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1 Key Laboratory of Agro-Microbial Resource and Application, Ministry of Agriculture/Department of Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100094, PR China
2 Key Laboratory of Microbial Diversity Research and Application of Hebei Province, College of Life Sciences, Hebei University, Baoding 071002, PR China
3 Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, PR China
Correspondence
Li-Ping Zhang
zhlping{at}mail.hbu.edu.cn
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ABSTRACT |
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, and its members typically exhibit a branched substrate mycelium that fragments into rod-shaped and coccoid elements and abundant aerial hyphae that frequently form a zigzag morphology. The genus Nocardiopsis was subsequently shown to exhibit distinct chemotaxonomic characteristics: cell-wall chemotype III/C (meso-diaminopimelic acid and no diagnostic sugars in whole-cell hydrolysates; Lechevalier & Lechevalier, 1970), phospholipid type PIII (phosphatidylcholine and phosphatidylmethylethanolamine as characteristic phospholipids; Lechevalier et al., 1977), menaquinone MK-10 with variable degrees of saturation in the side chain as the predominant isoprenoid quinone (Kroppenstedt, 1992), fatty acid type 3d (containing iso-branched, anteiso-branched and 10-methyl-branched chain fatty acids; Kroppenstedt, 1985) and DNA G+C contents between 64 and 71 mol% (Grund & Kroppenstedt, 1990). At the time of writing, 25 species and subspecies have validly published names (Euzéby, 2007). Most of them were isolated from habitats such as saline or alkaline soil and marine sediments. We isolated a novel strain, HBUM 20038T, from an alkaline soil in western China. The aim of the study was to identify whether or not the strain represents a novel species within the genus Nocardiopsis by using phenotypic, genotypic and phylogenetic approaches.
Strain HBUM 20038T was isolated on agar plates of DSMZ medium no. 852 (GYP medium, adjusted to pH 10 with sterile saturated sodium carbonate solution after sterilization of the medium). Cultural characteristics of strain HBUM 20038T were observed by using 3-, 5-, 7- and 14-day-old cultures grown on GYP agar at 28 °C. The morphological properties and colours of aerial and substrate mycelium, as well as of diffusible pigments, were tested as described by Shirling & Gottlieb (1966). Spore-chain morphology and spore-surface ornamentation of 7-day-old cultures grown on GYP agar were observed using an Olympus BH-2 light microscope and a KYKY-AMRAY-100B scanning electron microscope. Strain HBUM 20038T formed powdery-surfaced colonies. The strain produced abundant grey–white aerial mycelium which divided into rod-shaped, smooth-surfaced and non-motile spores (0.3–0.5x1.0–2.0 µm). It formed long, well-developed and branched substrate mycelium (Fig. 1). The substrate mycelium was light-yellowish. No diffusible pigments were produced on GYP agar medium. These morphological characteristics are consistent with those described for Nocardiopsis species (Kroppenstedt, 1992).
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and Hasegawa et al. (1983). Menaquinones were determined according to the methods of Collins (1985). Analysis of phospholipids was based on the procedure of Komagata & Suzuki (1987). The cellular fatty acid composition was determined as described by Sasser (1990) using the Microbial Identification System (MIDI, Inc.). The chemical properties of the strain are consistent with those of the genus Nocardiopsis (Lechevalier & Lechevalier, 1970). Cell walls of strain HBUM 20038T contained meso-diaminopimelic acid and no characteristic sugars were found in the strain. The diagnostic phospholipids contained quantities of diphosphatidylglycerol, phosphatidylcholine and phosphatidylmethylethanolamine (phospholipid type III; Lechevalier et al., 1977). The predominant menaquinones were MK-10(H2), MK-10(H4) and MK-10(H6). The major cellular fatty acids were iso-C16 : 0 (41.03 %), C18 : 1
9c (14.73 %) and tuberculostearic acid (10-methyl-C18 : 0) (9.69 %). These chemical properties offer evidence that the strain should be classified in the genus Nocardiopsis (Collins & Jones, 1981; Lechevalier & Lechevalier, 1970; Lechevalier et al., 1977).
The physiological characteristics of the strain was tested using the methods described for the genus Nocardiopsis in Bergey's Manual of Determinative Bacteriology (Holt et al., 1994) and the methods described by Evtushenko et al. (2000). Growth of strain HBUM 20038T on agar was observed at pH 8.5–13.0, while optimum growth occurred at pH 8.5–9.5, and there was no growth at pH 7.0. The other results are listed in Table 1 or in the species description.
