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Streptomyces alni sp. nov., a daidzein-producing endophyte isolated from a root of Alnus nepalensis D. Don

Ning Liu, Haibin Wang, Mei Liu, Qiang Gu, Wen Zheng, Ying Huang

  • 1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
  • 2Center of Technology, Zhejiang Hisun Pharmaceutical Co., Ltd, Taizhou 318000, PR China
  • Correspondence
    Ying Huang
    huangy{at}im.ac.cn

    • International Journal of Systematic and Evolutionary Microbiology 2009; 59(2):254 · https://doi.org/10.1099/ijs.0.65769-0

    View at publisher PubMed

    Abstract

    A filamentous actinomycete, designated strain D65T, was isolated from a root of a wild tree, Alnus nepalensis D. Don (Nepalese Alder), collected in Xishuangbanna, China. It produced the bioactive agents daidzein and N-acetyltyramine and had morphological and chemical properties characteristic of streptomycetes. Pink to brownish red diffusible pigments were produced on several ISP media. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain D65T formed a distinct phyletic line that was most closely, albeit loosely, associated with Streptomyces hebeiensis YIM 001T, Streptomyces aurantiogriseus NRRL B-5416T, Streptomyces griseoviridis NBRC 12874T, Streptomyces niveoruber NBRC 15428T and Streptomyces thermovulgaris NBRC 13473. A number of phenotypic properties allowed differentiation of the strain from related Streptomyces species. Therefore strain D65T represents a novel species of the genus Streptomyces, for which the name Streptomyces alni sp. nov. is proposed. The type strain is D65T (=CGMCC 4.3510T=NRRL B-24611T).

    • ↵†These authors contributed equally to this paper.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain D65T is DQ460470.

    • Scanning electron micrographs showing the spore chains and spore-surface ornamentation of cells of strain D65T are presented in a supplementary figure available with the online version of this paper.

    Soil actinomycetes, especially streptomycetes, are ubiquitous and isolates have been screened for decades because of their particular capacity to produce bioactive molecules (e.g. antibiotics) (Labeda et al., 1997; Dietera et al., 2003; Bérdy, 2005). Nevertheless, the investigation of endophytic actinomycetes for the purpose of producing bioactive metabolites did not attract attention until Gurney & Mantle (1993) isolated 1-N-methylalbonoursin from an Acremonium-like Streptomyces species living in perennial ryegrass, although there had been previous reports of actinomycetes being isolated from plant samples (Evtushenko et al., 1989; Kudo et al., 1998). During screening for antimicrobial and antitumour agents, an active streptomycete was isolated from the surface-sterilized root of a higher plant, Alnus nepalensis D. Don (Nepalese Alder). The aim of the present study was to characterize the isolate by using a polyphasic approach.

    Root samples of Alnus nepalensis D. Don, a plant traditionally used in Chinese medicine, were collected in the rainforest of Nannuo Mountain, Xishuangbanna, Yunnan Province, China, and were surface-sterilized according to the method of Coombs & Franco (2003). Strain D65T was isolated from the root elongation zone by using the procedure and medium described by Gu et al. (2006).

    Aerial spore-mass colour, substrate mycelial pigmentation and the production of diffusible pigments of strain D65T were determined on a number of ISP agar media (Shirling & Gottlieb, 1966) following incubation at 28 °C for 14 days. The morphological characteristics were studied by examining gold-coated dehydrated specimens with a model FEI Quanta 200 scanning electron microscope, using cultures grown on ISP 2 agar for 14 days. Physiological and biochemical characteristics of strain D65T and its phylogenetically close neighbours were tested together, using the well-established procedures of Williams et al. (1983) and Kämpfer et al. (1991). Temperature and pH tolerance was tested on ISP 2 agar plates incubated for 7–14 days. The isomers of diaminopimelic acid and the whole-cell sugar composition were analysed using TLC according to the procedures described by Hasegawa et al. (1983) and Lechevalier & Lechevalier (1980). Menaquinones were extracted and determined by HPLC using the methods of Collins (1985). A phospholipid analysis was carried out by means of two-dimensional TLC, using the method of Minnikin et al. (1984). The G+C content of genomic DNA was determined using the thermal denaturation method of Marmur & Doty (1962) with Escherichia coli K-12 as a control.

    The morphological and chemical properties of strain D65T 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, with abundant aerial hyphae that differentiated into smooth cylindrical spores (0.42–0.47×0.70–0.75 μm) in flexuous (rectiflexibiles) spore chains (see Supplementary Fig. S1, available in IJSEM Online) and had a white to grey spore mass and pink to brownish red diffusible pigments on various ISP media. It contained ll-diaminopimelic acid in whole-organism hydrolysates, hexa- and octa-menaquinones with nine isoprene units [MK-9(H6, H8)] as the predominant menaquinones and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides as major polar lipids (phospholipid type II sensu Lechevalier et al., 1977), but lacked characteristic sugars and mycolic acids. The DNA G+C content was 70.2 mol%. In addition, the organism was found to produce two bioactive compounds, N-acetyltyramine and daidzein, in ISP 2 fermentation broth.

