A Report of 38 Unrecorded Bacterial Species in Korea, Belonging to the Phylum Actinobacteria

Journal of Species Research 5(2):223-234, 2016

A report of 38 unrecorded bacterial species in Korea, belonging to the phylum Actinobacteria

Mi-Sun Kim1, Ji-Hee Lee1, Joo-Won Kang1, Seung-Bum Kim2, Jang-Cheon Cho3, Jung-Hoon Yoon4, Ki-seong Joh5, Chang-Jun Cha6, Wan-Taek Im7, Jin-Woo Bae8, Kwang-Yeop Jahng9, Che-Ok Jeon10 and Chi-Nam Seong1,*

1Department of Biology, Sunchon National University, Suncheon 57922, Korea 2Department of Microbiology, Chungnam National University, Daejeon 34134, Korea 3Department of Biological Sciences, Inha University, Incheon 22212, Korea 4Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Korea 5Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Gyeonggi 17035, Korea 6Department of Biotechnology, Chung-Ang University, Anseong 17546, Korea 7Department of Biotechnology, Hankyong National University, Anseong 17579, Korea 8Department of Biology, Kyung Hee University, Seoul 02447, Korea 9Department of Life Sciences, Chonbuk National University, Jeonju 28644, Korea 10Department of Life Science, Chung-Ang University, Seoul 06974, Korea

*Correspondent: [email protected]

As a subset work for the collection of indigenous prokaryotic species in Korea, 38 actinobacterial strains were isolated from various environmental samples obtained from plant root, ginseng cultivating soil, mud flat, freshwater and seawater. Each strain showed higher 16S rRNA gene sequence similarity (>99.1%) and formed a robust phylogenetic clade with closest actinobacterial species which were defined and validated with nomenclature, already. There is no official description on these 38 actinobacterial species in Korea. Consequently, unrecorded 37 species of 24 genera in the 12 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 sequence, Actinobacteria, Actinomycetales, unrecorded species

IntroductIon http://www.bacterio.net/). Most Actinobacteria of med- ical or economic significance belong to the order Acti- In 2013, the authors isolated a great number of novel nomycetales (Miao and Davies, 2010). While many of and unrecorded prokaryotic species from diverse envi- these cause disease in humans, Streptomyces is notable ronmental samples in Korea. The identified species be- as a source of antibiotics (Gupte et al., 2002; Mahajan, longed to the phyla Proteobacteria, Bacteroidetes, Fir- 2012). Actinobacteria, especially Streptomyces spp., are micutes, Actinobacteria, Deinococci and Verrucomicro- recognized as the producers of many bioactive metabo- bia. As a subset of this study, the present report focuses lites that are useful to humans in medicine, such as an- on the description of unrecorded species belonging to tibacterials, antifungals, antivirals, antithrombotics, im- the phylum Actinobacteria. munomodifiers, anti-tumor drugs and enzyme inhibitors; Actinobacteria is a phylum of Gram-stain-positive and in agriculture, including insecticides, herbicides, bacteria with high G+C content in their DNA (Ventura fungicides and growth promoting substances for plants et al., 2007). Members of Actinobacteria are distribut- and animals (Bressan, 2003). Actinobacteria also con- ed in terrestrial or aquatic environments (Servin et al., tributes to the biogeochemical cycling of soil and then 2008). Actinobacteria is one of the dominant phyla and to the crop production. contains one of the largest genera, Streptomyces (LPSN; Until 2013, 228 Korean indigenous species belonging 224 JOURNAL OF SPECIES RESEARCH Vol. 5, No. 2 to the phylum Actinobacteria were isolated, described results and dIscussIon and validated according to the List of Prokaryotic name with Standing in Nomenclature (LPSN; http://www. All the 38 strains belonged to order Actinomycetales bacterio.net/). As a part of results obtained from the re- and affiliated to 12 families; 1 strain for Corynebacte- search program supported by NIBR, the present report riaceae, 7 strains for Mycobacteriaceae, 2 strains for focuses on the description of bacterial species belonging Nocardiaceae, 1 strain for Cellulomonadaceae, 4 strains to the phylum Actinobacteria except suborder Strepto- for Intrasporangiaceae, 12 strains for Microbacteriace- mycineae, which have not been previously isolated in ae, 2 strains for Micrococcaceae, 2 strains for Promicro- Korea. Here we report 38 unrecorded Actinobacterial monosporaceae, 1 strain for Sanguibacteraceae, 1 strain strains in Korea. for Micromonosporaceae, 4 strains for Nocardioidaceae and 1 strain for Geodermatophilaceae (Table 1). All the strains were isolated from diverse environ- MaterIals and Methods ments and regions: 17 strains from fresh water, 6 strains from rhizosphere, 6 strains from ginseng cultivated soil, A total of 45 bacterial strains assigned to the phylum 4 strains from brackish water or lagoon, 3 strains from Actinobacteria were isolated from various environmen- tidal flat sediment, each one strain from seawater dust; tal samples collected from plant root, ginseng cultivating 13 strains from Gyeongnam, 9 strains from Daejeon, 6 soil, mud flat, freshwater and seawater (Table 1). Each strains from Gyeonggi, 3 strains from Jeonnam, each 2 environmental sample was processed separately, spread strains from Jeonbuk, Ganfwon and Jeju, and one strain onto diverse culture media (Becton Dickinson) including from Gyeongbuk. R2A, glucose yeast extract (Gordon and Mihm, 1962), These strains were Gram-staining-positive and chemo- marine, tryptic soy and nutrient agars and incubated at heterotrophic. Fig. 2 shows phylogenetic assignment of 25-30°C for 2-5 days. All strains were purified as sin- the strains based on 16S rRNA gene sequences. gle colonies and stored as 10-20% glycerol suspension Here we report 38 unrecorded bacterial species in Ko- -80°C as well as lyophilized ampoules. Among them 7 rea belonging to the phylum Actinobacteria. strains produced aerial mycelium, belonged to the suborder Streptomycineae and were excluded in this work. Description of Corynebacterium doosanense W2-3-5 Colony morphology of the strains was observed on Cells are Gram-staining-positive, non-flagellated, non- agar plates with a magnifying glass after cells grew up to pigmented and rod-shaped. Colonies are circular, convex stationary phase. Cellular morphology and cell size were and entire after 2 days of incubation on R2A at 25°C. examined by either transmission electron microscopy Positive for nitrate reduction and oxidase in API 20NE or scanning electron microscopy (Fig. 1). Biochemical but negative for esculin hydrolysis, urease, gelatinase, characteristics were tested by using API 20NE galleries β-galactosidase, indole production, glucose fermenta- (bioMérieux) according to the manufacturer’s instruc- tion and arginine dihydrolase. D-Glucose, L-arabinose, tions. D-mannose, D-mannitol, N-acetyl-glucosamine, D-mal- Bacterial DNA extraction, PCR amplification and 16S tose, potassium gluconate, malic acid, trisodium citrate, rRNA gene sequencing were performed using the stan- capric acid, adipic acid and phenylacetic acid are not dard procedures described elsewhere. The 16S rRNA utilized. Strain W2-3-5 ( =NIBRBA0000114209) has gene sequences of the strains assigned to the phylum been isolated from mineral water, Daejeon, Korea. Actinobacteria were compared with the sequences held in GenBank by BLASTN and also analyzed using the Description of Mycobacterium aurum WM31 EzTaxon-e server (Kim et al., 2012). For phylogenetic analyses, multiple alignments were performed using the Cells are Gram-staining-positive, non-flagellated and Clustal_W program (Thompson et al., 1994) and gaps rod-shaped. Colonies are entire, opaque and orange col- were edited in the BioEdit program (Hall, 1999). Evolu- ored after 7 days on R2A at 25°C. Negatives for nitrate tionary distances were calculated using the Jukes-Cantor reduction, Indole production, glucose fermentation, gel- model (Jukes and Cantor, 1969). The phylogenetic trees atin hydrolysis, arginine dihydrolase, urease, esculin hy- were constructed by using the neighbour-joining (Saitou drolysis and β-galactosidase in API 20NE. D-Glucose, and Nei, 1987), the maximum-likelihood (Felsenstein, L-arabinose, D-mannose, D-mannitol, N-acetyl-glu- 1981) and the maximum-parsimony (Fitch, 1971) meth- cosamine, D-maltose, potassium gluconate, trisodium ods with the MEGA 6.0 Program (Tamura et al., 2013) citrate and malic acid are not utilized. Strain WM31 with bootstrap values based on 1,000 replications (Fel- (=NIBRBA0000114426) has been isolated from fresh- senstein, 1985). water, Woopo wetland, Gyeongnam Province, Korea. June 2016 KIM ET AL.-UNRECORDED ACTINOBACTERIAL SPECIES 225 30℃, 3 d 30℃, 3 d 25℃, 3 d 25℃, 3 d 30℃, 3 d 25℃, 3 d 30℃, 2 d 30℃, 3 d 25℃, 3 d 25℃, 3 d 25℃, 7 d 30℃, 3 d 25℃, 5 d 25℃, 2 d 25℃, 2 d 30℃, 3 d 25℃, 2 d 25℃, 3 d 25℃, 2 d 30℃, 3 d 30℃, 2 d 25℃, 2 d 25℃, 7 d 25℃, 3 d 25℃, 7 d 25℃, 7 d 30℃, 2 d condition Incubation MA MA MA MA MA MA MA R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A TSA R2A R2A R2A R2A R2A TSA Medium Isolation

