Introduction

UV radiation has a major impact on the environment and damages living organisms. UV radiation is readily absorbed by DNA and leads to mutations (D’Orazio et al. 2013). Some UV radiation can trigger the formation of reactive oxygen species and causes cell or tissue damage in the form of oxidation of DNA, proteins and lipids (Choi et al. 2018; Jang et al. 2017). Nevertheless, living cells have developed the ability to self-repair their own DNA damage (Yu and Lee 2017). Overall, the level of ionizing radiation resistance is related to genetic and physiological characteristics of organisms. Systems such as the enzymatic machinery of DNA repair, perform important roles in cell recovery after exposure to ionizing radiation (Krisko and Radman 2013). Studies on microbial diversity and isolation of novel radiation resistant bacteria are widely reported, as well as the complete genome sequences of UV resistant bacteria such as Hymenobacter sedentarius, Hymenobacter spp., Methylobacterium spp., Microvirga spp., Nibribacter radioresistens and Spirosoma pulveris, (Kang and Srinivasan 2018; Kim et al. 2017a, b; Sathiyaraj et al. 2018a, b; Srinivasan et al. 2017), in order describe their role on ionizing radiation resistance.

The genus Hymenobacter was first described by Hirsch et al. (1998) and belongs to the family Hymenobacteraceae. At the time of writing, the genus comprises 67 species (http://www.bacterio.net/hymenobacter.html). Hymenobacter species have been found to survive in extreme conditions such as sandstone sediments (Hymenobacter qilianensis; Han et al. 2014), freshwater (Hymenobacter. aquatilis; Kang et al. 2018), desiccation (Hymenobacter deserti; Zhang et al. 2009), radiation (Hymenobacter sedentarius; Kim et al. 2017a), heavy metals (Hymenobacter flocculans; Chung et al. 2010) and glacial ice (Hymenobacter antarcticus; Klassen and Foght 2011). In general, members of Hymenobacter are rod-shaped, Gram-negative, red to pink-coloured and aerobic with menaquinone-7 (MK7) as their major isoprenoid quinone and phosphatidylethanolamine (PE) as the major polar lipid. Moreover, according to previous iso-C15 : 0, anteiso-C15 : 0, C16 : 1ω5c, summed feature 3 (C 16 : 1ω7c and/or C16 : 1ω6c) and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B) as the major fatty acids (Zhu et al. 2017). In this study we isolated a novel UV radiation tolerant strain, 17J68-5T, from a soil sample collected on Jeju Island, South Korea and the 16S rRNA sequence similarity determined that strain 17J68-5T belongs to the genus Hymenobacter. The strain was subjected to a polyphasic analysis was conducted to determine the precise taxonomic position of strain 17J68-5T.

Materials and methods

Strain isolation and maintenance

Strain 17J68-5T was isolated from a soil sample collected in Jeju Island (33.450049°N, 126.321168°E), South Korea. 0.2 g soil sample was added to 0.9 mL of sterile water (10−1 dilution) and 100 µL of the suspension were spread on the surface of R2A agar plates at 28 °C and incubated for 1 week. An isolate which formed red to pink coloured colonies was isolated and purified using R2A medium (Difco). The plates were incubated at 25 °C for 3 days. The colonies were maintained as glycerol stocks (20%, w/v) in R2A medium and the ampoules were stored at − 80 °C. The purified colonies were identified using partial 16S rRNA gene sequences using the EzTaxon server (https://www.ezbiocloud.net/) (Yoon et al. 2017).

The reference strain Hymenobacter daecheongensis KCTC 22258T, obtained from Korean Collection of Type cultures and was cultured in R2A medium at 25 °C for 3 days and included for experiments for comparative purposes. All the strains were grown in the same conditions for the experiments.

