A Report on 53 Unrecorded Bacteria Species in Korea in the Class Gammaproteobacteria

Home , Acinetobacter towneri, Agarivorans, Alteromonadales, Amphritea, Citrobacter, Citrobacter europaeus

Journal of Species Research 8(4):319-336, 2019

A report on 53 unrecorded bacteria species in Korea in the class Gammaproteobacteria

Rungravee Kanjanasuntree1, Chang-Jun Cha2, Jang-Cheon Cho3, Wan-Taek Im4, Myung Kyum Kim5, Che-Ok Jeon6, Kiseong Joh7, Seung-Bum Kim8, Chi-Nam Seong9, Hana Yi10, Soon Dong Lee11, Jin-Woo Bae12 and Wonyong Kim1,*

1Department of Microbiology, Chung-Ang University College of Medicine, Seoul 06974, Republic of Korea 2Department of Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 3Department of Biological Science, Inha University, Incheon 22212, Republic of Korea 4Department of Biotechnology, Hankyong National University, Anseong 17579, Republic of Korea 5Division of Chemistry and Bio-Environmental Sciences, Seoul Women’s University College of Natural Sciences, Seoul 01797, Republic of Korea 6Department of Life Sciences, 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 Narional University, Suncheon 57922, Republic of Korea 10School of Biosystem and Biomedical Science, Department of Public Health Science, Korea University, Seoul 02841, Republic of Korea 11Department of Science Education, Jeju National University, Jeju 63243, Republic of Korea 12Department of Biology, Kyung Hee University, Seoul 02447, Republic of Korea

*Correspondent: [email protected]

During an investigation of unrecorded prokaryotic species in Republic of Korea, a total of 53 bacterial strains belonging to the class Gammaproteobacteria were isolated from soil, seawater, tidal flats, rhizosphere, salt ponds, beach sand, urine, manure, sediment, and animal intestine (Russian grayling butterfly [Hipparchia autonoe], mouse [Mus musculus], and sea bass [Lateolabrax japonicus]). Strains were identified to species using the 16S rRNA gene sequence, showing high similarity (>98.7%) with the closest bacterial species and forming a robust clade in the neighbor-joining phylogenetic tree. The 53 strains of Gammaproteobacteria in this study have not been report previously in Korea. Therefore, we describe 27 genera of 16 families in 7 orders: 13 strains in the order Alteromonadales, 1 strain in the order Chromatiales, 11 strains in the order Enterobacterales, 7 strains in the order Oceanospirillales, 10 strains in the order Pseudomonadales, 8 strains in the order Vibrionales, and 3 strains in the order Xanthomonadales. Gram reaction, strain ID, isolation source, and morphological and basic biochemical characteristics are described for each species. Keywords: ‌16S rRNA, Gammaproteobacteria, unrecorded species

Introduction of the microbial community (Lauber et al., 2009; Whon et al., 2012; Pascault et al., 2014). The understanding of the bacterial phylogeny has rap- The phylum Proteobacteria constitutes the largest idly transformed over past decades. The introduction phylogenetic lineage (Kersters et al., 2006) that contains of small subunit ribosomal RNA gene (Woese and Fox, many of pathogenic bacteria. Gammaproteobacteria has 1977), followed by the development of next-generation the taxonomic rank of class within the phylum Proteo- sequencing techniques and bioinformatics expanded to bacteria that was first proposed by Garrity et al. (2005a) large-scale, cost-effective multiplex analyses, producing and ammended by Williams and Kelly (2013). Gram- new data to study the taxonomy and functional diversity maproteobacteria contains a large and diverse group of 320 Journal of Species Research Vol. 8, No. 4 bacteria that exhibits wide variation in term of pheno- out using a genomic DNA extraction kit (Intron). The types, morphologies, metabolic capabilities, and trophism 16S rRNA gene was amplified by PCR as described pre- (phototrophs and chemolithotrophs). Members of the viously using two universal primers, 8F (5ʹ-AGAGTTT- class Gammaproteobacteria are Gram-staining-nega- GATCCTTGGCTCAG-3ʹ) and 1525R (5ʹ-AAGGAGGT- tive with different morphologies including rods, cocci, GWTCCARCC-3ʹ) (Lane, 1991). The BigDye Terminator spirilla, and filaments, and isolated from wide range of Cycle Sequencing Kit (Applied Biosystems) and the 3730 environments. At the time of this writing, the class has automatic DNA sequencer (Applied Biosystems) were been divided into 20 orders: Acidithiobacillales (Kojima used to sequence the 16S rRNA gene amplicons. Multiple et al., 2015), Aeromonadales (Martin-Carnahan and Jo- sequence alignments were constructed using CLUST- seph, 2005), Alteromonadales (Bowman and McMeekin, AL-X (Thompson et al., 1997) and calculations of gene 2005), Arenicellales (Teramoto, 2005), Cardiobacteria- sequence similarity between each strain and most closely les (Garrity et al., 2005b), Cellvibrionales (Spring et al., related strains were performed by using EzTaxon-e - Ez- 2015), Chromatiales (Imhoff, 2005), Enterobacterales BioCloud.net (http://www.ezbiocloud.net/eztaxon) (Kim (Adeolu et al., 2016), Immundisolibacterales (Corteselli et al., 2012). A phylogenetic tree was constructed using et al., 2017), Legionellales (Garrity et al., 2005d), Meth- neighbor-joining (Saitou and Nei, 1987), maximum-likeli- ylococcales (Bowman, 2005), Nevskiales (Naushad et al., hood (Felsenstein, 1981), and maximum-parsimony (Fitch 2015), Oceanospirillales (Garrity et al., 2005e), Orbales and Margoliash, 1967), algorithms in MEGA7 program (Kwong and Moran, 2013), Pasteurellales (Garrity et al., (Kumar et al., 2016). Evolutionary distance matrices were 2005f), Pseudomonadales (Orla-Jensen, 1921), Salinis- generated by the neighbor-joining described by Jukes and phaerales (Skerman et al., 1980), Thiotrichales (Garrity et Cantor (1969). Branch support in the neighbor-joining al., 2005c), Vibrionales (Skerman et al., 1980), and Xan- tree was estimated by the bootstrap resampling method thomonadales (Saddler and Bradbury, 2005). (Felsenstein, 1985) with 1000 replicates. In 2017, we collected diverse environmental samples Colony morphology was observed for morphological from habitats in Korea and isolated novel and unrecorded features such as appearance, pigmentation, size, shape, bacteria species in Korea. The isolated bacteria species and texture on agar plates incubated at optimal conditions. belong to the class Actinobacteria, Alphaproteobacteria, Cell morphology was examined under JEM 1010 trans- Bacteroidetes, Deinococci, Deltaproteobacteria, Epsi- mission electron microscope (JEOL) using cells grown lonproteobacteria, Firmicutes, Fusobacteria, and Gam- in the exponential phase. Gram staining was performed maproteobacteria. In this study, we focused on the de- using Gram staining kit (bioMérieux) according to the scription of 53 unreported strains belonging to 16 families manufacturer̓s instructions. Biochemical properties and of 7 orders in the class Gammaproteobacteria. enzyme activities were determined for each strain using API 20NE (except for one strain that was determined by using API 20A) according to the manufacturer̓s instruc-

Materials and Methods tions (bioMérieux) and read after incubation for 48 hours.

