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1 School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
2 Microbial Screening Technologies, Building A, 28–54 Percival Road, Smithfield, New South Wales 2164, Australia
Correspondence
Geok Yuan Annie Tan
gyatan{at}um.edu.my
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Present address: Division of Microbiology, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia. 
Present address: Department of Microbiology, Chung-Ang University College of Medicine, 221 Huksuk-Dong, Dongjak-ku, Seoul 156-756, Republic of Korea.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains GY080T, GY091, GY246, GY248, GY249, GY250 and GY293 are AY129760, AY129761, AY129766, AY129768, AY129767, AY129763 and AY129769, respectively.
A consensus dendrogram showing relationships between the A. regifaucium isolates and A. orientalis based on SJ-UPGMA analysis of restriction endonuclease-generated fingerprints and an extended 16S rRNA gene sequence-based neighbour-joining tree are available as supplementary material with the online version of this paper.
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; Saintpierre-Bonaccio et al., 2005; Tan et al., 2006a). It currently encompasses 37 species with validly published names that form a distinct phyletic line within the Pseudonocardiaceae 16S rRNA gene tree (Lee et al., 2006; Majumdar et al., 2006). The genus can be distinguished from the other genera classified in the family Pseudonocardiaceae by using a combination of chemical and morphological properties (Kim & Goodfellow, 1999). Similarly, Amycolatopsis species can be separated from one another using a range of phenotypic properties (Labeda et al., 2003; Tan et al., 2006a). The improved classification of the genus provides a sound basis for the circumscription of additional Amycolatopsis species, as exemplified by the description of novel species of clinical (Labeda et al., 2003; Huang et al., 2004), ecological (Goodfellow et al., 2001; Lee et al., 2006) and biotechnological (Goodfellow et al., 1997; Wink et al., 2003) interest, notably as a source of novel antibiotics (Tan et al., 2006a).
The aim of the present study was to establish the taxonomic position of seven Amycolatopsis isolates that produce a light-grey aerial spore mass, dark yellow–brown substrate mycelium and dark grey–brown diffusible pigments on modified Bennett's agar supplemented with mannitol and soybean flour (Tan et al., 2006b). The seven test strains were the subject of a polyphasic taxonomic study, which showed that they merited recognition as representatives of a novel species.
Strains GY080T, GY091, GY246, GY248, GY249, GY250 and GY293 were isolated on SM2 agar plates that had been inoculated with tenfold dilutions of a composite Australian soil sample and incubated at 28 °C for 21 days, as described by Tan et al. (2006b). The organisms were maintained on modified Bennett's agar slopes (Jones, 1949) at room temperature and as suspensions of mycelial fragments in glycerol (20 %, v/v) at –20 °C. Biomass for molecular systematic studies was grown and prepared according to Tan et al. (2006b). All of the isolates produced an amplification product of the expected size when treated with a set of genus-specific 16S rRNA oligonucleotide primers (Tan et al., 2006b).
Extraction of chromosomal DNA, PCR amplification and sequencing of 16S rRNA genes from the isolates was achieved using established procedures (Kim et al., 2002; Tan et al., 2006a). The resultant 16S rRNA gene sequences were aligned manually using the program PHYDIT (available at http://plaza.snu.ac.kr/
jchun/phydit/) against corresponding sequences of representatives of genera in the family Pseudonocardiaceae as retrieved from the GenBank/EMBL/DDBJ databases. Phylogenetic trees were inferred by using the least-squares (Fitch & Margoliash, 1967), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) algorithms drawn from the PHYLIP suite of programs (Felsenstein, 1993); evolutionary distance matrices were generated for the least-squares and neighbour-joining algorithms after Jukes & Cantor (1969). Topologies of the resultant trees were evaluated by bootstrap analysis (Felsenstein, 1985) of the neighbour-joining method based upon 1000 replicates using the programs CONSENSE and SEQBOOT from the PHYLIP package. Prauserella rugosa DSM 43194T was used as the outgroup organism to root the neighbour-joining tree. Phylogenetic comparison of almost complete 16S rRNA gene sequences of the tested strains with corresponding data on representatives of the family Pseudonocardiaceae showed that they belonged to the genus Amycolatopsis (data not shown).
