Introduction

The genus Hymenobacter of the family Cytophagaceae was first proposed by Hirsch et al. (1998) and its description was later emended by Buczolits et al. (2006) and Han et al. (2014). Recently, the genus was placed together with Adhaeribacter, Nibribacter, Pontibacter, and Rufibacter in the new family Hymenobacteraceae (Munoz et al. 2016). Currently, the genus Hymenobacter comprises 65 species with validly published names (http://www.bacterio.net/hymenobacter.html). Members of this genus are Gram-stain negative, rod-shaped, red-pigmented, non-motile, aerobic bacteria that contain phosphatidylethanolamine as the major polar lipid, MK-7 as the predominant menaquinone, and iso-C15:0, anteiso-C15:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 4 (iso-C17:1 I/anteiso-C17:1 B) as the major fatty acids. Hymenobacter species occupy a wide range of ecological niches, such as air (Buczolits et al. 2002), fresh water (Ten et al. 2017), soil (Buczolits et al. 2006), and extreme environments like arid land (Reddy and Garcia-Pichel 2013), glacier (Klassen and Foght 2011), and continental Antarctic soils (Hirsch et al. 1998).

During a screening for novel bacteria, strain 1-3-3-3T was isolated from a soil sample collected in Jeollabuk-do province, South Korea. Based on 16S rRNA gene sequence analysis, this isolate was assigned to the genus Hymenobacter. The strain was subjected to detailed investigation using a polyphasic taxonomic analysis that included genotypic, chemotaxonomic and phenotypic analyses. The results indicate that strain 1-3-3-3T should be placed in the genus Hymenobacter as the type strain of a novel species, here named Hymenobacter persicinus sp. nov.

Materials and methods

Isolation of bacterial strain and culture conditions

Strain 1-3-3-3T was isolated from a soil sample collected in Jeollabuk-do province, (35°41′04.50″N, 127°25′53.43″E), South Korea. The sampling location is a public area; therefore, no specific permissions were required to collect the material that was necessary for the present study, and the activities did not involve endangered or protected species. The soil texture was sandy clay with a pH of 7.2. The isolation was performed at 25 °C using the dilution plating technique with Reasoner’s 2A (R2A) medium (Difco, Sparks, MD, USA), as described previously (Han et al. 2018). The novel strain was routinely cultured on R2A agar at 25 °C, maintained at 4 °C and stored in a 20% (w/v) glycerol suspension at − 70 °C. Strain 1-3-3-3T has been deposited in the Korean Collection for Type Cultures (KCTC 52742T) and the Japan Collection of Microorganisms (JCM 32191T). Hymenobacter algoricola JCM 17214T, Hymenobacter fastidious JCM17224T, and Hymenobacter elongatus JCM 17223T were obtained from the Japan Collection of Microorganisms. Hymenobacter knuensis KCTC 52538 and Hymenobacter aquaticus KCTC 32554T were purchased from the Korean Collection for Type Cultures and Hymenobacter chitinivorans KACC 12068T was obtained from the Korean Agricultural Culture Collection.

Sequencing of the 16S rRNA gene and phylogenetic analysis

Genomic DNA was extracted from strain 1-3-3-3T using a commercial genomic DNA extraction kit (SolGent, Daejeon, South Korea). The 16S rRNA gene was amplified from chromosomal DNA using the universal bacterial primers 9F and 1512R, as described previously (Weisburg et al. 1991), and purified PCR products were sequenced by Genotech (Daejeon, South Korea). A nearly complete 16S rRNA gene sequence was compiled using SeqMan software (DNASTAR, Madison, WI, USA) and compared with sequences of closely related species using the EzBioCloud server (Yoon et al. 2017). The 16S rRNA gene sequences of related taxa were obtained from GenBank and assembled in BioEdit (Hall 1999). Multiple sequence alignments were performed with Clustal X 2.0 (Larkin et al. 2007). Phylogenetic trees were constructed using the neighbor-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981), and maximum-parsimony (MP) (Fitch 1971) methods in the program MEGA7 (Kumar et al. 2016). The NJ and ML phylogenetic trees were constructed using Kimura’s two-parameter model (Kimura 1980) and the ML tree was constructed using the nearest neighbor interchange heuristic search method. The MP tree was constructed using subtree pruning and regrafting. The option of complete deletion of gaps was applied for NJ, ML and MP tree construction. A bootstrap analysis with 1000 replicate datasets was performed to assess support for groups.

