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A Report of 14 Unrecorded Bacterial Species in Korea Isolated in 2017

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A Report of 14 Unrecorded Bacterial Species in Korea Isolated in 2017 Journal of Species Research 7(2):161-180, 2018 A report of 14 unrecorded bacterial species in Korea isolated in 2017 Ju-Young Kim1, Jun Hwee Jang1, Soohyun Maeng2, Myung-Suk Kang3 and Myung Kyum Kim1,* 1Department of Bio & Environmental Technology, College of Natural Science, Seoul Women’s University, Seoul 01797, Republic of Korea 2Department of Public Health Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea 3Biological Resources Utilization Department, National Institute of Biological Resources, Incheon 22689, Republic of Korea *Correspondent: [email protected] Fourteen bacterial strains, low10-4-1, J11015, 17J27-22, 17G22-9, 17G9-4, 17Bio_15, 17gy_33, 17SD1_21, Strain8, 17Sr1_17, J21014T, H31021, 17J49-9, and 17J80-6 assigned to the phylum Actinobacteria, Bacteroidetes, Deinococcus-Thermus, and Firmicutes were isolated from soil samples. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strains low10-4-1, J11015, 17J27-22, 17G22-9, 17G9-4, 17Bio_15, 17gy_33, 17SD1_21, Strain8, 17Sr1_17, J21014T, H31021, 17J49-9, and 17J80-6 were most closely related to Marmoricola aurantiacus (98.9%), Calidifontibacter indicus (99.8%), Gordonia soli (98.8%), Rhodococcus globerulus (99.5%), Pseudarthrobacter siccitolerans (99.1%), Hymenobacter qilianensis (98.7%), Hymenobacter terrae (99.0%), Deinococcus yunweiensis (99.2%), Deinococcus proteolyticus (99.7%), Domibacillus indicus (99.2%), Exiguobacterium mexicanum (100.0%), Kurthia senegalensis (99.1%), Lysinibacillus composti (99.6%), and Bacillus loiseleuriae (99.3%). These fourteen species have never been reported in Korea, therefore we report them here for the first time. Keywords: 16S rRNA, Actinobacteria, bacterial diversity, Bacteroidetes, Deinococcus-Thermus, Firmicutes, unreported species Ⓒ 2018 National Institute of Biological Resources DOI:10.12651/JSR.2018.7.2.161 INTRODUCTION non-spore-forming bacteria (Madigan et al., 2005). They have DNA with 40-48% of GC content. Bacteroides are There are massive numbers of bacteria on earth present in the environments such as in soil, sediments, uniquely adapted to many environments. We isolated sea water and in the gastrointestinal system of animals species that have not been officially reported in Korea. (Johnson et al., 2016). This genus, tends to attract more In 2017, we collected diverse soil samples in Seoul, and more attention because of its abundance in the guts Jeju-do, and Gyeonggi-do in Korea and isolated unre- of human (Scarpellini et al., 2015). Several novel spe- ported bacterial species. The identified bacterial species cies have been recently proposed in the genus Bacteroi- belonged to the phyla Actinobacteria, Bacteroidetes, des (Hayashi et al., 2007; Sakamoto et al., 2011). These Deinococcus-Thermus, and Firmicutes. taxonomic studies were mainly based on 16S rRNA The Actinobacteria are the largest phyla and ubiq- gene sequence analysis (Shah et al., 2009). uitously distributed in both aquatic and terrestrial eco- Deinococcus-Thermus is a phylum of gram-positive systems (Barka et al., 2016). They are Gram-positive bacteria, also known as extremophiles (Griffith et al., filamentous bacteria with a high G+C content in their 2007). The group is distinguished by species that resist genomes. They are mostly mesophilic and can grow at the lethal effects of exposure to ionizing radiation and temperatures between 25 and 30°C however, thermo- UV light, and by species that thrive at high temperature philic Actinobacteria can grow at temperatures ranging (Battista, 2016). Deinococcus-Thermus is comprised of from 50 to 60°C (Edwards, 1993). They have numerous two orders, the Deinococcales and the Thermales. Gen- potential benefits for humans as some sources of antibi- erally, mesophilic species of Deinococcales demonstrate otics, antifungals, anticancer agents, and their secondary uncommon resistance to electromagnetic radiations metabolites are resistant to diseases (Barka et al., 2016). (Battista, 2016). The Thermales are thermophilic, can Bacteroides are a genus of Gram-negative, rod-shaped, grow at 60 and 80°C, but show no evidence of resistance 162 JOURNAL OF SPECIES RESEARCH Vol. 7, No. 2 to electromagnetic radiation. Despite these prominent Biochemical characteristics were performed by using and distinctive phenotypic differences, 16S ribosomal Biolog Microstation with GEN III microplate system. A ribonucleic acid sequences verify that members of these single colony was selected and emulsified into ‘inoculat- orders are evolved from same ancestor (Ho et al., 2016). ing fluid A’ (Biolog) for subsequent inoculation on to the So far, many Deinococcus sp. has been isolated based MicroPlate test