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1 Laboratoire de Biologie et Physiologie Végétales Appliquées, Université de la Nouvelle-Calédonie, BP 4477 98847, Nouméa, New Caledonia
2 School of Biology, King George VIth Building, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
Correspondence
Michael Goodfellow
m.goodfellow{at}ncl.ac.uk
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Published online ahead of print on 31 October 2003 as DOI 10.1099/ijs.0.02881-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SBHR OA6T is AY282603.
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). At the time of writing, the taxon contains 29 species with validly published names that form a clade within the evolutionary radiation occupied by mycolic-acid-containing actinomycetes classified in the suborder Corynebacterineae Stackebrandt et al. 1997. The genus can be distinguished from the other genera assigned to the suborder by a combination of chemical and morphological properties (Goodfellow et al., 1999). Similarly, Nocardia species can be separated from one another by using a range of phenotypic properties (Yassin et al., 2001; Zhang et al., 2003). The improved classification of the genus is providing an invaluable framework for the recognition of novel Nocardia species, as exemplified by the description of novel species of clinical (Yassin et al., 2000; Hamid et al., 2001), ecological (Chun et al., 1998; Maldonado et al., 2000; Albuquerque de Barros et al., 2003) and industrial (Isik et al., 1999b; Kinoshita et al., 2001) significance.
Ultramafic soils account for about a third of the landmass of the main island of New Caledonia (Jaffré, 1976). There is evidence that these arid, infertile soils, which have high metal toxicity due to the presence of chromium, cobalt, iron and nickel, provide a rich habitat for unusual actinomycetes of industrial significance (Saintpierre, 2001; Saintpierre et al., 2003; D. Saintpierre-Bonaccio, H. Amir, R. Pineau and M. Goodfellow, unpublished results). During the course of a screening programme designed to isolate novel actinomycetes from New-Caledonian ultramafic soils, an actinomycete, designated SBHR OA6T, was isolated and presumptively assigned to the genus Nocardia (Saintpierre, 2001). The aim of the present study was to establish the taxonomic position of this organism using genotypic and phenotypic procedures. It is evident from the results that the organism represents a novel species in the genus Nocardia, for which the name Nocardia neocaledoniensis sp. nov. is proposed.
Strain SBHR OA6T was isolated from a suspension of a brown hypermagnesian ultramafic soil that was used to inoculate an oatmeal agar plate (ISP 3 medium; Shirling & Gottlieb, 1966) supplemented with cycloheximide (100 µg ml-1) and incubated at 30 °C for 10 days. The soil sample was from the ‘Plum’ region at the southern end of the main island of New Caledonia [see Institut National Geographique, map no. 38 (Mont Dore), 669x7536·5, série orange]. The isolate was purified and maintained on modified Bennett's agar (MBA; Jones, 1949) and preserved as a suspension of mycelial fragments in glycerol (20 %, v/v) at -20 °C. Biomass for chemotaxonomic studies was prepared by growing the strain in shake flasks of glucose/yeast extract broth (Gordon & Mihm, 1962) at 100 r.p.m. for 10 days at 30 °C. Cultures were checked for purity, killed by shaking with formalin (1 %, v/v), harvested by centrifugation and freeze-dried.
Morphological and staining properties of strain SBHR OA6T were detected following growth on MBA plates that had been incubated for 2 weeks at 30 °C. Additional phenotypic properties were determined using well-established methods (Williams et al., 1983; Isik et al., 1999a). Standard procedures were used for the extraction and analysis of mycolic acids (Minnikin et al., 1975), whole-organism sugars (Schaal, 1985) and isoprenoid quinones and polar lipids (Minnikin et al., 1984), using appropriate controls. The isomeric form of diaminopimelic acid (A2pm) was determined after Staneck & Roberts (1974), using a modified solvent system consisting of methanol/water/10 M HCl/pyridine (85 : 15 : 5 : 10, by vol.).
Biomass for 16S rDNA nucleotide sequencing was obtained by growing strain SBHR OA6T on an MBA plate for 7 days at 30 °C. Isolation of chromosomal DNA, PCR amplification and direct sequencing of the purified product were carried out as described previously (Kim et al., 1999). The resultant 16S rRNA gene sequence was aligned manually with corresponding sequences of representatives of the genera classified in the suborder Corynebacterineae retrieved from the DDBJ/EMBL/GenBank databases using the PHYDIT program (Chun, 1995). Evolutionary trees were inferred using the least-squares (Fitch & Margoliash, 1967), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) treeing algorithms from the PHYLIP suite of programs (Felsenstein, 1993). Evolutionary-distance matrices for the least-squares and neighbour-joining methods were generated after Jukes & Cantor (1969). The topologies of the resultant trees were evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining datasets on 1000 resamplings using the SEQBOOT and CONSENSE options from the PHYLIP package. DNA–DNA relatedness values between strain SBHR OA6T and related strains were determined by the identification service at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany), as described by Kim et al. (1999).
