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1 Leibniz-Institut für Meereswissenschaften, IFM-GEOMAR, Düsternbrooker Weg 20, D-24105 Kiel, Germany
2 Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People's Republic of China
Correspondence
Johannes F. Imhoff
jimhoff{at}ifm-geomar.de
Li-Hua Xu
lihxu{at}ynu.edu.cn
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ABSTRACT |
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and emended by Rainey et al. in Stackebrandt et al., 1997). Kim et al. (2003) proposed the emendation of the description of the family Streptomycetaceae, which comprises the genera Streptomyces, Kitasatospora (Zhang et al., 1997) and Streptacidiphilus (Kim et al., 2003). Members of the family Streptomycetaceae have attracted great attention because of their production of various natural products of considerable commercial value (Berdy, 2005). In the course of our research on new sources of actinomycetes, we obtained morphological, physiological, chemical and phylogenetic data for isolate YIM 47672T. Strain YIM 47672T was shown to represent a novel species of the genus Streptomyces.
Strain YIM 47672T was isolated, using the dilution plating method, from a soil sample collected in a forest (comprising evergreen broadleaved trees) on Wuzhi Mountain in Hainan Province, China. Selective isolation was achieved using trehalose-proline agar (Jiang et al., 2006) and incubation at 28 °C for 35 days. Strain YIM 47672T was maintained on YIM 38 medium [10 g malt extract, 4 g yeast extract, 4 g glucose, vitamin mixture (0.5 mg each of thiamine-HCl, riboflavin, niacin, pyridoxine-HCl, inositol, calcium pantothenate and p-aminobenzoic acid and 0.25 mg biotin), 20 g agar; pH 7.2] (Hayakawa & Nonomura, 1987) at 4 °C and preserved in 20 % (v/v) glycerol at –20 °C. Biomass for chemical and molecular systematic studies was obtained by growing the strain in shake flasks (at 180 r.p.m.) using YIM 38 broth at 28 °C for 4–7 days. Cultural characteristics were determined, after 2 weeks incubation at 28 °C, with the methods used in the International Streptomyces Project (Shirling & Gottlieb, 1966). Czapek's agar and nutrient agar were prepared as described by Dong & Cai (2001). Morphological properties were examined using a light microscope (BH-2; Olympus) and a scanning electron microscope (XL30 ESEM-TMP; Philips). Colour determination was performed using colour chips from the ISCC–NBS colour charts (standard samples, no. 2106) (Kelly, 1964).
Analyses of the amino acids and sugars in whole-cell hydrolysates were performed using the procedures described by Staneck & Roberts (1974) and analysis of the phospholipids was carried out according to Lechevalier et al. (1981). Menaquinones were determined using the procedures of Collins et al. (1977). Biomass for a quantitative fatty acid analysis was prepared by scraping cell mass from plates containing trypticase soy broth (BBL; 3 %, w/v) and Bacto agar (Difco; 1.5 %, w/v), that had been incubated for 5 days at 28 °C. The fatty acids were extracted, methylated and analysed by using the standard MIDI (Microbial Identification) system (Kämpfer & Kroppenstedt, 1996; Sasser, 1990). Chromosomal DNA of strain YIM 47672T was extracted as described by Marmur (1961). The DNA G+C content was determined by HPLC (Tamaoka & Komagata, 1984) with an Agilent 1100 LC system (IRIS Technologies).
The cell wall of strain YIM 47672T contained LL-diaminopimelic acid, traces of meso-diaminopimelic acid and glycine. Whole-cell hydrolysates consisted of galactose and xylose. The analysis of the polar lipids revealed the presence of phosphatidylethanolamine and diphosphatidylglycerol, indicating that the phospholipid was of type II sensu Lechevalier et al. (1977). The major components of the fatty acid profile included iso-branched hexadecanoic acid (iso-C16 : 0), hexadecanoic acid (C16 : 0) and anteiso-branched heptadecanoic acid (C17 : 0). These characteristics are consistent with membership of the family Streptomycetaceae. According to Kim et al. (2003), the major menaquinones of the genus Streptomyces are MK-9(H6) and MK-9(H8). Strain YIM 47672T also contained MK-9(H6) (14.0 %) and MK-9(H8) (13.6 %), but the predominant menaquinones were MK-9(H4) (45.4 %) and MK-10(H0) (27.0 %). The G+C content of genomic DNA from strain YIM 47672T was 73.4 mol%.
