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Amycolatopsis Rubida Sp. Nov., a New Amycolatopsis Species from Soil

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Amycolatopsis Rubida Sp. Nov., a New Amycolatopsis Species from Soil International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1093–1097 Printed in Great Britain Amycolatopsis rubida sp. nov., a new NOTE Amycolatopsis species from soil 1 Institute of Microbiology, Ying Huang,1 Weihong Qi,1 Zhitang Lu,1† Zhiheng Liu1 Chinese Academy of 2 Sciences, Beijing 100080, and Michael Goodfellow People’s Republic of China 2 Department of Author for correspondence: Zhiheng Liu. Tel: j86 10 6255 3628 Fax: j86 10 6256 0912. Agricultural and e-mail: zhliu!sun.im.ac.cn Environmental Science, University of Newcastle, Newcastle upon Tyne T NE1 7RU, UK The taxonomic position of a soil isolate, strain 13.4 , was established using a polyphasic approach. The organism was found to have chemical and morphological properties consistent with its classification in the genus Amycolatopsis. Phylogenetic analysis of the strain based on its 16S rDNA sequence showed that it forms a distinct phyletic line within members of the genus Amycolatopsis. The organism was also readily distinguished from the type strains of all validly described Amycolatopsis species by its phenotypic features. The name Amycolatopsis rubida sp. nov. is proposed for this new species. The type strain is strain 13.4T (l AS 4.1541T l JCM 10871T). Keywords: Amycolatopsis rubida sp. nov., polyphasic taxonomy, 16S rDNA sequencing The genus Amycolatopsis was proposed by Lechevalier passed by the family Pseudonocardiaceae (Embley et al. (1986) to accommodate nocardioform actino- et al., 1988; Warwick et al., 1994). In the present mycetes having type IV cell wall composition, type PII investigation, a soil isolate, strain 13.4T, was subject to phospholipid pattern, and lacking mycolic acid. This a polyphasic study designed to establish its taxonomic taxon has been classified in the family Pseudono- position. Genotypic and phenotypic data show that cardiaceae since the application of the polyphasic strain 13.4T represents a new species of Amycolatopsis, taxonomic approach to actinomycete systematics for which we propose the name Amycolatopsis rubida. (Embley et al., 1988; Warwick et al., 1994; A soil sample was collected from a conifer forest in Stackebrandt et al., 1997; Kim & Goodfellow, 1999). T Meanwhile, another two genera, Actinosynnema and Guangxi Province, China. Strain 13.4 was isolated on Saccharothrix, have been excluded from the family a glucose-asparagine agar (GAA; glucose, 10 g; - Pseudonocardiaceae (Warwick et al., 1994), and been asparagine, 0n5g;K#HPO%,0n5 g; distilled water, 1 l; classified in a new family, Actinosynnemataceae,by pH 7n2) plate, which had been seeded with a soil Labeda & Kroppenstedt (2000). suspension and incubated at 28 mC for 14 d. The isolate was maintained on modified Sauton’s agar (Mor- The genus Amycolatopsis was established with four darska et al., 1972) slants at 4 mC and as a glycerol species: Amycolatopsis orientalis, Amycolatopsis suspension (20%, v\v) at k20 mC. Biomass for studies mediterranei, Amycolatopsis rugosa and Amycolatopsis was prepared according to Chun et al. (1999). The sulphurea. Since then, another seven species and one cultural and morphological properties were examined subspecies have been validly published (Henssen et al., by using standard procedures and procedures of 1987; De Boer et al., 1990; Mertz & Yao, 1993; Williams et al. (1983) after growth on GAA and Labeda, 1995; Goodfellow et al., 1997, 2001; Chun et glucose-yeast extract agar (GYEA; Gordon & Mihm, al., 1999), and Amycolatopsis rugosa has been re- 1962) for 7–10 d at 28 mC. Phenotypic tests were carried classified as Prauserella rugosa (Kim & Goodfellow, out following the procedures of Goodfellow et al. 1999). Representatives of the genus form a distinct (1997) and Gordon et al. (1974). The ability of the test phyletic line within the evolutionary radiation encom- strain to grow in the presence of five antibiotics was ................................................................................................................................................. examined by placing 6 mm sensitivity discs (Difco) † Present address: Department of Biology, HeBei University, Baoding centrally in plates of Sauton’s agar seeded with a 071002, People’s Republic of China. loopful of 7 d Sauton’s broth culture and inhibition The GenBank accession number for the 16S rDNA sequence of strain 13.4T zones observed after 3–5 d incubation at 28 mC were (l AS 4.1541T l JCM 10871T) is AF222022. scored negative. Chemotaxonomy characters were 01700 # 2001 IUMS 1093 Y. Huang and others ‘’ ..................................................................................................... Fig. 1. Neighbour-joining tree (Saitou & Nei, 1987) based on almost complete 16S rDNA sequences showing relationships between Amycolatopsis rubida sp. nov. and repre- sentatives of the family Actinosynnema- taceae, Pseudonocardiaceae and related taxa. Asterisks indicate branches that were also recovered using the least-squares and maximum-likelihood methods. The numbers at the nodes indicate the levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled data sets; only values over 50% are given. Scale bar, 0n01 substitutions per nucleotide position. determined as described previously (Minnikin et al., (Felsenstein, 1993) was used for all phylogenetic 1980, 1984; Lechevalier & Lechevalier, 1980; Hase- analyses. gawa et al., 1983; Collins, 1985; Wu et al., 1989). An almost complete 16S rDNA sequence was de- T Genomic DNA was extracted employing the method termined for strain 13.4 (1402 nucleotides). Com- of Chun & Goodfellow (1995). The GjC content of parison of this sequence with those of representative the DNA was determined using the thermal reference strains of the families Actinosynnemataceae denaturation (Tm) method (Marmur & Doty, 1962) (Labeda & Kroppenstedt, 2000) and Pseudono- with Escherichia coli AS1.365 as the control. PCR of cardiaceae shows that the organism belongs to the the 16S rDNA was performed as described by Chun & genus Amycolatopsis (Fig. 1). The nucleotide sequence also serves to distinguish it from other Amycolatopsis Goodfellow (1995). The amplified product was directly T sequenced using a Taq DyeDeoxy Terminator Cycle in that strain 13.4 forms a monophyletic clade in the Sequencing kit (Applied Biosystems) and universal phylogenetic tree, supported by the 80% bootstrap primers named 27f (5h-GAGAGTTTGATCCTGGC- value recorded using the neighbour-joining method. The 16S rDNA sequence similarity between strain TCAG-3h), 704f (5h-GTACGGTGAARTGCGCA- T GA-3h), 765r (5h-CTGTTCGCTCCCCACGCTTTC- 13.4 and its nearest neighbour, Amycolatopsis 3h) and 1495r (5h-CTACGGCTACCTTGTTACGA- orientalis,is97n18%; this value corresponds to 39 3h). The nucleotide sequence was automatically differences out of 1383 nucleotide positions compared. obtained by using an Applied Biosystems 373A DNA Similarity values with the type strains of the remaining sequencer according to the manufacturer’s protocols. validly published Amycolatopsis species range from The resultant 16S rDNA sequence was aligned manu- 93n76% to 96n91% (Table 1). ally using the program (version 1.64b; The chemotaxonomic data of the test strain are Thompson et al., 1997) against corresponding consistent with its assignment to the genus Amycola- sequences retrieved from the GenBank database. topsis (Lechevalier et al., 1986). Strain 13.4T contains Evolutionary trees were inferred by using three tree- meso-diaminopimelic acid as the wall diamino acid, making algorithms, namely, neighbour-joining (Saitou arabinose and galactose as major wall sugars, and & Nei, 1987), least-squares (Fitch & Margoliash, 1967) hexahydrogenated menaquinones with nine isoprene and maximum-likelihood (Felsenstein, 1981). Evol- units as the predominant isoprenoid quinone. It also utionary distance matrices were generated as described contains phosphatidylethanolamine (taxonomically by Jukes & Cantor (1969). The resultant unrooted tree significant phospholipids), diphosphatidylglycerol, topologies were evaluated by bootstrap analyses phosphatidylinositol mannosides and phosphatidyl- (Felsenstein, 1985) of the neighbour-joining method methylethanolamine as diagnostic polar lipids but it based on 1000 resamplings. The package lacks mycolic acids; the GjC content of the DNA is 1094 International Journal of Systematic and Evolutionary Microbiology 51 Amycolatopsis rubida sp. nov. Table 1. 16S rDNA similarity values between Table 2. Phenotypic characters of Amycolatopsis rubida Amycolatopsis rubida sp. nov. and representatives of the sp. nov. genus Amycolatopsis ................................................................................................................................................. j, Positive or present; , weak positive; k, negative or Amycolatopsis species Similarity No. nucleotide absent. Source of data on Amycolatopsis species: Chun et al. to differences/total (1999). A. rubida no. nucleotides (%) compared Character Result T Acid production from: A. alba DSM 44262 96n91 43\1383 T Adonitol A. azurea JCM 3275 96n50 44\1257 j T ( )Arabinose A. coloradensis DSM 44225 96n86 43\1369 j j T ( )Cellobiose A. fastidiosa IFO 14105 93n76 80\1282 j j T Dextrin A. japonica DSM 44213 96n41 50\1393 k T meso-Erythritol A. mediterranei JCM 4789 96n43 48\1345 j T ( )Fructose A. methanolica IFO 15065 94n63 71\1322 j j ( )Galactose A. orientalis subsp. orientalis 97n18 39\1383 j j JCM 4235T meso-Inositol j T ( )Lactose A. sulphurea DSM 46092 96n71 46\1398 j k T ( )Maltose A. thermoflava IFO
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