was studied using light microscopy and transmission electron microscopy. Motility was observed at 12 and 36 h in wet mounts with a light microscope (Nikon E600). The flagellum type was examined by transmission electron microscopy using cells from the exponential growth phase. Cells were mounted on Formvar-coated copper grids and negatively stained with 1 % potassium phosphotungstate (pH 7·0). Grids were examined in a JEOL JEM-1010 transmission electron microscope operated at 60 kV. Endospores were stained according to the method of Schaeffer-Fulton (Smibert & Krieg, 1981).
Gram staining was determined using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Catalase activity was determined by production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck). Hydrolysis of aesculin, casein, starch, Tween 80, urea, hypoxanthine, tyrosine, gelatin and xanthine was determined on MA according to previously described methods (Cowan & Steel, 1965; Lanyi, 1987; Smibert & Krieg, 1994). Nitrate reduction was performed according to the method of Lanyi (1987). Acid production from carbohydrates was tested as described by Leifson (1963); all suspension media were supplemented with ASW containing 2·36 % (w/v) NaCl.
For quantitative analysis of whole cell fatty acids, strains BH030062T, BH030049 and BH030080 were cultivated on either MA or MA plus 3 % (w/v) NaCl for 2 days at 35 °C. Fatty acid methyl esters were prepared from 40 mg wet cells and analysis was carried out using GC/MS chromatography according to the instructions of the Microbial Identification System (MIDI; Microbial ID). Preparation of cell walls from the test strains and analysis of peptidoglycan structures were carried out using methods described by Schleifer & Kandler (1972), with the modification that TLC on cellulose sheets was performed instead of paper chromatography. Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987) using HPLC fitted with a reversed-phase column (GROM-SIL 100 ODS-2FE; GROM). Methanol/2-propanol (2 : 1, v/v) was used as the mobile phase and isoprenoid quinones were detected at 270 nm. The DNA G+C contents of strains BH030062T, BH030049 and BH030080 were determined by reversed-phase HPLC using the method of Tamaoka & Komagata (1984). DNA–DNA hybridization was carried out to evaluate the genomic DNA relatedness of the three isolates. The isolates were grown aerobically on MA for 2 days at 35 °C and chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996). Randomly primed DNA labelling with digoxigenin (DIG)-dUTP and detection of hybrids by enzyme immunoassay on nylon membranes were performed using the DIG High Prime DNA Labelling and Detection Starter kit II (Roche Applied Science) according to the manufacturer's instructions.
PCR amplification, sequencing and assembling of the 16S rRNA gene were carried out as described previously (Jeon et al., 2004). The resultant sequences were compared with 16S rRNA gene sequences available from GenBank using the BLAST program to determine the approximate phylogenetic affiliation and aligned with members of the group classically defined as the genus bacillus using the software CLUSTAL W (Thompson et al., 1994). Sequence similarity values were computed using Similarity Matrix version 1.1 (Ribosomal Database Project II; http://rdp.cme.msu.edu/html/analyses.html; Cole et al., 2003). Gaps at the 5' and 3' ends of the alignment were omitted from further analyses. The phylogenetic trees were constructed using three different methods: neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) algorithms available in the PHYLIP software, version 3.6 (Felsenstein, 2002). Evolutionary distance matrices were calculated according to the algorithm of the Kimura two-parameter model (Kimura, 1980) for the neighbour-joining method. To evaluate the stability of the phylogenetic tree, a bootstrap analysis (1000 replications) was performed with the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE in the PHYLIP package.
Colonies of strain BH030062T were yellow, low convex, smooth and circular/slightly irregular after 2 days incubation at 35 °C on MA. The strain grew in media containing 1–15 % (w/v) NaCl; optimum growth occurred on media containing 2–5 % (w/v) NaCl. It did not grow without NaCl or in the presence of more than 15 % (w/v) NaCl. It grew in nutrient agar (Difco) supplemented with ASW, but not in nutrient agar with just NaCl, indicating that the strain required salts other than NaCl for growth. Strain BH030062T grew at pH 6·5–9·5 in NB containing 3 % (w/v) NaCl; optimal growth occurred at pH 7·5–8·5. Growth was observed at temperatures between 15 and 45 °C, with optimum growth temperatures of 35–40 °C.
Strain BH030062T was Gram-positive and strictly aerobic. Cells were rod-shaped with a width of 0·6–0·9 µm and length of 2·3–3·0 µm and motile by means of peritrichous flagella after 2 days incubation at 35 °C on MA (see the transmission electron micrograph available as supplementary material in IJSEM Online). Spherical endospores were formed at terminal positions in swollen sporangia, enabling this strain to be distinguished from some other closely related Gram-positive bacteria (Table 1).