Source Ginseng cultivating filed Ginseng cultivating filed Freshwater Lagoon Plant root Freshwater Plant root Mud flat Freshwater Freshwater Freshwater Ginseng cultivating filed Fresh water Freshwater Dust Ginseng cultivating filed Mineral water Mud flat Plant root Mineral water Freshwater Freshwater Freshwater Freshwater Plant root Mineral water Freshwater 100 100 (%) 99.7 99.1 99.9 99.5 99.9 99.6 99.3 99.0 99.9 99.9 99.4 99.3 99.8 99.9 99.9 99.2 99.6 99.3 99.9 99.6 99.2 99.3 99.2 99.8 99.6 Similarity T T T T T T T T T DSM T DSM DSM T T T Gsoil CafT13 T CAU CF36 T T NCCP-11 MG13 ATCC ATCC JCM 14558 ATCC ATCC cerinus CCUG YIM T ATCC 27023 ATCC ATCC 43909 ATCC T ATCC 23366 ATCC DSM 20308 A5E-52 THG-e54 wged11 Ac-1401 DSM 9580 DSM 7486 NBRC 100374 subsp. P 229/10 P N1350 . T T T T T T T T Most closely related species T Microbacterium phyllosphaerae Microbacterium Rathayibacter tritici Microbacterium lacus Microbacterium paraoxydans Microbacterium 13468 Herbiconiux ginsengi Leifsonia poae VKM dextranolyticum Microbacterium 8607 hatanonis Microbacterium Terrabacter tumescens Terrabacter pratensis Agreia jenensis Agrococcus cerinus Agromyces 8595 cladoniiphilus Frondihabitans Williamsia faeni Williamsia Arsenicicoccus bolidensis 47306 ginsenosidimutans Terrabacter 3082 koreensis Terrabacter Mycobacterium aurum Mycobacterium chubuense Mycobacterium flavescens Mycobacterium gilvum Mycobacterium llatzerense Mycobacterium obuense Mycobacterium sediminis M13028 abscessus Nocardia Cellulomonas pakistanensis Corynebacterium doosanense 212 27278 14474 Actinobacteria NIBR NO. NIBRBA0000114274 NIBRBA0000114439 NIBRBA0000114307 NIBRBA0000114429 NIBRBA0000114394 NIBRBA0000114192 NIBRBA0000114277 NIBRBA0000114200 NIBRBA0000114440 NIBRBA0000114281 NIBRBA0000114437 NIBRBA0000114154 NIBRBA0000114146 NIBRBA0000114276 NIBRBA0000114323 NIBRBA0000114438 NIBRBA0000114210 NIBRBA0000114305 NIBRBA0000114179 NIBRBA0000114441 NIBRBA0000114426 NIBRBA0000114431 NIBRBA0000114428 NIBRBA0000114427 NIBRBA0000114183 NIBRBA0000114211 NIBRBA0000114209 Strain ID MK6Y-6-1 03SU14 BM24 WM113 HME9262 DR7-12 MMD3Y-15-3 SR5-11 WM26 MS4Y-2-4 WW45 JJ9003 MM3 MMD3Y-10-4 TMIL-2 01SU3 W4-2-4 BM14 DT5-10 WM79 W2-3-5 WM31 WS50 WM81 WM69 ST5-11 W2-2-3 Genus Rathayibacter Leifsonia Microbacterium Herbiconiux Terrabacter Agreia Agrococcus Agromyces Frondihabitans Nocardia Williamsia Cellulomonas Arsenicicoccus Corynebacterium Mycobacterium Family Microbacteriaceae Nocardiaceae Cellulomonadaceae Intrasporangiaceae Corynebacteriaceae Mycobacteriaceae Taxonomic affiliation and isolation information of the isolates belonging to phylum Taxonomic Suborder Micrococcineae Corynebacterineae Table 1. Table 226 JOURNAL OF SPECIES RESEARCH Vol. 5, No. 2

