Isolation and Identification of 18 Unrecorded Prokaryotic Species from the Intestinal Tracts of Aquatic Animals in Korea

Journal of Species Research 10(1):1-11, 2021

Isolation and identification of 18 unrecorded prokaryotic species from the intestinal tracts of aquatic animals in Korea

Jae-Yun Lee§, Yun-Seok Jeong§, Pil Soo Kim, Dong-Wook Hyun and Jin-Woo Bae*

Department of Biology and Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea

§These authors contributed equally to this work. *Correspondent: [email protected]

Evidence highlighting the importance of gut microbiota in biodiversity conservation is growing; however, gut bacteria in South Korean wildlife have not been well identified. Using a culture-dependent isolation method, we identified the gut bacteria from Korean aquatic wildlife: the gazami crab (Portunus trituberculatus), Korean striped bitterling (Acheilognathus yamatsutae), oily bitterling (Acheilognathus koreensis), leopard mandarin fish (Siniperca scherzeri), Korean dark chub (Zacco koreanus), diving beetle (Cybister lewisianus), spotted steed (Abbottina springeri), and Korean spotted sleeper (Odontobutis obscura interrupta). We identified 18 strains previously unrecorded in South Korea by comparing 16S rRNA gene sequences of isolates against the EzBioCloud and National Institute of Biological Resources (NIBR) databases. The isolated strains belong to the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. We also assessed for phylogenetic relatedness, Gram-stain reaction, colony and cell morphology, and biochemical characteristics. Basic information and 16S rRNA gene sequences of the isolates were registered in NIBR, and NIBR accession numbers are provided. Keywords: ‌16S rRNA gene sequences, gut microbiota, Korean aquatic wildlife, unidentified Korean gut bacteria

Introduction that comprise the gut microbiome of Korean wildlife have not yet been fully studied. We aimed to identify domesti Since the adoption of the Convention on Biological cally unrecorded bacterial species from the gastrointestinal Diversity (CBD) at the United Nations Conference on tracts of Korean aquatic animals, including fish, crusta Environment and Development (UNCED) in 1992, the ceans, and insects, using a culture-dependent isolation urgency of conserving biodiversity and protecting biologi method. Furthermore, using the 16S rRNA gene sequence- cal resources is being globally realized. Furthermore, in based identification, we isolated 18 strains previously un 2010, the Nagoya Protocol was adopted by the CBD given recorded in South Korea belonging to the phyla Actino the growing economic value of the genetic resources de bacteria, Bacteroidetes, Firmicutes, and Proteobacteria. fined as “genetic material of actual or potential value.” Hence, understanding indigenous biodiversity is essential to appropriate protection of a nation’s sovereign rights Materials and Methods over biological resources. Culture-dependent isolation and identification Owing to its wide-ranging potential functional capacities (Qin et al., 2010), the microbiome has received most atten Gastrointestinal tracts of eight aquatic species were dis tion as a biological resource. In particular, the gut micro- sected and their contents were immediately resuspended in biome is considered crucial because it has been shown to sterile phosphate-buffered saline (PBS) to prepare sample be closely related to host health (Shin et al., 2014), devel inoculums: Korean dark chub (Zacco koreanus), gazami opment (Koropatnick et al., 2004), and reproductive beha crab (Portunus trituberculatus), Korean striped bitterling vior (Sharon et al., 2010). However, bacterial members (Acheilognathus yamatsutae), oily bitterling (Acheilog 2 JOURNAL OF SPECIES RESEARCH Vol. 10, No. 1 nathus koreensis), spotted steed (Abbottina springeri), responsiveness, we used a commercial Gram-staining kit leopard mandarin fish (Siniperca scherzeri), Korean spot (bioMérieux). All the experiments were conducted in ted sleeper (Odontobutis obscura interrupta), and diving accordance with the manufacturers’ instructions. Cell mor beetle (Cybister lewisianus). Then, the inoculums were phology was observed using LIBRA120, an energy-filter - serially diluted with sterile PBS at concentrations of 10 1, ing transmission electron microscope (Carl Zeiss). Colony - - 10 2, and 10 3. Each concentration of the inoculum was in morphology was determined by observing a single colony dividually spread onto Reasoner’s 2A (R2A) agar, Marine of each strain incubated on appropriate media at isolation agar (MA), Tryptic soy agar (TSA; supplemented with 0.5 temperatures for 48 h. % yeast extract or not), Lactobacilli MRS agar (MRSA), Brain heart infusion (BHI), and MacConkey agar plates; these were incubated at 10℃, 15℃, 25℃, and 30℃. Results and Discussion Emerging colonies were separately transferred to fresh On the basis of 16S rRNA gene-based phylogenetic media and were sub-cultured at least 3 times to obtain pure analysis, 18 strains, designated K33R8, K33T6, G12R3, cultures. Each pure culture was routinely cultivated under R13S1, R23R4, R33M6-1, R33M4, K11M4, O13M9, isolation conditions and used for further experiments. 719, 176, 775, 765, 684, A52, 771, A79 and 772, were We used the 16S rRNA gene-based identification to assigned to the phyla Actinobacteria, Bacteroidetes, Firmi identify the bacterial pure cultures. The 16S rRNA gene cutes and Proteobacteria. The strains were confirmed as was amplified using C1000 Touch Thermal Cycler (Bio- chemoheterotrophic rod- or coccoid shaped bacteria (Fig. Rad) with 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) 1). The detailed phylogenetical, morphological and bio and 1492R (5′-GGYTACCTTGTTACGACTT-3′) primers, chemical characteristics of the strains are described below. and the PCR products were sequenced using the PRISM In the 16S rRNA gene sequence comparison among 3730xl DNA analyzer (Applied Biosystems) with univer the 18 isolated strains, K33R8, K33T6, G12R3, R13S1, sal bacterial primers (Lane, 1991). The 16S rRNA gene R23R4, R33M6-1, R33M4, K11M4, O13M9, 719, 176, sequence fragments were then assembled using SeqMan 775, 765, 684, A52, 771, A79 and 772 showed the highest 5.0 (DNASTAR) to obtain full-length 16S rRNA gene 16S rRNA gene sequence similarities with Chryseobacte sequences. EzBioCloud and National Institute of Biologi rium oleae DSM 25575T (jgi.1085839; 99.42% similarity), cal Resources database were used to assign the obtained Buttiauxella agrestis ATCC 33320T (JMPI01000079; 99.93 16S rRNA gene sequences to corresponding taxa (Yoon et %), Leucobacter triazinivorans JW-1T (KT439069; 100%), al., 2017). Lysinibacillus macroides DSM 54T (LGCI01000008; 99.52%), Flavobacterium cutihirudinis DSM 25795T (jgi. Phylogenetic analysis 1108034; 99.58%), Gordonia didemni B204T (JN615417; To understand the phylogenetic relationships of the 98.92%), Mycolicibacterium fortuitum subsp. acetami isolates, phylogenetic trees were reconstructed using the dolyticum JCM 6368T (BCSZ01000080; 100%), Yersinia isolates in the same phylum, the most closely related kristensenii subsp. rochesterensis EPLC-04T (KJ606916; species, and the type species in the genus using MEGA 99.86%), Paracoccus sanguinis 5503T (JRKQ01000154; 7 software (Kumar et al., 2016) using the neighbor join 99.86%), Aerococcus viridans ATCC 11563T (ADNT ing (NJ) (Saitou and Nei, 1987), maximum likelihood 01000041; 99.93%), Alcaligenes faecalis subsp. parafae (ML) (Tamura and Nei, 1993), and maximum parsimony calis GT (AJ242986; 100%), Aliarcobacter faecis AF1078T (MP) (Nei and Kumar, 2000) algorithms. Multiple se (JARS01000021; 100%), Citrobacter portucalensis A60T quence alignment of 16S rRNA gene sequences was per (MVFY01000035; 100%), Citrobacter gillenii CDC 4693- formed using ClustalW (Thompson et al., 1994). Clade 86T (AF025367; 99.66%), Cryobacterium arcticum GCJ credibility was assessed with a bootstrap test with 1000 02T (GQ406814; 99.56%), Comamonas odontotermitis replicates. Dant 3-8T (DQ453128; 99.86%), Comamonas nitrativo rans 23310T (AJ251577; 99.24%) and Enterococcus phoe Assessment of morphologic and biochemical niculicola ATCC BAA-412T (AJAT01000017; 100%), characteristics respectively (Table 1). Phylogenetic consensus trees of the 18 strains and related taxa also robustly support that the For biochemical characterization of the isolated strains, isolated strains are most closely related to the strains which we tested the cells from the strain isolates using API showed the highest 16S rRNA gene sequence similarities, 20NE, API ID 32GN, API ZYM (bioMérieux) and GEN establishing monophyletic clades each other (Figs. 2-5). III MicroPlate (Biolog) kits. Specifically, API 20NE, API ID 32GN, and GEN III MicroPlate demonstrated carbon Description of Chryseobacterium oleae K33R8 source utilization, and API 20NE and API ZYM demon strated enzymatic activity. To verify their Gram-staining Cells are Gram-stain-negative, non-flagellated and rod February 2021 Lee et al. Unrecorded gut bacteria from Korean aquatic animals 3

