Journal of Species Research 6(2):101-118, 2017

A report on 57 unrecorded bacterial species in Korea in the classes and

Hyun Sik Kim1, Chang-Jun Cha2, Jang-Cheon Cho3, Wan-Taek Im4, Kwang Yeop Jahng5, Che Ok Jeon6, Kiseong Joh7, Seung Bum Kim8, Chi Nam Seong9, Wonyong Kim10, Hana Yi11, Soon Dong Lee12, Jung-Hoon Yoon13 and Jin-Woo Bae1,*

1Department of Biology, Kyung Hee University, Seoul 02447, Republic of Korea 2Department of Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 3Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea 4Department of Biotechnology, Hankyong National University, Anseong 17579, Republic of Korea 5Department of Life Sciences, Chonbuk National University, Jeonju 54896, Republic of Korea 6Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea 7Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Geonggi 17035, Republic of Korea 8Department of Microbiology, Chungnam National University, Daejeon 34134, Republic of Korea 9Department of Biology, Sunchon National University, Suncheon 57922, Republic of Korea 10Department of Microbiology, Chung-Ang University College of Medicine, Seoul 06974, Republic of Korea 11School of Biosystem and Biomedical Science, Department of Public Health Science, Korea University, Seoul 02841, Republic of Korea 12Department of Science Education, Jeju National University, Jeju 63243, Republic of Korea 13Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea

*Correspondent: [email protected]

In an investigation of indigenous prokaryotic species in Korea, a total of 57 bacterial strains assigned to the classes Betaproteobacteria and Gammaproteobacteria were isolated from diverse environments. Samples were collected from fresh water, natural caves, soil, paddy fields, lakes, sea water, jeotgal (fermented seafood), salt flats, soil from abandoned mines, plant roots, digestive organs of both Japanese crested ibis (Nipponia nippon) and Burmese python (Python molurus bivittatus) and tidal flats. From the high 16S rRNA gene sequence similarity (>98.7%) and formation of robust phylogenetic clades with closely related species, it was determined that each strain belonged to an independent and predefined bacterial species within either the Betaproteobacteria or Gammaproteobacteria. There is no official report or publication that describes these 57 proteobacterial species in Korea. Overall, in the class Betaproteobacteria there were 16 species in 12 genera of 4 families in the order and two species in two genera of one family in the order Neisseriales. Within the class Gammaproteobacteia, there were five species in four genera of four families in the order Alteromonadales, 12 species in 11 genera of one family in the order Enterobacteriales, four species in four genera of three families in the order Oceanospirillales, 11 species in four genera of two families in the order , two species in the order Vibrionales and five species in five genera of one family in the order Xanthomonadales. Gram reaction, colony and cell morphology, basic biochemical characteristics, isolation source and strain IDs are described in the species description section. Keywords: 16S rRNA, Bacterial diversity, Betaproteobacteria, Gammaproteobacteria, Unrecorded species

Ⓒ 2017 National Institute of Biological Resources DOI:10.12651/JSR.2017.6.2.101

Introduction and bioinformatics-driven analysis of sequence data, have been applied to microbial study and transformed Recent advances in sequencing techniques, particu- our understanding of the interactions between microbial larly the powerful combination of genome sequencing communities and their niches (Lauber et al., 2009; Pas- 102 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2 cault et al., 2014; Loman and Pallen, 2015). Sequencing various bacterial species, which were collected from methods have undergone remarkable innovation, and the fresh water, natural caves, soil, paddy fields, lakes, sea transition of NGS techniques and bioinformatics into water, jeotgal (fermented seafood), salt flats, abandoned various fields are imminent. However, current culture-in- mines, plant roots, tidal flats, and digestive organs of dependent approaches have limitations, especially with both Japanese crested ibis (Nipponia nippon) and Bur- respect to sequencing depth and resolution of the 16S mese python (Python molurus bivittatus). Homogenized rRNA gene sequence as well as accurate microbial de- samples were separately spread onto diverse culture scriptions from diverse environments. In sequence-based media, including R2A, Tryptic Soy Agar (TSA), Marine techniques, insufficient sequencing depth leads to match Agar 2216 (MA), 0.1% of diluted MA, irrelevant populations. Moreover, a major short coming (NA) and International Streptomyces Project yeast malt of culture-independent approaches is the impossibility extract (ISP2), which were incubated at 25-30°C for of providing specific metabolic function information of 1-10 days. To obtain pure cultures, single colonies were the functions of each bacterial species or strain (Shige- transferred repeatedly to fresh media. All strains were matsu et al., 2009). Therefore, culture-dependent ap- preserved at -80°C in optimal media containing 10- proaches as putative building blocks for applied science 20% (v/v) glycerol as well as lyophilized ampoules. remain important preliminary tools in microbial studies. To confirm morphological, physiological and bio- A major advantage of this approach is that a pure culture chemical characterization, Gram-staining, electron mi- can be obtained and deposited in a culture collection as croscopy, and API 20NE tests were performed after cells biomaterial. Cultivation, isolation and characterization were grown to a stationary phase. A Gram-staining kit of these organisms will provide a comprehensive under- (bioMérieux), was used according to the manufacturer’s standing of their physiology and their complex biogeo- instructions. To observe detailed cellular morphology chemical characteristics will be better understood. and surface characteristics, cellular morphology and The phylum is the largest bacterial cell size were examined by either transmission electron phylogenetic clade, comprising 116 validated families microscopy or scanning electron microscopy. Enzyme according to the List of Prokaryotic Names with Stand- oxidase activity was determined by examining for a blue ing in Nomenclature (http://www.bacterio.net/) and is colour change upon exposure to 1% (w/v) tetrameth- highly abundant in various ecological niches such as soil yl-p-phenylenediamine (bioMérieux). Amino acid hy- (36.5%), plants (62%), the atmosphere (77.9%), seawa- drolysis and assimilation reactions were tested using ter (57.9%) and freshwater (61.3%) (Shin et al., 2015). API 20NE test strips (bioMérieux) with API AUX medi- Moreover, despite being represented by only a minor um, according to the manufacturer’s instructions. proportion of this phylum, an imbalanced gut microbio- For the phylogenetic analysis, amplification of the 16S ta in mammals was recognized as a potential diagnostic rRNA gene from strains were performed using a standard signature of dysbiosis and risk of other diseases related PCR method with a PCR pre-mix and two universal bac- to metabolism and inflammation. Therefore, identifying teria-specific primers (Bac 8F (5′-AGAGTTTGATCCT novel bacterial species in the phylum Proteobacteria, GGCTCAG-3′) and Bac 1492R (5′-GGYTACCTTGT describing their phylogenetic relationships and inves- TACGACTT-3′)) (Lane, 1991). The 16S rRNA gene tigating their various functions in different ecological amplicons were sequenced using a BigDye Terminator niches are of preeminent importance. Cycle Sequencing Ready Reaction kit (Applied Biosys- In 2015, we collected diverse environmental sam- tems), according to the manufacturer’s instructions. The ples from the Korean Peninsula and isolated myriads gene fragments of the 16S rRNA sequences of strains of novel bacterial species and unrecorded bacterial were then assembled. To determine theirs taxonomic po- species in Korea. We isolated bacterial species in the sition, 16S rRNA gene sequences were compared with classes/phyla , Betaproteobacteria, those of closely related strains in the EzTaxon-e server Gammaproteobacteria, Deltaproteobacteria, (Kim et al., 2012), then those sequences were aligned Bacteroidetes, Firmicutes, Actinobacteria, Deinococci using the multiple alignment program CLUSTAL W and Verrucomicrobia. As a subset of this study, we (Thompson et al., 1997). Phylogenetic consensus trees present a report focused on the description of unrecord- were obtained using the programme MEGA version 5 ed species belonging to the Betaproteobacteria and (Tamura et al., 2011). The neighbour-joining (NJ) al- Gammaproteobacteria. gorithm (Saitou and Nei, 1987) was the primary tool for tree construction. The maximum parsimony (MP) (Kluge and Farris, 1969) and maximum likelihood (ML) Materials and Methods algorithms were used for additional comparisons. The robustness of the phylogenetic trees was confirmed by Various environmental samples were used to isolate bootstrap analyses based on 1000 random replicates. June 2017 KIM ET AL.-UNRECORDED SPECIES OF THE CLASSES BETAPROTEOBACTERIA AND GAMMAPROTEOBACTERIA 103

Results and Discussion NJ-1-5, GF5, JF-5, 2015-3, NDH-10 and RDH4 were assigned to the Betaproteobacteria. They were all Gram- Strains assigned to the Betaproteobacteria staining-negative, chemoheterotrophic, non-pigmented and rod-shaped (Fig. 1). Detailed morphology Based on the comparative 16S rRNA gene sequence and physiological characteristics are shown in the spe- analyses and phylogeny, 18 strains, designated HMF cies description section. 3907, HMF4008, HMF4075, JD2, JD5, JD24, JD50, Strains HMF3907, HMF4008, HMF4075, JD2, JD5, IMCC25601, IMCC25614, IMCC25617, C1-1, C1-11, JD24, JD50, IMCC25601, IMCC25614, IMCC25617, C1-