Description of Mycobacterium chubuense WS50 Cells are Gram-staining-positive, non-flagellated and 30℃, 3 d 30℃, 3 d 30℃, 3 d 30℃, 3 d 25℃, 2 d 25℃, 3 d 25℃, 3 d 30℃, 3 d 30℃, 3 d 30℃, 3 d 30℃, 3 d condition Incubation rod-shaped. Colonies are entire, convex, opaque and yellow colored after 3 days on R2A at 25°C. Positive for MA MA nitrate production in API 20NE, but negative for glucose R2A R2A R2A R2A R2A R2A R2A R2A R2A

Medium fermentation, urease, esculin hydrolysis β-galactosidase, Isolation indole production, arginine dihydrolase and gelatinase. D- Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl- glucosamine, D-maltose, potassium gluconate, malic acid, Source trisodium citrate, capric acid, adipic acid and phenyl- Ginseng cultivating filed Lagoon Freshwater Seawater Brackish water Plant root Mud flat Plant root Ginseng cultivating filed Freshwater Brackish water acetic acid are not utilized. Strain WS50 ( =NIBRBA 0000114431) has been isolated from freshwater, Woopo (%) 99.6 99.8 99.1 99.6 99.6 99.6 99.3 99.8 99.4 99.1 99.2 wetland, Gyeongnam Province, Korea. Similarity

T Description of Mycobacterium flavescens WM81 T T T Cells are Gram-staining-positive, non-flagellated and T T SH2-13 T IFO

T rod shaped. Colonies are entire, convex, opaque and yel- YIM 65004 H17 low colored after 7 days on R2A at 25°C. In API 20NE KLBMP 1279 KLBMP DSM 10542 Sco-A36 Cr7-14 HKI 0478 gallery, negative for urease, esculin hydrolysis, glucose DSM 20550 fermentation and β-galactosidase, nitrate reduction, indole production, arginine dihydrolase and gelatinase. T Does not utilize L-arabinose, capric acid, adipic acid, T

Most closely related species malic acid, trisodium citrate and phenylacetic acid but Sanguibacter keddieii psychrotolerans Nocardioides RHLT2-1 Micrococcus yunnanensis Micrococcus Isoptericola nanjingensis sukumoe Promicromonospora 14650 sediminicola Micromonospora Kribbella aluminosa Marmoricola korecus alpinus Nocardioides Modestobacter roseus Arthrobacter agilis Arthrobacter utilize D-glucose, D-mannose, D-mannitol, N-acetyl- glucosamine, D-maltose and potassium gluconate. Strain WM81 (=NIBRBA0000114428) has been isolated from freshwater, Woopo wetland, Gyeongnam Province, Ko- rea. NIBR NO. NIBRBA0000114278 NIBRBA0000114396 NIBRBA0000114191 NIBRBA0000114436 NIBRBA0000114442 NIBRBA0000114313 NIBRBA0000114312 NIBRBA0000114197 NIBRBA0000114308 NIBRBA0000114219 NIBRBA0000114311 Description of Mycobacterium gilvum WM69 Cells are Gram-staining-positive, non-flagellated and rod shaped. Colonies are entire, convex, opaque and yellow colored after 7 days on R2A at 25°C. In API 20NE Strain ID MMD3Y-15-4 HME9278 DR7-01 WW39 WW41 GDM10 CR9 CDR5 SR4-01 BM28 Gsoil012 gallery, negative for urease, esculin hydrolysis, glucose fermentation and β-galactosidase, nitrate reduction, indole production, arginine dihydrolase and gelatinase. D- Glucose, L-arabinose, D-mannose, N-acetyl-glucosamine,

Genus D-maltose, D-Mannitol, potassium gluconate, malic acid, trisodium citrate, capric acid, adipic acid and phenyl- Promicromonospora Sanguibacter Micromonospora Marmoricola Nocardioides Modestobacter Arthrobacter Micrococcus Isoptericola Kribbella acetic acid are not utilized. Strain WM69 ( =NIBRBA 0000114427) has been isolated from freshwater, Woopo wetland, Gyeongnam Province, Korea.

