IJSEM Papers in Press. Published April 3, 2014 as doi:10.1099/ijs.0.048264-0
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Streptomyces catbensis sp. nov. isolated in Vietnam Yayoi Sakiyama1, Nguyen M. Giang 2, Shinji Miyadoh1, Dao Thi Luong2, Duong Van Hop2, and Katsuhiko Ando1 1
NITE-Biological Resource Center, National Institute of Technology and Evaluation (NBRC-NITE), Chiba, Japan 2 Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam (IMBT-VNUH)
Corresponding author: Yayoi Sakiyama NITE-Biological Resource Center, National Institute of Technology and Evaluation (NBRCNITE), 2-5-8 Kazusakamatarti, Kisarazu, Chiba, 292-0818, Japan Tel: +81-438-20-5763 Fax: +81-438-52-2329 E-mail: [email protected] Running head: Streptomyces catbensis sp. nov. Keywords: Streptomyces, new species, Vietnam, actinomycetes DDBJ accession number for the 16S rRNA gene sequence of Streptomyces catbensis VN07A0015T (= VTCC-A-1889T = NBRC 107860T) is AB705486.
Abstract Strain VN07A0015T was isolated from soil collected on Cat Ba Island, Vietnam. The taxonomic position of strain VN07A0015T was near Streptomyces aomiensis (98.5% similarity) and Streptomyces scabrisporus (95.6%), and it clustered within them; however, this cluster was distant from the type strains of other Streptomyces species. The aerial mycelia of strain VN07A0015T were greyish and formed imperfect spiral spore chains (retinaculiaperti type) with smooth-surfaced spores. The morphological features of strain VN07A0015T were different from those of S. aomiensis and S. scabrisporus. The chemotaxonomic characteristics of strain VN07A0015T were typical for all members of the genus Streptomyces, which possessed LL-type diaminopimelic acid, menaquinone MK-9(H6, H8) and the major fatty acids iso-C16:0 and iso-C15:0. The result of DNA relatedness between strain VN07A0015T and S. aomiensis NBRC 106164T was less than 30%. In addition, some physiological and biochemical traits differed from those of S. aomiensis. Therefore, we propose that strain VN07A0015T be classified in the genus Streptomyces as a representative of Streptomyces catbensis sp. nov. (Type strain VN07A0015T = VTCC-A-1889T = NBRC 107860T).
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Strains of genus Streptomyces produce a wide variety of antibiotics and enzymes (Goodfellow et al., 2010). It is easy to screen for strains of this genus because of their good growth and ease of isolation from nature. Consequently, many novel species have been proposed, and the genus Streptomyces contains about 600 validly published species at the time of writing this manuscript (List of Prokaryotic Names with Standing in Nomenclature; http://www.bacterio.cict.fr/). The genus Streptomyces also has distinctive morphological traits. Cultures produce colourful substrate mycelia, aerial mycelia, and soluble pigments. Sporulating aerial hyphae typically have a fibrous sheath (Wildermuth & Hopwood, 1970; Hardisson & Manzanal, 1976). Moreover, aerial mycelia have variously shaped spore chains (including short-long types, straight, rectiflexibiles, hook, loop, rectinaculiaperti, and spiral types) and spore surfaces (including smooth, warty, rugose, spiny, and hairy) (Pridham et al., 1958). Shirling & Gottlieb (1966) described the methods using the International Streptomyces Project (ISP) and introduced the above morphological characterizations. This project included ISP media No. 2–7, which are basic items for the observation of Streptomyces. Because morphology remains important in taxonomical classification of Streptomyces species, in this study, we identify and propose strain VN07A0015T as a novel species in the genus Streptomyces. Strain VN07A0015T was isolated from soil collected on Cat Ba Island in Vietnam. The soil sample was dried at room temperature for 3–5 days, and used by the sodium dodecyl sulphate-yeast extract dilution method (Hayakawa & Nonomura et al., 1989) on a humic acid-vitamin medium (Hayakawa & Nonomura, 1987) containing nalidixic acid (20 mg l-1) and kabicidine (7.5 mg l-1). Strain VN07A0015T was isolated from the humic acid-vitamin medium after incubation at room temperature for approximately 10 days. When strain VN07A0015T was cultured on ISP No. 2 medium at 28°C (Shirling & Gottlieb, 1966), the greyish aerial mycelia, the imperfect spiral spore chains (retinaculiaperti type), and the smooth surface spores were observed with light microscopy and scanning electron microscopy (Fig. 1). The cultural characteristics of strain VN07A0015T were observed on ISP No. 2–7 media (Table 1). The investigation of optimum temperature (5, 10, 15, 20, 25, 28, and 37°C), pH (3, 4, 