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1 Laboratoire de Biologie et Physiologie Végétales Appliquées, Université de la Nouvelle-Calédonie, BP4477 98847, Nouméa, New Caledonia
2 School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
Correspondence
Michael Goodfellow
m.goodfellow{at}ncl.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Amycolatopsis plumensis SBHS Strp1T is AY262825.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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(as circumscribed by Warwick et al., 1994), a taxon that is closely related to the family Actinosynnemataceae Labeda and Kroppenstedt 2000. Amycolatopsis species can be assigned to two multimembered 16S rRNA gene groups, the Amycolatopsis methanolica and Amycolatopsis orientalis subclades (Kim et al., 2002; Huang et al., 2004); the type strain of Amycolatopsis fastidiosa forms a single-membered phyletic line at the base of the Amycolatopsis tree (Goodfellow et al., 2001; Huang et al., 2001, 2004). A combination of chemical and morphological properties are available to distinguish Amycolatopsis from other genera classified in the family Pseudonocardiaceae (Kim & Goodfellow, 1999). Similarly, Amycolatopsis species can be separated from one another using a range of phenotypic properties (Goodfellow et al., 2001; Kim et al., 2002; Labeda et al., 2003). The genus Amycolatopsis is grossly underspeciated and is a source of novel bioactive compounds (Tan, 2002).
Ultramafic soils account for about one-third of the land mass in New Caledonia (Jaffré, 1976). These infertile soils have a high metal toxicity due to the presence of significant concentrations of chromium, cobalt, iron and nickel, and seem to provide habitats for unusual actinomycetes (Saintpierre, 2001; Saintpierre et al., 2003; Saintpierre-Bonaccio et al., 2004a, b). In the course of a screening programme designed to isolate novel actinomycetes from ultramafic soils, an actinomycete, SBHS Strp1T, was isolated and shown to have properties typical of the genus Amycolatopsis. The aim of the present study was to determine the taxonomic status of this organism by using a polyphasic approach. It is evident from the resultant data that the organism represents a novel species in the genus Amycolatopsis.
Strain SBHS Strp1T was isolated from a suspension of a brown hypermagnesian ultramafic soil, which was used to inoculate a yeast extract/malt extract agar plate (ISP 2 medium; Shirling & Gottlieb, 1966) supplemented with 10 µg streptomycin sulphate ml–1 and 100 µg cycloheximide ml–1 (Vobis, 1992) and incubated at 30 °C for 2 weeks. The soil sample had been collected from the ‘Plum’ region at the southern end of the main island of New Caledonia (see Institut National Géographique, map no. 4836, série orange [Mont Doré] 668·5x7535). The isolate was purified and maintained on modified Bennett's agar (Jones, 1949) and preserved as a mixture of hyphae and fragmentation spores in glycerol (20 %, v/v) at –20 °C.
Morphological and staining properties of strain SBHS Strp1T were detected using growth from modified Bennett's agar plates that had been incubated for 2 weeks at 30 °C. The same medium and growth conditions were used to establish the ability of the organism to grow at different temperature and pH values. Additional phenotypic properties were determined by using standard media and methods (Williams et al., 1983; Goodfellow et al., 1997). The ability of the test strain to grow in modified Bennett's agar supplemented with a range of antibiotics and chemical inhibitors was determined as described by Saintpierre et al. (2003). A number of the phenotypic tests were also carried out on the type strains of Amycolatopsis tolypomycina and Amycolatopsis vancoresmycina, as shown in Table 1.
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) at 100 r.p.m. for 7 days at 30 °C. Cultures were checked for purity, killed by shaking with formalin (1 %, v/v), harvested by centrifugation and freeze-dried. The isomeric form of diaminopimelic acid was determined by TLC of a whole-organism hydrolysate on cellulose acetate sheets according to Staneck & Roberts (1974) with a modified solvent system, namely methanol/water/HCl/10 M pyridine (85 : 15 : 5 : 10, by vol.). TLC of major whole-organism sugars was carried out according to the method described by Staneck & Roberts (1974). A standard procedure was used to establish the predominant menaquinones (Minnikin et al., 1984).
Extraction of chromosomal DNA and PCR amplification of the 16S rRNA gene from strain SBHS Strp1T were achieved using previously described procedures (Kim et al., 1999). Similarly, a 16S rRNA gene from the isolate was amplified by PCR and sequenced directly using a Taq Dye Deoxy Terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). The resultant rRNA gene sequence was aligned manually using the PHYDIT program (available at http://plaza.snu.ac.kr/
jchun/phydit/) against corresponding sequences of representatives of the families Actinosynnemataceae and Pseudonocardiaceae retrieved from the EMBL and GenBank databases. Unrooted evolutionary trees were inferred by using the least-squares (Fitch & Margoliash, 1967), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms. An evolutionary distance matrix was generated using the distance model of Jukes & Cantor (1969). The topologies of the resultant trees were evaluated in bootstrap analyses of the neighbour-joining method, based on 1000 replicates, using the CONSENSE and SEQBOOT programs. All of the phylogenetic analyses were carried out using the PHYLIP suite of programs (Felsenstein, 1993).