Phenotypic and biochemical characteristics

The Gram staining of strain 1-3-3-3T was examined using a previously described method (Smibert and Krieg 1994). The morphology of the isolate, grown for 3 days at 25 °C on R2A agar, was observed under a light microscope at 1000× magnification (Olympus, Tokyo, Japan) and a HT7700 transmission electron microscope (Hitachi, Tokyo, Japan). Motility was tested by stab inoculation of semi-solid R2A medium containing 0.6% (w/v) agar (Tittsler and Sandholzer 1936) and gliding motility was investigated by phase-contrast microscopy of cells incubated for 24 h on microscope slides coated with R2A medium (0.7% agar) (Bowman 2000). The anaerobic growth test was performed as described previously (Ten et al. 2006). Catalase activity was determined by bubble production in 3% (v/v) H2O2 and oxidase activity was examined using 1% (w/v) tetramethyl-p-phenylenediamine (Cappuccino and Sherman 2010). Aesculin hydrolysis was tested on R2A agar, as reported previously (Aslam et al. 2006). The ability to grow on different bacteriological media was assessed using R2A agar, Luria–Bertani (LB) agar, nutrient agar (NA), and trypticase soy agar (TSA) (all purchased from Difco). Growth at 4, 10, 15, 20, 25, 30, 37, 42 and 45 °C was assessed on R2A agar. Growth at different pH values (pH 4–10, in increments of 1 unit) and NaCl concentrations (0.5, 1, 1.5, 2, 3, 4, 5 and 10% [w/v]) was evaluated in R2A broth for 7 days as described previously (Lee et al. 2017a). Tests for nitrate and nitrite reduction were performed as described by La et al. (2005). Enzyme activities, assimilation of carbon sources, acid production from substrates and other physiological characteristics of the novel isolate and the reference strains were determined using the API ZYM, API 20 NE, API ID 32 GN, and API 50 CH kits according to the manufacturer’s instructions (bioMérieux, Marcy l’Etoile, France). The API 50 CH strips were inoculated with cells suspended in API 50 CHB/E medium (bioMérieux). The API 20 NE, API ID 32 GN, and API 50CH strips were inspected after 5 days of incubation at 18 °C for H. algoricola JCM 17214T, H. fastidious JCM17224T, and H. elongatus JCM 17223T or at 25 °C for other strains. The same temperature conditions and incubation time of 18 h were used to performed API ZYM tests with the above-mentioned strains.

Chemotaxonomic analyses

Following the growth conditions that were used for fatty acid analysis of the six references strains (Lee et al. 2017b; Ten et al. 2017), strain 1-3-3-3T was grown on R2A agar at 18 °C for 7 days. Cellular fatty acid methyl esters were prepared according to a Sherlock Microbial Identification System (MIDI) protocol. Fatty acid methyl esters were then analysed by gas chromatography and Microbial Identification Software (Sherlock TSBA, version 6.0) (Sasser 1990). For the analysis of polar lipids and respiratory isoprenoid quinones, cells of strain 1-3-3-3T were cultured, collected, and freeze-dried. Polar lipids were extracted using the procedure described by Minnikin et al. (1984) and identified by two-dimensional thin-layer chromatography, followed by spraying with appropriate detection reagents (Komagata and Suzuki 1987). Isoprenoid quinones were extracted and purified as described previously (Ten et al. 2008) and analysed according to the method of Hiraishi et al. (1996) with the following modifications. High-performance liquid chromatography (HPLC) analysis was performed on an Agilent 1100 series HPLC (Agilent Technologies, Waldbronn, Germany) equipped with G1315A photodiode array detector and ChemStation software. The chromatographic separation was achieved using Atlantis T3 C18 column (4.6 mm × 250 mm, 5.0 µm; Waters, Milford, USA) as the stationary phase and methanol/isopropanol in the proportion of 7:5 (v/v) as the mobile phase at a flow rate of 1.2 ml/min.

