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1 College of Life Sciences, Hebei University, Baoding 071002, PR China
2 The Key Laboratory for Microbial Resources of Ministry of Education, PR China, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, PR China
3 Department of Microbiology, College of Life Sciences, China Agricultural University, Beijing 100094, PR China
Correspondence
Li-Ping Zhang
zhlping{at}mail.hbu.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains CY-11007T and CY-15110T are AF191733 and AF191735.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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proposed the family Streptosporangiaceae, containing the type genus Streptosporangium Couch 1955 as well as the genera Herbidospora Kudo et al. 1993, Microbispora Nonomura and Ohara 1957 emend. Zhang et al. 1998, Microtetraspora Thiemann et al. 1968 emend. Zhang et al. 1998, Planobispora Thiemann and Beretta 1968, Planomonospora Thiemann et al. 1967, Planotetraspora Hu et al. 1993 and Nonomuraea Zhang et al. 1998. In addition, the genus Acrocarpospora was proposed by Tamura et al. (2000) as another member of the Streptosporangiaceae. Description of taxa of each genus was based on phenotypic, genotypic and phylogenetic properties. In the course of research on unknown microbial resources and microbiological biodiversity of actinomycetes, we isolated some strains of Streptosporangium from Yunnan Province, a region of south-western China that has specific geographical conditions that contribute to its great microbiological diversity. Strains CY-11007T and CY-15110T were identified by a polyphasic approach. Comparative studies of morphology, physiology and biochemical composition of cells and phylogenetic analysis based on 16S rRNA gene sequences were carried out among isolated strains CY-11007T and CY-15110T and type strains of the genus Streptosporangium and of related genera. The results indicated that the two strains are different from known members of the genus Streptosporangium. Therefore, we consider that CY-11007T and CY-15110T represent two novel species, Streptosporangium yunnanense sp. nov. (CY-11007T) and Streptosporangium purpuratum sp. nov. (CY-15110T).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Morphology.
Strains CY-11007T, CY-15110T and other type strains were cultured for 3, 5, 7 and 14 days at 28 °C on International Streptomyces project medium 3 (ISP3; oatmeal agar) (Shirling & Gottlieb, 1966) and HV agar (Hayakawa & Nonomura, 1987) and observed by light microscopy (Olympus) and scanning electron microscopy (model KYKY-AMRAY-100B).
Cultural, physiological and biochemical tests.
Cultural characteristics were studied by using 14-day-old cultures grown at 28 °C on various agar media. Colours were determined by comparing the cultures with colour chips from the ISCC-NBS colour charts standard samples no. 2106 (Kelly, 1964). The physiological characteristics of the strains were tested according to the standards for Streptosporangium (Nonomura, 1989).
Analysis of chemotaxonomic characteristics.
Cultures of the test strains were grown in shake flasks containing Bennett's broth to prepare biomass. These cultures were incubated for 5–7 days at 28 °C. Cell walls were purified from 10 g wet biomass. Amino acids of purified cell walls were analysed by the methods of Lechevalier & Lechevalier (1980). Amino acids and sugars of whole-cell hydrolysates were determined by the methods of Becker et al. (1964). Phospholipids were obtained from freeze-dried biomass (approx. 100 mg) and analysed by the methods of Lechevalier et al. (1981). Menaquinone analysis (from 100 mg freeze-dried biomass) was performed as described previously by Collins (1985). Methyl esters of cellular fatty acids (from 10 mg freeze-dried biomass) were determined by the methods of Miller (1982) and Kuykendall et al. (1989).
DNA base composition.
The G+C content of the chromosomal DNA was determined from the melting point value of the thermal denaturation profile using a spectrophotometer (Ultrospec 2000) equipped with a programmable temperature-control unit by the equation of Marmur & Doty (1962), as modified by De Ley (1970).
DNA–DNA hybridization.
Chromosomal DNA of strains CY-11007T and CY-15110T was prepared as described by Marmur (1961). The initial reassociation rate method (De Ley et al., 1970) was used for determining percentage DNA–DNA hybridization.
16S rRNA gene sequencing.