The strains were isolated from samples collected from soil, seawater, tidal flats, rhizosphere, salt ponds, beach Results and Discussion sand, urine, manure, sediment, and animal intestine (Rus- sian grayling butterfly [H. autonoe], mouse [M. muscu- On the basis of 16S rRNA sequence comparison and lus], and sea bass [Lateolabrax japonicus]). Each sample phylogenetic analysis, a total of 53 strains were assigned was separately homogenized and suspended in the appro- to the class Gammaproteobacteria and were classified priate solution based on its source. The suspensions were into 16 families of 7 orders: 13 species in 6 genera of 6 serially diluted and an aliquot (100 μL) of each sample families within the order Alteromonadales, 1 species in was placed on various culture media including marine genus Wenzhouxiangella of family Wenzhouxiangellaceae agar 2216 (MA), trypticase soy agar (TSA), Luria-Bertani within the order Chromatiales, 11 species in 7 genera of (LB), MacConkey (MAC), nutrient agar (NA), brain heart 3 families within the order Enterobacterales, 7 species in infusion (BHI), yeast malt agar (ISP medium No.2), R2A, 5 genera of 2 families within the order Oceanospirillales, R5A, R8A, R10A, R12A, R15A, and R17A. The plates 10 species in 2 genera of 2 families within the order Pseu- were incubated at 25-37℃ for 2-5 days (Table 1). All domonadales, 8 species in 3 genera of family Vibrionace- strains were purified by subculturing a single colony on ae within the order Vibrionales, and 3 species in 3 genera fresh media and pure cultures were stored in optimal me- of family Xanthomonadaceae within the order Xanthomo- dia supplemented with 25% glycerol (v/v) at -80℃ as nadales. All strains were Gram-staining-negative, chemo- lyophilized ampules. heterotrophic, and rod-shaped, except for 10 strains that Extraction of genomic DNA of each strain was carried were coccoid or ovoid-shaped (Figs. 1 and 2). Details of November 2019 Kanjanasuntree et al. Unrecorded species of Gammaproteobacteria 321 30℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 30℃, 3d 25℃, 3d 30℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 30℃, 3d 30℃, 2d 30℃, 2d 25℃, 3d 30℃, 3d 30℃, 3d 37℃, 2d 37℃, 2d 37℃, 2d 37℃, 2d 25℃, 3d 37℃, 2d 20℃, 2d 37℃, 2d 30℃, 5d 37℃, 2d 30℃, 3d 30℃, 3d 37℃, 2d 25℃, 3d condition Incubation Medium MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA R12A MA R10A MA MA R2A R2A MA R2A R2A TSA TSA R2A MacConkey LB MacConkey TSA TSA ISP 2, pH5 ISP BHI R2A R2A MacConkey R5A Soil Soil Soil Soil Soil U rine Mouse Manure Manure Sea sand Tidal flat Tidal flat Tidal Tidal flat Tidal Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Salt-pond Salt-pond Rhizosphere Isolation source Russian grayling butterfly Russian grayling butterfly Russian grayling butterfly Russian grayling butterfly Russian grayling butterfly Russian grayling butterfly Russian grayling butterfly Russian grayling butterfly 99 100 100 100 (%) 99.5 98.9 99.3 99.7 99.7 98.9 98.8 99.3 99.6 98.9 99.9 99.9 99.7 98.9 99.9 99.9 99.6 99.3 99.6 99.6 99.72 99.72 99.79 99.93 99.71 99.92 99.15 99.79 99.93 99.28 99.72 99.72 99.42 98.85 99.78 99.93 Similarity

T T T T T T T T T T T T T T T T T T T T T T T pneumoniae T T T T T marcescens T T T T T T T T NT U -104 T T 908033 subsp. HRKA1 DSM 14971 E B4 HAW- CIP 110307 CIP subsp. ATCC BAA-803 ATCC LMG 2864 97/99 KCTC 2190 HT073016 63.46 CIP XDB06 CIP 105588 CIP KCTC 12086 T CIP 64.6 CIP KMM 300 T DSM 15968 CGMCC 1.8499 DSM 18887 JCM 605 JAMM 1866 U ST010723-006 DSM 7027 JAMM 1525 DSM 16037 CDC 2991-81 DSM 14962 NIPH 2168 CIP 110367 CIP fav-2-10-05 LMG 5345 CBMAI 722 OS145 M1-18 WH0801 KMM 3582 ATCC 11775 ATCC Most closely related species DSM 30104 M. szutsaonensis I. baltica R. variigena S. corallii sediminis W. C. europaeus A. atlantica A. beijerinckii A. gilvus I. donghaiensis phenolica P. ruthenica P. spongiae P. S. fidelis S. halifaxensis S. schlegeliana C. farmeri E. aerogenes E. asburiae E. coli E. marmotae K. pneumoniae K. variicola S. marcescens L. salina A. balenae A. japonica M. stanieri M. communis A. guillouiae A. indicus A. johnsonii A. tjernbergiae A. ursingii A. vivianii P. lipolytica P. A. towneri P. septica P. T. loyana T. H. sulfidaeris ATCC 1388 ATCC NIBR ID NIBRBAC000501021 NIBRBAC000501173 NIBRBAC000501016 NIBRBAC000501193 NIBRBAC000501234 NIBRBAC000501023 NIBRBAC000501009 NIBRBAC000501012 NIBRBAC000501014 NIBRBAC000501172 NIBRBAC000501139 NIBRBAC000501140 NIBRBAC000501141 NIBRBAC000501142 NIBRBAC000501300 NIBRBAC000501143 NIBRBAC000501144 NIBRBAC000501145 NIBRBAC000501174 NIBRBAC000501017 NIBRBAC000501013 NIBRBAC000501019 NIBRBAC000501129 NIBRBAC000501074 NIBRBAC000501072 NIBRBAC000501010 NIBRBAC000501071 NIBRBAC000501073 NIBRBAC000501087 NIBRBAC000501198 NIBRBAC000501097 NIBRBAC000501126 NIBRBAC000501098 NIBRBAC000501076 NIBRBAC000501075 NIBRBAC000501137 NIBRBAC000501127 NIBRBAC000501128 NIBRBAC000501130 NIBRBAC000501043 and their taxonomic affiliations. Strain ID LPB0300 HMF8561 LPB0188 MMS17-GJ020 CA U 1471 LPB0308 LPB0180 LPB0184 LPB0186 HMF8517 BT3507 BT3301 BR3501 BC3307 LPB0191 BC3202 BT2501 BT3409 HMF9021 LPB0189 LPB0185 LPB0208 KYW1404 16_S3_M11 16_H2_V4 LPB0181 16_H3_M12 16_H2_M14 IMCC34138 LPB0182 IMCC34178 KYW1326 IMCC34174 16_H6_M1 16_H5_M7 BC3402 KYW1346 KYW1359 KYW1415 GH3-6 Gammaproteobacteria Genus Marinobacter Idiomarina Idiomarina Rahnella Serratia Shewanella Shewanella Shewanella Shewanella Wenzhouxiangella Citrobacter Citrobacter Enterobacter Enterobacter Escherichia Escherichia Klebsiella Klebsiella Amphritea Amphritea Amphritea Marinobacterium Marinomonas Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Agarivorans Pseudoalteromonas Pseudoalteromonas Pseudoalteromonas Pseudoalteromonas Acinetobacter Acinetobacter Acinetobacter Pantoea Thalassotalea Halomonas Larsenimonas Family Marinobacter Idiomarinaceae Yersiniaceae Wenzhouxiangellaceae Enterobacteriaceae Moraxellaceae Shewanellaceae Oceanospirillaceae Agarivorans Pseudoalteromonadaceae Erwiniaceae Halomonadaceae Colwelliaceae Lists of isolated strain belonging to the class Order Chromatiales Enterobacterales Pseudomonadales Alteromonadales Table 1. Table Oceanospirillales 322 Journal of Species Research Vol. 8, No. 4

colony morphology and physiology are reported in the species description section. 25℃, 5d 30℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 3d 25℃, 5d 25℃, 5d 30℃, 2d 37℃, 2d 25℃, 4d 30℃, 3d 30℃, 1d condition Incubation Comparison of 16S rRNA gene sequences showed high similarities (>98.6%) with the closest related strains. Thirteen strains of the order Alteromonadales

Medium (IMCC34138, KYW1359, HMF8561, LPB0188, R2A MA MA R15A MA R17A R2A R8A R2A MacConkey R2A NA R2A LPB0300, LPB0182, IMCC34178, KYW1326, IMCC34174, LPB0308, LPB0180, LPB0184 and LPB0186), one strain of the order Chromatiales (HMF8517), and eleven strains of the order Enterobac- Soil Soil Soil terales (BT3507, BT3301, BR3501, BC3307, LPB0191, Sea bass Tidal flat Tidal flat Tidal Seawater Seawater Seawater Seawater Sediment Beach sand

Isolation source BC3202, BT2501, BT3409, KYW1346, MMS17-GJ020, and CAU 1471) had the highest similarities to Agarivorans Russian grayling butterfly gilvus WH0801T (CP013021; 99.00%), Thalassotalea loyana CBMAI 722T (AY643537; 99.50%), Idiomarina balti- 100 100 100 (%) T 99.4 99.5 99.3 99.9 99.1 99.1 99.65 99.92 99.33 99.71 ca OS145 (AJ440214; 98.90%), Idiomarina donghaiensis Similarity 908033T (EU600204; 99.72%), Marinobacter szutsaonen- T sis NTU-104T (EU164778; 99.772%), Pseudoalteromonas T

T T lipolytica CGMCC 1.8499 (FJ40472; 100%), Pseudoal- T T T T

T teromonas phenolica KCTC 12086 (CP013187; 99.30%) T T T T T T piscicida Pseudoalteromonas ruthenica KMM 300 (AF316891; J36 JCM 13310 WZBFD3-5A2 T

LMG 19158 99.70%), Pseudoalteromonas spongiae UST010723-006 T XM415 DSM 14398 LMG 13240 subsp. GHG21 JCM 16456 (AHCE02000026; 99.70%), Shewanella corallii fav-2-10- JCM 18803 DSM 18327 DSM 19584 05T (FJ041083; 98.90%), Shewanella fidelis KMM 3582T

