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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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for alkaliphilic strains isolated from potato-processing effluent (Gee et al., 1980). The first species of this genus to be described was Exiguobacterium aurantiacum, the type species. The second species of the genus, Exiguobacterium acetylicum, was created by reclassification of Brevibacterium acetylicum (Farrow et al., 1994). Recently, three more Exiguobacterium species, Exiguobacterium antarcticum and Exiguobacterium undae (Frühling et al., 2002) and Exiguobacterium oxidotolerans (Yumoto et al., 2004), have been described. 16S rRNA gene sequence analyses have shown that the genus Exiguobacterium is related phylogenetically to the genus Bacillus and related taxa (Farrow et al., 1992, 1994; Yumoto et al., 2004). In this study, three slightly halophilic bacterial strains, TF-16T, TF-19 and TF-80T, which were isolated from a tidal flat of Daepo Beach (Yellow Sea), near Mokpo City in Korea, are described. 16S rRNA gene sequence comparisons indicated that the three isolates are Exiguobacterium-like organisms. The aim of the present work was to determine the exact taxonomic positions of the three isolates by detailed characterization combining phenotypic, chemotaxonomic and genetic analyses. Tidal flat sediment was collected from a tidal flat and used to isolate bacterial strains. Strains TF-16T, TF-19 and TF-80T were isolated by the usual dilution plating method on marine agar 2216 (MA; Difco) at 30 °C. To investigate their morphological and physiological characteristics, strains TF-16T, TF-19 and TF-80T were routinely cultivated at 30 °C on MA under aerobic conditions. Reference strains E. aurantiacum KCTC 9244T and E. acetylicum KCTC 3255T were obtained from the Korean Collection for Type Cultures (KCTC), Taejon, Korea. For chemical and genetic analyses, cell mass of strains TF-16T, TF-19 and TF-80T and E. aurantiacum KCTC 9244T was obtained from marine broth 2216 (MB; Difco) cultures at 30 °C. For fatty acid methyl ester analysis, cell mass of strains TF-16T, TF-19 and TF-80T and E. aurantiacum KCTC 9244T was harvested from MA plates after 3 days cultivation at 30 °C and cell mass of E. acetylicum KCTC 3255T was obtained from trypticase soy agar (TSA; BBL) plates under the same conditions.
Cell morphology was examined by light microscopy (Nikon E600) and TEM. Flagellum type was examined by TEM using cells from exponentially growing cultures. The Gram reaction was determined using a Gram Stain kit (bioMérieux) according to the manufacturer's instructions. Growth at various temperatures, pH and NaCl concentrations was investigated on MA or in MB. Growth in the absence of NaCl was investigated in trypticase soy broth lacking NaCl. Catalase activity was tested by adding 3 % (v/v) hydrogen peroxide solution to colonies grown on MA. Cytochrome oxidase activity was determined using Kovac's reagent. Anaerobic growth was determined after incubation in an anaerobic chamber with MA that had been prepared anaerobically. Nitrate reduction, urease activity and hydrolysis of casein, starch, aesculin, xanthine, hypoxanthine, tyrosine, gelatin and Tween 80 were analysed as described by Cowan & Steel (1965) and Lanyi (1987). Acid production from carbohydrates was determined as described by Leifson (1963). Preparation of cell wall peptidoglycan was carried out using the method described by Schleifer & Kandler (1972) and peptidoglycan structure was determined using an automated amino acid analyser (Hitachi model L-8500A). Menaquinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250x4·6 mm) column. Fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Polar lipids were extracted using procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with the appropriate detection reagent (Komagata & Suzuki, 1987). Chromosomal DNA isolation and purification were carried out according to the method described by Yoon et al. (1996), with the exception that RNase T1 was applied in combination with RNase A. DNA G+C content was determined using the procedure of Tamaoka & Komagata (1984), with the modification that DNA was hydrolysed using nuclease P1 (Boehringer Mannheim) 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. Of the values obtained, the highest and lowest values in each sample were excluded. DNA–DNA relatedness values are the means of the remaining three values. Amplification of the 16S rRNA gene was carried out according to the method described previously using two universal primers (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene was performed as described by Yoon et al. (2003). Alignment of sequences was carried out using the program CLUSTAL_W (Thompson et al., 1994) and gaps at the 5' and 3' ends of the alignment were omitted from further analysis. Evolutionary distances were calculated using the Kimura two-parameter correction with the CLUSTAL_W package (Thompson et al., 1994). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) on the basis of distance matrix data. The grouping reliability was assessed by 1000 bootstrap resamplings of the neighbour-joining dataset using the CLUSTAL_W package.