Fig. 1. The energy-filtering transmission electron micrographs of the isolated strains. Strains: 1, K33R8; 2, K33T6; 3, G12R3; 4, R13S1; 5, R23R4; 6, R33M6-1; 7, R33M4; 8, K11M4; 9, O13M9; 10, 719; 11, 176; 12, 775; 13, 765; 14, 684; 15, A52; 16, 771; 17, A79; 18, 772. shaped. Colonies are circular, convex, entire and yellowish phosphatase, leucine arylamidase, valine arylamidase, on R2A agar medium after 48 h incubation at 30℃. Cells trypsin, acid phosphatase, naphthol-AS-BI-phosphohy reduce nitrate to nitrogen and are positive for arginine drolase, β-galactosidase, α-glucosidase, β-glucosidase, dihydrolase, β-glucosidase, protease, alkaline phosphatase, N-acetyl-β-glucosaminidase (API 20NE/ZYM). Assimi esterase (C4), esterase lipase (C8), leucine arylamidase, va late D-glucose, L-arabinose, D-mannose, D-mannitol, line arylamidase, trypsin, acid phosphatase, naphthol-AS- N-acetyl-D-glucosamine, D-maltose, gluconate, malate, BI-phosphohydrolase, α-glucosidase, N-acetyl-β-glucosa citrate, phenyl-acetate, salicin, D-melibiose, L-fucose, minidase (API 20NE/ZYM). Assimilate D-glucose, L-ara L-histidine, 2-ketogluconate, 4-hydroxy-benzoate, L-pro binose, D-mannose, D-mannitol, D-maltose, gluconate, line, L-rhamnose, D-ribose, D-sucrose, malonate, acetate, adipate, malate, citrate, L-histidine, L-proline, D-sucrose, lactate, L-alanine, 5-ketogluconate, L-serine (API 20NE/ acetate, glycogen (API 20NE/ID 32GN) and utilize dextrin, ID 32GN) and utilize dextrin, D-maltose, D-trehalose, gentiobiose, D-turanose, D-melibiose, α-D-glucose, D-cellobiose, gentiobiose, D-turanose, stachyose, D-raffi D-mannose, D-fructose, D-galactose, 6-methyl-glucose, nose, α-D-lactose, D-melibiose, β-D-glucoside, D-salicin, D-fucose, L-fucose, L-rhamnose, D-glucose, D-fructose, N-glucosamine, β-mannosamine, N-galactosamine, neura gelatin, L-histidine, galacturonic acid, galactonic lactone, minic acid, α-D-glucose, D-mannose, D-fructose, D-gal D-glucuronic acid, glucuronamide, Tween40, acetoacetic actose, 6-methyl-glucose, D-fucose, L-fucose, L-rham acid and acetic acid as sole carbon sources (Biolog GEN nose, inosine, D-sorbitol, D-mannitol, glycerol, D-glucose, Ⅲ). D-fructose, D-serine, glycyl-L-proline, L-alanine, L-aspar Strain K33R8 (=NIBRBAC000503075) was isolated tic acid, L-glutamic acid, L-histidine, L-serine, pectin, gal from the gut of Korean dark chub (Nipponocypris korea acturonic acid, galactonic lactone, D-gluconic acid, D-glu nus) at Hwasun, Jeollanam Province, Korea. The DNA curonic acid, glucuronamide, mucic acid, quinic acid, G+C content of the type strain of the species is 38 mol%. methyl pyruvate, L-lactic acid, citric acid, L-malic acid, succinic acid, α-butyric acid and acetic acid as sole car

