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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, South Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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Published online ahead of print on 10 June 2005 as DOI 10.1099/ijs.0.63582-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KSL-107T is AY822044.
A table detailing the cellular fatty acid composition of strain KSL-107T is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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with a single species, Aeromicrobium erythreum. Subsequently, Nocardioides fastidiosa was transferred to the genus as Aeromicrobium fastidiosum (Tamura & Yokota, 1994). One further species, Aeromicrobium marinum, was described recently (Bruns et al., 2003). Phylogenetic analyses based on 16S rRNA gene sequences showed that the genus Aeromicrobium belongs to the family Nocardioidaceae (Stackebrandt et al., 1997; Yoon et al., 1998; Bruns et al., 2003). In this study, we report on the taxonomic characterization of an Aeromicrobium-like strain, KSL-107T, which was isolated from an alkaline soil in Korea.
Alkaline soil (approx. pH 10·0) collected from Kwangchun, Korea, was used as the source for the isolation of bacterial strains. Strain KSL-107T was isolated by the standard dilution plating technique at 25 °C on 10x diluted nutrient agar (NA; Difco) with pH adjusted to 10·0 by using Na2CO3. A. erythreum DSM 8599T and A. marinum DSM 15272T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). A. fastidiosum KCTC 9576T was obtained from the Korean Collection for Type Cultures (KCTC). The morphological, physiological and biochemical characteristics of strain KSL-107T were investigated using routine cultivation on trypticase soy agar (TSA; Difco) at 25 °C. Cell morphology was examined by light microscopy (E600; Nikon) and transmission electron microscopy (TEM). The presence of flagella was examined by TEM using cells from exponentially growing cultures. The Gram-reaction was determined by using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Growth at various NaCl concentrations was investigated in trypticase soy broth (TSB; Difco) and TSB lacking NaCl. Growth at various temperatures (4–40 °C) was measured on TSA. The pH range for growth was determined in TSB adjusted to various pH values (pH 4·5–12·0 at intervals of 0·5 pH units). The pH was adjusted prior to sterilization by the addition of HCl or Na2CO3 (below pH 10·5) and KOH (above pH 10·5). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on TSA and TSA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, hypoxanthine, starch, tyrosine, xanthine and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and nitrate reduction were studied according to Lanyi (1987). Utilization of various substrates as sole carbon and energy sources was determined as described by Shirling & Gottlieb (1966). Enzyme activity was determined by using the API ZYM system (bioMérieux). The need for vitamins for growth was investigated using the liquid medium used for substrate utilization tests supplemented with 0·4 % (w/v) glucose as the sole carbon and energy sources. Vitamins were added to the medium at the following concentrations (per litre): biotin (2 mg), thiamin hydrochloride (2 mg) and nicotinic acid (2 mg). Other physiological and biochemical tests were performed with the API 20E system (bioMérieux). Morphological, cultural, physiological and biochemical properties of strain KSL-107T are shown in Table 1 or are given in the species description (see below).
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with the exception that RNase T1 was applied in combination with RNase A. 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 Yoon et al. (2004). An almost-complete 16S rRNA gene sequence of strain KSL-107T was determined in this study and comprised 1473 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain KSL-107T falls within the cluster with Aeromicrobium species (Fig. 1). The same tree topologies were found using the maximum-likelihood and maximum-parsimony algorithms (data not shown). Similarity values between the 16S rRNA gene sequences of strain KSL-107T and the type strains of Aeromicrobium species ranged from 97·9 % (with A. erythreum and A. fastidiosum) to 98·2 % (with A. marinum). Sequence similarities to other species used in the phylogenetic analysis were lower than 93·5 %. 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 means of the remaining three values were quoted as DNA–DNA relatedness values. Mean DNA–DNA relatedness levels between strain KSL-107T and the type strains of three Aeromicrobium species with validly published names were in the range of 11–17 %, indicating that strain KSL-107T represents a distinct genomic species.
