Journal of Species Research 8(1):97-108, 2019

Unrecorded prokaryotic species belonging to the class in Korea

Mi-Sun Kim1, Seong-Hwa Jeong1, Joo-Won Kang1, Seung-Bum Kim2, Jang-Cheon Cho3, Chang-Jun Cha4, Wan-Taek Im5, Jin-Woo Bae6, Soon-Dong Lee7, Won-Yong Kim8, Myung-Kyum Kim9 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 Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 5Department of Biotechnology, Hankyong National University, Anseong17579, Republic of Korea 6Department of Biology, Kyung Hee University, Seoul 02447, Republic of Korea 7Faculty of Science Education, Jeju National University, Jeju 63243, Republic of Korea 8Department of Microbiology, College of Medicine, Chung-Ang University, Seoul 06974, Republic of Korea 9Department of Bio & Environmental Technology, Division of Environmental & Life Science, College of Natural Science, Seoul Women’s University, Seoul 01797, Republic of Korea

*Correspondent: [email protected] For the collection of indigenous prokaryotic species in Korea, 35 strains within the class Actinobacteria were isolated from various environmental samples (animals and clinical specimens) in 2017. Each strain showed high 16S rRNA gene sequence similarity (>98.8%) and formed a robust clade with recognized actinobacterial species. The isolates were assigned to 35 species, 22 genera, 15 families, and 8 orders of the class Actinobacteria. There are no official descriptions of these 35 bacterial species in Korea. Morphological properties, basic biochemical characteristics, isolation source, and strain IDs are included in the species descriptions. Keywords: 16S‌ rRNA sequence, Actinobacteria, unrecorded species

Ⓒ 2019 National Institute of Biological Resources DOI:10.12651/JSR.2019.8.1.097

Introduction phytic microorganisms but several genera are pathogenic to humans, animals, and plants. They are major producers The class Actinobacteria is one of the dominant class- of medically important antibiotics, particularly members es in the phylum Actinobacteria, which accommodates of the genus Streptomyces, the most abundant group in Gram-stain-positive with high G +C content in this phylum (Barka et al., 2015). Also, members can have their DNA (Ventura et al., 2007). As of August 2018, the polymer-degradation activities and contribute to the bio- class Actinobacteria consisted of 18 orders, 48 families, geochemical cycling (Jendrossek et al., 1997). and 379 genera (NCBI; https://www.ncbi.nlm.nih.gov/ In 2017, we isolated a great number of unrecorded pro- /). Also, 252 novel Korean indigenous species karyotic species from diverse environmental samples, belonging to the class Actinobacteria were described and artificial sources, and clinical specimens in Korea. The validated according to the List of Prokaryotic name with present report focuses on the description 35 strains be- Standing in Nomenclature (LPSN) since February 2016 longing to the class Actinobacteria which have not been (Bae et al., 2016). previously isolated in Korea. Members of the class Actinobacteria are abundantly distributed in terrestrial and aquatic environments (Servin et al., 2008) and are morphologically diverse, ranging Materials and Methods from coccoid, fragmenting hyphal forms to those with a highly differentiated branched mycelium (Goodfellow A total of 35 bacterial strains assigned to the class Ac- and Williams, 1983). Many of these bacteria also produce tinobacteria were isolated from various environmental external spores. Most actinobacterial species are sapro- samples, animals and clinical specimens in 2017 (Table 1). 98 JOURNAL OF SPECIES RESEARCH Vol. 8, No. 1 25°C 3d 25°C, 3d 25°C, 3d 25°C, 4d 25°C, 2d 37°C, 7d 25°C, 3d 30°C, 3d 20°C, 7d 25°C, 3d 20°C, 7d 30°C, 2d 30°C, 1d 30°C, 2d 30°C, 4d 25°C, 4d 25°C, 4d 25°C, 4d 25°C, 4d 25°C, 4d 30°C, 2d 30°C, 7d 30°C, 2d 30°C, 3d 37°C, 3d 30°C, 2d 37°C, 7d condition 30°C, 12d 30°C, 10d Incubation NA NA NA MA MA MA MA MA R2A R2A TSA R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A TSA R2A BHIA BHIA Medium* with seawater Isolation Seaweed Seawater Soil Seawater Chicken intestine Freshwater sediment Chicken intestine Freshwater sediment Chicken intestine Soil Soil Soil Soil Soil Soil Soil Soil Soil Tidal flat sediment Tidal Soil Soil Tidal flat sediment Tidal flat sediment Tidal Mammal feces Soil Soil Soil Human urine Human urine Source

