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A Report of 17 Unrecorded Bacterial Species of Korea Belonging to the Phylum Bacteroidetes

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A Report of 17 Unrecorded Bacterial Species of Korea Belonging to the Phylum Bacteroidetes Journal of Species Research 7(3):231-239, 2018 A report of 17 unrecorded bacterial species of Korea belonging to the phylum Bacteroidetes Jang-Cheon Cho1, Chi Nam Seong2, Kiseong Joh3, Chang-Jun Cha4, Jin-Woo Bae5, Hana Yi6, , Soon Dong Lee7, Myung Kyum Kim8 and Jung-Hoon Yoon9 * 1Department of Biological Sciences, Inha University, Incheon 22201, Republic of Korea 2Department of Biology, Sunchon National University, Suncheon 57922, Republic of Korea 3Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 17035, Republic of Korea 4Department of Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea 5Department of Biology, Kyung Hee University, Seoul 02447, Republic of Korea 6Department of Public Health Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea 7Faculty of Science Education, Jeju University, Jeju 63243, Republic of Korea 8Department of Bio & Environmental Technology, Seoul Women’s University, Seoul 01797, Republic of Korea 9Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea *Correspondent: [email protected] While screening indigenous prokaryotic species in Republic of Korea in 2017, a total of 17 bacterial strains assigned to the phylum Bacteroidetes were isolated from a variety of environmental habitats including water of fountain, tidal flat, plant root, soil, the gut of Russian grayling butterfly, ginseng field, seawater, lagoon and seashore sand. From the 16S rRNA gene sequence similarity of more than 98.7% and the formation of a robust phylogenetic clade with the closest species, it was found that the 17 strains belong to independent and recognized bacterial species. There has been no official report that the identified 17 species have been previously isolated in the Republic of Korea. Thus, 15 species in 10 genera of one family in the order Flavobacteriales, one species in one genus of one family in the order Cytophagales, and one species in one genus of one family in the order Sphingobacteriales are proposed as unrecorded species of the phylum Bacteroidetes found in the Republic of Korea. Gram reaction, colony and cell morphology, basic phenotypic characteristics, isolation source, taxonomic status, strain ID and other information are described in the species descriptions. Keywords: ‌prokaryote, 16S rRNA gene, Bacteroidetes, unrecorded species, prokaryotic diversity Ⓒ 2018 National Institute of Biological Resources DOI:10.12651/JSR.2018.7.3.231 INTRODUCTION of microbial diversity is becoming increasingly import- ant, a good deal of effort has been dedicated to discover Microorganisms, including prokaryotes, have been es- novel microorganisms that have not been yet discovered timated to occupy more than approximately 60% of the (Connon and Giovannoni, 2002; Cho and Giovannoni, total biomass on earth and are known to play important 2004; Yoon et al., 2011). roles in sustaining ecological system. Microorganisms While screening indigenous prokaryotic species from are arguably the most valuable resources in biotechnol- a variety of environmental samples in the Republic of ogy, and thus the demand for novel and useful micro- Korea in 2017, we isolated a number of novel and unre- organisms is expected to increase in the future due to corded bacterial species. The identified bacterial species their high economic value. Nevertheless, the majority of belonged to the classes/phyla Proteobacteria, Bacteroi- microorganisms in nature have not been cultured in the detes, Firmicutes and Actinobacteria. Of these bacterial laboratory, because the current methods can cultivate species, the present study focuses on the descriptions of only a small fraction (< 0.1%) of microbial cells (Delong unrecorded species belonging to the phylum Bacteroi- et al., 1989; Giovannoni et al., 1990). Since the value detes. The phylum Bacteroidetes encompasses a pheno- 232 JOURNAL OF SPECIES RESEARCH Vol. 7, No. 3 typically diverse group of Gram-negative and non-en- (1969) with the program DNADIST. The stability of re- dospore-forming bacteria (Krieg et al., 2010). Members lationships was assessed by bootstrap analysis based on of the phylum Bacteroidetes have been described from 1000 re-samplings of the neighbor-joining dataset by us- a variety of habitats including terrestrial, marine and ing the programs SEQBOOT, DNADIST, NEIGHBOR aquatic environments, gastrointestinal tracts of animals and CONSENSE of the PHYLIP package. and other environments (Thomas et al., 2011; Sun et al., 2016). It this study, we report 17 unrecorded bacterial species, isolated from Republic of Korea, belonging to RESULTS AND DISCUSSION three families in three orders in the phylum