plate (Biolog). More fastidious organisms, on 16S rRNA sequencing and proved for its radiation re- including capnophilic strains, were cultured on alterna- sistance (Srinivasan et al., 2012a; 2012b). tive media, according to the manufacturer’s instructions, The phylum Firmicutes is one of the most predominant and the inoculate was prepared to a specified transmit- groups of prokaryotes in the microbiota of human’s guts. tance using a turbidimeter, as specified in the user guide. They include genera of outstanding relevance in biomed- For each isolate, 100 μL of the cell suspension was in- icine, health care, and industry (Lanza et al., 2015). Fir- oculated into each well of the MicroPlate, using a multi- micutes are gram-positive with low G+C content and channel pipette and incubated at 37°C for 24 h, according has specific property to form heat resistant endo-spore to growth characteristics. MicroPlates were read in the (Galperin, 2015). There is a consensus that they have di- MicroStation semi-automated reader after 24 h and re- verged from other bacterial phyla at an early stage (Lake sults interpreted by the identification system’s software et al., 2009). Later, the evolution of Firmicutes obviously (GEN III database, version 5.2.1). The system indicates included numerous events of lateral gene transfer to and the isolates which could not be identified after 20 h, are from representatives of other phyla and hence certain subjected to further incubation. Consequently, such iso- gene families are shared Firmicutes with Fusobacteria, lates were re-incubated and re-read between 3 and 6 h Thermotogae, and other groups (Mira et al., 2004). later (Wragg et al., 2014). Genomic DNA was extracted and 16S rRNA gene was amplified by PCR with 9F and 1492R universal bacterial MATERIAL AND METHODS primers (Weisburg et al., 1991). The 16S rRNA gene sequences of the closely related strains were obtained Bacteria were isolated from different soil samples then from EzTaxon-e (http://eztaxon-e.ezbiocloud.net) (Kim were suspended on distilled water and serially diluted. et al., 2012) and edited using the BioEdit program (Hall, The aliquot was inoculated onto R2A agar and incubat- 1999). Multiple alignments were performed with the ed at 25°C for 3 days. The designated strain name, iso- MUSCL program (Edgar, 2004). Using the two-param- lation sources, growth media, and incubation conditions eter model (Kimura, 1983), calculated the evolutionary are summarized in Table 1. All strains were purified as distances. Phylogenetic trees were constructed using single colonies and stored as 20% glycerol suspension at the neighbor-joining (Saitou and Nei, 1987) in MEGA5 -80°C as well as freeze dried ampoules. Colony mor- program (Tamura, 2011) with bootstrap values based on phology and cell size of the strains were observed by 1,000 replications (Felsenstein, 1985). transmission electron microscopy (LIBRA 120, Carl Zeiss) using cells grown for 3 days at 25°C on R2A agar. Transmission electron micrograph of the strains are RESULTS AND DISCUSSION shown in Fig. 1. Gram reaction was tested following the classic Gram procedure described by Doetsch (1981). Based on 16S rRNA gene sequence similarity, four- Table 1. List of 16S rRNA gene sequence similarity, isolation source, medium, and incubation conditions of unrecorded strains. Strain ID Most closely related species Similarity (%) Isolation source Medium Incubation conditions low10-4-1 Marmoricola aurantiacus 98.9 Soil R2A 25°C, 3d J11015 Calidifontibacter indicus 99.8 Soil R2A 25°C, 3d 17J27-22 Gordonia soli 98.8 Soil R2A 25°C, 3d 17G22-9 Rhodococcus globerulus 99.5 Soil R2A 25°C, 3d 17G9-4 Pseudarthrobacter siccitolerans 99.1 Soil R2A 25°C, 3d 17Bio_15 Hymenobacter qilianensis 98.7 Soil R2A 25°C, 3d 17gy_33 Hymenobacter terrae 99.0 Soil R2A 25°C, 3d 17SD1_21 Deinococcus yunweiensis 99.2 Soil R2A 25°C, 3d Strain8 Deinococcus proteolyticus 99.7 Soil R2A 25°C, 3d 17Sr1_17 Domibacillus indicus 99.2 Soil R2A 25°C, 3d J21014T Exiguobacterium mexicanum 100.00 Soil R2A 25°C, 3d H31021 Kurthia senegalensis 99.13 Soil R2A 25°C, 3d 17J49-9 Lysinibacillus composti 99.66 Soil R2A 25°C, 3d 17J80-6 Bacillus loiseleuriae 99.32 Soil R2A 25°C, 3d May 2018 Kim et al. Unrecorded bacterial species from Korea 2017 163 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) Fig. 1. Transmission electron micrographs of the strains isolated in this study. Strains: 1, low10-4-1; 2, J11015; 3, 17J27-22; 4, 17G22-9; 5, 17G9-4; 6, 17Bio_15; 7, 17gy_33; 8, 17SD1_21; 9, Strain8; 10, 17Sr1_17; 11, J21014T; 12, H31021; 13, 17J49-9; 14, 17J80-6. teen of previously unreported bacterial species were uobacteriaceae (1 strain), and Bacillaceae (1 strain) of identified. The taxonomic composition and identifica- the phylum Firmicutes. At generic level, the strains be- tion results were summarized in Table 1.
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