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Home , Betaproteobacteria, Curvibacter, Neisseriaceae, Spirillaceae, Undibacterium

Journal of Species Research 6(1):15-24, 2017

Report of 21 unrecorded bacterial species in Korea belonging to and

Min-Kyeong Kim1, Chi-Nam Seong2, Kwangyeop Jahng3, Chang-Jun Cha4, Ki-seong Joh5, Jin-Woo Bae6, Jang-Cheon Cho7, Wan-Taek Im8 and Seung-Bum Kim1,*

1Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, Republic of Korea 2Department of Biology, Sunchon National University, Suncheon 57922, Republic of Korea 3Department of Biological Sciences, Chonbuk National Universty, Jeonju 54896, Republic of Korea 4Department of Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 5Department of Biotechnology, Hankuk University of Foreign Studies, Yongin 17035, Republic of Korea 6Department of Biology, Kyung Hee University, Seoul 02447, Republic of Korea 7Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea 8Department of Biotechnology, Hankyoung National University, Anseong 17579, Republic of Korea

*Correspondent: [email protected]

During the extensive survey of the prokaryotic species diversity in Korea, bacterial strains belonging to Betaproteobacteria and Epsilonproteobacteria were isolated from various sources including freshwater, sediment, soil and fish. A total of 23 isolates were obtained, among which 22 strains were assigned to the class Betaproteobacteria and one strain to the class Epsilonproteobacteria. The 22 betaproteobacterial strains were further assigned to (11 strains), (6 strains), (2 strains), (1 strain) and unclassified family groups (2 strains). For the strains of Burkholderiaceae, 3 strains were identified as 3 species of , and 2 strains were as 2 species of . For the strains of Comamonadaceae, 4 strains were identified as 2 species of the , 2 strains as 2 species of , 2 strains as 2 species of Limnohabitans, and each of the remaining strains as single species of , and , respectively. For the strains of Oxalobacteraceae, 1 strain was identified as a species of , and the other strain as a species of . The strain belonging to Neisseriaceae was identified as a species of Iodobacter. The remaining strains of Betaproteobacteria were identified as species of Sphaerotilus and Methylibium respectively (family unassigned). The epsilonproteobacterial strain was identified as a species of Arcobacter of the family Camplyobacteraceae. The detailed description of each unrecorded species is provided. Keywords: 16S rRNA, API20NE, Betaproteobacteria, Epsilonproteobacteria, Fatty acid, unrecorded species

Ⓒ 2017 National Institute of Biological Resources DOI:10.12651/JSR.2017.6.1.015

Introduction Hydrogenophilales, Methylophilales, Neisseriales, , “Procabacteriales” and , The understanding on the prokaryotic diversity on 13 families, , Burkholderiaceae, earth is still at large despite the fact that more than 600 Comamonadaceae, Oxalobacteraceae, , new species are published every year (Euzéby, 2016). , , Neisseriaceae, The study on prokaryotic diversity is significant in tax­ Gallionellaceae, Nitrosomonadaceae, , onomic and ecological aspects as well as in industrial “Procabaceriaceae” and , and 161 gen­ aspects as an invaluable source of useful bioactive com­ era (Garrity et al., 2005a; Euzéby, 2016). pounds. The Epsilonproteobacteria is a relatively small group The class Betaproteobacteria is one of the representa­ currently containing 2 orders, tive and largest Gram negative bacterial groups cur­ and Nautiliales, 4 families, Campylobacteraceae, rently encompassing 7 orders, namely , Helicobacteraceae, “Hydrogenimonaceae” and Nautiliaceae, 16 Journal of Species Research Vol. 6, No. 1 and 17 genera (Garrity et al., 2005a; Euzéby, 2016). rea, and the detailed descriptions are given below. Betaproteobacteria include physiologically wide group of , for example phototrophs, animal or plant , hydrogen , , nitro­ Materials and Methods gen fixing bacteria and methylotrophs (Garrity et al., 2005a). Some betaproteobacteria are known as parasites A total of 23 bacterial strains assigned to the classes or symbionts to invertebrates (Gruwell et al., 2010; Lund Betaproteobacteria and one strain assigned to Epsilon­ et al., 2010), and as degraders of recalcitrants (Aguirre were isolated from diverse environmental de Cárcer et al., 2007; Callaghan et al., 2009; Heinzel samples collected from soil, sediment, freshwater, sea­ et al., 2009). Epsilonproteobacteria include intestinal water and fish gut (Table 1). The samples were processed pathogens such as Campylobacter and Helicobacter, but or treated separately, diluted and spread onto diverse a number of species have also been found in natural en­ culture media R2A, marine agar (MA), tryptic soy agar vironment (Garrity et al., 2005b; Euzéby, 2016). (TSA) and (NA) (BD), and incubated at 25- In this study, bacterial strains belonging to Betaproteo­ 30°C for 2-5 days. The designated strain IDs, sources of bacteria and Epsilonproteobacteria were isolated from isolation, culture media, and incubation conditions are various sources including freshwater, sediment, soil and provided in the description and Table 1. All strains were fish during the extensive survey of the prokaryotic spe­ purified as single colonies and stored as 10-20% glycerol cies diversity in Korea. As a result, a total of 23 strains suspension at -80°C as well as lyophilized ampoules. are proposed to represent 21 unrecorded species of Ko­ Colony characteristics of the isolates were observed

