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

+ 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) methods 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 KIM et al.-Unrecorded actinobacterial species 27 condition Incubation 25℃, 3d 30℃, 2d 30℃, 2d 25℃, 3d 26℃, 1d 25℃, 4d 30℃, 2d 30℃, 2d 30℃, 7d 30℃, 2d 30℃, 4d 30℃, 7d 30℃, 3d 25℃, 3d 20°C, 10d 26℃, 1d 30℃, 2d 25℃, 3d 25℃, 2d 20°C, 10d 30℃, 3d 25℃, 3d 20°C, 10d 26℃, 2d 37℃, 3d 30℃, 5d 26℃, 2d 25℃, 3d 30℃, 3d 20°C, 10d 25℃, 3d Medium R2A R2A R2A MA MA MA R2A R2A IS P 2 R2A TSA TSA MA NA R2A MA R2A NA R2A R2A R2A TSA R2A MA BHIA TSA MA R2A R2A 1/10 MA MA Isolation Source Gut of japanese crested ibis F resh water Activated sludge Sea water Sea water Sea water Ginseng cultivated soil Ginseng cultivated soil Natural cave Ginseng cultivated soil Natural cave Natural cave F ermented food Gut of red- crowned crane F resh water Sea water Activated sludge Gut of red- crowned crane F resh water F resh water Soil Gut of red- crowned crane F resh water Soil Gut of korean native cattle P lant root Sea water Gut of japanese crested ibis Natural cave F resh water Sea water

(%) Similarity 99.64 99.64 99.93 100.0 99.92 99.65 99.08 99.58 99.72 98.75 99.25 99.22 99.92 99.79 100 100.0 99.64 99.57 99.69 99.50 99.93 99.78 99.86 99.85 99.08 100 99.23 99.86 99.16 99.86 99.86 T T T

T T T T T T T T T

T T

C F 36 DSM 20754 T T KV-492 T DSM 16091 T

108036 W6929 CC 0387 L1b-b9 I F O 15075 CCUG 47306 H101 T DSM 20123 DSM 17612 T MD-T1-10-2 T MT2 T CI P ST26 H85-3 DSM 11926 0704 P 10-1 Kc1 2Sb DSM 11925 D7-27 T KMM 3905 TG-S248 T T T T T . Most closely related species AM EL-30 DSM 8609 Cellulomonas soli Leifsonia soli arborescens Microbacterium esteraromaticum Microbacterium ketosireducens Microbacterium DSM 12510 marinum Microbacterium oleivorans Microbacterium paraoxydans Microbacterium schleiferi Microbacterium nicotianae Arthrobacter Kocuria palustris Kocuria rhizophila Cellulomonas denverensis LMG 19861 YIM 61095 Brachybacterium paraconglomeratum Dermacoccus barathri Arsenicicoccus bolidensis Janibacter indicus Phycicoccus ochangensis lahaulensis Agrococcus Humibacter antri Leifsonia lichenia aoyamense Microbacterium Mycetocola miduiensis bergerei Arthrobacter Kocuria marina lacusekhoensis Nesterenkonia I F Zhihengliuella flava funkei Cellulosimicrobium BAA-886 ATCC flava Promicromonospora Sanguibacter suarezii endophytica Saccharopolyspora Actinobacteria NIBR NO. NIBRBAC000498079 NIBRBAC000498033 NIBRBAC000497916 NIBRBAC000497842 NIBRBAC000498002 NIBRBAC000497980 NIBRBAC000498106 NIBRBAC000497857 NIBRBAC000498052 NIBRBAC000497856 NIBRBAC000497975 NIBRBAC000497862 NIBRBAC000497852 NIBRBAC000498105 NIBRBAC000497927 NIBRBAC000497985 NIBRBAC000497943 NIBRBAC000498111 NIBRBAC000498117 NIBRBAC000498029 NIBRBAC000498109 NIBRBAC000498021 NIBRBAC000498001 NIBRBAC000497900 NIBRBAC000498005 NIBRBAC000498084 NIBRBAC000497981 NIBRBAC000497851 NIBRBAC000498031 NIBRBAC000497994 NIBRBAC000497925 Strain ID RDH8 C7-7 HM F 4427 KHC15 IMCC25612 L P B0100 Ho-14 KHG7 DO214 KHK4 L P B0092 bT304 KHH20 Ho-10 KYW950 L P B0110 KYW1206 HKS12 HKS25 C4-1 HKS09 C1-46 IMCC25611 HM F 3875 IMCC25615 4-03 UT L P B0101 KHC19 C6-16 IMCC25604 KYW998 Genus Cellulomonas Brachybacterium Dermacoccus Arsenicicoccus Janibacter Phycicoccus Agrococcus Humibacter Leifsonia Microbacterium Mycetocola Arthrobacter Kocuria Nesterenkonia Zhihengliuella Cellulosimicrobium Promicromonospora Sanguibacter Saccharopolyspora F amily Cellulomonadaceae Dermabacteraceae Dermacoccaceae Intrasporangiaceae Microbacteriaceae Micrococcaceae Promicromonosporaceae Sanguibacteraceae Pseudonocardiaceae Pseudonocardiaceae Taxonomic affiliation and isolation information of the isolates belonging to phylum Taxonomic Suborder Micrococcineae Pseudonocardineae Table 1. Table 28 Journal of Species Research Vol. 6, No. 1 condition Incubation 30℃, 2d 30℃, 2d 20°C, 10d 30℃, 10d 30℃, 2d 30℃, 3d 30℃, 7d 30℃, 16d 30℃, 2d 25℃, 3d 30℃, 3d 30℃, 5d 30℃, 9d 20°C, 7d 30℃ 30℃, 3d 30℃, 3d 30℃, 1d 30℃, 7d 30℃, 7d 25℃, 2d 30℃, 2d 30℃, 7d 25℃, 2d 30℃, 7d 30℃, 7d 30℃, 1d 30℃, 3d 30℃, 7d 30℃, 3d 30℃, 3d Medium R2A MA R2A TSA R2A IS P 2 agar R2A R2A R2A NA TSA IS P 2 agar TSA R2A IS P 2 MA MA R2A IS P 2 IS P 2 R2A R2A IS P 2 R2A IS P 2 IS P 2 R2A MA IS P 2 MA MA Isolation Source Ginseng cultivated soil flat Tidal F resh water Natural cave Ginseng cultivated soil Natural cave Natural cave Natural cave flat Tidal Gut of japanese crested ibis Natural cave Natural cave Natural cave F resh water P lant root Ginseng cultivated soil Ginseng cultivated soil Ginseng cultivated soil Soil Soil Black biotite Soil Soil Black biotite Soil Soil Ginseng cultivated soil Ginseng cultivated soil Soil Ginseng cultivated soil Ginseng cultivated soil

(%) Similarity 99.63 99.85 99.50 99.29 99.72 99.44 99.00 99.30 99.48 99.00 99.72 99.50 99.71 100.00 98.96 100 100 99.24 99.56 99.92 99.79 99.86 100 99.93 99.11 99.41 99.37 99.71 99.14 99.79 99.16 T T T T T T T T T T T T T T T T T T T T 104941 T T T T T T T