5, 6, 7, 8, and 9), and NaCl concentration (1%, 2%, 3%, 4%, and 5%) were observed on a yeast extractsoluble starch medium (YS medium; 2 g yeast-extract, 10 g soluble starch and 15 g agar per 1 litre of distilled water; pH 7.3). Good growth of strain VN07A0015T was observed on ISP No. 2 medium; moderate growth on ISP No. 3, 5, 6, and 7 media; and no growth on ISP No. 4 medium. Strain VN07A0015T formed greyish aerial mycelia on ISP No. 2, 3, 5, and 7 media and did not produce a soluble pigment. Strain VN07A0015 T grew at temperatures of 15°C, 20°C, 25°C, and 28°C and at pH levels of 5, 6, 7, 8, and 9. The optimal temperature was 28°C and the optimal pH was 6–7. Strain VN07A0015T grew on YS media with a NaCl concentration of 0–2%. The biomass of VN07A0015T was cultured in a yeast extract-glucose medium (10 g yeast-extract, 10 g glucose per 1 litre of distilled water; pH 7.3) for 3–5 days at 28°C, harvested by centrifugation, rinsed by sterilized water, and then lyophilized. The types of diaminopimelic acids and whole-cell sugars were analysed as described by Staneck & Roberts (1974), isoprenoid quinines and phospholipids by Minnikin et al. (1984),
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cellular fatty acids by Sasser (1990) and by using the MIDI Sherlock Microbial Identification system (Microbial ID), respectively. Strain VN07A0015T possessed LLdiaminopimelic acid, arabinose, glucose, rhamnose and ribose in whole cell. The major isoprenoid quinones were MK-9(H6) and MK-9(H8), and the minor isoprenoid quinone was MK9-(H4). Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), six unknown lipids and one ninhydrin-positive unknown lipid were detected (Fig. 2), but not phosphatidylcholine (PC) and phosphatidylglycerol (PG). The predominant cellular fatty acids were iso-C16:0 (28.0%) and iso-C15:0 (22.2%) based on MIDI system ver. 4.02 (Table 2). The major fatty acids composition of the strain VN07A0015T and Streptomyces aomiensis NBRC 106164T were branched type and pattern 2a, however Streptomyces scabrisporus NBRC 100760T were non-branched type and pattern 2c. These aspects of the morphology and chemotaxonomy of strain VN07A0015T conformed to the characteristics of the genus Streptomyces. DNA of strain VN07A0015T was extracted, amplified, and its 16S rRNA gene was sequenced according to the techniques of Sakiyama et al. (2009). Sequence analysis was carried out with an ABI Prism BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems) and an automatic DNA sequencer (model 3130 Genetic Analyzer; PE Applied Biosystems). The 16S rRNA gene sequences were aligned by the CLUSTAL X program (Thompson et al., 1997) with corresponding sequences from some type strains of Streptomyces species available in the DDBJ/EMBL/GenBank database. Subsequently, phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987), maximum-likelihood method (Felsenstein, 1981), and maximumparsimony method (Kluge & Farris, 1969) using MEGA 5 (Tamura et al., 2011). The topology of the constructed tree was evaluated by bootstrap analysis with 1000 replicates (Felsenstein, 1985). DNA–DNA hybridization and G+C contents of the DNA were examined by the method described previously (Sakiyama et al., 2009). In the phylogenetic analysis based on the 16S rRNA gene, strain VN07A0015T was most similar to S. aomiensis (98.5% similarity) and S. scabrisporus (95.6%). Strain VN07A0015T was clustered with them in a phylogenetic tree (Fig. 3), and the taxonomic position of this cluster was distant from that of the other type strains of the genus Streptomyces. The DNA relatedness between strain VN07A0015T and S. aomiensis NBRC 106164T were 29.5% (probe VN07A0015T) and 8.4% (probe S. aomiensis NBRC 106164T). The values are well below the 70% cut-off point recommended for the delineation of genomic species (Wayne et al., 1987). Therefore, strain VN07A0015T was different from S. aomiensis. The G+C content of the DNA was 73.2%. In biochemical and physiological tests, the utilization of different carbon sources and enzyme reaction by strain VN07A0015T, S. aomiensis NBRC 106164T and S. scabrisporus NBRC 100760T was examined using methods described previously (Sakiyama et al., 2009) and API Zym (REF 25 207, BioMérieux Inc.) and API Coryne (REF 20 907). The API examines of the strain VN07A0015T and S. aomiensis NBRC 106164T were almost same results, but the results of S. scabrisporus NBRC 100760T was fairly different with them (Table 3). Strain VN07A0015T showed some differences from S. aomiensis NBRC 106164T in the utilization of meso-erythritol, D-raffinose, and
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D-xylose.
