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Int J Syst Evol Microbiol 53 (2003), 317-321; DOI  10.1099/ijs.0.02055-0
© 2003 International Union of Microbiological Societies

Note

Nocardiopsis xinjiangensis sp. nov., a halophilic actinomycete isolated from a saline soil sample in China

Ming-Gang Li, Wen-Jun Li, Ping Xu, Xiao-Long Cui, Li-Hua Xu and Cheng-Lin Jiang

The Key Laboratory for Microbial Resources of the Ministry of Education, Institute of Microbiology, Yunnan University, Kunming 650091, Yunnan, China

Correspondence
Cheng-Lin Jiang
linxu{at}ynu.edu.cn


   ABSTRACT
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An actinomycete strain (YIM 90004T) was isolated from a saline soil sample from the Xinjiang Province, People's Republic of China. The strain displayed abundant aerial and substrate mycelia, and short spore chains were borne on the aerial mycelium. The spore chains were composed of non-motile, smooth-surfaced, rod-shaped spores. The cell wall of strain YIM 90004T contained mainly meso-diaminopimelic acid, alanine and glutamic acid (cell wall type III). Glucose, ribose, galactose, xylose and arabinose were the whole-cell sugars of the strain. The predominant phospholipids were phosphatidylinositol and phosphatidylglycerol; MK-10(H2) and MK-10(H4) were the predominant menaquinones. The DNA G+C content of strain YIM 90004T was 74·3 mol%. Chemotaxonomic properties and 16S rDNA analysis placed strain YIM 90004T in the genus Nocardiopsis. On the basis of phenotypic and phylogenetic analyses and DNA–DNA hybridization results, strain YIM 90004T (=CCRC 16285T =CCTCC AA99004T =DSM 44589T) is considered to represent a novel species of the genus Nocardiopsis, for which the name Nocardiopsis xinjiangensis is proposed.

Published online ahead of print on 19 September 2002 as DOI 10.1099/ijs.0.02055-0.

The GenBank accession number for the 16S rDNA sequence of Nocardiopsis xinjiangensis YIM 90004T is AF251709.


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At the time of writing, there were 15 validly described species of the genus Nocardiopsis (Al-Tai & Ruan, 1994Go; Kroppenstedt, 1992Go; Yassin et al., 1993Go, 1997Go; Evtushenko et al., 2000Go; Chun et al., 2000Go; Peltola et al., 2001Go; Al-Zarban et al., 2002Go). Of these species, only Nocardiopsis halophila and Nocardiopsis kunsanensis are halophilic actinomycetes. Here, we report the characterization of a novel halophilic member of the genus Nocardiopsis that was isolated from a saline soil sample from the Xinjiang Province, People's Republic of China.

Strain YIM 90004T was isolated after 4 weeks incubation at 28 °C on glycerol/asparagine agar (ISP5) containing 10 % (w/v) NaCl. Stock cultures were maintained on ISP2 and ISP5 slants containing 10 % NaCl and kept at 4 °C. The micromorphological characteristics of strain YIM 90004T were observed by light microscopy (model BH2; Olympus) after 24–28 days growth on ISP2 and ISP5 agar containing 10 % NaCl. Spore characteristics were determined by examination of cells using a model EPMA-8705 scanning electron microscope (Shimadzu), following desiccation of cells and coating with gold.

Strain YIM 90004T was Gram-positive. Its vegetative hyphae were long, slim (0·1–0·2 µm in width), well-developed and fragmented. Short spore chains were borne on the aerial hyphae. Spores (dimensions 0·3–0·5x0·5–1·0 µm) were rod-shaped, smooth-surfaced and non-motile (Fig. 1Go).



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  		Fig. 1. Light micrograph (top panel) and scanning electron micrographs (middle and bottom panels) of strain YIM 90004T grown on ISP5 plates for 28 days at 28 °C.