CI P K04-0144

NBRC 13414 BK166 S77 7015

NBRC 15399 NBRC 12779 NEAU-ML12 103464 NBRC 15401 LMG 20091 NBRC 12891 NBRC 13407 T IS P B 6695110 NBRC 13075 ATCC 27023 ATCC DSM 44076 BK168 DSM 44183 TA4-8 NBRC 13450 LMG 3623 NBRC 13056 C5 CI P MA140/96 ID05-A0528 SJS0289-JS1 T T T Most closely related species Corynebacterium marinum Mycobacterium hodleri Mycobacterium obuense Mycobacterium senegalense Mycobacterium sphagni Rhodococcus fascians Rhodococcus kronopolitis Rhodococcus wratislaviensis NBRC 100605 muralis Williamsia coelicoflavus Streptomyces colombiensis Streptomyces B-1990 NRRL crystallinus Streptomyces cyslabdanicus Streptomyces griseoplanus Streptomyces kanamyceticus Streptomyces laculatispora Streptomyces lannensis Streptomyces lienomycini Streptomyces lucensis Streptomyces mirabilis Streptomyces misakiensis Streptomyces prunicolor Streptomyces puniciscabiei Streptomyces rishiriensis Streptomyces turgidiscabies Streptomyces 700248 ATCC Corynebacterium freneyi Dietzia timorensis Mycobacterium alvei Rhodococcus cerastii maris Williamsia cocklensis Streptomyces NIBR NO. NIBRBAC000498060 NIBRBAC000498114 NIBRBAC000498030 NIBRBAC000498035 NIBRBAC000498032 NIBRBAC000497838 NIBRBAC000498022 NIBRBAC000498026 NIBRBAC000497997 NIBRBA0000114269 NIBRBA0000114270 NIBRBAC000498113 NIBRBAC000498097 NIBRBAC000498098 NIBRBA0000114215 NIBRBAC000498080 NIBRBAC000498099 NIBRBA0000114216 NIBRBAC000498100 NIBRBAC000498101 NIBRBAC000498112 NIBRBA0000114268 NIBRBAC000498102 NIBRBA0000114279 NIBRBA0000114273 NIBRBAC000498118 NIBRBAC000498003 NIBRBAC000498028 NIBRBAC000498066 NIBRBAC000498023 NIBRBAC000498091 Strain ID Cip10 HKS22 C6-12 C10-13 C6-18 KHO6 C1-60 C3-42 IMCC25607 MGS3Y-3-1 MGS3Y-3-4 HKS20 TW1K13 TW1K14 BBT-4 Tri-200-1 TW1K20 BBT-7 TW1M1 TW1K17 HKS13 MEC3Y-3-1 TW1S1 MMD3Y-3-3 MK6Y-2-3 HKS28 IMCC25613 C3-50 G1 C2-18 BK1I29 Genus Corynebacterium Dietzia Mycobacterium Rhodococcus Williamsia Streptomyces F amily Corynebacteriaceae Dietziaceae Mycobacteriaceae Nocardiaceae Streptomycetaceae Continued. Suborder Corynebacterineae Streptomycineae Table 1. Table February 2017 KIM et al.-Unrecorded actinobacterial species 29

Fig. 1. Transmission electron micrographs or scanning electron micrographs of cells of the strains isolated in this study. Strains: 1, KHH20; 2, RDH8; 3, Ho-10; 4, KYW950; 5, LPB0110; 6, KYW1206; 7, HKS12; 8, HKS25; 9, C4-1; 10, HKS09; 11, C7-7; 12, C1-46; 13, HMF4427; 14, KHC15; 15, IMCC25612; 16, LPB0100; 17, Ho-14; 18, KHG7; 19, DO214; 20, IMCC25611; 21, HMF3875; 22, KHK4; 23, IMCC25615; 24, LPB0092; 25, bT304; 26, UT 4-03; 27, LPB0101; 28, KHC19; 29, C6-16; 30, IMCC25604; 31, KYW998; 32, HKS28; 33, Cip10; 34, IMCC25613; 35, C3-50; 36, HKS22; 37, C6-12; 38, C10-13; 39, C6-18; 40, G1; 41, KHO6; 42, C1-60; 43, C3-42; 44, C2- 18; 45, IMCC25607; 46, BK1I29; 47, MGS3Y-3-1; 48, MGS3Y-3-4; 49, HKS20; 50, TW1K13; 51, TW1K14; 52, BBT-4; 53, Tri-200-1; 54, TW1K20; 55, BBT-7; 56, TW1M1; 57, TW1K17; 58, HKS13; 59, MEC3Y-3-1; 60, TW1S1; 61, MMD3Y-3-3; 62, MK6Y-2-3. 30 Journal of Species Research Vol. 6, No. 1

Fig. 2. Neighbor-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationship between the strains isolated in this study and their relatives of the phylum Actinobacteria. Bootstrap values (>70%) are shown above nodes. Filled circles indicate the nodes recovered by three other treeing methods including maximum likelihood, maximum parsimony, and neighbor joining. Bar, 0.02 sub- stitutions per nucleotide position. February 2017 KIM et al.-Unrecorded actinobacterial species 31

Fig. 2. Continued. 32 Journal of Species Research Vol. 6, No. 1 ible pigment is produced. Positive for esculin hydroly- L-arabinose, capric acid, D-glucose, malic acid, D-malt- sis, β-galactosidase and nitrate reduction in API 20NE, ose, D-mannitol, D-mannose, phenylacetic acid, potas- but negative for arginine dihydrolase, glucose fermenta- sium gluconate and trisodium citrate. Strain LPB0110 tion, gelatinase, indole production and urease. L-Arab- (=NIBRBAC000497985) has been isolated from a sea inose, D-glucose, D-maltose, D-mannose are utilized. water sample, Busan, Korea. Does not utilize N-acetyl-glucosamine, adipic acid, cap ric acid, malic acid, D-mannitol, phenylacetic acid, po- Description of Janibacter indicus KYW1206 tassium gluconate and trisodium citrate. Strain RDH8 Cells are Gram-staining-positive, non-flagellated, ( =NIBRBAC000498079) has been isolated from a non-pigmented and coccoid-shaped. Colonies are circu- fresh water lake at Chung-Ang University, Anseong, lar, convex and light yellow-colored after 4 days on MA Gyeonggi Province, Korea. at 25°C. Negative for arginine dihydrolase, esculin hydrolysis, β-galactosidase, glucose fermentation, indole Description of Brachybacterium paraconglomeratum production, nitrate reduction and urease, but positive for Ho-10 gelatinase in API 20NE. D-Glucose, malic acid and po- Cells are Gram-staining-positive, non-flagellated and tassium gluconate are utilized. Does not utilize N-acetyl- round or oval shaped. Colonies are circular, round, en- glucosamine, adipic acid, L-arabinose, capric acid, D- tire and light yellow colored after 2 days on R2A at maltose, D-mannitol, D-mannose, phenylacetic acid 30°C. Positive for esculin hydrolysis, glucose fermenta- and trisodium citrate. Strain KYW1206 ( =NIBRBAC tion and nitrate reduction in API 20NE, but negative for 000497943) has been isolated from a sea water sample, arginine dihydrolase, β-galactosidase, gelatinase, indole Gwangyang Bay, Gwangyang, Jeonnam province, Ko- production and urease. D-Glucose, malic acid, D-malt- rea. ose, D-mannitol and D-mannose are utilized. Does not utilize N-acetyl-glucosamine, adipic acid, L-arabinose, Description of Phycicoccus ochangensis HKS12 capric acid, phenylacetic acid, potassium gluconate Cells are Gram-staining-positive and round or rod- and trisodium citrate and are not utilized. Strain Ho-10 shaped. Colonies are circular, round, entire and milky ( =NIBRBAC000498105) has been isolated from acti- white colored after 2 days on R2A at 30°C. Negative for vated sludge, Daejeon, Korea. arginine dihydrolase, β-galactosidase, indole production, nitrate reduction and urease, but positive for esculin hy- Description of Dermacoccus barathri KYW950 drolysis, gelatinase and glucose fermentation in API Cells are Gram-staining-positive, non-flagellated and 20NE. Utilize N-acetyl-glucosamine, D-glucose, malic coccoid-shaped. Colonies are circular, entire, convex acid, D-maltose, D-mannitol, D-mannose and potassium and light yellow-colored after 3 days on MA at 25°C. gluconate. Does not utilize adipic acid, L-arabinose, cap Negative for arginine dihydrolase, esculin hydrolysis, ric acid, phenylacetic acid and trisodium citrate. Strain β-galactosidase, gelatinase, glucose fermentation, indole HKS12 ( =NIBRBAC000498111) has been isolated production, nitrate reduction and urease in API 20NE. Ginseng cultivated soil, Anseong, Gyeonggi Province, D-Glucose, malic acid, D-maltose and potassium gluco- Korea. nate are utilized. Does not utilize N-acetyl-glucosamine, adipic acid, L-arabinose, capric acid, D-mannitol, D- Description of Agrococcus lahaulensis HKS25 mannose, phenylacetic acid and trisodium citrate. Strain Cells are Gram-staining-positive, non-flagellated and KYW950 ( =NIBRBAC000497927) has been isolated coccus shaped. Colonies are wrinkled circular, convex, from a sea water sample, Gwangyang Bay, Gwangyang, opaque and pale yellow colored after 2 days on R2A at Jeonnam Province, Korea. 30°C. Positive for gelatinase and urease, but negative for arginine dihydrolase, esculin hydrolysis, β-galactos Description of Arsenicicoccus bolidensis LPB0110 idase, glucose fermentation, indole production and ni- Cells are Gram-staining-positive, non-flagellated and trate reduction in API 20NE. N-Acetyl-glucosamine, coccus shaped. Colonies are circular and light yellow- adipic acid, L-arabinose, capric acid, D-glucose, malic colored after 1 day on MA at 26°C. Positive for esculin acid, D-maltose, D-mannitol, D-mannose, phenylacetic hydrolysis and nitrate reduction in API 20NE, but neg- acid, potassium gluconate and trisodium citrate are not ative for arginine dihydrolase, β-galactosidase, gelati- utilized. Strain HKS25 ( =NIBRBAC000498117) has nase, glucose fermentation, indole production and ure- been isolated from Ginseng cultivated soil, Anseong, ase. Does not utilize N-acetyl-glucosamine, adipic acid, Gyeonggi Province, Korea. February 2017 KIM et al.-Unrecorded actinobacterial species 33