Strain SBHR OA6T has phenotypic properties consistent with its classification in the genus Nocardia (Goodfellow et al., 1999). The organism is an aerobic, Gram-positive, partially acid–alcohol-fast actinomycete that forms an extensively branched substrate mycelium that fragments into irregular, rod-shaped, non-motile elements and supports pale-orange aerial hyphae when grown on glucose/yeast extract agar and MBA plates. The organism was also shown to yield whole-organism hydrolysates rich in meso-A2pm, arabinose and galactose (wall chemotype IV sensu Lechevalier & Lechevalier, 1970) and to have major proportions of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides (phospholipid type II sensu Lechevalier et al., 1977). It also contained predominant amounts of hexahydrogenated menaquinones with eight isoprene units where the end two were cyclized; this menaquinone is characteristic of the genera Nocardia and Skermania (Chun et al., 1997; Goodfellow et al., 1999). It is also characterized by the presence of mycolic acids that co-migrated (Rf value around 0·47) with those from marker strains of Nocardia.
The almost complete 16S rRNA gene sequence (1474 nt) obtained for strain SBHR OA6T was compared with sequences of representatives of the suborder Corynebacterineae and found to have the signature sequences expected for members of this taxon and the family Nocardiaceae (Stackebrandt et al., 1997). The high 16S rRNA gene sequence similarities found between the tested strain and representatives of the genus Nocardia (95·4–98·7 %) support its inclusion in this taxon. The 16S rRNA gene sequence of strain SBHR OA6T also has the signature nucleotides characteristic of the genus Nocardia (Chun & Goodfellow, 1995).
It is evident from Fig. 1 that strain SBHR OA6T is most closely associated with the type strain of Nocardia asteroides, a relationship that is supported by all of the treeing algorithms and by a moderately high bootstrap value. The two strains share 98·7 % 16S rRNA gene sequence similarity, a value that corresponds to 17 nt differences at 1470 locations. However, it is evident from the DNA–DNA relatedness study that the two strains should not be classified in the same species, as they were found to share 40·8 % DNA–DNA relatedness, a value well below the 70 % cut-off point recommended for the delineation of genomic species (Wayne et al., 1987). The organism also has a profile of phenotypic properties that distinguish it from representatives of all species of Nocardia with validly published names, including N. asteroides (Table 1).
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Description of Nocardia neocaledoniensis sp. nov.
Nocardia neocaledoniensis (ne.o.ca.le.do.ni.en'sis. N.L. fem. adj. neocaledoniensis pertaining to New Caledonia, the source of the isolate).
Aerobic, Gram-positive, catalase-positive, slightly acid–alcohol-fast, non-motile actinomycete that forms an extensively branched orange substrate mycelium that fragments in situ into irregular rod-shaped elements and which carries abundant pale-orange aerial hyphae on MBA. Melanin pigments are produced on peptone/yeast extract/iron agar. Chemotaxonomic properties are typical of Nocardia. Grows at 10–45 °C and from pH 4 to 12. Degrades DNA and Tween 80 but not gelatin, guanine, starch or xylan. Produces hydrogen sulphide. Utilizes (+)-D-fructose, (+)-D-mannose, (+)-D-raffinose and (+)-D-trehalose as sole carbon sources for energy and growth but not adonitol, (-)-D-arabinose, (+)-D-cellobiose, (+)-D-galactose, (+)-D-lactose, (+)-D-maltose, (+)-D-melibiose, (+)-D-sucrose (all at 1 %, w/v) or sodium succinate (0·1 %, w/v). Growth occurs in the presence of erythromycin (4 µg ml-1), gentamicin sulphate (10 µg ml-1), penicillin G (25 µg ml-1), rifampicin (6 µg ml-1), streptomycin sulphate (5 µg ml-1), vancomycin hydrochloride (10 µg ml-1), crystal violet (0·0002 %, w/v), phenol (0·01 %, w/v) and sodium chloride (3 %, w/v), but not in the presence of tetracycline hydrochloride (30 µg ml-1), potassium tellurite (0·005 %, w/v) or 5 % (w/v) sodium chloride. Additional phenotypic properties are shown in Table 1
. The species description is based on a single strain, which hence serves as the type strain.
The type strain, SBHR OA6T (=DSM 44717T=NCIMB 13955T), was isolated from a brown hypermagnesian ultramafic soil at the southern end of the main island of New Caledonia.
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ACKNOWLEDGEMENTS |
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