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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 National Research Laboratory of Molecular Ecosystematics, Institute of Probionic, Probionic Corporation, Bio-venture Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
3 Department of Food and Life Science, Sungkyunkwan University, Chunchun-dong 300, Jangan-gu, Suwon, Korea
Correspondence
Yong-Ha Park
yhpark{at}kribb.re.kr
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Published online ahead of print on 8 August 2003 as DOI 10.1099/ijs.0.02585-0.
The GenBank accession number for the 16S rDNA sequence of strain GW-9T is AF529063.
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, 1989). The type strain of Nocardioides simplex was isolated from rice soil (Jensen, 1934) and atypical N. simplex strains were isolated from soil of the petroleum zone in Japan (Iizuka & Komagata, 1964). Subsequently, Nocardioides species have been found in habitats other than soils, such as herbage (Nocardioides plantarum; Collins et al., 1994), an oil shale column (Nocardioides pyridinolyticus; Yoon et al., 1997) and industrial wastewater (Nocardioides nitrophenolicus; Yoon et al., 1999). Recently, Nocardioides aquaticus was isolated from an aquatic rather than a terrestrial environment. It was isolated from water of the hypersaline and meromictic Ekho Lake, located in the ice-free area of the Vestfold Hills in East Antarctica (Lawson et al., 2000). These facts indicate that Nocardioides species are widely distributed in nature. In this study, an LL-2,6-diaminopimelic acid (DAP)-containing actinomycete, GW-9T, isolated from groundwater in Korea, is described. This isolate was considered to be a Nocardioides-like strain from 16S rDNA sequence comparisons, as well as the presence of LL-DAP. Accordingly, the aim of the present study was to determine the exact taxonomic status of strain GW-9T using a combination of phenotypic properties, detailed phylogenetic analysis based on 16S rDNA sequence and genomic relatedness. On the basis of the data presented below, it is proposed that strain GW-9T represents a novel species in the genus Nocardioides, for which the name Nocardioides aquiterrae sp. nov. is proposed.
Strain GW-9T was isolated by the usual dilution plating technique on nutrient agar (NA; Difco) at 30 °C. Type strains of Nocardioides species used as reference organisms for DNA–DNA hybridization were obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and KCTC (Korean Collection for Type Cultures). Strain GW-9T was cultivated on NA and in nutrient broth (NB; Difco) at 30 °C to investigate its morphological and physiological characteristics. Cell mass of strain GW-9T for analyses of the cell wall and menaquinones was obtained from NB culture at 30 °C. Cell mass of strain GW-9T and reference strains for DNA extraction was produced in NB. For fatty acid methyl ester analysis, cell mass of strain GW-9T was obtained from agar plates after 6 days cultivation at 30 °C on NA. Cell morphology was examined by light microscopy (Nikon E600) and TEM. The presence or absence of flagella was determined using TEM with cells from exponentially growing cultures. For TEM observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air drying, the grids were examined with a model CM-20 TEM (Philips). Oxidase activity was determined by oxidation of 1 % (w/v) p-aminodimethylaniline oxalate. Catalase activity was determined by bubble production in a 3 % (v/v) hydrogen peroxide solution. Hydrolysis of aesculin and nitrate reduction were determined as described previously (Lanyi, 1987). Hydrolysis of casein, gelatin, hypoxanthine, starch, Tween 80, tyrosine and xanthine and urease activity were determined as described by Cowan & Steel (1965). Utilization of various substrates as sole carbon and energy sources was determined as described by Shirling & Gottlieb (1966). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with NA that had been prepared anaerobically. Growth at various temperatures (4–55 °C) was determined on NA.
The isomer type of the diamino acid in the cell-wall peptidoglycan was analysed using TLC according to the method described by Komagata & Suzuki (1987). Menaquinones were analysed as described previously (Komagata & Suzuki, 1987) using reversed-phase HPLC. For quantitative analysis of the cellular fatty acid content, a loopful of cell mass was harvested and fatty acid methyl esters were prepared and identified according to the instructions of the Microbial Identification System (MIDI). Chromosomal DNA was isolated and purified according to Yoon et al. (1996), with the exception that ribonuclease T1 was used together with ribonuclease A. The G+C content was determined by the method of Tamaoka & Komagata (1984). DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
16S rDNA was amplified by PCR using two universal primers as described previously (Yoon et al., 1998). The PCR product was purified with a QIAquick PCR purification kit (Qiagen). Sequencing of the purified 16S rDNA was performed using an ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) as recommended by the manufacturer. Purified sequencing reaction mixtures were electrophoresed automatically using an Applied Biosystems model 377 automatic DNA sequencer. Alignment of sequences was carried out with CLUSTAL W software (Thompson et al., 1994). Gaps at the 5' and 3' ends of the alignment were omitted from further analysis. Phylogenetic trees were inferred using three tree-making algorithms, the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) methods in the PHYLIP package (Felsenstein, 1993). Evolutionary distance matrices for the neighbour-joining method were calculated using the algorithm of Jukes & Cantor (1969) with the program DNADIST. The stability of relationships was assessed by bootstrap analysis based on 1000 resamplings of the neighbour-joining dataset using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package. 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 and the means of the remaining three values were taken as DNA relatedness values.
