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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
2 BCCM/LMG Bacteria Collection, Department of Biochemistry, Physiology and Microbiology, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
Ying Huang
huangy{at}im.ac.cn
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-region of this molecule showed that it formed a distinct (but closely associated) line with Streptomyces griseoaurantiacus DSM 40430T in Streptomyces trees. However, the DNA–DNA relatedness between the two strains was only 48·8 %. A number of phenotypic properties also readily distinguished the isolate from S. griseoaurantiacus and related Streptomyces species with validly published names. It is proposed, therefore, that this organism be classified as a novel species of the genus Streptomyces, for which the name Streptomyces jietaisiensis sp. nov. is proposed. The type strain is FXJ46T (=AS 4.1859T=JCM 12279T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains FXJ46T and S. griseoaurantiacus DSM 40430T are AY314783 and AY450561, respectively.
A micrograph showing aerial hyphae with rectiflexibiles spore chains and a neighbour-joining tree based on the 120 nt -region are available as supplementary material in IJSEM Online.
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; S. B. Kim et al., 1998, 2004; Al-Tai et al., 1999; B. Kim et al., 2000; Li et al., 2002; Saintpierre et al., 2003). However, due to the large number of species, the complete 16S rRNA gene sequences have not been determined for many type strains of this genus; incomplete sequence data may result in misclassification (Kataoka et al., 1997; Anderson & Wellington, 2001). In this study, therefore, the partial sequence (120 nucleotides ) covering the variable -region of the 16S rRNA gene was also analysed phylogenetically to classify strain FXJ46T. While phylogenetic data showed that it was closely related to S. griseoaurantiacus DSM 40430T, polyphasic studies based on a judicious combination of genotypic and phenotypic features revealed a novel species of Streptomyces.
Strain FXJ46T was isolated on a yeast extract-starch agar (Emerson, 1958) plate supplemented with 50 µg cycloheximide ml–1, which had been seeded with a soil sample suspension and incubated for 14 days at 28 °C. The soil sample was collected from a cypress forest, at Jietaisi, Beijing, China. The isolate was maintained on yeast extract-starch slopes at 4 °C and as glycerol suspensions (20 %, v/v) at –20 °C. Biomass for the chemotaxonomic and molecular systematic studies was prepared as described previously (Li et al., 2002).
The morphological characteristics of strain FXJ46T were examined by light and scanning electron microscopy of 14-day cultures grown on oatmeal agar and inorganic salts-starch agar (ISP medium 4). The coverslip technique (Zhou et al., 1998; Kawato & Shinobu, 1959) was used to observe the hyphae and spore-chain characters by light microscope. Spore-chain morphology and spore-surface ornamentation were studied by examining gold-coated dehydrated specimens with a model FEI QUANTA electron microscope. The cultural features were observed on a number of standard media (Table 1) after 14 days incubation at 28 °C. The test strain was examined for a range of biochemical and physiological properties as described by Williams et al. (1983) and Kämpfer et al. (1991).
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and Lechevalier & Lechevalier (1980). Menaquinones were extracted and purified according to Collins (1985) and analysed by HPLC. Polar lipids were examined and identified using the method of Minnikin et al. (1984). The fatty acids were extracted, methylated and analysed by GC using the standard Sherlock MIDI (Microbial Identification) system (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The G+C content of the DNA of the test strain was determined using the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli AS 1.365 as a control.
Genomic DNA preparation and PCR amplification of the 16S rRNA gene of strain FXJ46T were carried out using the procedure of Chun & Goodfellow (1995). The PCR products were sequenced using the method of Huang et al. (2001). CLUSTAL X version 1.8 (Thompson et al., 1997) was used for multiple alignment with available almost-complete sequences of type strains of the family Streptomycetaceae and then with corresponding sequences of representative Streptomyces species; in each case, the reference sequences were retrieved from the DDBJ/EMBL/GenBank databases. The least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) algorithms from the PHYLIP package version 3.5c (Felsenstein, 1993) were used to infer the phylogenetic trees. Evolutionary distance matrices were generated as described by Kimura (1980). Tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. The partial sequence covering the variable -region (120 nt, positions 158–277) of the 16S rRNA gene sequence of strain FXJ46T was also compared with corresponding nucleotide sequences of 485 Streptomyces type strains retrieved from GenBank. A phylogenetic tree based on these partial sequences was constructed using the neighbour-joining algorithm (Saitou & Nei, 1987).
Levels of DNA–DNA relatedness between strain FXJ46T and S. griseoaurantiacus DSM 40430T were determined according to the fluorometric micro-well method (Ezaki et al., 1989), with the modification that detection of the DNA hybridization rate was tested by the addition of streptavidin-conjugate alkaline phosphatase acting on the substrate 4-methylumbelliferyl phosphate (Christensen et al., 2000). Fluorescence intensities were measured using a Fluostar Optima microplate reader (BMG LABTECH) at a wavelength of 360 nm for excitation and 460 nm for emission. The DNA hybridization rate was calculated from quadruplicated hybridization experiments and expressed as a mean of the corresponding reciprocal values.