Table 1. List of strains, isolation sources and taxonomic affiliations. Culture medium 16S rRNA gene Strain ID NIBR no. Isolation source Closest match (Identification) (temperature) similarity (%) Betaproteobacteria Burkholderiales Burkholderiaceae LB-1 BA0000114802 Artificial pond TSA (30°C) Burkholderia caryophylli 98.8 R1-16 BA0000114814 soil R2A (30°C) Burkholderia jiangsuensis 99.9 RG 3Y-10-2 BA0000114876 Field soil R2A (30°C) Burkholderia phytofirmans 98.8 NGS 3Y-15-3 BA0000114886 Field soil R2A (30°C) Cupriavidus campinensis 99.9 Oil1-9 BA0000114824 Oil-contaminated soil R2A (25°C) 99.3 IMCC26232 BA0000114870 Freshwater R2A (20°C) necessaries 99.8 subsp. asymbioticus Comamonadaceae WS97 BA0000115011 Freshwater R2A (25°C) 99.3 61DPR29 BA0000114796 Freshwater R2A (25°C) Acidovorax radicis 99.3 WS99 BA0000115015 Freshwater R2A (25°C) Comamonas jiangduensis 98.9 HMF2824 BA0000115005 Sediment R2A (30°C) Curvibacter fontanus 98.9 MG2F 9 BA0000114784 Freshwater R2A (25°C) Hydrogenophaga atypica 99.0 03SU8 BA0000115023 Freshwater R2A (25°C) Hydrogenophaga atypica 99.4 MR22 BA0000114927 Fish gut R2A (25°C) Hydrogenophaga taeniospiralis 98.1 WS11 BA0000115016 Freshwater R2A (25°C) Hydrogenophaga taeniospiralis 99.3 LIN8 BA0000114804 Freshwater R2A (25°C) Limnohabitans curvus 99.9 63ED25-2 BA0000114801 Freshwater R2A (25°C) Limnohabitans parvus 99.4 MC2F19 BA0000114774 Freshwater R2A (25°C) Rhodoferax saidenbachensis 99.7 Oxalobacteraceae LR-14 BA0000114808 Freshwater pond TSA (30°C) Herbaspirillum aquaticum 99.2 WA5 BA0000114770 Freshwater R2A (25°C) Undibacterium pigrum 99.1 Unclassified Burkholderiales HMF2787 BA0000115004 Sediment R2A (30°C) Methylibium petroleiphilum 99.9 HMF2472 BA0000114996 Freshwater R2A (25°C) subsp. 99.9 natans Neisseriales Neisseriaceae 61DPR38 BA0000114797 Freshwater R2A (25°C) Iodobacter arcticus 99.7 Epsilonproteobacteria Camplyobacterales Campylobacteraceae 63ED13 BA0000114800 Freshwater R2A (25°C) Arcobacter butzleri 100 February 2017 Kim et al.-Unrecorded species of Beta- and Epsilonproteobacteria 17 on the same agar media for cultivation. Cellular mor­ including freshwater, sediment, soil and fish. As a result, a phology and cell size were examined using either trans­ total of 22 isolates belonging to the class Betaproteobac­ mission electron microscope or scanning electron micro­ teria and one strain belonging to the class Epsilonproteo­ scope. Gram staining was performed using the standard bacteria were isolated (Table 1). The 22 betaproteobacte­ procedures. Biochemical characteristics were tested by rial strains were further assigned to Comamonadaceae (11 using API 20NE galleries (bioMérieux) according to the strains), Burkholderiaceae (6 strains), Oxalobacteraceae manufacturer’s instructions. (2 strains) and Neisseriaceae (1 strain), but 2 strains could The extraction of genomic DNA, PCR amplification of not be assigned to any known family. All families belong 16S rRNA gene and sequencing were performed using to the order Burkholderiales except for Neisseriaceae, the standard procedures as described previously (Shin which belongs to the order Neisseriales. For the strains of et al., 2011; Choi et al., 2015). The 16S rRNA gene se­ Burkholderiaceae, 3 strains were identified as 3 species quences of the strains assigned to the Betaproteobacteria of Burkholderia, 2 strains as 2 species of Cupriavidus, and Epsilonproteobacteria were compared with the ref­ and 1 strain as a species of Polynucleobacter (Fig. 1). For erence strains using the EzBioCloud (Kim et al., 2012). the strains of Comamonadaceae, 4 strains were identi­ Phylogenetic trees were generated by using neighbor- fied as 2 species of the genusHydrogenophaga , 2 strains joining (Saitou and Nei, 1987) and maximum-likelihood as 2 species of Acidovorax, 2 strains as 2 species of (Felsenstein, 1981) algorithms using MEGA 6.0 (Tamura Limnohabitans, and each of the remaining strains as sin­ et al., 2013). The phylogenetic trees were evaluated using gle species of Comamonas, Curvibacter and Rhodoferax, bootstrap analyses based on 1,000 resampled dataset. respectively (Fig. 2). For the strains of Oxalobacteraceae, 1 strain was identified as a species of Undibacterium, and the other as a species of Herbaspirillum (Fig. 3). Results and Discussion The remaining strains were identified as single species of Sphaerotilus and Methylibium respectively, which The bacterial strains belonging to Betaproteobacteria were unassigned members of the order Burkholderiales and Epsilonproteobacteria were isolated from sources (Fig. 3). For the strain belonging to Neisseriales, the

