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Streptomyces Siamensis Sp. Nov., and Streptomyces Similanensis Sp. Nov., Isolated from Thai Soils

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Streptomyces Siamensis Sp. Nov., and Streptomyces Similanensis Sp. Nov., Isolated from Thai Soils The Journal of Antibiotics (2013) 66, 633–640 & 2013 Japan Antibiotics Research Association All rights reserved 0021-8820/13 www.nature.com/ja ORIGINAL ARTICLE Streptomyces siamensis sp. nov., and Streptomyces similanensis sp. nov., isolated from Thai soils Paranee Sripreechasak1, Atsuko Matsumoto2, Khanit Suwanborirux3, Yuki Inahashi2, Kazuro Shiomi2,4, Somboon Tanasupawat1 and Yoko Takahashi2,4 Three actinomycete strains, KC-038T, KC-031 and KC-106T, were isolated from soil samples collected in the southern Thailand. The morphological and chemotaxonomic properties of strains KC-038T, KC-031 and KC-106T were consistent with the characteristics of members of the genus Streptomyces, that is, the formation of aerial mycelia bearing spiral spore chains; the presence of LL-diaminopimelic acid in the cell wall, MK-9 (H6), MK-9 (H4) and MK-9 (H8) as the predominant menaquinones; and C16:0, iso-C16:0 and anteiso-C15:0 as the major cellular fatty acids. 16S rRNA gene sequence analyses indicated that strains KC-038T and KC-031 were highly similar (99.9%), and they were closely related to S. olivochromogenes NBRC 3178T (98.1%) and S. psammoticus NBRC 13971T (98.1%). Strain KC-106T was closely related to S. seoulensis NBRC 16668T (98.9%), S. recifensis NBRC 12813T (98.9%), S. chartreusis NBRC 12753T (98.7%) and S. griseoluteus NBRC 13375T (98.4%). The values of DNA–DNA relatedness between the isolates and the type strains of the related species were below 70%. On the basis of the polyphasic evidence, the isolates should be classified as two novel species, namely Streptomyces siamensis sp. nov. (type strain, KC-038T ¼ NBRC 108799T ¼ PCU 328T ¼ TISTR 2107T) and Streptomyces similanensis sp. nov. (type strain, KC-106T ¼ NBRC 108798T ¼ PCU 329T ¼ TISTR 2104T). The Journal of Antibiotics (2013) 66, 633–640; doi:10.1038/ja.2013.60; published online 12 June 2013 Keywords: new species; polyphasic taxonomy; Streptomycetaceae; Streptomyces siamensis; Streptomyces similanensis INTRODUCTION KC-038T, KC-031 and KC-106T, which were isolated from soils in the The genus Streptomyces belonging to the family Streptomycetaceae was south of Thailand. proposed by Waksman and Henrici1 to accommodate aerobic, Gram- positive and spore-forming actinomycetes. The Streptomyces strains MATERIALS AND METHODS represent a group of actinomycetes that are widely distributed in Strains KC-038T and KC-031 were isolated from soil samples collected from the nature. At present, the genus comprises more than 550 recognized Krung Ching Waterfall, Khao Luang National Park, Nakhon Si Thammarat species with validly published names, and recently some novel species, Province, Thailand, and strain KC-106T was isolated from the Similan Island 2 3 4 5 including S. cocklensis, S. gramineus, S. nanhaiensis, S. panacagri, National Park (813900900N9713802700E), Phanga Province, Thailand. The soil S. pharmamarensis,6 S. qinglanensis7 and S. staurosporininus,8 have samples were serially diluted with distilled water, heated at 55 1C for 5 min and been described. Strains of the genus Streptomyces are superior to other plated onto potato starch-glycerol agar10 and starch casein nitrate agar11 actinomycete strains in their ability to produce various bioactive containing nystatin (25 mg l À1) and tetracycline (10 mg l À1). The resulting metabolites, especially antibiotics. Well-known antibiotics derived pure isolates were maintained on SYM agar (starch 1.0%, NZ amine 0.3%, from Streptomyces strains include tetracycline, streptomycin, yeast extract 0.1%, meat extract 0.1%, CaCO3 0.3%, agar 1.2%, pH 7.0). chloramphenicol, neomycin, nystatin, amphotericin, kanamycin and Genomic DNA of each isolate was obtained by sonication of a suspension of cells12 grown in YD broth (yeast extract 1.0%, dextrose 1.0%, pH 7.0). The 16S cycloheximide. Streptomyces strains are still a rich source of rRNA gene was amplified using the primers described by Takahashi et al.13 The commercially significant compounds, such as antibiotics, enzymes, PCR products were sequenced on a DNA sequencer (model 3130 Genetic 9 enzyme inhibitors and other pharmacologically active agents. Analyzer; Applied Biosystems, Foster City, CA, USA) using a BigDye