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

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

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

(16) (17) (18)

Fig. 1. Transmission electron micrographs or scanning electron micrographs of cells of the species belonging to the Betaproteobacteria iso- lated in this study. Strains: 1, HMF3907; 2, JD2; 3, JD5; 4, JD24; 5, JD50; 6, IMCC25601; 7, IMCC25614; 8, C1-1; 9, C1-11; 10, NJ-1-5; 11, GF5; 12, JF-5; 13, 2015-3; 14, IMCC25617; 15, NDH-10; 16, RDH4; 17, HMF4008; 18, HMF4075. 104 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2 30°C, 3d 30°C, 3d 30°C, 2d 30°C, 3d 20°C, 10d 25°C, 2d 25°C, 2d 20°C, 10d 20°C, 10d 30°C, 2d 30°C, 3d 25°C, 2d 26°C, 2d 25°C, 3d 25°C, 3d 25°C, 3d 25°C, 3d 25°C, 3d 20°C, 10d 25°C, 2d 25°C, 2d 30°C, 2d 30°C, 3d 25°C, 2d 26°C, 2d 26°C, 2d 25°C, 2d 30°C, 3d 25°C, 2d 25°C, 2d 25°C, 2d 25°C, 4d 25°C, 7d 30°C, 3d 25°C, 3d 30°C, 3d 30°C, 2d 30°C, 2d 25°C, 2d conditions Incubation Medium TSA R2A R2A R2A R2A R2A R2A R2A R2A R2A R2A MA R2A R2A R2A R2A R2A R2A R2A R2A ISP2 R2A R2A MA MA R2A TSA R2A R2A R2A R2A R2A R2A MA MA R2A NA R2A MA Isolation source Natural cave Paddy field Fresh water Fresh water Fresh water Fresh water Fresh water Fresh water Lake Fresh water Fresh water Sea water Burmese python Japanese crested ibis Japanese crested ibis Japanese crested ibis Burmese python Fresh water Sea water Fresh water Soil of mine Soil Sea water Sea water Sea water Soil Natural cave Fresh water Fresh water Fresh water Fresh water Fresh water Fresh water Sea water Sea water Plant root Soil of mine Fresh water Salt

(%) 99.11 98.95 99.93 99.72 99.93 99.79 99.50 99.92 99.51 99.01 99.59 99.90 99.19 98.87 99.39 99.27 99.93 99.89 99.72 99.50 99.60 99.26 99.90 99.80 99.66 99.19 99.02 98.72 98.92 99.86 99.51 98.75 99.18 99.58 99.10 99.06 99.30 100.00 100.00 Similarity T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 10-17 IAM 14942 JCM 6096 T IAM 12670 JA-1 CFBP 4730 CFBP YIM 31327 NS21 NBRC 15126 5CRT DSM 3493 BZ59 CS-K2 T DSM 4540 CK47 ATCC 33852 ATCC KMM 1561 CCUG 11779 HT073016 A62-14BT ATCC 19706 ATCC KMM 3893 ATCC 27126 ATCC ATCC 35998 ATCC KMM 3899 LMG 24198 CECT 5335 CECT ATCC 29927 ATCC CC-AFH3 CCUG 35299 107300 CIP MWU205 N3 Ruye-90 DSM 1522 DSM 4542 DSM 14288 CCUG 52222 SUEMI10 Most closely related species and their taxonomic affiliations K. japonica L. thiooxidans D. zoogloeoides H. autotrophicum J. lividum J. svalbardensis M. varians N. malthae N. soli R. terrae A. macleodii R. ornithinolytica K. cowanii E. marmotae E. xiangfangensis C. youngae E. persicina rettgeri P. H. paralvei agglomerans P. S. plymuthica C. amphilecti H. fontilapidosi M. arenicola P. violaceinigra P. C. bauzanensis H. hiltneri R. gummiphilus A. temperans A. valerianellae C. vaccinii indigofera V. M. maritimus arctica P. E. americana P. eucalypti P. A. xylosoxidans R. texasensis R. tilapiae NIBR ID Gammaproteobacteria NIBRBAC000497979 NIBRBAC000498058 NIBRBAC000497904 NIBRBAC000498039 NIBRBAC000497938 NIBRBAC000497939 NIBRBAC000498004 NIBRBAC000498007 NIBRBAC000498055 NIBRBAC000498046 NIBRBAC000497923 NIBRBAC000497837 NIBRBAC000497843 NIBRBAC000497846 NIBRBAC000497849 NIBRBAC000497850 NIBRBAC000497992 NIBRBAC000498041 NIBRBAC000498045 NIBRBAC000498083 NIBRBAC000498088 NIBRBAC000498040 NIBRBAC000497917 NIBRBAC000497972 NIBRBAC000498038 NIBRBAC000498018 NIBRBAC000497991 NIBRBAC000498078 NIBRBAC000497934 NIBRBAC000497935 NIBRBAC000497911 NIBRBAC000497908 NIBRBAC000497907 NIBRBAC000497973 NIBRBAC000497902 NIBRBAC000498086 NIBRBAC000498019 NIBRBAC000498051 NIBRBAC000498056 and Strain ID LPB0099 NDH-10 HMF3907 GF5 JD24 JD50 IMCC25614 IMCC25617 2015-3 JF-5 KYW1005 KHL6 KHN5 KHM5 KHN16 KHL16 IMCC25602 KM3 DF-5 TW2C3 8-07 GT KM2 HMF4436 LPB0063 NJ-1-5 C1-1 IMCC25601 RDH4 JD2 JD5 HMF4075 HMF4008 HMF3946 LPB0073 HMF3896 TW2I3 C1-11 TJB7 JF6 Betaproteobacteria Genus Kangiella Limnobacter Duganella Herbaspirillum Janthinobacterium Janthinobacterium Massilia Noviherbaspirillum Noviherbaspirillum Roseateles Alteromonas Raoultella Kosakonia Escherichia Enterobacter Erwinia Hafnia Pantoea Serratia Cobetia Halomonas Marinomonas Pseudoduganella Candidimonas Herbaspirillum Rhizobacter Acidovorax Chromobacterium Vogesella Marinobacter Psychromonas Ewingella Pantoea Achromobacter Rheinheimera Rheinheimera f f f f f Family Kangiella_ Lautropia_ Alteromonadaceae Enterobacteraceae Halomonadaceae Oceanospirillaceae Sphaerotilus_ Marinobacter_ Psychromonadaceae Alcaligenaceae Alishewanella_ Continued Summary of strains isolated belonging to the Phylum Table 1. Table Betaproteobacteria Gammaproteobacteria June 2017 KIM ET AL.-UNRECORDED SPECIES OF THE CLASSES BETAPROTEOBACTERIA AND GAMMAPROTEOBACTERIA 105

1, C1-11, NJ-1-5, GF5, JF-5, 2015-3, NDH-10 and RDH4 were most closely related to Duganella zoogloeoides T 25°C, 3d 20°C, 7d 25°C, 2d 30°C, 2d 25°C, 3d 25°C, 4d 25°C, 2d 25°C, 2d 25°C, 3d 26°C, 2d 20°C, 15d 25°C, 3d 30°C, 13d 30°C, 2d 30°C, 3d 26°C, 1d 25°C, 3d 30°C, 2d conditions Incubation IAM 12670 (D14256; 99.70% 16S rRNA gene sequence similarity), Vogesella indigofera ATCC 19706T (AB 021385; 99.60%), Chromobacterium vaccinia MWU205T Medium TSA 0.1% MA R2A MA MA MA R2A R2A R2A MA MA R2A TSA R2A MA MA R2A R2A (JZJL01000120; 99.10%), Acidovorax temperans CCUG 11779T (AF078766; 99.51%), Acidovorax valerianellae CFB 4730T (AJ431731; 98.75%), Janthinobacterium lividum DSM 1522T (Y08846; 99.8%), Janthinobacterium svalbardensis JA-1T (DQ355146; 99.50%), Herbaspirillum hiltneri N3T (DQ150563; 99.93%), Massilia varians Isolation source T Japanese crested ibis Fresh water Fresh water flat Tidal Sea water Sea water Fresh water Fresh water Fresh water Sea water Fresh water Japanese crested ibis Natural cave Plant root Jeotgal Sea water Fresh water Plant root CCUG 35299 (AM774587; 99.85%), Noviherbaspirillum

malthae CC-AFH3T (DQ490985; 99.42%), Candidimonas T

(%) bauzanensis BZ59 (GQ246953; 98.95%), Achromobacter 98.79 99.45 99.60 99.47 99.24 99.47 99.10 99.51 99.86 99.48 99.93 99.72 98.91 99.41 99.50 99.03 98.87 99.71 T

Similarity xylosoxidans DSM 10346 (Y14908; 99.09%), T Pseudoduganella violaceinigra Y9T (AY376163; 98.90%), Herbaspirillum autotrophicum IAM 14942T