Phenotypic characteristics

The morphological characteristics of strain 17J68-5T were examined using cells grown on R2A agar for 2–3 days at 25 °C by transmission electron microscopy (JEOL, JEM1010). Colony characteristics were observed after incubation of the bacterial cells at 25 °C for 3 days on R2A agar (Difco). Growth on different bacteriological culture media was evaluated by using R2A agar (Difco), nutrient agar (NA; Difco), tryptone soya agar (TSA; Difco) and Luria–Bertani agar (LBA; Difco). Gram-staining was performed using a commercial kit, following the manufacturer’s instruction (bioMérieux). A motility test was performed in SIM medium (Oxoid) and R2A medium supplemented with 0.4% agar. Oxidase and catalase activity were examined by the addition of 1% (w/v) tetramethyl-p-phenylene diamine and 3% (w/v) H2O2 solution, respectively (Cappuccino and Sherman 2002). Growth at different temperatures (4, 10, 15, 18, 20, 25, 30, 37, 42 and 45 °C) was assessed on R2A agar for 3 days at 25 °C. NaCl tolerance of cells was tested up to 2% salt concentration. Growth pH (4 to 10, 0.5 pH intervals) and different salt concentrations was assessed on R2A medium for 3 days and at 25 °C. For the pH test, the R2A medium was adjusted to pH 4.0–11.0 (at intervals of 0.5 pH units) prior to autoclaving using 0.1 M citrate/NaH2PO4 buffer (for pH range 4.0–5.5), 0.1 M phosphate buffer (for pH range 6–7.5), 0.2 M Tris buffer (for pH range 8–10) and 5 M NaOH (for pH range 10.5–11.0). API 20NE, 32GN and API ZYM tests were performed according to the instructions of the manufacturer (bioMérieux).

Genome sequencing and phylogenetic analysis

The genomic DNA was extracted using a commercial genomic DNA extraction kit (Solgent, Korea). The draft genome sequence of strain 17J68-5T was sequenced using a PacBio RS II platform at DNA Link (www.dnalink.com), Korea. The assembly of the genome sequence was done using a hierarchical genome assembly process (SMRT Analysis HGAP.3 Version 2.3) including consensus polishing with Quiver (Chin et al. 2013). The genome sequence has been deposited in GenBank (www.ncbi.nlm.nih.gov/) database and annotated using the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016). To estimate the overall similarity among compared genomes, average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) was used. ANI values were calculated using the Orthologous Average Nucleotide Identity Tool version 0.98 (Lee et al. 2015) and dDDH using the Genome-to Genome Distance Calculator (GGDC) web server (http://ggdc.dsmz.de).

The 16S rRNA genes of strain 17J68-5T were amplified using the 27F and 1492R universal bacterial primer set (Weisburg et al. 1991), then sequenced by Macrogen (Korea) using the 337F, 518R, 785F, and 926R universal bacterial primer sets (Kim et al. 2005). The 16S rRNA sequences of related taxa were obtained from EzBioCloud and edited with the EzEditor2 program (Jeon et al. 2013). Multiple alignments were performed with the CLUSTAL X program (Thompson et al. 1997). The gaps at 5′ and 3′ends was cut using Bioedit (Hall 1999). Phylogenetic trees were constructed using the MEGA7 program (Tamura et al. 2016) using the neighbour-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) methods. Evolutionary distances were calculated with the Kimura two-parameter model (Kimura 1983). The bootstrap values were calculated based on 1000 replicates (Felsenstein 1985). The GenBank accession number for the 16S rRNA gene sequence of strain 17J68-5T is MH588267.

Chemotaxonomic studies

To identify fatty acids, cells were incubated on R2A for 3 days at 25 °C. Fatty acids were then purified by saponification, methylation and extraction procedures, as described previously (Sasser 1990). The fatty acid methyl esters were identified using the Sherlock Microbial Identification System V6.01 (MIS, database TSBA6, MIDI Inc., Newark, DE, USA). Polar lipids were extracted (Minnikin et al. 1984), separated using two-dimensional thin layer chromatography and the different spots were observed by spraying with the appropriate detection reagents (Komagata and Suzuki 1987). Respiratory quinones were extracted with Sep-Pak Vac cartridges (Waters, USA) and menaquinones were analysed by high performance lipid chromatography based on previous methods (Hiraishi et al. 1996).

UV radiation survival test

For identification of radiation resistant bacteria, pure cultures of isolates were used. The UV resistance survival test was studied according to Im et al. (2013) and Selvam et al. (2013). Cells were irradiated with a UVC cross-linker (UVP, CX-2000, USA) at 254 nm and was used with different dose adjustments. After being exposed to UV radiation, the survival rates of strain 17J68-5T, Deinococcus radiodurans R1T (DSM 20539T), positive control) and Escherichia coli K-12 (KCTC 1116T, negative control) were measured using cells in the early stationary phase (≈ 109 c.f.u. ml−1) on R2A agar medium (Difco). The numbers of colony-forming units (CFU) of the strains were counted and the survival rate was calculated based on CFU values.

