International Journal of Systematic and Evolutionary Microbiology (2001), 51, 319–325 Printed in Great Britain Saccharopolyspora flava sp. nov. and Saccharopolyspora thermophila sp. nov., novel actinomycetes from soil Zhitang Lu,1† Zhiheng Liu,1 Liming Wang,1 Yamei Zhang,1 Weihong Qi1 and Michael Goodfellow2 Author for correspondence: Zhiheng Liu. Tel: j86 10 6255 3628. Fax: j86 10 6256 0912. e-mail: zhliu!sun.im.ac.cn 1 Institute of Microbiology, The generic position of two aerobic, Gram-positive, non-acid–alcohol-fast Chinese Academy of actinomycetes was established following the isolation of their PCR-amplified Sciences, Beijing 100080, People’s Republic of China 16S rRNA genes and alignment of the resultant sequences with the corresponding sequences from representatives of the families 2 Department of Agricultural and Actinosynnemataceae and Pseudonocardiaceae. The assignment of the Environmental Science, organisms to the genus Saccharopolyspora was strongly supported by University of Newcastle, chemotaxonomic and morphological data. The strains were distinguished both Newcastle upon Tyne NE1 7RU, UK from one another and from representatives of validly described Saccharopolyspora species on the basis of a number of phenotypic properties. It is proposed that the organisms, strains 07T (l AS4.1520T l IFO 16345T l JCM 10665T) and 216T (l AS4.1511T l IFO 16346T l JCM 10664T), be classified in the genus Saccharopolyspora as Saccharopolyspora flava sp. nov. and Saccharopolyspora thermophila sp. nov., respectively. Keywords: Saccharopolyspora flava sp. nov., Saccharopolyspora thermophila sp. nov., polyphasic taxonomy INTRODUCTION these taxa were excluded from the family by Warwick et al. (1994), who considered that they might form a The application of the polyphasic taxonomic approach ‘sister’ group to the Pseudonocardiaceae clade. This to actinomycete systematics led to the circumscription now seems to be the case, as Labeda & Kroppenstedt of the family Pseudonocardiaceae Embley et al. 1988 (2000) have proposed that members of the genera for mycolateless actinomycetes characterized by the Actinokineospora, Actinosynnema, Lentzea and presence of meso-diaminopimelic acid (meso-A#pm), Saccharothrix be classified in a new taxon, i.e. the arabinose and galactose in their peptidoglycan (wall family Actinosynnemataceae. Members of both clades chemotype IV sensu Lechevalier & Lechevalier, 1970). can be distinguished from one another using chemical Members of this family are currently assigned to the and morphological markers (Kim & Goodfellow, genera Actinobispora, Amycolatopsis, Kibdelo- 1999; Labeda & Kroppenstedt, 2000). sporangium, Prauserella, Pseudonocardia (including Amycolata), Saccharomonospora, Saccharopolyspora The genus Saccharopolyspora contains eight validly and Thermocrispum (Kim & Goodfellow, 1999; Xu et described species: Saccharopolyspora erythraea al., 1999; Labeda & Kroppenstedt, 2000). Labeda 1987; Saccharopolyspora gregorii Goodfellow et al. 1989; Saccharopolyspora hordei Goodfellow et al. The family Pseudonocardiaceae recognized by 1989; Saccharopolyspora rectivirgula (Krasilhnikov Stackebrandt et al. (1997) also included the genera and Agre 1964) Korn-Wendisch et al. 1989; Actinosynnema, Saccharothrix and Streptoalloteichus; Saccharopolyspora spinosa Mertz and Yao 1990; Sac- charopolyspora spinosporotrichia Zhou et al. 1998; ................................................................................................................................................. Saccharopolyspora taberi (Labeda 1987) Korn- † Present address: Department of Biology, HeBei University, Baoding 071002, People’s Republic of China. Wendisch et al. 1989; and Saccharopolyspora hirsuta The GenBank accession numbers for the 16S rDNA sequences of strains 07T Lacey and Goodfellow 1975, the type species. Repre- (l AS4.1520T) and 216T (l AS4.1511T) are AF154128 and AF127526, sentatives of these species form a distinct phyletic line respectively. within the evolutionary radiation encompassed by the 01453 # 2001 IUMS 319 Z. Lu and others family Pseudonocardiaceae and can be distinguished fresh cultures and examined for growth after 10 d at either using a combination of biochemical, morphological 28 mCor50mC. The antibiotic-sensitivity tests were carried and physiological properties (Zhou et al., 1998). out by placing impregnated filter-paper discs (Goodfellow & Genus-specific primers are available for the PCR Orchard, 1974) over Sauton’s agar and incubating the identification of Pseudonocardia and Saccharopoly- samples for 7–10 d at the incubation temperatures cited above. The two organisms were also examined for their spora strains (Moro! n et al., 1999). T ability to grow at either 20, 25, 37 and 45 mC (strain 07 )or T Two