Therefore, we propose that strain VN07A0015T is a novel species in the genus Streptomyces, which is named Streptomyces catbensis sp. nov. The type strain is VN07A0015T (= VTCC-A-1889T = NBRC 107860T). Description of Streptomyces catbensis sp. nov. Streptomyces catbensis (cat.ben’en.sis. N.L. masc. adj., catbensis of or belonging to Cat Ba Island in Vietnam, from which the strain was isolated). The substrate and aerial mycelia are developed on ISP No. 2 medium. The colour of the substrate mycelium and aerial mycelium are strong brown and grey, respectively, on ISP No. 2 medium. It grows moderately on ISP Nos. 3, 5, 6, and 7 but not 4. Aerial mycelia are found on ISP Nos. 2, 3, 5, and 7. Spore chains are imperfect spiral (retinaculiaperti type), and the spore surface is smooth. It grows at 15–28°C (optimum 25–28°C) and pH 5–9 (optimum 6–8). It does not hydrolyse starch, liquefy gelatin, and coagulate milk. Urease activity is negative, and nitrate reduction is positive. Decomposition occurs on tyrosine but not on hypoxanthine (0.4%), adenine, and xanthine. As sole carbon sources, L-arabinose, D-cellobiose, D-fructose, D-glucose, lactose, maltose, D-mannitol, melibiose, L-rhamnose, ribose, D-salicin, D-sorbitol, sucrose, and D-trehalose are utilized, but meso-erythritol, myo-inositol, D-raffinose, and D-xylose are not. Menaquinones are MK-9(H6, H8), and the major fatty acids are iso-C16:0 and iso-C15:0 (pattern 2a). Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), six unknown lipids and one ninhydrin-positive unknown lipid were detected (pattern PⅡ). The G+C contents of the DNA of the type strain are 73.2%. Strain VN07A0015T (= VTCC-A-1889T = NBRC 107860T) was isolated from soil collected on Cat Ba Island in Vietnam. Acknowledgements This work was conducted as a joint research project between NBRC-NITE and IMBTVNUH. The authors thank Dr. Tomohiko Tamura, Ms. Kozue Anzai, Mr. Nobuyuki Goto, Mr. Shinpei Ino, Ms. Ayako Hashimoto, Ms. Mayuko Sukisaki (NBRC), and Ms. Nguyen Thi Van (Vietnam type culture collection) for experimental assistance. We are grateful to Dr. Dinh Thuy Hang, Dr. Nguyen Lan Dung, and all VTCC members for supporting our joint project. References Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Goodfellow, M. & Fiedler, H.P. (2010). A guide to success bioprospecting: informed by actinobacterial systematic. Antonie van Leeuwenhoek 98, 119–142. Hardisson, C. & Manzanal, M. B. (1976). Ultrastructural studies of sporulation in Streptomyces. J Bacteriol 127, 1443–1454. Hayakawa, M. & Nonomura, H. (1989). A new method for the intensive isolation of Actinomycetes from soil. Actinomycetologica 3, 95-104. Hayakawa, M. & Nonomura, H. (1987). Humic acid-vitamin agar, a new medium for
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selective isolation of soil actinomycetes. J Ferment Technol 65, 501–509. Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the evolution of anurans. Syst Zool 18, 1–32. Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated producer for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241. Ping, X., Takahashi, Y., Seino, A., Iwai, Y. & Ōmura, S. (2004). Streptomyces scabrisporus sp. nov.. Int J Syst Evol Microbiol 54, 577–581. Pridham, T. G., Hesseltine, C. W. & Benedict, R. G. (1958). A Guide for the Classification of Streptomycetes According to Selected Groups. Placement of Strains in Morphological Sections. Appl. Microbiol. 6 (1), 52. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Sakiyama, Y., Nguyen K. N. T., Nguyen M. G., Miyadoh, S., Duong V. H. & Ando, K. (2009). Kineosporia babensis sp. nov., isolated from plant litter in Vietnam. Int J Syst Evol Microbiol 59, 550–554. Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340. Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28, 226–231. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731– 2739. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463-464. Wildermuth, H. & Hopwood, D. A. (1970). Septation during sporulation in Streptomyces coelicolor. J gen Microbiol 60, 51–59.
225 226 227 228 229
Table 1 Culture characteristics of S. catbensis VN07A0015T, S. aomiensis NBRC 106164T, and S. scabrisporus NBRC 100760T. The described data were placed in the order: growth, colour of aerial mycelium, and colour of substrate mycelium.