Strain YIM 47672T grew well on most of the media tested (Table 1). Aerial mycelia developed well, were long and branched and ranged in colour from white to pink–white and pink–grey. Vegetative mycelia developed well, and ranged in colour from a pale to a deep orange–yellow. Light brown to yellowish orange soluble pigments were produced. Spore chains were spiral or retinaculiaperti (looped at the top) in nature (Fig. 1). Spores were elliptical or shaped like short rods (0.4–0.6x0.7–1.0 µm. The spore surfaces were smooth.
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; Williams et al. 1983). In addition, acid production from carbohydrates was tested using the media and methods described by Gordon et al. (1974). The utilization of carbon and nitrogen sources was determined by using the methods of Gordon & Mihm (1962) and Tsukamura (1966). Strain YIM 47672T was negative in tests for gelatin liquefaction, milk coagulation and peptonization, arginase activity, phenylalanine deaminase activity, DNase activity and melanin production. The strain was positive in tests for starch hydrolysis, arginine decarboxylase activity, nitrate reduction, gas production from nitrate, growth on cellulose and H2S production. Growth occurred at pH 6.0–9.0 (optimum, pH 7.0) and with 0–10 % NaCl (optimum, 0 % NaCl). Glucose, cellobiose, starch, aesculin, trehalose, 
-galactoside and urea were utilized; acids were not produced from these carbon sources. A broad range of additional carbon sources, as well as three nitrogen sources, were tested and shown not to be utilized.
The almost-complete 16S rRNA gene sequence of strain YIM 47672T was aligned with sequences of related type strains, as well as with sequences of representative Streptomyces species (obtained from the GenBank/EMBL/DDBJ and RDPII databases). The CLUSTAL_X program (Thompson et al., 1997) was used for multiple alignments (corrected manually). In addition, a second alignment method, ARB, was used. Both alignment methods revealed the same tree topology (data not shown). The phylogenetic analysis was performed with the ARB alignment and the neighbour-joining method (Saitou & Nei, 1987) using the Kimura two-parameter model (Kimura, 1980) using the MEGA3.1 software package (Kumar et al., 2004). The PHYML software package (Guindon et al., 2005) was used to construct the maximum-likelihood tree, using the general time reversible (GTR) model. The topologies of the trees were evaluated by using bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings (neighbour-joining) and 500 resamplings (maximum-likelihood), respectively. NJPLOT software (Perrière & Gouy, 1996) was used to display the phylogenetic trees.
The phylogenetic analysis demonstrated that strain YIM 47672T belongs to the genus Streptomyces. The rooted phylogenetic tree for YIM 47672T and related and representative type strains of the genus Streptomyces indicated that this strain formed a distinct clade with an undescribed isolate, Streptomyces sp. D44 (Q. Gu, Y. Huang & Z. Liu, unpublished; GenBank/EMBL/DDBJ accession no. DQ460471), derived from a traditional Chinese medicinal plant. Streptomyces sp. D44 was found to be the organism most closely related to strain YIM 47672T (showing 99.9 % gene sequence similarity). The separate branching of this clade was supported by the neighbour-joining tree (Fig. 2) and by the maximum-likelihood tree (data not shown). An analysis of the 
-region sequences of the 43 Streptomyces strains shown in the phylogenetic trees (positions 158–277 on the 16S rRNA gene; Kataoka et al., 1997) confirmed the affiliation of strain YIM 47672T to a separate branch (data not shown). Streptomyces sodiiphilus CCTCC AA 203015T clustered with Streptomyces rimosus subsp. rimosus JCM 4667T, Streptomyces albus subsp. albus DSM 40313T and Streptomyces albofaciens JCM 4342T. Streptomyces cheonanensis JCM 14549T belonged to a cluster consisting of Streptomyces carpaticus NRRL B-16359T, Streptomyces armeniacus JCM 3070T, the suggested type strain of ‘Streptomyces cattleya’ JCM 4925, Streptomyces macrosporus DSM 41449T and Streptomyces thermolineatus DSM 41451T.
