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1 The National Key Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing 100050, People's Republic of China
2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China
Correspondence
Ying Huang
huangy{at}im.ac.cn
Yue-Qin Zhang
zyq_0525{at}yahoo.com.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 4776T is DQ462649.
Light and scanning electron micrographs showing the spore-chain morphology and spore-surface ornamentation of cells of strain 4776T are available with the online version of this paper.
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MAIN TEXT |
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; Dietera et al., 2003; Bérdy, 2005). Despite the large number of species with validly published names, the genus Streptomyces as a whole is underspeciated (Kim & Goodfellow, 2002; Saintpierre et al., 2003; Huang et al., 2004; Xu et al., 2006). In the course of searching for new anti-atherosclerosis drugs targeted on the scavenger receptor BI (SR-BI), an active streptomycete-like strain, strain 4776T, was isolated from a soil sample collected from Emei Mountain in Sichuan province, China. A polyphasic taxonomic investigation based on a judicious combination of genotypic and phenotypic characteristics revealed that the new isolate represents a novel species of the genus Streptomyces.
Strain 4776T was isolated after two weeks incubation at 28 °C on yeast-malt extract agar (ISP 2; Shirling & Gottlieb, 1966), which had been seeded with a soil sample suspension. The soil sample was collected from Emei Mountain, Sichuan, China. The isolate was maintained on ISP 2 slopes at 4 °C and as glycerol suspensions (20 %, v/v) at –20 °C. Biomass for molecular systematic and most of the chemotaxonomic studies was obtained after incubation in shake flasks of ISP 2 broth at 28 °C for 4–7 days.
The spore-chain morphology and spore-surface ornamentation of strain 4776T were recorded by examining gold-coated dehydrated specimens of 10 to 14-day cultures grown on ISP 2 agar by scanning electron microscopy (SEM) (Quanta; FEI). The coverslip technique (Zhou et al., 1998; Kawato & Shinobu, 1959) was used to observe the hyphae and spore-chain characteristics. Aerial spore-mass colour, substrate mycelial pigmentation and the production of diffusible pigments were estimated on a number of agar media (Table 1) following incubation at 28 °C for 14 days.
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and Lechevalier & Lechevalier (1980). Menaquinones were extracted and determined using the methods of Collins (1985). The phospholipid analysis was carried out using the technique described by Minnikin et al. (1984). The fatty acids were extracted, methylated and estimated by GC using the standard Sherlock MIDI (Microbial Identification) system (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The G+C content of genomic DNA was determined using the thermal denaturation method of Marmur & Doty (1962) with Escherichia coli K12 as a control.
Strain 4776T was examined for a broad range of biochemical and physiological characteristics according to the established methods of Williams et al. (1983) and Kämpfer et al. (1991). Tolerance to temperature and pH was tested on ISP 2 agar plates incubated for 7–14 days. Resistance to antibiotics was examined as described by Al-Tai et al. (1999). Readings were taken at 1, 3, 7 and 14 days and inhibition zones observed were scored as negative.
Isolation of chromosomal DNA and PCR amplification of the 16S rRNA gene were carried out after Chun & Goodfellow (1995). Sequencing of the PCR product was performed as described by Gu et al. (2006). The resultant sequence was aligned manually using CLUSTAL_X version 1.8 (Thompson et al., 1997) with available, almost complete sequences of type strains of the family Streptomycetaceae and then with corresponding sequences of representative species of the genus Streptomyces. Phylogenetic trees were constructed using the least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) algorithms from the PHYLIP package version 3.5c (Felsenstein, 1993) and the TREECON program version 1.3b (Van de Peer & De Wachter, 1994). Evolutionary distance matrices were generated according to the method of Kimura (1980). Tree topologies were evaluated by performing bootstrap analysis based on 1000 resamplings of the neighbour-joining dataset using the SEQBOOT and CONSENSE programs provided in the PHYLIP package (Felsenstein, 1993).
Levels of DNA–DNA relatedness between strain 4776T and related type strains were determined using the fluorometric micro-well method (Ezaki et al., 1989), with the modifications described by He et al., (2005).
The chemical and morphological properties of strain 4776T were consistent with its assignment to the genus Streptomyces (Williams et al., 1989; Manfio et al., 1995). The organism formed an extensively branched substrate mycelium, aerial hyphae which carried spiny-surfaced spores in rectiflexibiles and hooked spore chains (Fig. 1) and a greenish aerial spore mass on various standard media (Table 1
). The novel strain contained LL-diaminopimelic acid in whole-organism hydrolysates, hexa-, octa- and a minor amount of tetra-hydrogenated menaquinones with nine isoprene units [MK-9 (H6, H8 and H4)] as isoprenologues and phosphatidylethanolamine and phosphatidylinositol and phosphatidylinositol mannosides as the major polar lipids (phospholipid type II sensu Lechevalier et al., 1977), but lacked characteristic sugars and mycolic acids. The fatty acid profile included mainly saturated iso- and anteiso-branched-chain and straight-chain fatty acids (fatty acid type 2c sensu Kroppenstedt, 1985).