Description of Humibacter antri C4-1 ose, D-mannitol, D-mannose and potassium gluconate are utilized. Weakly utilize the L-arabinose. Does not Cells are Gram-staining-positive, non-flagellated and utilize adipic acid, capric acid, malic acid, phenylacetic rod shaped. Colonies are circular, entire, convex and acid and trisodium citrate. Strain C1-46 ( =NIBRBAC cream colored after 7 days on ISP2 agar at 30°C. Nega- 000498021) has been isolated from a natural cave, Jeju, tive for arginine dihydrolase, β-galactosidase, gelatinase, Korea. glucose fermentation, indole production and urease, but weakly positive for esculin hydrolysis and nitrate reduc- Description of Microbacterium arborescens HMF4427 tion in API 20NE. N-Acetyl-glucosamine, adipic acid, L-arabinose, capric acid, D-glucose, malic acid, D-malt- Cells are Gram-staining-positive, non-flagellated and ose, D-mannitol, D-mannose, phenylacetic acid, potas- rod shaped. Colonies are circular, entire, convex and sium gluconate and trisodium citrate are not utilized. yellow colored after 3 days on MA at 30°C. Positive Strain C4-1 (=NIBRBAC000498029) has been isolated for esculin hydrolysis, β-galactosidase, gelatinase and from a natural cave, Jeju, Korea. glucose fermentation in API 20NE, but negative for arginine dihydrolase, indole production, nitrate reduction Description of Leifsonia lichenia HKS09 and urease. N-Acetyl-glucosamine, L-arabinose, D-glucose, malic acid, D-maltose, D-mannitol, D-mannose, Cells are Gram-staining-positive, non-flagellated and potassium gluconate and trisodium citrate are utilized. rod or oval rod shaped. Colonies are circular, raised, en Does not utilize adipic acid, capric acid and phenylacetic tire and dark yellow colored after 2 days on R2A at 30°C. acid. Strain HMF4427 ( =NIBRBAC000497916) has Positive for esculin hydrolysis and glucose fermentation, been isolated from Korean fermented food (jeotgal), but negative for arginine dihydrolase, β-galactosidase, Jeonbuk Province, Korea. gelatinase, indole production, nitrate reduction and urease in API 20NE. Utilize N-acetyl-glucosamine, D-glu- Description of Microbacterium esteraromaticum cose, D-maltose, D-mannitol, D-mannose and potassium KHC15 gluconate. Does not utilize adipic acid, L-arabinose, cap ric acid, malic acid, phenylacetic acid and trisodium ci- Cells are Gram-staining-positive, flagellated and coc- trate. Strain HKS09 (=NIBRBAC000498109) has been cibacillus. Colonies are circular and cream-colored after isolated from Ginseng cultivated soil, Anseong, Gyeong- 3 days on NA at 25°C. Positive for esculin hydrolysis, gi Province, Korea. β-galactosidase and nitrate reduction in API 20NE, but negative for arginine dihydrolase, gelatinase, glucose Description of Leifsonia soli C7-7 fermentation, indole production and urease. Utilize N-acetyl-glucosamine, L-arabinose, D-glucose, D-malt- Cells are Gram-staining-positive, non-flagellated and ose, D-mannitol, D-mannose and potassium gluconate, rod shaped. Colonies are circular, entire, convex and but not utilize adipic acid, capric acid, malic acid, phe- cream colored after 4 days on TSA at 30°C. Positive for nylacetic acid and trisodium citrate. Strain KHC15 esculin hydrolysis and β-galactosidase, but negative for (=NIBRBAC000497842) has been isolated from Gut of arginine dihydrolase, gelatinase, glucose fermentation, Red-crowned Crane (Grus japonensis), Gyeonggi Prov- indole production, nitrate reduction and urease in API ince, Korea. 20NE. Does not utilize N-acetyl-glucosamine, adipic acid, L-arabinose, capric acid, D-glucose, malic acid, Description of Microbacterium ketosireducens D-maltose, D-mannitol, D-mannose, phenylacetic acid, IMCC25612 potassium gluconate and trisodium citrate. Strain C7-7 (=NIBRBAC000498033) has been isolated from a nat- Cells are Gram-staining-positive, non-flagellated and ural cave, Jeju, Korea. rod shaped. Colonies are circular, raised, entire and red colored after 10 days on R2A at 20°C. Positive for esculin hydrolysis, β-galactosidase, urease and oxidase, Description of Microbacterium aoyamense C1-46 but negative for arginine dihydrolase, gelatinase, glucose Cells are Gram-staining-positive, non-flagellated and fermentation, indole production and nitrate reduction in rod shaped. Colonies are circular, entire, convex and light API 20NE. N-Acetyl-glucosamine, L-arabinose, D-glu- yellow colored after 7 days on TSA at 30°C. Positive cose, D-maltose, D-mannitol, D-mannose and potassi- for esculin hydrolysis and β-galactosidase, but negative um gluconate are utilized. Does not utilize adipic acid, for arginine dihydrolase, gelatinase, glucose fermenta- capric acid, malic acid, phenylacetic acid and trisodium tion, indole production, nitrate reduction and urease in citrate. Strain IMCC25612 ( =NIBRBAC000498002) API 20NE. N-Acetyl-glucosamine, D-glucose, D-malt- has been isolated from a fresh water sample, Inkyong 34 Journal of Species Research Vol. 6, No. 1 lake, Incheon, Korea. glucose fermentation, indole production and urease. Uti- lize N-acetyl-glucosamine, L-arabinose, D-glucose, ma- Description of Microbacterium marinum LPB0100 lic acid, D-maltose, D-mannitol, D-mannose and trisodium citrate, but not utilize adipic acid, capric acid, phe- Cells are Gram-staining-positive, non-flagellated and nylacetic acid and potassium gluconate. Strain DO214 rod-shaped. Colonies are circular and light yellow col- ( =NIBRBAC000498052) has been isolated from a ored after 1 day on MA at 26°C. Negative for arginine fresh water sample, Jeonju, Jeonbuk Province, Korea. dihydrolase, gelatinase, glucose fermentation, indole production, nitrate reduction and urease in API 20NE, but positive for esculin hydrolysis and β-galactosidase. Description of Mycetocola miduiensis IMCC25611 Positive for utilization of L-arabinose, D-glucose, D- Cells are Gram-staining-positive, non-flagellated and maltose, D-mannitol and potassium gluconate. Does not rod shaped. Colonies are circular, raised, entire and utilize N-acetyl-glucosamine, adipic acid, capric acid, white colored after 10 days on R2A at 20°C. Positive for malic acid, D-mannose, phenylacetic acid and trisodium nitrate reduction and oxidase, but negative for arginine citrate. Strain LPB0100 ( =NIBRBAC000497980) has dihydrolase, esculin hydrolysis, β-galactosidase, gelati- been isolated from a sea water sample, Busan, Korea. nase, glucose fermentation, indole production and urease in API 20NE. Does not utilize N-acetyl-glucosamine, Description of Microbacterium oleivorans Ho-14 adipic acid, L-arabinose, capric acid, D-glucose, malic Cells are Gram-staining-positive, non-flagellated and acid, D-maltose, D-mannitol, D-mannose, phenylacetic irregular rod shaped. Colonies are circular, round, entire acid, potassium gluconate and trisodium citrate. Strain = and yellow colored after 2 days on R2A at 30°C. Posi- IMCC25611 ( NIBRBAC000498001) has been isolat- tive for esculin hydrolysis, but negative for arginine di- ed from a fresh water sample, Inkyong lake, Incheon, hydrolase, β-galactosidase, gelatinase, glucose fermen- Korea. tation, indole production, nitrate reduction and urease in API 20NE. Does not utilize N-acetyl-glucosamine, adip- Description of Arthrobacter bergerei HMF3875 ic acid, L-arabinose, capric acid, D-glucose, malic acid, Cells are Gram-staining-positive, non-flagellated and D-maltose, D-mannitol, D-mannose, phenylacetic acid coccus-shaped. Colonies are circular, convex, entire and and trisodium citrate, but utilize potassium gluconate. yellow colored after 3 days on R2A at 30°C. Negative = Strain Ho-14 ( NIBRBAC000498106) has been isolat- for arginine dihydrolase, esculin hydrolysis, β-galacto- ed from activated sludge, Daejeon, Korea. sidase, gelatinase, glucose fermentation, indole production, nitrate reduction and urease in API 20NE. Utilize Description of Microbacterium paraoxydans KHG7 L-arabinose, D-glucose, malic acid, D-maltose, D-man- Cells are Gram-staining-positive, non-flagellated and nose, phenylacetic acid, potassium gluconate and triso- streptobacilli shaped. Colonies are circular and cream dium citrate, but not utilize N-acetyl-glucosamine, adip- colored after 3 days on NA at 25°C. Positive for esculin ic acid, capric acid and D-mannitol. Strain HMF3875 hydrolysis, β-galactosidase and gelatinase in API 20NE, ( =NIBRBAC000497900) has been isolated from soil but negative for arginine dihydrolase, glucose fermen- sample, Yongin, Gyeonggi Province, Korea. tation, indole production, nitrate reduction and urease. Utilize N-acetyl-glucosamine, D-glucose, D-maltose, Description of Arthrobacter nicotianae KHK4 D-mannitol, D-mannose, potassium gluconate and tri- Cells are Gram-staining-positive, non-flagellated and sodium citrate, but not utilize L-arabinose, adipic acid, bacillus shaped. Colonies are circular, cream colored capric acid, malic acid and phenylacetic acid. Strain after 3 days on TSA at 25°C. Positive for gelatinase and KHG7 (=NIBRBAC000497857) has been isolated from nitrate reduction in API 20NE, but negative for arginine Gut of Red-crowned Crane (Grus japonensis), Gyeonggi dihydrolase, esculin hydrolysis, β-galactosidase, glucose Province, Korea. fermentation, indole production and urease. Utilizes adi pic acid, L-arabinose, D-glucose, malic acid, D-maltose, Description of Microbacterium schleiferi DO214 phenylacetic acid, potassium gluconate and trisodium Cells are Gram-staining-positive, non-flagellated and citrate, but not utilize N-acetyl-glucosamine, capric acid, rod shaped. Colonies are circular, convex and yellow D-mannitol and D-mannose. Strain KHK4 (=NIBRBAC colored after 2 days on R2A at 25°C. Positive for esculin 000497856) has been isolated from Gut of Red-crowned hydrolysis, β-galactosidase and nitrate reduction in API Crane (Grus japonensis), Gyeonggi Province, Korea. 20NE, but negative for arginine dihydrolase, gelatinase, February 2017 KIM et al.-Unrecorded actinobacterial species 35