Fig. 1. Neighbor-joining tree of the isolates and related taxa belonging to the family Burkholderiaceae. Numbers at nodes indicate level of bootstrap support (%) based on 1,000 resamplings. Branches Scale bar, 0.01 substitutions per nucleotide position. 18 Journal of Species Research Vol. 6, No. 1

Fig. 2. Neighbor-joining tree of the isolates and related taxa belonging to the family Comamonadaceae. Numbers at nodes indicate level of bootstrap support (%) based on 1,000 resamplings. Scale bar, 0.01 substitutions per nucleotide position. isolate was identified as a species of Iodobacter (Fig. 4). acid, malic acid, trisodium citrate and phenylacetic acid. The epsilonproteobacterial strain was identified as a spe­ Strain LB-1 (=NIBRBA0000114802) was isolated from cies of Arcobacter of the family Campylobacteraceae artificial pond. The 16S rRNA gene sequence accession (Fig. 5). The detailed description of each unrecorded number is KP182159. species is as given below. The electron micrographic im­ ages of each isolate were also provided in Fig. 6. Description of Burkholderia jiangsuensis R1-16 Cells are Gram negative and rod shaped. Grows on Family Burkholderiaceae R2A at 30°C. The colonies are bright yellow colored, Description of Burkholderia caryophylli LB-1 and circular, convex with entire margin. Diffusible pig­ Cells are Gram negative and rod shaped. Grows on ment is not produced. Based on API20NE, reduc­ TSA at 30°C. The colonies are light brown colored, and tion is positive, but indole production, glucose fermen­ circular, glistening and moist. Diffusible pigment is not tation, arginine dihydrolase, urease, β-glucosidase, pro­ produced. Based on API20NE, cytochrome oxidase, β- tease and β-galactosidase activities are negative. D-Glu­ glucosidase, protease and β-galactosidase activities are cose, D-mannose, D-mannitol, N-acetyl-glucosamine, positive, but nitrate reduction, indole production, glucose maltose, potassium gluconate, and phenylacetic acid are fermentation, arginine dihydrolase and urease activities assimilated, but not L-arabinose, D-maltose, capric acid, are negative. D-Glucose, L-arabinose, D-mannitol, D- adipic acid and trisodium citrate. Strain R1-16 (=NIBR maltose and potassium gluconate are assimilated, but not BA0000114814) was isolated from soil. The 16S rRNA D-mannose, N-acetyl-glucosamine, capric acid, adipic gene sequence accession number is KP182171. February 2017 Kim et al.-Unrecorded species of Beta- and Epsilonproteobacteria 19

Fig. 3. Neighbor-joining tree of the isolates and related taxa belonging to the family Comamonadaceae. Numbers at nodes indicate level of bootstrap support (%) based on 1,000 resamplings. Scale bar, 0.01 substitutions per nucleotide position.