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and Kutchma et al. (1998). The 16S rRNA gene was amplified by PCR with the universal primers 27f (Escherichia coli positions 8–27; 5'-GAGTTTGATCCTGGCTCAG-3') and 1525r (E. coli positions 1525–1545; 5'-AGAAAGGAGGTGTACCAGCC-3') (Lane et al., 1985). The PCR conditions used were an initial denaturation at 95 °C for 10 min, rapid cooling in an ice bath, addition of Taq DNA polymerase and instantaneous centrifugation followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s and extension at 72 °C for 90 s with a final extension at 72 °C for 5 min. The resultant PCR products were purified and sequenced by Sangon Biological Engineering Co. Ltd (Shanghai, China). The G+C content was determined by HPLC of deoxyribonucleosides by using the method of Mesbah et al. (1989). The initial reassociation-rate method (De Ley et al., 1970) was used for determining the percentage of DNA–DNA hybridization by using a spectrophotometer (model CE9500; Cecil Instruments) equipped with a programmable melting-temperature control unit.
The 16S rRNA gene sequence of the strain was aligned manually with corresponding sequences of representatives of the genus Nocardiopsis retrieved from the GenBank database using CLUSTAL_X version 1.64b (Thompson et al., 1997). Phylogenetic trees were inferred by using the neighbour-joining (Saitou & Nei, 1987), Fitch–Margoliash (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1981) methods. Evolutionary distance matrices for the neighbour-joining and Fitch–Margoliash methods were generated according to the algorithm of Jukes & Cantor (1969) and the robustness of the tree topology of the neighbour-joining data was evaluated by bootstrap analysis (Felsenstein, 1993) based on 1000 resamplings using the SEQBOOT and CONSENSE options from the PHYLIP suite of programs.
The nearly complete 16S rRNA gene sequence determined for strain HBUM 20038T (1434 nt) was aligned with corresponding sequences of representative reference strains of Nocardiopsis. It was evident from the phylogenetic tree (Fig. 2) that Nocardiopsis prasina DSM 43845T, N. metallicus KBS6T, N. exhalans ES10.1T, N. alkaliphila DSM 44657T, N. listeri DSM 40297T, N. alba DSM 43377T, N. tropica VKM Ac-1457T, N. umidischolae 66/93T, N. aegyptia DSM 44442T, N. lucentensis DSM 44048T and strain HBUM 20038T formed a phylogenetic clade. Pairwise similarity values of 16S rRNA gene sequences >97 % were found between HBUM 20038T and related strains (Table 2). The DNA–DNA relatedness of strain HBUM 20038T to related type strains was 21.2–52.6 % (Table 2). These values were far below the value of 70 % recommended by Wayne et al. (1987) for strains of the same species. The DNA G+C content of strain HBUM 20038T is 71.1 mol% (determined by HPLC).
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) show that there are clear differences between strain HBUM 20038T and other species of the genus Nocardiopsis. DNA–DNA hybridization is the standard for designation of species (Wayne et al., 1987) and the criterion for membership of the same species is 
70 % DNA–DNA relatedness (Wayne et al., 1987). The levels of DNA–DNA relatedness between strain HBUM 20038T and related type strains are 21.2–52.6 % (Table 2), which were <70 %. It is therefore proposed that strain HBUM 20038T be classified as the type strain of the novel species Nocardiopsis ganjiahuensis sp. nov.
Description of Nocardiopsis ganjiahuensis sp. nov.
Nocardiopsis ganjiahuensis (gan.jia.hu.en'sis. N.L. fem. adj. ganjiahuensis pertaining to Ganjiahu Natural Reserve, Xinjiang, north-west China, where the type strain was isolated).
Aerobic and Gram-positive. The surface of colonies is powdery. The colour of the aerial mycelium is grey–white and the substrate mycelium is light-yellowish. The aerial mycelium divides into rod-shaped, irregular-sized spores (0.3–0.5x1.0–2.0 µm). Spores are smooth and non-motile. The substrate mycelium is long, well-developed and branched. No diffusible pigments are produced. Whole-cell hydrolysates contain meso-diaminopimelic acid, but no diagnostic sugars. Polar lipids are diphosphatidylglycerol, phosphatidylcholine and phosphatidylmethylethanolamine. The predominant menaquinones are MK-10(H2) and MK-10(H4); in addition, there are substantial amounts of MK-10(H6). Major cellular fatty acids are iso-C16 : 0 (41.03 %), C18 : 19c (14.73 %) and tuberculostearic acid (10-methyl C18 : 0) (9.69 %). The G+C content of the DNA of the type strain is 71.1 mol%. Grows occurs at pH 8.5–13, while optimum growth occurs at pH 8.5–9.5. No growth at pH 7.0. Grows optimally at 28–32 °C; growth occurs at 10 °C, with no growth at 42 °C. Grows well on medium without NaCl and with 1, 3 and 5 % NaCl, but no growth occurs at 10 % (w/v) NaCl. L-Arabinose, D-xylose, myo-inositol, glycerol and L-rhamnose are utilized as sole carbon sources, but not sucrose, lactose or cellobiose. Positive reactions are found for reduction of nitrate and production of urease. Tween 80 can be degraded, but not Tween 85.
The type strain is strain HBUM 20038T (=DSM 45031T =CGMCC 4.3500T), isolated from soil.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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