Most closely related species (AF420312; 99.79%), Shewanella halifaxensis HAW- T L. ximonensis P. mohnii P. P. nitritireducens P. damselae P. comitans V. hispanicus V. nereis V. penaeicida V. scophthalmi V. tritonius V. kaohsiungensis P. S. acidaminiphila NCIMB 2058 A. fischeri EB4 (CP000931; 99.93%), Shewanella schlegeliana HR- KA1T (AB081760; 99.71%), Wenzhouxiangella sediminis XDB06T (KU645983; 98.80%), Citrobacter europaeus 97/99T (FLYB01000015; 99.92%), Citrobacter farmeri CDC 2991-81T (AF025371; 99.15%), Enterobacter aero- NIBR ID genes KCTC 2190T (CP002824; 99.79%), Enterobacter T NIBRBAC000501324 NIBRBAC000501210 NIBRBAC000501080 NIBRBAC000500996 NIBRBAC000501147 NIBRBAC000501090 NIBRBAC000501008 NIBRBAC000501301 NIBRBAC000501102 NIBRBAC000501018 NIBRBAC000501103 NIBRBAC000501104 NIBRBAC000501105 asburiae JCM 6051 (BBED01000197; 99.30%), Esch- and their taxonomic affiliations. erichia coli ATCC 11775T (X80725; 99.93%), Esche- richia marmotae HT073016T (JNBP01000188; 99.28%), Klebsiella pneumoniae subsp. pneumoniae DSM 30104T Strain ID (AJJI01000018; 99.72), Klebsiella variicola DSM 17J9-2 MMS17-SY254 16H1 E 7 Gsoil 1532 BC3406 IMCC34228 LPB0174 LPB0190 IMCC34186 LPB0202 IMCC34213 IMCC34225 IMCC34148 15968T (CP010523; 99.72), Pantoea septica LMG 5345T (MLJJ01000077; 99.60%), Rahnella variigena CIP T Gammaproteobacteria 105588 (GQ148969; 98.90%), Serratia marcescens subsp. marcescens ATCC 13880T (JMPQ01000005; Genus 99.90%). Eight strains of the order Oceanospirillales Lysobacter Pseudoxanthomonas Stenotrophomonas Pseudomonas Pseudomonas Aliivibrio Photobacterium Vibrio Vibrio Vibrio Vibrio Vibrio Vibrio (KYW1415, GH3-6, HMF9021, LPB0189, LPB0185, LPB0208, and KYW1404), 10 strains of the order Pseu- domonadales (16_S3_M11, 16_H2_V4, LPB0181, 16_H3_M12, 16_H2_M14, 16_H6_M1, 16_H5_M7, BC3402, Gsoil 1532, and BC3406), 8 strains of the or- Family der Vibrionales (IMCC34228, LPB0174, LPB0190, IMCC34186, LPB0202, IMCC34213, IMCC34225, Xanthomonadaceae Pseudomonadaceae Vibrionaceae and IMCC34148), and 3 strains of the order Xanthomo- nadales (17J9-2, MMS17-SY254, and 16H1E7), had the highest similarities to Halomonas sulfidaeris ATCC Continued. Lists of isolated strain belonging to the class BAA-803T (AF212204; 100%), Larsenimonas salina Order M1-18T (HF678441; 99.90%), Amphritea atlantica DSM T Table 1. Table Xanthomonadales Pseudomonadales Table 1. Table Vibrionales 18887 (FOGB01000032; 99.70%), Amphritea balenae November 2019 Kanjanasuntree et al. Unrecorded species of Gammaproteobacteria 323

Fig. 1. Transmission electron micrographs of cells of the species in the order Alteromonadales, Chromatiales and Enterobacterales belonging to the class Gammaproteobacteria in this study. Strain: 1, IMCC34138; 2, KYW1359; 3, HMF8561; 4, LPB0188; 5, LPB0300; 6, LPB0182; 7, IMCC34178; 8, KYW1326; 9, IMCC34174; 10, LPB0308; 11, LPB0180; 12, LPB0184; 13, LPB0186; 14, HMF8517; 15, BT3507; 16, BT3301; 17, BR3501; 18, BC3307; 19, LPB0191; 20, BC3202; 21, BT2501; 22, BT3409; 23, KYW1346; 24, MMS17-GJ020; 25, CAU 1471.

JAMM 1525T (AB330883; 100%), Amphritea japonica 01000028; 99.90%), Acinetobacter indicus CIP 110367T JAMM 1866T (AB330881; 99.42%), Marinobacterium (KI530754; 99.78%), Acinetobacter johnsonii CIP 64.6T stanieri DSM 7027T (AB021367; 98.85%), Marino- (APON01000005; 99.60%), Acinetobacter tjernbergiae monas communis LMG 2864T (DQ011528; 98.90%), DSM 14971T (ARFU01000016; 99.30%), Acinetobacter Acinetobacter beijerinckii CIP 110307T (APQL01000005; towneri DSM 14962T (APPY01000064; 99.60%), Acine- 99.90%), Acinetobacter guillouiae CIP 63.46T (APOS tobacter ursingii DSM 16037T (AIEA01000080; 99.60%), 324 Journal of Species Research Vol. 8, No. 4

Fig. 2. Transmission electron micrographs of cells of the species in the order Oceanospirillales, Pseudomonadales, Vibrionales and Xanthomo- nadales belonging to the class Gammaproteobacteria in this study. Strain: 1, KYW1415; 2, GH3-6; 3, HMF9021; 4, LPB0189; 5, LPB0185; 6, LPB0208; 7, KYW1404; 8, 16_S3_M11; 9, 16_H2_V4; 10, LPB0181; 11, 16_H3_M12; 12, 16_H2_M14; 13, 16_H6_M1; 14, 16_H5_M7; 15, BC3402; 16, Gsoli 1532; 17, BC3406; 18, IMCC34228; 19, LPB0174; 20, LPB0190; 21, IMCC34186; 22, LPB0202; 23, IMCC34213; 24, IMCC34225; 25, IMCC34148; 26, 17J9-2; 27, MMS17-SY254; 28, 16H1E7. November 2019 Kanjanasuntree et al. Unrecorded species of Gammaproteobacteria 325