100 100 100 100 100 100 100 (%) 99.4 99.8 99.7 99.9 99.8 99.7 99.7 99.8 99.4 99.7 98.8 99.6 99.9 99.7 99.4 99.8 98.9 99.9 99.6 99.0 99.0 99.9 Similarity . anitratus subsp Micrococcus flavus Micrococcus Kocuria oceani Kocuria salsicia Glutamicibacter arilaitensis Glutamicibacter nicotianae Citricoccus nitrophenolicus luteolus Arthrobacter russicus Arthrobacter Arthrobacter halodurans Arthrobacter Arthrobacter gandavensis Arthrobacter Okibacterium fritillariae resistens Microbacterium Leucobacter humi Leucobacter margaritiformis Agromyces ulmi Agromyces Agromyces salentinus Agromyces Knoellia flava Cellulomonas cellasea Serinibacter salmoneus Williamsia phyllosphaerae Williamsia Nocardia rhamnosiphila Nocardia Mycobacterium poriferae Mycobacterium pyrenivorans Mycobacterium brisbanense Dietzia maris Dietzia aerolata Corynebacterium ureicelerivorans Corynebacterium freneyi Actinomyces neuii Most closely related species . Actinobacteria NIBRBAC000501123 NIBRBAC000501131 NIBRBAC000501337 NIBRBAC000501125 NIBRBAC000501182 NIBRBAC000501120 NIBRBAC000501177 NIBRBAC000501118 NIBRBAC000501212 NIBRBAC000501176 NIBRBAC000501083 NIBRBAC000501086 NIBRBAC000501082 NIBRBAC000501085 NIBRBAC000501345 NIBRBAC000501344 NIBRBAC000501339 NIBRBAC000501330 NIBRBAC000501099 NIBRBAC000501341 NIBRBAC000500993 NIBRBAC000501032 NIBRBAC000501045 NIBRBAC000501036 NIBRBAC000501067 NIBRBAC000500997 NIBRBAC000501240 NIBRBAC000501001 NIBRBAC000501230 NIBR ID ECSD18 KYW1377 J1101 WD9 LM3301 JMn10 LT2304 JMn2 MMS17-SY291 LR2314 Ibu_O_21 AMX_O_4 Ibu_O_11 Ibu_W2_3 17J49-11 17J49-8 17J28-11 17J27-1 IMCC34147 17J72-9 Gsoil 1173 GH1-18 GH1-39 D7-24 HC_48 NA_1 CAU 1475 1_5_R2A CAU 1470 Strain ID Micrococcus Kocuria Glutamicibacter Citricoccus Arthrobacter Okibacterium Microbacterium Leucobacter Agromyces Knoellia Cellulomonas Serinibacter Williamsia Nocardia Mycobacterium Dietzia Corynebacterium Actinomyces Genus The taxonomic affiliations of isolated strains belonging to the phylum Corynebacteriales Actinomycetales Intrasporangiaceae Cellulomonadaceae Beutenbergiaceae Order Williamsiaceae Nocardiaceae Mycobacteriaceae Dietziaceae Corynebacteriaceae Order Actinomycetaceae Order Table 1. Table Family February 2019 Kim. et al. Unrecorded Actinobacteria in Korea 99 30°C, 2d 30°C, 5d 30°C, 2d 30°C, 2d 30°C, 3d 30°C, 5d 30°C, 2d 30°C, 2d 30°C, 2d 25°C, 4d 30°C, 8d 30°C, 2d 30°C, 2d condition Incubation NA NA NA R2A R2A R2A R2A R2A R2A R2A R2A R2A Medium* ISP2, pH5 Isolation Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil flat sediment Tidal Soil Source

100 (%) 99.7 98.9 99.9 99.3 99.4 99.4 99.8 98.9 98.8 99.7 99.5 99.7 Similarity Microbispora bryophytorum Microbispora Streptomyces scabrisporus Streptomyces seranimatus Streptomyces Streptomyces mauvecolor Streptomyces Streptomyces chartreusis Streptomyces Streptomyces caeruleatus Streptomyces Lentzea cavernae Amycolatopsis speibonae Nocardioides exalbidus Nocardioides Nocardioides aromaticivorans Nocardioides Micromonospora endophytica Micromonospora wenchangensis Micromonospora Dactylosporangium fulvum Most closely related species . Actinobacteria NIBRBAC000500986 NIBRBAC000500989 NIBRBAC000500995 NIBRBAC000500981 NIBRBAC000501209 NIBRBAC000501213 NIBRBAC000501004 NIBRBAC000500982 NIBRBAC000501342 NIBRBAC000500992 NIBRBAC000500994 NIBRBAC000501034 NIBRBAC000500984 NIBR ID Gsoil 554 Gsoil 961 Gsoil 1526 MMS17-GJ001 MMS17-SY227 MMS17-SY284 Gsoil 262 Gsoil 006 17J48-16 Gsoil 1130 Gsoil 1175 BE2-18 Gsoil 335 Strain ID Microbispora Streptomyces Lentzea Amycolatopsis Nocardioides Micromonospora Dactylosporangium Genus Continued. The taxonomic affiliations of isolated strains belonging to the phylum Streptosporangiales Streptomycetales Pseudonocardiales Propionibacteriales Micromonosporales Streptosporangiaceae Order Streptomycetaceae Order Pseudonocardiaceae Order Nocardioidaceae Order Micromonosporaceae Order Table 1. Table Family TSA, tryptic soy agar; NA, nutrient agar. 2A; ISP2, international streptomyces project medium 2; BHIA, brain heart infusion; MA, marine agar; *Abbreviations: R2A, Reasoner’s 100 JOURNAL OF SPECIES RESEARCH Vol. 8, No. 1

(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) (39) (40) (41) (42)

Fig. 1. Transmission electron micrographs or scanning electron micrographs of cells of the strains isolated in this study. Strains: 1, CAU 1470; 2, CAU 1475; 3, NA_1; 4, HC_48; 5, D7-24; 6, GH1-18; 7, GH1-39; 8, Gsoil 1173; 9, 17J72-9; 10, IMCC34147; 11, 17J28-11; 12, 17J49-8; 13, 17J49-11; 14, Ibu_O_11; 15, Ibu_O_21; 16, MMS17-SY291; 17, LT2304; 18, JMn2; 19, JMn10; 20, WD9; 21, LM3301; 22, KYW1377; 23, Gsoil 335; 24, Gsoil 1175; 25, BE2-18; 26, Gsoil 1130; 27, 17J48-16; 28, Gsoil 006; 29, Gsoil 262; 30, MMS17-SY284; 31, MMS17-SY227; 32, MMS17-GJ001; 33, Gsoil 961; 34, Gsoil 1526; 35, Gsoil 554.