T T JF3835 (AB074524; 98.7%), Roseateles terrae CCUG 52222 T T T T T (AB003626; 99.9%), Noviherbaspirillum soli SUEM T T T

T T piscium T T T 110 (HQ830498; 100%), Limnobacter thiooxidans CS- T T Rhizobacter gummiphilus

E414 K2 (AJ289885; 99.93%) and T HL22-2 NEAU-ST5-5 IPL-1 T subsp. T DSM 18974 NS21 (AB609313; 99.01%), respectively (Table 1). ATCC 19867 ATCC T DSM 12411 DSM 14405 LMG 2352 NCTC 10465 Vb 11.11 DSM 11363 From the high 16S rRNA gene sequence similarity NCTC 5866 P15 CIP 64.5 CIP ATCC 4973 ATCC

DCY45 and robust formation of phylogenetic clades, it was Most closely related species concluded that strains HMF3907, JD2, JD5, JD24, P. frigidicola P. M. osloensis A. junii indoloxydans P. kunmingensis P. chlororaphis P. fragi P. viridiflava P. graminis P. zhanjiangensis V. S. rhizophila R. soli indica P. X. translucens P. songnenensis P. atlanticus V. hibiscicola P. A. lwoffii JD50, IMCC25601, IMCC25614, C1-1, C1-11, NJ-1- 5, GF5, JF-5, 2015-3, IMCC25617, NDH-10 and RDH4 were members of the order Burkholderiales and strains HMF4008 and HMF4075 were members of the order Neisseriales, respectively (Fig. 4). At present, there has NIBR ID been no official report that these 18 species have been NIBRBAC000497995 NIBRBAC000498036 NIBRBAC000497887 NIBRBAC000497989 NIBRBAC000497928 NIBRBAC000498042 NIBRBAC000497936 NIBRBAC000497937 NIBRBAC000497940 NIBRBAC000498008 NIBRBAC000497840 NIBRBAC000498024 NIBRBAC000498087 NIBRBAC000498090 NIBRBAC000497924 NIBRBAC000497974 NIBRBAC000497913 NIBRBAC000497835 isolated in Korea; therefore 16 species in 12 genera of four families in the order Burkholderiales and two spe- cies in tow genera of one family in the order Neisseriales are reported as betaproteobacterial species found in Ko- Strain ID IMCC25605 YH-5 S6-6 LPB0123 KYW1051 YF5 JD11 JD16 JD57 IMCC25618 KHC14 C2-29 NR 4-01 NR 5-07 KYW941 LPB0083 HMF4401 KHN15 rea.

Strains assigned to the Gammaproteobacteria On the basis of 16S rRNA gene sequence comparisons Genus and phylogenetic analyses, a total of 39 strains, designat- ed TJB7, JF6, KYW1005, HMF3946, LPB0073, KHL6, Psychrobacter Moraxella Acinetobacter Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Vibrio Stenotrophomonas Rhodanobacter Pseudoxanthomonas Xanthomonas Pseudomonas Vibrio Pseudomonas Acinetobacter KHN5, KHM5, KHN16, KHL16, IMCC25602, KM3, DF-5, TW2C3, TW2I3, GT 8-07, HMF3896, KM2, HMF4436, LPB0099, LPB0063, KHN15, IMCC25605, YH-5, S6-6, LPB0123, KYW941, KYW1051, YF5, Family JD11, JD16, JD57, LPB0083, IMCC25618, KHC14, Moraxellaceae C2-29, NR 4-01, NR 5-07 and HMF4401 were assigned to the Gammaproteobacteria. The 39 strains were dis- tributed in six orders of the Gammaproteobacteria; five strains in the order Alteromonadales, 12 strains in the

Continued Enterobacteriales, four strains in the Oceanospirillales, Phylum 11 strains for the Pseudomonadales, two strain in the Vibrionales and five strains in the Xanthomonadales Table 1. Table Gammaproteobacteria 106 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2

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

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

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

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

(21)

Fig. 2. Transmission electron micrographs or scanning electron micrographs of cells of the species in the order Alteromonadales, Entero- bacteriales and Oceanospirillales belonging to the Gammaproteobacteria isolated in this study. Strains: 1, TJB7; 2, JF6; 3, KYW1005; 4, HMF3946; 5, LPB0073; 6, KHL6; 7, KHN5; 8, KHM5; 9, KHN16; 10, KHL16; 11, IMCC25602; 12, KM3; 13, DF-5; 14, TW2C3; 15, TW2I3; 16, GT 8-07; 17, HMF3896; 18, KM2; 19, HMF4436; 20, LPB0099; 21, LPB0063.

(Table 1). These strains were Gram-staining-negative, LPB0073, KHL6, KHN5, KHM5, KHN16, KHL16, chemoheterotrophic and rod- or coccoid-shaped bacte- IMCC25602, KM3, DF-5, TW2C3, TW2I3, GT 8-07, ria (Fig. 2). Colony size, morphology and physiological HMF3896, KM2, HMF4436, LPB0099, LPB0063, characteristics are also shown in the species descrip- KHN15, IMCC25605, YH-5, S6-6, LPB0123, KYW941, tion section. Strains TJB7, JF6, KYW1005, HMF3946, KYW1051, YF5, JD11, JD16, JD57, LPB0083, June 2017 KIM ET AL.-UNRECORDED SPECIES OF THE CLASSES BETAPROTEOBACTERIA AND GAMMAPROTEOBACTERIA 107

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

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

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

(16) (17) (18)

Fig. 3. Transmission electron micrographs or scanning electron micrographs of cells of the species in the order Pseudomonadales, Vibrio- nales and Xanthomonadales belonging to the Gammaproteobacteria isolated in this study. Strains: 1, KHN15; 2, IMCC25605; 3, YH-5; 4, S6-6; 5, LPB0123; 6, KYW941; 7, KYW1051; 8, YF5; 9, JD11; 10, JD16; 11, JD57; 12, LPB0083; 13, IMCC25618; 14, KHC14; 15, C2- 29; 16, NR 4-01; 17, NR 5-07; 18, HMF4401.

IMCC25618, KHC14, C2-29, NR 4-01, NR 5-07 and Kosakonia cowanii CIP 107300T (AJ508303; 99.39%), HMF4401 were most closely related to Rheinheimera Escherichia marmotae HT073016T (JNBP01000188; texasensis A62-14BT (AY701891; 99.30% 16S rRNA 99.27%), Enterobacter xiangfangensis 10-17T (HF679035; gene sequence similarity), Rheinheimera tilapiae Ruye- 99.93%), Citrobacter youngae CECT 5335T (AJ564736; 90T (HQ111524; 99.90%), Alteromonas macleodii ATCC 99.89%), Erwinia persicina ATCC 35998T (U80205; 27126T (CP003841; 99.58%), Marinobacter maritimus 99.72%), Providencia rettgeri DSM 4542T (AM040492; CK47T (AJ704395; 99.10%), Psychromonas arctica 99.50%), Hafnia paralvei ATCC 29927T (FM179943; DSM 14288T (AXWP01000011; 99.19%), Raoultella 99.60%), Pantoea agglomerans DSM 3493T (AJ233423; ornithinolytica JCM 6096T (AJ251467; 98.87%), 99.11%), Pantoea eucalypti LMG 24198T (EF688009; 108 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2

Fig. 4. Neighbour-joining (NJ) tree based on the 16S rRNA gene sequence comparisons, showing the taxonomic position between the 18 strains isolated in this study and the notable type species from the class Betaproteobacteria. The tree was primarily reconstructed using a neighbor-joining algorithm (NJ). Maximum parsimony (MP) and maximum likelihood (ML) algorithms were applied for additional com- parisons. Corresponding branches are presented as filled diamonds; at the nodes, only bootstrap values of more than 70% are shown, which are expressed as a percentage of 1000 replicates (NJ/MP/ML). ATCC 11775T (JMST01000030) was established as an out- group. The bar indicates 0.02 substitutions per nucleotide.

99.26%), Serratia plymuthica DSM 4540T (AJ233433; frigidicola DSM 12411T (AJ609556; 99.45%), Moraxella 99.06%), Ewingella americana ATCC 33852T (JMPJ osloensis NCTC 10465T (X74897; 99.60%), Acinetobacter 01000013; 99.90%), Cobetia amphilecti KMM 1561T junii CIP 64.5T (APPX01000010; 99.47%), Pseudomonas (AB646236; 99.80%), Halomonas fontilapidosi 5CRT indoloxydans IPL-1T (DQ916277; 99.03%), Pseudomonas (EU541349; 100%), Kangiella japonica KMM 3899T songnenensis NEAU-ST5-5T (JQ762269; 99.24%), (AB505051; 99.66%), Marinomonas arenicola KMM Pseudomonas kunmingensis HL22-2T (JQ246444; 99.47 3893T (AB467281; 99.19%), Acinetobacter lwoffii %), subsp. piscium JF3835T NCTC 5866T (AIEL01000120; 98.79%), Psychrobacter (FJ168539; 99.10%), ATCC 4973T June 2017 KIM ET AL.-UNRECORDED SPECIES OF THE CLASSES BETAPROTEOBACTERIA AND GAMMAPROTEOBACTERIA 109

Fig. 5. Neighbour-joining (NJ) tree based on the 16S rRNA gene sequence comparisons, showing the taxonomic position between the 21 strains isolated in this study and the notable type species from the orders Alteromonadales, Enterobacteriales and Oceanospirillales in the class Gammaproteobacteria. The tree was primarily reconstructed using a neighbor-joining algorithm (NJ). Maximum parsimony (MP) and maximum likelihood (ML) algorithms were applied for additional comparisons. Corresponding branches are presented as filled diamonds; at the nodes, only bootstrap values of more than 70% are shown, which are expressed as a percentage of 1000 replicates (NJ/MP/ML). Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001) was established as an out-group. The bar indicates 0.02 substitutions per nucleotide.