Therefore, new species in the genus Streptomyces remains a focus of Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to efficient research for the discovery of new bioactive compounds. the manufacturer’s instructions. The closest phylogenetic neighbors were In this paper, we report the taxonomic status of Streptomyces strains identified by BLAST searches using the EzTaxon-e server.14 The clustalw2 1Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand; 2Kitasato Institute for Life Sciences, Kitasato University, Minato-ku, Tokyo, Japan; 3Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand and 4Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo, Japan Correspondence: Dr S Tanasupawat, Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand. E-mail: [email protected] or Dr Y Takahashi, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108 8641, Japan. E-mail: [email protected] Received 4 January 2013; revised 2 May 2013; accepted 9 May 2013; published online 12 June 2013 Streptomyces siamensis and S. similanensis P Sripreechasak et al 634 program was used for multiple alignments with selected sequences for and maximum-parsimony19 methods. Data were resampled with 1000 calculating evolutionary distances15 with SeaView version 4.2.16 Phylogenetic bootstrap replications.20 Values for sequence similarity among the closely trees were constructed using the neighbour-joining,17 maximum-likelihood18 related strains were determined using the EzTaxon-e server.14 Table 1 Cellular fatty acid compositions (%) of strains KC-038T, KC-031 and closely related type strains Table 2 Cellular fatty acid compositions (%) of strain KC-106T and closely related type strains S. olivochromogenes S. psammoticus T T T Fatty acid KC-038 KC-031 NBRC 3178 NBRC 13971 S. seoulensis S. recifensis S. griseoluteus S. chartreusis Saturated straight chain NBRC NBRC NBRC NBRC C14:0 4.0 3.0 0.7 3.2 Fatty acid KC-106T 16668T 12813T 13375T 12753T C16:0 23.5 19.7 7.6 23.9 C17:0 1.2 1.0 0.5 1.0 Saturated straight chain C18:0 —— — 0.5 C14:0 0.6 0.8 — 0.8 2.0 C17:0cyclo 0.5 0.7 2.9 3.7 C16:0 5.4 4.4 5.7 8.5 9.7 C18:0 1.1 2.1 0.6 — — Unsaturated straight chain C17:0 cyclo 2.0 2.4 3.3 1.8 0.4 C 8c 0.6 0.6 — — 17:1 o Unsaturated straight chain C o9c — 2.2 — — — Saturated branched chain 18:1 iso-C14:0 8.6 8.5 8.0 2.0 Saturated branched chain iso-C15:0 7.7 8.9 13.0 10.8 iso-C14:0 5.8 10.2 6.7 4.2 7.3 iso-C16:0 18.4 22.3 22.1 10.5 iso-C15:0 8.6 8.9 10.6 9.3 11.6 iso-C 23.2 20.1 19.3 17.9 19.8 iso-C17:0 1.7 2.7 4.9 2.9 16:0 iso-C —— 0.9 0.6 iso-C17:0 3.3 1.1 3.6 2.8 2.4 18:0 iso-C 1.5 1.0 1.5 1.1 — anteiso-C 0.5 — — — 18:0 13:0 anteiso-C13:0 ND — — 0.6 1.3 anteiso-C15:0 17.7 16.6 25.2 22.6 anteiso-C15:0 25.0 26.2 29.3 29.9 2.8 anteiso-C17:0 4.2 4.6 6.7 9.8 anteiso-C17:0 10.3 4.2 8.0 13.6 5.8 Unsaturated branched chain Unsaturated branched chain iso-C H 4.4 5.9 2.2 1.3 1.6 iso-C16:1 H 0.6 0.9 1.0 — 16:1 iso-C17:1 o9c 1.6 1.7 2.2 1.7 1.4 iso-C17:1 o9c 0.6 1.0 1.9 0.5 anteiso-C17:1 4.1 4.3 3.2 2.6 1.2 anteiso-C17:1 0.5 0.6 1.2 0.7 o9c o9c Summed 0.9 2.0 1.4 2.2 6.9 Summed 6.0 5.5 1.2 4.4 featurea 3 featurea 3 Abbreviation: ND, not detected. —, the amount of fatty acid less than 0.5% was omitted. —, the amount of fatty acid less than 0.5% was omitted. a a Summed feature 3 comprises C16:1 o7c and/or C16:1 o6c. Summed feature 3 comprises C16:1 o7c and/or C16:1 o6c. Figure 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strains KC-038T, KC-031 and closely related type strains of the genus Streptomyces. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. (K), branches were also recovered in the maximum-parsimony tree; (*), branches were also recovered in the maximum-likelihood tree; Bar, 0.005 nucleotide substitutions persite. The Journal of Antibiotics Streptomyces siamensis and S. similanensis P Sripreechasak et al 635 Strains KC-038T,KC-031andKC-106T were cultivated at 27 1C for 2 weeks using a CAPCELL PAK C18 UG120 column (Shiseido, Tokyo, Japan) with on ISP (International Streptomyces Project) 2, 3, 4, 5, 6 and 7 media,21 YS agar methanol/2-propanol (7:3). The N-acyl types of muramic acid were (yeast extract 2.0%, starch 1.0%, agar 1.5%, pH 7.0) and nutrient agar. The determined by using the method of Uchida and Aida.27 Phospholipids were Color Harmony Manual22 was used to determine the color of aerial and extracted and identified by using the method of Minnikin et al.28 The presence substrate mycelia and soluble pigment.
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