Description of Mycobacterium llatzerense ST5-11 Family Cells are Gram-staining-positive, non-flagellated and Sanguibacteraceae Micromonosporaceae Nocardioidaceae Geodermatophilaceae Micrococcaceae Promicromonosporaceae rod-shaped. Colonies are entire, glistening and orange colored after 2 days on TSA at 30°C. Positive for and urease and esculin hydrolysis in API 20NE, but negative for nitrate reduction, indole production, glucose fermen- Continued. tation, arginine dihydrolase, gelatinase and β-galactosi- Suborder dase. Utilize D-glucose, L-arabinose, D-mannitol, D-mal- Micromonosporineae Frankineae Propionibacterineae Micrococcineae Table 1. Table tose, but not utilize D-mannose, N-acetyl-glucosamine, June 2016 KIM ET AL.-UNRECORDED ACTINOBACTERIAL SPECIES 227

(1) (2) (3) (4) (5)

(6) (7) (8) (9) (10)

(11) (12) (13) (14) (15)

(16) (17) (18) (19) (20)

(21) (22) (23) (24) (25)

(26) (27) (28) (29) (30)

(31) (32) (33) (34) (35)

(36) (37) (38)

Fig. 1. Transmission electron micrographs or scanning electron micrographs of cells of the strains isolated in this study. Strains: 1, W2-3- 5; 2, WM31; 3, WS50; 4, WM81; 5, WM69; 6, ST5-11; 7, W2-2-3; 8, 01SU3; 9, W4-2-4; 10, BM14; 11, DT5-10; 12, WM79; 13, WM26; 14, MS4Y-2-4; 15, WW45; 16, JJ9003; 17, MM3; 18, MMD3Y-10-4; 19, TMIL-2; 20, SR5-11; 21, BM24; 22, WM113; 23, HME9262; 24, DR7-12; 25, MMD3Y-15-3; 26, MK6Y-6-1; 27, 03SU14; 28, CDR5; 29, SR4-01; 30, BM28; 31, MMD3Y-15-4; 32, HME9278; 33, DR7- 01; 34, Gsoil012; 35, WW39; 36, WW41; 37, GDM10; 38, CR9. 228 JOURNAL OF SPECIES RESEARCH Vol. 5, No. 2

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, 0.01 sub- stitutions per nucleotide position. Suborder: A, Micromonosporineae; B, Frankineae; C, Propionibacterineae. June 2016 KIM ET AL.-UNRECORDED ACTINOBACTERIAL SPECIES 229 capric acid, adipic acid, potassium gluconate, malic acid, β-galactosidase in API 20NE, but negative for indole trisodium citrate and phenylacetic acid. Strain ST5-11 production, glucose fermentation, arginine dihydrolase ( =NIBRBA0000114183) has been isolated from plant and gelatinase. Does not utilize D-glucose, L-arabinose, root, Daejeon, Korea. D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid, capric acid, adip- Description of Mycobacterium obuense W2-2-3 ic acid, trisodium citrate and phenylacetic acid. Strain BM14 (=NIBRBA0000114305) has been isolated from Cells are Gram-staining-positive, non-flagellated and mud flat, Wando, Jeonnam Province, Korea. rod-shaped. Colonies are round, convex and yellow colored after 2 days on R2A at 25°C. Negative for nitrate reduction, indole production, glucose fermentation, ar- Description of Cellulomonas pakistanensis DT5-10 ginine dihydrolase, gelatinase, esculin hydrolysis and β- Cells are Gram-staining-positive, non-flagellated, non- galactosidase but positive for urease. L-arabinose, D- pigmented and rod-shaped. Colonies are round, glisten- mannitol, potassium gluconate and D-maltose are util- ing, convex and yellow-colored after 2 days incubation on ized. Does not utilize D-glucose, D-mannose, capric TSA at 30°C. Positive for glucose fermentation, esculin acid, adipic acid and N-acetyl-glucosamine. Strain W2- hydrolysis, β-galactosidase and gelatinase in API 20NE.

2-3 ( =NIBRBA0000114211) has been isolated from Negative for nitrate reduction, indole production, arginine mineral water, Daejeon, Korea. dihydrolase and urease. Utilize D-glucose, L-arabinose, N-acetyl-glucosamine, D-maltose, potassium gluconate Description of Mycobacterium sediminis 01SU3 and D-mannitol, but not utilize D-mannose, capric acid, Cells are Gram-staining-positive, non-flagellated and adipic acid, phenylacetic acid, malic acid and trisodium = rod shaped. Colonies are entire, convex, opaque and or- citrate. Strain DT5-10 ( NIBRBA0000114179) has been ange colored after 3 days of incubation on R2A at 25°C. isolated from a plant root, Daejeon, Korea. Negatives for nitrate reduction, Indole production, glucose fermentation, gelatin hydrolysis, arginine dihydrol- Description of Arsenicicoccus bolidensis WM79 ase, esculin hydrolysis and β-galactosidase in API 20NE, Cells are Gram-staining-positive, flagellated, non-pig- but positive for urease. D-Glucose, L-arabinose, D-man- mented and coccoid. Colonies are opaque, round, smooth, nose, D-mannitol, N-acetyl-glucosamine, D-mal tose, po- umbonate and yellow-colored after 3 days on R2A at tassium gluconate, trisodium citrate and malic acid are 25°C. Positive for nitrate reduction, esculin hydrolysis = not utilized. Strain 01SU3 ( NIBRBA0000114438) has and gelatinase in API 20NE, but negative for indole pro- been isolated from a freshwater sample, Woopo wetland, duction, glucose fermentation, arginine dihydrolase, ure- Gyeongnam Province, Korea. ase and β-galactosidase. Does not utilize D-glucose, D- mannose, potassium gluconate, capric acid, adipic acid, Description of Nocardia abscessus W4-2-4 trisodium citrate, malic acid and phenylacetic acid, but Cells are Gram-staining-positive, non-flagellated, non- utilize D-mannitol, N-acetyl-glucosamine and D-maltose. pigmented and rod-shaped. Colonies are circular, convex Strain WM79 (=NIBRBA0000114441) has been isolat- and entire after 2 days on R2A at 25°C. In API 20NE ed from a freshwater sample, Woopo wetland, Gyeong- gallery, negative for nitrate reduction, arginine dihydro- nam Province, Korea. lase, urease, indole production, glucose fermentation, esculin hydrolysis, gelatinase and β-galactosidase. D- Description of Terrabacter ginsenosidimutans WM26 glucose, D-mannitol, potassium gluconate and malic acid Cells are Gram-staining-positive, non-flagellated and are utilized, but L-arabinose, D-mannose, trisodium cit- rod shaped. Colonies are glossy, entire, smooth and pale rate, N-acetyl-glucosamine, D-maltose, capric acid, adi- yellow colored after 7 days on R2A at 25°C. Positive for pic acid and phenylacetic acid are not utilized. Strain W4- nitrate reduction, esculin hydrolysis and β-galactosidase 2-4 ( =NIBRBA0000114210) has been isolated from a in API 20NE, but negative for indole production, arginine mineral water sample, Daejeon, Korea. dihydrolase glucose fermentation, urease and gelatinase. Utilizes D-glucose, D-mannose, D-mannitol, D-maltose, Description of Williamsia faeni BM14 potassium gluconate and malic acid, but not L-arabinose, Cells are Gram-staining-positive, non-flagellated and N-acetyl-glucosamine, adipic acid, capric acid, trisodium rod-shaped. Colonies are circular, raised, entire, glisten- citrate and phenylacetic acid. Strain WM26 (=NIBRBA ing and yellow colored after 3 days on MA at 30°C. Pos- 0000114440) has been isolated from a freshwater sam- itive for nitrate reduction, urease, esculin hydrolysis and ple, Woopo wetland, Gyeongnam Province, Korea. 230 JOURNAL OF SPECIES RESEARCH Vol. 5, No. 2