Bacteroide- tes. Strains assigned to the phylum Bacteroidetes On the basis of 16S rRNA gene sequence comparisons MATERIALS AND METHODS and phylogenetic analyses, a total of 17 strains were as- signed to the phylum Bacteroidetes. The 17 strains were A total of 17 bacterial strains were isolated from di- distributed in three orders of the phylum Bacteroidetes; verse environmental samples including water of foun- 15 strains in the Flavobacteriales, one strain in the Cy- tain, tidal flat, plant root, soil, gut of Russian grayling tophagales, and one strain in the Sphingobacteriales butterfly, ginseng field, seawater, lagoon and seashore (Table 1). These strains were Gram-staining-negative, sand (Table 1). Each sample was processed separately, chemoheterotrophic, and rod-shaped bacteria (Fig. 1). spread onto diverse culture media including R2A, ma- Colony size, morphology, and phenotypic characteristics rine agar 2216 (MA; Difco), trypticase soy agar (TSA; are shown in the species descriptions. Difco) or seawater agar, and incubated at 25-30℃ for A total of 15 strains assigned to the order Flavobacte- 2-5 days (Table 1). The designated strain ID, taxonomic riales belonged to the family Flavobacteriaceae (Fig. 2, information, isolation sources, culture media and incu- Table 1). The strains assigned to the family Flavobacte- bation conditions are summarized in Table 1. All strains riaceae belonged to the genera Bizionia, Chryseobacte- were purified as single colonies and stored as 10-20% rium, Cellulophaga, Dokdonia, Flavimarina, Flavobac- glycerol suspension at -80℃ as well as lyophilized terium, Maribacter, Mesoflavibacter, Salegentibacter ampoules. and Zobellia, and were isolated mainly from a variety of Colony morphology of isolated strains was observed habitats (Fig. 2, Table 1). Phylogenetic analyses based on agar plates after their cells were grown to stationary on 16S rRNA gene sequences showed that the 15 strains phase. Cellular morphology and cell size were examined are members of the following species: Bizionia para- by light microscopy, transmission electron microscopy gorgiae (Nedashkovskaya et al., 2005a), Chryseobacte- or scanning electron microscopy. Gram staining was rium culicis (Kämpfer et al., 2010), Chryseobacterium performed using a Gram-staining kit or the standard pro- formosense (Young et al., 2005), Chryseobacterium cedures. Phenotypic characteristics were tested by using sediminis (Kämpfer et al., 2015), Chryseobacterium API 20NE galleries (bioMérieux) according to the man- shandongense (Yang et al., 2015), Chryseobacterium ufacturer’s instructions. viscerum (Zamora et al., 2012), Cellulophaga lytica (Jo- DNA extraction, PCR amplification of 16S rRNA hansen et al., 1999), Dokdonia diaphoros (Yoon et al., gene and 16S rRNA gene sequencing were performed 2012), Flavimarina pacifica (Nedashkovskaya et al., using the standard procedures described elsewhere 2014), Flavobacterium hibernum (McCammon et al., (Yoon et al., 2012). The 16S rRNA gene sequences of 1998), Maribacter spongiicola (Jackson et al., 2015), the strains assigned to the phylum Bacteroidetes were Mesoflavibacter zeaxanthinifaciens (Asker et al., 2007), compared with those of other bacterial species with val- Salegentibacter echinorum (Xia et al., 2013), Salegenti- idly published names using the EzBioCloud database bacter mishustinae (Nedashkovskaya et al., 2005b) and (Yoon et al., 2017). For phylogenetic analyses, align- Zobellia russellii (Nedashkovskaya et al., 2004). ment of sequences was carried out with CLUSTAL W One strain (JH2) assigned to the family Cytophaga- software (Thompson et al., 1994). Phylogenetic trees ceae of the order Cytophagales belonged to the genus were inferred by using three tree-making algorithms: the Runella and was isolated from a water of fountain (Fig. neighbor-joining (Saitou and Nei, 1987), maximum-like- 2, Table 1). From phylogenetic analysis based on 16S lihood (Felsenstein, 1981) and maximum-parsimony rRNA gene sequences, strain JH2 was identified as a (Kluge and Farris, 1969) methods implemented within member of Runella slithyformis (Larkin and Williams, the PHYLIP package (Felsenstein, 1993). Evolutionary 1978). distance matrices for the neighbor-joining method were One strain (Amx_S_17) assigned to the family Chiti- calculated by using the algorithm of Jukes and Cantor nophagaceae of the order Sphingobacteriales belonged August 2018 Table 1. Summary of isolates belonging to the phylum Bacteroidetes and their taxonomic affiliations. 16S rRNA gene Medium Condition Order Family Genus Strain no. NIBR ID Closest species sequence Isolation source for for similarity incubation incubation (%) Cytophagales Cytophagaceae Runella JH2 VYWGBAC000000145 Runella slithyformis 99.3 Water of fountain R2A 25°C, 3d Bizionia GH1-5 VYWGBAC000000063 Bizionia paragorgiae 99.8
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