(AF094733; 99.51%), Pseudomonas viridiflava LMG (EF424397; 99.41%), Xanthomonas translucens DSM 2352T (Z76671; 99.86%), Pseudomonas graminis DSM 18974T (CAPJ01000550; 99.71%) and Pseudomonas 11363T (Y11150; 98.87%), Vibrio atlanticus Vb 11.11T hibiscicola ATCC 19867T (AB021405; 99.50%), re- (EF599163; 99.48%), Vibrio zhanjiangensis E414T (JF spectively (Table 1). In the conjoined, phylogenetic 931139; 99.93%), Stenotrophomonas rhizophila DSM consensus tree, isolated strains provided a robust phy- 14405T (CP007597; 99.72%), Rhodanobacter soli DCY logenetic clade with the most closely related species 45T (FJ605268; 98.91%), Pseudoxanthomonas indica P15T in the orders Alteromonadales, Enterobacteriales, 110 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2

Fig. 6. Neighbour-joining (NJ) tree based on the 16S rRNA gene sequence comparisons, showing the taxonomic position between the iso- lated 18 strains in this study and the notable type species from the orders Pseudomonadales, Vibrionales and Xanthomonadales in the class Gammaproteobacteria. The tree was primarily reconstructed using a neighbor-joining algorithm (NJ). Maximum parsimony (MP) and max- imum likelihood (ML) algorithms were applied for additional comparisons. Corresponding branches are presented as filled diamonds; at the nodes, only bootstrap values of more than 70% are shown, which are expressed as a percentage of 1000 replicates (NJ/MP/ML). Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001) was established as an out-group. The bar indicates 0.05 substitutions per nucleotide.

Oceanospirillales (Fig. 5), Pseudomonadales, and five species in five genera of one family in the order Vibrionales and Xanthomonadales (Fig. 6) within the Xanthomonadales are reported for gammaproteobacteri- class Gammaproteobacteria, as expected from high 16S al species found in Korea. rRNA gene sequence similarities. There is no official report that these 39 species have been isolated in Korea; Description of Duganella zoogloeoides HMF3907 therefore five species in four genera of four families in the order Alteromonadales, 12 species in 11 genera of one Cells are Gram-staining-negative, flagellated, non-pig- family in the order Enterobacteriales, four species in four mented and rod-shaped. Colonies are circular, convex, genera of three families in the order Oceanospirillales, entire and pale yellow-colored after 3 days of incuba- 11 species in four genera of two families in the order tion on R2A at 30°C. Positive for cytochrome oxidase, Pseudomonadales, two species in the order Vibrionales esculin hydrolysis and β-galactosidase, but negative June 2017 KIM ET AL.-UNRECORDED SPECIES OF THE CLASSES BETAPROTEOBACTERIA AND GAMMAPROTEOBACTERIA 111 for arginine dihydrolase, gelatinase, glucose fermenta- raised and white-colored after 7 days of incubation on tion, indole production, nitrates reduction and urease in R2A at 25°C. Positive for urease, but negative for argi- API 20NE test. L-Arabinose, D-maltose, malic acid, D- nine dihydrolase, cytochrome oxidase, esculin hydroly- mannose, phenylacetic acid and trisodium citrate are uti- sis, β-galactosidase, gelatinase, glucose fermentation, lized. Strain HMF3907 ( =NIBRBAC000497904) has indole production and nitrates reduction in API 20NE been isolated from fresh water sample, Yongin, Gyeong- test. L-Arabinose, D-glucose, malic acid, D-mannitol, gi-do, Korea. D-mannose and potassium gluconate are utilized. Strain JD5 ( =NIBRBAC000497935) has been isolated from Description of Vogesella indigofera HMF4008 fresh water sample, Changwon, Gyeongsangnam-do, Korea. Cells are Gram-staining-negative, flagellated, non-pig- mented and rod-shaped. Colonies are circular, convex, Description of Janthinobacterium lividum JD24 entire and white-colored after 3 days of incubation on R2A at 30°C. Positive for cytochrome oxidase, but neg- Cells are Gram-staining-negative, non-flagellated, ative for arginine dihydrolase, esculin hydrolysis, β-ga- non-pigmented and rod-shaped. Colonies are circular, lactosidase, gelatinase, glucose fermentation, indole pro- convex, smooth and purple-white-colored after 2 days duction, nitrates reduction and urease in API 20NE test. of incubation on R2A at 25°C. Positive for cytochrome N-Acetyl-glucosamine, capric acid, malic acid, D-maltose oxidase, esculin hydrolysis, gelatinase and nitrates re- and potassium gluconate are utilized. Strain HMF4008 duction, but negative for arginine dihydrolase, β-galac­ (=NIBRBAC000497908) has been isolated from fresh tosidase, glucose fermentation, indole production and water sample, Gyeongju, Gyeongsangbuk-do, Korea. urease in API 20NE test. Adipic acid, L-arabinose, capric acid, D-glucose, malic acid, D-maltose, D-mannitol, Description of Chromobacterium vaccinia HMF4075 D-mannose, phenylacetic acid and trisodium citrate are utilized. Strain JD24 (=NIBRBAC000497938) has been Cells are Gram-staining-negative, flagellated, non-pig- isolated from fresh water sample, Changwon, Gyeong- mented and rod-shaped. Colonies are circular, convex, sangnam-do, Korea. entire and violet-colored after 3 days of incubation on R2A at 30°C. Positive for arginine dihydrolase, cyto- Description of Janthinobacterium svalbardensis JD50 chrome oxidase, esculin hydrolysis, gelatinase, glucose fermentation, nitrates reduction and urease, but negative Cells are Gram-staining-negative, non-flagellated, for β-galactosidase and indole production in API 20NE non-pigmented and rod-shaped. Colonies are circular, test. N-Acetyl-glucosamine, capric acid, D-glucose, convex, smooth and pale yellow-colored after 2 days of potassium gluconate and trisodium citrate are utilized. incubation on R2A at 25°C. Positive for esculin hydroly- Strain HMF4075 ( =NIBRBAC000497911) has been sis, β-galactosidase and nitrates reduction, but negative isolated from fresh water sample, Jeju, Jeju-do, Korea. for arginine dihydrolase, cytochrome oxidase, gelatinase, glucose fermentation, indole production and urease in Description of Acidovorax temperans JD2 API 20NE test. Adipic acid, L-arabinose, D-glucose, malic acid, D-maltose, D-mannitol, D-mannose, phenyl­ Cells are Gram-staining-negative, non-flagellated, acetic acid and trisodium citrate are utilized. Strain JD50 non-pigmented and rod-shaped. Colonies are convex, (=NIBRBAC000497939) has been isolated from fresh smooth and white-colored after 4 days of incubation on water sample, Changwon, Gyeongsangnam-do, Korea. R2A at 25°C. Positive for nitrates reduction and urease, but negative for arginine dihydrolase, cytochrome ox- Description of Herbaspirillum hiltneri IMCC25601 idase, esculin hydrolysis, β-galactosidase, gelatinase, glucose fermentation and indole production in API Cells are Gram-staining-negative, non-flagellated, 20NE test. L-Arabinose, D-glucose, malic acid, D-man- non-pigmented and rod-shaped. Colonies are circular, nitol, D-mannose, potassium gluconate and trisodium raised, entire and white-colored after 10 days of incu- citrate are utilized. Strain JD2 (=NIBRBAC000497934) bation on R2A at 20°C. Positive for esculin hydrolysis, has been isolated from fresh water sample, Changwon, β-galactosidase and indole production, but negative Gyeongsangnam-do, Korea. for arginine dihydrolase, cytochrome oxidase, gelati- nase, glucose fermentation, nitrates reduction and ure- ase in API 20NE test. N-Acetyl-glucosamine, L-arab- Description of Acidovorax valerianellae JD5 inose, D-glucose, malic acid, D-maltose, D-mannitol, Cells are Gram-staining-negative, non-flagellated, D-mannose and potassium gluconate are utilized. Strain non-pigmented and rod-shaped. Colonies are circular, IMCC25601 (=NIBRBAC000497991) has been isolat- 112 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2 ed from fresh water sample, Chuncheon, Gangwon-do, 20NE test. Adipic acid, malic acid, phenylacetic acid Korea. and trisodium are utilized. Strain C1-11 ( =NIBRBAC 000498019) has been isolated from natural cave, Jeju, Description of Massilia varians IMCC25614 Jeju-do, Korea. Cells are Gram-staining-negative, non-flagellated, Description of Pseudoduganella violaceinigra NJ-1-5 non-pigmented and rod-shaped. Colonies are circular, raised, entire and white-colored after 10 days of incu- Cells are Gram-staining-negative, flagellated, non-pig- bation on R2A at 20°C. Positive for gelatinase and ure- mented and rod-shaped. Colonies are circular, convex ase, but negative for arginine dihydrolase, cytochrome and purple-colored after 2 days of incubation on R2A oxidase, esculin hydrolysis, β-galactosidase, glucose at 25°C. Negative for arginine dihydrolase, cytochrome fermentation, indole production and nitrates reduction oxidase, esculin hydrolysis, β-galactosidase, gelatinase, in API 20NE test. N-Acetyl-glucosamine, adipic acid, glucose fermentation, indole production, nitrates reduc- L-arabinose, capric acid, D-glucose, malic acid, D-malt- tion and urease in API 20NE test. D-Glucose is utilized. ose, D-mannitol, D-mannose, phenylacetic acid, potas- Strain NJ-1-5 (=NIBRBAC000498038) has been isolat- sium gluconate and trisodium citrate are utilized. Strain ed from fresh water sample, Naju, Jeollanam-do, Korea. IMCC25614 (=NIBRBAC000498004) has been isolat- ed from fresh water sample, Nam-gu, Incheon, Korea. Description of Herbaspirillum autotrophicum GF5 Cells are Gram-staining-negative, flagellated, non-pig- Description of Noviherbaspirillum malthae mented and rod-shaped. Colonies are circular, convex IMCC25617 and white-colored after 2 days of incubation on R2A at Cells are Gram-staining-negative, non-flagellated, 25°C. Positive for cytochrome oxidase and urease, but non-pigmented and rod-shaped. Colonies are circular, negative for arginine dihydrolase, esculin hydrolysis, β- raised, entire and yellow-colored after 10 days of incu- galactosidase, gelatinase, glucose fermentation, indole bation on R2A at 20°C. Positive for cytochrome oxidase production and nitrates reduction in API 20NE test. N- and esculin hydrolysis, but negative for arginine dihy- Acetyl-glucosamine, L-arabinose, D-glucose, malic acid, drolase, β-galactosidase, gelatinase, glucose fermenta- D-mannose, phenylacetic acid, potassium gluconate and tion, indole production, nitrates reduction and urease trisodium citrate are utilized. Strain GF5 (=NIBRBAC in API 20NE test. Strain IMCC25617 ( =NIBRBAC 000498039) has been isolated from fresh water sample, 000498007) has been isolated from fresh water sample, Jeju, Jeju-do, Korea. Nam-gu, Incheon, Korea. Description of Roseateles terrae JF-5 Description of Candidimonas bauzanensis C1-1 Cells are Gram-staining-negative, flagellated, non-pig- Cells are Gram-staining-negative, non-flagellated, mented and rod-shaped. Colonies are circular, flat and non-pigmented and rod-shaped. Colonies are circular, white-colored after 2 days of incubation on R2A at convex, entire and cream-colored after 3 days of incu- 25°C. Positive for cytochrome oxidase, β-galactosidase bation on TSA at 30°C. Positive for cytochrome oxi- and gelatinase, but negative for arginine dihydrolase, dase and urease, but negative for arginine dihydrolase, esculin hydrolysis, glucose fermentation, indole pro- esculin hydrolysis, β-galactosidase, gelatinase, glucose duction, nitrates reduction and urease in API 20NE test. fermentation, indole production and nitrates reduction D-Glucose, L-arabinose, D-mannose, D-mannitol, D- in API 20NE test. Adipic acid, malic acid, phenylacetic maltose and potassium gluconate are utilized. Strain acid and trisodium citrate are utilized. Strain C1-1 JF-5 ( =NIBRBAC000498046) has been isolated from (=NIBRBAC000498018) has been isolated from natu- fresh water sample, Jeonju, Jeollabuk-do, Korea. ral cave, Jeju, Jeju-do, Korea. Description of Noviherbaspirillum soli 2015-3 Description of Candidimonas bauzanensis C1-11 Cells are Gram-staining-negative, flagellated, non-pig- Cells are Gram-staining-negative, non-flagellated, mented and rod-shaped. Colonies are circular, flat and non-pigmented and rod-shaped. Colonies are circular, white-colored after 2 days of incubation on R2A at 25°C. convex, entire and cream-colored after 3 days of incu- Positive for cytochrome oxidase, but negative for argi- bation on TSA at 30°C. Positive for cytochrome oxidase nine dihydrolase, esculin hydrolysis, β-galactosidase, and urease, but negative for arginine dihydrolase, esculin gelatinase, glucose fermentation, indole production, hydrolysis, β-galactosidase, gelatinase, glucose fermen- nitrates reduction and urease in API 20NE test. Potas- tation, indole production and nitrates reduction in API sium gluconate is utilized. Strain 2015-3 (=NIBRBAC June 2017 KIM ET AL.-UNRECORDED SPECIES OF THE CLASSES BETAPROTEOBACTERIA AND GAMMAPROTEOBACTERIA 113