 
Procedures and media used for determination of the physiological properties of and carbon utilization by strain YIM 90004T were based on described methods (Shirling & Gottlieb, 1966Go; Williams et al., 1989Go). Strain YIM 90004T could grow on almost all of the test media (Table 1Go). Phenotypic properties useful for distinguishing strain YIM 90004T from related species are shown in Table 2Go. Strain YIM 90004T did not produce any diffusible pigment during incubation. Its optimum pH, cultivation temperature and NaCl concentration were 7·2, 28 °C and 10 %, respectively. Colour of the strain's mycelium was determined by comparing the culture with the most suitable colour chips from the ISCC–NBS colour charts (standard sample, no. 2106) (Kelly, 1964Go).


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  		Table 1. Growth of strain YIM 90004T on different media

 

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  		Table 2. Phenotypic properties useful for distinguishing strain YIM 90004T from the halophilic members of the genus Nocardiopsis

Species: 1, N. halophila; 2, N. kunsanensis; 3, Nocardiopsis sp. YIM 90004T. Data for N. halophila and N. kunsanensis are from this study and studies by Yassin et al. (1997)Go and Chun et al. (2000)Go. +, Positive; W, weak positive; -, negative; V, variable result. meso-DAP, meso-diaminopimelic acid. PC, Phosphatidylcholine; DPG, diphosphatidylglycerol; PI, phosphatidylinositol; PG, phosphatidylglycerol; DAP, diaminopimelic acid.

 
DNA extraction was performed according to the methods of Hopwood et al. (1985)Go and Jiang & Xu (1995)Go. The DNA G+C base content of strain YIM 90004T, determined by the thermal denaturation method (Mandel & Marmur, 1968Go), was found to be 74·3 mol%. Amplification and analysis of the 16S rDNA sequence of strain YIM 90004T were done as described previously (Cui et al., 2001Go). The 16S rDNA sequence of strain YIM 90004T was aligned with previously published sequences obtained from GenBank. A more-detailed comparison was performed with members of the genus Nocardiopsis using the Knuc value of Kimura (1980Go, 1983)Go and the neighbour-joining method of Saitou & Nei (1987)Go. The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985)Go with 1000 replicates. Similarities between strain YIM 90004T and other actinomycetes indicated strain YIM 90004T to be a member of the genus Nocardiopsis (94·8–98·1 %). Strain YIM 90004T was phylogenetically most closely related to N. kunsanensis (98·1 %). Fig. 2Go shows a phylogenetic dendrogram displaying the relatedness of strain YIM 90004T to type strains of the genus Nocardiopsis.



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  		Fig. 2. Neighbour-joining tree, based on 16S rDNA sequences, showing the phylogenetic relationship of strain YIM 90004T with recognized members of the genus Nocardiopsis. Streptomyces megasporus was used as the outgroup. Only bootstrap values >50 % (expressed as percentages of 1000 replications) are shown at the nodes of the tree. Bar, one nucleotide substitution per 100 nt.

 
For chemotaxonomic analyses, strain YIM 90004T was grown for 2 weeks in 100 ml ISP2 liquid medium. The mycelium was harvested by centrifugation at 3500 r.p.m. for 10 min and then washed several times with distilled water. Cell wall was purified and diaminopimelic acid analysed by the method of Lechevalier & Lechevalier (1980)Go. Whole-cell sugar analysis was determined by GC and MS (GC-MS model HP-6890; Hewlett Packard) (Saddler et al., 1991Go; Takahashi et al., 1992Go). The analysis of phospholipids and menaquinones followed the methods of Lechevalier & Lechevalier (1980)Go and Collins (1985)Go, respectively. meso-Diaminopimelic acid was the diagnostic amino acid of the peptidoglycan. Whole-cell sugars were quantitatively analysed and found to be glucose (32 %), ribose (23 %), xylose (22 %), arabinose (14 %) and galactose (8 %). Major menaquinones were MK-10(H2) and MK-10(H4), and phospholipid included phosphatidylglycerol and phosphatidylinositol. Some of these properties were consistent with the phylogenetic placement of strain YIM 90004T into the genus Nocardiopsis.