Description of Buttiauxella agrestis K33T6 bon sources (Biolog GEN Ⅲ). Strain K33T6 (=NIBRBAC000503076) was isolated Cells are Gram-stain-negative, flagellated and coccus from the gut of Korean dark chub (Nipponocypris korea shaped. Colonies are circular, convex, entire and beige nus) at Hwasun, Jeollanam Province, Korea. The DNA colored on TSA medium after 48 h incubation at 30℃. G+C content of the type strain of the species is 47-51 Cells reduce nitrate to nitrite and are positive for argi mol%. nine dihydrolase, β-glucosidase, β-galactosidase, alkaline 4 JOURNAL OF SPECIES RESEARCH Vol. 10, No. 1 ) ) ) ) ) ) ) ) )

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Isolation source Portunus trituberculatus Acheilognathus yamatsutae Acheilognathus yamatsutae scherzeri Siniperca Zacco koreanus Acheilognathus yamatsutae Acheilognathus yamatsutae scherzeri Siniperca Cybister lewisianus Zacco koreanus Acheilognathus koreensis Abbottina springeri scherzeri Siniperca Cybister lewisianus Cybister lewisianus Odontobutis obscura interrupta Odontobutis obscura interrupta Odontobutis obscura interrupta Leopard mandarin fish Leopard mandarin fish Korean spotted sleeper Korean striped bitterling Diving beetle Korean striped bitterling Korean striped bitterling Oily bitterling Spotted steed Korean spotted sleeper Leopard mandarin fish Gazami crab Korean dark chub Diving beetle Korean dark chub Korean spotted sleeper Diving beetle ( ( Korean striped bitterling ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

100 100 100 (%) 100 100 100 99.56 99.93 98.92 99.58 99.52 99.86 99.86 99.66 99.42 99.86 99.93 99.24 Similarity Accession number GQ406814 ADNT01000041 MVFY01000035 JN615417 AJAT01000017 jgi.1108034 LGCI01000008 KJ606916 JRKQ01000154 JARS01000021 AF025367 KT439069 jgi.1085839 DQ453128 JMPI01000079 AJ242986 AJ251577 BCSZ01000080

EPLC - 04(T) G(T) acetamidolyticum - 1(T) subsp. DSM 25795(T) ATCC BAA - 412(T) ATCC rochesterensis Dant 3-8(T) parafaecalis JW 23310(T) A60(T) DSM 54(T) GCJ02(T) DSM 25575(T) Most closely related species 5503(T) ATCC 33320(T) ATCC ATCC 11563(T) ATCC subsp. AF1078(T) subsp. CDC 4693-86(T) Closest type strain B204(T) Gordonia didemni Gordonia Mycolicibacterium fortuitum Cryobacterium arcticum macroides Lysinibacillus viridans Aerococcus faecis Aliarcobacter portucalensis Citrobacter gillenii Citrobacter Leucobacter triazinivorans Chryseobacterium oleae phoeniculicola Enterococcus Comamonas odontotermitis Flavobacterium cutihirudinis Buttiauxella agrestis kristensenii Yersinia Paracoccus sanguinis Alcaligenes faecalis Comamonas nitrativorans JCM 6368(T) NIBR accession number NIBRBAC000503080 NIBRBAC000503081 NIBRBAC000503090 NIBRBAC000503078 NIBRBAC000503085 NIBRBAC000503087 NIBRBAC000503088 NIBRBAC000503089 NIBRBAC000503077 NIBRBAC000503075 NIBRBAC000503082 NIBRBAC000503091 NIBRBAC000503079 NIBRBAC000503076 NIBRBAC000503083 NIBRBAC000503084 NIBRBAC000503086 NIBRBAC000503092 ID Strain R33M6 - 1 R33M4 A52 R13S1 719 775 765 684 G12R3 K33R8 772 771 R23R4 K33T6 K11M4 O13M9 176 A79 Genus Gordonia Mycolicibacterium Cryobacterium Lysinibacillus Aerococcus Aliarcobacter Citrobacter Citrobacter Leucobacter Chryseobacterium Enterococcus Comamonas Flavobacterium Buttiauxella Yersinia Paracoccus Alcaligenes Comamonas Family Gordoniaceae Mycobacteriaceae Microbacteriaceae Planococcaceae Aerococaceae Campylobacteraceae Enterobacteriaceae Enterobacteriaceae Microbacteriaceae Flavobacteriaceae Enterococcaceae Comamonadaceae Flavobacteriaceae Enterobacteriaceae Yersiniaceae Rhodobacteraceae Alcaligenaceae Comamonadaceae Order Actinomycetales Corynebacteriales Micrococcales Bacillales Lactobacillales Campylobacterales Enterobacterales Enterobacterales Actinomycetales Flavobacteriales Lactobacillales Burkholderiales Flavobacteriales Enterobacteriales Enterobacteriales Rhodobacterales Burkholderiales Burkholderiales Class Actinomycetes Actinobacteria Actinobacteria Bacilli Bacilli Epsilonproteobacteria Gammaproteobacteria Gammaproteobacteria Actinobacteria Flavobacteriia Bacilli Betaproteobacteria Flavobacteriia Gammaproteobacteria Gammaproteobacteria Alphaproteobacteria Betaproteobacteria Betaproteobacteria Taxonomic affiliations and isolation sources of the 18 isolated strains. Taxonomic Phylum Actinobacteria Actinobacteria Actinobacteria Firmicutes Firmicutes Proteobacteria Proteobacteria Proteobacteria Actinobacteria Table 1. Table Bacteroidetes Firmicutes Proteobacteria Bacteroidetes Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria February 2021 Lee et al. Unrecorded gut bacteria from Korean aquatic animals 5