Description of Kocuria marina IMCC25615 acid, D-maltose, D-mannitol, D-mannose, phenylacetic acid, potassium gluconate and trisodium citrate. Strain Cells are Gram-staining-positive, non-flagellated and UT 4-03 ( =NIBRBAC000498084) has been isolated rod shaped. Colonies are circular, raised, entire and yel- from a plant root, Daejeon, Korea. low colored after 10 days on R2A at 20°C. Negative for arginine dihydrolase, esculin hydrolysis, β-galactosidase, Description of Zhihengliuella flava LPB0101 gelatin hydrolysis, glucose fermentation, Indole production, nitrate reduction and urease in API 20NE. D-Glu- Cells are Gram-staining-positive, non-flagellated and cose, D-maltose, D-mannitol, D-mannose and potassium rod-shaped. Colonies are circular and yellow colored gluconate are utilized. Does not utilize N-acetyl-glucos- after 2 days on R2A at 26°C. Positive for esculin hy- amine, L-arabinose, adipic acid, capric acid, malic acid, drolysis, β-galactosidase and nitrate production in API phenylacetic acid and trisodium citrate. Strain IMCC 20NE, but negative for arginine dihydrolase, gelatinase, 25615 (=NIBRBAC000498005) has been isolated from glucose fermentation, indole production and urease. a fresh water sample, Inkyong lake, Incheon, Korea. Does not utilize N-acetyl-glucosamine, L-arabinose, adipic acid and capric acid, but utilize D-glucose, malic Description of Kocuria palustris LPB0092 acid, D-maltose, D-mannitol, D-mannose, phenylacetic acid, potassium gluconate and trisodium citrate. Strain Cells are Gram-staining-positive, non-flagellated, LPB0101 ( =NIBRBAC000497981) has been isolated non-pigmented and coccus-shaped. Colonies are circular from a sea water sample, Busan, Korea. and light yellow colored after 2 days on MA medium at 26°C. Positive for nitrate reduction and urease in API Description of Cellulosimicrobium funkei KHC19 20NE, but negative for arginine dihydrolase, esculin hydrolysis, β-galactosidase, gelatinase, glucose fermen- Cells are Gram-staining-positive, flagellated and coc- tation and Indole production. Does not utilize N-acetyl- cibacillus shaped. Colonies are circular and yellow col- glucosamine, adipic acid, L-arabinose, capric acid, D- ored after 3 days on R2A at 25°C. Negative for arginine glucose, malic acid, D-maltose, D-mannitol, D-mannose, dihydrolase, gelatinase, glucose fermentation, indole phenylacetic acid, potassium gluconate and trisodium production and urease in API 20NE, but positive for citrate. Strain LPB0092 ( =NIBRBAC000497975) has esculin hydrolysis, β-galactosidase and nitrate reduction. been isolated from soil sample, Bukhan Mountain, Seoul, Does not utilize N-acetyl-glucosamine, adipic acid, cap Korea. ric acid, malic acid, D-mannitol, phenylacetic acid and trisodium citrate, but utilize L-arabinose, D-glucose, Description of Kocuria rhizophila BT304 D-mannose, D-maltose and potassium gluconate. Strain KHC19 ( =NIBRBAC000497851) has been isolated Cells are Gram-staining-positive, non-flagellated, non- from Gut of Japanese crested ibis (Nipponia nippon), pigmented and coccus-shaped. Colonies are circular Gyeonggi Province, Korea. and lighy yellow colored after 3 days on BHIA at 37°C. Negative for arginine dihydrolase, esculin hydrolysis, β- Description of Promicromonospora flava C6-16 galactosidase, gelatinase, glucose fermentation, indole production, nitrate reduction and urease in API 20NE. Cells are Gram-staining-positive, non-flagellated and Utilize N-acetyl-glucosamine, adipic acid, D-glucose, rod shaped. Colonies are circular, convex, entire and malic acid, D-maltose, D-mannose, phenylacetic acid, white cream colored after 3 days on R2A at 30°C. Posi- potassium gluconate and trisodium citrate are utilized. tive for esculin hydrolysis, β-galactosidase and oxidase, Does not utilize L-arabinose, capric acid and D-mannitol. weakly positive for urease, but negative for arginine di- Strain BT304 (=NIBRBAC000497862) has been isolat- hydrolase, gelatinase, glucose fermentation, Indole pro- ed from Gut of Korean native cattle, Korea. duction and nitrate reduction. In API 20NE, positive assimilates for N-acetyl-glucosamine, L-arabinose, D-glu- Description of Nesterenkonia lacusekhoensis UT 4-03 cose, malic acid, D-maltose, D-mannitol, D-mannose and trisodium citrate, but negative for adipic acid, capric Cells are Gram-staining-positive, non-flagellated and acid, phenyl acetic acid and potassium gluconate. Strain rod-shaped. Colonies are punctiform, flat, entire and C6-16 (=NIBRBAC000498031) has been isolated from white colored on TSA medium at 30°C. Positive for ni- a natural cave, Jeju, Korea. trate reduction and urease in API 20NE, but negative for arginine dihydrolase, esculin hydrolysis, β-galacto- Description of Sanguibacter suarezii IMCC25604 sidase, gelatin hydrolysis, glucose fermentation and In- dole production. Does not utilize N-acetyl-glucosamine, Cells are Gram-staining-positive, non-flagellated and adipic acid, L-arabinose, capric acid, D-glucose, malic rod-shaped. Colonies are circular, raised, entire and yel- 36 Journal of Species Research Vol. 6, No. 1 low colored after 10 days on 1/10 MA at 20°C. Positive acid, L-arabinose, capric acid, D-glucose, D-maltose, for esculin hydrolysis and β-galactosidase in API 20NE, D-mannitol, D-mannose, phenylacetic acid, potassium but negative for arginine dihydrolase, gelatinase, glu- gluconate and trisodium citrate, but utilize malic acid. cose fermentation, indole production, nitrate reduction Strain Cip10 (=NIBRBAC000498060) has been isolat- and urease. Utilize L-arabinose, D-glucose, D-maltose ed from a tidal flat, Incheon, Korea. and D-mannose, but not utilize N-acetyl-glucosamine, adipic acid, capric acid, malic acid, D-mannitol, phenyl Description of Dietzia timorensis IMCC25613 acetic acid, potassium gluconate and trisodium citrate. Strain IMCC25604 (=NIBRBAC000497994) has been Cells are Gram-staining-positive, non-flagellated and isolated from a fresh water sample, Chuncheon, Gang- cocci-shaped. Colonies are circular, entire, raised and won Province, Korea. red colored after 10 days on R2A at 20°C. Positive for gelatinase and urease in API 20NE, but negative for ar- Description of Saccharopolyspora endophytica ginine dihydrolase, esculin hydrolysis, β-galactosidase, KYW998 glucose fermentation, indole production and nitrate production. Utilize N-acetyl-glucosamine, adipic acid, L- Cells are Gram-staining-positive, non-flagellated. Col- arabinose, capric acid, D-glucose, malic acid, D-maltose, onies are circular, opaque and white colored after 3 days D-mannitol, D-mannose, phenylacetic acid, potassium on MA at 25°C. Positive for esculin hydrolysis, gelati- gluconate and trisodium citrate. Strain IMCC25613 nase and urease, weakly positive for β-galactosidase, (=NIBRBAC000498003) has been isolated from a fresh but negative for arginine dihydrolase, glucose fermen- water sample, Inkyong lake, Incheon, Korea. tation, Indole production and nitrate reduction. In API 20NE, positive assimilates for D-glucose, malic acid, Description of Mycobacterium alvei C3-50 D-maltose, D-mannitol, D-mannose and potassium gluconate, weakly positive for N-acetyl-glucosamine and Cells are Gram-staining-positive, non-flagellated and adipic acid, but negative for L-arabinose, capric acid, rod-shaped. Colonies are circler, convex, entire and phenylacetic acid and trisodium citrate. Strain KYW998 cream colored after 10 days on TSA at 30°C. Negative (=NIBRBAC000497925) has been isolated from a sea for arginine dihydrolase, esculin hydrolysis, β-galacto- water sample, Gwangyang Bay, Gwangyang, Jeonnam sidase, gelatinase, glucose fermentation, indole produc- Province, Korea. tion, nitrate reduction and urease in API 20NE. Does not utilize N-acetyl-glucosamine, adipic acid, L-arabinose, Description of Corynebacterium freneyi HKS28 capric acid, D-glucose, malic acid, D-maltose, D-mannitol, phenylacetic acid, potassium gluconate and triso- Cells are Gram-staining-positive, non-flagellated, non- dium citrate. Weakly utilize the D-mannose. Strain C3- pigmented and rod-shaped. Colonies are circular, round, 50 ( =NIBRBAC000498028) has been isolated from a entire and yellow colored after 2 days on R2A at 30°C. natural cave, Jeju, Korea. Positive for esculin hydrolysis, glucose fermentation and urease in API 20NE, but negative for arginine dihy- Description of Mycobacterium hodleri HKS22 drolase, β-galactosidase, gelatin hydrolysis, Indole production and nitrate reduction. Does not utilize N-acetyl- Cells are Gram-staining-positive, non-flagellated and glucosamine, adipic acid, L-arabinose, capric acid, malic rod shaped. Colonies are entire, circular, round and saf- acid, D-mannitol, phenylacetic acid and trisodium citrate, fron yellow colored after 2 days on R2A at 30°C. Posi- but utilize D-glucose, D-maltose, D-mannose and potas- tive for arginine dihydrolase, esculin hydrolysis, glucose sium gluconate. Strain HKS28 (=NIBRBAC000498118) fermentation and urease in API 20NE, but negative for has been isolated from Ginseng cultivated soil, Anseong, β-galactosidase, gelatinase, indole production and ni- Gyeonggi Province, Korea. trate reduction. Utilize L-arabinose, D-glucose, malic acid, D-maltose, D-mannitol, D-mannose, potassium Description of Corynebacterium marinum Cip10 gluconate and trisodium citrate, but not utilize N-acetyl- glucosamine, adipic acid, capric acid and phenylacetic Cells are Gram-staining-positive, non-flagellated and acid. Strain HKS22 (=NIBRBAC000498114) has been rod-shaped. Colonies are circular, convex, erose and isolated from Ginseng cultivated soil, Anseong, Gyeong yellow colored after 2 days on MA at 30°C. Positive for gi Province, Korea. nitrate reduction and urease in API 20NE, but negative for arginine dihydrolase, esculin hydrolysis, β-galacto- Description of Mycobacterium obuense C6-12 sidase, gelatinase, glucose fermentation and indole production. Does not utilize N-acetyl-glucosamine, adipic Cells are Gram-staining-positive, non-flagellated and February 2017 KIM et al.-Unrecorded actinobacterial species 37 rod-shaped. Colonies are entire, circular, convex and acid. Strain G1 (=NIBRBAC000498066) has been iso- light orange colored after 3 days on ISP2 agar at 30°C. lated from a tidal flat sample, Incheon, Korea. Negative for arginine dihydrolase, esculin hydrolysis, β-galactosidase, gelatinase, glucose fermentation, indole Description of Rhodococcus fascians KHO6 production, nitrate reduction and urease in API 20NE. Cells are Gram-staining-positive, non-flagellated and Does not utilize N-acetyl-glucosamine, adipic acid, L- coccus-shaped. Colonies are circular and white colored arabinose, capric acid, D-glucose, malic acid, D-malt- after 3 days on NA at 25°C. Positive for arginine dihydro- ose, D-mannitol, D-mannose, phenylacetic acid and tri- lase, esculin hydrolysis, β-galactosidase, gelatinase, ni- sodium citrate. Weakly utilize the potassium gluconate. trate reduction and urease in API 20NE, but negative for Strain C6-12 (=NIBRBAC000498030) has been isolat- glucose fermentation and indole production. Utilize D- ed from a natural cave, Jeju, Korea. glucose, D-maltose and D-mannose, but not utilize N- acetyl-glucosamine, adipic acid, L-arabinose, capric acid, Description of Mycobacterium senegalense C10-13 malic acid, D-mannitol, phenylacetic acid, potassium glu-