Fig. 4. Neighbor-joining tree of the isolate and related taxa belonging to the family Neisseriaceae. Numbers at nodes indicate level of boot­ strap support (%) based on 1,000 resamplings. Scale bar, 0.01 substitutions per nucleotide position.

Description of Burkholderia phytofirmans RG but not maltose. Strain RG 3Y-10-2 (=NIBRBA000011 3Y-10-2 4876) was isolated from agricultural field soil. The 16S rRNA gene sequence accession number is KP126810. Cells are Gram negative. Grows on R2A at 30°C. Mo­ tile by flagella. The colonies are pale yellow colored, and Description of Cupriavidus campinensis NGS 3Y-15-3 circular, convex with entire margin. Based on API20NE, β-galactosidase activity is positive, but cytochrome ox­ Cells are Gram negative and rod shaped. Grows on idase, nitrate reduction, indole production, glucose fer­ R2A at 30°C. Colonies are white colored, and puncti­ mentation, arginine dihydrolase, urease, β-glucosidase form, raised with entire margin. Based on API20NE, and protease activities are negative. D-Glucose, L-arab­ nitrate reduction is positive, but cytochrome oxidase, in­ inose, D-mannose, D-mannitol, N-acetyl-glucosamine, dole production, glucose fermentation, arginine dihydro­ potassium gluconate, capric acid, adipic acid, malic acid, lase, urease, β-glucosidase, protease and β-galactosidase trisodium citrate and phenylacetic acid are assimilated, activities are negative. Potassium gluconate, capric acid, 20 Journal of Species Research Vol. 6, No. 1

Fig. 5. Neighbor-joining tree of the isolate and related taxa belonging to the family Campylobacteraceae. Numbers at nodes indicate level of bootstrap support (%) based on 1,000 resamplings. Scale bar, 0.01 substitutions per nucleotide position.

adipic acid, malic acid, citrate and phenylacetic acid are glucosamine, D-maltose, potassium gluconate, capric assimilated, but not D-glucose, L-arabinose, D-mannose, acid, adipic acid, malic acid, trisodium citrate and phe­ D-mannitol, N-acetyl-glucosamine, maltose and triso­ nylacetic acid are not assimilated. Strain IMCC26232 dium citrate. Strain NGS 3Y-15-3 (=NIBRBA0000114 (=NIBRBA0000114870) was isolated from freshwater. 886) was isolated from agricultural field soil. The 16S rRNA gene sequence accession number is KP126819. Comamonadaceae Description of Acidovorax delafieldii WS97 Description of Cupriavidus necator Oil1-9 Cells are Gram negative and rod shaped. Grows on Cells are Gram negative and rod shaped. Grows on R2A at 25°C. Colonies are beige colored, and round, R2A at 25°C. Colonies are white colored, and circular, circular and convex. Diffusible pigment is not produced. convex with entire margin. Diffusible pigment is not pro­ Based on API20NE, cytochrome oxidase and nitrate duced. Based on API20NE, cytochrome oxidase, nitrate reduction activities are positive, but indole production, reduction, arginine dihydrolase and urease activities are glucose fermentation, arginine dihydrolase, urease, β- positive, but indole production, glucose fermentation, β- glucosidase, protease and β-galactosidase activities are glucosidase, protease and β-galactosidase activities are negative. D-Glucose, L-arabinose, D-mannose, D-man­ negative. Potassium gluconate, capric acid, adipic acid, nitol, potassium gluconate, capric acid and malic acid malic acid, citrate and phenylacetic acid are assimilated, are assimilated, but not N-acetyl-glucosamine, D-malt­ but not D-glucose, L-arabinose, D-mannose, D-mannitol, ose, adipic acid, trisodium citrate and phenylacetic acid. N-acetyl-glucosamine, maltose and trisodium citrate. Strain WS97 ( =NIBRBA0000115011) was isolated Strain Oil1-9 ( =NIBRBA0000114824) was isolated from freshwater. from oil-contaminated soil. Description of Acidovorax radicis 61DPR29 Description of Polynucleobacter necessarius subsp. Cells are Gram negative and rod shaped. Grows on asymbioticus IMCC26232 R2A at 25°C. Colonies are light brown colored, and cir­ Cells are Gram negative and rod shaped. Grows on cular, smooth with entire margin. Diffusible pigment is R2A at 20°C. Colonies are cream colored, and circular, not produced. Based on API20NE, cytochrome oxidase, convex and smooth. Diffusible pigment is not produced. urease and protease activities are positive, but indole Based on API20NE, cytochrome oxidase activity is pos­ production, glucose fermentation, arginine dihydrolase, itive, but nitrate reduction, indole production, glucose β-glucosidase and β-galactosidase activities are negative. fermentation, arginine dihydrolase, urease, β-glucosidase, D-Glucose, L-arabinose, D-mannose, D-mannitol, po­ protease and β-galactosidase activities are negative. D- tassium gluconate, adipic acid and malic acid are assim­ Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl- ilated, but not N-acetyl-glucosamine, D-maltose, capric February 2017 Kim et al.-Unrecorded species of Beta- and Epsilonproteobacteria 21