000498055) has been isolated from soil sample, Wanjoo, test. Strain HMF3946 ( =NIBRBAC000497907) has Jeollabuk-do, Korea. been isolated from sea water sample, Pohang, Gyeong- sangbuk-do, Korea. Description of Limnobacter thiooxidans NDH-10 Description of Pseudomonas hibiscicola HMF4401 Cells are Gram-staining-negative, flagellated, non-pig- mented and rod-shaped. Colonies are circular, entire, Cells are Gram-staining-negative, flagellated, non-pig- smooth, flat and cream-colored after 2 days of incu- mented and rod-shaped. Colonies are circular, convex, bation on R2A at 30°C. Positive for cytochrome oxi- entire and beige-colored after 3 days of incubation on dase, esculin hydrolysis, β-galactosidase and nitrates MA at 30°C. Positive for cytochrome oxidase, esculin reduction, but negative for arginine dihydrolase, gelati- hydrolysis, β-galactosidase and gelatinase, but negative nase, glucose fermentation, indole production and ure- for arginine dihydrolase, glucose fermentation, indole ase in API 20NE test. Strain NDH-10 ( =NIBRBAC production, nitrates reduction and urease in API 20NE 000498058) has been isolated from paddy field, An- test. N-Acetyl-glucosamine, capric acid, D-glucose, ma- seong, Gyeonggi-do, Korea. lic acid, D-maltose, D-mannose and trisodium citrate are utilized. Strain HMF4401 (=NIBRBAC000497913) has Description of Rhizobacter gummiphilus RDH4 been isolated from jeotgal sample, Buan, Jeollabuk-do, Korea. Cells are Gram-staining-negative, flagellated, non-pig- mented and rod-shaped. Colonies are smooth, shiny, Description of Halomonas fontilapidosi HMF4436 convex and yellow-colored after 2 days of incubation on R2A at 30°C. Positive for esculin hydrolysis and β-ga- Cells are Gram-staining-negative, flagellated, non-pig- lactosidase and urease, but negative for arginine dihy- mented and rod-shaped. Colonies are circular, convex, drolase, cytochrome oxidase, gelatinase, glucose fermen- entire and beige-colored after 3 days of incubation on tation, indole production and nitrates reduction in API MA at 30°C. Positive for cytochrome oxidase, nitrates 20NE test. Adipic acid, L-arabinose, D-glucose, D-malt- reduction, but negative for arginine dihydrolase, esculin ose, D-mannitol, D-mannose and potassium gluconate hydrolysis, β-galactosidase, gelatinase, glucose fermen- are utilized. Strain RDH4 ( =NIBRBAC000498078) tation, indole production and urease in API 20NE test. has been isolated from lake sample, Dongjak-gu, Seoul, Strain HMF4436 ( =NIBRBAC000497917) has been Korea. isolated from salt sample, Buan, Jeollabuk-do, Korea.