Chromosomal DNA was extracted as described by Marmur (1961) and Jiang & Xu (1990). 16S rRNA genes were amplified by PCR (Saiki et al., 1988) using a PCR kit (Sino-American Biotechnology Co.) with primers A 8–38f (5'-CGGGATCCAGAGTTTGATCCTGGCTCAGAACGAACGCT-3') and B 1479–1506r (5'-CGGGATCCTACGGCTACCTTGTTACGACTTCACCCC-3') and the 1·5 kb amplified fragment was purified by 0·8 % low-melting-point agarose gel electrophoresis, by the methods of Wieslander (1979). Purified PCR products and plasmid pUC18 vector were cut with BamHI and ligated at 18 °C for 20 h. Ligated plasmids were transformed into Escherichia coli DH5
(Wieslander, 1979) and transformants were selected by blue-white selection (Sambrook et al., 1989). Plasmids were extracted and purified according to the methods of Tiesman & Rizzino (1991). Purified plasmids containing PCR products were sequenced with a model 377 Prism automatic sequencer by American Cybersyn Company. The sequencing primer was 5'-TTCAGCAGGGACGAAGTTGA-3'. The 16S rRNA gene sequences of type strains of species of related genera were obtained from GenBank.
Phylogenetic analysis.
The 16S rRNA gene sequences were aligned by the CLUSTAL X program (Thompson et al., 1997) with corresponding nucleotide sequences of representatives of the genus Streptosporangium retrieved from GenBank (Benson et al., 1997). Phylogenetic trees were constructed using the neighbour-joining (Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967) and maximum-parsimony (Fitch, 1971) algorithms from the PHYLIP package (Felsenstein, 1993). Evolutionary distance matrices were generated according to the model of Kimura (1980). Tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings of the neighbour-joining dataset using SEQBOOT and CONSENSE options from the PHYLIP suite of programs.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Strain CY-15110T produced globose, single or clustered sporangia, usually 2–5 µm in diameter on the aerial mycelium. Sporangiospores were formed by septation of a coiled, unbranched mycelium. Sporangiospores within the sporangium were spherical and non-motile (Fig. 1c, d).
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shows the degree of growth and colour of both aerial and vegetative hyphae of strains CY11007T and CY-15110T on various media. The aerial mycelium of CY-11007T was abundant and pale-pink (7. p. Pink; Research Group of Actinomycetes, 1970) to yellowish-pink (31. p.y. Pink) on most of the media used. The substrate mycelium was brownish and produced pigment on some media tested. Colonies of strain CY-15110T were deep red (13. deep Red) to deep purplish-red (257. v.deep p. R), lacking visible aerial mycelium by eye on organic media and poor aerial mycelium on inorganic media used. The strain did not produce soluble pigment.
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. The optimal growth temperatures of strains CY-11007T and CY-15110T were respectively 30 and 28 °C and optimal pH for both strains was 7·2.
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. The major menaquinones of strain CY-11007T were MK-9(H0), MK-9(H2) and MK-9(H4) (in descending order of abundance) and for strain CY-15110T were MK-9(H2), MK-9(H0) and MK-9(H4). The predominant fatty acids in whole-cell methyl esters were C16 : 0 (14·73 %), C17 : 0 (4·95 %), C18 : 0 (10·24 %), C19 : 0 (29·64 %) and C16 : 3 (2·54 %) (strain CY-11007T) and C15 : 0 (23·74 %), C16 : 0 (42·41 %), C17 : 0 (24·84 %), C18 : 0 (0·88 %) and C17 : 3 (0·66 %) (strain CY-15110T).
DNA base composition and DNA hybridization
The G+C contents of the DNA of strains CY-11007T and CY-15110T were respectively 71·06 and 69·10 mol%. The levels of DNA–DNA relatedness with type strains of Streptosporangium ranged from 9 to 56·8 % (Table 3).
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shows a neighbour-joining phylogenetic tree constructed on the basis of evolutionary distances calculated by using the method of Kimura (1980).
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).
The two strains CY-11007T and CY-15110T should be placed in the genus Streptosporangium based on their morphological characteristics, menaquinones, predominant fatty acids in whole-cell methyl esters and DNA base composition and the similarity of their 16S rRNA gene sequences. The results of phylogenetic analysis based on 16S rRNA gene sequences show that strains CY-11007T and CY-15110T were clustered into a group, and the evolutionary distance between them was below 5 %. The novel isolates and other representatives of Streptosporangium appear to be relatively distantly related. DNA–DNA hybridization is the standard criterion for designation of species, and the criterion for a species is 
70 % DNA–DNA relatedness (Wayne et al., 1987). The DNA–DNA relatedness of strains CY-11007T and CY-15110T with related type strains was <70 %. Cultural characteristics, physiological properties and biochemical reactions indicated that strain CY-11007T is different from strain CY-15110T. We therefore propose that the two isolates represent novel species, Streptosporangium yunnanense sp. nov. (CY-11007T) and Streptosporangium purpuratum sp. nov. (CY-15110T). Differences between isolates CY-11007T and CY-15110T and related strains of Streptosporangium are shown in Table 4.