Description of Terrabacter koreensis MS4Y-2-4 hydrolase, indole production, glucose fermentation and urease. D-Glucose, D-mannose, L-arabinose, D-manni- Cells are Gram-staining-positive, non-flagellated and tol, N-acetyl-glucosamine, potassium gluconate, capric rod shaped. Colonies are glossy, entire, smooth and pale acid, malic acid, trisodium citrate, D-maltose, adipic yellow colored after 3 days on MA at 30°C. Positive for acid and phenylacetic acid are not utilized. Strain MM3 nitrate reduction, esculin hydrolysis and β-galactosidase ( =NIBRBA0000114146) has been isolated from dust in API 20NE, but negative for glucose fermentation, ure- sample, Jeonbuk National University, Korea. ase, indole production, arginine dihydrolase and gelatinase. Positive for utilization of D-glucose, L-arabinose, Description of Agromyces cerinus subsp. cerinus D-mannose, D-mannitol, N-acetyl-glucosamine, D-malt- MMD3Y-10-4 ose, potassium gluconate, malic acid, trisodium citrate and adipic acid, but negative for capric acid and phenyl- Cells are Gram-staining-positive, non-flagellated and acetic acid. Strain MS4Y-2-4 (=NIBRBA0000114281) rod-shaped. Colonies are circular, entire, convex and has been isolated from Ginseng cultivated soil, Anseong, pale yellow-colored after 3 days on marine agar at 30°C. Gyeonggi Province, Korea. Positive for esculin hydrolysis and β-galactosidase in API 20NE, but negative for nitrate reduction, arginine Description of Terrabacter tumescens WW45 dihy drolase, glucose fermentation, urease, gelatinase and indole production. D-Glucose, L-arabinose, D-man- Cells are Gram-staining-positive, non-flagellated and nose, malic acid, N-acetyl-glucosamine and D-maltose rod shaped. Colonies are glossy, entire, smooth and pale are utilized. Does not utilize D-mannitol, potassium yellow colored after 5 days on R2A at 25°C. Positive for gluconate, trisodium citrate, capric acid, adipic acid and nitrate reduction, esculin hydrolysis, gelatinase and β- phenylacetic acid. Strain MMD3Y-10-4 ( =NIBRBA galactosidase in API 20NE, but negative for indole pro- 0000114276) has been isolated from a Ginseng cultivat- duction, glucose fermentation, urease and arginine dihy- ed soil, Anseong, Gyeonggi Province, Korea. drolase. Utilizes L-arabinose and potassium gluconate, but not capric acid, malic acid, trisodium citrate, D-glu- Description of Frondihabitans cladoniiphilus TMIL-2 cose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, adipic acid and phenylacetic acid. Strain Cells are Gram-staining-positive, non-flagellated and WW45 (=NIBRBA0000114437) has been isolated from rod shaped. Colonies are irregular, smooth, glistening a freshwater sample, Woopo wetland, Gyeongnam Prov- and yellow-colored after 3 days on R2A at 25°C. Posi- ince, Korea. tive for nitrate reduction, esculin hydrolysis and β-galactosidase in API 20NE, but negative for indole produc- Description of Agreia pratensis JJ9003 tion, urease, glucose fermentation, arginine dihydrolase and gelatinase. Utilize D-glucose, L-arabinose, D-man- Cells are Gram-staining-positive, non-flagellated and nose, D-mannitol, N-acetyl-glucosamine, D-maltose and rod to coccus-shaped. Colonies are circular, raised and potassium gluconate, but not utilize capric acid, malic yellow colored after 2 days on R2A at 25°C. Diffusible acid, trisodium citrate, phenylacetic acid and adipic acid. pigment is not produced. Positive for esculin hydrolysis, Strain TMIL-2 ( =NIBRBA0000114323) has been iso- β-galactosidase and oxidase, but negative for nitrate related from a freshwater sample, Milyang, Gyeongnam duction glucose fermentation, arginine dihydrolase, ure- Province, Korea. ase, gelatinase and indole production. Does not utilize D- glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl- Description of Herbiconiux ginsengi SR5-11 glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenyl- Cells are Gram-staining-positive, non-flagellated, non- acetic acid. Strain JJ9003 (=NIBRBA0000114154) has pigmented and short rod-shaped. Colonies are circular, been isolated from a freshwater sample, Wanju, Jeonbuk convex and entire after 3 days on R2A at 30°C. Positive Province, Korea. for arginine dihydrolase, esculin hydrolysis, β-galactosidase, urease and gelatinase, but negative for nitrate reduction, glucose fermentation and indole production in Description of Agrococcus jenensis MM3 API 20NE. Positive for utilization of D-glucose, D-man- Cells are Gram-staining-positive, non-flagellated and nose, D-mannitol and L-arabinose, weakly positive for rod to coccus-shaped. Colonies are circular, raised, entire N-acetyl-glucosamine, malic acid and D-maltose, but and yellow colored after 2 days on MA at 25°C. Positive negative for potassium gluconate, trisodium citrate, phe- for esculin hydrolysis, gelatinase, β-galactosidase and nylacetic acid, capric acid and adipic acid. Strain SR5- oxidase, but negative for nitrate reduction, arginine di- 11 ( =NIBRBA0000114200) has been isolated from a June 2016 KIM ET AL.