Phylogenetically, strain YIM 90004T was most closely related to N. kunsanensis (98·1 %), one of the two halophilic species of the genus Nocardiopsis. The second species, N. halophila, shared only 95·8 % 16S rDNA sequence similarity with strain YIM 90004T, and can thus be clearly distinguished from strain YIM 90004T by the primary structure of its 16S rDNA. At this level of 16S rDNA relatedness, strain YIM 90004T and N. kunsanensis belong to different DNA–DNA reassociation groups and should not be combined in a single species (Stackebrandt & Goebel, 1994Go). These two taxa also differ phenotypically, morphologically and chemotaxonomically. Strain YIM 90004T differs from the type strain of N. kunsanensis (KTCT 9831T) in some of its chemotaxonomic properties (Table 2Go): the predominant menaquinones of strain YIM 90004T are MK-10(H2, H4), while that of N. kunsanensis is MK-10(H8); the phospholipids of strain YIM 90004T included phosphatidylglycerol and phosphatidylinositol, while those of N. kunsanensis are composed of phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol (phospholipid type III) (Chun et al., 2000Go). Strain YIM 90004T did not grow well in media above pH 8·0, had an optimum growth temperature of 28 °C and did not utilize glucose, xylose or alanine, whereas N. kunsanensis has optimum growth at pH 9·0 and 37 °C, and utilizes glucose, xylose and alanine. The DNA–DNA reassociation value determined for strains YIM 90004T and N. kunsanensis KTCT 9831T (only 20 %) reinforces the genomic differences between the two strains.

Based on the above results, we consider strain YIM 90004T to represent a novel species of the genus Nocardiopsis, for which we propose the name Nocardiopsis xinjiangensis.

Description of Nocardiopsis xinjiangensis sp. nov
Nocardiopsis xinjiangensis (xin.ji.ang.en'sis. N.L. adj. xinjiangensis pertaining to Xinjiang, the Province of western China in which the sample was collected).

On most test medium, the fragmented substrate and aerial mycelia are well developed. Short spore chains are borne on the aerial mycelium; spores are rod-shaped with a smooth surface. No diffusible pigment is produced. Whole-cell hydrolysates contain meso-diaminopimelic acid; whole-cell sugars include glucose, ribose, xylose, arabinose and galactose. Predominant menaquinones are MK-10(H2, H4), and the diagnostic phospholipids are phosphatidylglycerol and phosphatidylinositol. Utilizes cellobiose, galactose, alanine, proline and serine, but not glucose, xylose, maltose, mannitol, raffinose, rhamnose or sucrose. Gelatin liquefaction, urease activity and melanin production are positive. Milk coagulation, milk peptonization, starch hydrolysis, nitrate reduction and H2S production are negative. Optimum growth temperature is 28 °C; optimum salt concentration for growth is 10 %; optimum pH for growth is 7·2. DNA G+C content is 74·3 mol%. Isolated from a saline soil sample in the west of China. The type strain is YIM 90004T (=CCRC 16285T =CCTCC AA99004T =DSM 44589T).


   ACKNOWLEDGEMENTS
 
The authors would like to thank Professor Sang-Jin Kim for kindly providing the type strain of N. kunsanensis. We also thank Professor Tseng Min for assistance in obtaining the scanning electron micrographs. This research was supported by the Key Laboratory for Microbial Resources of the Ministry of Education, People's Republic of China, the Yunnan Provinacial Educational Administration and the Yunnan Provinacial Natural Science Foundation.


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Collins, M. D. (1985). Isoprenoid quinone analysis in bacterial classification and identification. In Chemical Methods in Bacterial Systematics, pp. 267–287. London: Academic Press.

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Kroppenstedt, R. M. (1992). The genus Nocardiopsis. In The Prokaryotes, pp. 1139–1156. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer-Verlag.

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