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1 Microbial Resources Laboratory, Division 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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ABSTRACT |
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Present address: Microbiology Division, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia. 
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for aerobic, filamentous actinomycetes that contained meso-diaminopimelic acid, arabinose and galactose (wall chemotype IV sensu Lechevalier & Lechevalier, 1970), phosphatidylethanolamine as the diagnostic polar lipid (phospholipid type 2 sensu Lechevalier et al., 1977) and di- and tetra-hydrogenated menaquinones with nine isoprene units as the predominant isoprenologues, but lacked mycolic acids. The genus currently encompasses 31 species with validly published names, which form a distinct phyletic line within the 16S rRNA gene tree for the Pseudonocardiaceae (Lee, 2006). Amycolatopsis species fall into several multimembered 16S rRNA gene groups, such as the Amycolatopsis methanolica and Amycolatopsis orientalis clades (Kim et al., 2002; Saintpierre-Bonaccio et al., 2005); the type strain of Amycolatopsis fastidiosa forms a single-membered phyletic line at the foot of the Amycolatopsis tree (Goodfellow et al., 2001; Huang et al., 2001, 2004).
Amycolatopsis strains can be distinguished from members of the other genera classified in the family Pseudonocardiaceae by using genus-specific oligonucleotide primers (Tan et al., 2006) and chemical and morphological markers (Kim & Goodfellow, 1999). Similarly, a range of phenotypic properties are available for the identification of Amycolatopsis strains (Goodfellow et al., 2001; Wink et al., 2003; Saintpierre-Bonaccio et al., 2005). Members of the genus have been isolated from vegetable matter (Lechevalier et al., 1986) and clinical material (Labeda et al., 2003; Huang et al., 2004) but it is evident from the application of improved selective isolation and characterization procedures that they are common in soil (Tan et al., 2006). Amycolatopsis strains are a rich source of secondary metabolites, including ansamycins (rifamycins and tolypomycins) and glycopeptides (balhimycins and vancomycins). Their commercial significance has led to an intensive search of Amycolatopsis strains for sources of novel pharmaceutical activities – an activity that has promoted a better understanding of the taxonomic structure of the genus (Wink et al., 2003).
The present polyphasic study was designed to establish the taxonomic status of seven Amycolatopsis isolates, of which three, strains GY048T, GY056 and GY064, form a distinct lineage in the Amycolatopsis 16S rRNA gene tree (Tan et al., 2006). The resultant data show that all of the isolates represent a novel species of the genus Amycolatopsis.
Strains GY048T, GY049, GY056, GY064, GY269, GY276 and GY286 were isolated on SM2 agar plates that had been inoculated with suspensions of a composite arid Australian soil sample and incubated at 28 °C for 3 weeks, as described by Tan et al. (2006). The organisms were maintained on modified Bennett's agar slants (Jones, 1949) at room temperature and as suspensions of mycelial fragments in glycerol (20 %, v/v) at –20 °C. Biomass samples for the molecular systematic studies were prepared by growing the isolates in glucose/yeast extract broth shake flasks for 3 days at 28 °C. Cultures were checked for purity, killed by shaking with formalin (1 %, v/v) and harvested by centrifugation. Cells were washed in NaCl/EDTA buffer (0.1 M EDTA, pH 8.0, 0.1 M NaCl) and stored at –20 °C until needed.