Acinetobacter vivianii NIPH 2168T (KB850133; 99.93%), Strain is positive for esculin hydrolysis, β-galactosidase, Pseudomonas mohnii DSM 18327T (FNRV01000001; and cytochrome oxidase; but negative for nitrate reduc- 99.40%), Pseudomonas nitritireducens WZBFD3-5A2T tion, glucose fermentation, indole production, arginine (HM246143; 99.65%), Aliivibrio fischeri JCM 18803T dihydrolase, urease, gelatinase, and utilization of glucose, (BBEE01000115; 100%), Photobacterium damselae sub- arabinose, mannose, mannitol, N-acetyl-glucosamine, sp. piscicida NCIMB 2058T (X78105; 99.92%), Vibrio maltose, potassium gluconate, malic acid, capric acid, comitans GHG21T (DQ922915; 99.33%), Vibrio hispan- adipic acid, trisodium citrate, and phenylacetic acid. icus LMG 13240T (AY254039; 99.50%), Vibrio nereis Strain KYW1359 (=NIBRBAC000501128) was isolated DSM 19584T (LHPJ01000025; 99.71%), Vibrio penae- from seawater from Gwangyang, Jeollanam-do, Republic icida DSM 14398T (AJ421444; 99.30%), Vibrio scoph- of Korea. thalmi LMG 19158T (AFWE01000105; 99.90%), Vibrio tritonius JCM 16456T (AP014635; 99.10%), Lysobacter Idiomarina baltica HMF8561 ximonensis XM415T (EU237492; 100%), Pseudoxan- thomonas kaohsiungensis J36T (AY650027; 99.10%) Cells are Gram-staining-negative, flagellated, and rod and Stenotrophomonas acidaminiphila JCM 13310T shaped. Colonies are beige color, circular, convex, and (LDJO01000053; 100%). smooth after incubation on MA at 30℃ for 3 days. Strain Phylogenetic analyses showed that isolated strains is positive for esculin hydrolysis and cytochrome oxidase; formed a robust clade with the most closely related spe- but negative for nitrate reduction, indole production, ar- cies in the orders Alteromonadales, Chromatiales, Entero- ginine dihydrolase, urease, gelatinase, β-galactosidase, bacterales (Fig. 3), Oceanospirillales, Pseudomonadales, glucose fermentation, and utilization of glucose, arabi- N- Vibrionales, and Xanthomonadales (Fig. 4). There are nose, mannose, mannitol, acetyl-glucosamine, maltose, potassium gluconate, malic acid, trisodium citrate, capric no official report of these 53 strains in Korea. Therefore, Gammaproteobacteria acid, adipic acid, and phenylacetic acid. Strain HMF8561 these 53 strains in the class are = new records to Korea: 13 species in the order Altero- ( NIBRBAC000501173) was isolated from a salt pond monadales, 1 species in the order Chromatiales, 11 spe- from Sinan-gun, Jeollanam-do, Republic of Korea. cies in the order Enterobacterales, 7 species in the order Oceanospirillales, 10 species in the order Pseudomonad- Idiomarina donghaiensis LPB0188 ales, 8 species in the order Vibrionales, and 3 species in Cells are Gram-staining-negative, flagellated, and rod the order Xanthomonadales. shaped. Colonies are cream color, circular, convex, and smooth after incubation on MA at 25℃ for 3 days under aerobic conditions. Strain is positive for gelatinase (weak) Species Descriptions and cytochrome oxidase; but negative for nitrate reduction, indole production, glucose fermentation, arginine Agarivorans gilvus IMCC34138 dihydrolase, esculin hydrolysis, β-galactosidase, urease, Cells are Gram-staining-negative, non-flagellated, and and utilization of glucose, arabinose, mannose, manni- rod shaped. Colonies are cream to yellow color, circular, tol, N-acetyl-glucosamine, maltose, potassium gluconate, convex, and entire margin after incubation on R2A at adipic acid, malic acid, capric acid, trisodium citrate, and 25℃ for 3 days under aerobic conditions. Strain is pos- phenylacetic acid in API 20NE. Strain LPB0188 (=NI- itive for esculin hydrolysis, gelatinase, cytochrome oxi- BRBAC000501016) was isolated from seawater from dase, and utilization of glucose, mannitol, maltose, malic Munseom, Jeju-do, Republic of Korea. acid, and trisodium citrate; but negative for nitrate reduction, indole production, glucose fermentation, arginine Marinobacter szutsaonensis LPB0300 dihydrolase, urease, β-galactosidase, and utilization of Cells are Gram-staining-negative, non-flagellated, and arabinose, mannose, N-acetyl-glucosamine, adipic acid, rod shaped. Colonies are cream color, circular, convex, potassium gluconate, capric acid, and phenylacetic acid in and smooth after incubation on MA at 25℃ for 3 days API 20NE. Strain IMCC34138 (=NIBRBAC000501087) under aerobic conditions. Strain is positive for nitrate was isolated from tidal flat from Jangbong-do, Incheon, reduction; but negative for indole production, glucose Republic of Korea. fermentation, arginine dihydrolase, esculin hydrolysis, β-galactosidase, gelatinase, urease, cytochrome oxidase, Thalassotalea loyana KYW1359 and utilization of glucose, arabinose, mannose, manni- Cells are Gram-staining-negative, non-flagellated, and tol, N-acetyl-glucosamine, maltose, potassium gluconate, rod shaped. Colonies are cream color, circular, convex, adipic acid, malic acid, capric acid, trisodium citrate, and and smooth after incubation on MA at 25℃ for 3 days. phenylacetic acid in API 20NE. Strain LPB0300 (=NI- 326 Journal of Species Research Vol. 8, No. 4

Fig. 3. Neighbor-joining phylogenetic tree (NJ) based on nearly complete 16S rRNA gene sequences showing the relationships between 25 isolated strains and their most closely related from the order Alteromonadales, Chromatiales and Enterobacterales of the class Gammaproteo- bacteria. Dots indicate that the corresponding nodes that were also recovered in the trees created with the maximum likelihood (ML) and maximum parsimony (MP) algorithms. Bootstrap values are indicated as percentages of 1000 resampled datasets, when greater than 70% (NJ/ML/ MP). Bar, 0.01 substitutions per nucleotide position. Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001) was used as the outgroup. November 2019 Kanjanasuntree et al. Unrecorded species of Gammaproteobacteria 327

Fig. 4. Neighbor-joining phylogenetic tree (NJ) based on nearly complete 16S rRNA gene sequences showing the relationships between 28 isolated strains and their most closely related from the order Oceanospirillales, Pseudomonadales, Vibrionales and Xanthomonadales of the class Gammaproteobacteria. Dots indicate that the corresponding nodes that were also recovered in the trees created with the maximum likelihood (ML) and maximum parsimony (MP) algorithms. Bootstrap values are indicated as percentages of 1000 resampled datasets, when greater than 70% (NJ/ML/MP). Bar, 0.01 substitutions per nucleotide position. Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001) was used as the outgroup. 328 Journal of Species Research Vol. 8, No. 4

BRBAC000501021) was isolated from seawater from raised, and entire margin after incubation on R2A at 25℃ Incheon, Ganghwa-do, Republic of Korea. for 3 days under aerobic conditions. Strain is positive for esculin hydrolysis, gelatinase, and cytochrome oxidase; Pseudoalteromonas lipolytica LPB0182 but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, β-galac- Cells are Gram-staining-negative, non-flagellated, and tosidase, and utilization of glucose, arabinose, mannose, rod shaped. Colonies are cream color, circular, convex, mannitol, N-acetyl-glucosamine, maltose, potassium and smooth after incubation on MA at 25℃ for 3 days gluconate, adipic acid, capric acid, malic acid, trisodi- under aerobic conditions. Strain is positive for nitrate re- um citrate, and phenylacetic acid in API 20NE. Strain duction, indole production, glucose fermentation, esculin IMCC34174 ( =NIBRBAC000501098) was isolated hydrolysis, gelatinase, β-galactosidase, cytochrome ox- from tidal flat from Jangbong-do, Incheon, Republic of idase, and utilization of arabinose and maltose; but neg- Korea. ative for arginine dihydrolase, urease, and utilization of glucose, mannose, mannitol, N-acetyl-glucosamine, po- Shewanella corallii LPB0308 tassium gluconate, adipic acid, malic acid, capric acid, trisodium citrate, and phenylacetic acid in API 20NE. Strain Cells are Gram-staining-negative, flagellated, and rod LPB0182 ( =NIBRBAC000501198) was isolated from shaped. Colonies are orange color, circular, convex, and seawater from Munseom, Jeju-do, Republic of Korea. smooth after incubation on MA at 25℃ for 3 days under aerobic conditions. Strain is positive for nitrate reduction,