Cells are Gram-staining-positive, non-flagellated and conate and trisodium citrate. Strain KHO6 (=NIBRBAC rod-shaped. Colonies are circler, convex, entire and 000497838) has been isolated from Gut of Japanese cream colored after 7 days on R2A at 30°C. Positive for crested ibis (Nipponia nippon), Gyeonggi Province, Ko- nitrate reduction in API 20NE, but negative for arginine rea. dihydrolase, esculin hydrolysis, β-galactosidase, gelatinase, glucose fermentation, indole production and ure- Description of Rhodococcus kronopolitis C1-60 ase. Utilize N-acetyl-glucosamine, D-mannitol, malic Cells are Gram-staining-positive, non-flagellated, non- acid and potassium gluconate, but not utilize adipic acid, pigmented and rod- or coccoid-shaped. Colonies are cir- L-arabinose, capric acid, D-glucose, D-maltose, D-man- cler, convex, entire and light pink-colored after 3 days on nose, phenylacetic acid and trisodium citrate. Strain TSA at 30°C. Negative for arginine dihydrolase, esculin = C10-13 ( NIBRBAC000498035) has been isolated hydrolysis, β-galactosidase, gelatinase, glucose fermen- from a natural cave, Jeju, Korea. tation, indole production, nitrate reduction and urease in API 20NE. Does not utilize N-acetyl-glucosamine, adip- Description of Mycobacterium sphagni C6-18 ic acid, L-arabinose, capric acid, D-glucose, malic acid, Cells are Gram-staining-positive, non-flagellated and D-maltose, D-mannitol, D-mannose, phenylacetic acid rod shaped. Colonies are circular, convex, entire and and trisodium citrate. Weakly utilize the potassium glu- = cream colored after 16 days on R2A at 30°C. Negative conate. Strain C1-60 ( NIBRBAC000498022) has been for arginine dihydrolase, esculin hydrolysis, β-galacto- isolated from a natural cave, Jeju, Korea. sidase, gelatinase, glucose fermentation, indole production, nitrate reduction and urease in API 20NE. Does not Description of Rhodococcus wratislaviensis C3-42 utilize N-acetyl-glucosamine, adipic acid, L-arabinose, Cells are Gram-staining-positive, non-flagellated, non- capric acid, D-glucose, malic acid, D-maltose, D-man- pigmented and rod shaped. Colonies are circular, convex, nitol, D-mannose, phenylacetic acid, potassium gluco- entire and light apricot-colored after 2 days on ISP2 agar nate and trisodium citrate. Strain C6-18 ( =NIBRBAC at 30°C. Positive for β-galactosidase and nitrate reduc- 000498032) has been isolated from a natural cave, Jeju, tion in API 20NE, but negative for arginine dihydrolase, Korea. esculin hydrolysis, gelatinase, glucose fermentation, indole production and urease. Utilize N-acetyl-glucos- Description of Rhodococcus cerastii G1 amine, adipic acid, D-glucose, malic acid, D-mannitol, phenylacetic acid, potassium gluconate and trisodium ci- Cells are Gram-staining-positive, non-flagellated, non- trate, but not utilize L-arabinose, capric acid, D-maltose pigmented and rod-shaped. Colonies are circular, con- and D-mannose. Strain C3-42 (=NIBRBAC000498026) vex and yellow colored after 2 days on R2A at 30°C. has been isolated from a natural cave, Jeju, Korea. Positive for β-galactosidase and urease in API 20NE, but negative for arginine dihydrolase, esculin hydrolysis, Description of Williamsia maris C2-18 gelatinase, glucose fermentation, indole production and nitrate reduction. Utilize N-acetyl-glucosamine, adipic Cells are Gram-staining-positive, non-flagellated and acid, L-arabinose, D-glucose, malic acid, D-maltose, rod shaped. Colonies are circular, convex, entire and D-mannitol, D-mannose, phenylacetic acid, potassium light yellow colored after 9 days on TSA at 30°C. Pos- gluconate and trisodium citrate, but not utilize capric itive for β-galactosidase in API 20NE, but negative for 38 Journal of Species Research Vol. 6, No. 1 arginine dihydrolase, esculin hydrolysis, gelatinase, glu- Description of Streptomyces colombiensis MGS3Y-3-4 cose fermentation, indole production, nitrate reduction Cells are Gram-staining-positive, non-flagellated and and urease. Does not utilize N-acetyl-glucosamine, adip- rod-shaped. Colonies are circular, entire, rough, raised ic acid, L-arabinose, capric acid, D-glucose, malic acid, and yellow colored after 3 days on MA at 30°C. Positive D-maltose, D-mannose, phenylacetic acid, potassium for esculin hydrolysis, β-galactosidase and gelatinase in gluconate and trisodium citrate. Weakly utilize the D- API 20NE, but negative for arginine dihydrolase, glu- mannitol. Strain C2-18 ( =NIBRBAC000498023) has cose fermentation, indole production, nitrate reduction been isolated from a natural cave, Jeju, Korea. and urease. Utilize N-acetyl-glucosamine, adipic acid, L-arabinose, D-glucose, malic acid, D-maltose, D-man- Description of Williamsia muralis IMCC25607 nitol, D-mannose, phenylacetic acid, potassium gluco- Cells are Gram-staining-positive, non-flagellated and nate and trisodium citrate, but not utilize capric acid. rod shaped. Colonies are circular, raised, entire and red- Strain MGS3Y-3-4 ( =NIBRBA0000114270) has been yellow colored after 7 days on R2A at 20°C. Positive for isolated from Ginseng cultivated soil, Anseong, Gyeong nitrate reduction, urease and oxidase in API 20NE, but gi Province, Korea. negative for arginine dihydrolase, esculin hydrolysis, β- galactosidase, gelatinase, glucose fermentation and indole Description of Streptomyces crystallinus HKS20 production. Positive for utilization of L-arabinose, D- glucose, malic acid, D-mannitol, D-mannose and potas- Cells are Gram-staining-positive, non-flagellated, non- sium gluconate, but negative for N-acetyl-glucosamine, pigmented and rod-shaped. Colonies are round, wrin- adipic acid, capric acid, D-maltose, phenylacetic acid kled circular, penet and white grey-colored after 1 day and trisodium citrate. Strain IMCC25607 (=NIBRBAC on R2A at 30°C. Positive for esculin hydrolysis, glucose 000497997) has been isolated from a fresh water sam- fermentation and urease in API 20NE. Negative for ar- ple, Chuncheon, Gangwon Province, Korea. ginine dihydrolase, β-galactosidase, gelatinase, indole production and nitrate reduction. Utilize adipic acid, Description of Streptomyces cocklensis BK1I29 L-arabinose, D-glucose, malic acid, D-maltose, D-mannitol, D-mannose, phenylacetic acid, potassium gluco- Cells are Gram-staining-positive, non-flagellated and nate and trisodium citrate, but not utilize N-acetyl-glu- filamentous. Colonies are rhizoid, circular, aggregated cosamine and capric acid. Strain HKS20 (=NIBRBAC and bright brown colored on ISP2 media at 30°C. Nega 000498113) has been isolated from Ginseng cultivated tive for arginine dihydrolase, esculin hydrolysis, β-galac soil, Anseong, Gyeonggi Province, Korea. tosidase, gelatinase, glucose fermentation, indole production, nitrate reduction and urease in API 20NE. Does not Description of Streptomyces cyslabdanicus TW1K13 utilize N-acetyl-glucosamine, adipic acid, L-arabinose, capric acid, D-glucose, malic acid, D-maltose, D-man- Cells are Gram-staining-positive, non-flagellated and nitol, D-mannose, phenylacetic acid, potassium gluco- filamentous. Colonies are rhizoid, umbonate, curled and nate and trisodium citrate. Strain BK1I29 (=NIBRBAC brown-colored after 7 days on ISP2 at 30°C. Diffusible 000498091) has been isolated from a plant root, Naejang pigment is produced. Negative for arginine dihydrolase, Mountain, Jeonbuk Province, Korea. esculin hydrolysis, β-galactosidase, gelatinase, glucose fermentation, indole production, nitrate reduction and Description of Streptomyces coelicoflavus MGS3Y-3-1 urease in API 20NE. Utilize N-acetyl-glucosamine, adi pic acid, L-arabinose, D-glucose, malic acid, D-maltose, Cells are Gram-staining-positive, non-flagellated and D-mannitol, D-mannose and potassium gluconate, but rod-shaped. Colonies are circular, entire, rough, raised not utilize capric acid, phenylacetic acid and trisodium and white colored after 3 days on MA at 30°C. Positive citrate. Strain TW1K13 ( =NIBRBAC000498097) has for esculin hydrolysis, β-galactosidase, nitrate reduction been isolated from soil sample, Chungbuk Province, and urease in API 20NE, but negative for arginine dihy- Korea. drolase, gelatinase, glucose fermentation and indole pro duction. N-Acetyl-glucosamine, adipic acid, L-arabinose, Description of Streptomyces griseoplanus TW1K14 D-glucose, malic acid, D-maltose, D-mannitol, D-mannose and potassium gluconate are utilized, but capric Cells are Gram-staining-positive, non-flagellated and acid, phenylacetic acid and trisodium citrate are not uti- filamentous. Colonies are rhizoid, umbonate, curled and lized. Strain MGS3Y-3-1 (=NIBRBA0000114269) has greyish colored after 7 days on ISP2 at 30°C. Diffusible been isolated from Ginseng cultivated soil, Anseong, pigment is produced. Positive for esculin hydrolysis, Gyeonggi Province, Korea. β-galactosidase and oxidase in API 20NE, but negative February 2017 KIM et al.