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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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Present address: Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México (UNAM), CP 04510, México DF, Mexico. 
Present address: Centro Interdisciplinario de Investigaciones y Estudios en Medio Ambiente y Desarrollo (CIIEMAD), Instituto Politécnico Nacional (IPN), CP 07700, México DF, Mexico.
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for a group of thermophilic actinomycetes that formed short chains of spores on short sporophores. The species was subsequently associated with a number of other genera, including Nocardia (Becker et al., 1965; Lechevalier et al., 1966), Actinomadura (Cross & Goodfellow, 1973; Lacey et al., 1978), Micropolyspora (Krassilnikov et al., 1968), Microtetraspora (Kroppenstedt et al., 1990) and latterly Nonomuraea (Zhang et al., 1998). The present polyphasic study was undertaken to clarify the taxonomic position of Nonomuraea flexuosa DSM 41386T. The resultant data show that the strain should be classified in a new genus within the family Streptosporangiaceae Goodfellow et al. 1990, emend. Ward-Rainey et al. 1997. It is proposed that the name Thermopolyspora flexuosa be used for this taxon, a proposal advanced by Greiner-Mai et al. (1987) on the basis of chemotaxonomic and morphological data.
Nonomuraea flexuosa DSM 41386T was maintained on glucose-yeast extract-malt extract agar plates (ISP2; Shirling & Gottlieb, 1966) at 45 °C and as a glycerol suspension (20 %, v/v) at –20 °C. Biomass for the molecular systematic studies was prepared from shake flasks of ISP2 broth incubated at 45 °C for 15 days and then washed in NaCl/EDTA buffer (0·1 M EDTA, pH 8·0, 0·1 M NaCl) and stored at –20 °C until needed.
Isolation of chromosomal DNA, PCR amplification and direct sequencing of the purified products of the test strain were carried out after Quintana et al. (2003). An almost full-length 16S rRNA gene sequence was aligned manually with corresponding sequences of representatives of genera classified in the suborder Streptosporangineae 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-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) tree-making 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 robustness of the resultant trees was evaluated by bootstrap analyses (Felsenstein, 1985) of the neighbour-joining dataset on 1000 resamplings using the SEQBOOT and CONSENSE options from the PHYLIP package.
DNA from the organism was also examined using the primers Sm6F and Sm5R which yield a product that is specific for members of the family Streptosporangiaceae (Monciardini et al., 2002).
Strain DSM 41386T formed the PCR product characteristic of members of the family Streptosporangiaceae when examined with the family-specific set of oligonucleotide primers developed by Monciardini et al. (2002) for the recognition of members of this taxon (data not shown). Comparison of the almost complete 16S rRNA gene sequence (1493 nt) of the tested strain with corresponding sequences of representatives of taxa classified in the suborder Streptosporangineae showed that the organism forms a distinct phyletic line within the evolutionary radiation occupied by the family Streptosporangiaceae (Fig. 1). The organism showed 16S rRNA gene sequence similarities to other members of the family within the range 92·7 to 94·7 %, values consistent with the assignment of the strain to a novel genus. It is apparent from Table 1 that strain DSM 41386T shows a range of phenotypic properties that distinguish it from representatives of the established genera classified in the family Streptosporangiaceae.
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Description of Thermopolyspora gen. nov.
Thermopolyspora [Ther.mo.po'ly.spo.ra. Gr. n. thermos heat; Gr. adj. poly many; Gr. n. spora a seed; N.L. fem. n. Thermopolyspora the heat (-loving) many-spored organism].
The description is taken from this and earlier studies (Krassilnikov & Agre, 1964; Greiner-Mai et al., 1987; Meyer, 1989; Kroppenstedt et al., 1990). Aerobic, Gram-positive, non-acid–alcohol-fast, thermophilic actinomycetes which form non-fragmenting substrate and aerial mycelia. Hooked or irregular spiral chains of 4 to 10 warty to spiny ornamented spores (1·2–1·5 µm in diameter) are arranged in clusters on long, moderately branched aerial hyphae on potato carrot agar; the organism forms a light blue aerial mycelium and a brown soluble pigment on this medium. White to yellowish white aerial hyphae are borne on a light-brown substrate mycelium on oatmeal agar, but diffusible pigments are not produced. Growth occurs from pH 6·0 to 9·0 and from 40 to 60 °C; particularly good growth occurs between 45 and 55 °C. The wall peptidoglycan contains meso-diaminopimelic acid and N-acetylated muramic acid. Glucose and ribose are found in whole-organism hydrolysates from the type species. The type species contains MK9, MK9(H2) and MK9(H4) as predominant isoprenologues and minor amounts of MK9(H6), phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositolmannosides, ninhydrin- and sugar-positive phospholipids and uncharacterized glycolipids as major polar lipids. It also contains fatty acids rich in saturated, unsaturated and branched chain components, but lacks mycolic acids. The G+C content of the DNA of the type species is 77 mol%. The type and only species is Thermopolyspora flexuosa. 16S rRNA gene sequence data show that the organism belongs to the family Streptosporangiaceae Goodfellow et al. 1990, emend. Ward-Rainey et al. 1997.