It is apparent from Fig. 1 that the seven isolates share identical 16S rRNA gene sequences and form a distinct phyletic line in the Amycolatopsis tree together with the type strains of Amycolatopsis japonica and Amycolatopsis orientalis. The relationship between these organisms is supported by all of the tree-making algorithms and by a bootstrap value of 81 % in the neighbour-joining analysis. The seven isolates and A. orientalis IMSNU 20058T shared 99.86 % 16S rRNA gene sequence similarity, a value that corresponds to 2 nt differences at 1437 locations. Similarly, the isolates and A. japonica DSM 44213T had a 16S rRNA gene sequence similarity of 99.45 %, equivalent to 8 nt differences at 1453 sites. High 16S rRNA gene sequence similarities were also found between the isolates and the type strains of Amycolatopsis alba (99.04 %), Amycolatopsis azurea (98.96 %), Amycolatopsis coloradensis (98.68 %), Amycolatopsis decaplanina (99.10 %), Amycolatopsis keratiniphila subsp. nogabecina (99.10 %) and Amycolatopsis lurida (99.05 %).
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), as modified by Goris et al. (1998). Photobiotin-labelled DNA from the isolate was hybridized with single-stranded unlabelled DNA from A. orientalis IMSNU 20058T that had been non-covalently bound to microtitre wells. The hybridization experiments were carried out under stringent conditions in 50 % formamide at 43 °C. Fluorescence intensities were measured using a Labtech F1 fluorescent plate reader (Labtech International) at a wavelength of 360 nm for excitation and 450 nm for emission. The DNA–DNA relatedness between the two strains based on triplicate samples was 42.5±4.0 % (mean±SD), a value well below the 70 % cut-off point recommended for assignment of strains to the same genomic species (Wayne et al., 1987).
Isolate GY080T was examined for key chemical markers following preparation of biomass as described by Tan et al. (2006a). Standard HPLC and TLC procedures were used to determine the predominant menaquinones (Collins, 1994), muramic acid residue type (Uchida et al., 1999), major polar lipids (Minnikin et al., 1984) and diagnostic whole-organism sugars (Schaal, 1985) of the test strain and appropriate control organisms. Strain GY080T, which had been shown to contain meso-diaminopimelic acid but not mycolic acids (Tan et al., 2006a), contained the following: arabinose and galactose in whole-organism hydrolysates; N-acetylated muramic acid; diphosphatidylglycerol, phosphatidylethanolamine (taxonomically significant phospholipid), phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and phosphatidylmethylethanolamine as major polar lipids [phospholipid pattern type II sensu Lechevalier et al. (1977)]; and tetrahydrogenated menaquinones with nine isoprene units [MK-9(H4)] as the predominant isoprenologue (71 % of total menaquinone composition), with the balance formed by MK-9(H6). This chemical profile distinguished isolate GY080T from members of all other wall chemotype IV actinomycetes with the exception of those classified in the genus Amycolatopsis (Kim & Goodfellow, 1999).
Genomic DNA was extracted from the isolates and from type strains of representative Amycolatopsis species with validly published names using a standard protocol (Sambrook et al., 1989). Enzymically amplified 16S rRNA genes obtained by PCR (Tan et al., 2006a) were analysed by restriction digestion using EcoRV, HhaI, RsaI and TaqI (MBI Fermentas). The restriction patterns of the strains were normalized and the combined patterns were examined with BIONUMERICS version 2 software (Applied Maths) using the Jaccard coefficient (SJ) and the unweighted pair group method with arithmetic averages (UPGMA) clustering algorithm. The isolates were assigned to a homogeneous cluster in a numerical analysis of the combined amplified rDNA restriction analysis patterns, a taxon that was distinctly separate from the Amycolatopsis reference strains, including the type strains of A. japonica and A. orientalis (Supplementary Fig. S1, available in IJSEM Online).
Colony and pigmentation properties of the isolates were observed following growth on modified Bennett's agar supplemented with mannitol and soybean flour and on peptone-yeast extract-iron and tyrosine agars (Shirling & Gottlieb, 1966) for 14 days at 28 °C using a Nikon Optiphot binocular light microscope fitted with long-working-distance objectives. In addition, a range of phenotypic tests known to be of value in Amycolatopsis systematics were carried out using established procedures (de Boer et al., 1990; Goodfellow et al., 1997, 2001). The isolates shared a broad range of phenotypic properties that enabled them to be distinguished from representatives of phylogenetically closely related species, including A. japonica and A. orientalis (Table 1).