-UNRECORDED ACTINOBACTERIAL SPECIES 231 plant root, Daejeon, Korea. nitrate reduction, glucose fermentation, arginine dihydrolase, indole production and gelatinase in API 20NE. Description of Leifsonia poae BM24 D-maltose, D-mannose and D-mannitol are utilized. Does not utilize D-glucose, L-arabinose, N-acetyl-glu- Cells are Gram-staining-positive and rod-shaped. Col- cosamine, potassium gluconate, malic acid, trisodium onies are circular, smooth, convex, glistening and yellow citrate, phenylacetic acid, capric acid and adipic acid. colored after 3 days on MA at 30°C. Negative for nitrate Strain DR7-12 ( =NIBRBA0000114192) has been iso- reduction, indole production, glucose fermentation, argi- lated from plant root, Daejeon, Korea. nine dihydrolase, urease and gelatinase, but positive for esculin hydrolysis and β-galactosidase in API 20NE. D- Description of Microbacterium paraoxydans Glucose, L-arabinose, D-mannose, D-maltose and N- MMD3Y-15-3 acetyl-glucosamine are utilized. Does not utilize D-mannitol, potassium gluconate, malic acid, trisodium citrate, Cells are Gram-staining-positive, non-flagellated and phenylacetic acid, capric acid and adipic acid. Strain rod shaped. Colonies are entire, convex and golden yel- BM24 (=NIBRBA0000114307) has been isolated from low colored after 3 days on MA at 30°C. Positive for mud flat, Wando, Jeonnam Province, Korea. esculin hydrolysis, β-galactosidase and gelatinase, but negative for nitrate reduction, glucose fermentation, ar- Description of Microbacterium dextranolyticum ginine dihydrolase, indole production and urease and in WM113 API 20NE. D-Maltose, D-mannose, D-mannitol, N-acetyl-glucosamine and potassium gluconate are utilized. Cells are Gram-staining-positive, non-flagellated and Does not utilize D-glucose, L-arabinose, malic acid, tri- rod shaped. Colonies are entire, convex, opaque and yel- sodium citrate, phenylacetic acid, capric acid and adipic low colored after 3 days on R2A at 25°C. Positive for acid. Strain MMD3Y-15-3 (=NIBRBA0000114277) has glucose fermentation, esculin hydrolysis and β-galacto- been isolated from Ginseng cultivated soil, Anseong, sidase, but negative for nitrate reduction, arginine dihy- Gyeonggi Province, Korea. drolase, indole production, urease and gelatinase in API 20NE. D-Glucose, L-arabinose, D-mannose, D-mannitol, Description of Microbacterium phyllosphaerae N-acetyl-glucosamine, potassium gluconate, D-maltose, MK6Y-6-1 malic acid and trisodium citrate are utilized. Does not utilize phenylacetic acid, capric acid and adipic acid. Cells are Gram-staining-positive, non-flagellated and Strain WM113 ( =NIBRBA0000114429) has been iso- rod shaped. Colonies are entire, convex and golden yel- lated from a freshwater sample, Woopo wetland, Gyeong- low colored after 3 days on MA at 30°C. Positive for nam Province, Korea. esculin hydrolysis and β-galactosidase, but negative for nitrate reduction, glucose fermentation, arginine dihy- Description of Microbacterium hatanonis HME9262 drolase, indole production, urease and gelatinase in API 20NE. D-Glucose, D-mannose, D-mannitol, potassium Cells are Gram-staining-positive, non-flagellated and gluconate, phenylacetic acid and malic acid are utilized. rod shaped. Colonies are entire, convex and yellow col- Does not utilize L-arabinose, N-acetyl-glucosamine, ored after 2 days on R2A at 30°C. Negative for nitrate D-maltose, trisodium citrate, capric acid and adipic acid. reduction, glucose fermentation, arginine dihydrolase, Strain MK6Y-6-1 ( =NIBRBA0000114274) has been indole production, urease and gelatinase, but positive isolated from Ginseng cultivated soil, Anseong, Gyeong- for esculin hydrolysis and β-galactosidase in API 20NE. gi Province, Korea. D-Glucose, L-arabinose, D-mannose, D-maltose D-mannitol, N-acetyl-glucosamine, potassium gluconate and Description of Rathayibacter tritici 03SU14 malic acid are utilized. Does not utilize trisodium citrate, phenylacetic acid, capric acid and adipic acid. Strain Cells are Gram-staining-positive, non-flagellated and HME9262 ( =NIBRBA0000114394) has been isolated short rod shaped. Colonies are entire, convex, opaque and from lagoon, Gangneung, Gangwon Province, Korea. yellow colored after 3 days of incubation on R2A at 25 °C. In API 20NE, positive for esculin hydrolysis and β- galactosidase, but negative for nitrate reduction, glucose Description of Microbacterium lacus DR7-12 fermentation, indole production, arginine dihydrolase, Cells are Gram-staining-positive, non-flagellated and gelatinase and urease. D-Glucose, L-arabinose, D-man- rod shaped. Colonies are entire, convex and yellow col- nose, D-mannitol, D-maltose and potassium gluconate ored after 3 days on R2A at 30°C. Positive for esculin are utilized. Des not utilize N-acetyl-glucosamine, capric hydrolysis, β-galactosidase and urease, but negative for acid, adipic acid, malic acid, trisodium citrate and phenyl- 232 JOURNAL OF SPECIES RESEARCH Vol. 5, No. 2 acetic acid. Strain 03SU14 ( =NIBRBA0000114439) rod shaped. Colonies are entire, convex and pale yellow has been isolated from a freshwater sample, Woopo wet- colored after 3 days on MA at 30°C. Positive for esculin land, Gyeongnam Province, Korea. hydrolysis and β-galactosidase, but negative for nitrate reduction, glucose fermentation, arginine dihydrolase, Description of Arthrobacter agilis CDR5 indole production, urease and gelatinase in API 20NE. D-Glucose, L-arabinose, D-mannose, potassium