SGM Journals
Actinobacteria

Streptomyces tateyamensis sp. nov., Streptomyces marinus sp. nov. and Streptomyces haliclonae sp. nov., isolated from the marine sponge Haliclona sp.

Shams Tabrez Khan, Tomohiko Tamura, Motoki Takagi, Kazuo Shin-ya

  • 1Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), 2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan
  • 2NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
  • 3Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-42 Aomi, Koto-ku, Tokyo, 135-0064, Japan
  • Correspondence
    Kazuo Shin-ya
    k-shinya{at}aist.go.jp
    Shams Tabrez Khan
    shamsalig75{at}gmail.com

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2775–2779 · https://doi.org/10.1099/ijs.0.019869-0

    View at publisher PubMed

    Abstract

    Three Gram-positive, NaCl-requiring actinobacteria were isolated from a marine sponge, Haliclona sp., collected from the coast of Tateyama City, Japan. Comparison of 16S rRNA gene sequences indicated that these strains represent novel members of the genus Streptomyces, exhibiting low 16S rRNA gene sequence similarities of 98.3–97.4 % with recognized members of the genus. The cell hydrolysates contained the LL-isomer of diaminopimelic acid and the predominant quinones were MK-9 (H6 and/or H8). The DNA G+C contents were in the range 72–75 mol%. A polyphasic study of the strains and comparison of the characters with related species of the genus show that these strains represent three novel species of the genus Streptomyces. Therefore, the names Streptomyces tateyamensis sp. nov., Streptomyces haliclonae sp. nov. and Streptomyces marinus sp. nov. are proposed for strains Sp080513SC-30T (=NBRC 105048T =DSM 41969T), Sp080513SC-31T (=NBRC 105049T =DSM 41970T) and Sp080513GE-26T (=NBRC 105047T =DSM 41968T), respectively.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences of strains Sp080513SC-30T, Sp080513SC-31T and Sp080513GE-26T are AB473555, AB473556 and AB473554, respectively.

    • Scanning electron micrographs showing spore chain morphology, cellular fatty acid profiles, and a comparison of 16S rRNA gene sequences with closely related species are available with the online version of this paper.

    Marine sponges are an attractive source of novel chemicals and bioactive compounds (Zhang et al., 2005). These members of the phylum Porifera are filter feeders and can accumulate as much as 2.76×106 bacteria (g sponge wet weight)−1 (Wehrl et al., 2007), therefore concentrating bacteria found otherwise diluted in the seawater. A number of reports are available on the isolation of actinobacteria from marine sponges (Mincer et al., 2002; Ward & Bora, 2006; Khan et al., 2010). Bacteria, especially the members of the genera Streptomyces and Micromonospora, produce a number of novel compounds (Lam, 2006), and are therefore a promising source of novel compounds for therapeutic use.

    In our studies on the isolation of actinobacteria from a marine sponge, Haliclona sp., three strains, Sp080513SC-30T, Sp080513SC-31T and Sp080513GE-26T, were isolated. A polyphasic phylogenetic study shows that these strains represent three novel species of the genus Streptomyces, for which the names S. tateyamensis sp. nov., S. haliclonae sp. nov., and S. marinus sp. nov. are proposed, respectively.

    A sample of the marine sponge Haliclona sp. was collected from the pacific coastline of Tateyama City, Chiba prefecture, Japan, and was transferred to the laboratory within 3 h of collection at room temperature. The sponge sample was then rinsed with sterile seawater, cut into tiny pieces with sterile scissors and resuspended in sterile seawater. An aliquot of 100 μl from this suspension was spread on Jewfish (Ruditapes philippinarum) extract agar (Khan et al., 2010) and starch casein nitrate agar plates prepared with 50 % (v/v) seawater and supplemented with 35 μg nalidixic acid ml–1 and 50 μg cycloheximide ml–1. Strains Sp080513SC-30T and Sp080513SC-31T were isolated from starch casein nitrate agar and Sp080513GE-26T was isolated from Jewfish extract agar. ISP2 (International Streptomyces project; Shirling & Gottlieb, 1966) medium prepared with 50 % (v/v) artificial seawater (Naigai Chemicals; ISP2 M) was used for further purification and maintenance of the strains. For long-term preservation, strains were stored at −80 °C in 50 % (v/v) artificial seawater supplemented with 15 % glycerol (v/v).

