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Unrecorded Bacterial Species Belonging to the Phylum Actinobacteria Originated from Republic of Korea

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Unrecorded Bacterial Species Belonging to the Phylum Actinobacteria Originated from Republic of Korea Journal of Species Research 6(1):25-41, 2017 Unrecorded bacterial species belonging to the phylum Actinobacteria originated from Republic of Korea Mi-Sun Kim1, Ji-Hee Lee1, Seung-Bum Kim2, Jang-Cheon Cho3, Soon Dong Lee4, Ki-seong Joh5, Chang-Jun Cha6, Wan-Taek Im7, Jin-Woo Bae8, Kwangyeop Jahng9, Hana Yi10 and Chi-Nam Seong1,* 1Department of Biology, Sunchon National University, Suncheon 57922, Republic of Korea 2Department of Microbiology, Chungnam National University, Daejeon 34134, Republic of Korea 3Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea 4Department of Science Education, Jeju National University, Jeju 63243, Republic of Korea 5Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Gyeonggi 17035, Republic of Korea 6Department of Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 7Department of Biotechnology, Hankyong National University, Anseong 17579, Republic of Korea 8Department of Biology, Kyung Hee University, Seoul 02447, Republic of Korea 9Department of Life Sciences, Chonbuk National University, Jeonju 54896, Republic of Korea 10Department of Public Health Science & Guro Hospital, Korea University, Seoul 02841, Republic of Korea *Correspondent: [email protected] As a subset study for the collection of Korean indigenous prokaryotic species, 62 bacterial strains belonging to the phylum Actinobacteria were isolated from various sources. Each strain showed higher 16S rRNA gene sequence similarity (>98.75%) and formed a robust phylogenetic clade with closest species of the phylum Actinobacteria which were defined with valid names, already. There is no official description on these 62 actinobacterial species in Korea. Consequently, unrecorded 62 species of 25 genera in the 14 families belonging to the order Actinomycetales of the phylum Actinobacteria were found in Korea. Morphological properties, basic biochemical characteristics, isolation source and strain IDs are described in the species descriptions. Keywords: 16S rRNA gene sequence, Actinobacteria, Actinomycetales, unrecorded species Ⓒ 2017 National Institute of Biological Resources DOI:10.12651/JSR.2017.6.1.025 + INTRODUCTION with high G C content in their DNA (Ventura et al., 2007). Members of the phylum Actinobacteria are abun- During 2013 and 2014, the authors isolated a great dantly distributed in terrestrial or aquatic environments number of unrecorded actinobacterial species from di- and are involving in the decomposition of the organic verse environments in Korea. Natural environments matter, and then in promoting crop production (Servin et which were used for the isolation sources were soils al., 2008). Most of the species which have medical or eco- such as ginseng cultivating soil, biotite and natural cave, nomic significance belong to the order Actinomycetales fresh water, seawater including tidal flat sediment, plant (Miao and Davies, 2010). Many actinobacterial species, root and gut of birds and cattle. In addition, artificial en- especially members of the genus Streptomyces are rec- vironments such as fermented food and activated sludge ognized as the producers of many bioactive metabolites were used for the actinobacterial isolation sources. The that are useful to humans in medicine, such as antibac- present report focuses on the description of unrecord- terials, antifungals, antivirals, antithrombotics, immuno- ed Korean indigenous species belonging to the phylum modifiers, anti-tumor drugs and enzyme inhibitors; and Actinobacteria. in agriculture, including insecticides, herbicides, fungi- The phylum Actinobacteria is one of the greatest cides and growth promoting substances for plants and groups in the domain Bacteria (Ludwig et al., 2012). animals (Bressan, 2003). These bacteria are Gram-stain-positive microorganisms Until 2015, more than 250 Korean indigenous spe- 26 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 1 cies belonging to the phylum Actinobacteria have been RESULTS AND DISCUSSION isolated, described and validated according to the List of Prokaryotic name with Standing in Nomenclature All 62 strains belonged to order Actinomycetales and (LPSN; http://www.bacterio.net/). As a part of results affiliated to 4 suborders and 15 families; 2 strains for obtained from the research program supported by NIBR, Cellulomonadaceae, 1 strain for Dermabacteraceae, 1 the present report focuses on the description of bacterial strain for Dermacoccaceae, 3 strains for Intrasporangiaceae, species belonging to the phylum Actinobacteria, which 13 strains for Microbacteriaceae, 7 strains for have not been previously isolated in Korea. Here we re- Micrococcaceae, 2 strains for Promicromonosporaceae and port 62 unrecorded actinobacterial species in Korea. 