1 2 3 4

5 6 7 8

9 10 11 12

13 14 15 16

17 18 19 20

21 22 23

Fig. 6. Electron micrographic images of cells. Strains: 1, LB-1; 2, R1-16; 3, RG 3Y-10-2; 4, NGS 3Y-15-3; 5, Oil1-9; 6, IMCC26232; 7, 61DPR29; 8, WS97; 9, WS99; 10, HMF2824; 11, MG2F 9; 12, 03SU8; 13, MR22; 14, WS11; 15, LIN8; 16, 63ED25-2; 17, MC2F19; 18, 61DPR38; 19, LR-14; 20, WA5; 21, HMF2787; 22, HMF2472; 23, 63ED13. acid, trisodium citrate and phenylacetic acid. Strain 61D Description of Comamonas jiangduensis WS99 PR29 (=NIBRBA0000114796) was isolated from fresh­ water. The 16S rRNA gene sequence accession number Cells are Gram negative and rod shaped. Grows on is KP182153. R2A at 25°C. Colonies are white colored, and irregular, umbonate and lobate. Diffusible pigment is not produced. 22 Journal of Species Research Vol. 6, No. 1

Based on API20NE, cytochrome oxidase and nitrate nate and malic acid are assimilated, but not L-arabinose, reduction activities are positive, but indole production, D-mannose, N-acetyl-glucosamine, D-maltose, capric glucose fermentation, arginine dihydrolase, urease, β- acid, adipic acid, trisodium citrate and phenylacetic acid. glucosidase, protease and β-galactosidase activities are Strain 03SU8 ( =NIBRBA0000115023) was isolated negative. Capric acid and malic acid are assimilated, but from freshwater. not D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, Description of Hydrogenophaga taeniospiralis MR22 adipic acid, trisodium citrate and phenylacetic acid. Cells are Gram negative and rod shaped. Grows on Strain WS99 ( =NIBRBA0000115015) was isolated R2A at 25°C. Colonies are cream colored and circular. from freshwater. The 16S rRNA gene sequence acces­ Based on API20NE, cytochrome oxidase, nitrate reduc­ sion number is KP099964. tion, indole production, glucose fermentation, arginine dihydrolase, urease, β-glucosidase, protease and β-galac­ Description of Curvibacter fontanus HMF2824 tosidase activities are negative. D-Glucose, L-arabinose, Cells are Gram negative and rod shaped. Grows on D-mannose, D-mannitol, N-acetyl-glucosamine, D-malt­ R2A at 30°C. Colonies are beige colored, and circular, ose, potassium gluconate, capric acid, adipic acid, malic convex with entire margin. Diffusible pigment is not acid, trisodium citrate and phenylacetic acid are not as­ produced. Based on API20NE, cytochrome oxidase and similated. Strain MR22 ( =NIBRBA0000114927) was urease activities are positive, but nitrate reduction, indole isolated from the gut of Mugil cephalus (grey mullet). production, glucose fermentation, arginine dihydrolase, The 16S rRNA gene sequence accession number is KP β-glucosidase, protease and β-galactosidase activities 172206. are negative. D-Glucose and potassium gluconate are as­ similated, but not L-arabinose, D-mannose, D-mannitol, Description of Hydrogenophaga taeniospiralis WS11 N-acetyl-glucosamine, D-maltose, capric acid, adipic