Extraction of chromosomal DNA, PCR amplification and sequencing of 16S rRNA genes from isolates GY049, GY269, GY276 and GY286 were achieved using procedures described previously (Kim et al., 2002; Tan et al., 2006). The resultant 16S rRNA gene sequences were aligned manually, using the PHYDIT program (available at http://plaza.snu.ac.kr/
jchun/phydit/), against corresponding sequences of representatives of genera classified in the family Pseudonocardiaceae (data not shown) and of type species of the genus Amycolatopsis (as retrieved from the GenBank database). Unrooted trees were inferred by using the least-squares (Fitch & Margoliash, 1967), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) tree-making algorithms. Evolutionary distance matrices were generated for the least-squares and neighbour-joining algorithms after Jukes & Cantor (1969). The analyses were carried out using the PHYLIP suite of programs (Felsenstein, 1993) and the topologies of the resultant trees were evaluated by using a bootstrap analysis (Felsenstein, 1985) of the neighbour-joining dataset with the CONSENSE and SEQBOOT programs from the PHYLIP package. The root position of the unrooted Amycolatopsis tree based on the neighbour-joining method was estimated by using Pseudonocardia thermophila ATCC 19285T (GenBank accession no. X53195) as the outgroup.
It is evident from Fig. 1 that the strains tested share identical (100 % similarity) 16S rRNA gene sequences both with each other and with isolates GY048T, GY056 and GY064. These strains form a distinct phyletic line within the Amycolatopsis mediterranei clade, a taxon that encompasses the type strains of nine Amycolatopsis species with validly published names; the taxonomic integrity of this clade was supported by the results from all of the tree-making algorithms and by a bootstrap value of 86 % in the neighbour-joining analysis. The isolates showed the highest level of 16S rRNA gene similarity with the type strain of A. mediterranei (98.6 %, a value that corresponded to 20 nucleotide differences at 1411 locations). Relatively high similarity values were shown with the type strains of Amycolatopsis balhimycina (97.8 %), Amycolatopsis kentuckyensis (98.2 %), Amycolatopsis lexingtonensis (98.0 %), Amycolatopsis plumensis (98.0 %), Amycolatopsis pretoriensis (98.2 %), Amycolatopsis rifamycinica (98.2 %), Amycolatopsis tolypomycina (98.5 %) and Amycolatopsis vancoresmycina (98.1 %).
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for the circumscription of strains that belong to the same genomic species. The type strains of A. lexingtonensis and A. pretoriensis, for example, share a 16S rRNA gene similarity of 99.9 % and a DNA–DNA relatedness value of 54.1 % (Labeda et al., 2003). Similarly, the type strain of A. rifamycinica has only six mismatches (99.6 % similarity) with respect to its closest neighbour, A. kentuckyensis, but shows a DNA–DNA hybridization value of only 67 % with the latter (Bala et al., 2004).
Isolate GY048T was examined for key chemical markers to determine whether it had a chemical profile consistent with its classification in the genus Amycolatopsis. Biomass for the chemotaxonomic studies was prepared by growing the isolate in glucose/yeast extract broth shake flasks (150 r.p.m.) for 3 days at 28 °C; the cells were washed in distilled water and then freeze-dried. Standard HPLC and TLC procedures were used to determine the predominant menaquinones (Collins, 1994), the type of muramic acid residue (Uchida et al., 1999), the major polar lipids (Minnikin et al., 1984) and the predominant whole-organism sugars (Schaal, 1985) of strain GY048T. The control organisms were A. orientalis KCTC 9412T, Gordonia bronchialis N654T and Nocardia brasiliensis N318T.
Strain GY048T, which is known to contain meso-diaminopimelic acid but which lacks mycolic acids (Tan et al., 2006), contained arabinose and galactose in whole-organism hydrolysates, contained N-acetylated muramic acid, possessed diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylmethylethanolamine as the major polar lipids (phospholipid pattern type 2 sensu Lechevalier et al., 1977) and contained tetrahydrogenated menaquinone with nine isoprene units [MK-9(H4)] as the predominant isoprenologue (66 % of the total menaquinone composition) and smaller proportions of MK-8(H2) (26 % of the total) and MK-9(H6) (8 % of the total). These chemical markers serve to distinguish strain GY048T from all other actinomycetes with wall chemotype IV, apart from those classified in the genus Amycolatopsis (Lechevalier et al., 1986; Embley et al., 1988; Yassin et al., 1991, 1993; Kim & Goodfellow, 1999).