Each sample was processed separately by spread onto a py or scanning electron microscopy (Fig. 1). Biochemical diverse culture agar media (Becton Dickinson) including characteristics were tested by using API 20NE galleries Reasoner’s 2A (R2A), international streptomyces project (bioMérieux) according to the manufacturer’s instruc- medium 2 (ISP2), brain heart infusion (BHIA), marine tions. (MA), tryptic soy (TSA) and nutrient (NA) and incubated DNA extraction, PCR amplification, and 16S rRNA at 20-37°C for 1-12 days. All strains were purified as sin- gene sequencing were carried out as described previous- gle colonies and stored as 10-20% glycerol suspension at ly (Chun and Goodfellow, 1995). The 16S rRNA gene -80°C as well as lyophilized ampoules. sequences of the strains assigned to the class Actinobac- Colony morphology of the strains was observed on teria were compared with the sequences in GenBank by agar plates with a magnifying glass after cells grew up BLASTN and analyzed using the EzTaxon-e server (Kim to a stationary phase. Cellular morphology and cell size et al., 2012). For phylogenetic analyses, multiple align- were examined by either transmission electron microsco- ments were performed using Clustal_W (Thompson et al., February 2019 Kim. et al. Unrecorded Actinobacteria in Korea 101

1994) and gaps were edited in the BioEdit program (Hall, Kyunghee University Hospital, Korea. 1999). Evolutionary distances were calculated using the Jukes-Cantor model (Jukes and Cantor, 1969). Phyloge- Description of Corynebacterium ureicelerivorans netic trees were constructed by using the neighbour-join- CAU 1475 ing (Saitou and Nei, 1987), maximum-likelihood (Fel- Cells are Gram-stain-positive, non-flagellated, and senstein, 1981), and maximum-parsimony (Fitch, 1971) rod-shaped. Colonies are circular, flat, smooth, opaque, methods in MEGA 6.0 (Tamura et al., 2013) with 1,000 and cream colored after 3 days on BHIA at 37°C. Ni- bootstrap replicates (Felsenstein, 1985). trate is not reduced and esculin is not hydrolyzed. The strain shows negative reactions for enzyme activities of Results and Discussion gelatinase, β-galactosidase, and arginine dihydrolase, whereas the strain shows positive reaction for enzyme The 35 Actinobacteria strains are classified into 8 or- activity of urease. Indole is not produced and glucose is ders, 15 families, and 22 genera. The orders Micrococ- not fermented. The strain utilizes D-glucose, whereas the cales (13 strains) and Corynebacteriales (8 strains) were strain does not utilize L-arabinose, D-mannose, D-man- most common. The remaining orders are Streptomycetales nitol, N-acetyl-glucosamine, D-maltose, potassium glu- (5 strains), Micromonosporales (3 strains), Propionibac- conate, capric acid, adipic acid, malic acid, trisodium teriales (2 strains), Pseudonocardiales (2 strains), Actino- citrate, and phenylacetic acid. Strain CAU 1475 (=NI- mycetales (1 strain), and Streptosporangiales (1 strain). BRBAC000501240) was isolated from human urine, Many isolates were affiliated with the families Micrococ- Kyunghee University Hospital, Korea. caceae (7 strains) and Microbacteriaceae (4 strains) of the order Micrococcales and the family Streptomycetaceae (5 Description of Dietzia aerolata NA_1 strains) of the order Streptomycetales (Table 1). Cells are Gram-stain-positive, non-flagellated, and oval All the strains were isolated from diverse sources: 22 shaped. Colonies are circular, convex, and pale-orange strains from soil, 4 strains from tidal flat sediment, 3 colored after 2 days on NA at 30°C. Nitrate is reduced strains from animal intestine or feces, and 2 strains each and esculin is not hydrolyzed. The strain shows nega- from fresh water sediment, seawater, and clinical spec- tive reactions for enzyme activities of urease, gelatinase, imens. The geographic region where the strains were β-galactosidase, and arginine dihydrolase. Indole is not found is as follows: 14 strains from Gyeonggi Province, produced and glucose is not fermented. The strain utilizes 6 strains from Jeju Province, 4 strains from Jeollannam D-glucose, whereas the strain does not utilize L-arabi- Province, 3 strains each from Jeollabuk Province and nose, D-mannose, D-mannitol, N-acetyl-glucosamine, Incheon City, 2 strains from Seoul City, and each one D-maltose, potassium gluconate, capric acid, adipic acid, strain from Daejeon City, Busan City, and Gangwon malic acid, trisodium citrate, and phenylacetic acid. Strain Province. NA_1 (=NIBRBAC000500997) was isolated from a soil These strains were Gram-stain-positive and chemohet- sample, Anseong, Gyeonggi Province, Korea. erotrophic. Figure 2 shows phylogenetic assignment of the strains based on 16S rRNA gene sequences. Description of Dietzia maris HC_48 Here we report in detail about the 35 Actinobacteria Cells are Gram-stain-positive, non-flagellated, and species previously unrecorded in Korea. rod shaped. Colonies are circular and light-yellow col- ored after 3 days on TSA at 30°C. Nitrate is reduced and Description of Actinomyces neuii subsp. anitratus esculin is weakly hydrolyzed. The strain shows negative CAU 1470 reactions for enzyme activities of oxidase, urease, β-ga- Cells are Gram-stain-positive, non-flagellated, and rod- lactosidase, and arginine dihydrolase, whereas the strain shaped. Colonies are circular, convex, opaque, shiny, shows positive reaction for enzyme activity of gelatinase. and cream colored after 7 days of incubation on BHIA at Indole is not produced and glucose is not fermented. The 37°C. Nitrate is reduced and esculin is not hydrolyzed. strain utilizes