    Prepman Ultra (Applied Biosystems) was used to prepare template DNA for 16S rRNA gene amplification. The genes were amplified by using a universal primer set (9f and 1492r; Brosius et al., 1978) and sequenced directly using a BigDye Terminator v3.1 Cycle Sequencing kit and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). In blast searches (Altschul et al., 1990) against the sequences available in the DDBJ and EzTaxon server (Chun et al., 2007), strains shared a maximum of 98.35 % 16S rRNA gene sequence similarity with both recognized and unpublished members of the genus Streptomyces. Results of the sequence comparison are summarized in Supplementary Table S1, available in IJSEM Online. Closely related sequences with validly published names were downloaded from DDBJ and pair-wise sequence similarities were checked using the Needleman-Wunsch alignment algorithm (Needleman & Wunsch, 1970; ). Strain Sp080513SC-30T shared a maximum similarity of 98.35 % with Streptomyces sioyaensis NRRL B-5408T (DQ026654) amongst the close relatives with validly published names. Strains Sp080513SC-31T and Sp080513GE-26T shared maximum similarities of 97.66 % and 97.4 % with Streptomyces cacaoi subsp. cacaoi NBRC 12748T (AB184115) and Streptomyces albiaxialis NBRC 101002T (AY999901), respectively. These two novel strains share 98.02 % sequence similarity with each other and share low sequence similarities (96.4–96.9 %) with strain Sp080513SC-30T.

    Downloaded sequences were aligned using the clustal x program (Thompson et al., 1997), and phylogenetic trees were reconstructed by using the neighbour-joining algorithm (Saitou & Nei, 1987). The robustness of the tree topology was evaluated by bootstrap analysis using 1000 resamplings of the sequences (Felsenstein, 1985). Strain Sp080513SC-30T shared maximum 16S rRNA gene sequence similarity with S. sioyaensis, but formed an independent clade (Fig. 1⇓). Strains Sp080513GE-26T and Sp080513SC-31T formed an independent clade supported by a high bootstrap value of 97.5 % in a neighbour-joining phylogenetic tree (Fig. 2⇓). Almost similar results were obtained with maximum-parsimony analysis (data not shown).

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree based on almost complete 16S rRNA gene sequences showing the phylogenetic position of strain Sp080513SC-30T and related species. Bootstrap values from 1000 replications are indicated at branches when the values are significant (>500). Nodes recovered in maximum-parsimony analysis are marked with closed circles. Accession numbers of sequences downloaded from DDBJ are shown in parentheses. The sequence of Paraoerskovia marina (AB445007) was used as an outgroup. Bar, 0.01 Knuc.

    Figure image not available in archive
    Fig. 2.

    Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic positions of strains Sp080513SC-31T and Sp080513GE-26T and related species. Bootstrap values from 1000 replications are indicated at branches when the values are significant (>500). Nodes recovered in maximum-parsimony analysis are marked with closed circles. Accession numbers of sequences downloaded from DDBJ are shown in parentheses. The sequence of Paraoerskovia marina (AB445007) was used as an outgroup. Bar, 0.01 Knuc.

    As the phylogenetic trees for the novel strains were inconclusive, phylogenetic neighbours identified from the blast searches (EzTaxon server) were compared in the detailed polyphasic study.

    The arrangement of hyphae, spore chains and spore surface were observed under light (Olympus CX41LF) and scanning electron (JEOL JSM-6060) microscopes after growing the cells on ISP2 M or on water agar [1.6 % agar in 50 % (v/v) seawater] at 28 °C for 15–20 days. Growth of standard strains used in this study (S. cacaoi subsp. cacaoi NBRC 12748T, S. albiaxialis NBRC 101002T, and S. sioyaensis NBRC 12820T) and that of novel strains was checked on ISP2 and ISP2 M media. Standard strains grew well on both media, while the novel strains isolated during this study showed poor growth on ISP2 medium. Therefore, media (Shirling & Gottlieb, 1966) prepared with 50 % (v/v) seawater were used to determine the cultural and physiological characteristics. DNA was extracted from cells grown to late exponential growth phase using the protocol of Minamisawa (1990), and the method described by Mesbah et al. (1989) was used to determine the G+C content of the genomic DNA. Commercially available API ZYM and API Coryne systems (bioMérieux) were used following the instructions of the manufacturer for the biochemical characterization of the strains. Menaquinones, cellular fatty acids and the diaminopimelic acid isomer in whole cell hydrolysates were determined as described previously (Tamura et al., 1994).