Pseudoalteromonas phenolica IMCC34178 arginine dihydrolase (weak), esculin hydrolysis (weak), and utilization of N-acetyl-glucosamine and maltose; but Cells are Gram-staining-negative, flagellated, and cocci negative for indole production, glucose fermentation, shaped. Colonies are beige to brown color, circular, con- β-galactosidase, gelatinase, urease, cytochrome oxidase, vex, and entire margin after incubation on R2A at 25℃ and utilization of glucose, arabinose, mannose, mannitol, for 3 days under aerobic conditions. Strain is positive for potassium gluconate, adipic acid, malic acid, capric acid, esculin hydrolysis, gelatinase, and cytochrome oxidase; trisodium citrate, and phenylacetic acid in API 20NE. but negative for nitrate reduction, indole production, glu- Strain LPB0308 (=NIBRBAC000501023) was isolated cose fermentation, arginine dihydrolase, urease, β-galac- from seawater from Incheon, Ganghwa-do, Republic of tosidase, and utilization of glucose, arabinose, mannose, Korea. mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, adipic acid, capric acid, malic acid, trisodi- Shewanella fidelis LPB0180 um citrate, and phenylacetic acid in API 20NE. Strain IMCC34178 (=NIBRBAC000501097) was isolated from Cells are Gram-staining-negative, non-flagellated, and tidal flat from Jangbong-do, Incheon, Republic of Korea. rod shaped. Colonies are ivory color, circular, convex, and smooth after incubation on MA at 25℃ for 3 days under Pseudoalteromonas ruthenica KYW1326 aerobic conditions. Strain is positive for nitrate reduction, esculin hydrolysis (weak), gelatinase, and cytochrome Cells are Gram-staining-negative, flagellated, and oxidase; but negative for glucose fermentation, indole rod shaped. Colonies are cream color, circular, flat, and production, arginine dihydrolase, urease, β-galactosidase, smooth after incubation on MA at 25℃ for 3 days. Strain and utilization of glucose, arabinose, mannose, manni- is positive for nitrate reduction, glucose fermentation, tol, N-acetyl-glucosamine, maltose, potassium gluconate, esculin hydrolysis, gelatinase, β-galactosidase, cyto- malic acid, capric acid, adipic acid, trisodium citrate, and chrome oxidase, and utilization of glucose, arabinose, phenylacetic acid in API 20NE. Strain LPB0180 (=NI- mannose, N-acetyl-glucosamine, maltose, potassium glu- BRBAC000501009) was isolated from seawater from conate, and malic acid; but negative for indole production, Munseom, Jeju-do, Republic of Korea. arginine dihydrolase, urease, and utilization of mannitol, capric acid, adipic acid, trisodium citrate, and pheny- Shewanella halifaxensis LPB0184 lacetic acid. Strain KYW1326 (=NIBRBAC000501126) was isolated from seawater from Gwangyang, Jeolla- Cells are Gram-staining-negative, flagellated, and rod nam-do, Republic of Korea. shaped. Colonies are cream color, circular, convex, and smooth after incubation on MA at 25℃ for 3 days under aerobic conditions. Strain is positive for nitrate reduc- Pseudoalteromonas spongiae IMCC34174 tion, esculin hydrolysis, gelatinase, β-galactosidase, and Cells are Gram-staining-negative, non-flagellated, and cytochrome oxidase; but negative for indole production, rod shaped. Colonies are slightly orange color, circular, glucose fermentation, arginine dihydrolase, urease, and November 2019 Kanjanasuntree et al. Unrecorded species of Gammaproteobacteria 329 utilization of glucose, arabinose, mannose, mannitol, Citrobacter farmeri BT3301 N-acetyl-glucosamine, maltose, potassium gluconate, Cells are Gram-staining-negative, flagellated, and adipic acid, malic acid, capric acid, trisodium citrate, and short-rod shaped. Colonies are beige color, circular, and phenylacetic acid in API 20NE. Strain LPB0184 (=NI- convex after incubation on TSA at 37℃ for 2 days. Strain BRBAC000501012) was isolated from seawater from is positive for nitrate reduction, indole production, glu- Munseom, Jeju-do, Republic of Korea. cose fermentation, arginine dihydrolase, esculin hydrolysis, β-galactosidase, and utilization of glucose, arabinose, Shewanella schlegeliana LPB0186 mannose, mannitol, N-acetyl-glucosamine, maltose, po- Cells are Gram-staining-negative, non-flagellated, and tassium gluconate, malic acid, and trisodium citrate; but rod shaped. Colonies are cream color, circular, convex, negative for gelatinase, urease, cytochrome oxidase, and and smooth after incubation on MA at 25℃ for 3 days utilization of capric acid, adipic acid, and phenylacetic under aerobic conditions. Strain is positive for nitrate re- acid. Strain BC3301 (=NIBRBAC000501140) was iso- duction, esculin hydrolysis (weak), gelatinase, and cyto- lated from intestines of Russian grayling butterfly (H. au- chrome oxidase; but negative for indole production, glu- tonoe) from Jeju, Jeju-do, Republic of Korea. cose fermentation, arginine dihydrolase, β-galactosidase, urease, and utilization of glucose, arabinose, mannose, Enterobacter aerogenes BR3501 mannitol, N-acetyl-glucosamine, maltose, potassium glu- Cells are Gram-staining-negative, flagellated, and coc- conate, adipic acid, malic acid, capric acid, trisodium ci- ci or ovoid shaped. Colonies are white color, circular, trate, and phenylacetic acid in API 20NE. Strain LPB0186 and convex after incubation on R2A at 37℃ for 2 days. = ( NIBRBAC000501014) was isolated from seawater Strain is positive for nitrate reduction, glucose fermenta- from Munseom, Jeju-do, Republic of Korea. tion, esculin hydrolysis, β-galactosidase, and utilization of glucose, arabinose, mannose, mannitol, N-acetyl-glu- Wenzhouxiangella sediminis HMF8517 cosamine, maltose, potassium gluconate, malic acid, tri- Cells are Gram-staining-negative, non-flagellated, and sodium citrate, capric acid, adipic acid, and phenylacetic rod shaped. Colonies are yellow color, circular, convex, acid; but negative for indole production, arginine dihydro- and smooth after incubation on MA at 30℃ for 3 days. lase, gelatinase, urease, and cytochrome oxidase. Strain BC3501 ( =NIBRBAC000501141) was isolated from Strain is positive for esculin hydrolysis, gelatinase, and cytochrome oxidase; but negative for nitrate reduction, intestines of Russian grayling butterfly (H. autonoe) from indole production, arginine dihydrolase, urease, β-galac- Jeju, Jeju-do, Republic of Korea. tosidase, glucose fermentation, and utilization of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, Enterobacter asburiae BC3307 maltose, potassium gluconate, malic acid, trisodium ci- Cells are Gram-staining-negative, flagellated, and rod trate, capric acid, adipic acid, and phenylacetic acid. shaped. Colonies are pink color, circular and convex after

Strain HMF8517 (=NIBRBAC000501172) was isolated incubation on MacConkey at 37℃ for 2 days. Strain is from a salt pond from Sinan-gun, Jeollanam-do, Republic positive for nitrate reduction, indole production, arginine of Korea. dihydrolase, glucose fermentation, esculin hydrolysis, gelatinase, β-galactosidase, and utilization of glucose, Citrobacter europaeus BT3507 arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, adipic acid, malic acid, Cells are Gram-staining-negative, flagellated, and rod trisodium citrate, and phenylacetic acid; but negative for shaped. Colonies are white color, circular, and convex cytochrome oxidase, and utilization of capric acid in API after incubation on TSA at 37℃ for 2 days. Strain is pos- 20NE. Strain BC3307 (=NIBRBAC000501142) was iso- itive for nitrate reduction, glucose fermentation, arginine lated from intestine of Russian grayling butterfly (H. au- dihydrolase, β-galactosidase, and utilization of glucose, tonoe) from Jeju, Jeju-do, Republic of Korea. arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, malic acid and trisodium Escherichia coli LPB0191 citrate; but negative for indole production, esculin hydrolysis, gelatinase, urease, cytochrome oxidase, and utili- Cells are Gram-staining-negative, flagellated, and rod zation of capric acid, adipic acid, and phenylacetic acid. shaped. Colonies are cream color, circular, convex, and Strain BC3507 ( =NIBRBAC000501139) was isolated smooth after incubation on LB at 25℃ for 3 days under from intestines of Russian grayling butterfly (H. autonoe) aerobic condition. Strain is positive for nitrate reduction, from Jeju, Jeju-do, Republic of Korea. indole production, glucose fermentation, β-galactosidase, and utilization of glucose, arabinose, mannose, mannitol, 330 Journal of Species Research Vol. 8, No. 4

N-acetyl-glucosamine, potassium gluconate, and maltose; from Jeju, Jeju-do, Republic of Korea. but negative for arginine dihydrolase, esculin hydrolysis, gelatinase, urease, cytochrome oxidase, and utilization of Pantoea septica KYW1346 adipic acid, malic acid, capric acid, trisodium citrate, and Cells are Gram-staining-negative, non-flagellated, and phenylacetic acid in API 20NE. Strain LPB0191 (=NI- rod shaped. Colonies are yellow color, circular, convex, BRBAC000501300) was isolated from intestine of mouse and smooth after incubation on MA at 25℃ for 3 days. (M. musculus) from Seongbuk-gu, Seoul, Republic of Strain is positive for nitrate reduction, glucose fermenta- Korea. tion, esculin hydrolysis, β-galactosidase, and utilization of glucose, arabinose, mannose, mannitol, N-acetyl-glu- Escherichia marmotae BC3202 cosamine, maltose, potassium gluconate, malic acid, and Cells are Gram-staining-negative, non-flagellated, and trisodium citrate; but negative for indole production, argi- rod shaped. Colonies are magenta color, circular, and con- nine dihydrolase, urease, gelatinase, cytochrome oxidase, vex after incubation on MacConkey at 37℃ for 2 days. and utilization of capric acid, adipic acid, and pheny-

Strain is positive for nitrate reduction, indole production, lacetic acid. Strain KYW1346 (=NIBRBAC000501127) arginine dihydrolase, glucose fermentation, esculin hy- was isolated from seawater from Gwangyang, Jeolla- drolysis, β-galactosidase, and utilization of glucose, arabi- nam-do, Republic of Korea. nose, mannose, mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, malic acid, and phenylacetic acid; Rahnella variigena MMS17-GJ020 but negative for gelatinase, urease, cytochrome oxidase, Cells are Gram-staining-negative, flagellated, and rod and utilization of capric acid, adipic acid, and trisodium shaped. Colonies are pale yellow color, circular, convex, = citrate. Strain BC3202 ( NIBRBAC000501143) was smooth, and glistening after incubation on ISP2 at pH5 at isolated from intestines of Russian grayling butterfly (H. 30℃ for 5 days under aerobic conditions. Strain is pos- autonoe) from Jeju, Jeju-do, Republic of Korea. itive for nitrate reduction, glucose fermentation, esculin hydrolysis, β-galactosidase, and utilization of glucose, Klebsiella pneumoniae subsp. pneumoniae BT2501 mannose, mannitol, N-acetyl-glucosamine, maltose, po- Cells are Gram-staining-negative, non-flagellated, and tassium gluconate, malic acid, and trisodium citrate; but cocci or ovoid shaped. Colonies are white color, circular, negative for indole production, arginine dihydrolase, and convex after incubation on TSA at 20℃ for 2 days. urease, gelatinase, cytochrome oxidase, and utiliza- Strain is positive for nitrate reduction, glucose fermen- tion of arabinose, capric acid, adipic acid, and pheny- = tation, esculin hydrolysis, urease, β-galactosidase, and lacetic acid in API 20NE. Strain MMS17-GJ020 ( NI- utilization of glucose, arabinose, mannose, mannitol, BRBAC000501193) was isolated from soil from Daejeon, N-acetyl-glucosamine, maltose, potassium gluconate, Republic of Korea. malic acid, trisodium citrate, capric acid, adipic acid, and phenylacetic acid; but negative for indole production, ar- Serratia marcescens subsp. marcescens CAU 1471 ginine dihydrolase, gelatinase and cytochrome oxidase. Cells are Gram-staining-negative, flagellated, and rod