D-glucose, N-acetyl-glucosamine, D-malt- The strain shows negative reactions for enzyme activi- ose, and potassium gluconate, and weakly utilizes adipic ties of urease, gelatinase, β-galactosidase, and arginine acid and malic acid, whereas the strain does not utilize dihydrolase. Indole is not produced and glucose is not L-arabinose, D-mannose, D-mannitol, capric acid, triso- fermented. The strain utilizes D-glucose, D-mannitol, and dium citrate, and phenylacetic acid. Strain HC_48 (=NI- D-maltose, whereas the strain does not utilize L-arab- BRBAC000501067) was isolated from a soil sample, inose, D-mannose, N-acetyl-glucosamine, potassium Hwacheon, Gangwon Province, Korea. gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain CAU 1470 (=NI- Description of Mycobacterium brisbanense D7-24 BRBAC000501230) was isolated from human urine, Cells are Gram-stain-positive, non-flagellated, non-pig- 102 JOURNAL OF SPECIES RESEARCH Vol. 8, 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 class 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 substitu- tions per nucleotide position. February 2019 Kim. et al. Unrecorded Actinobacteria in Korea 103 mented, and rod-shaped. Colonies are punctiform, con- lyzed. The strain shows negative reactions for enzyme vex, entire, and cream colored after 12 days on NA at activities of gelatinase and β-galactosidase, whereas the 30°C. Nitrate is reduced and esculin is not hydrolyzed. strain shows positive reactions for enzyme activities of The strain shows negative reactions for enzyme activities urease, arginine dihydrolase, and oxidase. Indole is not of urease, gelatinase, β-galactosidase and arginine dihy- produced and glucose is not fermented. The strain utilizes drolase, whereas the strain shows positive reaction for D-glucose, whereas the strain does not utilize D-glucose, enzyme activity of oxidase. Indole is not produced and L-arabinose, D-mannose, N-acetyl-glucosamine, D-malt- glucose is not fermented. The strain utilizes N-acetyl-glu- ose, D-mannitol, potassium gluconate, capric acid, adipic cosamine, potassium gluconate and malic acid, and weak- acid, malic acid, trisodium citrate, and phenylacetic acid. ly utilizes D-mannitol, whereas the strain does not utilize Strain Gsoil 1173 (=NIBRBAC000500993) was isolated L-arabinose, D-mannose, D-glucose, D-maltose, capric from a soil sample, Pocheon, Gyeonggi Province, Korea. acid, adipic acid, trisodium citrate, and phenylacetic acid. Strain D7-24 ( =NIBRBAC000501036) was isolated Description of Williamsia phyllosphaerae 17J72-9 from mammal feces, Jeju, Korea. Cells are Gram-stain-positive, non-flagellated, and rod- shaped. Colonies are circular, convex, smooth, and orange Description of Mycobacterium poriferae GH1-18 colored after 4 days on R2A at 25°C. Nitrate is not re- Cells are Gram-stain-positive, non-flagellated, non-pig- duced and esculin is hydrolyzed. The strain shows nega- mented, and rod-shaped. Colonies are circular, convex, tive reactions for enzyme activities of gelatinase and β-ga- entire, and orange colored after 7 days on MA at 30°C. lactosidase, whereas the strain shows positive reactions Nitrate is not reduced and esculin is not hydrolyzed. The for enzymes activities of urease and arginine dihydrolase. strain shows negative reactions for enzyme activities of Indole is not produced and glucose is not fermented. The urease, gelatinase, β-galactosidase, and arginine dihy- strain utilizes D-glucose, D-maltose, D-mannitol, potas- drolase, whereas the strain shows positive reaction for sium gluconate, malic acid, and trisodium citrate, where- enzyme activity of oxidase. Indole is not produced and as the strain does not utilize L-arabinose, D-mannose, glucose is not fermented. The strain utilizes malic acid N-acetyl-glucosamine, capric acid, adipic acid, and phe- and weakly utilizes D-mannitol, whereas the strain does nylacetic acid. Strain 17J72-9 (=NIBRBAC000501341) not utilize D-glucose, L-arabinose, D-mannose, N-ace- was isolated from a soil sample, Jeju, Korea. tyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. Description of Serinibacter salmoneus IMCC34147 Strain GH1-18 ( =NIBRBAC000501032) was isolated Cells are Gram-stain-positive, non-flagellated, and rod- from tidal flat sediment, Incheon, Korea. shaped. Colonies are circular, entire, raised, and cream beige-colored after 3 days incubation on R2A with sea- Description of Mycobacterium pyrenivorans GH1-39 water at 25°C. Nitrate is not reduced and esculin is not Cells are Gram-stain-positive, non-flagellated, non-pig- hydrolyzed. The strain shows negative reactions for en- mented, and rod shaped. Colonies are circular, convex, zyme activities of urease, gelatinase, and arginine dihy- entire, and pale-yellow colored after 10 days of incuba- drolase, whereas the strain shows positives reactions for tion on R2A at 30°C. Nitrate is reduced and esculin is enzymes activities of β-galactosidase and oxidase. Indole not hydrolyzed. The strain shows negative reactions for is not produced and glucose is not fermented. The strain enzyme activities of urease, gelatinase, β-galactosidase does not utilize D-glucose, L-arabinose, N-acetyl-glu- and arginine dihydrolase, whereas the strain shows pos- cosamine, D-maltose, potassium gluconate, D-mannitol, itive reaction for enzyme activity of oxidase. Indole is D-mannose, malic acid, capric acid, adipic acid, trisodium not produced and glucose is not fermented. The strain citrate, and phenylacetic acid. Strain IMCC34147 (=NI- does not utilize D-glucose, L-arabinose, D-mannose, BRBAC000501099) was isolated from tidal flat