Streptomyces emeiensis sp. nov., a novel streptomycete from soil in China

Streptomycetes continue to be a rich source of novel bioactive, commercially significant compounds (Labeda et al., 1997; 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 4776^T, 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 4776^T 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 4776^T 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.

The isomers of diaminopimelic acid and whole-cell sugar composition were analysed by TLC following procedures described by Hasegawa et al. (1983) 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 4776^T 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 4776^T 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 4776^T 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).

The almost complete 16S rRNA gene sequence (1400 nt) was determined for strain 4776^T. The sequence data clearly showed that strain 4776^T is a member of genus Streptomyces. It was evident from Fig. 2⇓ that strain 4776^T formed a distinct phyletic line with Streptomyces prasinopilosus DSM 40098^T. 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 4776^T–S. prasinopilosus line was consistently in the same clade along with Streptomyces prasinopilosus DSM 40098^T, Streptomyces prasinus JCM 4603^T, Streptomyces bambergiensis DSM 40590^T, Streptomyces hirsutus DSM 40095^T and Streptomyces cyanoalbus DSM 40198^T. 16S rRNA gene sequence similarities between strain 4776^T 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 40098^T and S. prasinus JCM 4603^T, although it showed a lower 16S rRNA gene sequence identity with S. prasinopilosus DSM 40098^T 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).

DNA–DNA hybridization tests were carried out between strain 4776^T and the phylogenetically close type strains. The DNA–DNA relatedness between strain 4776^T and S. prasinopilosus DSM 40098^T (62.7 %), S. prasinus JCM 4603^T (55.5 %), S. hirsutus DSM 40095^T (46.4 %), S. bambergiensis DSM 40590^T (31.7 %) and S. cyanoalbus DSM 40198^T (26.1 %) are all well below the 70 % cut-off point generally recognized for genomic species (Wayne et al., 1987), thus suggesting that the novel strain should be considered as a separate species. Comparison of the phenotypic characteristics of strain 4776^T and its close phylogenetic neighbours also revealed significant differences (Table 2⇓). S. prasinopilosus was selected for a detailed cultural characteristic comparison with strain 4776^T 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.

A combination of genotypic and phenotypic data show that strain 4776^T merits recognition as the type strain of a novel species in the genus Streptomyces, for which we propose the name Streptomyces emeiensis sp. nov.