Description of Ewingella americana HMF3896 Description of Pseudomonas hibiscicola IMCC25602 Cells are Gram-staining-negative, non-flagellated, Cells are Gram-staining-negative, non-flagellated, non-pigmented and rod-shaped. Colonies are circular, non-pigmented and rod-shaped. Colonies are circular, convex, entire and cream-colored after 3 days of incu- raised, entire and white-colored after 10 days of incu- bation on R2A at 30°C. Positive for esculin hydrolysis, bation on R2A at 20°C. Positive for esculin hydrolysis, β-galactosidase, glucose fermentation and nitrates re- β-galactosidase and indole production, but negative duction, but negative for arginine dihydrolase, cyto- for arginine dihydrolase, cytochrome oxidase, gelati- chrome oxidase, gelatinase, indole production and ure- nase, glucose fermentation, nitrates reduction and ure- ase in API 20NE test. N-Acetyl-glucosamine, D-glucose, ase in API 20NE test. N-Acetyl-glucosamine, L-arab- malic acid, D-maltose, D-mannitol, D-mannose, potas- inose, D-glucose, malic acid, D-maltose, D-mannitol, sium gluconate and trisodium citrate are utilized. Strain D-mannose and potassium gluconate are utilized. Strain HMF3896 ( =NIBRBAC000497902) has been isolated IMCC25602 (=NIBRBAC000497992) has been isolat- from soil sample, Yongin, Gyeonggi-do, Korea. ed from fresh water sample, Chuncheon, Gangwon-do, Korea. Description of Marinobacter maritimus HMF3946 Description of Psychrobacter frigidicola IMCC25605 Cells are Gram-staining-negative, flagellated, non-pig- mented and rod-shaped. Colonies are circular, convex, Cells are Gram-staining-negative, non-flagellated, entire and cream-colored after 3 days of incubation on non-pigmented and rod-shaped. Colonies are circular, MA at 30°C. Positive for cytochrome oxidase and ni- raised, entire and white-colored after 7 days of incu- trates reduction, but negative for arginine dihydrolase, bation on 0.1% of diluted MA at 20°C. Positive for esculin hydrolysis, β-galactosidase, gelatinase, glucose cytochrome oxidase and urease, but negative for argi- fermentation, indole production and urease in API 20NE nine dihydrolase, esculin hydrolysis, β-galactosidase, 114 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2 gelatinase, glucose fermentation, indole production and Description of Providencia rettgeri KM3 nitrates reduction in API 20NE test. Strain IMCC25605 Cells are Gram-staining-negative, non-flagellated, (=NIBRBAC000497995) has been isolated from fresh non-pigmented and rod-shaped. Colonies are opaque, water sample, Chuncheon, Gangwon-do, Korea. flat and white-colored after 2 days of incubation on Description of Vibrio zhanjiangensis IMCC25618 R2A at 25°C. Positive for arginine dihydrolase, esculin hydrolysis, glucose fermentation, indole production, ni- Cells are Gram-staining-negative, non-flagellated, trates reduction and urease, but negative cytochrome ox- non-pigmented and rod-shaped. Colonies are circular, idase, β-galactosidase and gelatinase in API 20NE test. raised, entire and white-colored after 15 days of incu- N-Acetyl-glucosamine, capric acid, D-glucose, malic bation on MA at 20°C. Positive for cytochrome oxidase acid, D-mannitol, D-mannose, potassium gluconate and and esculin hydrolysis, but negative for arginine dihy- trisodium citrate are utilized. Strain KM3 (=NIBRBAC drolase, β-galactosidase, gelatinase, glucose fermenta- 000498041) has been isolated from sea water sample, tion, indole production, nitrates reduction and urease Jeju, Jeju-do, Korea. in API 20NE test. Strain IMCC25618 ( =NIBRBAC 000498008) has been isolated from fresh water sample, Description of Pseudomonas chlororaphis subsp. Nam-gu, Incheon, Korea. piscium YF5

Description of Rhodanobacter soli C2-29 Cells are Gram-staining-negative, non-flagellated, non-pigmented and rod-shaped. Colonies are irregular, Cells are Gram-staining-negative, non-flagellated, transparent and yellow-colored after 2 days of incuba- non-pigmented and rod-shaped. Colonies are circular, tion on R2A at 25°C. Positive for arginine dihydrolase, convex, entire and light yellow-colored after 13 days of cytochrome oxidase, gelatinase, glucose fermentation incubation on TSA at 30°C. Positive for nitrates reduc- and urease, but negative for esculin hydrolysis and β-ga- tion, but negative for arginine dihydrolase, cytochrome lactosidase, indole production and nitrates reduction in oxidase, esculin hydrolysis, β-galactosidase, gelatinase, API 20NE test. N-Acetyl-glucosamine, L-arabinose, glucose fermentation, indole production and urease in capric acid, D-glucose, malic acid, D-mannitol, D-man- API 20NE test. N-Acetyl-glucosamine and D-maltose nose, potassium gluconate and trisodium citrate are uti- are utilized. Strain C2-29 (=NIBRBAC000498024) has lized. Strain YF5 ( =NIBRBAC000498042) has been been isolated from natural cave, Jeju, Jeju-do, Korea. isolated from fresh water sample, Jangsu, Jeollabuk-do, Korea. Description of Moraxella osloensis YH-5 Description of Hafnia paralvei DF-5 Cells are Gram-staining-negative, non-flagellated, non-pigmented and rod-shaped. Colonies are convex, Cells are Gram-staining-negative, non-flagellated, opaque and white-colored after 2 days of incubation on non-pigmented and rod-shaped. Colonies are irregular R2A at 25°C. Positive for cytochrome oxidase and ni- and white-colored after 2 days of incubation on R2A at trates reduction, but negative for arginine dihydrolase, 25°C. Positive for β-galactosidase, glucose fermentation esculin hydrolysis, β-galactosidase, gelatinase, glucose and nitrates reduction, but negative for arginine dihydro- fermentation, indole production and urease in API 20NE lase, cytochrome oxidase, esculin hydrolysis, gelatinase, test. N-Acetyl-glucosamine, D-glucose, D-mannose indole production and urease in API 20NE test. N-Ace- and adipic acid are utilized. Strain YH-5 (=NIBRBAC tyl-glucosamine, D-glucose, malic acid, D-maltose, 000498036) has been isolated from fresh water sample, D-mannitol, D-mannose and potassium gluconate are Jeonju, Jeollabuk-do, Korea. utilized. Strain DF-5 (=NIBRBAC000498045) has been isolated from fresh water sample, Jangsu, Jeollabuk-do, Description of Cobetia amphilecti KM2 Korea. Cells are Gram-staining-negative, flagellated, non-pig- Description of Rheinheimera texasensis TJB7 mented and rod-shaped. Colonies are transparent, shiny and white-colored after 2 days of incubation on R2A at Cells are Gram-staining-negative, flagellated, non-pig- 25°C. Positive for β-galactosidase, but negative for argi- mented and rod-shaped. Colonies are circular, trans- nine dihydrolase, cytochrome oxidase, esculin hydroly- parent and yellow-colored after 2 days of incubation sis, gelatinase, glucose fermentation, indole production, on R2A at 25°C. Positive for cytochrome oxidase and nitrates reduction and urease in API 20NE test. Strain gelatinase, but negative for arginine dihydrolase, esculin

KM2 (=NIBRBAC000498040) has been isolated from hydrolysis, β-galactosidase, glucose fermentation, in- sea water sample, Jeju, Jeju-do, Korea. dole production, nitrates reduction and urease in API June 2017 KIM ET AL.-UNRECORDED SPECIES OF THE CLASSES BETAPROTEOBACTERIA AND GAMMAPROTEOBACTERIA 115

20NE test. Potassium gluconate is utilized. Strain TJB7 mannose and are utilized. Strain NR 4-01 (=NIBRBAC (=NIBRBAC000498051) has been isolated from fresh 000498087) has been isolated from plant root sample, water sample, Yeonggwang, Jeollanam-do, Korea. Jeongeup, Jeollabuk-do, Korea.

Description of Rheinheimera tilapiae JF6 Description of Serratia plymuthica GT 8-07 Cells are Gram-staining-negative, flagellated, non-pig- Cells are Gram-staining-negative, flagellated, non-pig- mented and rod-shaped. Colonies are circular, raised mented and short rod-shaped. Colonies are circular, and yellow-colored after 2 days of incubation on R2A at pulvinate, entire and ivory-colored after 2 days of incu­ 25°C. Positive for cytochrome oxidase, esculin hydroly- bation on TSA at 30°C. Positive for cytochrome oxidase, sis, β-galactosidase and nitrates reduction, but negative esculin hydrolysis, β-galactosidase, gelatinase, glucose for arginine dihydrolase, gelatinase, glucose fermen- fermentation and nitrates reduction, but negative for ar- tation, indole production and urease in API 20NE test. ginine dihydrolase, indole production and urease in API Strain JF6 ( =NIBRBAC000498056) has been isolated 20NE test. N-Acetyl-glucosamine, L-arabinose, capric from fresh water sample, Jeonju, Jeollabuk-do, Korea. acid, D-glucose, malic acid, D-maltose, D-mannitol, D- mannose, phenylacetic acid, potassium gluconate and tri- Description of Pantoea agglomerans TW2C3 sodium citrate are utilized. Strain GT 8-07 (=NIBRBAC 000498088) has been isolated from plant root sample, Cells are Gram-staining-negative, non-flagellated, Gumi, Gyeongsangbuk-do, Korea. non-pigmented and coccoid-shaped. Colonies are cir- cular, convex, entire and yellow-colored after 2 days of Description of Xanthomonas translucens NR 5-07 incubation on NA at 30°C. Positive for gelatinase, but negative for arginine dihydrolase, cytochrome oxidase, Cells are Gram-staining-negative, non-flagellated, esculin hydrolysis, β-galactosidase, glucose fermenta- non-pigmented and short rod-shaped. Colonies are cir- tion, indole production, nitrates reduction and urease in cular, convex, smooth and yellow-colored after 2 days API 20NE test. D-Glucose, D-maltose and potassium of incubation on R2A at 30°C. Positive for cytochrome gluconate are utilized. Strain TW2C3 ( =NIBRBAC oxidase, esculin hydrolysis, β-galactosidase and nitrates 000498083) has been isolated from soil sample of aban- reduction, but negative for arginine dihydrolase, gelati- doned mine, Boeun, Chungcheongbuk-do, Korea. nase, glucose fermentation, indole production and ure- ase in API 20NE test. N-Acetyl-glucosamine, D-glucose,