Physiological and biochemical characteristics of strain GW-9T are given in the species description below and are compared with those of N. pyridinolyticus in Table 1.
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. The predominant isoprenoid quinone found in strain GW-9T was tetrahydrogenated menaquinone with eight isoprene units [MK-8(H4)]. Strain GW-9T had a cellular fatty acid profile containing large amounts of straight-chain, branched, unsaturated and 10-methyl fatty acids. The major fatty acid was iso-C16 : 0. This cellular fatty acid profile is similar to those of Nocardioides species (Yoon et al., 1997, 1999), particularly the type strain of N. pyridinolyticus (Table 2), although there are differences in the proportions of some fatty acids. The DNA G+C content of strain GW-9T was 73 mol%.
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). The same tree topology was also found in phylogenetic trees generated with maximum-parsimony and maximum-likelihood algorithms (data not shown). The level of 16S rDNA similarity between strain GW-9T and the type strain of N. pyridinolyticus was 99·2 %. Strain GW-9T exhibited 16S rDNA similarities of 94·9–96·0 % with the type strains of other Nocardioides species with validly published names and 89·9–94·2 % with members of other genera used in the phylogenetic analysis. Two independent levels of DNA–DNA relatedness between GW-9T and N. pyridinolyticus KCTC 0074BPT, its phylogenetic neighbour, were 32·5 and 28·7 %, when each of their DNAs was used separately as the labelled DNA probe. Strains GW-9T exhibited levels of DNA–DNA relatedness of 8·7–15·3 % with the type strains of other Nocardioides species with validly published names.
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). Results obtained in the chemotaxonomic analysis are consistent with the results of phylogenetic inference based on 16S rDNA sequences. The chemotaxonomic data obtained from strain GW-9T are most similar to those of members of the genus Nocardioides in the family Nocardioidaceae (Miller et al., 1991; Park et al., 1999; Urzì et al., 2000; Wang et al., 2001). Of genera belonging to the family Nocardioidaceae, two genera, Nocardioides and Marmoricola, have been known to contain MK-8(H4) as the predominant menaquinone. However, the genus Marmoricola contains C16 : 0 and C18 : 1 as the major fatty acids, which distinguishes it from the genus Nocardioides (Urzì et al., 2000). Therefore, both phylogenetic and chemotaxonomic results clearly indicate that strain GW-9T belongs to the genus Nocardioides. Strain GW-9T was very similar to N. pyridinolyticus in its morphological and physiological properties, as shown in the phylogenetic analysis. The levels of DNA–DNA relatedness, together with minor differential phenotypic properties and phylogenetic distinctiveness, reveal that strain GW-9T is distinct from previously described Nocardioides species, as well as N. pyridinolyticus, its closest phylogenetic neighbour (Wayne et al., 1987). Therefore, on the basis of the data presented, strain GW-9T should be placed in the genus Nocardioides as a representative of a novel species, for which the name Nocardioides aquiterrae sp. nov. is proposed.
Description of Nocardioides aquiterrae sp. nov.
Nocardioides aquiterrae (a.qui.ter'rae. L. n. aqua water; L. gen. fem. n. terrae of earth or ground; N.L. gen. fem. n. aquiterrae from/of groundwater).
Cells are aerobic, non-spore-forming, non-acid-fast rods, 0·8–1·0x1·7–2·0 µm in the exponential phase of growth after approximately 7 days incubation at 30 °C on NA. Motile by means of a single lateral flagellum. Cells show rod-to-coccus morphogenesis from the early exponential phase to the stationary phase. Gram-positive (Gram-variable in old cultures). Colonies are smooth, circular, convex, cream-coloured and 0·5–1·0 mm in diameter after 7 days incubation on NA. Neither substrate nor primary mycelium is formed. Optimal temperature for growth is 30 °C; grows at 15 and 42 °C, but not at 10 °C or temperatures above 43 °C. Optimal pH for growth is 6·0–7·0; no growth at pH 5·0. Oxidase-positive. Urease-negative. Casein and Tween 80 are hydrolysed. Hypoxanthine, starch, tyrosine and xanthine are not hydrolysed. The cell-wall peptidoglycan contains LL-DAP as the diagnostic diamino acid. The predominant menaquinone is MK-8(H4). The cellular fatty acids consist of straight-chain, branched, unsaturated and 10-methyl fatty acids. The major fatty acid is iso-C16 : 0. The DNA G+C content of the type strain is 73 mol% (determined by HPLC). Other characteristics are given in Table 1.
The type strain, strain GW-9T (=KCCM 41647T=JCM 11813T), was isolated from groundwater in Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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