230
Characteristics Spore chains Spore surface ISP medium Yeast extract/malt extract agar (ISP 2) Oatmeal agar (ISP 3) Inorganic salts/starch agar (ISP 4) Glycerol/asparagine agar (ISP 5) Peptone/yeast extract/iron agar (ISP 6) Tyrosine agar (ISP 7) 231
S. catbensis VN07A0015T Retinaculiaperti Smooth
S. aomiensis NBRC 106164T Rectiflexibles Smooth
S. scabrisporus NBRC 100760T Spiral Rugose
Good Light bluish grey Strong brown Moderate Light brownish grey Colourless Weak Light brownish gray Colourless Moderate Yellowish grey Colourless Moderate, Wet Moderate yellow Moderate Yellowish grey Pale yellow
Good White Vivid yellow Moderate White Light yellow Moderate White Colourless Moderate White Colourless Good, Wet White Moderate yellow Moderate White Pale yellow
Moderate, Wrinkled Light ivory Weak, Penetrating Colourless Weak, Penetrating Pearl Moderate, Penetrating Pearl Good, Wrinkled,Wet Pearl Moderate, Raised Bamboo
232 233 234 235 236
Table 2 Cellular fatty acid composition (%) of S. catbensis VN07A0015T, S. aomiensis NBRC 106164T and S. scabrisporus NBRC 100760T. Values less than 1% were omitted from this table. S. catbensis VN07A0015T
S. aomiensis NBRC 106164T
S. scabrisporus NBRC 100760T
11.0 22.2 4.1 12.4 28.0
9.0 19.0 16.0 4.9 25.6 6.3
5.2 6.3 14.8
13.1 4.3
6.1 7.4
24.9 18.7
4.9
5.8
3.7 5.8
Branched type iso-C14:0 iso-C15:0 anteiso-C15:0 iso-C16:1 H iso-C16:0 anteiso-C17:0 Non-branched type C16:1 cis 9 C16:0 Methyl type C16:0 9?Methyl Sum In Feature 237 238 239 240 241 242 243 244 245 246 247
10.9 6.1
Table 3 Biochemical and physiological characteristics of S. catbensis sp. nov. VN07A0015T, S. aomiensis NBRC 106164T, and S. scabrisporus NBRC 100760T.
Characteristics
S. catbensis VN07A0015T
S. aomiensis NBRC 106164T
S. scabrisporus NBRC 100760T
＋ -
＋ -
+
-
-
+ + +
+
-
+ +
API ZYM Esterase C4 α-cymotrypsin α-glucosidase α-fucosidase API Coryne Pyrrolidonyl arylamidase Pyrrolidonyl arylamidase α-glucosidase Utilization of D-xylose D-raffinose
+
+ +
inositol 248 249 250
+
-
-
251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271
272 273
Figure legends Figure 1 Scanning electron micrograph of strain VN07A0015T grown on humic acid-vitamin medium for 3 weeks at 28°C. Bar is 10 µm. Figure 2 Phospholipids analysis of S. catbensis sp. nov. VN07A0015T The detected regents are Molybdatophosphoric acid (plate 1), Dittmer-Lester (plate 2), Schiff (plate 3), and Anisaldehyde (plate 4). Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), unknown lipid (PL), and ninhydrin-positive unknown lipid (NL) are abbreviated. Figure 3 Phylogenetic tree of strain VN07A0015T with related type strains of Streptomyces species based on 16S rRNA gene sequences. The tree was constructed using the neighbour-joining method (Saitou & Nei, 1987). Scale bar = 0.005 Knuc in nucleotide sequences. The numbers on the branches are the confidence limits estimated by bootstrap analysis with 1,000 replicates (only values above 500 are presented). A closed circle indicates a branch that was identical in both maximum-likelihood method and maximum-parsimony method analyses; open circles indicate maximum-likelihood method analysis only.
275 0.005 Knuc
Streptomyces cacaoi subsp. cacaoi NBRC 12748T (AB184115)
Streptomyces sodiiphilus YIM 80305T (AY236339)
Streptomyces gibsonii NBRC 15415T (AB184663) 928 577
Streptomyces rangoonensis LMG 20295T (AJ781366)
Streptomyces almquistii NBRC 13015T (AB184258) 999
576
Streptomyces albus subsp. albus NRRL B-2365T (DQ026669)
Streptomyces flocculus NBRC 13041T (AB184272)
Streptomyces rimosus subsp. rimosus JCM 4667T (AB045883) 727
1000
Streptomyces chrestomyceticus DSM 40545T (AJ621609)
Streptomyces rimosus subsp. paromomycinus DSM 41429T (AJ621610) 616
Streptomyces catenulae ISP 5258T (AY999778)
Streptomyces mashuensis DSM 40221T (X79323)
994
1000 613
707
Streptomyces sparsogenes NBRC 13086T (AB184301)
Streptomyces cuspidosporus NBRC 12378T (AB184090)
Streptomyces hiroshimensis NBRC 3839T (AB184802)
Streptomyces iranensis HM 35T (FJ472862)
Streptomyces mobaraensis NBRC 13819T (AB184870)
Streptomyces xinghaiensis CCTCC AA 208049T (EF577247)
Streptomyces scabrisporus NBRC 100760T (AB249946) 965
Streptomyces aomiensis M24DS4T (AB522686) 1000
VN07A0015T (AB705486)
276
Nocardioides albus KCTC9186T (AF004988)
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Streptomyces catbensis sp. nov. isolated in Vietnam Yayoi Sakiyama1, Nguyen M. Giang 2, Shinji Miyadoh1, Dao Thi Luong2, Duong Van Hop2, and Katsuhiko Ando1 1
NITE-Biological Resource Center, National Institute of Technology and Evaluation (NBRC-NITE), Chiba, Japan 2 Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam (IMBT-VNUH)
Corresponding author: Yayoi Sakiyama NITE-Biological Resource Center, National Institute of Technology and Evaluation (NBRCNITE), 2-5-8 Kazusakamatarti, Kisarazu, Chiba, 292-0818, Japan Tel: +81-438-20-5763 Fax: +81-438-52-2329 E-mail: [email protected] Running head: Streptomyces catbensis sp. nov. Keywords: Streptomyces, new species, Vietnam, actinomycetes DDBJ accession number for the 16S rRNA gene sequence of Streptomyces catbensis VN07A0015T (= VTCC-A-1889T = NBRC 107860T) is AB705486.
