DOI: 10.15468/qmwq5e
title: Streptomyces krungchingensis sp. nov., isolated from soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Sripreechasak, Paranee, Phongsopitanun, Wongsakorn, Tamura, Tomohiko, Tanasupawat, Somboon (2017): Streptomyces krungchingensis sp. nov., isolated from soil. International Journal of Systematic and Evolutionary Microbiology 67 (1): 50-54, DOI: 10.1099/ijsem.0.001570, URL: http://dx.doi.org/10.1099/ijsem.0.001570AbstractA novel actinomycete, designated strain KC-035 T, was isolated from soil collected from Krung Ching Waterfall National Park, Nakhon Si Thammarat Province, Thailand. Its taxonomic position was determined using a polyphasic approach. The strain had morphological and chemotaxonomic properties typical of members of the genus Streptomyces: flexuous spore chain; LL-diaminopimelic acid in the cell-wall peptidoglycan; MK-9(H 8), MK-9(H 6) and MK-9(H 4) as menaquinones; diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside as phospholipids; anteiso-C 15: 0, C 16: 0, iso-C 16: 0, iso-C 15: 0 and iso-C 14: 0 as major cellular fatty acids; and DNA G+C content of 72 mol%. 16S rRNA gene sequence analysis revealed that strain KC-035 T showed high similarity to Streptomyces albiflavescens n20 T (99.16 %) and Streptomyces siamensis KC-038 T (98.43 %) as well as formed a monophyletic clade with them in the phylogenetic tree. On the basis of comparison of phenotypic properties and the low level of DNA–DNA relatedness, strain KC-035 T could be distinguished from its closely related Streptomyces species and is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces krungchingensis sp. nov. is proposed. The type strain is KC-035 T (= NBRC 110087 T = KCTC 29503 T = TISTR 2402 T).The genus Streptomyces, belonging to the family Streptomycetaceae, is the largest genus in the domain Bacteria and currently encompasses 787 species with validly published names (http://www.bacterio.net/streptomyces.html). Members of this genus are economically important because of their ability to produce antibiotics as well as other bioactive compounds [1]. During the course of our investigation into the diversity of actinomycetes in Thailand, we isolated a novel Streptomyces strain, designated KC-035 T, from soil collected from the southern part of the country. The aim of this work is to describe this new isolate and propose it as a representative of a novel species within the genus Streptomyces.Strain KC-035 T was isolated from a soil sample collected from Krung Ching Waterfall National Park, Nakhon Si Thammarat Province, Thailand, by using starch casein nitrate agar supplemented with nystatin (25 mg l ‒1) and tetracycline (10 mg l ‒1) as previous described by Sripreechasak et al. [2]. To observe the cultural characteristics, strain KC-035 T was cultured on various ISP (International Streptomyces Project) media as recommended by Shirling and Gottlieb [3]. The NBS/ISCC colour system [4] was used to determine the colour of aerial and substrate mycelia and soluble pigments. The morphology of spores was observed by light microscopy and scanning electron microscopy (model JSM-5410LV; JEOL) after the strain was cultivated on ISP 2 medium at 30 Ǫ C for 2 weeks. Nitrate reduction was determined using nitrate medium (beef extract 0.3 %, peptone 0.5 %, KNO 3 0.1 %, pH 7.0). Gelatin liquefaction was determined using glucose-peptone gelatin medium (glucose 2.0 %, peptone 0.5 %, gelatin 20 %, pH 7.0). The coagulation and peptonization of skimmed milk was tested using 10 % (w/v) skimmed milk [5, 6]. Enzymatic activities were determined using the API ZYM system (bioḾerieux). The temperature (15, 25, 30, 37 and 45 Ǫ C), pH (5–9) and NaCl concentration (0–7 %, w/v) for growth were determined on ISP 2 medium and observed after incubation at 30 Ǫ C for 2 weeks.The freeze-dried cells for chemotaxonomic studies were prepared