Abstract
Abbreviations: ISP, International Streptomyces Project
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 90010T is AY373031.
The genus Nocardiopsis was first described by Meyer (1976) and currently comprises 18 species with validly published names (Meyer, 1976; Grund & Kroppenstedt, 1990; Yassin et al., 1993, 1997; Al-Tai & Ruan, 1994; Evtushenko et al., 2000; Chun et al., 2000; Peltola et al., 2001; Al-Zarban et al., 2002; Kämpfer et al., 2002; Schippers et al., 2002; M. G. Li et al., 2003; Hozzein et al., 2004; Sabry et al., 2004). During our taxonomic study on extremophilic actinomycetes, we used phenotypic and genotypic approaches to facilitate the characterization of one moderately halophilic actinomycete, strain YIM 90010T.
Strain YIM 90010T was isolated from a saline soil sample by using modified International Streptomyces Project (ISP) 5 medium supplemented with 20 % (w/v) NaCl. The soil sample was collected from the same source as described previously (Cui et al., 2001; M. G. Li et al., 2003; W. J. Li et al., 2003a, b, c). The strain was maintained on ISP 2 and ISP 5 slants containing 10 % (w/v) NaCl at 4 °C and as 20 % (w/v) glycerol suspensions at 20 °C. Biomass for chemical and molecular studies was obtained by cultivation in shake flasks (about 150 r.p.m.) using modified ISP 5 medium [10 % (w/v) NaCl, pH 7·0] broth at 28 °C for 1 week.
The morphological characteristics of strain YIM 90010T were observed by using light microscopy (model BH 2; Olympus) and scanning electron microscopy with a JEOL model JSM5600LV after 14 days growth on ISP 5 medium containing 10 % (w/v) NaCl. Cultural characteristics were determined after 4 weeks at 28 °C by using the methods adopted in the ISP (Shirling & Gottlieb, 1966). All media were supplemented with 10 % (w/v) NaCl, and the colours of both substrate and aerial mycelia and the production of soluble pigments were determined by comparison with chips from the ISCCNBS colour charts (Kelly, 1964). The detailed results are shown in Table 1.
Table 1. Cultural characteristics of strain YIM 90010T All media were supplemented with 10 % (w/v) NaCl (pH 7·2); ISP (Shirling & Gottlieb, 1966). Colours were taken from ISCCNBS colour charts (Kelly, 1964).
Strain YIM 90010T was found to be Gram-positive. Its vegetative hyphae were long, well-developed and fragmented. Long spore chains were borne on the aerial hyphae. Spores (dimensions 0·40·6x0·81·2 µm) were rod-shaped, smooth and non-motile (Fig. 1).
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The media and procedures used for determining physiological and biochemical features and carbon-source utilization were as described by Shirling & Gottlieb (1966). The results are given in Table 2 or in the species description.
Table 2. Characteristics useful for differentiating between strain YIM 90010T and closely related Nocardiopsis species Abbreviations: PC, phosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol; DPG, diphosphatidylglycerol. All three strains shared the following characteristics: negative results for arabinose, meso-inositol, mannitol, rhamnose, trehalose, xylitol, xylose and maltose; positive results for fructose, sodium citrate and L-alanine; growth on media with NaCl concentrations between 3 and 20 % (optimum 10 %); H2S not produced; meso-diaminopimelic acid as the cell-wall peptidoglycan. +, Positive; , negative; w, weak growth or reaction.
Analyses of the amino acids and sugars of the cell walls were performed as described by Stanek & Roberts (1974). Polar lipids were extracted, examined by two-dimensional TLC and identified using published procedures (Minnikin et al., 1984). Menaquinones were isolated using the methods of Minnikin et al. (1984) and separated by HPLC (Kroppenstedt, 1982). The cellular fatty acid composition was determined as described by Sasser (1990), using the Microbial Identification System (MIDI). The cell walls of strain YIM 90010T contained meso-diaminopimelic acid and no diagnostic sugars. The phospholipids contained phosphatidylglycerol and phosphatidylinositol. The predominant menaquinones were MK-10(H6), MK-10(H8) and MK-12. The major cellular fatty acids were i-C16 : 0 (37·80 %), 10Me C18 : 0 (15·73 %) and C18 : 1ω9c (9·04 %).
Extraction of genomic DNA and amplification of the 16S rRNA gene were done as described by Cui et al. (2001). Phylogenetic analysis was performed using the software packages PHYLIP (Felsenstein, 1993) and MEGA (Molecular Evolutionary Genetics Analysis) version 2.1 (Kumar et al., 2001) after multiple alignment of data by CLUSTAL_X (Thompson et al., 1997). Distances (distance options according to the Kimura two-parameter model) (Kimura, 1980, 1983) and clustering were determined using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was used to evaluate the tree topology of the neighbour-joining data by performing 1000 resamplings (Felsenstein, 1985).