Abstract Strain VN07A0015T was isolated from soil collected on Cat Ba Island, Vietnam. The taxonomic position of strain VN07A0015T was near Streptomyces aomiensis (98.5% similarity) and Streptomyces scabrisporus (95.6%), and it clustered within them; however, this cluster was distant from the type strains of other Streptomyces species. The aerial mycelia of strain VN07A0015T were greyish and formed imperfect spiral spore chains (retinaculiaperti type) with smooth-surfaced spores. The morphological features of strain VN07A0015T were different from those of S. aomiensis and S. scabrisporus. The chemotaxonomic characteristics of strain VN07A0015T were typical for all members of the genus Streptomyces, which possessed LL-type diaminopimelic acid, menaquinone MK-9(H6, H8) and the major fatty acids iso-C16:0 and iso-C15:0. The result of DNA relatedness between strain VN07A0015T and S. aomiensis NBRC 106164T was less than 30%. In addition, some physiological and biochemical traits differed from those of S. aomiensis. Therefore, we propose that strain VN07A0015T be classified in the genus Streptomyces as a representative of Streptomyces catbensis sp. nov. (Type strain VN07A0015T = VTCC-A-1889T = NBRC 107860T).
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Strains of genus Streptomyces produce a wide variety of antibiotics and enzymes (Goodfellow et al., 2010). It is easy to screen for strains of this genus because of their good growth and ease of isolation from nature. Consequently, many novel species have been proposed, and the genus Streptomyces contains about 600 validly published species at the time of writing this manuscript (List of Prokaryotic Names with Standing in Nomenclature; http://www.bacterio.cict.fr/). The genus Streptomyces also has distinctive morphological traits. Cultures produce colourful substrate mycelia, aerial mycelia, and soluble pigments. Sporulating aerial hyphae typically have a fibrous sheath (Wildermuth & Hopwood, 1970; Hardisson & Manzanal, 1976). Moreover, aerial mycelia have variously shaped spore chains (including short-long types, straight, rectiflexibiles, hook, loop, rectinaculiaperti, and spiral types) and spore surfaces (including smooth, warty, rugose, spiny, and hairy) (Pridham et al., 1958). Shirling & Gottlieb (1966) described the methods using the International Streptomyces Project (ISP) and introduced the above morphological characterizations. This project included ISP media No. 2–7, which are basic items for the observation of Streptomyces. Because morphology remains important in taxonomical classification of Streptomyces species, in this study, we identify and propose strain VN07A0015T as a novel species in the genus Streptomyces. Strain VN07A0015T was isolated from soil collected on Cat Ba Island in Vietnam. The soil sample was dried at room temperature for 3–5 days, and used by the sodium dodecyl sulphate-yeast extract dilution method (Hayakawa & Nonomura et al., 1989) on a humic acid-vitamin medium (Hayakawa & Nonomura, 1987) containing nalidixic acid (20 mg l-1) and kabicidine (7.5 mg l-1). Strain VN07A0015T was isolated from the humic acid-vitamin medium after incubation at room temperature for approximately 10 days. When strain VN07A0015T was cultured on ISP No. 2 medium at 28°C (Shirling & Gottlieb, 1966), the greyish aerial mycelia, the imperfect spiral spore chains (retinaculiaperti type), and the smooth surface spores were observed with light microscopy and scanning electron microscopy (Fig. 1). The cultural characteristics of strain VN07A0015T were observed on ISP No. 2–7 media (Table 1). The investigation of optimum temperature (5, 10, 15, 20, 25, 28, and 37°C), pH (3, 4, 5, 6, 7, 8, and 9), and NaCl concentration (1%, 2%, 3%, 4%, and 5%) were observed on a yeast extractsoluble starch medium (YS medium; 2 g yeast-extract, 10 g soluble starch and 15 g agar per 1 litre of distilled water; pH 7.3). Good growth of strain VN07A0015T was observed on ISP No. 2 medium; moderate growth on ISP No. 3, 5, 6, and 7 media; and no growth on ISP No. 4 medium. Strain VN07A0015T formed greyish aerial mycelia on ISP No. 2, 3, 5, and 7 media and did not produce a