    Phenotypic and biochemical features of the novel strains are listed in Tables 1⇓ and 2⇓ and in the species descriptions. Comparison of the characteristics with previously described relatives shows that these strains represent novel species of the genus Streptomyces. Strain Sp080513SC-30T shares 98.35 % 16S rRNA gene sequence similarity with its closest phylogenetic neighbour, S. sioyaensis NBRC 12820T, but differs in a number of phenotypic characteristics (Table 1⇓), such as the production of soluble pigment on ISP media, growth at 40 °C, and the presence of α-mannosidase, α-chymotrypsin and urease. Based on the significant distance in 16S rRNA gene sequence (Stackebrandt & Ebers, 2006) and the phenotypic differences, we propose that strain Sp080513SC-30T should be classified as a new species of the genus Streptomyces for which the name Streptomyces tateyamensis sp. nov. is proposed.

    Table 1.

    Characteristics that differentiate strain Sp080513SC-30T from Streptomyces sioyaensis NBRC 12820T

    Strains: 1, Sp080513SC-30T; 2, S. sioyaensis NBRC 12820T. Both strains produced white and grey aerial mycelium on ISP2 medium. Data from this study. +, Positive; w, weakly positive; −, negative.

    Table 2.

    Characteristics that differentiate strains Sp080513SC-31T and Sp080513GE-26T and related species

    Strains: 1, Sp080513SC-31T; 2, Sp080513GE-26T; 3, S. albiaxialis NBRC 101002T; 4, S. cacaoi subsp. cacaoi NBRC 12748T. Data from this study. +, Positive; w, weakly positive; −, negative.

    Similarly, strains Sp080513GE-26T and Sp080513SC-31T also differ from their closest relatives and from each other in a number of characters summarized in Table 2⇑. Although the two strains cluster together in a clade supported by a high bootstrap value (97.5 %), they can be distinguished based on colony morphology, the production of soluble pigment, aerial mass, and the presence or absence of naphthol-AS-BI-phosphohydrolase and α-mannosidase. These novel strains can be differentiated from their phylogenetic neighbours by growth temperature range, nitrate reduction, soluble pigment production and absence of urease (Table 2⇑). Significantly low 16S rRNA gene sequence similarities (Stackebrandt & Ebers, 2006) and the differences in a number of phenotypic characteristics support the classification of these strains as novel species of the genus Streptomyces. Therefore, the names Streptomyces haliclonae sp. nov. and Streptomyces marinus sp. nov. are proposed for strains Sp080513SC-31T and Sp080513GE-26T, respectively.

    Description of Streptomyces tateyamensis sp. nov.

    Streptomyces tateyamensis (ta.te.ya.men′sis. N.L. masc. adj. tateyamensis pertaining to Tateyama, the place from where the type strain was isolated).

    Aerobic, Gram-positive and catalase-positive actinomycete. Spore chains are spiral with multiple turns and the spore surface is smooth. Predominant fatty acids are iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. Detailed fatty acid profile is given in Supplementary Table S2. The major respiratory quinones are MK-9 (H6) and MK-9 (H8). MK-9 (H2) and MK-9 (H4) are also present. Grows at 15–37 °C and optimally at 25–30 °C and at pH 6.0–8.0 (weakly at pH 9.0). Growth occurs with 2–7 % (w/v) NaCl. Weak growth is observed with 1 and 10 % (w/v) NaCl. Growth is good on all ISP media tested [tryptone yeast extract agar (ISP1), yeast extract malt agar (ISP2), oatmeal agar (ISP3), inorganic salts–starch agar (ISP4), glycerol–asparagine agar (ISP5), peptone–yeast extract iron agar (ISP6) and tyrosine agar (ISP7)]. Spores are grey and aerial mycelium is white, except on ISP1 where aerial mass is not produced. Melanin pigments are not produced on ISP6 and ISP7 media. Light brown soluble pigments are produced on most of the ISP media (except on ISP7). In API ZYM and API Coryne tests, positive for N-acetyl-β-glucosaminidase, acid phosphatase, alkaline phosphatase, α-chymotrypsin, gelatin hydrolysis, α-glucosidase, β-glucosidase, leucine arylamidase, α-mannosidase, naphthol-AS-BI-phosphohydrolase, pyrazinamidase, pyrolidonyl arylamidase, trypsin and urease. Negative activity was observed for esterase, esterase lipase, α-fucosidase, α-galactosidase, β-galactosidase, β-glucuronidase and lipase. Starch is not degraded and nitrate is not reduced. d-Mannitol, sucrose, d-glucose, sorbitol and myo-inositol are utilized as carbon sources. d-Fructose, l-arabinose, raffinose, l-rhamnose and d-xylose are not utilized. The DNA G+C content of the type strain is 74 mol%.