Results and discussion

Phenotypic characterisation

The images from transmission electron microscopy revealed the morphology of strain 17J68-5T to be short rod shaped with a size range of 0.6–0.8 × 0.8 µm (Supplementary Fig. S1). The colonies of strain 17J68-5T are convex, smooth, circular and pink-coloured after 3 days growth at 25 °C on R2A agar medium. Cells are Gram-negative and the strain can grow between 18 and 37 °C with pH range 6.0 and 7.5. No growth was observed in the presence of NaCl (up to 2%) in R2A broth. Growth was observed on NA and R2A but not on TSA agar. The other physiological characteristics of the strain 17J68-5T and related type strains are shown in Table 1. In API 20NE tests, esculin hydrolysis, gelatin hydrolysis, β-galactosidase are positive, but negative reactions were observed for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-d-glucosamine, d-maltose, gluconate, caprate, adipate, l-malate, citrate and phenyl acetate. In API ZYM tests, strain 17J68-5T was found to produce alkaline phosphatase, esterase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, starch hydrolysis, β-glucosidase, N–acetyl-β-glucosaminidase, esterase (C4) (weak, w), lipase (C14) (w), β-glucuronidase (w) and α-mannosidase (w). Does not produce α-chymotrypsin and α-fucosidase.

Table 1 Differential characteristics of strain 17J68-5T and closely related species of Hymenobacter. Strains: 1, 17J68-5T; 2, H. daecheongensis KCTC 22258T; 3, H. rutilus K2-33028T; 4, H. tibetensis
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Genome sequence and phylogenetic analysis

The draft genome sequence of strain 17J68-5T is 5.1 Mb and consists of 5996 total genes, among them 5746 coding genes (CDS), 123 RNA and 127 pseudogenes with G + C content of 59.6% (Table S1). The draft genome sequence of the strain 17J68-5T has been deposited in GenBank under the accession number CP040896. The PCR amplification and sequencing of the 16S rRNA gene was performed and the amplified product size was 1451 bp. The 16S rRNA gene sequences from PCR and the draft genome sequence were identical. Multiple sequence alignment was carried out with 16S rRNA sequences of 30 type strains in the genus Hymenobacter. The phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 17J68-5T is closely related to H. daecheongensis DSM 21074T (94.9% 16S rRNA gene sequence similarity), Hymenobacter rutilus K2-33028T (94.6%) and Hymenobacter tibetensis XTM003T (94.3%). The sequence similarities between strain 17J68-5T and other Hymenobacter species were less than 94%. The phylogenetic analysis presented by a neighbor-joining tree (Fig. 1), together with the topologies generated by the maximum likelihood and maximum parsimony algorithms (Supplementary Figs. S2 and S3), showed the relationship of strain 17J68-5T with other Hymenobacter species. A threshold of < 97% similarity in 16S rRNA gene sequence was proposed for bacterial species delineation by Stackebrandt and Goebel (1994). Later, the species delineation threshold was increased to < 98.7  % (Kim et al. 2014). On the basis of threshold values, the above data indicate that strain 17J68-5T represents a novel species of the genus Hymenobacter. For further confirmation, the whole genome sequence of strain 17J68-5T was analysed for ANI and dDDH with related available genomes of Hymenobacter species. The calculated orthoANI values between strain 17J68-5T with closely related H. daecheongensis Dae14T, H. norwichensis DSM 15439T, H. perfusus A1-12T and H. swuensis DY53T were 75.4, 73.8, 73.8 and 73.7%, respectively (Fig. S4). The dDDH values between strain 17J68-5T and H. daecheongensis Dae14T, H. norwichensis DSM 15439T, H. perfusus A1-12T and H. swuensis DY53T were 20.4, 19.5, 19.9 and 20.0% (Table S2). The information held in genome sequences provides objective and reliable methods for the taxonomy of prokaryotes (Chun et al. 2010). The ANI and dDDH values between the strain 17J68-5T and related taxa are below the cut-off values of ≥ 95–96% for ANI (Richter and Rosselló-Móra, 2009) and ≥ 70% for dDDH (Meier-Kolthoff et al. 2013) used to define bacterial species.