DNA-DNA hybridization and determination of DNA G + C content

The genomic DNA of strain 1-3-3-3T and the six closely related strains were extracted according to the standard cetyltrimethylammonium bromide/NaCl protocol (Wilson 1997). DNA-DNA hybridization was performed fluorometrically using the method described by Ezaki et al. (1989) with photobiotin-labelled DNA probes and microdilution wells. The DNA–DNA relatedness values presented are the mean ± standard deviation of five replicates.

The genomic DNA G + C content of strain 1-3-3-3T was analysed by reverse-phase HPLC of the individual nucleosides that resulted from DNA hydrolysis and dephosphorylation using nuclease P1 and alkaline phosphatase (Mesbah et al. 1989). HPLC analysis was carried out on the above-mentioned Agilent 1100 HPLC system. Nucleosides were separated using a SunFire C18 column (4.6 mm x 250 mm, 5 µm; Waters, Milford, USA) and a mobile phase of 0.2 M ammonium phosphate (pH 4.0)/acetonitrile (40:1, v/v) at flow rate of 0.7 ml/min. Single-stranded DNA from salmon testes (D7656; Sigma; DNA G + C content, 41.2 mol %) was used as a standard.

Results and discussion

Phylogenetic analysis

The nearly complete 16S rRNA gene sequence determined for strain 1-3-3-3T contains 1430 bp (NCBI GenBank/EMBL/DDBJ accession number LC315608). Based on 16S rRNA gene sequence similarity, the close relatives of the isolate were identified to be H. algoricola VUG-A23aT (98.0%), H. knuensis 16F7C-2 (97.9%), H. fastidiosus VUG-A124T (97.1%), H. elongatus VUG-A112T (97.0%), H. chitinivorans Txc1T (97.0%) and H. aquaticus 16F3PT (96.7%). The sequence similarities between strain 1-3-3-3T and other Hymenobacter species were less than 96.3%. The phylogenetic position of strain 1-3-3-3T, determined using three tree-making algorithms (ML, NJ, and MP), revealed the novel strain was positioned within the radiation comprising members of the genus Hymenobacter, joining H. algoricola VUG-A23aT in the ML and MP phylogenetic trees with moderate bootstrap values of 71 and 72%, respectively (Fig. 1).

Fig. 1
figure 1

Maximum-likelihood phylogenetic tree, based on 16S rRNA gene sequences, showing the phylogenetic position of strain 1-3-3-3T among related species of the genus Hymenobacter. Bootstrap values greater than 50% (percentages of 1000 replications) are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in trees generated with the neighbor-joining and maximum-parsimony algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the neighbor-joining or maximum-parsimony algorithms. Cytophaga hutchinsonii ATCC 33406T (M58768) was used as an outgroup. Bar, 0.02 substitutions per nucleotide position

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A threshold of 97% similarity in 16S rRNA gene sequence was proposed for bacterial species delineation (Stackebrandt and Goebel, 1994), then later re-evaluated as 98.7% by Stackebrandt and Ebers (2006) and Chun et al. (2018). On the basis of the re-evaluated threshold value, the above data indicate that strain 1-3-3-3T represents a novel species of the genus Hymenobacter. To confirm the taxonomic status of the novel isolate, DNA-DNA hybridization was performed with its five above-mentioned close phylogenetic neighbours. The DNA–DNA relatedness values of strain 1-3-3-3T with H. algoricola JCM 17214T (33.8 ± 6.0%), H. knuensis KCTC 52538 (26.9 ± 5.6%), H. fastidious JCM17224T (24.8 ± 5.5%), H. elongatus JCM 17223T (20.4 ± 3.9%), and H. chitinivorans KACC 12068T (18.4 ± 3.8%) were lower than the threshold value of 70% suggested for the delineation of novel prokaryotic species (Wayne et al. 1987).