Fig. 2. The phylogenetic tree based on 16S rRNA gene sequence showing the phylogenetic relatedness between the nine isolated strains be longing to the phylum Proteobacteria and the type species from each genus. The tree was mainly reconstructed using the neighbor joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) algorithms. The Filled diamonds indicate the branches present in the phylogenetic trees reconstructed using three different tree reconstruction methods. Numbers on the nodes represent bootstrap values with 1000 replicates (NJ/ ML/MP). Bacteroides fragilis NCTC 9434T was used as an outgroup. The bar indicates 0.05 accumulated substitutions per nucleotide.

Description of Leucobacter triazinivorans G12R3 Description of Lysinibacillus macroides R13S1 Cells are Gram-stain-positive, non-flagellated and rod Cells are Gram-stain-positive, flagellated and rod shaped. shaped. Colonies are circular, convex, entire and yellow Colonies are irregular, raised and beige colored on lacto colored on TSA medium after 48 h incubation at 25℃. bacilli MRS agar medium after 48 h incubation at 30℃. Cells are positive for esterase (C4), esterase lipase (C8), Cells are positive for arginine dihydrolase, protease, ester leucine arylamidase, valine arylamidase, cystine arylami ase (C4), esterase lipase (C8), leucine arylamidase, valine dase, acid phosphatase, naphthol-AS-BI-phosphohydro arylamidase, acid phosphatase, naphthol-AS-BI-phos lase (API ZYM) and assimilate D-glucose, L-arabinose, phohydrolase (API 20NE/ZYM) and assimilate N-acetyl- D-mannose, N-acetyl-D-glucosamine, D-maltose, adipate, D-glucosamine, gluconate, malate, propionate, valerate, citrate, phenyl-acetate, L-fucose, propionate, L-histidine, L-histidine, 3-hydroxy-butyrate, L-proline, acetate, lac L-proline, L-rhamnose, inositol, acetate, L-alanine (API tate, L-alanine, glycogen, L-serine (API 20NE/ID 32GN). 20NE/ID 32GN). Utilize dextrin, phenylacetic acid, methyl Utilize dextrin, D-maltose, D-trehalose, D-cellobiose, pyruvate, Tween40, α-butyric acid, acetoacetic acid, pro gentiobiose, sucrose, D-turanose, stachyose, D-raffinose, pionic acid and acetic acid as sole carbon sources (Biolog N-glucosamine, β-mannosamine, D-fructose, D-fucose, GEN Ⅲ). L-fucose, L-rhamnose, inosine, D-sorbitol, D-mannitol, Strain G12R3 (=NIBRBAC000503077) was isolated D-arabitol, glycerol, D-glucose, D-fructose, D-aspartic from the gut of gazami crab (Portunus trituberculatus) at acid, D-serine, gelatin, glycyl-L-proline, L-alanine, L-argi- Taean, Chungcheongnam Province, Korea. The DNA G+ nine, L-aspartic acid, L-glutamic acid, L-histidine, pyro C content of the type strain is 73 mol%. glutamic acid, L-serine, pectin, galacturonic acid, galac tonic lactone, D-gluconic acid, D-glucuronic acid, mucic 6 JOURNAL OF SPECIES RESEARCH Vol. 10, No. 1

Fig. 3. The phylogenetic tree based on 16S rRNA gene sequence showing the phylogenetic relatedness between the four isolated strains be longing to the phylum Actinobacteria and the type species from each genus. The tree was mainly reconstructed using the NJ, ML, and MP algo rithms. The filled diamonds indicate the branches present in the phylogenetic trees reconstructed with three different tree reconstruction methods. Numbers on the nodes represent bootstrap values with 1000 replicates (NJ/ML/MP). B. fragilis NCTC 9434T was used as an outgroup. The bar indicates 0.05 accumulated substitutions per nucleotide. acid, quinic acid, D-saccharic acid, phenylacetic acid, acetyl-D-glucosamine, D-maltose, malate, D-sucrose, methyl pyruvate, D-lactic acid, L-lactic acid, α-glutaric glycogen (API 20NE/ID 32GN) and utilize dextrin, acid, L-malic acid, Tween40, γ-butyric acid, α-butyric D-maltose, D-cellobiose, gentiobiose, sucrose, D-tura acid, β-butyric acid, α-butyric acid, acetoacetic acid, pro nose, stachyose, D-raffinose, α-D-lactose, D-melibiose, pionic acid, acetic acid and formic acid as sole carbon N-glucosamine, β-mannosamine, N-galactosamine, neura sources (Biolog GEN Ⅲ). minic acid, α-D-glucose, D-mannose, D-fructose, D-gal Strain R13S1 ( =NIBRBAC000503078) was isolated actose, 6-methyl-glucose, L-fucose, D-fructose, gelatin, from the gut of Korean striped bitterling (Acheilognathus glycyl-L-proline, L-arginine, L-aspartic acid, L-glutamic yamatsutae) at Yeongwol, Gangwon Province, Korea. The acid, L-histidine, L-serine, pectin, galacturonic acid, galac DNA G+C content of the type strain is 38 mol%. tonic lactone, D-glucuronic acid, glucuronamide, Tween40, acetoacetic acid and acetic acid as sole carbon sources Description of Flavobacterium cutihirudinis R23R4 (Biolog GEN Ⅲ). Strain R23R4 (=NIBRBAC000503079) was isolated Cells are Gram-stain-negative, non-flagellated and rod from the gut of Korean striped bitterling (Acheilognathus shaped. Colonies are irregular, raised and yellow colored yamatsutae) at Yeongwol, Gangwon Province, Korea. The on R2A agar medium after 48 h incubation at 30℃. Cells DNA G+C content of the type strain is 34 mol%. reduce nitrate to nitrogen and positive for β-glucosidase, β-galactosidase, alkaline phosphatase, esterase (C4), Description of Gordonia didemni R33M6-1 esterase lipase (C8), leucine arylamidase, valine arylami dase, cystine arylamidase, trypsin, α-chymotrypsin, acid Cells are Gram-stain-positive, non-flagellated and rod phosphatase, naphthol-AS-BI-phosphohydrolase, α-galac shaped. Colonies are circular, flat, entire and apricot col tosidase, β-galactosidase, α-glucosidase, β-glucosidase, ored on MA medium after 48 h incubation at 30℃. Cells N-acetyl-β-glucosaminidase, α-fucosidase (API 20NE/ are positive for alkaline phosphatase, leucine arylamidase, ZYM). Assimilate D-glucose, L-arabinose, D-mannose, N- valine arylamidase, cystine arylamidase, acid phosphatase, February 2021 Lee et al. Unrecorded gut bacteria from Korean aquatic animals 7