soluble pigment. Strain VN07A0015 T grew at temperatures of 15°C, 20°C, 25°C, and 28°C and at pH levels of 5, 6, 7, 8, and 9. The optimal temperature was 28°C and the optimal pH was 6–7. Strain VN07A0015T grew on YS media with a NaCl concentration of 0–2%. The biomass of VN07A0015T was cultured in a yeast extract-glucose medium (10 g yeast-extract, 10 g glucose per 1 litre of distilled water; pH 7.3) for 3–5 days at 28°C, harvested by centrifugation, rinsed by sterilized water, and then lyophilized. The types of diaminopimelic acids and whole-cell sugars were analysed as described by Staneck & Roberts (1974), isoprenoid quinines and phospholipids by Minnikin et al. (1984),
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cellular fatty acids by Sasser (1990) and by using the MIDI Sherlock Microbial Identification system (Microbial ID), respectively. Strain VN07A0015T possessed LLdiaminopimelic acid, arabinose, glucose, rhamnose and ribose in whole cell. The major isoprenoid quinones were MK-9(H6) and MK-9(H8), and the minor isoprenoid quinone was MK9-(H4). Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), six unknown lipids and one ninhydrin-positive unknown lipid were detected (Fig. 2), but not phosphatidylcholine (PC) and phosphatidylglycerol (PG). The predominant cellular fatty acids were iso-C16:0 (28.0%) and iso-C15:0 (22.2%) based on MIDI system ver. 4.02 (Table 2). The major fatty acids composition of the strain VN07A0015T and Streptomyces aomiensis NBRC 106164T were branched type and pattern 2a, however Streptomyces scabrisporus NBRC 100760T were non-branched type and pattern 2c. These aspects of the morphology and chemotaxonomy of strain VN07A0015T conformed to the characteristics of the genus Streptomyces. DNA of strain VN07A0015T was extracted, amplified, and its 16S rRNA gene was sequenced according to the techniques of Sakiyama et al. (2009). Sequence analysis was carried out with an ABI Prism BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems) and an automatic DNA sequencer (model 3130 Genetic Analyzer; PE Applied Biosystems). The 16S rRNA gene sequences were aligned by the CLUSTAL X program (Thompson et al., 1997) with corresponding sequences from some type strains of Streptomyces species available in the DDBJ/EMBL/GenBank database. Subsequently, phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987), maximum-likelihood method (Felsenstein, 1981), and maximumparsimony method (Kluge & Farris, 1969) using MEGA 5 (Tamura et al., 2011). The topology of the constructed tree was evaluated by bootstrap analysis with 1000 replicates (Felsenstein, 1985). DNA–DNA hybridization and G+C contents of the DNA were examined by the method described previously (Sakiyama et al., 2009). In the phylogenetic analysis based on the 16S rRNA gene, strain VN07A0015T was most similar to S. aomiensis (98.5% similarity) and S. scabrisporus (95.6%). Strain VN07A0015T was clustered with them in a phylogenetic tree (Fig. 3), and the taxonomic position of this cluster was distant from that of the other type strains of the genus Streptomyces. The DNA relatedness between strain VN07A0015T and S. aomiensis NBRC 106164T were 29.5% (probe VN07A0015T) and 8.4% (probe S. aomiensis NBRC 106164T). The values are well below the 70% cut-off point recommended for the delineation of genomic species (Wayne et al., 1987). Therefore, strain VN07A0015T was different from S. aomiensis. The G+C content of the DNA was 73.2%. In biochemical and physiological tests, the utilization of different carbon sources and enzyme reaction by strain VN07A0015T, S. aomiensis NBRC 106164T and S. scabrisporus NBRC 100760T was examined using methods described previously (Sakiyama et al., 2009) and API Zym (REF 25 207, BioMérieux Inc.) and API Coryne (REF 20 907). The API examines of the strain VN07A0015T and S. aomiensis NBRC 106164T were almost same results, but the results of S. scabrisporus NBRC 100760T was fairly different with them (Table 3). Strain VN07A0015T showed some differences from S. aomiensis NBRC 106164T in the utilization of meso-erythritol, D-raffinose, and
140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
D-xylose.