1 strain for Sanguibacteraceae (suborder Micrococcineae), 1 strain for Pseudonocardiaceae (suborder Pseudonocardineae), 2 strains for Corynebacteriaceae, 1 strain for Dietziaceae, MATERIALS AND METHODS 5 strains for Mycobacteriaceae and 6 strains for Nocardiaceae (suborder Corynebacterineae), and 17 A total of 62 bacterial strains assigned to the phylum strains for Streptomycetaceae (suborder Streptomycineae) Actinobacteria were isolated from various environmen- (Table 1). tal samples collected from soils such as ginseng culti- Isolation sources of the strains were as follows: 12 vated soil, biotite and natural cave, fresh water, seawater strains from ginseng cultivated soil, 11 strains from nat- including tidal flat sediment, plant root and gut of birds ural cave, 9 strains from soil, 8 strains from fresh water, and cattle. In addition, artificial environments such as 6 strains from sea water, 6 strains from gut of birds, fermented food and activated sludge were used for the each 2 strains from rhizosphere, black biotite, activated isolation sources (Table 1). Each sample was processed sludge and tidal flat sediment and each one strain from separately and spread onto diverse culture media (Becton gut of Korean native cattle and Korean fermented food Dickinson) including R2A, international streptomyces (jeotgal). Regional origins of the isolates were as fol- project medium 2 (ISP2), brain heart infusion (BHIA), lows: 21 strains from Gyeonggi, 11 strains from Jeju, 6 1/10 marine (1/10 MA), marine (MA), tryptic soy (TSA) strains from Incheon, 5 strains from Chungbuk, 4 strains and nutrient (NA) agars. Agar plates were incubated at from Daejeon, each 3 strains from Busan, Jeonnam and 20-37°C for 1-16 days. All strains were purified as sin- Jeonbuk, each 2 strains from Gangwon and Chungnam gle colonies and stored as 10-20% glycerol suspension and each one strain from Gyeongbuk and Seoul. at -80°C as well as lyophilized ampoules. These strains were Gram-stain-positive and chemo- Colony morphology of the strains was observed on heterotrophic. Fig. 2 shows phylogenetic assignment of agar plates with a magnifying glass after cells grew up to the strains based on 16S rRNA gene sequences. stationary phase. Cellular morphology and cell size were Here we report 62 unrecorded bacterial species in Ko- examined by either transmission electron microscopy rea belonging to the phylum Actinobacteria. or scanning electron microscopy (Fig. 1). Biochemical characteristics were tested by using API 20NE galleries Description of Cellulomonas denverensis KHH20 (bioMérieux) according to the manufacturer’s instructions. Cells are Gram-staining-positive, non-flagellated and Bacterial DNA extraction, PCR amplification and 16S palisades shaped. Colonies are circular and yellow col- rRNA gene sequencing were performed using the stan- ored after 3 days on R2A at 25°C. Positive for esculin dard procedures described elsewhere. The 16S rRNA hydrolysis and β-galactosidase in API 20NE, but neg- gene sequences of the strains assigned to the phylum ative for arginine dihydrolase, gelatinase, glucose fer- Actinobacteria were compared with the sequences held mentation, indole production, nitrate reduction and ure- in GenBank by BLAST and also analyzed using the ase. Utilize N-acetyl-glucosamine, L-arabinose, D-glu- EzTaxon-e server (Kim et al., 2012). For phylogenetic cose, D-maltose, D-mannitol, D-mannose and potassium analyses, multiple alignments were performed using the gluconate, but not utilize adipic acid, capric acid, malic Clustal_W program (Thompson et al., 1994) and gaps acid, phenylacetic acid and trisodium citrate. Strain were edited in the BioEdit program (Hall, 1999). Evolu- KHH20 ( =NIBRBAC000497852) has been isolated tionary distances were calculated using the Jukes-Cantor from Gut of Japanese crested ibis (Nipponia nippon), model (Jukes and Cantor, 1969). The phylogenetic trees Gyeonggi Province, Korea. were constructed by using the neighbour-joining (Saitou and Nei, 1987), the maximum-likelihood (Felsenstein, Description of Cellulomonas soli RDH8 1981) and the maximum-parsimony (Fitch, 1971) meth- ods with the MEGA 6.0 Program (Tamura et al., 2013) Cells are Gram-staining-positive, non-flagellated and with bootstrap values based on 1,000 replications (Fel- rod-shaped. Colonies are glossy, watery, smooth and senstein, 1985). light yellow colored after 2 days on R2A at 3°C. Diffus- February 2017 Table 1. Taxonomic affiliation and isolation information of the isolates belonging to the phylumActinobacteria . Isolation Similarity Suborder Family Genus Strain ID NIBR NO. Most closely related species Incubation (%) Source Medium condition Micrococcineae Cellulomonadaceae Cellulomonas KHH20 NIBRBAC000497852 Cellulomonas denverensis W6929T 99.64 Gut
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