sediment, N-acetyl-glucosamine, D-maltose, D-mannitol, potassium Incheon, Korea. gluconate, capric acid, adipic acid, malic acid, trisodi- um citrate, and phenylacetic acid. Strain GH1-39 (=NI- Description of Knoellia flava 17J28-11 BRBAC000501045) was isolated from tidal flat sediment, Cells are Gram-stain-positive and cocci shaped. Colo- Incheon, Korea. nies are circular, convex, smooth and yellow colored after 4 days on R2A at 25°C. Nitrate is reduced and esculin Description of Nocardia rhamnosiphila Gsoil 1173 is hydrolyzed. The strain shows positive reactions for Cells are Gram-stain-positive, non-flagellated, and enzyme activities of urease, gelatinase, arginine dihy- branched mycelium-forming. Colonies are filamentous, drolase, and β-galactosidase. Indole is not produced and umbonate, and pale-orange colored after 2 days on R2A glucose is not fermented. The strain utilizes D-glucose, at 30°C. Nitrate is not reduced and esculin is not hydro- D-mannose, and D-maltose, whereas the strain does not 104 JOURNAL OF SPECIES RESEARCH Vol. 8, No. 1 utilize L-arabinose, D-mannitol, N-acetyl-glucosamine, Description of Okibacterium fritillariae Ibu_O_21 potassium gluconate, adipic acid, capric acid, malic acid, Cells are Gram-stain-positive, non-flagellated, and trisodium citrate, and phenylacetic acid. Strain 17J28-11 rod shaped. Colonies are circular, glistening, and pale (=NIBRBAC000501339) was isolated from a soil sam- yellow-colored after 2 days on R2A at 30°C. Nitrate is ple, Jeju, Korea. reduced and esculin is hydrolyzed. The strain shows posi- tive reactions for enzyme activities of oxidase, gelatinase, Description of Agromyces salentinus 17J49-8 and β-galactosidase, whereas the strain shows negative Cells are Gram-stain-positive, non-flagellated, and reactions for enzyme activities of arginine dihydrolase rod shaped. Colonies are circular, convex, smooth, and and urease. Indole is not produced and glucose is not pale-yellow colored after 4 days on R2A at 25°C. Nitrate fermented. The strain utilizes D-glucose, L-arabinose, is reduced and esculin is hydrolyzed. The strain shows D-mannose, D-mannitol, D-maltose, potassium gluco- positive reactions for enzyme activities of urease, argi- nate, malic acid, and trisodium citrate, and weakly utilizes nine dihydrolase, and β-galactosidase, whereas the strain N-acetyl-glucosamine, whereas the strain does not utilize shows negative reaction for enzyme activity of gelati- capric acid, phenylacetic acid, and adipic acid. Strain Ibu_ nase. Indole is not produced and glucose is not fermented. O_21 (=NIBRBAC000501083) was isolated from a soil The strain utilizes D-glucose, L-arabinose, D-mannose, sample, Anseong, Gyeonggi Province, Korea. D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid, and trisodium citrate, whereas the Description of Arthrobacter halodurans MMS17- strain does not utilize adipic acid, capric acid, and phe- SY291 nylacetic acid. Strain 17J49-8 (=NIBRBAC000501344) Cells are Gram-stain-positive and cocci-shaped. Col- was isolated from a soil sample, Jeju, Korea. onies are circular, raised, opaque, glistening, and yel- low colored after 3 days on NA at 30°C. Nitrate is not Description of Agromyces ulmi 17J49-11 reduced and esculin is weakly hydrolyzed. The strain Cells are Gram-stain-positive, non-flagellated, and rod shows positive reactions for enzyme activities of oxidase shaped. Colonies are circular, convex, smooth, and yellow and β-galactosidase, whereas the strain shows negative colored after 4 days on R2A at 25°C. Nitrate is reduced reactions for enzyme activities of arginine dihydrolase, and esculin is hydrolyzed. The strain shows positive reac- gelatinase, and urease. Indole is not produced and glucose tions for enzyme activities of urease and β-galactosidase, is not fermented. The strain utilizes D-glucose, D-man- whereas the strain shows negative reactions for enzyme nose, D-mannitol, D-maltose, potassium gluconate, malic activities of arginine dihydrolase and gelatinase. Indole acid, trisodium citrate, and phenylacetic acid, whereas the is not produced and glucose is not fermented. The strain strain does not utilize L-arabinose, N-acetyl-glucosamine, utilizes D-glucose, L-arabinose, D-mannose, D-mannitol, capric acid and adipic acid. Strain MMS17-SY21 (=NI- N-acetyl-glucosamine, D-maltose, potassium gluconate, BRBAC000501212) was isolated from a soil sample, adipic acid, malic acid, and trisodium citrate, whereas the Sunyudo, Gunsan, Jeollabuk Province, Korea. strain does not utilize capric acid and phenylacetic acid. Strain 17J49-11 (=NIBRBAC000501345) was isolated Description of Arthrobacter luteolus LT2304 from a soil sample, Jeju, Korea. Cells are Gram-stain-positive and coccus-ovoid- shaped. Colonies are circular, convex with entire margin, Description of Leucobacter humi Ibu_O_11 and beige colored after 7 days on TSA at 20°C. Nitrate Cells are Gram-stain-positive, non-flagellated, and rod is reduced and esculin is hydrolyzed. The strain shows shaped. Colonies are circular and white colored after 2 positive reaction for enzyme activity of β-galactosidase, days on R2A at 30°C. Nitrate is not reduced and esculin whereas the strain shows negative reactions for enzyme is hydrolyzed. The strain shows positive reactions for en- activities of arginine dihydrolase, gelatinase, urease, zyme activities of gelatinase and β-galactosidase, whereas and oxidase. Indole is not produced and glucose is not the strain shows negative reactions for enzyme activities fermented. The strain utilizes D-glucose, L-arabinose, of arginine dihydrolase, urease, and oxidase. Indole is not D-mannose, D-mannitol, N-acetyl-glucosamine, D-malt- produced