Fig. 4. The phylogenetic tree based on 16S rRNA gene sequence showing the phylogenetic relatedness between the two isolated strains be longing to the phylum Bacteroidetes and the type species from each genus. The tree was mainly reconstructed using the NJ, ML, and MP algorithms. The filled diamonds indicate the branches present in the phylogenetic trees reconstructed using three different tree reconstruction methods. Numbers on the nodes represent bootstrap values with 1000 replicates (NJ/ML/MP). Escherichia coli ATCC 11775T was used as an outgroup. The bar indicates 0.05 accumulated substitutions per nucleotide. naphthol-AS-BI-phosphohydrolase, α-galactosidase, rase (C4), esterase lipase (C8), leucine arylamidase, valine α-glucosidase, β-glucosidase (API ZYM) and D-glucose, arylamidase, trypsin, acid phosphatase, naphthol-AS-BI- L-arabinose, D-mannose, N-acetyl-D-glucosamine, D-mal- phosphohydrolase (API 20NE/ZYM). Assimilate D-glu tose, gluconate, adipate, citrate, salicin, D-melibiose, propi cose, L-arabinose, D-mannose, D-mannitol, gluconate, onate, valerate, 3-hydroxy-butyrate, 4-hydroxy-benzoate, adipate, malate, propionate, valerate, 3-hydroxy-butyrate, inositol, D-sucrose, acetate (API 20NE/ID 32GN). Utilize L-proline, acetate (API 20NE/ID 32GN) and utilize α-D- D-trehalose, D-cellobiose, gentiobiose, sucrose, D-tur glucose, D-fructose, D-fructose, L-aspartic acid, L-serine, anose, N-glucosamine, neuraminic acid, α-D-glucose, D-gluconic acid, D-glucuronic acid, glucuronamide, citric D-mannose, D-fructose, 6-methyl-glucose, D-fucose, myo- acid, L-malic acid, succinic acid, Tween40, β-butyric inositol, glycerol, D-glucose, gelatin, L-glutamic acid, acid, propionic acid and acetic acid as sole carbon sources pectin, D-glucuronic acid, citric acid, α-glutaric acid, (Biolog GEN Ⅲ). L-malic acid, succinic acid, α-butyric acid, β-butyric acid, Strain R33M4 (=NIBRBAC000503081) was isolated acetoacetic acid, propionic acid and acetic acid as sole from the gut of Korean striped bitterling (Acheilognathus carbon sources (Biolog GEN Ⅲ). yamatsutae) at Yeongwol, Gangwon Province, Korea. The Strain R33M6-1 (=NIBRBAC000503080) was isolated DNA G+C content of the type strain is 66 mol%. from the gut of Korean striped bitterling (Acheilognathus yamatsutae) at Yeongwol, Gangwon Province, Korea. The Description of Yersinia kristensenii subsp. DNA G+C content of the type strain is 71 mol%. rochesterensis K11M4 Cells are Gram-stain-negative, non-flagellated and rod Description of Mycolicibacterium fortuitum subsp. shaped. Colonies are circular, convex, entire and beige acetamidolyticum R33M4 colored on MA medium after 48 h incubation at 10℃. Cells are Gram-stain-positive, non-flagellated and rod Cells reduce nitrate to nitrite and are positive for β-glu shaped. Colonies are circular, flat and beige colored on cosidase, β-galactosidase, alkaline phosphatase, leucine MA medium after 48 h incubation at 30℃. Cells reduce arylamidase, acid phosphatase, naphthol-AS-BI-phospho nitrate to nitrite and are positive for β-glucosidase, este hydrolase, β-galactosidase, α-glucosidase, N-acetyl-β-glu 8 JOURNAL OF SPECIES RESEARCH Vol. 10, No. 1