Phenotypic characteristics

Cells of strain 1-3-3-3T were observed to be Gram-stain negative, aerobic, non-motile, red pink pigment-producing rods (0.9–1.2 μm wide and 1.8–3.8 μm long) (Supplementary Fig. S1). The isolate was found to be able to grow at 10–30 °C, but not at 4 or 37 °C, with optimum growth at 25 °C. Strain 1-3-3-3T can grow at a pH range between 6.0 and 8.0, with the optimum being pH 7.0. The strain was found to tolerate 1% (w/v) NaCl, but not 1.5%. Growth was observed on NA, R2A and TSA agar but not on LB agar. Other phenotypic features of the isolate are summarised in the species description. In API ZYM tests, the strain is positive for acid phosphatase, alkaline phosphatase, cystine arylamidase (weak, w), esterase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase (w) and valine arylamidase activities, but negative for N-acetyl-β-glucosaminidase, α-chymotrypsin, esterase (C4), α-fucosidase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase, lipase (C14), α-mannosidase and trypsin activities. In API ID 32 GN and API 20 NE tests, citrate, l-histidine, 3-hydroxybenzoate, 4-hydroxybenzoate and d-melibiose are utilised, but acetate, N-acetyl-d-glucosamine, adipate, l-alanine, l-arabinose, caprate, l-fucose, gluconate, d-glucose, glycogen, dl-3-hydroxybutyrate, inositol, itaconate, 2-ketogluconate, 5-ketogluconate, dl-lactate, l-malate, malonate, d-maltose, d-mannitol, d-mannose, phenyl acetate, l-proline, propionate, l-rhamnose, d-ribose, salicin, l-serine, d-sorbitol, suberate, d-sucrose and n-valerate are not utilised. In API 50 CH tests, acid is produced from arbutin, d-cellobiose (w), d-fructose, d-galactose, d-melibiose, d-melezitose, methyl-β-d-xylopyranoside (w), l-sorbose, d-raffinose (w) and d-trehalose, but not from N-acetylglucosamine, d-adonitol, amygdalin, d-arabinose, l-arabinose, d-arabitol, l-arabitol, dulcitol, erythritol, d-fucose, l-fucose, gentiobiose, gluconate, d-glucose, glycerol, glycogen, inositol, inulin, 2-ketogluconate, 5-ketogluconate, d-lactose, d-lyxose, d-maltose, d-mannitol, d-mannose, methyl-α-d-glucopyranoside, methyl-α-d-mannopyranoside, l-rhamnose, d-ribose, salicin, d-sorbitol, starch, d-sucrose, d-tagatose, d-turanose, xylitol, d-xylose or l-xylose.

The morphological, physiological and biochemical characteristics differentiating the novel strain from H. knuensis KCTC 52538, H. algoricola JCM 17214T, H. fastidious JCM17224T, H. elongatus JCM 17223T, H. chitinivorans KACC 12068T and H. aquaticus KCTC 32554T are listed in Table 1. In particular, strain 1-3-3-3T can be readily differentiated from the reference strains by its ability to grow on TSA agar, to utilise citrate and d-melibiose, and to produce acid from d-melibiose, d-melezitose and l-sorbose.