and glucose is not fermented. The strain utilizes ose, potassium gluconate, adipic acid, phenylacetic D-glucose, D-mannose, N-acetyl-glucosamine, D-malt- acid, and malic acid, whereas the strain does not utilize ose, malic acid, and trisodium citrate, whereas the strain capric acid and trisodium citrate. Strain LT2304 (=NI- does not utilize L-arabinose, D-mannitol, potassium glu- BRBAC000501177) was isolated from a chicken intes- conate, capric acid, adipic acid, and phenylacetic acid. tine, Seoul Grand Park, Gyeonggi Province, Korea. Strain Ibu_O_11 (=NIBRBAC000501082) was isolated from a soil sample, Anseong, Gyeonggi Province, Korea. Description of Arthrobacter russicus JMn2 Cells are Gram-stain-positive, non-flagellated, non-pig- February 2019 Kim. et al. Unrecorded Actinobacteria in Korea 105 mented, and rod shaped. Colonies are entire, smooth, Nitrate is reduced and esculin is hydrolyzed. The strain circular, and cream colored after 3 days on R2A at 25°C. shows positive reaction for enzyme activity of gelatinase, Nitrate is not reduced and esculin is hydrolyzed. The whereas the strain shows negative reactions for enzyme strain shows positive reactions for enzyme activities of activities of arginine dihydrolase, β-galactosidase, ure- gelatinase and β-galactosidase, whereas the strain shows ase, and oxidase. Indole is not produced and glucose is negative reactions for enzyme activities of arginine di- not fermented. The strain utilizes D-glucose, L-arabi- hydrolase, urease, and oxidase. Indole is not produced nose, D-maltose, potassium gluconate, adipic acid, phe- and glucose is not fermented. The strain utilizes D-glu- nylacetic acid, malic acid, and trisodium citrate, whereas cose, D-mannose, D-maltose D-mannitol, N-acetyl-glu- the strain does not utilize mannose, D-mannitol, N-ace- cosamine, potassium gluconate, malic acid, adipic acid, tyl-glucosamine, and capric acid. Strain LM3301 (=NI- and trisodium citrate, whereas the strain does not utilize BRBAC000501182) was isolated from a chicken intes- L-arabinose, phenylacetic acid and capric acid. Strain tine, Seoul Grand Park, Gyeonggi Province, Korea. JMn2 (=NIBRBAC000501118) was isolated from fresh- water sediment, Juam, Suncheon, Jeollanam Province, Description of Kocuria oceani KYW1377 Korea. Cells are Gram-stain-positive, non-flagellated, non-pig- mented, and coccoid shaped. Colonies are entire, circular, Description of Citricoccus nitrophenolicus JMn10 smooth, convex, and pale-orange colored after 3 days Cells are Gram-stain-positive, non-flagellated, non-pig- of incubation on MA at 25°C. Nitrate is not reduced and mented, and coccoid shaped. Colonies are circular, esculin is hydrolyzed. The strain shows positive reaction smooth, convex, entire, and pale-yellow colored after 3 for enzyme activity of β-galactosidase, whereas the strain days on R2A at 25°C. Nitrate is not reduced and esculin shows negative reactions for enzyme activities of arginine is not hydrolyzed. The strain shows negative reactions dihydrolase, gelatinase, urease, and oxidase. Indole is for enzyme activities of arginine dihydrolase, gelatinase, produced and glucose is not fermented. The strain utilizes β-galactosidase, urease, and oxidase. Indole is not pro- D-glucose, L-arabinose, D-mannitol, D-maltose, potas- duced and glucose is not fermented. The strain utilizes sium gluconate, adipic acid, and malic acid, whereas the D-glucose, malic acid, and trisodium citrate, whereas strain does not utilize D-mannose, N-acetyl-glucosamine, the strain does not utilize L-arabinose, N-acetyl-glucos- capric acid, phenylacetic acid, and trisodium citrate. amine, D-maltose, D-mannose, D-mannitol, potassium Strain KYW1377 (=NIBRBAC000501131) was isolated gluconate, phenylacetic acid, capric acid, and adipic acid. from seawater, Gwangyang, Jeollanam Province, Korea. Strain JMn10 ( =NIBRBAC000501120) was isolated from freshwater sediment, Juam, Suncheon, Jeollanam Description of Dactylosporangium fulvum Gsoil 335 Province, Korea. Cells are Gram-stain-positive, non-flagellated, and mycelium-forming. Colonies are filamentous, umbonate Description of Glutamicibacter arilaitensis WD9 and orange colored after 2 days on R2A at 30°C. Ni- Cells are Gram-stain-positive, non-flagellated, non-pig- trate is reduced and esculin is not hydrolyzed. The strain mented, and rod-coccus shaped. Colonies are circular, shows positive reaction for enzyme activity of oxidase, smooth, convex, opaque, and pale-yellow colored after 2 whereas the strain shows negative reactions for enzyme days on MA at 25°C. Nitrate is reduced and esculin is hy- activities of arginine dihydrolase, gelatinase, urease, and drolyzed. The strain shows positive reactions for enzyme β-galactosidase. Indole is not produced and glucose is activities of gelatinase and β-galactosidase, whereas the not fermented. The strain utilizes D-glucose, L-arabi- strain shows negative reactions for enzyme activities of nose, D-mannose, D-maltose, adipic acid, and malic acid, arginine dihydrolase, urease, and oxidase. Indole is not whereas the strain does not utilize N-acetyl-glucosamine, produced and glucose is not fermented. The strain utilizes D-mannitol, potassium gluconate, capric acid, trisodium D-glucose, L-arabinose, D-maltose, potassium gluconate, citrate, and phenylacetic acid. Strain Gsoil 335 ( =NI- adipic acid, malic acid, trisodium citrate, and pheny- BRBAC000500984) was isolated from a soil sample, lacetic acid, whereas the strain does not utilize D-man- Pocheon, Gyeonggi Province, Korea. nose, D-mannitol, N-acetyl-glucosamine, and capric acid. Strain