Fig. 5. The phylogenetic tree based on 16S rRNA gene sequence showing the phylogenetic relatedness between the three isolated strains belong ing to the phylum Firmicutes and the type species from each genus. The tree was mainly reconstructed using the NJ, ML, and MP algorithms. The filled diamonds indicate the branches present in the phylogenetic trees reconstructed using three different tree reconstruction methods. Numbers on the nodes represent bootstrap values with 1000 replicates(NJ/ML/MP). E. coli ATCC 11775T was used as an outgroup. The bar in dicates 0.05 accumulated substitutions per nucleotide. cosaminidase (API 20NE/ZYM). Assimilate D-glucose, shaped. Colonies are circular, convex, entire and yellow L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucos ish on MA medium after 48 h incubation at 30℃. Cells amine, D-maltose, gluconate, salicin, L-fucose, D-sorbitol, reduce nitrate to nitrogen and are positive for alkaline phos L-histidine, 2-ketogluconate, 3-hydroxy-butyrate, L-pro phatase, esterase (C4), esterase lipase (C8), leucine aryl line, L-rhamnose, D-ribose, inositol, D-sucrose, acetate, amidase, valine arylamidase, acid phosphatase, naphthol- lactate, L-alanine, 5-ketogluconate, glycogen (API 20NE/ AS-BI-phosphohydrolase (API ZYM). Assimilate D-glu ID 32GN) and utilize dextrin, D-maltose, D-trehalose, cose, L-arabinose, gluconate, adipate, malate, L-fucose, D-cellobiose, gentiobiose, sucrose, D-raffinose, D-melibi propionate, valerate, L-histidine, 3-hydroxy-butyrate, ose, β-D-glucoside, D-salicin, N-glucosamine, β-manno L-proline, D-ribose, itaconate, suberate, acetate, lactate, samine, N-galactosamine, α-D-glucose, D-mannose, L-alanine, 3-hydroxy-benzoate, L-serine (API 20NE/ID D-fructose, D-galactose, L-fucose, L-rhamnose, inosine, 32GN) and utilize β-D-glucoside, α-D-glucose, D-galac D-sorbitol, D-mannitol, myo-inositol, glycerol, D-glucose, tose, D-fucose, L-fucose, inosine, myo-inositol, glycerol, D-fructose, D-serine, glycyl-L-proline, L-alanine, L-argi gelatin, glycyl-L-proline, L-alanine, L-aspartic acid, nine, L-aspartic acid, L-glutamic acid, L-histidine, L-ser L-glutamic acid, pyroglutamic acid, L-serine, D-gluconic ine, pectin, galacturonic acid, galactonic lactone, D-glu acid, quinic acid, methyl pyruvate, L-lactic acid, α-glutaric conic acid, D-glucuronic acid, glucuronamide, mucic acid, acid, D-malic acid, L-malic acid, Tween40, β-butyric methyl pyruvate, D-lactic acid, citric acid, D-malic acid, acid, acetoacetic acid, acetic acid and formic acid as sole L-malic acid, succinic acid, Tween40, acetoacetic acid, carbon sources (Biolog GEN Ⅲ). acetic acid and formic acid as sole carbon sources (Biolog Strain O13M9 (=NIBRBAC000503084) was isolated GEN Ⅲ). from the gut of spotted steed (Biwia springeri) at Yeong Strain K11M4 (=NIBRBAC000503083) was isolated wol, Gangwon Province, Korea. The DNA G+C content from the gut of oily bitterling (Tanakia koreensis) at Cheong - of the type strain is 70 mol%. ju, Chungcheongbuk Province, Korea. The DNA G+C content of the type strain is 48 mol%. Description of Aerococcus viridans 719 Cells are Gram-stain-positive, flagellated and coccus Description of Paracoccus sanguinis O13M9 shaped. Colonies are circular, convex, entire and beige Cells are Gram-stain-negative, non-flagellated and rod colored on MA medium after 48 h incubation at 25℃. February 2021 Lee et al. Unrecorded gut bacteria from Korean aquatic animals 9

Cells reduce nitrate to nitrite and are positive for β-glu shaped. Colonies are circular, convex, entire and beige cosidase, β-galactosidase, alkaline phosphatase, esterase colored on TSA medium supplemented with 0.5% yeast (C4), leucine arylamidase, valine arylamidase, acid phos extract after 48 h incubation at 25℃. Cells reduce nitrate phatase, naphthol-AS-BI-phosphohydrolase, β-galactosi to nitrite and are positive for urease, alkaline phosphatase, dase, β-glucosidase (API 20NE/ZYM). Assimilate D-glu leucine arylamidase, acid phosphatase, naphthol-AS-BI- cose, L-arabinose, D-mannose, D-mannitol, N-acetyl-D- phosphohydrolase (API 20NE/ZYM). Assimilate acetate, glucosamine, D-maltose, gluconate, malate, phenyl-ace lactate, glycogen (API ID 32GN) and utilize D-fructose, tate, salicin, D-melibiose, L-fucose, D-sorbitol, 2-ketoglu pectin, galactonic lactone, D-gluconic acid, D-glucuronic conate, L-rhamnose, D-ribose, lactate, 5-ketogluconate, acid, glucuronamide, mucic acid, methyl pyruvate, D-lactic L-serine (API 20NE/ID 32GN) and utilize dextrin, D-malt acid, L-lactic acid and acetic acid as sole carbon sources ose, D-trehalose, D-cellobiose, gentiobiose, sucrose, sta- (Biolog GEN Ⅲ). chyose, D-raffinose, α-D-lactose, β-D-glucoside, D-salicin, Strain 775 (=NIBRBAC000503087) was isolated from α-D-glucose, D-mannose, D-fructose, D-galactose, D-man the gut of Leopard mandarin fish (Siniperca scherzeri) at nitol, glycerol, pectin, D-gluconic acid, methyl pyruvate Chungju, Chungcheongbuk Province, Korea. The DNA and acetoacetic acid as sole carbon sources (Biolog GEN G+C content of the type strain is 27 mol%. Ⅲ). Strain 719 (=NIBRBAC000503085) was isolated from Description of Citrobacter portucalensis 765 the gut of Leopard mandarin fish (Siniperca scherzeri) at Cells are Gram-stain-negative, flagellated and coccus Chungju, Chungcheongbuk Province, Korea. The DNA shaped. Colonies are circular, convex, entire and beige G+C content of the type strain is 39 mol%. colored on TSA medium supplemented with 0.5% yeast Description of Alcaligenes faecalis subsp. extract after 48 h incubation at 25℃. Cells reduce nitrate parafaecalis 176 to nitrite and are positive for β-galactosidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine Cells are Gram-stain-negative, flagellated and rod arylamidase, valine arylamidase, trypsin, acid phospha shaped. Colonies are circular, umbonate and beige colored tase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, on BHI agar medium after 48 h incubation at 25℃. Cells β-galactosidase, α-glucosidase (API 20NE/ZYM). Assim are positive for alkaline phosphatase, esterase (C4), leu ilate D-glucose, L-arabinose, D-mannose, D-mannitol, cine arylamidase, valine arylamidase, acid phosphatase, N-acetyl-D-glucosamine, D-maltose, gluconate, malate, naphthol-AS-BI-phosphohydrolase (API ZYM) and assi citrate, phenyl-acetate, D-melibiose, L-fucose, D-sorbitol, milate D-glucose, caprate, malate, citrate, phenyl-acetate, propionate, valerate, 2-ketogluconate, 3-hydroxy-butyrate, propionate, valerate, L-histidine, 3-hydroxy-butyrate, L-proline, L-rhamnose, D-ribose, inositol, D-sucrose, ace