Fig. 1
figure 1

Phylogenetic analysis of strain 17J68-5T (in bold type) with closely related members of the genus Hymenobacter based on 16S rRNA gene sequences available from NCBI database (published). The tree was constructed using neighbor joining method and the same topologies was recovered from the maximum-likelihood and maximum-parsimony algorithms respectively. Bootstrap values (> 50%) based on 1000 replications are shown at the branch nodes. Bar, 0.01 substitutions per nucleotide position. Fulvivirga kasyanovii KMM 6220T (DQ836305) is used as an outgroup

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Chemotaxonomic characteristics

The major fatty acids of strain 17J68-5T were identified as summed feature 3(C16:1ω6c/C16:1ω7c) (21.2%), C15:0 iso (19.1%), summed feature 4 (C17:1 iso I/C17:1 anteiso B) (17.9%) and C16:1ω5c (13.1%). The fatty acid profiles of strain 17J68-5T compared with related strains are shown in Table 2. The fatty acid profile of strain 17J68-5T is similar to those of phylogenetically related strains. Strain 17J68-5T was found to contain major amount of PE, two unidentified aminophospholipids and one phospholipid and minor amounts of two unidentified lipids (Supplementary figure S5). The predominant respiratory quinones of strain 17J68-5T were found to be MK-6 (33.7%) and MK-7 (66.2%). The results of these chemotaxonomic characteristics are consistent with those of most species in the genus Hymenobacter (Hirsch et al. 1999).

Table 2 Cellular fatty acid profiles of strain 17J68-5T and closely related species
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In conclusion, the results of 16S rRNA gene sequence analysis, the presence of PE, two unidentified aminophospholipids and a phospholipid as major polar lipids, MK-7 as the major quinone and the fatty acid profile showed that the strain 17J68-5T is a member of the genus Hymenobacter. However, the differences such as the additional respiratory quinone and in the fatty acids (C12:0, C15:1ω6c, C17:1ω6c, summed feature 1 and summed feature 3), together with ANI and dDDH values (< 95–96%; < 70%), indicated that strain 17J68-5T can be distinguished from closely related Hymenobacter spp. Therefore, it is concluded that strain 17J68-5T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter jejuensis sp. nov. is proposed. The Digital Protologue database (Rosselló-Móra et al. 2017) TaxoNumber of strain 17J68-5T is TA00996.

UV radiation resistance

In the UV radiation survival test, strain 17J68-5T showed resistance to UV radiation (Fig. 2). Strain 17J68-5T was tolerant of UV irradiation, showing a survival rate of 10% after exposure to UV light at a dose of 300 J/m2. At this dose, D. radiodurans (DSM 20539T), exhibited a 98% survival rate and Escherichia coli K12 xhibited a 0% survival rate, respectively.

Fig. 2
figure 2

Representative survival plot for strain 17J68-5T (filled circle) following exposure to UV radiation. Survival rates of D. radiodurans R1T (filled square) and E. coli K12 (filled daimond) are also shown. The survival rate after UV radiation was measured using early stationary phase (~ 109 CFU/ml) cells on R2A agar. Each increment on the y-axis represents a tenfold reduction in viability

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Description of Hymenobacter jejuensis sp. nov

Hymenobacter jejuensis (je.ju.en′sis. N.L. fem. adj. jejuensis pertaining to Jeju Island in the Republic of Korea, from where the type strain was isolated).

Cells are Gram-stain negative, non-motile and rod-shaped. Colonies on R2A agar are convex, smooth, circular, pink-coloured and 4 mm in diameter after 3 days of growth at 25 °C. Cells are approximately 0.6–0.8 µm wide and 0.8–1.2 µm long. Growth occurs at 18–37 °C (optimum 25 °C) and at pH 6.0–7.5 (optimum 6.5). Cells do not show NaCl tolerance. Grows well on R2A agar and nutrient agar, but not on TSA and MacConkey agar. Catalase and oxidase activities are positive. The major polar lipid is phosphatidylethanolamine. The predominant respiratory quinones are menaquinone 6 and 7. The main cellular fatty acids are C15:0 iso, C16:1ω5c, summed feature 3 (C16:1ω6c/C16:1ω7c) and summed feature 4 (C17:1 iso I/C17:1 anteiso B). The genomic G + C mol % derived from the draft genome of the type strain is 59.6.

The type strain, 17J68-5T (= KCTC 62224T = JCM 33182T), was isolated from soil in South Korea. The GenBank accession number for the 16S rRNA gene sequence of strain 17J68-5T is MH588267. The draft genome sequence of strain 17J68-5T has been deposited in GenBank/DDBJ/EMBL under the accession number CP040896.