Cells are Gram negative and rod shaped. Grows on acid, malic acid, trisodium citrate and phenylacetic acid. R2A at 25°C. Colonies are pale yellow colored, and cir­ Strain HMF2824 (=NIBRBA0000115005) was isolated cular, slightly convex, smooth with entire margin. Dif­ from sediment. The 16S rRNA gene sequence accession fusible pigment is not produced. Based on API20NE, number is KP099964. nitrate reduction is positive, but cytochrome oxidase, in­ dole production, glucose fermentation, arginine dihydro­ Description of Hydrogenophaga atypica MG2F 9 lase, urease, β-glucosidase, protease and β-galactosidase Cells are Gram negative and rod shaped. Grows on activities are negative. D-Glucose, L-arabinose, D-man­ R2A at 25°C. Colonies are cream colored, and circular, nose, D-mannitol, N-acetyl-glucosamine, D-maltose, po­ raised with entire margin. Based on API20NE, cyto­ tassium gluconate, capric acid, adipic acid, malic acid, chrome oxidase activity is positive, but nitrate reduction, trisodium citrate and phenylacetic acid are not assimilat­ indole production, glucose fermentation, arginine dihy­ ed. Strain WS11 (=NIBRBA0000115016) was isolated drolase, urease, β-glucosidase, protease and β-galacto­ from freshwater. sidase activities are negative. D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-malt­ Description of Limnohabitans curvus LIN8 ose, potassium gluconate, capric acid, adipic acid, malic Cells are Gram negative and vibroid shaped. Grows acid, trisodium citrate and phenylacetic acid are not as­ on R2A at 25°C. Colonies are beige colored, and punc­ similated. Strain MG2F 9 (=NIBRBA0000114784) was tiform, smooth with entire margin. Diffusible pigment is isolated from freshwater. The 16S rRNA gene sequence not produced. Based on API20NE, cytochrome oxidase accession number is KP196831. activity is positive, but nitrate reduction, indole produc­ tion, glucose fermentation, arginine dihydrolase, urease, Description of Hydrogenophaga atypica 03SU8 β-glucosidase, protease and β-galactosidase activities Cells are Gram negative and rod shaped. Grows on are negative. D-Glucose, L-arabinose, D-mannose, D- R2A at 25°C. Colonies are white colored, and raised-cir­ mannitol, N-acetyl-glucosamine, D-maltose, potassium cular, smooth, convex. Diffusible pigment is not pro­ gluconate, capric acid, adipic acid, malic acid, trisodium duced. Based on API20NE, cytochrome oxidase, nitrate citrate and phenylacetic acid are not assimilated. Strain reduction and glucose fermentation activities are posi­ LIN8 (=NIBRBA0000114804) was isolated from fresh­ tive, but indole production, arginine dihydrolase, urease, water. The 16S rRNA gene sequence accession number β-glucosidase, protease and β-galactosidase activities is KP182161. are negative. D-Glucose, D-mannitol, potassium gluco­ February 2017 Kim et al.-Unrecorded species of Beta- and Epsilonproteobacteria 23