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that strain 4776T formed a distinct phyletic line with Streptomyces prasinopilosus DSM 40098T. This line was supported by all four tree-making algorithms and by a high bootstrap value. Three tree-making algorithms and a 100 % bootstrap value supported the position that the strain 4776T–S. prasinopilosus line was consistently in the same clade along with Streptomyces prasinopilosus DSM 40098T, Streptomyces prasinus JCM 4603T, Streptomyces bambergiensis DSM 40590T, Streptomyces hirsutus DSM 40095T and Streptomyces cyanoalbus DSM 40198T. 16S rRNA gene sequence similarities between strain 4776T and these type strains were between 98.6 and 98.8 % (18–19 nucleotide differences at 1390–1402 sites). The novel isolate showed the shortest phylogenetic distance (0.012) with S. prasinopilosus DSM 40098T and S. prasinus JCM 4603T, although it showed a lower 16S rRNA gene sequence identity with S. prasinopilosus DSM 40098T than with the other four neighbours from the BLAST result. This is probably because the 16S rRNA gene sequence of S. prasinopilosus that has been deposited in the GenBank database is shorter than those of the other species and contains more gaps (4 gaps).
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), thus suggesting that the novel strain should be considered as a separate species. Comparison of the phenotypic characteristics of strain 4776T and its close phylogenetic neighbours also revealed significant differences (Table 2). S. prasinopilosus was selected for a detailed cultural characteristic comparison with strain 4776T on the basis of phylogenetic distance and DNA–DNA relatedness data and its close position to the novel isolate in all four phylogenetic trees. Despite the resemblance borne by the two strains, the two strains could be differentiated by several characteristics (Table 1), especially when using ISP 4, ISP 6 and yeast extract-starch agars.
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Description of Streptomyces emeiensis sp. nov.
Streptomyces emeiensis (e.mei.en'sis. N.L. masc. adj. emeiensis pertaining to Emei, a famous mountain in Sichuan Province, southern China, where the sample yielding the type strain was collected).
Aerobic, mesophilic, Gram-positive actinomycete that develops well-branched substrate and aerial mycelium. Diffusible pigments are not formed, nor are melanin pigments produced on peptone/yeast extract/iron or tyrosine agars. Additional cultural characteristics on various agar media are shown in Table 1. Rectiflexibiles and hooked spore chains of elliptic, spiny-surfaced spores are frequently arranged in a verticillate structure. Growth occurs between 15 and 40 °C and at pH values from 5.5 to 9.5, but not at pH 4.5 or 10.5. Growth occurs in the presence of 0.1 % phenol (w/v), 5 % NaCl (w/v) and 0.01 % NaN3 (w/v), but not in the presence of 7 % NaCl (w/v). Nitrate is reduced. Amylase and gelatinase are not produced. Shows weak antimicrobial activity against strains of Bacillus subtilis and Mycobacterium smegmatis, but not against strains of Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Candida albicans. The organism is sensitive to filter-paper discs soaked in the following (µg ml–1): novobiocin (5), streptomycin (10), oxacillin (1), chloramphenicol (30), ciprofloxacin (5) and erythromycin (15). Additional physiological properties are listed in Table 2. The cell-wall is of type I. Type II phospholipids and menaquinone MK-9 (H6, H8 and H4) are detected. The fatty acid profile is composed of anteiso-C15 : 0 (14.6 %), iso-C16 : 0 (13.3 %), anteiso-C17 : 0 (12.6 %), C16 : 0 (9.4 %), C16 : 1
7c (8.2 %), anteiso-C17 : 19c (7.0 %), C18 : 0 (3.7 %), C17 : 18c (3.6 %), iso H-C16 : 1 (3.6 %), C15 : 0 (3.1 %), iso-C15 : 0 (3.0 %), iso-C17 : 19c (3.0 %), iso-C15 : 0 (3.0 %), C14 : 0 (2.8 %), C18 : 19c (2.4 %), C18 : 17c (1.6 %), iso- C17 : 0 (1.3 %), C17 : 0 (1.1 %), iso-C14 : 0 (1.1 %) and iso I-C15 : 1 (1.1 %). The G+C content of the DNA is 70.8 mol%.
The type strain, strain 4776T (=CGMCC 4.3504T=DSM 41884T), was isolated from a soil sample collected from Emei Mountain, Sichuan Province, China.
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ACKNOWLEDGEMENTS |
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