Crude extracts of the isolates were prepared by methanol extraction of agar cultures grown on modified Bennett's agar (Jones, 1949) and yeast extract-malt extract agar (ISP 2; Shirling & Gottlieb, 1966) at 28 °C for 7 days. Antibacterial and anti-yeast activities were assessed using Bacillus subtilis ATCC 6633 and Saccharomyces cerevisiae ATCC 9763, respectively. All strains produced extracts that exhibited low titres of antibacterial or anti-yeast activity when grown on either medium.
The morphological properties of the isolates were observed, using a Nikon Optiphot light microscope fitted with a long-working-distance objective, after growth on modified Bennett's agar (Jones, 1949) supplemented with mannitol (0.5 %, w/v) and soybean flour (0.5 %, w/v), and after growth on tyrosine agar (ISP 7; Shirling & Gottlieb, 1966) for 14 days at 28 °C. In addition, a range of phenotypic tests were carried out using media and methods described previously (de Boer et al., 1990; Goodfellow et al., 1997, 2001). The strains shared a combination of phenotypic properties that serve to distinguish them from the type strains of Amycolatopsis species classified in the A. mediterranei clade (Table 1).
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Description of Amycolatopsis australiensis sp. nov.
Amycolatopsis australiensis (aus.tra.li.en'sis. N.L. fem. adj. australiensis pertaining to Australia, the source of the soil from which the first strains were isolated).
Aerobic, Gram-positive, non-acid–alcohol-fast, non-motile actinobacteria that form extensively branched substrate mycelia that fragment into squarish, rod-shaped elements. Abundant, white aerial hyphae and pale-yellow substrate mycelia are formed on modified Bennett's agar supplemented with mannitol and soybean flour; a medium-yellow diffusible pigment is evident on this medium. Melanin pigments are not produced on peptone-yeast extract-iron or tyrosine agars. Good growth occurs between 28 and 45 °C, and from pH 5 to 7. Nitrate is reduced to nitrite; elastin, Tween 40 and uric acid are degraded. D(+)-Arabitol, D(+)-cellobiose, dextrin, D(+)-galactose, D(+)-glucose, glycerol, glycogen, myo-inositol, 
-lactose, D(+)-maltose, D(+)-mannitol, D(+)-melezitose, D(+)-raffinose, sucrose, D(+)-trehalose, xylitol, D(+)-xylose (all at 1 %, w/v) and citric acid, oxalic acid and propionic acid (all at 0.1 %, w/v) are used as sole carbon sources, but adonitol, meso-erythritol, D(+)-melibiose, methyl -D-glucoside, D(+)-ribose, D(–)-sorbitol (all at 1 %, w/v) and sodium benzoate, sodium lactate and sodium mucate (at 0.1 %, w/v) are not. Acid is formed from D(+)-arabitol, D(+)-glucose, glycerol, glycogen, D(+)-mannose and D(+)-turanose, but not from dulcitol, D(+)-ribose or xylitol. The organism is sensitive to novobiocin (10 µg ml–1), penicillin G (20 µg ml–1), rifampicin (10 µg ml–1) and streptomycin sulphate (16 µg ml–1), but resistant to gentamicin sulphate (5 µg ml–1), neomycin sulphate (8 µg ml–1), polymyxin B sulphate (50 µg ml–1) and tobramycin sulphate (8 µg ml–1). Activity is shown against B. subtilis ATCC 6633 and S. cerevisiae ATCC 9763 but not against Candida albicans ATCC 10231. Additional phenotypic properties are shown in Table 1
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The type strain, GY048T (=DSM 44671T=NCIMB 14142T), was isolated from arid Australian soils.
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ACKNOWLEDGEMENTS |
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1460 nt), showing the relationships between the novel strains and representatives of the genus Amycolatopsis. Asterisks indicate branches of the tree that were also found using the least-squares (Fitch & Margoliash, 1967