L-proline, malonate, acetate, lactate, L-alanine (API 20NE/ tate, lactate, L-alanine, 5-ketogluconate, 3-hydroxy-benzo- ID 32GN). Utilize dextrin, D-cellobiose, gentiobiose, D- ate, L-serine (API 20NE/ID 32GN) and utilize dextrin, D- turanose, stachyose, D-melibiose, β-mannosamine, N-gal maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, actosamine, α-D-glucose, D-mannose, D-fructose, D-gal stachyose, D-raffinose, α-D-lactose, D-melibiose, β-D- actose, 6-methyl-glucose, D-fucose, L-fucose, L-rham glucoside, N-glucosamine, β-mannosamine, N-galactosa nose, D-mannitol, D-arabitol, myo-inositol, glycerol, D- mine, neuraminic acid, α-D-glucose, D-mannose, D-fruc fructose, D-serine, gelatin, glycyl-L-proline, L-alanine, tose, D-galactose, 6-methyl-glucose, D-fucose, L-fucose, L-arginine, L-glutamic acid, L-histidine, pyroglutamic L-rhamnose, inosine, D-sorbitol, D-mannitol, myo-inositol, acid, L-serine, pectin, galacturonic acid, galactonic lac glycerol, D-glucose, D-fructose, D-aspartic acid, D-serine, tone, D-gluconic acid, D-glucuronic acid, glucuronamide, glycyl-L-proline, L-alanine, L-arginine, L-aspartic acid, phenylacetic acid, methyl pyruvate, D-lactic acid, L-lactic L-glutamic acid, L-histidine, L-serine, pectin, galacturonic acid, citric acid, α-glutaric acid, D-malic acid, L-malic acid, galactonic lactone, D-gluconic acid, D-glucuronic acid, succinic acid, Tween40, β-butyric acid, acetoacetic acid, glucuronamide, mucic acid, D-saccharic acid, methyl acid, propionic acid and acetic acid as sole carbon sources pyruvate, D-lactic acid, L-lactic acid, citric acid, α-glutaric (Biolog GEN Ⅲ). acid, D-malic acid, L-malic acid, succinic acid, α-butyric = Strain 176 ( NIBRBAC000503086) was isolated from acid, β-butyric acid, α-butyric acid, propionic acid, acetic the gut of Leopard mandarin fish (Siniperca scherzeri) at acid and formic acid as sole carbon sources (Biolog GEN Chungju, Chungcheongbuk Province, Korea. The DNA Ⅲ). + G C content of the type strain is 56 mol%. Strain 765 (=NIBRBAC000503088) was isolated from the gut of Leopard mandarin fish (Siniperca scherzeri) at Description of Aliarcobacter faecis 775 Chungju, Chungcheongbuk Province, Korea. The DNA Cells are Gram-stain-negative, flagellated and rod G+C content of the type strain is 52 mol%. 10 JOURNAL OF SPECIES RESEARCH Vol. 10, No. 1