Table 1 Differential phenotypic characteristics of strain 1-3-3-3T and phylogenetically related species of the genus Hymenobacter
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Chemotaxonomic characteristics

The major fatty acids of strain 1-3-3-3T were identified as iso-C15:0 (19.9%), summed feature 3 (C16:1ω7c/C16:1ω6c) (19.7%), summed feature 4 (iso-C17:1 I/anteiso-C17:1 B) (17.8%), C16:1ω5c (12.5%), and anteiso-C15:0 (11.2%), together representing 81.1% of the total fatty acids present. As shown in Table 2, this fatty acid profile is similar to those of this strain’s phylogenetically close relatives. However, under the same growth conditions, the isolate differed from the six reference strains in terms of the proportions of the major and some minor fatty acids. The major polar lipid in strain 1-3-3-3T was observed to be phosphatidylethanolamine (Supplementary Fig. S2), which has also been identified as the main polar lipid in other Hymenobacter species (Buczolits et al. 2006; Lee et al. 2017b; Han et al. 2018; Ohn et al. 2018). In addition, the polar lipid profile of the isolate included three unidentified aminophospholipids, an unidentified aminoglycolipid, two unidentified lipids and an unidentified phospholipid. The predominant isoprenoid quinone of strain 1-3-3-3T was found to be a menaquinone with seven isoprene units (MK-7), which is the major respiratory quinone found in other members of the genus Hymenobacter (Hirsch et al. 1998; Buczolits et al. 2006; Jin et al. 2018; Ten et al. 2018).

Table 2 Fatty acid profiles of strain 1-3-3-3T and phylogenetically related species of the genus Hymenobacter
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DNA G + C content

The genomic DNA G + C content of strain 1-3-3-3T was determined to be 61.6 mol %, which lies within the range of values recorded for recognised members of the genus Hymenobacter (55.0–70.0 mol%) (Buczolits et al. 2006; Han et al. 2014).

Taxonomic conclusion

The DNA G + C content, respiratory quinone composition, polar lipid profile and major cellular fatty acids of strain 1-3-3-3T are generally in agreement with those of the members of the genus Hymenobacter, consistent with the assignment of the novel isolate into the genus. However, based on its phylogenetic distance from established species within the genus Hymenobacter, the low level of DNA relatedness with closely related species and the specific combination of phenotypic characteristics (Table 1), it is clear that strain 1-3-3-3T is distinct from any recognised Hymenobacter species. These results support the conclusion that strain 1-3-3-3T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter persicinus sp. nov. is proposed. The Digital Protologue Database (Rosselló-Móra et al. 2017) TaxoNumber for strain 1-3-3-3T is TA00817.

Description of Hymenobacter persicinus sp. nov.

Hymenobacter persicinus (per.si.ci’nus. N.L. masc. adj. persicinus peach-coloured because of its red-pink colour).

Cells are Gram-stain negative, non-motile, aerobic rods without flagella that are 0.9–1.2 μm wide and 1.8–3.8 μm long. After 3 days of incubation at 25 °C on R2A agar, colonies are convex, smooth, circular, red-pink, and approximately 0.9-1.1 mm in diameter. Growth occurs at 10–30 °C and pH 6–8. Grows on NA, R2A, and TSA agar but not on LB agar; tolerates 1% but not 1.5% NaCl. No growth is observed under anaerobic conditions. Positive for catalase, oxidase, aesculin and gelatin hydrolysis but negative for nitrate reduction, urea hydrolysis and indole production. The major cellular fatty acids are iso-C15:0, summed feature 3 (C16:1ω7c/C16:1ω6c), summed feature 4 (iso-C17:1 I/anteiso-C17:1 B), C16:1ω5c, and anteiso-C15:0. The predominant respiratory quinone is MK-7. The major polar lipid is phosphatidylethanolamine. The genomic DNA G + C content of the type strain is 61.6 mol %.

The type strain 1-3-3-3T (= KCTC 52742T = JCM 32191T) was isolated from a soil sample collected in Jeollabuk-do province, (35°41′04.50″N, 127°25′53.43″E), South Korea. The 16S rRNA gene sequence of strain 1-3-3-3T has been deposited in NCBI GenBank/EMBL/DDBJ under the accession number LC315608.