gluco- Cells are Gram-staining-positive, flagellated and coc- nate and malic acid are utilized. Does not utilize D-malt- coid. Colonies are circular, entire, smooth, glistening, ose, D-mannitol, N-acetyl-glucosamine, trisodium citrate, convex and deep yellowish pink-colored after 3 days on phenylacetic acid, capric acid and adipic acid. Strain R2A at 30°C. Positive for esculin hydrolysis, gelatinase MMD3Y-15-4 ( =NIBRBA0000114278) has been iso- and β-galactosidase in API 20NE, but negative for ni- lated from Ginseng cultivated soil, Anseong, Gyeonggi trate reduction, indole production, glucose fermentation, Province, Korea. arginine dihydrolase and urease. Utilize D-glucose, D- mannose, D-mannitol, D-maltose and potassium gluconate, but not utilize capric acid, but L-arabinose, N-ace- Description of Sanguibacter keddieii HME9278 tyl-glucosamine, adipic acid, malic acid, trisodium ci- Cells are Gram-staining-positive, non-flagellated and trate and phenylacetic acid. Strain CDR5 ( =NIBRBA rod shaped. Colonies are entire, convex and yellow col- 0000114311) has been isolated from brackish water sam- ored after 3 days on R2A at 30°C. Positive for glucose ple, Jeju, Korea. fermentation, esculin hydrolysis and β-galactosidase in API 20NE, but negative for nitrate reduction, indole Description of Micrococcus yunnanensis SR4-01 production, arginine dihydrolase, urease and gelatinase. Cells are Gram-staining-positive, non-flagellated and Utilizes D-glucose, L-arabinose, D-mannose, N-acetyl- rod shaped. Colonies are entire, convex and yellow col- glucosamine and D-maltose, but not capric acid, malic acid, trisodium citrate, D-mannitol, potassium gluco- ored after 3 days on R2A at 30°C. Positive for gelatinase, but negative for nitrate reduction, indole production, glu- nate, adipic acid and phenylacetic acid. Strain HME9278 = cose fermentation, esculin hydrolysis, arginine dihydro- ( NIBRBA0000114396) has been isolated from lagoon, lase, urease and β-galactosidase in API 20NE. D-Glu- Gangneung, Gangwon Province, Korea. cose and D-maltose are utilized. Weakly utilize the capric acid and malic acid. Does not utilize L-arabinose, Description of Micromonospora sediminicola DR7-01 D-mannose, D-mannitol, N-acetyl-glucosamine, potas- Cells are Gram-staining-positive, non-flagellated and sium gluconate, adipic acid, trisodium citrate and phe- rod shaped. Colonies are entire and rough after 3 days = nylacetic acid. Strain SR4-01 ( NIBRBA0000114197) on R2A at 30°C. Orange colored colony darkened when has been isolated from plant root, Daejeon, Korea. aged. Positive for nitrate reduction, esculin hydrolysis, gelatinase and β-galactosidase in API 20NE, but negative Description of Isoptericola nanjingensis BM28 for glucose fermentation, urease, indole production, argi- Cells are Gram-staining-positive, non-flagellated and nine dihydrolase and. Weakly utilizes D-glucose, L-ara- oval-shaped. Colonies are circular, smooth, convex, glis- binose, N-acetyl-glucosamine and D-maltose, but not uti- tening and yellow colored after 3 days on MA at 30°C. lize capric acid, malic acid, trisodium citrate, D-mannose, Negative for nitrate reduction, indole production, glu- D-mannitol, potassium gluconate, adipic acid and phenyl- cose fermentation and arginine dihydrolase in API 20NE acetic acid. Strain DR7-01 (=NIBRBA0000114191) has but positive for oxidase, esculin hydrolysis urease, gel- been isolated from plant root, Daejeon, Korea. atinase and β-galactosidase. Positive for utilization of D-glucose, L-arabinose, D-mannose, N-acetyl-glucos- Description of Kribbella aluminosa Gsoil012 amine, D-maltose and potassium gluconate; weakly pos- Cells are Gram-staining-positive and non-flagellated. itive for adipic acid. Does not utilize D-mannitol, capric White aerial mycelium and yellow substrate mycelium is acid, malic acid, trisodium citrate and phenylacetic acid. produced after 2 days on R2A at 25°C. Positive for escu- Strain BM28 (=NIBRBA0000114308) has been isolat- lin hydrolysis and β-galactosidase, but negative for nitrate ed from a mud flat sample, Wando, Jeonnam Province, reduction, glucose fermentation, urease indole production, Korea. arginine dihydrolase and gelatinase. D-Glucose, D-mannose, L-arabinose, D-mannitol, N-acetyl-glucosamine, Description of Promicromonospora sukumoe D-maltose, potassium gluconate and malic acid are uti- MMD3Y-15-4 lized. Does not utilize adipic acid, capric acid and triso- Cells are Gram-staining-positive, non-flagellated and dium citrate. Strain Gsoil012 (=NIBRBA0000114219) June 2016 KIM ET AL.