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), 95.95 % (for S. carpaticus; Gause et al., 1983, 1986) and 95.81 % (for S. cheonanensis; Kim et al., 2006). The levels of gene sequence similarity with respect to other members of the genus Streptomyces ranged from 93.40 to 95.50 %. Strain YIM 47672T showed some characteristics that differed from those of its closest relatives. Whole-cell hydrolysates of S. sodiiphilus contain mainly galactose and glucose, the predominant menaquinones are MK-9(H4) (13 %), MK-9(H6) (68 %) and MK-9(H8) (19 %) and the G+C content is 70.5 mol%. S. cheonanensis shows rectiflexibiles spore chains, the aerial mycelium is grey and the substrate mycelium is light yellow; it produces brown and dark grey soluble pigments, produces melanin, utilizes arabinose, mannitol, raffinose and xylose and the DNA G+C content is 75.5 mol%. The description of S. carpaticus focused mainly on its morphological and physiological characteristics (Spirales spore chains dark brown mycelium on ISP 4 agar, utilization of rhamnose, arabinose, mannitol, raffinose, fructose and xylose).
It can be concluded, therefore, that strain YIM 47672T represents a novel member of the genus Streptomyces, for which the name Streptomyces hainanensis sp. nov. is proposed.
Description of Streptomyces hainanensis sp. nov.
Streptomyces hainanensis (hai.nan.en'sis. N.L. adj. hainanensis pertaining to Hainan, a province of south China, from where the type strain was isolated).
Aerial mycelia are white and pink–white to pink–grey. Spore chains are spiral or looped. Spores are elliptical or shaped like short rods. Spore surface is smooth. Vegetative mycelia are pale to deep orange–yellow. Produces light brown to orange–yellowish soluble pigments. Negative in tests for gelatin liquefaction, milk coagulation and peptonization, arginase activity, phenylalanine deaminase activity, DNase and melanin production. Positive for starch hydrolysis, arginine decarboxylase activity, nitrate reduction, gas production from nitrate, growth on cellulose and H2S production. Optimal growth occurs at pH 7.0 (range, pH 6.0–9.0) and without NaCl (range, 0–10 % NaCl). Glucose, cellobiose, starch, aesculin, trehalose, -galactoside and urea are utilized; acids are not produced from these carbon sources. Galactose, mannose, fructose, arabinose, xylose, ribose, rhamnose, sucrose, lactose, maltose, melibiose, raffinose, turanose, melezitose, sorbin, dextrin, salicin, adonitol, inositol, mannitol, sorbitol, xylitol, galactitol, erythritol, amygdaloside, sodium citrate, sodium acetate, gluconate, malonate, tartrate, lysine, ornithine and acetamide are not utilized. Resistant to the following antibiotics (µg, unless indicated otherwise): penicillin G (10 U), amoxycillin/clavulanic acid (20 : 10), novobiocin (30), rifampicin (5) and ampicillin (10). Sensitive to the following antibiotics (µg, unless indicated otherwise): erythromycin (15), gentamicin (10), kanamycin (30), tetracycline (30), vancomycin (30), midecamycin (15), clindamycin (2), sulfamethoxazole/trimethoprim (23.75 : 1.25), chloramphenicol (30), polymyxin B (300 U) and norfloxacin (10). Cell wall contains L-diaminopimelic acid, traces of meso-diaminopimelic acid and glycine. Whole-cell hydrolysates contain galactose and xylose. The diagnostic phospholipid is phosphatidylethanolamine. The predominant menaquinones are MK-9(H4) (45.4 %), MK-9(H6) (14.0 %), MK-9(H8) (13.6 %) and MK-10(H0) (27.0 %). Fatty acids comprise iso-C15 : 0 (1.1 %), iso-C16 : 0 (30.9 %), C16 : 1
7c/C16 : 16c (2.1 %), C16 : 0 (13.7 %), iso-C17 : 19c (1.4 %), iso-C17 : 0 (2.8 %), anteiso-C17 : 0 (10.8 %), C17 : 18c (7.1 %), C17 : 0 cyclo (1.4 %), C17 : 0 (5.6 %), anteiso-C18 : 0/C18 : 26,9c (9.7 %), C18 : 19c (4.9 %) and C18 : 0 (1.2 %). In addition, the sum of C16 : 17c/C16 : 16c was 2.1 %, the sum of anteiso-C18 : 0/C18 : 26,9c was 9.7 % and the sum of iso-C17 : 19c and/or C16 : 0 10-methyl was 1.4 %. The G+C content of genomic DNA is 73.4 mol%.
The type strain, YIM 47672T (=CCTCC AA 205017T=DSM 41900T), was isolated from soil collected from a forest (of evergreen broadleaved trees) on Wuzhi Mountain, Hainan Province, China.
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ACKNOWLEDGEMENTS |
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