actinomycetes isolated from soil samples collected at 40, 45, 50, 55 and 60 mC (strain 216 ). in China were provisionally assigned to the genus Chemotaxonomy. Established procedures were used to Saccharopolyspora, using chemical and morphologi- determine the diagnostic isomers of A#pm, the predominant cal properties. In the present investigation, these whole-organism sugars and the major polar lipids organisms, namely strains 07T and 216T, were (Lechevalier & Lechevalier, 1980; Hasegawa et al., 1983). examined for a range of genotypic and phenotypic Isoprenoid quinones were extracted and purified according properties and found to form distinct new centres of to Collins (1985) and were analysed by HPLC (Wu et al., taxonomic variation within the genus Saccharo- 1989), with Streptomyces griseus as the control. The acid polyspora; the names Saccharopolyspora flava and methanolysis procedure was used to screen for mycolic acids (Minnikin et al., 1980). The DNA base ratios of the two Saccharopolyspora thermophila are proposed for these strains were determined using the thermal denaturation strains, respectively. method of Marmur & Doty (1962), with Escherichia coli AS1.365 as the control. METHODS 16S rDNA sequencing. Genomic DNA was isolated from the T strains by following the procedure described by Kim et al. Organisms and cultural conditions. Strain 07 was isolated (1995). PCR amplification of the 16S rDNA preparations on an oatmeal agar plate (Shirling & Gottlieb, 1966) seeded was carried out as described previously (Kim et al., 1996). with a soil suspension; the latter was prepared from a garden The resultant PCR products were purified by using the soil collected from Xishan Mountain, Beijing, and incubated T Wizard PCR purification system (Promega) according to the at 28 mC for 5 d. Similarly, strain 216 was isolated on procedure provided by the manufacturer. The purified selective medium 2 (Lu et al., 2000) inoculated with a soil products were directly sequenced using a Taq DyeDeoxy suspension prepared from a grassland soil sample taken Terminator Cycle Sequencing kit (Applied Biosystems) and from Nanning, Guangxi Province, Southern China, and the universal primers 27f (5h-GAGAGTTTGATCCTGGC- incubated at 45 mC for 7 d; the selective medium consisted of TCAG-3h), 704f (5h-GTACGGTGAARTGCGCAGA-3h) Stevenson’s basal medium (Stevenson, 1967) supplemented " 765r (5h-CTGTTCGCTCCCCACGCTTTC-3h) and 1495r µ − with melezitose (1%, w\v) and neomycin (4 gml ). The (5h-CTACGGCTACCTTGTTACGA-3h). Sequence gel strains were maintained on glucose yeast-extract agar electrophoresis was carried out and nucleotide sequences (GYEA) (Gordon & Mihm, 1962) at room temperature and automatically obtained by using an Applied Biosystems as glycerol suspensions (20%, v\v) at k20 mC. Biomass for DNA sequencer (model 373A) and software provided by the the chemotaxonomic and molecular systematic studies was manufacturer. prepared by growing the strains in shake flasks of modified Sauton’s broth (Mordarska et al., 1972) at either 28 mC Analysis of sequence data. The 16S rDNA sequences of the (strain 07T)or50mC (strain 216T) for 5 d. At maximum strains were aligned manually using the program growth, the broth cultures were checked for purity, killed (version 1.64b, Thompson et al., 1997) against correspond- with formaldehyde (1%, v\v), washed three times with ing nucleotide sequences of representatives of the families distilled water and freeze-dried. Actinosynnemataceae (Labeda & Kroppenstedt, 2000) and Pseudonocardiaceae (Kim & Goodfellow, 1999) retrieved Cultural and morphological properties. The undisturbed from the DDBJ (Thompson et al., 1997) and GenBank arrangement of hyphae, notably aerial hyphae and spore- (Benson et al., 1997) databases. Evolutionary trees were chain morphology, was observed on modified Sauton’s agar inferred by using the neighbour-joining (Saitou & Nei, 1987) (Mordarska et al., 1972) and ISP medium 2 (Shirling & and least-squares (Fitch & Margoliash, 1967) treeing Gottlieb, 1966) after 7 d, using the coverslip technique of algorithms from the package (Felsenstein, 1993). Kawato & Shinobu (1959). Growth on the coverslips was Evolutionary distance matrices were generated as described fixed and examined according to the procedure described by by Kimura (1980). Tree topologies were evaluated by Zhou et al. (1998). Additional morphological properties bootstrap analysis based on 1000 resamplings of the were observed by examining gold-coated, dehydrated neighbour-joining dataset using the and specimens of the organisms, using a Hitachi S-570 scanning options from the suite of programs (Felsenstein, electron microscope. 1993). Biochemical and physiological properties. The strains were examined for a range of phenotypic properties following