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. Isoprenoid quinones were analysed according to Komagata & Suzuki (1987) using reverse-phase HPLC. For fatty acid methyl ester (FAME) analysis, a loop of cell mass was harvested after cultivation for 6 days on NA and TSA at 25 °C. FAMEs were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with a modification that DNA was hydrolysed and the resultant nucleotides were analysed using reverse-phase HPLC and a YMC ODS-A (250x4·6 mm) column. Strain KSL-107T contained LL-2,6-diaminopimelic acid (LL-DAP) as the diagnostic diamino acid in the cell-wall which is characteristic of wall chemotype I sensu Lechevalier & Lechevalier (1970). The isolate contained a tetrahydrogenated menaquinone with nine isoprene units [MK-9(H4)] as the predominant isoprenoid quinone. This menaquinone type was different from that found in the genera Nocardioides, Marmoricola and Actinopolymorpha (Park et al., 1999; Urzì et al., 2000; Wang et al., 2001). When strain KSL-107T was cultivated on TSA and NA, the fatty acid profiles were characterized by a common core of straight-chain, unsaturated, hydroxy and 10-methyl fatty acids in varying amounts (see Supplementary Table S1 in IJSEM Online). When cultivated on TSA, the fatty acids (>1·0 %) detected were C16 : 0 (34·1 %), C16 : 0 2-OH (17·6 %), 10-methyl C18 : 0 (16·3 %), C18 : 1
9c (5·7 %), 10-methyl C17 : 0 (5·0 %), 10-methyl C16 : 0 (4·0 %), C17 : 0 (3·5 %), C16 : 17c and/or iso-C15 : 0 2-OH (3·4 %), C15 : 0 (3·2 %), C17 : 18c (2·4 %), C18 : 0 (1·2 %) and C14 : 0 (1·0 %). No significant differences in the fatty acid profiles were found between strain KSL-107T and Aeromicrobium species (Park et al., 1999; Bruns et al., 2003). The fatty acid profiles of strain KSL-107T were different from those of members of the genus Nocardioides, the closest phylogenetic neighbour (Park et al., 1999; Bruns et al., 2003). Branched fatty acids, particularly iso-C16 : 0, which are major components in Nocardioides species, were minor components in strain KSL-107T and Aeromicrobium species as determined by most authors (Park et al., 1999; Urzì et al., 2000; Bruns et al., 2003; Yi & Chun, 2004; Yoon et al., 2004). Fatty acid C16 : 0, a minor component in Nocardioides species, was a major component in strain KSL-107T and Aeromicrobium species. The tuberculostearic acid (10-methyl C18 : 0) content of strain KSL-107T was greater than that of Nocardioides species in most experiments (Park et al., 1999; Bruns et al., 2003; Yi & Chun, 2004; Yoon et al., 2004). The DNA G+C content of strain KSL-107T was 71·5 mol%.
Comparative 16S rRNA gene sequence analyses showed that strain KSL-107T is most closely related to the genus Aeromicrobium of the family Nocardioidaceae (Fig. 1
). Chemotaxonomic properties support the monothetic phylogenetic classification of strain KSL-107T as a member of the genus Aeromicrobium. The phenotypic differentiation and phylogenetic and genetic distinctiveness are sufficient to categorize strain KSL-107T as a species that is distinct from the recognized Aeromicrobium species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain KSL-107T should be placed in the genus Aeromicrobium as a member of a novel species, for which the name Aeromicrobium alkaliterrae sp. nov. is proposed.
Description of Aeromicrobium alkaliterrae sp. nov.
Aeromicrobium alkaliterrae (al.ka.li.ter'rae. N.L. n. alkali alkali; L. gen. n. terrae of the soil or earth; N.L. gen. n. alkaliterrae of alkaline soil).
Cells are rods (0·3–0·5x0·8–1·4 µm) or cocci. Gram-positive. Non-motile. Non-endospore-forming. Colonies are circular, convex, smooth, glistening, cream-coloured and 1·0–1·5 mm in diameter after 7 days cultivation at 25 °C on NA. Aerial mycelium is not formed. Growth occurs at 4 °C, but not at 36 °C. Optimal initial pH for growth is 7·0–7·5; growth occurs at initial pH 6·0 and 11·0, but not at initial pH 5·5 and 11·5. Optimal growth occurs in the absence of NaCl; growth does not occur in the presence of >9 % (w/v) NaCl. Biotin, thiamin and nicotinic acid are not required for growth. Growth does not occur under anaerobic conditions on TSA or on TSA supplemented with nitrate. Oxidase-negative. Tweens 20, 40, 60 and 80 are hydrolysed. Hypoxanthine, tyrosine and xanthine are not hydrolysed. H2S and indole are not produced. Voges–Proskauer reaction is negative. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. D-Galactose, D-cellobiose, succinate and L-malate are utilized. D-Ribose, acetate, benzoate and formate are not utilized. The cell-wall peptidoglycan contains LL-DAP as the diagnostic diamino acid. The predominant menaquinone is MK-9(H4). The major fatty acids are C16 : 0, C16 : 0 2-OH and 10-methyl C18 : 0. The DNA G+C content is 71·5 mol% (determined by HPLC). Other phenotypic properties are given in Table 1.
The type strain, KSL-107T (=KCTC 19073T=DSM 16824T), was isolated from an alkaline soil in Kwangchun, Korea.
Emended description of the genus Aeromicrobium Miller et al. 1991
The emended description is based on the original description given by Miller et al. (1991), the data compiled by Collins & Stackebrandt (1989), Tamura & Yokota (1994) and Bruns et al. (2003) and those generated in this study. Cells are aerobic, non-endospore-forming, Gram-positive, cultures contain rods and cocci or rods. Non-motile or motile. Mesophilic; optimal temperature for growth is about 25–35 °C. Heterotrophic; a wide range of carbon sources are used for growth and various organic compounds are hydrolysed. The cell-wall peptidoglycan contains LL-DAP as the diagnostic diamino acid. The predominant menaquinone is MK-9(H4). The major fatty acids are 10-methyl C18 : 0, C16 : 0 and both or either of C18 : 19c and C16 : 0 2-OH. The DNA G+C content is 70·6–73 mol%. Members of this genus form a coherent group within the radiation of the family Nocardioidaceae on the basis of 16S rRNA gene sequence data. Members of the genus occur in various habitats, including soils, herbage and sea water. The type species is Aeromicrobium erythreum.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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