WD9 (=NIBRBAC000501125) was isolated from Description of Micromonospora endophytica seawater, Wando, Jeollanam Province, Korea. Gsoil 1175 Cells are Gram-stain-positive, non-flagellated, and Description of Glutamicibacter nicotianae LM3301 mycelium-forming with spore bearing. Colonies are cir- Cells are Gram-stain-positive, non-flagellated, and cular, convex and orange colored after 2 days on R2A at coccus shaped. Colonies are circular, convex with entire 30°C. Nitrate is reduced and esculin is not hydrolyzed. margin, and beige colored after 7 days on MA at 37°C. The strain shows negative reactions for enzyme activities 106 JOURNAL OF SPECIES RESEARCH Vol. 8, No. 1 of oxidase, arginine dihydrolase, gelatinase, urease, and drolase, urease, gelatinase, and β-galactosidase. Indole β-galactosidase. Indole is not produced and glucose is is not produced and glucose is not fermented. The strain not fermented. The strain utilizes D-glucose, L-arabinose, utilizes D-glucose, D-mannose, L-arabinose, D-mannitol, D-mannose, D-maltose, and potassium gluconate, where- N-acetyl-glucosamine, D-maltose, potassium gluconate, as the strain does not utilize D-mannitol, N-acetyl-glucos- and malic acid, whereas the strain does not utilize adipic amine, malic acid, trisodium citrate, phenylacetic acid, acid, capric acid, trisodium citrate, and phenylacetic acid. capric acid, and adipic acid. Strain Gsoil 1175 ( =NI- Strain 17J48-16 (=NIBRBAC000501342) was isolated BRBAC000500994) was isolated from a soil sample, from a soil sample, Jeju, Korea. Pocheon, Gyeonggi Province, Korea. Description of Amycolatopsis speibonae Gsoil 006 Description of Micromonospora wenchangensis Cells are Gram-stain-positive, non-flagellated, and BE2-18 branched mycelium-forming. Colonies are filamentous, Cells are Gram-stain-positive, non-flagellated, non-pig- umbonate, and cream colored after 2 days incubation on mented, and mycelium-forming with spore bearing. Col- R2A at 30°C. Nitrate is not reduced and esculin is hydro- onies are circular, entire, convex, and orange colored lyzed. The strain shows negative reactions for enzyme after 8 days on NA at 30°C. Nitrate is not reduced and activities of arginine dihydrolase, urease, gelatinase, esculin is hydrolyzed. The strain shows positive reactions β-galactosidase, and oxidase. Indole is not produced and for enzyme activities of gelatinase, oxidase, and β-ga- glucose is not fermented. The strain utilizes D-glucose, lactosidase, whereas the strain shows negative reactions L-arabinose, D-mannose, D-mannitol, N-acetyl-glucos- for enzyme activities of arginine dihydrolase and urease. amine, potassium gluconate, adipic acid, malic acid, and Indole is not produced and glucose is not fermented. The phenylacetic acid, whereas the strain does not utilize strain utilizes L-arabinose, potassium gluconate, and D-maltose, capric acid and trisodium citrate. Strain Gsoil weakly utilizes D-glucose and D-maltose, whereas the 006 (=NIBRBAC000500982) was isolated from a soil strain does not utilize D-mannose, D-mannitol, N-ace- sample, Pocheon, Gyeonggi Province, Korea. tyl-glucosamine, capric acid, malic acid, trisodium citrate, adipic acid, and phenylacetic acid. Strain BE2-18 (=NI- Description of Lentzea cavernae Gsoil 262 BRBAC000501034) was isolated from tidal flat sediment, Cells are Gram-stain-positive, non-flagellated, and Eulsukdo, Busan, Korea. branched mycelium-forming. Colonies are filamentous, umbonate, and white colored after 2 days incubation on Description of Nocardioidesa romaticivorans R2A at 30°C. Nitrate is not reduced and esculin is hy- Gsoil 1130 drolyzed. The strain shows positive reactions for enzyme Cells are Gram-stain-positive, flagellated, and rod activities of oxidase and β-galactosidase, whereas the shaped. Colonies are circular, umbonate, and cream col- strain shows negative reactions for enzyme activities of ored after 2 days on R2A at 30°C. Nitrate is not reduced arginine dihydrolase, gelatinase, and urease. Indole is not and esculin is hydrolyzed. The strain shows positive produced and glucose is not fermented. The strain uti- reactions for enzyme activities of gelatinase, oxidase, lizes D-glucose, L-arabinose, D-mannose, D-mannitol, and β-galactosidase, whereas the strain shows negative N-acetyl-glucosamine, D-maltose, and malic acid, where- reactions for enzyme activities of arginine dihydrolase as the strain does not utilize potassium gluconate, capric and urease. Indole is not produced and glucose is not fer- acid, trisodium citrate, adipic acid, and phenylacetic acid. mented. The strain utilizes L-arabinose and potassium Strain Gsoil 262 (=NIBRBAC000501004) was isolated gluconate, and weakly utilizes D-glucose, L-arabinose, from a soil sample, Pocheon, Gyeonggi Province, Korea. potassium gluconate, and malic acid, whereas the strain does not utilize N-acetyl-glucosamine, D-maltose, capric Description of Streptomyces caeruleatus MMS17- acid, trisodium citrate, D-mannose, D-mannitol, adipic SY284 acid, and phenylacetic acid. Strain Gsoil 1130 ( =NI- Cells are Gram-stain-positive, non-flagellated, and BRBAC000500992) was isolated from a soil sample, branched mycelium-forming. Colonies are circular, brit- Pocheon, Gyeonggi Province, Korea. tle, umbonate, opaque, and gray colored after 5 days incu- bation on NA at 30°C. Nitrate is not reduced and esculin Description of Nocardioides exalbidus 17J48-16 is hydrolyzed. The strain shows positive reactions for Cells are Gram-stain-positive, non-flagellated, and enzyme activities of gelatinase, oxidase, and β-galactosi- rod shaped. Colonies are circular, convex, smooth, and dase, whereas the strain shows negative reactions for en- cream colored after 4 days on R2A at 25°C. Nitrate is zyme activities of arginine dihydrolase and urease. Indole not reduced and esculin is hydrolyzed. The strain shows is not produced and glucose is not fermented. The strain positive reactions for enzyme activities of arginine dihy- utilizes D-glucose, L-arabinose, D-mannose, D-mannitol, February 2019 Kim. et al. Unrecorded Actinobacteria in Korea 107