Description of Citrobacter gillenii 684 curonic acid, glucuronamide, D-lactic acid, Tween40, ace toacetic acid, propionic acid and acetic acid as sole carbon Cells are Gram-stain-negative, flagellated and rod sources (Biolog GEN Ⅲ). shaped. Colonies are circular, raised, entire and pink col Strain A52 (=NIBRBAC000503090) was isolated from ored on MacConkey agar medium after 48 h incubation the gut of Leopard mandarin fish (Siniperca scherzeri) at at 25℃. Cells reduce nitrate to nitrite and are positive for Chungju, Chungcheongbuk Province, Korea. The DNA urease, alkaline phosphatase, esterase (C4), leucine aryl G+C content of the type strain is 68 mol%. amidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase (API 20NE/ZYM). Description of Comamonas odontotermitis 771 Assimilate D-glucose, D-mannose, N-acetyl-D-glucos amine, gluconate, malate, propionate, L-histidine, L-pro Cells are Gram-stain-negative, flagellated and rod line, D-ribose, lactate, L-alanine, L-serine (API 20NE/ID shaped. Colonies are irregular, raised and beige colored on 32GN) and utilize dextrin, D-maltose, D-trehalose, D-cel TSA medium supplemented with 0.5% yeast extract after lobiose, D-raffinose, α-D-lactose, D-melibiose, β-D-glu 48 h incubation at 25℃. Cells reduce nitrate to nitrite and coside, D-salicin, N-glucosamine, β-mannosamine, N-gal are positive for alkaline phosphatase, esterase (C4), ester actosamine, neuraminic acid, α-D-glucose, D-mannose, ase lipase (C8), leucine arylamidase, valine arylamidase, D-fructose, D-galactose, 6-methyl-glucose, D-fucose, acid phosphatase, naphthol-AS-BI-phosphohydrolase (API L-fucose, L-rhamnose, inosine, D-sorbitol, D-mannitol, ZYM). Assimilate D-mannitol, gluconate, caprate, adi myo-inositol, glycerol, D-glucose, D-fructose, D-serine, pate, malate, phenyl-acetate, propionate, valerate, L-his glycyl-L-proline, L-alanine, L-arginine, L-aspartic acid, tidine, 2-ketogluconate, 3-hydroxy-butyrate, 4-hydroxy- L-glutamic acid, L-histidine, pyroglutamic acid, L-serine, benzoate, L-proline, D-ribose, itaconate, suberate, acetate, pectin, galacturonic acid, galactonic lactone, D-gluconic lactate, L-alanine (API 20NE/ID 32GN) and utilize dex acid, D-glucuronic acid, glucuronamide, mucic acid, trin, D-galactose, 6-methyl-glucose, D-fucose, L-fucose, D-saccharic acid, methyl pyruvate, D-lactic acid, L-lactic L-rhamnose, D-mannitol, D-arabitol, myo-inositol, glyce acid, citric acid, α-glutaric acid, L-malic acid, succinic acid, rol, D-glucose, D-fructose, D-aspartic acid, L-alanine, L- γ-butyric acid, α-butyric acid, β-butyric acid, α-butyric arginine, L-aspartic acid, L-glutamic acid, L-histidine, acid, propionic acid and acetic acid as sole carbon sources pyroglutamic acid, L-serine, galacturonic acid, galactonic (Biolog GEN Ⅲ). lactone, D-gluconic acid, D-glucuronic acid, glucurona Strain 684 (=NIBRBAC000503089) was isolated from mide, phenylacetic acid, methyl pyruvate, D-lactic acid, the gut of Leopard mandarin fish (Siniperca scherzeri) at L-lactic acid, α-glutaric acid, D-malic acid, L-malic acid, Chungju, Chungcheongbuk Province, Korea. succinic acid, Tween40, γ-butyric acid, α-butyric acid, β-butyric acid, α-butyric acid, acetoacetic acid, propionic Description of Cryobacterium arcticum A52 acid, acetic acid and formic acid as sole carbon sources (Biolog GEN Ⅲ). Cells are Gram-stain-positive, non-flagellated and rod Strain 771 (=NIBRBAC000503091) was isolated from shaped. Colonies are circular, convex, entire and yellow the gut of Leopard mandarin fish (Siniperca scherzeri) at colored on TSA medium supplemented with 0.5% yeast Chungju, Chungcheongbuk Province, Korea. The DNA extract after 48 h incubation at 15℃. Cells reduce nitrate G+C content of the type strain is 62 mol%. to nitrite and are positive for β-glucosidase, β-galactosi dase, esterase (C4), esterase lipase (C8), leucine arylami Description of Comamonas nitrativorans A79 dase, valine arylamidase, acid phosphatase, naphthol-AS- BI-phosphohydrolase, α-galactosidase, β-galactosidase, Cells are Gram-stain-negative, flagellated and rod β-glucuronidase, α-glucosidase, β-glucosidase (API 20NE/ shaped. Colonies are circular, convex, entire and pink ZYM). Assimilate D-glucose, L-arabinose, D-mannose, colored on MacConkey agar medium after 48 h incuba D-mannitol, D-maltose, gluconate, salicin, D-melibiose, tion at 15℃. Cells are positive for esterase (C4), leucine L-fucose, 2-ketogluconate, 3-hydroxy-butyrate, L-rham arylamidase, valine arylamidase, α-chymotrypsin, acid nose, D-ribose, D-sucrose, lactate (API 20NE/ID 32GN) phosphatase, naphthol-AS-BI-phosphohydrolase, N-ace and utilize dextrin, D-maltose, D-trehalose, D-cellobiose, tyl-β-glucosaminidase (API ZYM) and assimilate caprate, gentiobiose, sucrose, D-turanose, stachyose, D-raffinose, malate, propionate, valerate, 3-hydroxy-butyrate, L-pro α-D-lactose, D-melibiose, β-D-glucoside, D-salicin, β- line, acetate, lactate, L-alanine (API 20NE/ID 32GN). mannosamine, α-D-glucose, D-mannose, D-fructose, D- Utilize dextrin, gentiobiose, D-melibiose, α-D-glucose, galactose, 6-methyl-glucose, D-fucose, L-fucose, L-rham D-mannose, D-fructose, D-galactose, 6-methyl-glucose, nose, D-sorbitol, D-mannitol, glycerol, D-fructose, L-argi D-fucose, L-fucose, L-rhamnose, D-fructose, D-serine, nine, L-glutamic acid, pyroglutamic acid, pectin, galac gelatin, glycyl-L-proline, L-aspartic acid, L-glutamic turonic acid, galactonic lactone, D-gluconic acid, D-glu acid, pectin, galacturonic acid, galactonic lactone, D-glu February 2021 Lee et al. Unrecorded gut bacteria from Korean aquatic animals 11 curonic acid, glucuronamide, L-lactic acid, L-malic acid genetics. Oxford; New York, Oxford University Press. and acetoacetic acid as sole carbon sources (Biolog GEN Qin, J., R. Li, J. Raes, M. Arumugam, K.S. Burgdorf, C. Ⅲ). Manichanh, T. Nielsen, N. Pons, F. Levenez, T. Yamada, Strain A79 (=NIBRBAC000503092) was isolated from D.R. Mende, J. Li, J. Xu, S. Li, D. Li, J. Cao, B. Wang, H. the gut of Leopard mandarin fish (Siniperca scherzeri) at Liang, H. Zheng, Y. Xie, J. Tap, P. Lepage, M. Bertalan, Chungju, Chungcheongbuk Province, Korea. J.M. Batto, T. Hansen, D. Le Paslier, A. Linneberg, H.B. Nielsen, E. Pelletier, P. Renault, T. Sicheritz-Ponten, K. Description of Enterococcus phoeniculicola 772 Turner, H. Zhu, C. Yu, S. Li, M. Jian, Y. Zhou, Y. Li, X. Zhang, S. Li, N. Qin, H. Yang, J. Wang, S. Brunak, J. Cells are Gram-stain-positive, non-flagellated and Dore, F. Guarner, K. Kristiansen, O. Pedersen, J. Parkhill, coccus shaped. Colonies are circular, convex, entire and J. Weissenbach, H.I.T.C. Meta, P. Bork, S.D. Ehrlich and J. white colored on TSA medium supplemented with 0.5% Wang. 2010. A human gut microbial gene catalogue estab yeast extract after 48 h incubation at 25℃. Cells are posi lished by metagenomic sequencing. Nature 464(7285):59- tive for arginine dihydrolase, β-galactosidase, esterase 65.

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