Description of Thermopolyspora flexuosa comb. nov., nom. rev.
Thermopolyspora flexuosa (flex.u'o.sa. L. fem. adj. flexuosa full of turns or windings, tortuous, flexuous referring to the morphology of the spore chains).
Basonym: Actinomadura flexuosa (ex Krasil'nikov and Agre 1964) Meyer 1989.
The description is based on the results of this and previous studies (Krassilnikov & Agre, 1964; McCarthy & Cross, 1984; Greiner-Mai et al., 1987; Meyer, 1989; Kroppenstedt et al., 1990; Stackebrandt et al., 2001).
General chemotaxonomic, growth and morphological properties are included in the genus description. Grows well on peptone glucose agar, producing a brown substrate mycelium but no aerial hyphae. Melanin pigments are formed from tyrosine. Growth poor on Bennett's sucrose and glycerol asparagine agars. Degrades agar, casein, DNA, elastin, gelatin, keratin, starch, testosterone, tyrosine, Tweens 20 and 80, xanthine and xylan, but not carboxymethylcellulose, chitin, guanine, hypoxanthine or pectin. Aesculin, allantoin and arbutin are hydrolysed but not urea. Produces arginine dihydrolase, catalase, citrate lyase, 
-galactosidase, -glucosidase and oxidase, but not acetoin, hydrogen sulphide, lysine decarboxylase, ornithine decarboxylase or phosphatase. (+)-L-Arabinose, (+)-D-fructose, (+)-D-galactose, (+)-D-maltose, (+)-D-glucose, (+)-D-glycerol, (+)-D-mannitol, (+)-D-mannose, (+)-D-sucrose, (+)-D-trehalose and (+)-D-xylose are used as sole carbon sources for energy and growth, but not dulcitol, inulin, (+)-D-lactose, (+)-D-melezitose, (+)-D-melibiose, myo-inositol, (+)-D-raffinose, (+)-D-rhamnose, (+)-D-ribose, (+)-D-sorbitol or (+)-D-sorbose (all at 1 %, w/v). Sodium citrate is used as a sole carbon source, but not sodium oxalate (both at 0·1 %, w/v). Grows well on alanine, asparagine, arginine, glutamic acid, histidine, tyrosine and tryptophan as sole nitrogen sources. Nitrates and ammonia salts are not utilized. Resistant (µg per filter paper disc) to chloramphenicol (25), cephaloridine (5), cephalexin (30), cefoxin (30), gentamicin sulphate (2), kanamycin sulphate (30), tobramycin sulphate (10) and penicillin (5 U), but sensitive to cephazolin (30) and gentamicin sulphate (10). Grows in the presence of bile salts (0·02 % w/v), crystal violet (0·00002 %, w/v), potassium tellurite (0·02 %, w/v), sodium azide (0·01 %, w/v) and thallous acetate (0·001 %, w/v), but is sensitive to bile salts (0·5 %, w/v), brilliant green (0·0005 %, w/v), crystal violet (0·00005 %, w/v), lysozyme (0·0025 %, w/v), sodium azide (0·02 %, w/v), sodium chloride (3 %, w/v), tetrazolium chloride (0·002 %, w/v), thallous acetate (0·005 %, w/v) and novobiocin (50 µg ml–1). Produces major proportions of MK-9, MK-9(H2) and MK-9(H4) (points of saturation: III and VIII) and minor proportions of MK-9(H6) (points of saturation: II, III, VIII). Major fatty acids are iso-15 : 0 (12·5 %), 15 : 0 (5·7 %), iso-16 : 0 (13·8 %), iso-17 : 0 (18·7 %), anteiso-17 : 0 (12·4 %) and 10-methyl-branched 17 : 0 (6·9 %) components; minor peaks include anteiso-15 : 0 (4·3 %), cis-16 : 1 (2·8 %), iso-2-OH 15 : 0 (1·4 %), 16 : 0 (3·4 %), 10-methyl 16 : 0 (2·4 %), iso-2-OH 16 : 0 (2·5 %), 17 : 0 (2·0 %), iso-2-OH 17 : 0 (3·0 %) and 18 : 0 (2·5 %).
The type strain, DSM 43186T (=NRRL B-24348T), was isolated from soil from the Pamir mountains.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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