Strains TF-16T and TF-19 had similar phenotypic characteristics. However, strain TF-80T could be differentiated from strains TF-16T and TF-19 by the following characteristics: flagellum type, colony colour, maximum growth temperature, NaCl tolerance and acid production from some substrates (Table 1). Other phenotypic characteristics are given in the species descriptions (see below). Some phenotypic characteristics that differentiated strains TF-16T, TF-19 and TF-80T from other Exiguobacterium species are indicated in Table 1. Cell wall analysis revealed that the peptidoglycan type in strains TF-16T, TF-19 and TF-80T was based on L-Lys–Gly, as described by Schleifer & Kandler (1972). The major menaquinone of strains TF-16T, TF-19 and TF-80T was MK-7. The three isolates and E. aurantiacum KCTC 9244T contained iso-C17 : 0, anteiso-C13 : 0, iso-C13 : 0 and iso-C15 : 0 as the major fatty acids (Table 2). The cellular fatty acid profile of E. acetylicum KCTC 3255T differed from the profiles of TF-16T, TF-19, TF-80T and E. aurantiacum KCTC 9244T; this may be because of different cultivation conditions, e.g. growth media. E. acetylicum KCTC 3255T contained large amounts of the unsaturated fatty acid C16 : 1
11c and the straight-chain fatty acids C14 : 0 and C16 : 0, which were minor components in TF-16T, TF-19, TF-80T and E. aurantiacum KCTC 9244T (Table 2). Polar lipids found in strains TF-16T, TF-19 and TF-80T were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and an unidentified phospholipid; ninhydrin-positive amino-group-containing lipids were also detected. The DNA G+C contents of strains TF-16T, TF-19 and TF-80T were 48·6, 48·4 and 48·0 mol%, respectively.
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). Sequences from strains TF-16T, TF-19 and TF-80T exhibited highest similarity values to that of the type strain of E. aurantiacum (98·3, 98·3 and 98·1 %, respectively). 16S rRNA gene sequence similarity levels between the three novel strains and the type strains of other Exiguobacterium species were 94·0–94·6 %. The relationship between the cluster comprising the three isolates and E. aurantiacum NCDO 2321T and the cluster comprising other Exiguobacterium species was supported by a bootstrap confidence value of 100 % (Fig. 1). DNA–DNA hybridization was performed to determine the genetic relatedness between strains TF-16T, TF-19 and TF-80T and between these three novel isolates and E. aurantiacum KCTC 9244T. The mean level of DNA–DNA relatedness between strains TF-16T and TF-19 was 98 %. Strain TF-80T showed mean DNA–DNA relatedness levels of 10–22 % to strains TF-16T and TF-19. Strains TF-16T and TF-19 and strain TF-80T showed DNA–DNA relatedness levels of 3–11 and 12 % to E. aurantiacum KCTC 9244T, respectively.