-Unrecorded actinobacterial species 39 for arginine dihydrolase, gelatinase, glucose fermen- Description of Streptomyces lienomycini BBT-7 tation, indole production, nitrate reduction and urease. Cells are Gram-staining-positive and rod-shaped. Col- Does not utilize N-acetyl-glucosamine, adipic acid, L- onies are circular, raised, convex and yellow colored arabinose, capric acid, D-glucose, malic acid, D-malt- after 2 days on R2A at 25°C. Positive for arginine di- ose, D-mannitol, D-mannose, phenylacetic acid, potas- hydrolase, esculin hydrolysis, β-galactosidase, nitrate sium gluconate and trisodium citrate. Strain TW1K14 reduction and urease in API 20NE, but negative for (=NIBRBAC000498098) has been isolated from from gelatinase, glucose fermentation and indole production. soil sample, Chungbuk Province, Korea. Utilizes N-acetyl-glucosamine, L-arabinose, D-glucose, malic acid, D-maltose, D-mannitol, D-mannose, phenyl Description of Streptomyces kanamyceticus BBT-4 acetic acid, potassium gluconate and trisodium citrate, Cells are Gram-staining-positive, non-flagellated and but not utilize adipic acid and capric acid. Strain BBT-7 rod-shaped. Colonies are circular, raised, entire and (=NIBRBA0000114216) has been isolated from Black white-colored after 2 days on R2A at 25°C. Positive for biotite, Chungnam Province, Korea. esculin hydrolysis and β-galactosidase in API 20NE, but negative for arginine dihydrolase, gelatinase, glucose Description of Streptomyces lucensis TW1M1 fermentation, indole production, nitrate reduction and urease. Utilize N-acetyl-glucosamine, L-arabinose, D- Cells are Gram-staining-positive, non-flagellated, non- glucose, malic acid, D-maltose, D-mannitol, D-mannose pigmented and filamentous. Colonies are rhizoid, um- and potassium gluconate. Does not utilize adipic acid, bonate, curled and bright brown colored after 7 days capric acid, phenylacetic acid and trisodium citrate. on ISP2 at 30°C. Positive for esculin hydrolysis, β-ga- Strain BBT-4 (=NIBRBA0000114215) has been isolat- lactosidase and oxidase in API 20NE, but negative for ed from Black biotite, Chungnam Province, Korea. arginine dihydrolase, gelatinase, glucose fermentation, indole production, nitrate reduction and urease. Does not Description of Streptomyces laculatispora Tri-200-1 utilize N-acetyl-glucosamine, adipic acid, L-arabinose, capric acid, D-glucose, malic acid, D-maltose, D-man- Cells are Gram-staining-positive, non-flagellated and nitol, D-mannose, phenylacetic acid, potassium gluco- rod shaped. Colonies are circular, rough, dry and gray- nate and trisodium citrate. Strain TW1M1 (=NIBRBAC ish-colored after 2 days on R2A at 30°C. Positive for ar- 000498100) has been isolated from soil sample, Cheong ginine dihydrolase, esculin hydrolysis, β-galactosidase, Ju, Chungbuk Province, Korea. gelatinase, nitrate reduction and urease in API 20NE, but negative for glucose fermentation and indole production. Description of Streptomyces mirabilis TW1K17 Utilize N-acetyl-glucosamine, adipic acid, L-arabinose, D-glucose, malic acid, D-maltose, D-mannitol, D-man- Cells are Gram-staining-positive, non-flagellated, non- nose, potassium gluconate and trisodium citrate, but not pigmented and filamentous. Colonies are rhizoid, um- utilize capric acid and phenylacetic acid. Strain Tri-200-1 bonate, curled and brown colored after 7 days on ISP2 ( =NIBRBAC000498080) has been isolated from soil at 30°C. Positive for esculin hydrolysis, β-galactosidase sample, Anseong, Gyeonggi Province, Korea. and oxidase in API 20NE, but negative for arginine dihydrolase, gelatinase, glucose fermentation, indole pro- Description of Streptomyces lannensis TW1K20 duction, nitrate reduction and urease. Utilize N-acetyl- glucosamine, adipic acid, L-arabinose, D-glucose, malic Cells are Gram-staining-positive, non-flagellated, non- acid, D-maltose, D-mannitol, D-mannose and potassium pigmented and filamentous. Colonies are rhizoid, um- gluconate, but not utilize capric acid, phenylacetic acid bonate, curled and brown colored after 7 days on ISP2 and trisodium citrate. Strain TW1K17 ( =NIBRBAC at 30°C. Positive for esculin hydrolysis, β-galactosidase, 000498101) has been isolated from soil sample, Cheong gelatinase and oxidase, but negative for arginine dihy- Ju, Chungbuk Province, Korea. drolase, glucose fermentation, indole production, nitrate reduction and urease in API 20NE. Positive for utiliza- Description of Streptomyces misakiensis HKS13 tion of N-acetyl-glucosamine, L-arabinose, D-glucose, malic acid, D-mannitol, D-mannose and potassium glu- Cells are Gram-staining-positive, non-flagellated and conate, but negative for adipic acid, capric acid, D-malt- rod or cocci rod shaped. Colonies are circular, round, ose, phenylacetic acid and trisodium citrate. Strain penetrating, entire and white yellow colored after 1 days TW1K20 ( =NIBRBAC000498099) has been isolated on R2A at 30°C. Positive for β-galactosidase, gelatinase from soil sample, CheongJu, Chungbuk Province, Ko- and oxidase in API 20NE, but negative for arginine di- rea. hydrolase, esculin hydrolysis, glucose fermentation, 40 Journal of Species Research Vol. 6, No. 1 indole production, nitrate reduction and urease. Utilize soil, Anseong, Gyeonggi Province, Korea. N-acetyl-glucosamine, adipic acid, L-arabinose, D-glucose, malic acid, D-maltose, D-mannitol, D-mannose Description of Streptomyces turgidiscabies MK6Y-2-3 and potassium gluconate, but not utilize capric acid, Cells are Gram-staining-positive, non-flagellated and phenylacetic acid and trisodium citrate. Strain HKS13 rod-shaped. Colonies are irregular, undulate, rough, raised ( =NIBRBAC000498112) has been isolated from Gin- and white colored after 3 days on MA at 30°C. Positive seng cultivated soil, Anseong, Gyeonggi Province, Ko- for esculin hydrolysis, β-galactosidase and nitrate reduc rea. tion in API 20NE, but negative for arginine dihydrolase, gelatinase, glucose fermentation, indole production and Description of Streptomyces prunicolor MEC3Y-3-1 urease. Utilize N-acetyl-glucosamine, adipic acid, L- Cells are Gram-staining-positive, non-flagellated and arabinose, D-glucose, malic acid, D-maltose, D-mannitol, rod-shaped. Colonies are circular, entire, dry, raised and D-mannose and potassium gluconate, but not utilize cap orange colored after 3 days on MA at 30°C. Positive for ric acid, phenylacetic acid and trisodium citrate. Strain esculin hydrolysis and β-galactosidase in API 20NE, MK6Y-2-3 ( =NIBRBA0000114273) has been isolated but negative for arginine dihydrolase, gelatinase, glu- from Ginseng cultivated soil, Anseong, Gyeonggi Prov- cose fermentation, indole production, nitrate reduction ince, Korea. and urease. Utilize N-acetyl-glucosamine, adipic acid, L-arabinose, D-glucose, malic acid, D-maltose, D-mannitol, D-mannose and potassium gluconate, but not uti- Acknowledgements lize capric acid, phenylacetic acid and trisodium citrate. Strain MEC3Y-3-1 ( =NIBRBA0000114268) has been This study was supported by the research grant “The isolated from Ginseng cultivated soil, Anseong, Gyeong Survey of Korean Indigenous Species” from the Nation- gi Province, Korea. al Institute of Biological Resources of the Ministry of Environment in Korea. Description of Streptomyces puniciscabiei TW1S1

