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1 School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, People's Republic of China
2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
3 School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
Correspondence
Zhiheng Liu
zhliu{at}sun.im.ac.cn
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). At the time of writing the genus contains 37 species with validly published names, most of which have been described within the last 5 years. Members of many of the novel species have been implicated as agents of human disease (Hamid et al., 2001, Yassin et al., 2000, 2001a, b, 2003; Kageyama et al., 2004a, b) though it is evident that nocardial diversity is grossly underestimated in environmental samples, notably soil (Orchard & Goodfellow, 1980; Wang et al., 1999; Maldonado et al., 2000). Nocardiae form part of the soil microflora and play a role in the turnover of organic matter (Orchard, 1981); there is also evidence that some strains produce secondary metabolites of potential industrial value (Isik et al., 1999; Kinoshita et al., 2001). The present investigation was designed to clarify the taxonomic position of a soil isolate, strain 276T, using a polyphasic approach.
Strain 276T was isolated on modified Sauton's agar plate (Mordarska et al., 1972) that had been incubated at 28 °C for 8 days following inoculation with a suspension of a soil sample collected from Xishan Mountain, Beijing, China. The isolate was maintained on modified Sauton's agar slants at 4 °C and as suspensions of mycelial fragments in glycerol (20 %, v/v) at –20 °C. Biomass for chemotaxonomic and molecular genetic studies was prepared as described previously (Zhang et al., 2002).
The colonial and micromorphological properties of the isolate were observed using standard procedures following inoculation on modified Sauton's agar plates for 7 days at 28 °C. The remaining phenotypic tests were determined using procedures described by Zhang et al. (2003).
Standard procedures were used for the extraction and analysis of the diagnostic isomers of diaminopimelic acid (A2pm; Hasegawa et al., 1983), mycolic acids (Minnikin et al., 1975), polar lipids (Minnikin et al., 1984) and whole-organism sugars (Lechevalier & Lechevalier, 1980), using appropriate controls. Isoprenoid quinones were extracted and purified after Collins et al. (1987); purified menaquinones were determined by reverse-phase HPLC (Wu et al., 1989). The fatty acids were extracted, purified, methylated and quantified by GC using the standard MIDI (Microbial Identification) system (Sasser, 1990; Kämpfer & Kroppenstedt, 1996).
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of the PCR product from strain 276T were carried out after Rainey et al. (1996) and the purified PCR product was sequenced directly using the method of Lu et al. (2001). The resultant 16S rRNA gene sequence was aligned manually with corresponding sequences of representatives of the genera classified in the suborder Corynebacterineae, including all of the type strains of Nocardia species, retrieved from the DDBJ/EMBL/GenBank databases using the CLUSTAL X 1.8 program (Thompson et al., 1997). Phylogenetic trees were generated using the least-squares, maximum-likelihood and neighbour-joining algorithms from the PHYLIP suite of programs (Felsenstein, 1993). Evolutionary distance matrices for the least-squares and neighbour-joining methods were generated after Kimura (1980). The topologies of the resultant unrooted trees were evaluated by bootstrap analyses of the neighbour-joining dataset, based on 1000 resamplings, using the SEQBOOT and CONSENSE options from the PHYLIP package.
Chromosomal DNA, extracted from biomass of strain 276T following growth of the organism in modified Sauton's broth for 3 days at 28 °C, was purified using established procedures (Saito & Miura, 1963; Whipple et al., 1987). The G+C content of the DNA was determined using the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli AS 1.365 as control. The extent of DNA–DNA relatedness between strain 276T and Nocardia abscessus DSM 44432T and Nocardia asteroides ATCC 19247T was determined from the DNA–DNA liquid reassociation rate (De Ley et al., 1970; Huß et al., 1983) using a UV-1206 spectrophotometer (Shimadzu) fitted with a TB-85 thermal controller and standard software (Jahnke, 1992); the results were expressed as the mean of two determinations.
Comparison of the almost complete 16S rRNA gene sequence (1411 nt) of isolate 276T with those of representatives of the genera classified in the suborder Corynebacterineae show that it contains all of the signature nucleotides expected for members of this taxon (Stackebrandt et al., 1997) and, more specifically, those characteristic of members of the family Nocardiaceae (Stackebrandt et al., 1997) and the genus Nocardia (Chun & Goodfellow, 1995). The high 16S rRNA gene sequence similarities found between the tested organism and representatives of the species of the genus Nocardia with validly published names, namely 95·1–98·2 %, provide further evidence of its assignment to this taxon.
Strain 276T has phenotypic properties typical of members of the genus Nocardia (Goodfellow et al., 1999; Wang et al., 2004). The organism is an aerobic, Gram-positive, slightly acid–alcohol-fast actinomycete which forms an extensively branched substrate mycelium that fragments into irregular, non-motile, coccoid and rod-shaped elements and which supports sparse to abundant white aerial hyphae on modified Sauton's agar. Whole-organism hydrolysates were rich in meso-A2pm, arabinose and galactose (wall chemotype IV sensu Lechevalier & Lechevalier, 1970) and contained major amounts of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides (phospholipid type II sensu Lechevalier et al., 1977). The isolate also contained predominant amounts of hexahydrogenated menaquinone with eight isoprene units in which the two terminal isoprene moieties were cyclized; this menaquinone is characteristic of members of the genera Nocardia and Skermania (Chun et al., 1997; Goodfellow et al., 1999). One-dimensional TLC of whole-organism acid methanolysates revealed the presence of a lower spot that corresponded to mycolic acids and a higher one corresponding to non-hydroxylated fatty acids. The mycolic acids of the tested strain co-migrated (Rf value around 0·47) with those from marker strains of Nocardia. The non-hydroxylated fatty acid profile consisted of straight-chain saturated, unsaturated and 10-methyl-branched components.