Description of Pantoea eucalypti TW2I3 D-maltose and D-mannose are utilized. Strain NR 5-07 (=NIBRBAC000498090) has been isolated from plant Cells are Gram-staining-negative, non-flagellated, root sample, Jeongeup, Jeollabuk-do, Korea. non-pigmented and rod-shaped. Colonies are circular, umbonate, entire and yellow-colored after 2 days of Description of Alteromonas macleodii KYW1005 incubation on ISP2 media at 30°C. Positive for gelati- nase, but negative for arginine dihydrolase, cytochrome Cells are Gram-staining-negative, non-flagellated, oxidase, esculin hydrolysis, β-galactosidase, glucose non-pigmented and rod-shaped. Colonies are circular, fermentation, indole production, nitrates reduction and smooth, raised and cream-colored after 3 days of incu- urease in API 20NE test. D-Glucose, D-maltose, D-man- bation on MA at 25°C. Positive for cytochrome oxidase, nitol and potassium gluconate are utilized. Strain TW2I3 esculin hydrolysis and β-galactosidase, but negative for ( =NIBRBAC000498086) has been isolated from soil arginine dihydrolase, gelatinase, glucose fermentation, sample of abandoned mine, Boeun, Chungcheongbuk- indole production, nitrates reduction and urease in API do, Korea. 20NE test. Strain KYW1005 (=NIBRBAC000497923) has been isolated from sea water sample, Gwangyang, Description of Pseudoxanthomonas indica NR 4-01 Jeollanam-do, Korea.

Cells are Gram-staining-negative, flagellated, non-pig- Description of Pseudomonas songnenensis KYW941 mented and rod-shaped. Colonies are circular, convex, entire and yellow-colored after 2 days of incubation on Cells are Gram-staining-negative, non-flagellated, R2A at 30°C. Positive for cytochrome oxidase, esculin non-pigmented and rod-shaped. Colonies are radial wrin- hydrolysis, β-galactosidase and nitrates reduction, but kled and light yellow-colored after 3 days of incubation negative for arginine dihydrolase, gelatinase, glucose on MA at 25°C. Positive for cytochrome oxidase and fermentation, indole production and urease in API 20NE nitrates reduction, but negative for arginine dihydrolase, test. N-Acetyl-glucosamine, D-glucose, D-maltose and D- esculin hydrolysis, β-galactosidase, gelatinase, glucose 116 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2 fermentation, indole production and urease in API 20NE cent, convex and cream-colored after 3 days of incuba- test. Adipic acid, capric acid, D-glucose, malic acid, tion on R2A at 25°C. Positive for arginine dihydrolase D-maltose, D-mannitol, potassium gluconate and triso- and cytochrome oxidase, but negative for esculin hydro- dium citrate are utilized. Strain KYW941 (=NIBRBAC lysis, β-galactosidase, gelatinase, glucose fermentation, 000497924) has been isolated from sea water sample, indole production, nitrates reduction and urease in API Gwangyang, Jeollanam-do, Korea. 20NE test. L-Arabinose, capric acid, D-glucose, malic acid, D-mannitol, D-mannose, potassium gluconate and Description of Pseudomonas kunmingensis KYW1051 trisodium citrate are utilized. Strain JD57 (=NIBRBAC 000497940) has been isolated from fresh water sample, Cells are Gram-staining-negative, non-flagellated, Changwon, Gyeongsangnam-do, Korea. non-pigmented and rod-shaped. Colonies are circular, opaque and pale yellow-colored after 4 days of incuba- tion on MA at 25°C. Positive for cytochrome oxidase and Description of Acinetobacter lwoffii KHN15 nitrates reduction, but negative for arginine dihydrolase, Cells are Gram-staining-negative, non-flagellated, esculin hydrolysis, β-galactosidase, gelatinase, glucose non-pigmented and coccoid-shaped. Colonies are circu- fermentation, indole production and urease in API 20NE lar and cream-colored after 3 days of incubation on TSA test. Adipic acid, capric acid, D-glucose, malic acid, at 25°C. Negative for arginine dihydrolase, cytochrome D-maltose, D-mannitol, potassium gluconate and trisodi- oxidase, esculin hydrolysis, β-galactosidase, gelatinase, um citrate are utilized. Strain KYW1051 (=NIBRBAC glucose fermentation, indole production, nitrates reduc- 000497928) has been isolated from sea water sample, tion and urease in API 20NE test. Capric acid, malic Gwangyang, Jeollanam-do, Korea. acid and phenylacetic acid are utilized. Strain KHN15 ( =NIBRBAC000497835) has been isolated from gut Description of Pseudomonas kunmingensis JD11 of Japanese crested ibis (Nipponia nippon), Gwacheon, Cells are Gram-staining-negative, non-flagellated, non- Gyeonggi-do, Korea. pigmented and rod-shaped. Colonies are circular, smooth and white-colored after 2 days of incubation on R2A Description of Raoultella ornithinolytica KHL6 at 25°C. Positive for arginine dihydrolase, cytochrome Cells are Gram-staining-negative, flagellated, non-pig- oxidase and urease, but negative for esculin hydrolysis, mented and rod-shaped. Colonies are irregular and β-galactosidase, gelatinase, glucose fermentation, in- cream-colored after 3 days of incubation on R2A at dole production and nitrates reduction in API 20NE 25°C. Positive for esculin hydrolysis, β-galactosidase, test. Capric acid, D-glucose, malic acid, D-mannitol, glucose fermentation, indole production and nitrates D-mannose, phenylacetic acid, potassium gluconate and reduction, but negative for arginine dihydrolase, cyto- = trisodium citrate are utilized. Strain JD11 ( NIBRBAC chrome oxidase, gelatinase and urease in API 20NE test. 000497936) has been isolated from fresh water sample, N-Acetyl-glucosamine, L-arabinose, D-glucose, malic Changwon, Gyeongsangnam-do, Korea. acid, D-maltose, D-mannitol, D-mannose, phenylacetic acid and potassium gluconate are utilized. Strain KHL6 Description of Pseudomonas viridiflava JD16 (=NIBRBAC000497837) has been isolated from gut of

Cells are Gram-staining-negative, non-flagellated, pig- burmese python (Python molurus bivittatus), Gwacheon, mented and rod-shaped. Colonies are circular, smooth Gyeonggi-do, Korea. and pale yellow-colored after 3 days of incubation on R2A at 25°C. Positive for esculin hydrolysis, gelatinase Description of Stenotrophomonas rhizophila KHC14 and glucose fermentation, but negative for arginine di- Cells are Gram-staining-negative, non-flagellated, hydrolase, cytochrome oxidase, β-galactosidase, indole non-pigmented and rod-shaped. Colonies are circular, production, nitrates reduction and urease in API 20NE convex and light yellow-colored after 3 days of incuba- test. L-Arabinose, capric acid, D-glucose, malic acid, tion on R2A at 25°C. Positive for cytochrome oxidase, D-mannitol, D-mannose, potassium gluconate and tri- esculin hydrolysis, β-galactosidase and gelatinase, but sodium citrate are utilized. Strain JD16 ( =NIBRBAC negative for arginine dihydrolase, glucose fermentation, 000497937) has been isolated from fresh water sample, indole production, nitrates reduction and urease in API Changwon, Gyeongsangnam-do, Korea. 20NE test. N-Acetyl-glucosamine, D-glucose, malic acid, D-maltose, D-mannose and trisodium citrate are Description of Pseudomonas graminis JD57 utilized. Strain KHC14 ( =NIBRBAC000497840) has Cells are Gram-staining-negative, non-flagellated, been isolated from gut of Japanese crested ibis (Nipponia non-pigmented and rod-shaped. Colonies are translu- nippon), Gwacheon, Gyeonggi-do, Korea. June 2017 KIM ET AL.-UNRECORDED SPECIES OF THE CLASSES BETAPROTEOBACTERIA AND GAMMAPROTEOBACTERIA 117