The organism exhibited a range of chemotaxonomic and phenotypic properties typical of members of the genus Streptomyces. It formed an extensively branched substrate mycelium, aerial hyphae which carried smooth-surfaced spores in rectiflexibiles spore chains (see Supplementary Fig. S1 available in IJSEM Online) and a greyish aerial spore mass on several standard media (Table 1
). It contained LL-diaminopimelic acid in whole-organism hydrolysates, hexa-, octa- and a minor amount of tetrahydrogenated menaquinones with nine isoprene units [MK-9 (H6, H8 and H4)] as isoprenologues, and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides as major polar lipids (phospholipid type II sensu Lechevalier et al., 1977). The fatty acid profile was composed mainly of saturated straight-chain and iso- and anteiso-branched-chain fatty acids (fatty acid type 2c sensu Kroppenstedt, 1985).
An almost complete 16S rRNA gene sequence (1426 nt) was determined for the organism. Primary sequence analysis with sequences of representatives of the family Streptomycetaceae confirmed that the unknown isolate was closely related to species of the genus Streptomyces. The highest 16S rRNA gene sequence similarity value was found with Streptomyces nogalater JCM 4799T (97·5 %). However, in the phylogenetic tree based on the 120 nt -region, the strain showed a very close affinity with S. griseoaurantiacus, supported by a 97 % bootstrap level, and was loosely related to other species (see Supplementary Fig. S2). The almost-complete 16S rRNA gene sequence (1458 nt) of S. griseoaurantiacus DSM 40430T was therefore also determined in this study, and added to the 16S rRNA gene analysis. It is clear from Fig. 1 that strain FXJ46T consistently formed a distinct phyletic line with S. griseoaurantiacus DSM 40430T, suported by all four tree-making algorithms and by high bootstrap values. A 100 % bootstrap value with the neighbour-joining method was indicated. The divergence of the 16S rRNA gene sequences between the two strains was 0·2 % (3 nt differences at 1421 sites). The isolate also showed moderately low sequence divergence from other Streptomyces type strains, namely S. nogalater JCM 4799T (2·2 %), Streptomyces ambofaciens ATCC 23877T (2·2 %), Streptomyces rutgersensis subsp. rutgersensis DSM 40077T (2·3 %), Streptomyces intermedius DSM 40372T (2·3 %), Streptomyces paradoxus DSM 43350T (2·3 %), Streptomyces gougerotii DSM 40324T (2·4 %), Streptomyces violaceolatus DSM 40438T (2·4 %), Streptomyces collinus DSM 40129T (2·5 %) and Streptomyces eurythermus DSM 40014T (2·6 %), respectively. Therefore, well-selected phenotypic traits and DNA–DNA pairing data are needed (Kim et al., 1998; Labeda, 1988) to clarify the finer relationships between the isolate and the phylogenetically close species.
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), thus suggesting that the test strain should be considered as a separate species. Furthermore, the strain was distinguished from S. griseoaurantiacus DSM 40430T by cultural characteristics on a number of standard media (Table 1), by spore-chain characters and by a set of physiological features (Table 2). The test strain can also be distinguished from all of the other phylogenetically close relatives using a combination of phenotypic properties (Table 2).
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Description of Streptomyces jietaisiensis sp. nov.
Streptomyces jietaisiensis (jie.tai.si.en'sis. N.L. masc. adj. jietaisiensis pertaining to Jietaisi, a place in a suburb of Beijing, where the type strain was isolated).
The organism is aerobic, Gram-positive and mesophilic. The characteristics of aerial and substrate mycelium on various solid media is given in Table 1. Spore chains with 10 to 20 cylindrical spores are rectiflexibiles. The spore surface is smooth. Diffusible pigments are not produced, nor are melanin pigments formed on peptone-yeast extract-iron or tyrosine agars. Growth occurs between 10 and 40 °C, and between pH 5·0 and 10·0, but not at pH 4·0 or 11·0 or in the presence of streptomycin (10 µg ml–1) or novobiocin (5 µg ml–1). Adonitol, D-cellobiose, dextrin, D-galactose, D-glucose, inulin, glycogen, D-maltose, D-mannitol, D-mannose, D-melezitose, salicin, D-trehalose, D-xylose, (all at 1 %, w/v), L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-glutamic acid, L-histidine, L-isoleucine, L-phenylalanine, sodium oxalate, sodium pyruvate, L-threonine and L-valine (all at 0·1 %, w/v) are used as sole carbon sources for energy and growth, but not glycerol, glycine, xylitol (all at 1 %, w/v) or DL-aminobutyric acid (at 0·1 %, w/v). L-alanine, L-arginine, L-aspartic acid, L-glutamic acid and L-phenylalanine (all at 0·1 %, w/v) are metabolized as sole carbon and nitrogen sources, but not L-isoleucine (at 0·1 %, w/v). Cell wall type I, phospholipid type II and menaquinone MK-9 (H6, H8 and H4). The fatty acid profile is composed of ai-C15 : 0 (35·7 %), ai-C17 : 0 (18·9 %), i-C16 : 0 (14·8 %), ai-C17 : 1
9c (8·1 %), C16 : 0 (6·2 %), i-C16 : 1 (4·5 %), i-C15 : 0 (4·3 %), C16 : 17c (2·33 %), i-C17 : 19c (1·8 %), i-C17 : 0 (1·7 %) and i-C14 : 0 (1·7 %). The G+C content of the DNA is 72·3 mol%. Additional characteristics are listed in Table 2.
The type strain is FXJ46T (=AS 4.1859T=JCM 12279T), isolated from cypress forest soil collected at Jietaisi, Beijing, China.
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ACKNOWLEDGEMENTS |
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