Therefore, we propose that strain VN07A0015T is a novel species in the genus Streptomyces, which is named Streptomyces catbensis sp. nov. The type strain is VN07A0015T (= VTCC-A-1889T = NBRC 107860T). Description of Streptomyces catbensis sp. nov. Streptomyces catbensis (cat.ben’en.sis. N.L. masc. adj., catbensis of or belonging to Cat Ba Island in Vietnam, from which the strain was isolated). The substrate and aerial mycelia are developed on ISP No. 2 medium. The colour of the substrate mycelium and aerial mycelium are strong brown and grey, respectively, on ISP No. 2 medium. It grows moderately on ISP Nos. 3, 5, 6, and 7 but not 4. Aerial mycelia are found on ISP Nos. 2, 3, 5, and 7. Spore chains are imperfect spiral (retinaculiaperti type), and the spore surface is smooth. It grows at 15–28°C (optimum 25–28°C) and pH 5–9 (optimum 6–8). It does not hydrolyse starch, liquefy gelatin, and coagulate milk. Urease activity is negative, and nitrate reduction is positive. Decomposition occurs on tyrosine but not on hypoxanthine (0.4%), adenine, and xanthine. As sole carbon sources, L-arabinose, D-cellobiose, D-fructose, D-glucose, lactose, maltose, D-mannitol, melibiose, L-rhamnose, ribose, D-salicin, D-sorbitol, sucrose, and D-trehalose are utilized, but meso-erythritol, myo-inositol, D-raffinose, and D-xylose are not. Menaquinones are MK-9(H6, H8), and the major fatty acids are iso-C16:0 and iso-C15:0 (pattern 2a). Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), six unknown lipids and one ninhydrin-positive unknown lipid were detected (pattern PⅡ). The G+C contents of the DNA of the type strain are 73.2%. Strain VN07A0015T (= VTCC-A-1889T = NBRC 107860T) was isolated from soil collected on Cat Ba Island in Vietnam. Acknowledgements This work was conducted as a joint research project between NBRC-NITE and IMBTVNUH. The authors thank Dr. Tomohiko Tamura, Ms. Kozue Anzai, Mr. Nobuyuki Goto, Mr. Shinpei Ino, Ms. Ayako Hashimoto, Ms. Mayuko Sukisaki (NBRC), and Ms. Nguyen Thi Van (Vietnam type culture collection) for experimental assistance. We are grateful to Dr. Dinh Thuy Hang, Dr. Nguyen Lan Dung, and all VTCC members for supporting our joint project. References Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Goodfellow, M. & Fiedler, H.P. (2010). A guide to success bioprospecting: informed by actinobacterial systematic. Antonie van Leeuwenhoek 98, 119–142. Hardisson, C. & Manzanal, M. B. (1976). Ultrastructural studies of sporulation in Streptomyces. J Bacteriol 127, 1443–1454. Hayakawa, M. & Nonomura, H. (1989). A new method for the intensive isolation of Actinomycetes from soil. Actinomycetologica 3, 95-104. Hayakawa, M. & Nonomura, H. (1987). Humic acid-vitamin agar, a new medium for
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selective isolation of soil actinomycetes. J Ferment Technol 65, 501–509. Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the evolution of anurans. Syst Zool 18, 1–32. Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated producer for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241. Ping, X., Takahashi, Y., Seino, A., Iwai, Y. & Ōmura, S. (2004). Streptomyces scabrisporus sp. nov.. Int J Syst Evol Microbiol 54, 577–581. Pridham, T. G., Hesseltine, C. W. & Benedict, R. G. (1958). A Guide for the Classification of Streptomycetes According to Selected Groups. Placement of Strains in Morphological Sections. Appl. Microbiol. 6 (1), 52. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Sakiyama, Y., Nguyen K. N. T., Nguyen M. G., Miyadoh, S., Duong V. H. & Ando, K. (2009). Kineosporia babensis sp. nov., isolated from plant litter in Vietnam. Int J Syst Evol Microbiol 59, 550–554. Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340. Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28, 226–231. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731– 2739. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463-464. Wildermuth, H. & Hopwood, D. A. (1970). Septation during sporulation in Streptomyces coelicolor. J gen Microbiol 60, 51–59.
225 226 227 228 229
Table 1 Culture characteristics of S. catbensis VN07A0015T, S. aomiensis NBRC 106164T, and S. scabrisporus NBRC 100760T. The described data were placed in the order: growth, colour of aerial mycelium, and colour of substrate mycelium.