Cells are Gram-staining-positive, non-flagellated and References filamentous. Colonies are rhizoid, umbonate, curled and bright brown colored after 7 days on ISP2 at 30°C. Bressan, W. 2003. Biological control of maize seed patho- Positive for esculin hydrolysis, β-galactosidase, gelati- genic fungi by use of actinomycetes. Biocontrol 48(2): nase and oxidase in API 20NE, but negative for arginine 233-240. dihydrolase, glucose fermentation, indole production, Felsenstein, J. 1981. Evolutionary trees from DNA sequences: nitrate reduction and urease. Does not utilize capric a maximum likelihood approach. J. Mol. Evol. 17(6): acid, phenylacetic acid and trisodium citrate, but utilize 368-376. N-acetyl-glucosamine, adipic acid, L-arabinose, D-glu- Felsenstein, J. 1985. Confidence limit on phylogenies: an cose, malic acid, D-maltose, D-mannitol, D-mannose approach using the bootstrap. Evolution 39(4):783-791. and potassium gluconate. Strain TW1S1 (=NIBRBAC Fitch, W.M. 1971. Toward defining the course of evolution: 000498102) has been isolated from soil, Daejeon, Ko- minimum change for a specific tree topology. Syst. Zool. rea. 20(4):406-416. Hall, T.A. 1999. BioEdit: a user-friendly biological sequence Description of Streptomyces rishiriensis MMD3Y-3-3 alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95-98. Cells are Gram-staining-positive, non-flagellated and Jukes, T.H. and C.R. Cantor. 1969. Evolution of protein rod shaped. Colonies are filamentous, undulate, rough, molecules. In: H.N. Munro (ed.), Mammalian Protein raised and pale yellow colored after 3 days on MA at Metabolism, Academic Press, New York. pp. 21-132. 30°C. Positive for esculin hydrolysis, β-galactosidase Kim, O.S., Y.J. Cho, K. Lee, S.H. Yoon, M. Kim, H. Na, and gelatinase in API 20NE, but negative for arginine S.C. Park, Y.S. Jeon, J.H. Lee, H. Yi, S. Won and J. dihydrolase, glucose fermentation, indole production, Chun. 2012. Introducing EzTaxon-e: a prokaryotic 16S nitrate reduction and urease. Utilize N-acetyl-glucos- rRNA gene sequence database with phylotypes that rep- amine, adipic acid, L-arabinose, D-glucose, malic acid, resent uncultured species. Int. J. Syst. Evol. Microbiol. D-maltose, D-mannitol, D-mannose, potassium gluco- 62(3):716-721. nate and trisodium citrate, but not utilize capric acid Ludwig, W., J. Euzéby, P. Schumann, H.J. Busse, M.E. Tru- and phenylacetic acid. Strain MMD3Y-3-3 (=NIBRBA jillo, P. Kämpfer and W.B. Whiteman. 2012. Road map 0000114279) has been isolated from Ginseng cultivated of the phylum Actinobacteria. In: M. Goodfellow, P. February 2017 KIM et al.-Unrecorded actinobacterial species 41