Strain BT2501 ( =NIBRBAC000501144) was isolated shaped. Colonies are cream color, circular, convex, opa from intestines of Russian grayling butterfly (H. autonoe) que, and shiny after incubation on BHI at 30℃ for 2 days from Jeju, Jeju-do, Republic of Korea. under aerobic conditions. Strain is positive for esculin hydrolysis, and utilization of glucose, mannose, mannitol, Klebsiella variicola BT3409 and maltose; but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, ure- Cells are Gram-staining-negative, non-flagellated, and ase, gelatinase, β-galactosidase, cytochrome oxidase, and rod shaped. Colonies are white color, circular, convex, utilization of arabinose, N-acetyl-glucosamine, potassium and massive after incubation on TSA at 37℃ for 2 days. gluconate, capric acid, adipic acid, malic acid, trisodium Strain is positive for nitrate reduction, glucose fermen- citrate, and phenylacetic acid in API 20NE. Strain CAU tation, esculin hydrolysis, urease, β-galactosidase, and 1471 (=NIBRBAC000501234) was isolated from urine utilization of glucose, arabinose, mannose, mannitol, from Kyunghee University Hospital, Dongdaemun-gu, N-acetyl-glucosamine, maltose, potassium gluconate, Seoul, Republic of Korea. malic acid, trisodium citrate, capric acid, adipic acid, and phenylacetic acid; but negative for indole production, ar- Halomonas sulfidaeris KYW1415 ginine dihydrolase, gelatinase and cytochrome oxidase. Strain BT3409 ( =NIBRBAC000501145) was isolated Cells are Gram-staining-negative, flagellated, and short from intestines of Russian grayling butterfly (H. autonoe) rod shaped. Colonies are pale yellow color, circular, en- November 2019 Kanjanasuntree et al. Unrecorded species of Gammaproteobacteria 331 tire margin, opaque, and smooth after incubation on MA acid, capric acid, trisodium citrate, and phenylacetic acid at 25℃ for 3 days. Strain is positive for cytochrome ox- in API 20NE. Strain LPB0189 (=NIBRBAC000501017) idase; but negative for nitrate reduction, glucose fermen- was isolated from seawater from Munseom, Jeju-do, Re- tation, indole production, arginine dihydrolase, urease, public of Korea. gelatinase, esculin hydrolysis, β-galactosidase, and utilization of glucose, arabinose, mannose, mannitol, N-ace- Amphritea japonica LPB0185 tyl-glucosamine, maltose, potassium gluconate, malic Cells are Gram-staining-negative, non-flagellated, and acid, capric acid, adipic acid, trisodium citrate, and pheny- rod shaped. Colonies are cream color, circular, convex, lacetic acid. Strain KYW1415 (=NIBRBAC000501130) and smooth after incubation on MA at 25℃ for 3 days was isolated from seawater from Gwangyang, Jeolla- under aerobic conditions. Strain is positive for nitrate re- nam-do, Republic of Korea. duction and cytochrome oxidase; but negative for indole production, glucose fermentation, arginine dihydrolase, Larsenimonas salina GH3-6 esculin hydrolysis, gelatinase, β-galactosidase, urease, Cells are Gram-staining-negative, flagellated, and rod and utilization of glucose, arabinose, mannose, manni- shaped. Colonies are light yellow color, irregular, and tol, N-acetyl-glucosamine, maltose, potassium gluconate, convex with entire margin after incubation on MA at adipic acid, malic acid, capric acid, trisodium citrate, and