by cultivating the organism in yeast-dextrose broth (yeast extract 1.0 %, glucose 1.0 %, pH 7.0–7.2) under shaking conditions at 30 Ǫ C for 5 days. The isomers of diaminopimelic acids and sugars were determined by TLC using whole-cell hydrolysates [7]. Isoprenoid quinones were extracted according to the method of Collins et al. [8] and analysed by HPLC using a Cosmosil 5 C18 (4.6× 150 mm; chains were observed on aerial mycelia. Spore surfaces were smooth (Fig. 1). The substrate mycelia appeared greyish yellow on ISP 2, ISP 7 and nutrient agar, light olive brown on ISP 3 and ISP 4, brilliant yellow on ISP 5 and greyish olive green on ISP 6. A yellow to brown pigment was observed on most tested media except ISP 5. Melanin production was observed on ISP 6 medium. The cultural characteristics of strain KC-035 T are summarized in Table S1 (available in the online Supplementary Material). Other phenotypic properties are described in the description of the species.Author affiliations: 1 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; 2 NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan.*Correspondence: Somboon Tanasupawat, Somboon. T @chula.ac.thKeywords: Actinomycetes; Streptomycetaceae; Streptomyces krungchingensis; soil.Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbour-joining.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KC-035 T is LC008304.One supplementary figure and two supplementary tables are available with the online Supplementary Material.Strain KC-035 T exhibited typical characteristics of the genus Streptomyces. LL- Diaminopimelic acid, glucose and ribose were detected in whole-cell hydrolysates. The menaquinones Nacalai Tesque). The N -acyl types of muramic acids were determined using the method of Uchida and Aida [9]. Phospholipids were extracted and identified by the method of Minnikin et al. [10]. The cellular fatty acid composition was analysed by GC according to the instructions of the Microbial Identification System (MIDI) Sherlock version 6.0 with the RTSBA 6 MIDI database as described by Sasser [11]. The DNA G+C content was determined by HPLC according to the method of Tamaoka and Komagata [12]. DNA–DNA hybridization was carried out in microdilution well plates, as reported by Ezaki et al. [13].Table 1. Cellular fatty acid composition of strain KC-035 T and related Streptomyces speciesFig. 1. Scanning electron micrograph showing the flexuous spore chains and smooth spore surface of strain KC-035 T after growing on ISP2 medium at 30 Ǫ C for 14 days. Bar, 1 µm.Strains: 1, KC-035 T; 2, Streptomyces albiflavescens KCTC 29196 T; 3, Streptomyces siamensis KC-038 T. –, Not detected or &lt;0.5 %.Fatty acid 1 2 3 Saturated C12:0 0.7 – 0.7 C13:0 0.5 – – C14:0 3.7 1.3 3.1 C16:0 15.6 7.5 13.8 C17:0 1.3 0.8 1.0 C17:0 2-OH – – 1.2 C17:0cyclo 0.6 0.7 0.6 C18:0 0.5 – – Unsaturated C17:1 Ɯ 8 c 0.9 0.5 1.1 C18:1 Ɯ 9 c 1.3 – – Unsaturated branched iso-C16: 1 H 0.9 1.0 1.4 anteiso-C17: 1 Ɯ 9 c 0.9 1.2 1.4 Saturated branched iso-C12: 0 0.7 – – iso-C13: 0 0.8 0.6 0.6 anteiso-C13: 0 0.7 0.6 0.5 iso-C14: 0 9.3 10.0 7.7 iso-C15: 0 9.8 14.2 10.7 anteiso-C15: 0 18.9 21.0 22.3 iso-C16: 0 14.5 21.7 14.6 iso-C17: 0 2.0 3.7 1.9 iso-C17: 0 3-OH 0.6 0.7 – anteiso-C17: 0 3.8 5.1 4.3 iso-C18: 0 – 0.5 – iso-C19: 0 – 0.5 – Summed feature 3* 5.2 1.5 7.0 Summed feature 8† 0.5 – – Summed feature 9‡ 0.8 2.2 2.0Amplification of the 16S rRNA gene was carried out as described by Takahashi et al. [14]. The PCR product was sequenced using universal 16S rRNA gene primers [15]. A BLAST search analysis was performed against the EzTaxon-e server [16]. The alignment was manually verified and adjusted (BioEdit software; Ibis Biosciences) prior to reconstructing the phylogenetic tree. BioEdit software was used for multiple alignments with selected sequences. Phylogenetic trees were reconstructed based on the neighbour-joining (NJ) [17], maximum-likelihood (ML) [18] and maximum-parsimony (MP) [19] methods using the MEGA6 software [20]. Gaps and ambiguous nucleotides were eliminated from the calculations. Evolutionary distances among the strains were evaluated based on Kimura’ s two-parameter method [21], for NJ and ML, and subtree-pruning-regrafting method, for MP. Data were resampled with 1000 bootstrap replications [22]. Similarity values of the 16S rRNA gene sequence among closely related Streptomyces species were obtained from the EzTaxon server.Morphological observation revealed that stain KC-035 T grew well and formed extensively branched substrate mycelia on all test media. It produced white to light greenish grey aerial mycelia on ISP 2, ISP 3, ISP 4 and nutrient agar. Aerial mycelia were absent on ISP 5, ISP 6 and ISP 7 media. Flexuous spore detected were MK-9(H 8) (56 %), MK-9(H 6) (36 %) and MK-9 (H 4) (8 %). The N -acyl type of muramic acid was acetyl. It contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, two unknown lipids and an unidentified aminolipid as polar lipids (Fig. S1). The major cellular fatty acids (&gt;5 %) were anteiso-C 15: 0, C 16: 0, iso-C 16: 0, iso-C 15: 0 and iso-C 14: 0. The fatty acid composition of strain KC-035 T was similar to those of related Streptomyces species but there were minor differences in the proportions of the various fatty acids (Table 1). The DNA G+C content was 72 mol%.*Summed feature 3 comprised C 16: 1 Ɯ 7 c or C 16: 1 Ɯ 6 c.†Summed feature 8 comprised C 18: 1 Ɯ 7 c or C 18: 1 Ɯ 6 c.‡Summed feature 9 comprised C 16: 0 10-methyl or iso-C 17: 1 Ɯ 9 c.Fig. 2. Phylogenetic relationships based on an NJ analysis of 16S rRNA gene sequences of strain KC-035 T and closely related Streptomyces species. Kitasatospora setae JCM 3304 T was used as the outgroup. Symbols indicate that branches were also recovered in the ML (*) and MP (#) trees. The numbers at branch nodes indicate bootstrap percentages derived from 1000 replications (only values&gt;50 % are shown). Bar, 0.005 substitutions per nucleotide position.The result of 16S rRNA gene sequence analysis (1494 nt) showed that strain KC-035 T was affiliated to the genus Streptomyces. This strain showed highest 16S rRNA gene sequence similarity to Streptomyces albiflavescens n20 T (= KCTC 29196 T) (99.16 %). Phylogenetic analysis using the almost-complete 16S rRNA gene sequence showed that strain KC-035 T was placed in a monophyletic cluster with S. albiflavescens n20 T and Streptomyces siamensis KC-038 T (= NBRC 108799 T = TISTR 2107 T) (Fig. 2). Based on the highest 16S rRNA gene sequence similarity and phylogenetic analysis, S. albiflavescens KCTC 29196 T and S. siamensis KC-038 T (98.43 %) were selected for comparison with strain KC-035 T.Strain KC-035 T could be distinguished from S. albiflavescens KCTC 29196 T and S. siamensis KC-038 T based on its cultural characteristics when grown on various ISP media and nutrient agar as shown in Table S1, and based on its phenotypic characteristics. For example, strain KC-035 T produced melanin and was positive for gelatin liquefaction (weakly), and utilized raffinose and salicin (weakly), but it did not show activities of cystine arylamidase, a- galactosidase, a-glucosidase, a- mannosidase or trypsin (Table 2). Moreover, the DNA–DNA relatedness values between strain KC-035 T and the closest type strains, S. albiflavescens KCTC 29196 T and S. siamensis KC-038 T, were 14–52 % (Table S2). These values were below the 70 % cutoff point recommended by Wayne et al. [23] for assigning strains to the same species, and these results thus confirm that strain KC-035 T is distinct from its closely related phylogenetic neighbours. Therefore, strain KC-035 T is considered to represent a novel species within the genus Streptomyces, for which the name Streptomyces krungchingensis sp. nov. is proposed.