    The type strain is Sp080513SC-30T (=NBRC 105048T =DSM 41969T), isolated from the marine sponge Haliclona sp.

    Description of Streptomyces haliclonae sp. nov.

    Streptomyces haliclonae (ha.li.clo′nae. N.L. gen. n. haliclonae of Haliclona, isolated from the marine sponge Haliclona sp.).

    Cells are Gram-positive, aerobic and catalase-positive. Spores are smooth-surfaced and borne in spiral chains. The predominant respiratory quinone is MK-9 (H8). Predominant cellular fatty acids are anteiso-C15 : 0, iso-C16 : 0 and iso-C15 : 0. Detailed fatty acid profile is given in Supplementary Table S2. Grows at 15–37 °C (optimally at 25–30 °C) and at pH 6.0−8.0 (weakly at pH 9.0). Growth occurs with 2–7 % (w/v) NaCl. Weak growth is observed with 1 and 10 % (w/v) NaCl. Grows well on ISP1–ISP7 media except on ISP6 medium where growth was poor. Diffusible pigments are not produced. The spore mass is white and the reverse-side colour is buff to dull white on most media. Melanin pigments are not produced on ISP6 or ISP7 media. Starch is not hydrolysed. Positive for N-acetyl-β-glucosaminidase, acid phosphatase, alkaline phosphatase, β-galactosidase, α-glucosidase, β-glucosidase, gelatin hydrolysis, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and pyrazinamidase in API ZYM and API Coryne test systems, and negative for α-chymotrypsin, α-fucosidase, α-galactosidase, β-glucuronidase, lipase, α-mannosidase, pyrolidonyl arylamidase, trypsin and urease. Nitrate is not reduced. Starch is not degraded. d-Fructose, d-mannitol, sucrose, d-glucose, raffinose, sorbitol and myo-inositol are used as carbon sources. l-Arabinose, l-rhamnose, d-xylose and lactose are not utilized. The DNA G+C content of the type strain is 73 mol%.

    The type strain is Sp080513SC-31T (=NBRC 105049T =DSM 41970T), isolated from the marine sponge Haliclona sp.

    Description of Streptomyces marinus sp. nov.

    Streptomyces marinus (ma.ri′nus. L. masc. adj. marinus of the sea, marine).

    Aerobic, Gram-positive and non-motile actinomycete. Spore chains are spiral with multiple turns and spore surface is smooth. Predominant fatty acids are iso-C16 : 0, anteiso-C15 : 0, iso-C15 : 0 and iso-C14 : 0. Detailed fatty acid profile is given in Supplementary Table S2. Major respiratory quinone is MK-9 (H8); MK-9 (H6) and MK-9 (H10) are also present. Grows at 20–30 °C (optimally at 25–28 °C); grows weakly at 15 and 37 °C. Grows well at pH 6.0–9.0, while no growth is observed at pH 5.0. Growth occurs with 2–7 % (w/v) NaCl. Weak growth is observed with 1 and 10 % (w/v) NaCl. Nitrate is not reduced. Starch is not hydrolysed. Growth is good on ISP1 and ISP2 media and fair on other ISP media (ISP3–6). Grows poorly on ISP7 medium. Produces soluble vinaceous (violet red) pigment on most of the ISP media. Vegetative growth is dark brick-red and spores are not produced on most of the ISP media tested. Melanin pigments are not produced on ISP6 or ISP7 media. Spores are produced only on water agar. In API ZYM and API Coryne test systems, positive for N-acetyl-β-glucosaminidase, alkaline phosphatase, acid phosphatase, β-galactosidase, α-glucosidase, β-glucosidase, gelatin hydrolysis, leucine arylamidase, α-mannosidase and pyrazinamidase; negative for α-chymotrypsin, α-fucosidase, α-galactosidase, β-glucuronidase, naphthol-AS-BI-phosphohydrolase, lipase (C14), pyrolidonyl arylamidase, trypsin and urease. d-Fructose, d-mannitol, sucrose, d-glucose, l-arabinose and d-xylose are utilized as sole carbon sources; l-rhamnose, raffinose, sorbitol and myo-inositol are not utilized. The DNA G+C content of the type strain is 72 mol%.

    The type strain is Sp080513GE-26T (=NBRC 105047T =DSM 41968T), isolated from the marine sponge Haliclona sp.

    Acknowledgments

    This work was supported by a grant from the New Energy and Industrial Technology Department Organization (NEDO) of Japan. The authors would like to thank Mr Takeshi Fujiwara, OP BIO FACTORY Co., Ltd for his help in collecting the sponge sample.

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