230
Characteristics Spore chains Spore surface ISP medium Yeast extract/malt extract agar (ISP 2) Oatmeal agar (ISP 3) Inorganic salts/starch agar (ISP 4) Glycerol/asparagine agar (ISP 5) Peptone/yeast extract/iron agar (ISP 6) Tyrosine agar (ISP 7) 231
S. catbensis VN07A0015T Retinaculiaperti Smooth
S. aomiensis NBRC 106164T Rectiflexibles Smooth
S. scabrisporus NBRC 100760T Spiral Rugose
Good Light bluish grey Strong brown Moderate Light brownish grey Colourless Weak Light brownish gray Colourless Moderate Yellowish grey Colourless Moderate, Wet Moderate yellow Moderate Yellowish grey Pale yellow
Good White Vivid yellow Moderate White Light yellow Moderate White Colourless Moderate White Colourless Good, Wet White Moderate yellow Moderate White Pale yellow
Moderate, Wrinkled Light ivory Weak, Penetrating Colourless Weak, Penetrating Pearl Moderate, Penetrating Pearl Good, Wrinkled,Wet Pearl Moderate, Raised Bamboo
232 233 234 235 236
Table 2 Cellular fatty acid composition (%) of S. catbensis VN07A0015T, S. aomiensis NBRC 106164T and S. scabrisporus NBRC 100760T. Values less than 1% were omitted from this table. S. catbensis VN07A0015T
S. aomiensis NBRC 106164T
S. scabrisporus NBRC 100760T
11.0 22.2 4.1 12.4 28.0
9.0 19.0 16.0 4.9 25.6 6.3
5.2 6.3 14.8
13.1 4.3
6.1 7.4
24.9 18.7
4.9
5.8
3.7 5.8
Branched type iso-C14:0 iso-C15:0 anteiso-C15:0 iso-C16:1 H iso-C16:0 anteiso-C17:0 Non-branched type C16:1 cis 9 C16:0 Methyl type C16:0 9?Methyl Sum In Feature 237 238 239 240 241 242 243 244 245 246 247
10.9 6.1
Table 3 Biochemical and physiological characteristics of S. catbensis sp. nov. VN07A0015T, S. aomiensis NBRC 106164T, and S. scabrisporus NBRC 100760T.
Characteristics
S. catbensis VN07A0015T
S. aomiensis NBRC 106164T
S. scabrisporus NBRC 100760T
＋ -
＋ -
+
-
-
+ + +
+
-
+ +
API ZYM Esterase C4 α-cymotrypsin α-glucosidase α-fucosidase API Coryne Pyrrolidonyl arylamidase Pyrrolidonyl arylamidase α-glucosidase Utilization of D-xylose D-raffinose
+
+ +
inositol 248 249 250
+
-
-
251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271
272 273
Figure legends Figure 1 Scanning electron micrograph of strain VN07A0015T grown on humic acid-vitamin medium for 3 weeks at 28°C. Bar is 10 µm. Figure 2 Phospholipids analysis of S. catbensis sp. nov. VN07A0015T The detected regents are Molybdatophosphoric acid (plate 1), Dittmer-Lester (plate 2), Schiff (plate 3), and Anisaldehyde (plate 4). Phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), unknown lipid (PL), and ninhydrin-positive unknown lipid (NL) are abbreviated. Figure 3 Phylogenetic tree of strain VN07A0015T with related type strains of Streptomyces species based on 16S rRNA gene sequences. The tree was constructed using the neighbour-joining method (Saitou & Nei, 1987). Scale bar = 0.005 Knuc in nucleotide sequences. The numbers on the branches are the confidence limits estimated by bootstrap analysis with 1,000 replicates (only values above 500 are presented). A closed circle indicates a branch that was identical in both maximum-likelihood method and maximum-parsimony method analyses; open circles indicate maximum-likelihood method analysis only.
275 0.005 Knuc
Streptomyces cacaoi subsp. cacaoi NBRC 12748T (AB184115)
Streptomyces sodiiphilus YIM 80305T (AY236339)
Streptomyces gibsonii NBRC 15415T (AB184663) 928 577
Streptomyces rangoonensis LMG 20295T (AJ781366)
Streptomyces almquistii NBRC 13015T (AB184258) 999
576
Streptomyces albus subsp. albus NRRL B-2365T (DQ026669)
Streptomyces flocculus NBRC 13041T (AB184272)
Streptomyces rimosus subsp. rimosus JCM 4667T (AB045883) 727
1000
Streptomyces chrestomyceticus DSM 40545T (AJ621609)
Streptomyces rimosus subsp. paromomycinus DSM 41429T (AJ621610) 616
Streptomyces catenulae ISP 5258T (AY999778)
Streptomyces mashuensis DSM 40221T (X79323)
994
1000 613
707
Streptomyces sparsogenes NBRC 13086T (AB184301)
Streptomyces cuspidosporus NBRC 12378T (AB184090)
Streptomyces hiroshimensis NBRC 3839T (AB184802)
Streptomyces iranensis HM 35T (FJ472862)
Streptomyces mobaraensis NBRC 13819T (AB184870)
Streptomyces xinghaiensis CCTCC AA 208049T (EF577247)
Streptomyces scabrisporus NBRC 100760T (AB249946) 965
Streptomyces aomiensis M24DS4T (AB522686) 1000
VN07A0015T (AB705486)
276
Nocardioides albus KCTC9186T (AF004988)