The genomic DNA of strain YIM 90010T for the determination of G+C content was prepared according to the method of Marmur (1961). The G+C content was determined using the thermal denaturation method of Marmur & Doty (1962) and produced a value of 73·1 mol%.
The almost-complete 16S rRNA gene sequence (1449 bp) of strain YIM 90010T was determined. Phylogenetic analyses based on a dataset consisting of 1430 unambiguous nucleotides between positions 53 and 1482 (Escherichia coli positions; Brosius et al., 1978) showed that the novel isolate falls into a distinct clade with two other recognized Nocardiopsis species, Nocardiopsis kunsanensis (KCTC 9831T) and Nocardiopsis xinjiangensis (YIM 90004T). A phylogenetic tree based on the 16S rRNA gene sequences of strain YIM 90010T, the two aforementioned Nocardiopsis species and other related species is shown in Fig. 2. The 16S rRNA gene sequence of strain YIM 90010T exhibited 97·6 % similarity with that of N. kunsanensis KCTC 9831T and 98·1 % with that of N. xinjiangensis YIM 90004T.
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There are three recognized halophilic species of the genus Nocardiopsis: N. kunsanensis, N. xinjiangensis and Nocardiopsis halophila. However, the level of 16S rRNA gene sequence similarity between strain YIM 90010T and N. halophila DSM 44494T was low (below 96 %). In addition to this low level of similarity, the two strains lie in different clades within the phylogenetic tree based on the 16S rRNA gene sequences of all recognized Nocardiopsis species (Fig. 2).
Accordingly, comparative taxonomic studies were performed with strain YIM 90010T, N. kunsanensis KCTC 9831T and N. xinjiangensis YIM 90004T to determine whether strain YIM 90010T could be considered as a novel species of the genus Nocardiopsis or should be assigned to one of the two species.
Strain YIM 90010T differed greatly from the two species in terms of some of its physiological and biochemical characteristics and some chemotaxonomic data (Table 2). DNADNA relatedness tests were performed with strain YIM 90010T, N. kunsanensis KCTC 9831T and N. xinjiangensis YIM 90004T, using the optical renaturation method (De Ley et al., 1970; Huß et al., 1983; Jahnke, 1992). DNADNA reassociation similarity values between strain YIM 90010T and N. kunsanensis KCTC 9831T, and strain YIM 90010T and N. xinjiangensis YIM 90004T were 38·6 and 45·5 %, respectively, while the value for N. kunsanensis KCTC 9831T and N. xinjiangensis YIM 90004T was 22·4 % (repeated twice). DNADNA relatedness provided decisive evidence that the novel isolate, YIM 90010T, and the other two related type strains, N. kunsanensis KCTC 9831T and N. xinjiangensis YIM 90004T, are members of different genomic species (Wayne et al., 1987).
Therefore, on the basis of the above-mentioned phenotypic and genotypic results, we consider that strain YIM 90010T represents a novel species of the genus Nocardiopsis, for which we propose the name Nocardiopsis salina sp. nov.
Description of Nocardiopsis salina sp. nov.
Nocardiopsis salina (sa.li'na. N.L. fem. adj. salina salty, saline).
Cells are aerobic, Gram-positive, non-acid-fast and non-motile. The colour of the aerial mycelium is white on most media tested and the substrate mycelium is pale yellow to light orangeyellow or yellowwhite. No diffusible pigments are produced. The vegetative hyphae are long, well-developed and fragmented. Long or short spore chains are borne on the aerial hyphae. Spores (dimensions 0·40·6x0·81·2 µm) are rod-shaped, smooth and non-motile. Cell walls contain meso-diaminopimelic acid and have no diagnostic sugars. Polar lipids are phosphatidylglycerol and phosphatidylinositol. Major menaquinones are MK-10(H6), MK-10(H8) and MK-12. Major cellular fatty acids are i-C16 : 0 (37·80 %), C18 : 1ω9c (9·04 %) and 10Me C18 : 0 (15·73 %). Ribose, sucrose, fructose, raffinose, sodium citrate and sodium acetate are utilized as carbon sources, while arabinose, glucose, cellobiose, galactose, inositol, mannitol, melibiose, rhamnose, trehalose, xylitol, xylose and maltose are not. Almost all nitrogen sources tested, such as asparagine, phenylalanine, serine, histidine, methionine, valine, threonine, arginine, adenine, hypoxanthine, glycine, proline and hydroxyproline, can be utilized. Negative in tests for milk coagulation, milk peptonization, starch hydrolysis, H2S production, urease activity and melanin production. Doubtful result for gelatin liquefaction; positive for nitrate reduction. Grows optimally at 28 °C and at pH 7·2 with 10 % (w/v) NaCl; the temperature, pH and NaCl tolerance range are 2040 °C, 6·09·0 and 320 % (w/v), respectively. The DNA G+C content is 73·1 mol%. Isolated from a saline soil sample in the west of China.
The type strain is YIM 90010T (=KCTC 19003T=CCTCC AA 204009T).
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