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Aerobic, Gram-positive. Abundant aerial mycelium is pale-pink (7.p. Pink) to yellowish-pink (31. p.y. Pink) on most of the media tested such as glycerol-asparagine agar (ISP5), oatmeal agar (ISP3), oatmeal-yeast extract agar and Bennett's agar. Substrate mycelium is brownish and pigment is produced on various media tested. Spherical sporangia are produced on aerial mycelium on HV agar and oatmeal agar. Sporangiospores are formed by septation of coiled, unbranched hyphae and are spherical and non-motile. Cell walls contain meso-DAP; whole-cell hydrolysates contain madurose, glucose and rhamnose. Phospholipids consist of PE, PME, DPG, PI and GluNus; PG is not detected. Major menaquinones are MK-9(H0), MK-9(H2) and MK-9(H4). Predominant fatty acids in whole-cell methyl esters are 16 : 0 (14·73 %), 17 : 0 (4·95 %), 18 : 0 (10·24 %), 19 : 0 (29·64 %) and 16 : 3 (2·54 %). G+C content of the DNA of the type strain is 71·06 mol%. Optimal growth temperature is 30 °C; growth occurs at 10 and 42 °C but not at 50 °C. Optimal pH for growth is 7·2. D-Glucose, D-sucrose and D-cellobiose are utilized; L-arabinose, D-galactose, D-fructose, D-mannose, D-mannitol, L-rhamnose, inositol, D-raffinose, D-xylose, sorbitol, sorbose, erythrose, lactose, melibiose, methyl -D-glucoside and dextrin are not utilized. Degradation of cellulose and starch is positive. Reduction of nitrate and liquefaction of gelatin are positive. Vitamin B is not required for growth and iodinin production is negative. Melanin is produced. Uricase is negative. Succinate, malate, citrate, uric acid and hippurate are hydrolysed.
The type strain is CY-11007T (=CCTCC AA 97009T=CCRC 16307T=DSM 44663T).
Description of Streptosporangium purpuratum sp. nov.
Streptosporangium purpuratum (pur.pur.at'um. L. neut. adj. purpuratum clad in purple-violet, referring to the colony colour).
Aerobic, Gram-positive. Colonies are deep red (13. deep Red) to deep purplish-red (257. v. deep p. R) on most media tested such as glycerol-asparagine agar (ISP5), oatmeal agar (ISP3), oatmeal-yeast extract agar and Bennett's agar. Aerial mycelium is very poor so that it is invisible by eye. Substrate mycelium is deep red and soluble pigment is not produced on various media tested. Spherical sporangia are produced on aerial mycelium on HV agar and oatmeal agar, usually 2–5 µm in diameter. Sporangiospores are formed by septation of coiled, unbranched hyphae; they are spherical or oblate in shape and non-motile. Cell walls contain meso-DAP; whole-cell hydrolysates contain madurose, glucose and rhamnose. Phospholipids consist of PE, DPG, PI and GluNus. Major menaquinones are MK-9(H2), MK-9(H0) and MK-9(H4). Predominant fatty acids in whole-cell methyl esters are 15 : 0 (23·74 %), 16 : 0 (42·41 %), 17 : 0 (24·84 %), 18 : 0 (0·88 %) and 17 : 3 (0·66 %). Optimal growth temperature is 28 °C; grows at 42 and 50 °C. Optimal pH for growth is 7·2. D-Glucose, D-sucrose, D-cellobiose, D-fructose and D-xylose are utilized; L-arabinose, D-galactose, D-mannose, D-mannitol, inositol, D-raffinose, L-rhamnose, sorbitol, sorbose, erythrose, lactose, melibiose, methyl -D-glucoside and dextrin are not utilized. Degradation of cellulose and starch is positive. Reduction of nitrate and liquefaction of gelatin are negative. Vitamin B is not required for growth and iodinin and melanin are produced. Uricase is negative. Succinate, uric acid, malate, citrate and hippurate are hydrolysed.
The type strain is strain CY-15110T (=CCTCC AA 97010T=CCRC 16308T=DSM 44688T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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