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) and yeast extract-malt extract (ISP2; Shirling & Gottlieb, 1966) agars at 28 °C for 7 days. The agar was excised, sectioned and extracted with methanol to provide a crude extract that was analysed by gradient HPLC. The analytical gradient HPLC system consisted of an SCL-10AVP system controller, SIL-10ADVP auto-injector, LC-10ADVP pump, FCV-10ALVP mixer, DGU-14A degasser, CTO-10AVP column oven and SPD-10AVP diode-array detector. HPLC analysis was undertaken by injection of a sample (50 µl) onto a platinum EPS C18 rocket reverse phase column (Alltech Associates Australia). The solvent gradient was 10 to 100 % acetonitrile/water containing 0.01 % trifluoroacetic acid at 3 ml min–1 over 13 min. The electronically stored data were compiled and analysed using COMET, a metabolite recognition software package (Lacey & Tennant, 2003). This program takes the data generated by diode array detection-HPLC and creates a compact database of the chromatogram and UV spectra. The relationship between these profiles and the type strains of Amycolatopsis species was analysed using an in-house database comprising over 6000 type strains.
All of the strains produced a family of secondary metabolites with highly characteristic UV spectra. The presence of these metabolites was a consistent phenotypic feature of all the strains and they produced them on both media. The metabolite pattern was unique to these strains and was not shared by members of the genus Amycolatopsis with validly published names. Furthermore, in the analysis of over 2000 type species and published strains of actinobacteria, no cultures exhibited metabolites with comparable elution and UV spectral characteristics. The major analogue of the family of metabolites was isolated by preparative HPLC and identified as kigamicin C. The kigamicins are a family of metabolites that have been isolated and identified recently from an Amycolatopsis isolate (Kunimoto et al., 2003).
The extracts of all strains exhibited potent antibacterial and antitumour activity due to the presence of the kigamicins. Antitumour activity was determined in a microtitre plate cell proliferation assay. Briefly, murine NS-1 cells in RPMI 1640 medium (200 µl, 5x104 cells ml–1), supplemented with 1 mM sodium pyruvate and 5 % (v/v) newborn calf serum, were added to the wells of a microtitre plate containing serial twofold dilutions of the test compound. The plates were incubated at 37 °C in the presence of 5 % CO2. A qualitative assessment of cell proliferation was made at 72 h, with the LD99 determined as the lowest concentration of the test compound at which no cell proliferation was observed.
Antibacterial activity was determined in an agar-based microtitre plate assay. An aliquot of an overnight fermentation of Bacillus subtilis ATCC 6633 was applied to the surface of an agar matrix that contained the test compound, which was then incubated at 28 °C. Qualitative assessment of bacterial growth was made at 24 h, with the MIC determined as the lowest concentration of the test compound at which no growth of bacteria was observed.
It is apparent from this polyphasic study that the seven isolates form a homogeneous taxon that can be distinguished from representatives of all known species of Amycolatopsis. It is proposed that this taxon be recognized as a novel Amycolatopsis species, namely Amycolatopsis regifaucium sp. nov.
Description of Amycolatopsis regifaucium sp. nov.
Amycolatopsis regifaucium (re'gi.fau'ci.um. L. n. rex, regis king; L. gen. pl. n. faucium of a defile; N.L. gen. pl. n. regifaucium of King's Canyon, Australia, the source of the soil from which the first strains were isolated).
Aerobic, Gram-positive, non-acid–alcohol-fast, non-motile, catalase-positive actinomycete that forms an extensively branched substrate mycelium that fragments into squarish, rod-like elements. Abundant, light-grey aerial hyphae and dark yellow–brown substrate mycelium with filamentous edges are formed on modified Bennett's agar supplemented with mannitol and soybean flour; a dark grey–brown diffusible pigment is produced on this medium. Melanin pigments are not formed on peptone-yeast extract-iron or tyrosine agars. Good growth occurs between 28 and 37 °C, from pH 5 to 9 and in the presence of sodium chloride (5 %, w/v). Aesculin and arbutin are hydrolysed, but not urea. Nitrate is reduced to nitrite and hydrogen sulfide is produced. Grows on L-arabinose, D-arabitol, D-cellobiose, dextrin, D-galactose, D-glucose, glycerol, glycogen, myo-inositol, maltose, D-mannitol, methyl 
-D-glucoside, D-ribose, sucrose, trehalose and xylitol as sole carbon sources for energy and growth, but not on adonitol, meso-erythritol, D-melezitose, D-melibiose, D-raffinose or D-sorbitol. Resistant to (µg ml–1) gentamicin sulfate (5), neomycin sulfate (8), novobiocin (10), penicillin G (20), polymyxin B sulfate (50), rifampicin (10), streptomycin sulfate (16) and tobramycin sulfate (8). Additional phenotypic properties are shown in Table 1
. Chemotaxonomic characteristics are typical of members of the genus Amycolatopsis.
The type strain is GY080T (=DSM 45072T =NCIMB 14277T), isolated from Australian arid soil.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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