Kämpfer, H.J. Busse, M.E. Trujillo, K.I. Suzuki, W. Lud- Analysis version 6.0. Mol. Biol. Evol. 30(12):2725-2729. wig and W.B. Whitman (eds.), Bergey’s manual of sys- Thompson, J.D., D.G. Higgins and T.J. Gibson. 1994. tematic bacteriology, vol 5, Springer-Verlag, New York. CLUSTAL W: improving the sensitivity of progressive pp. 1-28. multiple sequence alignment through sequence weight- Miao, V. and J. Davies. 2010. Actinobacteria: the good, the ing, position-specific gap penalties and weight matrix bad and the ugly. Antonie Van Leeuwenhoek 98(2):143- choice. Nucleic Acids Res. 22(22): 4673-4680. 150. Ventura, M., C. Canchaya, A. Tauch, G. Chandra, G.F. Fitz- Saitou, N. and M. Nei. 1987. The neighbor-joining method: gerald, K.F. Chater and D. van Sinderen. 2007. Genom- a new method for reconstructing phylogenetic trees. ics of Actinobacteria: tracing the evolutionary history Mol. Biol. Evol. 4(4):406-425. of an ancient phylum. Microbiol. Mol. Biol. Rev. 71(3): Servin, J.A., C.W. Herbold, R.G. Skophammer and J.A. 495-548. Lake. 2008. Evidence excluding the root of the tree of life from the actinobacteria. Mol. Biol. Evol. 25(1):1-4. Submitted: August 30, 2016 Tamura, K., G. Stecher, D. Peterson, A. Filipski, and S. Ku- Revised: December 27, 2016 mar. 2013. MEGA6: Molecular Evolutionary Genetics Accepted: February 7, 2017