30℃ for 5 days under aerobic conditions. Strain is posi- phenylacetic acid in API 20NE. Strain LPB0185 (=NI- tive for esculin hydrolysis; but negative for nitrate reduc- BRBAC000501013) was isolated from seawater from tion, indole production, glucose fermentation, arginine di- Munseom, Jeju-do, Republic of Korea. hydrolase, urease, gelatinase, β-galactosidase, cytochrome oxidase, and utilization of glucose, arabinose, mannose, Marinobacterium stanieri LPB0208 mannitol, N-acetyl-glucosamine, maltose, potassium glu- Cells are Gram-staining-negative, flagellated, and rod conate, capric acid, adipic acid, malic acid, trisodium ci- shaped. Colonies are cream color, circular, convex, and trate, and phenylacetic acid in API 20NE. Strain GH3-6 smooth after incubation on MA at 25℃ for 3 days under = ( NIBRBAC000501043) was isolated from rhizosphere aerobic conditions. Strain is negative for nitrate reduction, from Ganghwa-gun, Incheon, Republic of Korea. indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, β-galactosidase, gelatinase, Amphritea atlantica HMF9021 urease, cytochrome oxidase, and utilization of glucose, Cells are Gram-staining-negative, flagellated, and rod arabinose, mannose, mannitol, N-acetyl-glucosamine, shaped. Colonies are white color, circular, convex, and maltose, potassium gluconate, adipic acid, malic acid, smooth after incubation on MA at 30℃ for 3 days. Strain capric acid, trisodium citrate, and phenylacetic acid in = is positive for utilization of malic acid and cytochrome API 20NE. Strain LPB0208 ( NIBRBAC000501019) oxidase; but negative for nitrate reduction, indole pro- was isolated from seawater from Incheon, Ganghwa-do, duction, arginine dihydrolase, esculin hydrolysis, urease, Republic of Korea. gelatinase, β-galactosidase, glucose fermentation, and utilization of glucose, arabinose, mannose, mannitol, N-ace- Marinomonas communis KYW1404 tyl-glucosamine, maltose, potassium gluconate, malic Cells are Gram-staining-negative, flagellated, and rod acid, trisodium citrate, capric acid, adipic acid, and pheny- shaped. Colonies are ivory color, circular, flat, opaque, lacetic acid. Strain HMF9021 (=NIBRBAC000501174) and smooth after incubation on MA at 25℃ for 3 days. was isolated from sea sand from Boseong-gun, Jeolla- Strain is positive for esculin hydrolysis, β-galactosidase, nam-do, Republic of Korea. and cytochrome oxidase; but negative for nitrate reduction, glucose fermentation, indole production, arginine Amphritea balenae LPB0189 dihydrolase, urease, gelatinase, and utilization of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, Cells are Gram-staining-negative, flagellated, and rod maltose, potassium gluconate, malic acid, capric acid, shaped. Colonies are cream color, circular, convex, and adipic acid, trisodium citrate, and phenylacetic acid. smooth after incubation on MA at 25℃ for 3 days under Strain KYW1404 (=NIBRBAC000501129) was isolated aerobic conditions. Strain is positive for cytochrome oxi- from seawater from Gwangyang, Jeollanam-do, Republic dase; but negative for nitrate reduction, indole production, of Korea. glucose fermentation, arginine dihydrolase, esculin hydrolysis, β-galactosidase, gelatinase, urease, and utilization of Acinetobacter beijerinckii 16_S3_M11 glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, adipic acid, malic Cells are Gram-staining-negative, non-flagellated, and 332 Journal of Species Research Vol. 8, No. 4 rod shaped. Colonies are cream color, circular, and con- tion, glucose fermentation, arginine dihydrolase, esculin vex after incubation on R2A at 30℃ for 2 days under hydrolysis, gelatinase, β-galactosidase, cytochrome ox- aerobic conditions. Strain is positive for utilization of idase, and utilization of glucose, arabinose, mannose, capric acid, malic acid, and trisodium citrate; but negative mannitol, N-acetyl-glucosamine, maltose, adipic acid, for nitrate reduction, indole production, glucose fermen- potassium gluconate, and phenylacetic acid in API 20NE. tation, arginine dihydrolase, urease, esculin hydrolysis, Strain 16_H3_M12 (=NIBRBAC000501071) was isolat- gelatinase, β-galactosidase, cytochrome oxidase, and uti- ed from soil from Seongdong-gu, Seoul, Republic of Ko- lization of glucose, arabinose, mannose, mannitol, N-ace- rea. tyl-glucosamine, maltose, potassium gluconate, adipic acid, and phenylacetic acid in API 20NE. Strain 16_S3_ Acinetobacter tjernbergiae 16_H2_M14 M11 ( =NIBRBAC000501074) was isolated from soil Cells are Gram-staining-negative, non-flagellated, and from Yeosu, Gyeonggi-do, Republic of Korea. cocci shaped. Colonies are cream color, circular, and convex after incubation on R2A at 30℃ for 3 days under aer- Acinetobacter guillouiae 16_H2_V4 obic conditions. Strain is positive for nitrate reduction, ar- Cells are Gram-staining-negative, non-flagellated, and ginine dihydrolase, gelatinase, and utilization of glucose, rod shaped. Colonies are pale white color, circular, con- arabinose, mannose, mannitol, N-acetyl-glucosamine, vex, and glistening after incubation on R2A at 30℃ for potassium gluconate, capric acid, adipic acid, malic acid, 2 days under aerobic conditions. Strain is positive for trisodium citrate, and phenylacetic acid; but negative for nitrate reduction, and utilization of glucose, mannose, indole production, glucose fermentation, urease, esculin N-acetyl-glucosamine, and potassium gluconate; but neg- hydrolysis, β-galactosidase, and cytochrome oxidase in ative for indole production, glucose fermentation, argi- API 20NE. However, utilization of maltose showed both nine dihydrolase, urease, esculin hydrolysis, gelatinase, positive and negative activity. Strain 16_H2_M14 (=NI- β-galactosidase, cytochrome oxidase, and utilization of BRBAC000501073) was isolated from manure from arabinose, mannitol, maltose, capric acid, adipic acid, Guri, Gyeonggi-do, Republic of Korea. malic acid, trisodium citrate, and phenylacetic acid in API 20NE. However, utilization pf maltose showed both Acinetobacter towneri 16_H6_M1 positive and negative activity. Strain 16_H2_V4 (=NI- Cells are Gram-staining-negative, non-flagellated, and BRBAC000501072) was isolated from manure from cocci shaped. Colonies are white color, circular, and con- Guri, Gyeonggi-do, Republic of Korea. vex after incubation on R2A at 30℃ for 3 days under aerobic condition. Strain is positive for nitrate reduction, Acinetobacter indicus LPB0181 esculin hydrolysis, gelatinase, β-galactosidase, and utili- Cells are Gram-staining-negative, non-flagellated, and zation of glucose, mannose, N-acetyl-glucosamine, malt- cocci shaped. Colonies are cream color, circular, convex, ose, capric acid, malic acid, and trisodium citrate; but and smooth after incubation on MA at 25℃ for 3 days negative for indole production, glucose fermentation, ar- under aerobic conditions. Strain is positive for utilization ginine dihydrolase, urease, cytochrome oxidase, and utili- of adipic acid; but negative for nitrate reduction, esculin zation of arabinose, mannitol, potassium gluconate, adipic hydrolysis, gelatinase, glucose fermentation, indole pro- acid, and phenylacetic acid in API 20NE. Strain 16_H6_ duction, arginine dihydrolase, urease, β-galactosidase, M1 (NIBRBAC000501076) was isolated from soil from cytochrome oxidase, and utilization of glucose, arabinose, Mapo-gu, Seoul, Republic of Korea. mannose, mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, malic acid, capric acid, trisodium ci- Acinetobacter ursingii 16_H5_M7 trate. and phenylacetic acid in API 20NE. Strain LPB0181 Cells are Gram-staining-negative, non-flagellated, and ( =NIBRBAC000501010) was isolated from seawater rod shaped. Colonies are white color, circular, and flat from Incheon, Ganghwa-do, Republic of Korea. after incubation on R2A at 30℃ for 3 days under aerobic conditions. Strain is positive for urease, and utilization of Acinetobacter johnsonii 16_H3_M12 capric acid, adipic acid, malic acid, and trisodium citrate; Cells are Gram-staining-negative, non-flagellated, and but negative for nitrate reduction, indole production, glu- rod shaped. Colonies are white color, circular, raised, and cose fermentation, arginine dihydrolase, esculin hydro- entire margin after incubation on R2A at 30℃ for 3 days lysis, gelatinase, β-galactosidase, cytochrome oxidase, under aerobic conditions. Strain is positive for urease, and and utilization of glucose, arabinose, mannose, mannitol, utilization of capric acid, malic acid, and trisodium citrate N-acetyl-glucosamine, maltose, potassium gluconate, and (waek); but negative for nitrate reduction, indole produc- phenylacetic acid in API 20NE. Strain 16_H5_M7 (=NI- November 2019 Kanjanasuntree et al. Unrecorded species of Gammaproteobacteria 333

BRBAC000501075) was isolated from soil from Ma- for 5 days under aerobic conditions. Strain is positive for po-gu, Seoul, Republic of Korea. nitrate reduction, indole production, glucose fermentation, esculin hydrolysis, cytochrome oxidase, and utilization of Acinetobacter vivianii BC3402 glucose, mannitol, potassium gluconate, and malic acid; but negative for arginine dihydrolase, gelatinase, urease, Cells are Gram-staining-negative, non-flagellated, and β-galactosidase, and utilization of arabinose, mannose, cocci or ovoid shaped. Colonies are pink color, circular, N-acetyl-glucosamine, maltose, adipic acid, capric acid, and convex after incubation on MacConkey at 37℃ for 2 trisodium citrate, and phenylacetic acid in API 20NE. days. Strain is positive for esculin hydrolysis, gelatinase, Strain IMCC34228 (=NIBRBAC000501090) was isolat- and utilization of capric acid, adipic acid, malic acid, tri- ed from seawater from Jeju, Jeju-do, Republic of Korea. sodium citrate and phenylacetic acid; but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, β-galactosidase, cytochrome Photobacterium damselae subsp. piscicida LPB0174 oxidase, and utilization of glucose, arabinose, mannose, Cells are Gram-staining-negative, flagellated, and rod mannitol, N-acetyl-glucosamine, maltose and potassium shaped. Colonies are translucent, circular, convex, and gluconate. Strain BC3402 (=NIBRBAC000501137) was smooth after incubation on MA at 30℃ for 3 days un- isolated from intestines of Russian grayling butterfly (H. der facultatively anaerobic conditions. Strain is positive autonoe) from Jeju, Jeju-do, Republic of Korea. for urease and acid production from glucose (weak) and mannose (weak); but negative for indole production, hy- Pseudomonas mohnii KA-176 drolysis of esculin and gelatin, and acid production from Cells are Gram-staining-negative, flagellated, and oval mannitol, lactose, saccharose, maltose, salicin, xylose, shaped. Colonies are cream color, circular, and convex arabinose, glycerol, cellobiose, mannose, melezitose, after incubation on R2A at 30℃ for 2 days under aerobic raffinose, sorbitol, rhamnose, and trehalose in API 20A. Strain LPB0174 (=NIBRBAC000501008) was isolated conditions. Strain is positive for nitrate reduction, arginine dihydrolase, gelatinase, cytochrome oxidase, and from intestine of sea bass (L. japonicus) from Incheon, utilization of glucose, arabinose, mannose, N-acetyl-glu- Ganghwa-do, Republic of Korea. cosamine, potassium gluconate, capric acid, malic acid, trisodium citrate, and phenylacetic acid; but negative for Vibrio comitans LPB0190 indole production, glucose fermentation, urease, esculin Cells are Gram-staining-negative, flagellated, and rod hydrolysis, β-galactosidase, and utilization of mannitol, shaped. Colonies are cream color, circular, convex, and maltose, and adipic acid in API 20NE. Strain KA-176 smooth after incubation on MA at 25℃ for 3 days under = ( NIBRBAC000500996) was isolated from soil from aerobic conditions. Strain is positive for esculin hydro- Pocheon, Gyeonggi-do, Republic of Korea. lysis and β-galactosidase; but negative for nitrate reduction, indole production, glucose fermentation, arginine Pseudomonas nitritireducens BC3406 dihydrolase, gelatinase, urease, cytochrome oxidase, and Cells are Gram-staining-negative, flagellated, and rod utilization of glucose, arabinose, mannose, mannitol, shaped. Colonies are pink color, circular, and convex af- N-acetyl-glucosamine, maltose, potassium gluconate, ter incubation on MacConkey at 37℃ for 2 days. Strain adipic acid, malic acid, capric acid, trisodium citrate, and is positive for nitrate reduction, arginine dihydrolase, phenylacetic acid in API 20NE. Strain LPB0190 (=NI- esculin hydrolysis, and utilization of glucose, arabinose, BRBAC000501301) was isolated from seawater from mannose, mannitol, N-acetyl-glucosamine, maltose, po- Munseom, Jeju-do, Republic of Korea. tassium gluconate, malic acid, trisodium citrate, capric acid, adipic acid, and phenylacetic acid; but negative for Vibrio hispanicus IMCC34186 indole production, glucose fermentation, urease, gelati- Cells are Gram-staining-negative, flagellated, and rod nase, β-galactosidase, and cytochrome oxidase. Strain shaped. Colonies are white color, circular, convex, and BC3406 ( =NIBRBAC000501147) was isolated from entire margin after incubation on R15A at 25℃ for 3 intestines of Russian grayling butterfly (H. autonoe) from days under aerobic conditions. Strain is positive for ni- Jeju, Jeju-do, Republic of Korea. trate reduction, arginine dihydrolase, esculin hydrolysis, β-galactosidase, cytochrome oxidase, and utilization of Aliivibrio fischeri IMCC34228 potassium gluconate; but negative for indole production, Cells are Gram-staining-negative, flagellated, and rod glucose fermentation, urease, gelatinase, and utilization of shaped. Colonies are cream to yellow color, circular, con- glucose, arabinose, mannose, mannitol, N-acetyl-glucos- vex, and entire margin after incubation on R2A at 25℃ amine, maltose, adipic acid, capric acid, malic acid, triso- 334 Journal of Species Research Vol. 8, No. 4 dium citrate, and phenylacetic acid in API 20NE. Strain shaped. Colonies are cream color, circular, convex, and IMCC34186 (=NIBRBAC000501102) was isolated from entire margin after incubation on R8A at 25℃ for 5 days tidal flat from Jangbong-do, Incheon, Republic of Korea. under aerobic conditions. Strain is positive for nitrate reduction, glucose fermentation, esculin hydrolysis, β-ga- Vibrio neresis LPB0202 lactosidase, and cytochrome oxidase; but negative for indole production, arginine dihydrolase, urease, gelatinase, Cells are Gram-staining-negative, flagellated, and cocci and utilization of glucose, arabinose, mannose, mannitol, shaped. Colonies are cream color, circular, convex, and N-acetyl-glucosamine, maltose, potassium gluconate, smooth after incubation on MA at 25℃ for 3 days under adipic acid, capric acid, malic acid, trisodium citrate, aerobic conditions. Strain is negative for nitrate reduction, and phenylacetic acid in API 20NE. Strain IMCC34148 indole production, glucose fermentation, arginine dihy- ( =NIBRBAC000501105) was isolated from tidal flat drolase, esculin hydrolysis, β-galactosidase, gelatinase, from Jangbong-do, Incheon, Republic of Korea. urease, cytochrome oxidase, and utilization of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, potassium gluconate, adipic acid, malic acid, Lysobacter ximonensis 17J9-2 capric acid, trisodium citrate, and phenylacetic acid in Cells are Gram-staining-negative, flagellated, and rod