N-acetyl-glucosamine, D-maltose, potassium gluconate, produced and glucose is not fermented. The strain utilizes adipic acid, and malic acid, and weakly utilizes trisodi- potassium gluconate, adipic acid, and malic acid, where- um citrate and phenylacetic acid, whereas the strain does as the strain does not utilize D-glucose, L-arabinose, not utilize capric acid. Strain MMS17-SY284 ( =NI- D-mannose, D-mannitol, N-acetyl-glucosamine, D-malt- BRBAC000501213) was isolated from a soil sample, ose, capric acid, trisodium citrate, and phenyl acetic acid. Sunyudo, Gunsan, Jeollabuk Province, Korea. Strain Gsoil 961 (=NIBRBAC000500989) was isolated from a soil sample, Pocheon, Gyeonggi Province, Korea. Description of Streptomyces chartreusis MMS17- SY227 Description of Streptomyces seranimatus Gsoil 1526 Cells are Gram-stain-positive, non-flagellated, non-pig- Cells are Gram-stain-positive, non-flagellated, and mented, and branched mycelium-forming. Colonies are branched mycelium-forming. Colonies are circular, con- circular, convex, rough, entire and beige colored after 3 vex, and cream colored after 2 days of incubation on R2A days of incubation on NA at 30°C. Nitrate is not reduced at 30°C. Nitrate is reduced and esculin is not hydrolyzed. and esculin is hydrolyzed. The strain shows positive re- The strain shows positive reaction for enzyme activity of actions for enzyme activities of gelatinase, oxidase and oxidase, whereas the strain shows negative reactions for β-galactosidase, whereas the strain shows negative re- enzyme activities of gelatinase, urease, β-galactosidase, actions for enzyme activities of arginine dihydrolase and arginine dihydrolase. Indole is not produced and and urease. Indole is not produced and glucose is not glucose is not fermented. The strain utilizes D-glucose, fermented. The strain utilizes D-glucose, L-arabinose, D-mannose and N-acetyl-glucosamine, whereas the strain D-mannose, D-mannitol, N-acetyl-glucosamine, D-malt- does not utilize L-arabinose, D-mannitol, D-maltose, po- ose, potassium gluconate, and malic acid, and weakly tassium gluconate, capric acid, adipic acid, malic acid, utilizes adipic acid, trisodium citrate and phenylacetic trisodium citrate, and phenylacetic acid. Strain Gsoil 1526 acid, whereas the strain does not utilize capric acid. Strain (=NIBRBAC000500995) was isolated from a soil sam- MMS17-SY227 (=NIBRBAC000501209) was isolated ple, Pocheon, Gyeonggi Province, Korea. from soil, Gunsan, Jeollabuk Province, Korea. Description of Microbispora bryophytorum Gsoil 554 Description of Streptomyces mauvecolor MMS17- Cells are Gram-stain-positive, non-flagellated, and my- GJ001 celium-forming. Colonies are filamentous, umbonate, and Cells are Gram-stain-positive, non-flagellated, pigment- brown colored after 2 days of incubation on R2A at 30°C. ed, and branched mycelium-forming. Colonies are irreg- Nitrate is reduced and esculin is hydrolyzed. The strain ular, wrinkled, rough, and light-yellow colored after 5 shows positive reactions for enzyme activities of β-galac- days of incubation on ISP2 with pH 5 at 30°C. Nitrate is tosidase and oxidase, whereas the strain shows negative reduced and esculin is hydrolyzed. The strain shows posi- reactions for enzyme activities of gelatinase, urease, and tive reactions for enzyme activities of gelatinase, oxidase, arginine dihydrolase. Indole is not produced and glucose urease, and β-galactosidase, whereas the strain shows is not fermented. The strain utilizes D-glucose, L-arab- negative reaction for enzyme activity of arginine dihydro- inose, N-acetyl-glucosamine, and potassium gluconate, lase. Indole is not produced and glucose is not fermented. whereas the strain does not utilize D-mannose, D-man- The strain utilizes D-glucose, N-acetyl-glucosamine and nitol, D-maltose, capric acid, adipic acid, malic acid, tri- potassium gluconate, and weakly utilizes D-mannose, sodium citrate, and phenyl acetic acid. Strain Gsoil 554 D-mannitol, and malic acid, whereas the strain does not (=NIBRBAC000500986) was isolated from a soil sam- utilize L-arabinose, D-maltose, capric acid, adipic acid, ple, Pocheon, Gyeonggi Province, Korea. trisodium citrate, and phenylacetic acid. Strain MMS17- GJ001 (=NIBRBAC000500981) was isolated from a soil sample, Daejeon, Korea. Acknowledgements

Description of Streptomyces scabrisporus Gsoil 961 This study was supported by the research grant “The Cells are Gram-stain-positive, non-flagellated, and Survey of Korean Indigenous Species” from the Nation- branched mycelium-forming. Colonies are filamentous, al Institute of Biological Resources of the Ministry of umbonate, and pale-orange colored after 2 days of incu- Environment in Korea. bation on R2A at 30°C. Nitrate is not reduced and esculin is not hydrolyzed. The strain shows positive reaction for enzyme activity of oxidase, whereas the strain shows neg- References ative reactions for enzyme activities of gelatinase, urease, β-galactosidase, and arginine dihydrolase. Indole is not Bae, K.S., M.S. Kim, J.H. Lee, J.W. Kang, D.I. Kim, J.H. 108 JOURNAL OF SPECIES RESEARCH Vol. 8, No. 1

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