Description of Kosakonia cowanii KHN5 negative for arginine dihydrolase, cytochrome oxidase, esculin hydrolysis, gelatinase and urease in API 20NE Cells are Gram-staining-negative, flagellated, non-pig- test. N-Acetyl-glucosamine, L-arabinose, D-glucose, mented and short rod-shaped. Colonies are circular malic acid, D-maltose, D-mannitol, D-mannose, potas- and cream-colored after 3 days of incubation on R2A sium gluconate and trisodium citrate are utilized. Strain at 25°C. Positive for esculin hydrolysis, β-galactosi- KHL16 ( =NIBRBAC000497850) has been isolated dase, glucose fermentation and nitrates reduction, but from gut of burmese python (Python molurus bivittatus), negative for arginine dihydrolase, cytochrome oxidase, Gwacheon, Gyeonggi-do, Korea. gelatinase, indole production and urease in API 20NE test. N-Acetyl-glucosamine, L-arabinose, D-glucose, Description of Citrobacter youngae S6-6 malic acid, D-maltose, D-mannitol, D-mannose, potas- sium gluconate and trisodium citrate are utilized. Strain Cells are Gram-staining-negative, non-flagellated, KHN5 (=NIBRBAC000497843) has been isolated from non-pigmented and rod-shaped. Colonies are circular, gut of Japanese crested ibis (Nipponia nippon), Gwa- raised, entire and pale yellow-colored after 2 days of in- cheon, Gyeonggi-do, Korea. cubation on MA at 30°C. Positive for esculin hydrolysis and nitrates reduction, but negative for arginine dihydro- Description of Escherichia marmotae KHM5 lase, cytochrome oxidase, β-galactosidase, gelatinase, glucose fermentation, indole production and urease in Cells are Gram-staining-negative, flagellated, non-pig- API 20NE test. Strain S6-6 ( =NIBRBAC000497887) mented and coccoid-shaped. Colonies are circular and has been isolated from tidal flat, Taean, Chungcheong­ white-colored after 3 days of incubation on R2A at nam-do, Korea. 25°C. Positive for β-galactosidase, glucose fermentation, indole production and nitrates reduction, but negative Description of Marinomonas arenicola LPB0063 for arginine dihydrolase, cytochrome oxidase, esculin hydrolysis, gelatinase and urease in API 20NE test. Cells are Gram-staining-negative, flagellated, non-pig- N-Acetyl-glucosamine, L-arabinose, D-glucose, malic mented and rod-shaped. Colonies are circular and acid, D-maltose, D-mannitol, D-mannose and potassi- white-colored after 2 days of incubation on MA at 26°C. um gluconate are utilized. Strain KHM5 (=NIBRBAC Negative for arginine dihydrolase, cytochrome oxidase, 000497846) has been isolated from gut of Japanese esculin hydrolysis, β-galactosidase, gelatinase, glucose crested ibis (Nipponia nippon), Gwacheon, Gyeong- fermentation, indole production, nitrates reduction and gi-do, Korea. urease in API 20NE test. Strain LPB0063 (=NIBRBAC 000497972) has been isolated from sea water sample, Description of Enterobacter xiangfangensis KHN16 Ongjin, Incheon, Korea.

Cells are Gram-staining-negative, non-flagellated, Description of Psychromonas arctica LPB0073 non-pigmented and diplococcirod-shaped. Colonies are circular and cream-colored after 3 days of incubation on Cells are Gram-staining-negative, flagellated, non-pig- R2A at 25°C. Positive for arginine dihydrolase, β-ga- mented and rod-shaped. Colonies are circular and lactosidase, glucose fermentation, nitrates reduction and white-colored after 2 days of incubation on MA at 26°C. urease, but negative for cytochrome oxidase, esculin hy- Positive for cytochrome oxidase, but negative for argi- drolysis, gelatinase and indole production in API 20NE nine dihydrolase, esculin hydrolysis, β-galactosidase, test. N-Acetyl-glucosamine, L-arabinose, malic acid, gelatinase, glucose fermentation, indole production, D-maltose, D-mannitol, D-mannose, phenylacetic acid, nitrates reduction and urease in API 20NE test. Strain potassium gluconate and trisodium citrate are utilized. LPB0073 ( =NIBRBAC000497973) has been isolated Strain KHN16 ( =NIBRBAC000497849) has been iso- from sea water sample, Ongjin, Incheon, Korea. lated from gut of Japanese crested ibis (Nipponia nip- pon), Gwacheon, Gyeonggi-do, Korea. Description of Vibrio atlanticus LPB0083 Cells are Gram-staining-negative, flagellated, non-pig- Description of Citrobacter youngae KHL16 mented and curved rod-shaped. Colonies are irregular Cells are Gram-staining-negative, non-flagellated, and beige-colored after 2 days of incubation on MA at non-pigmented and rod-shaped. Colonies are irregular 26°C. Positive for cytochrome oxidase, but negative and cream-colored after 3 days of incubation on R2A for arginine dihydrolase, esculin hydrolysis, β-galac- at 25°C. Positive for β-galactosidase, glucose fermen- tosidase, gelatinase, glucose fermentation, indole pro- tation, indole production and nitrates reduction, but duction, nitrates reduction and urease in API 20NE test. 118 JOURNAL OF SPECIES RESEARCH Vol. 6, No. 2

Strain LPB0083 ( =NIBRBAC000497974) has been uncultured species. International Journal of Systematic isolated from sea water sample, Ongjin, Incheon, Korea. and Evolutionary Microbiology 62(Pt3):716-721. Kluge, A.G. and F.S. Farris. 1969. Quantitative phyletics and Description of Kangiella japonica LPB0099 the evolution of anurans. Systematic Zoology 18(1):1- 32. Cells are Gram-staining-negative, non-flagellated, Lane, D.J. 1991. 16S/23S rRNA sequencing. In: Nucleic non-pigmented and rod-shaped. Colonies are circular, acid techniques in bacterial systematics John Wiley and glistening and yellowish-colored after 2 days of incu- Sons, New York, NY. bation on MA at 26°C. Positive for cytochrome oxidase Lauber, C.L., M. Hamady, R. Knight and N. Fierer. 2009. and gelatinase, but negative for arginine dihydrolase, Pyrosequencing-based assessment of soil pH as a pre- esculin hydrolysis, β-galactosidase, glucose fermenta- dictor of soil bacterial community structure at the conti- tion, indole production, nitrates reduction and urease nental scale. Applied and Environmental Microbiology in API 20NE test. Trisodium citrate is utilized. Strain 75(15):5111-5120.

LPB0099 ( =NIBRBAC000497979) has been isolated Loman, N.J. and M.J. Pallen. 2015. Twenty years of bacte- from sea water sample, Ongjin, Incheon, Korea. rial genome sequencing. Nature Reviews Microbiology 13(12):787-794. Description of Kangiella japonica LPB0123 Pascault, N., S. Roux, J. Artigas, S. Pesce, J. Leloup, R.D. Cells are Gram-staining-negative, flagellated, non-pig- Tadonleke, D. Debroas, A. Bouchez and J.F. Humbert. mented and rod-shaped. Colonies are circular and light 2014. A high-throughput sequencing ecotoxicology yellow-colored after 1 days of incubation on MA at study of freshwater bacterial communities and their re- sponses to tebuconazole. FEMS Microbiology Ecology 26°C. Positive for cytochrome oxidase, esculin hy- 90(3):563-574. drolysis and β-galactosidase, but negative for arginine Saitou, N. and M. Nei. 1987. The neighbor-joining method: dihydrolase, gelatinase, glucose fermentation, indole a new method for reconstructing phylogenetic trees. Mo- production, nitrates reduction and urease in API 20NE lecular Biology and Evolution 4(4):406-425. test. Capric acid, D-glucose, malic acid, D-maltose, Shigematsu, T., M. Hayashi, I. Kikuchi, S. Ueno, H. Masaki D-mannitol, D-mannose, potassium gluconate and triso- and T. Fujii. 2009. A culture-dependent bacterial com- dium citrate are utilized. Strain LPB0123 (=NIBRBAC munity structure analysis based on liquid cultivation and 000497989) has been isolated from sea water sample, its application to a marine environment. FEMS Microbi- Haeundae, Busan, Korea. ology Letters 293(2):240-247. Shin, N.R., T.W. Whon and J.W. Bae. 2015. Proteobacte- ria: microbial signature of dysbiosis in gut microbiota. Acknowledgements Trends in Biotechnology 33(9):496-503. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei This work was supported by the National Research and S. Kumar. 2011. MEGA5: molecular evolutionary Foundation of Korea, funded by the Ministry of Edu- genetics analysis using maximum likelihood, evolution- cation of Korea and the National Institute of Biological ary distance, and maximum parsimony methods. Molec- Resources (NIBR no. 2013-02-001) funded by the Min- ular Biology and Evolution 28(10):2731-2739. istry of Environment of Korea. Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin and D.G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence align- References ment aided by quality analysis tools. Nucleic Acids Re- search 25(24):4876-4882. Kim, O.S., Y.J. Cho, K. Lee, S.H. Yoon, M. Kim, H. Na, S.C. Park, Y.S. Jeon, J.H. Lee, H. Yi, S. Won and J. Chun Submitted: September 2, 2016 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA Revised: October 6, 2016 gene sequence database with phylotypes that represent Accepted: June 14, 2017