Description of Limnohabitans parvus 63ED25-2 indole production, glucose fermentation, arginine dihy­ drolase, urease, β-glucosidase, protease and β-galacto­ Cells are Gram negative and cocci shaped. Grows on sidase activities are negative. D-Glucose, L-arabinose, R2A at 25°C. Colonies are light brown colored, and cir­ D-mannose, D-mannitol, N-acetyl-glucosamine, D-malt­ cular, smooth with entire margin. Based on API20NE, ose, potassium gluconate, capric acid, adipic acid, ma­ cytochrome oxidase activity is positive, but nitrate re­ lic acid, trisodium citrate and phenylacetic acid are not duction, indole production, glucose fermentation, argi­ assimilated. Strain WA5 ( =NIBRBA0000114770) was nine dihydrolase, urease, β-glucosidase, protease and β- isolated from freshwater. The 16S rRNA gene sequence galactosidase activities are negative. D-Glucose, L-ara­ accession number is KP196819. binose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, UnclassifiedBurkholderiales malic acid, trisodium citrate and phenylacetic acid are Description of Methylibium petroleiphilum HMF2787 not assimilated. Strain 63ED25-2 ( =NIBRBA0000114 801) was isolated from freshwater. The 16S rRNA gene Cells are Gram negative and rod shaped. Grows on sequence accession number is KP182158. R2A at 30°C. Colonies are cream colored, and circular, convex with entire margin. Diffusible pigment is not Description of Rhodoferax saidenbachensis MC2F19 produced. Based on API20NE, cytochrome oxidase, ni­ trate reduction and arginine dihydrolase activities are Cells are Gram negative and rod shaped. Grows on positive, but indole production, glucose fermentation, R2A at 25°C. Colonies are white colored, and circular, urease, β-glucosidase, protease and β-galactosidase ac­ raised with entire margin. Based on API20NE, cyto­ tivities are negative. D-Glucose, D-mannose, potassium chrome oxidase, nitrate reduction and urease activities gluconate, capric acid, malic acid and trisodium citrate are positive, but indole production, glucose fermentation, are assimilated, but not L-arabinose, D-mannitol, N-ace­ arginine dihydrolase, β-glucosidase, protease and β-ga­ tyl-glucosamine, D-maltose, adipic acid and phenyl­ lactosidase activities are negative. D-Glucose, L-ara­ acetic acid. Strain HMF2787 ( =NIBRBA0000115004) binose, D-mannose, D-mannitol, N-acetyl-glucosamine, was isolated from sediment. The 16S rRNA gene se­ D-maltose, potassium gluconate, capric acid, adipic acid, quence accession number is KP099963. malic acid, trisodium citrate and phenylacetic acid are = not assimilated. Strain MC2F19 ( NIBRBA0000114 Description of Sphaerotilus natans subsp. natans 774) was isolated from freshwater. The 16S rRNA gene HMF2472 sequence accession number is KP196823. Cells are Gram negative and rod shaped. Grows on Oxalobacteraceae R2A at 25°C. Colonies are beige colored, and filamen­ Description of Herbaspirillum aquaticum LR-14 tous, flat and wooly. Diffusible pigment is not produced. Based on API20NE, cytochrome oxidase, urease, β-glu­ Cells are Gram negative and rod shaped. Grows on cosidase, protease and β-galactosidase activities are pos­ TSA at 30°C. Colonies are white colored, and undulate, itive, but nitrate reduction, indole production, glucose smooth. Based on API20NE, cytochrome oxidase, β- fermentation and arginine dihydrolase activities are neg­ glucosidase, protease and β-galactosidase activities are ative. D-Glucose, L-arabinose, D-mannose and N-acetyl- positive, but nitrate reduction, indole production, glu­ glucosamine are assimilated, but not D-mannitol, D- cose fermentation, arginine dihydrolase and urease ac­ maltose, potassium gluconate, capric acid, adipic acid, tivities are negative. D-Glucose, L-arabinose, D-man­ malic acid, trisodium citrate and phenylacetic acid. nose, D-mannitol, N-acetyl-glucosamine, potassium Strain HMF2472 (=NIBRBA0000114996) was isolated gluconate, capric acid, adipic acid, malic acid, trisodium from freshwater. The 16S rRNA gene sequence acces­ citrate and phenylacetic acid are assimilated, but not sion number is KP099955. D-maltose. Strain LR-14 (=NIBRBA0000114808) was isolated from freshwater pond. The 16S rRNA gene se­ Neisseriaceae quence accession number is KP182165. Description of Iodobacter arcticus 61DPR38 Cells are Gram negative and rod shaped. Grows on Description of Undibacterium pigrum WA5 R2A at 25°C. Colonies are white to violet colored, and Cells are Gram negative and rod shaped. Grows on circular, digging and viscid. Based on API20NE, cyto­ R2A at 25°C. Colonies are white colored, and circular, chrome oxidase, nitrate reduction and protease activities raised with entire margin. Based on API20NE, cyto­ are positive, but indole production, glucose fermenta­ chrome oxidase and nitrate reduction are positive, but tion, arginine dihydrolase, urease, β-glucosidase and β- 24 Journal of Species Research Vol. 6, No. 1 galactosidase activities are negative. D-Glucose, D-man­ 73(19):6224-6232. nose, N-acetyl-glucosamine, D-maltose and adipic acid Euzéby, J.P. 2016. List of Prokaryotic Names with Standing are assimilated, but not L-arabinose, D-mannitol, pota­ in Nomenclature, as of February 2016 (www.bacterio.net). ssium gluconate, capric acid, malic acid, trisodium ci­ Garrity, G.M., J.A. Bell and T. Lilburn. 2005a. Class II. trate and phenylacetic acid. Strain 61DPR38 ( =NIBR Betaproteobacteria class. nov. In: D.J. Brenner, N.R.