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). The chemotaxonomic characteristics of the three strains, including cell wall peptidoglycan type and menaquinone and fatty acid profiles, were similar to those of Exiguobacterium species (Collins et al., 1983; Frühling et al., 2002). The polar lipid profiles of the three strains were similar to those of E. undae and E. antarcticum, although they differed from those of E. aurantiacum and E. acetylicum in that phosphatidylinositol and phosphatidylserine were present (Collins et al., 1983; Frühling et al., 2002). Strains TF-16T and TF-19 had morphological and physiological characteristics that differed from those of strain TF-80T. Strains TF-16T, TF-19 and TF-80T could also be differentiated from other Exiguobacterium species by some phenotypic properties (Table 1). The phylogenetic and genetic distinctiveness indicated that strains TF-16T and TF-19 and strain TF-80T could be considered as members of two novel species that are separate from other Exiguobacterium species. Therefore, on the basis of the data presented, strains TF-16T and TF-19 and strain TF-80T should be classified in the genus Exiguobacterium as representatives of two distinct novel species, for which the names Exiguobacterium aestuarii sp. nov. and Exiguobacterium marinum sp. nov., respectively, are proposed.
Description of Exiguobacterium aestuarii sp. nov.
Exiguobacterium aestuarii (aes.tu'a.ri.i. L. gen. n. aestuarii of a part of the sea-coast which, during the flood-tide, is overflowed, but at the ebb-tide is left covered with mud or slime).
Cells are Gram-variable and facultatively anaerobic; growth is better under aerobic conditions. Colonies are circular, glossy, raised and 3–4 mm in diameter after incubation for 2 days on MA at 30 °C under aerobic conditions. White-coloured colonies are formed under anaerobic conditions. Cells are short rods (in the early growth phase) or cocci (exponential and stationary growth phases). Growth occurs at 10 and 47 °C, with optimum growth at 30–37 °C; growth does not occur at 4 °C or above 48 °C. Growth does not occur in the presence of >19 % NaCl. The optimal pH for growth is pH 6·5–8·5; growth is observed at pH 5·0 and 10·5, but not at pH 4·5 or 11·0. Hypoxanthine is not hydrolysed. Urease-negative. No acid is produced from D-sorbitol, myo-inositol, D-xylose, L-arabinose, D-melezitose, adonitol, L-rhamnose or lactose. The peptidoglycan type is L-Lys–Gly. The predominant menaquinone is MK-7. Major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. The major fatty acids are iso-C17 : 0, anteiso-C13 : 0, iso-C13 : 0 and iso-C15 : 0. The DNA G+C content is 48·4–48·6 mol% (48·6 mol% for the type strain). Other phenotypic properties are given in Table 1.
The type strain, TF-16T (=KCTC 19035T=DSM 16306T), was isolated from a tidal flat of Daepo Beach (Yellow Sea), near Mokpo City, Korea.
Description of Exiguobacterium marinum sp. nov.
Exiguobacterium marinum (ma.ri'num. L. neut. adj. marinum of the sea, marine).
Cells are Gram-variable and facultatively anaerobic; growth is better under aerobic conditions. Colonies are circular, glossy, raised and 3–4 mm in diameter after incubation for 2 days on MA at 30 °C under aerobic conditions. White-coloured colonies are formed under anaerobic conditions. Cells are short rods (in the early growth phase) or cocci (exponential and stationary growth phases). Growth occurs at 10 and 43 °C, with optimum growth at 30–37 °C; growth does not occur at 4 °C or above 44 °C. Growth does not occur in the presence of >17 % NaCl. The optimal pH for growth is pH 6·5–8·5; growth is observed at pH 5·0 and 10·5, but not at pH 4·5 or 11·0. Hypoxanthine is not hydrolysed. Urease-negative. No acid is produced from D-sorbitol, myo-inositol, D-xylose, L-arabinose, D-melezitose, adonitol, L-rhamnose or lactose. The peptidoglycan type is L-Lys–Gly. The predominant menaquinone is MK-7. Major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. The major fatty acids are iso-C17 : 0, anteiso-C13 : 0, iso-C13 : 0 and iso-C15 : 0. The DNA G+C content of the type strain is 48·0 mol%. Other phenotypic properties are given in Table 1.
The type strain, TF-80T (=KCTC 19036T=DSM 16307T), was isolated from a tidal flat of Daepo Beach (Yellow Sea), near Mokpo City, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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