It is apparent from Fig. 1 that strain 276T forms a distinct phyletic line within the 16S rRNA Nocardia gene tree. The organism is most closely related to the type strains of N. abscessus and N. asteroides. It shares 98·2 % 16S rRNA gene sequence similarity with the type strain of N. abscessus and 97·9 % with that of N. asteroides, values that correspond respectively to 25 and 29 nt differences at 1411 positions. Much higher 16S rRNA gene sequence similarities have been recorded between representatives of several Nocardia species with validly published names that have DNA–DNA relatedness values well below the 70 % cut-off point recommended for the delineation of genomic species by Wayne et al. (1987). The type strains of Nocardia cerradoensis and Nocardia veterana, for instance, share a 16S rRNA gene sequence similarity of 99 % and have a DNA–DNA relatedness value of 58 % (Albuquerque de Barros et al., 2003). In the present study, strain 276T showed a mean DNA–DNA relatedness value of 39 % with N. abscessus DSM 44432T and 35 % with N. asteroides ATCC 19247T. A range of phenotypic properties separated strain 276T from the type strains of phylogenetically close species (Table 1).
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Description of Nocardia xishanensis sp. nov.
Nocardia xishanensis (xi.shan.en'sis. N.L. fem. adj. xishanensis referring to Xishan Mountain, the source of the soil from which the type strain was isolated).
Aerobic, Gram-positive, catalase-positive, slightly acid–alcohol-fast, non-motile actinomycete that forms an extensively branched substrate mycelium which fragments in situ into coccoid and rod-shaped elements. A yellow to orange substrate mycelium carries sparse to abundant, white aerial hyphae on modified Sauton's agar. Diffusible pigments are not formed. Colony elevation is convex to irregular and colony margins are filamentous. Nitrate is reduced and aesculin, arbutin and urea are hydrolysed. Tweens 20, 60 and 80 are degraded, but not adenine, casein, elastin, guanine, hypoxanthine, starch, tyrosine or xanthine. Growth occurs between 22 and 38 °C and from pH 5·5 to 10. Chemotaxonomic properties are typical of Nocardia. Acid is formed from (+)-D-glucose and glycerol but not from arbutin, (+)-D-cellobiose, (+)-D-fructose, (+)-D-galactose, meso-inositol, inulin, (+)-D-maltose, (+)-D-mannose, (+)-D-melezitose, (+)-D-melibiose, (+)-D-raffinose, 
-L-rhamnose, (–)-D-ribose, starch, (+)-D-sucrose, (+)-D-trehalose, (+)-D-turanose or (+)-D-xylose. (+)-D-Cellobiose, (+)-D-fructose, (+)-D-fucose, (+)-D-galactose, (+)-D-glucose, glycerol, meso-inositol (weak), (+)-D-maltose, (+)-D-mannose (weak), (+)-D-melibiose, -L-rhamnose, (–)-D-ribose, (+)-D-salicin, (+)-D-sorbitol (weak), starch (weak), (+)-D-sucrose, (+)-D-trehalose, acetate, adipic acid, fumarate, lactate, malate, paraffin, pyruvate and succinate are used as sole carbon and energy sources, but not adonitol, (+)-L-arabinose, (+)-D-arabitol, arbutin, dulcitol, meso-erythritol, ethanol, glycogen, inulin, (+)-D-lactose, (+)-D-mannitol, (+)-D-melezitose, methyl -D-glucoside, (+)-D-raffinose, xylitol, (+)-D-xylose, benzoate, citrate, formate, hippurate, malonate, sebacic acid, oxalate or tartrate. L-Alanine, L-proline and L-valine are used as sole carbon and nitrogen sources but not acetamide, L-asparagine, L-aspartate, gelatin, D-glucosamine, L-leucine, L-phenylalanine or L-serine. Growth occurs in the presence of sodium chloride at 3 %, w/v but not at 5 %, w/v. Resistance is shown to lysozyme, gentamicin sulphate (10 µg ml–1) and penicillin G (10 U), but not to chloramphenicol (30 µg ml–1), erythromycin (15 µg ml–1), midecamycin (15 µg ml–1), minocycline hydrochloride (30 µg ml–1), rifampicin (5 µg ml–1), streptomycin sulphate (10 µg ml–1), tobramycin sulphate (10 µg ml–1) or vancomycin hydrochloride (30 µg ml–1). The fatty acid profile is composed of C16 : 0 (33·1 %), C18 : 0 (7·6 %), cis-9-C18 : 1 (32·7 %) and 10-methyl-C18 : 0 (11·3 %). The G+C content of the DNA is 68·8 mol%.
The type strain, 276T (=CGMCC 4.1860T=JCM 12160T), was isolated from a soil sample collected from Xishan Mountain, Beijing, China. The species description is based on a single strain and hence serves as the description of the species.
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ACKNOWLEDGEMENTS |
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