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-D-lactose, adonitol, D-raffinose, D-mannitol, D-fructose, arbutin, D-salicin, D-melibiose or D-mannose. Typical phenotypic and chemotaxonomic properties of strain BH030062T are summarized and compared with those of phylogenetically related type strains of the group classically defined as the genus Bacillus in Table 1
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Analysis of the cell wall peptidoglycan showed that strain BH030062T possessed A1-meso-diaminopimelic acid (DAP) as the diagnostic diamino acid, in common with the great majority of endospore-forming Gram-positive bacilli. The major isoprenoid quinone was menaquinone-7 (MK-7). The cellular fatty acid profile of strain BH030062T was characterized by anteiso-C15 : 0 (
31·1 %), iso-C15 : 0 (28·5 %) and iso-C16 : 0 (10·2 %) as the major fatty acids on MA. Strains BH030062T, BH030049 and BH030080 had very similar fatty acid profiles on both MA and MA supplemented with 3 % (w/v) NaCl (Table 2), indicating that they may possibly belong to the same species. The genomic DNA G+C contents of strains BH030062T, BH030049 and BH030080 were about 41·0, 40·8 and 41·5 %, respectively. The major fatty acid profiles, the major lipoquinone and the DNA G+C content are typical of the group classically defined as the genus Bacillus (Table 1). DNA–DNA hybridization was assessed to evaluate the genomic DNA relatedness among strains BH030062T, BH030049 and BH030080. The values obtained from DNA–DNA hybridization experiments of strain pairs BH030062T/BH030049, BH030062T/BH030080 and BH030049/BH030080 were about 97, 92 and 94 %, respectively. It was concluded that the DNA–DNA relatedness values were sufficiently high for these three strains to be classified as a single species (Stackebrandt et al., 2002).
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). The topologies of phylogenetic trees built using the maximum-likelihood and maximum-parsimony algorithms were similar to those of the tree constructed by neighbour-joining analysis (data not shown). The 16S rRNA gene sequence of strain BH030062T was highly similar to the sequences of BH030049 and BH030080 (99·9 and 99·3 % similarity, respectively). The 16S rRNA gene sequence similarity values of the three strains to sequences of closely related type strains of Gram-positive bacteria were as follows: Gracilibacillus (93·7–95·1 %), Virgibacillus (93·5–94·8 %), Halobacillus (94·8–95·9 %), Filobacillus (94·4–94·8 %) and Lentibacillus (93·3–93·7 %).
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Description of Pontibacillus gen. nov.
Pontibacillus (Pon.ti.ba.cil'lus. L. n. pontus the sea; L. dim. n. bacillus small rod; N.L. masc. n. Pontibacillus bacillus pertaining to the sea).
Cells are Gram-positive, spore-forming rods. Catalase-positive and oxidase-negative. Urease-negative. Spherical endospores are formed terminally in swollen sporangia. Strictly aerobic and moderately halophilic. Cells are motile by means of peritrichous flagella. Cell wall peptidoglycan contains A1 type meso-DAP. Major isoprenoid quinone is MK-7. DNA G+C content is 40·8–41·5 mol% (HPLC method). Predominant cellular fatty acids are anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0 on MA. Phylogenetically, the genus belongs to the family Bacillaceae.
The type species is Pontibacillus chungwhensis.
Description of Pontibacillus chungwhensis sp. nov.
Pontibacillus chungwhensis (chung.when'sis. N.L. masc. adj. chungwhensis belonging to Chungwha, where the organism was isolated).
Cells are approximately 0·6–0·9 µm wide and 2·3–3·0 µm long. Strictly aerobic rods. Colonies are yellow, low convex, smooth and circular/slightly irregular on MA. Grows at 15–45 °C (optimum: 30–35 °C), pH 6·5–9·5 (optimum: pH 7·5–8·5) and 1–15 % (w/v) NaCl (optimum: 2–5 %, w/v). No growth occurs without NaCl or in the presence of more than 15 % (w/v) NaCl. Nitrate is not reduced to nitrite. Casein, starch and Tween 80 are hydrolysed. Does not hydrolyse aesculin, L-tyrosine, hypoxanthine, xanthine, gelatin or urea. Acids are produced from D-glucose, D-ribose, maltose, glycerol and D-trehalose, but not from D-xylose, L-arabinose, L-rhamnose, -D-lactose, adonitol, D-raffinose, D-mannitol, D-fructose, arbutin, D-salicin, D-melibiose or D-mannose. Cellular fatty acid profiles on MA and MA plus 3 % (w/v) NaCl are given in Table 2.
The type strain is BH030062T (=KCTC 3890T=DSM 16287T), isolated from a solar saltern on the Yellow Sea in Korea. DNA G+C content of the type strain is 41·0 mol% (HPLC method).
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ACKNOWLEDGEMENTS |
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J.-M. Lim, C. O. Jeon, D.-J. Park, H.-R. Kim, B.-J. Yoon, and C.-J. Kim Pontibacillus marinus sp. nov., a moderately halophilic bacterium from a solar saltern, and emended description of the genus Pontibacillus Int J Syst Evol Microbiol, May 1, 2005; 55(3): 1027 - 1031. [Abstract] [Full Text] [PDF]
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C. O. Jeon, J.-M. Lim, J.-C. Lee, G. S. Lee, J.-M. Lee, L.-H. Xu, C.-L. Jiang, and C.-J. Kim Lentibacillus salarius sp. nov., isolated from saline sediment in China, and emended description of the genus Lentibacillus Int J Syst Evol Microbiol, May 1, 2005; 55(3): 1339 - 1343. [Abstract] [Full Text] [PDF]
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