BA0000114797) was isolated from freshwater. The 16S Krieg, J.T. Staley and G.M. Garrity (eds.), Bergey’s rRNA gene sequence accession number is KP182154. Manual of Systematic Bacteriology, second edition, vol. 2 (The Proteobacteria), part C (The Alpha-, Beta-, Epsilonproteobacteria Delta-, and Epsilonproteobacteria), Springer, New York, Description of Arcobacter butzleri 63ED13 2005, p. 575. Garrity, G.M., J.A. Bell and T. Lilburn. 2005b. Class V. Cells are Gram negative and rod shaped. Grows on Epsilonproteobacteria class. nov. In: D.J. Brenner, N.R. R2A at 25°C. Colonies are light brown colored, and Krieg, J.T. Staley and G.M. Garrity (eds.), Bergey’s Man­ circular, entire and smooth. Diffusible pigment is not ual of Systematic Bacteriology, second edition, vol. 2 produced. Based on API20NE, cytochrome oxidase and (The Proteobacteria), part C (The Alpha-, Beta-, Delta-, nitrate reduction are positive, but indole production, glu­ and Epsilonproteobacteria), Springer, New York, 2005, p. cose fermentation, arginine dihydrolase, urease, β-glu­ 1145. cosidase, protease and β-galactosidase activities are Gruwell, M.E., N.B. Hardy, P.J. Gullan and K. Dittmar. 2010. negative. D-Glucose and malic acid are assimilated, but Evolutionary relationships among primary endosymbi­ not L-arabinose, D-mannose, D-mannitol, N-acetyl-glu­ onts of the mealybug subfamily Phenacoccinae (Hemip­ cosamine, D-maltose, potassium gluconate, capric acid, tera: Coccoidea: Pseudococcidae). Applied and Environ­ adipic acid, trisodium citrate and phenylacetic acid. mental Microbiology 76(22):7521-7525.

Strain 63ED13 ( =NIBRBA0000114800) was isolated Heinzel, E., S. Hedrich, E. Janneck, F. Glombitza, J. Seifert from freshwater. The 16S rRNA gene sequence acces­ and M. Schlömann. 2009. Bacterial diversity in a mine sion number is KP182157. water treatment plant. Applied and Environmental Mi­ crobiology 75(3):858-861. Kim, O.S., Y.J. Cho, K. Lee, S.H. Yoon, M. Kim, H. Na, S.C. Acknowledgements Park, Y.S. Jeon, J.H. Lee, H. Yi, S. Won and J. Chun. 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA This study was supported by the research grant, “Sur­ gene sequence database with phylotypes that represent vey of Korean Indigenous Species” sponsored by the uncultured species. International Journal of Systematic National Institute of Biological Resources (NIBR) of the and Evolutionary Microbiology 62(3):716-721. Ministry of Environment, Korea. Lund, M.B., M. Holmstrup, B.A. Lomstein, C. Damgaard and A. Schramm. 2010. Beneficial effect of Verminephrobacter nephridial symbionts on the fitness of the earthworm References Aporrectodea tuberculata. Applied and Environmental Microbiology 76(14):4738-4743. Callaghan, A.V., M. Tierney, C.D. Phelps and L.Y. Young. Shin, Y.M., T.-U. Kim, A. Choi, J. Chun, S. Lee, H. Kim, 2009. Anaerobic biodegradation of n-hexadecane by a H. Yi, J.H. Jo, J.-C. Cho, K. Jahng, K. Kim, K. Joh, J. nitrate-reducing consortium. Applied and Environmental Chun, H.H. Lee and S.B. Kim. 2011. Species diversity Microbiology 75(5):1339-1344. of Betaproteobacteria in the Sumunmulbengdui wetland Choi, A., J.-W. Bae, C.-J. Cha, J. Chun, W.-T. Im, K.Y. area of Jeju Island and distribution of novel taxa. Korean Jahng, C.O. Jeon, K. Joh, S.B. Kim, C.N. Seong, J.-H. Journal of Environmental Biology 29(3):154-161. Yoon and J.-C. Cho. 2015. A report of 39 unrecorded Tamura, K., G. Stecher, D. Peterson, A. Filipski and S. Ku­ bacterial species in Korea, belonging to the Betaproteo­ mar. 2013. MEGA6: Molecular Evolutionary Genetics bacteria and . Journal of Species Analysis Version 6.0. Molecular Biology and Evolution Research 4(2):109-126. 30(12):2725-2729. de Cárcer, D.A., M. Martín, U. Karlson and R. Rivilla. 2007. Changes in bacterial populations and in biphenyl dioxy­ genase gene diversity in a polychlorinated biphenyl-pol­ Submitted: March 3, 2016 luted soil after introduction of willow trees for rhizore­ Revised: June 27, 2016 mediation. Applied and Environmental Microbiology Accepted: February 7, 2017