API 20NE. Strain LPB0202 ( =NIBRBAC000501018) shaped. Colonies are yellow color, circular, convex, and was isolated from seawater from Incheon, Ganghwa-do, smooth after incubation on R2A at 25℃ for 4 days under Republic of Korea. aerobic conditions. Strain is positive for arginine dihydrolase, urease, esculin hydrolysis, gelatinase, β-galac- Vibrio penaeicida IMCC34213 tosidase, and utilization of glucose, mannose, mannitol, Cells are Gram-staining-negative, flagellated, and rod N-acetyl-glucosamine, maltose, adipic acid, malic acid, shaped. Colonies are white color, circular, raised, and en- and trisodium citrate; but negative for nitrate reduction, tire margin after incubation on R17A at 25℃ for 3 days indole production, glucose fermentation, cytochrome ox- under aerobic conditions. Strain is positive for nitrate re- idase, and utilization of arabinose, capric acid, potassium duction, indole production, glucose fermentation, gelati- gluconate, and phenylacetic acid in API 20NE. Strain = nase, β-galactosidase, cytochrome oxidase, and utilization 17J9-2 ( NIBRBAC000501324) was isolated from soil of malic acid; but negative for arginine dihydrolase, escu from Jeju-do, Republic of Korea. lin hydrolysis, urease, and utilization of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, adipic Pseudoxanthomonas kaohsiungensis MMS17-SY254 acid, potassium gluconate, capric acid, trisodium citrate, Cells are Gram-staining-negative, non-flagellated, and and phenylacetic acid in API 20NE. Strain IMCC34213 rod shaped. Colonies are light yellow color, circular, con- = ( NIBRBAC000501103) was isolated from beach sand vex, and smooth with entire margin after incubation on from Wando, Jeollanam-do, Republic of Korea. NA at 30℃ for 3 days under aerobic conditions. Strain is positive for esculin hydrolysis, β-galactosidase, cyto- Vibrio scophthalmi IMCC34225 chrome oxidase, and utilization of glucose, arabinose, Cells are Gram-staining-negative, flagellated, and rod N-acetyl-glucosamine, and maltose; but negative for ni- shaped. Colonies are white color, circular, raised, and trate reduction, indole production, glucose fermentation, entire margin after incubation on R2A at 25℃ for 5 days arginine dihydrolase, urease, gelatinase, and utilization under aerobic conditions. Strain is positive for nitrate re- of mannose, mannitol, potassium gluconate, capric acid, duction, indole production, glucose fermentation, esculin adipic acid, malic acid, trisodium citrate, and pheny- hydrolysis, cytochrome oxidase, and utilization of glu- lacetic acid in API 20NE. Strain MMS17-SY254 (=NI- cose, mannose, N-acetyl-glucosamine, maltose, potassi- BRBAC000501210) was isolated from soil from Ok- um gluconate, and malic acid; but negative for arginine do-myeon, Gunsan, Jeollabuk-do, Republic of Korea. dihydrolase, gelatinase, urease, β-galactosidase, and utilization of arabinose, mannitol, adipic acid, capric acid, Stenotrophomonas acidaminiphila 16_H1_E7 trisodium citrate, and phenylacetic acid in API 20NE. Cells are Gram-staining-negative, flagellated, and rod Strain IMCC34225 ( =NIBRBAC000501104) was iso- shaped. Colonies are yellow color, circular, and convex lated from seawater from Gimnyeong, Jeju-do, Republic after incubation on R2A at 30℃ for 1 days under aerobic of Korea. conditions. Strain is positive for nitrate reduction, esculin hydrolysis, gelatinase, β-galactosidase, cytochrome oxi- Vibrio tritonius IMCC34148 dase, and utilization of glucose, mannose, N-acetyl-glu- Cells are Gram-staining-negative, flagellated, and rod cosamine, maltose, and malic acid; but negative for indole November 2019 Kanjanasuntree et al. Unrecorded species of Gammaproteobacteria 335 production, glucose fermentation, arginine dihydrolase, J.T. Staley and G.M. Garrity (eds). Bergey’s Manual of urease, and utilization of arabinose, mannitol, potassium Systematic Bacteriology, second edition, vol. 2 (The Pro- gluconate, capric acid, adipic acid, trisodium citrate, and teobacteria), Part B (The Gammaproteobacteria), Spring- phenylacetic acid in API 20NE. Strain 16_H1_E7 (=NI- er, New York. p. 1. BRBAC000501080) was isolated from sediment from Garrity, G.M., J.A. Bell and T. Lilburn. 2005b. Order IV. Car- Namyangju, Gyeonggi-do, Republic of Korea. diobacteriales ord. nov. In: D.J. Brenner, N.R. Krieg, J.T. Staley and G.M. Garrity (eds). Bergey’s Manual of Sys- tematic Bacteriology, second edition, vol. 2 (The Proteo- bacteria), Part B (The Gammaproteobacteria), Springer, Acknowledgements New York. p. 123. Garrity, G.M., J.A. Bell and T. Lilburn. 2005c. Order V. This work was supported by the project on survey of Thiotrichales ord. nov. In: D.J. Brenner, N.R. Krieg, J.T. indigenous species of Korea of the National Institute of Staley and G.M. Garrity (eds). Bergey’s Manual of Sys- Biological Resources (NIBR) under the Ministry of Envi- tematic Bacteriology, second edition, vol. 2 (The Proteo- ronment (MOE). bacteria), Part B (The Gammaproteobacteria), Springer, New York. p. 131. Garrity, G.M., J.A. Bell and T. Lilburn. 2005d. Order VI. References Legionellales ord. nov. In: D.J. Brenner, N.R. Krieg, J.T. Staley and G.M. Garrity (eds). Bergey’s Manual of Sys- Adeolu, M., S. Alnajar, S. Naushad and R.S. Gupta. 2016. 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