    Genomic DNA extraction and PCR amplification of the 16S rRNA gene from strain D65T were carried out as described by Chun & Goodfellow (1995). Purification and sequencing of the PCR product were performed as described by Gu et al. (2006). The resultant sequence (1426 nt) was aligned manually with corresponding sequences of available streptomycetes drawn from the GenBank/EMBL/DDBJ databases. The software mega, version 4.0 (Tamura et al., 2007), was used for both multiple alignment and phylogenetic tree construction using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) tree-making algorithms. Pairwise distances for the neighbour-joining algorithm were calculated using the Kimura two-parameter model (Kimura, 1980) and close-neighbour-interchange (search level=2, random additions=100) was applied to the maximum-parsimony analysis. The topologies of the resultant trees were evaluated using bootstrap analysis (Felsenstein, 1985) based on 1000 replicates. The evolutionary distances were computed using the Kimura two-parameter method; both transitions and transversions were included. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option).

    Preliminary comparison of the 16S rRNA gene sequence of D65T against the NCBI nucleotide collection database using the default blast algorithm indicated that the strain is a member of the genus Streptomyces. Phylogenetic analysis of the 16S rRNA gene sequences of representatives of related species confirmed the placement of strain D65T in the genus Streptomyces. Trees constructed using the neighbour-joining and maximum-parsimony methods gave very similar topologies (Fig. 1⇓). The organism formed a distinct lineage adjacent to a cluster comprising Streptomyces hebeiensis YIM 001T (97.6 % 16S rRNA sequence similarity), Streptomyces aurantiogriseus NRRL B-5416T (97.4 %), Streptomyces griseoviridis NBRC 12874T (97.4 %), Streptomyces niveoruber NBRC 15428T (97.4 %) and Streptomyces thermovulgaris NBRC 13473 (97.4 %), which were its closest neighbours based on the evolutionary distance matrix. DNA–DNA relatedness studies were not carried out between strain D65T and these organisms because of their relatively high evolutionary distances (0.024–0.026) and the distinct phylogenetic position of strain D65T, as representatives of a number of Streptomyces species with much lower 16S rRNA gene evolutionary distances have DNA–DNA relatedness values well below the 70 % cut-off point recommended for the delineation of genomic species. Moreover, a combination of physiological and biochemical characteristics enabled strain D65T to be distinguished from its closest phylogenetic neighbours (Table 1⇓).

    Figure image not available in archive
    Fig. 1.

    Unrooted neighbour-joining phylogenetic tree (Saitou & Nei, 1987), based on almost-complete 16S rRNA gene sequences, showing the relationships between strain D65T and related species in the genus Streptomyces. Accession numbers are given in parentheses. Neighbour-joining and maximum-parsimony algorithms were both applied using the software package mega, version 4.0 (Tamura et al., 2007). Bootstrap percentages (based on 1000 replications) are shown at branch points; those inferred from neighbour-joining are shown above the branches and those from maximum-parsimony are shown below. Bar, 0.005 substitutions per nucleotide position.

    Table 1.

    Physiological and biochemical characteristics of strain D65T and related Streptomyces species

    Strains: 1, D65T (S. alni sp. nov.); 2, S. hebeiensis YIM 001T; 3, S. niveoruber CGMCC 4.1395T (=NBRC 15428T); 4, S. griseoviridis CGMCC 4.1418T (=NBRC 12874T); 5, S. aurantiogriseus CGMCC 4.1450T (=NRRL B-5416T); 6, S. thermovulgaris CGMCC 4.1470 (=NBRC 13473). All strains are positive for assimilation of d-mannose, d-galactose, d-glucose and cellobiose as sole carbon sources but are negative for assimilation of l-aspartic acid. All strains degrade guanine and grow with 3 % NaCl but not with ≥7 % NaCl. +, Positive; −, negative; (+), weakly positive.

    The combined genotypic and phenotypic data show that strain D65T represents a novel species of the genus Streptomyces, for which the name Streptomyces alni sp. nov. is proposed.

    Description of Streptomyces alni sp. nov.

    Streptomyces alni (al′ni. L. gen. n. alni of the alder, referring to the isolation of the type strain from Alnus nepalensis).

    Aerobic, mesophilic, Gram-positive actinomycete that develops well-branched substrate and aerial mycelium. Smooth-surfaced spores are borne in rectiflexibiles spore chains. Light purple to purplish brown colonies that carry white to grey aerial spore mass are formed on ISP media 2, 3, 4, 5 and 7 and yeast-starch medium (DSMZ medium 1027). Pink to brownish red diffusible pigments are formed on the above media, but melanin pigments are not produced on peptone-yeast extract-iron medium (ISP 6) or tyrosine medium (ISP 7). Growth occurs between 10 and 45 °C and at pH 4.5–9.5, but not at pH 3.5 or 10.5. Physiological and biochemical profiles, including those relating to carbon-source utilization, biodegradation and growth, are listed in Table 1⇑. The organism exhibits antagonistic activity against Staphylococcus aureus and mouse melanoma cell line B16, and produces daidzein and N-acetyltyramine as its major bioactive metabolites. Chemotaxonomic properties are typical of the genus Streptomyces. The DNA G+C content of the type strain is 70.2 mol%.

    The type strain, D65T(=CGMCC 4.3510T=NRRL B-24611T), was isolated from a root of a wild tree, Alnus nepalensis D. Don (Nepalese Alder), collected in the rainforest of Nannuo Mountain, Xishuangbanna, Yunnan Province, China.

    Acknowledgments

    This research was supported by the Natural Science Foundation of China (grant 30670002) and by the National Basic Research Program of China (grant 2004CB719601). We also thank Professor Jisheng Ruan (Institute of Microbiology, Chinese Academy of Sciences) and Dr Jidong Wang (Zhejiang Hisun Pharmaceutical Co.) for their previous help with the chemical analysis.

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