An almost-complete 16S rRNA gene sequence (1488 nt) was generated for strain SBHS Strp1T. Comparison of this sequence with corresponding nucleotide sequences of representatives of the genera classified in the families Actinosynnemataceae and Pseudonocardiaceae showed the organism to be a member of the genus Amycolatopsis (data not shown). This assignment is supported by the chemotaxonomic data, as the organism contains meso-diaminopimelic acid, arabinose and galactose (wall chemotype IV sensu Lechevalier & Lechevalier, 1970) and tetrahydrogenated menaquinones with nine isoprene units as the major isoprenologue. The organism is an aerobic, non-motile, Gram-positive, non-acid–alcohol-fast, catalase-positive actinomycete which produces an extensively branched substrate mycelium that fragments into squarish, rod-like elements when grown on modified Bennett's agar. The substrate mycelium carries abundant pale-orange aerial hyphae, which undergo fragmentation yielding squarish, rod-like elements. All of these phenotypic properties are consistent with the classification of the strain in the genus Amycolatopsis (Lechevalier et al., 1986).
Strain SBHS Strp1T belongs to the A. orientalis subclade and is most closely related to the type strain of A. tolypomycina. The relationship between these organisms is supported by the results from all of the tree-making algorithms and by a bootstrap value of 82 % (Fig. 1). The two strains share a 16S rRNA gene sequence similarity of 99·4 %, a value which corresponds to 8 nt differences at 1384 sites. The organism also shares high 16S rRNA gene sequence similarity values with other organisms shown in Fig. 1, notably with the type strains of Amycolatopsis pretoriensis (99·2 %), Amycolatopsis lexingtonensis (99·0 %), Amycolatopsis kentuckyensis (98·9 %) and Amycolatopsis rifamycinica (98·9 %). DNA–DNA relatedness studies were not carried out between isolate SBHS Strp1T and these organisms, as some of the latter have almost identical 16S rRNA gene similarity values and have DNA–DNA relatedness values well below the cut-off point recommended by Wayne et al. (1987) for the delineation of strains that belong to the same genomic species. The type strains of A. lexingtonensis and A. pretoriensis, for instance, share a 16S rRNA gene similarity of 99·9 % and have a DNA–DNA relatedness value of 54·1 % (Labeda et al., 2003). In addition, the type strains of A. methanolica and Amycolatopsis thermoflava have almost identical 16S rRNA gene sequences but were found to have a DNA relatedness value of only 21 % when DNA from the former was used as the probe (Chun et al., 1999). The tested strain can be distinguished from the type strains of phylogenetically related Amycolatopsis species, including A. tolypomycina, using a range of phenotypic properties (Table 1
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Description of Amycolatopsis plumensis sp. nov.
Amycolatopsis plumensis (plum.en'sis. N.L. fem. adj. plumensis referring to the Plum region of the main island of New Caledonia, the source of the soil from which the type strain was isolated).
Aerobic, Gram-positive, non-acid–alcohol-fast, non-motile, catalase-positive actinomycete which forms extensively branched substrate hyphae that fragment into squarish, rod-like elements. A pale-orange substrate mycelium, but no diffusible pigments, is formed on modified Bennett's agar. The substrate mycelium carries abundant pale-orange aerial hyphae. Chemotaxonomic characteristics are typical for Amycolatopsis. Grows at pH 4–12 and at 20–37 °C, but not at 10 or 45 °C. Hydrogen sulphide is formed, and elastin, L-tyrosine, Tween 80 and xylan are degraded. Growth occurs in the presence of erythromycin (4 µg ml–1), gentamicin sulphate (10 µg ml–1), rifampicin (6 µg ml–1), streptomycin sulphate (5 µg ml–1), crystal violet (0·0002 %, w/v), phenol (0·01 %, w/v) and potassium tellurite (0·005 %, w/v), but the organism is susceptible to penicillin G (25 µg ml–1), tetracycline hydrochloride (30 µg ml–1), vancomycin hydrochloride (10 µg ml–1) and sodium chloride (5 %, w/v). Additional phenotypic properties are shown in Table 1. The organism shows activity against Candida albicans, Corynebacterium sp., Fusarium oxysporum, Klebsiella pneumoniae and Staphylococcus aureus strains but not against Bacillus, Escherichia or Staphylococcus epidermidis strains.
The type (and only) strain, SBHS Strp1T (=DSM 44776T=NRRL B-24324T), was isolated from brown hypermagnesian ultramafic soil at the southern end of the main island of New Caledonia.
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ACKNOWLEDGEMENTS |
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