-UNRECORDED ACTINOBACTERIAL SPECIES 233 has been isolated from Ginseng cultivated soil collected incubation on R2A medium at 30°C. Positive for esculin from Pocheon, Gyeonggi Province, Korea. hydrolysis, oxidase and gelatinase, weakly positive for glucose fermentation, but negative for nitrate reduction, Description of Marmoricola korecus WW39 Indole production, urease, arginine dihydrolase and β- galactosidase. In API 20NE, positive assimilates for L- Cells are Gram-staining-positive, non-flagellated and arabinose and N-acetyl-glucosamine, weakly positive for coccoid shaped. Colonies are opaque, entire, drop-like D-glucose, adipic acid and malic acid, but negative for and yellow colored after 3 days incubation on R2A at D-mannose, D-mannitol, D-maltose, D-maltose, potas- 25°C. Positive for glucose fermentation, esculin hydro- sium gluconate, trisodium citrate capric acid and phenyl lysis and gelatinase in API 20NE, but negative for ni- acetic acid. Strain CR9 ( =NIBRBA0000114312) has trate reduction, indole production, arginine dihydrolase been isolated from a brackish water sample, Jeju, Korea. urease and β-galactosidase. Does not utilize capric acid, malic acid, trisodium citrate, D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid and phenylacetic acknowledgeMents = acid. Strain WW39 ( NIBRBA0000114436) has been This study was supported by the research grant “The isolated from a freshwater sample, Woopo wetland, Survey of Korean Indigenous Species” from the Nation- Gyeongnam Province, Korea. al Institute of Biological Resources of the Ministry of Environment in Korea. Description of Nocardioides alpinus WW41 Cells are Gram-staining-positive, non-flagellated and rod shaped. Colonies are circular, entire, smooth pale references yellow colored after 3 days incubation on R2A at 25°C. Bressan, W. 2003. Biological control of maize seed patho- Positive for nitrate reduction, β-galactosidase and gelati- genic fungi by use of actinomycetes. Biocontrol 48(2): nase in API 20NE, but negative glucose fermentation, 233-240. urease, esculin hydrolysis and for indole production, Felsenstein, J. 1981. Evolutionary trees from DNA sequences: arginine dihydrolase. Utilizes D-glucose, L-arabinose, a maximum likelihood approach. Journal of Molecular D-mannitol, D-maltose and potassium gluconate, but Evolution 17(6):368-376. not utilizes capric acid, malic acid, trisodium citrate, D- Felsenstein, J. 1985. Confidence limit on phylogenies: an mannose, N-acetyl-glucosamine, adipic acid and phenyl- approach using the bootstrap. Evolution 39:783-791. acetic acid. Strain WW41 (=NIBRBA0000114442) has Fitch, W.M. 1971. Toward defining the course of evolution: been isolated from a freshwater sample, Woopo wetland, minimum change for a specific tree topology. Syst. Zool. Gyeongnam Province, Korea. 20:406-416. Gordon, R.E. and J.M. Mihm. 1962. Identification of Nocar- Description of Nocardioides psychrotolerans GDM10 dia caviae (Erikson) nov. comb. Ann. N. Y. Acad. Sci. Cells are Gram-staining-positive, non-flagellated and 98:628-636. rod shaped. Colonies are circular, entire, smooth and pale Gupte, M., P. Kulkarni and B.N. Ganguli. 2002. Antifungal yellow colored after 3-4 days incubation on R2A at 30 antibiotics. Appl Microbiol Biotechnol. 58(1):46-57. °C. Positive for nitrate reduction, esculin hydrolysis and Hall, T.A. 1999. BioEdit: a user-friendly biological sequence gelatinase in API 20NE, but negative for indole produc- alignment editor and analysis program for Windows 95/ tion, arginine dihydrolase glucose fermentation, urease 98/NT. Nucleic Acids Symp Ser 41:95-98. β Jukes, T.H. and C.R. Cantor. 1969. Evolution of protein mole- and -galactosidase. Does not utilize capric acid, malic acid, trisodium citrate, D-glucose, L-arabinose, D-man- cules. In: H. N. Munro (ed.), Mammalian Protein Meta- nose, D-mannitol, N-acetyl-glucosamine, D-maltose, bolism. Academic Press, New York, pp. 21-132. potassium gluconate, adipic acid and phenylacetic acid. Kim, O.S., Y.J. Cho, K. Lee, S.H. Yoon, M. Kim, H. Na, S.C. Strain GDM10 ( =NIBRBA0000114313) has been iso- Park, Y.S. Jeon, J.H. Lee, H. Yi, S. Won and J. Chun. lated from a seawater sample collected from seashore, 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA Pohang, Gyeongbuk Province, Korea. gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62(3): Description of Modestobacter roseus CR9 716-721. Mahajan, G.B. 2012. Antibacterial agents from actinomy- Cells are Gram-staining-positive, non-flagellated, non- cetes - a review. Frontiers in Bioscience 4:240-253. pigmented and long rod-shaped. Colonies are circular, Miao, V. and J. Davies. 2010. Actinobacteria: the good, the convex and light yellowish pink colored after 3 days of bad and the ugly. Antonie Van Leeuwenhoek 98(2):143- 234 JOURNAL OF SPECIES RESEARCH Vol. 5, No. 2

150. CLUSTAL W: improving the sensitivity of progressive Saitou, N. and M. Nei. 1987. The neighbor-joining method: a multiple sequence alignment through sequence weight- new method for reconstructing phylogenetic trees. Mole- ing, position-specific gap penalties and weight matrix cular Biology and Evolution 4(4):406-425. choice. Nucleic Acids Res 22:4673-4680. Servin, J.A., C.W. Herbold, R.G. Skophammer and J.A. Lake. Ventura, M., C. Canchaya, A. Tauch, G. Chandra, G.F. Fitz- 2008. Evidence excluding the root of the tree of life from gerald, K.F. Chater and D. van Sinderen. 2007. Genom- the actinobacteria. Molecular Biology and Evolution 25 ics of Actinobacteria: tracing the evolutionary history of (1):1-4. an ancient phylum. Microbiol Mol Biol Rev 71(3):495- Tamura, K., G. Stecher, D. Peterson, A. Filipski and S. Ku- 548. mar. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution Submitted: January 14, 2016 30(12):2725-2729. Revised: May 10, 2016 Thompson, J.D., D.G. Higgins and T.J. Gibson. 1994. Accepted: June 15, 2016