Introduction

The genus Hymenobacter was first described by Hirsch et al. (1998) and allocated to the family Hymenobacteraceae (Munoz et al. 2017). At the time of writing (November 2020), the genus comprised 87 validated species (http://www.bacterio.net/hymenobacter.html). Members of the genus Hymenobacter were found in various environments, including freshwater wetlands (Baik et al. 2006), grassy soils (Kim et al. 2008), sediments (Xu et al. 2009), an artificial lake (Joung et al. 2011) and soil (Li et al. 2019). Some species of the genus Hymenobacter were isolated from extreme environments, including Antarctica, glacial ice, permafrost, Antarctic lakes, sanitized clean-room facilities, leaves, snow, irradiated meat, and temperate desert (Klassen & Foght 2011).

Mostly, the cells of the genus Hymenobacter are rod shaped, red to pink in color, Gram-stain negative, and aerobic. The major cellular fatty acids of the genus were iso-C15:0, anteiso-C15:0, C16:1 ω5c, summed feature 3 (C16:1 ω7c/ C16:1 ω6c), and summed feature 4 (iso-C17:1 I / anteiso-C17:1 B). This genus has MK-7 as a major respiratory quinone and phosphatidylethanolamine as a main polar lipid (Zhu et al. 2017).

In this study, two newly isolated strains, BT442T and BT584, were characterized. According to the 16S rRNA sequence analysis, BT442T and BT584 showed less than 98.7% 16S rRNA gene sequence similarity to other strains in the genus Hymenobacter. Phenotypic, chemotaxonomic, and phylogenetic analyses showed that strains BT442T and BT584 were distinct from other previously reported Hymenobacter species.

Materials and methods

Strain isolation

Strain BT442T was isolated from a soil sample collected from mountain in Busan, Korea. And strain BT584 was isolated a soil sample collected from mountain in Guri, Korea during Winter. Isolation of strains from soil samples was performed using the standard dilution plating technique on an R2A medium (Difco) agar (Staley 1968) at 25 °C for 7 days. The 16S rRNA gene sequences of the purified colonies were identified using the EzBioCloud server (https://www.ezbiocloud.net/).

Morphology, physiology, and biochemical analysis

Cell morphology was tested using transmission electron microscopy (JEOL, JEM1010, Tokyo, Japan) with negative staining. The Gram stain was performed using a commercial kit (bioMérieux, Marcy-l'Étoile, France), following the manufacturer’s instructions. Catalase and oxidase activities were determined using 1% (w/v) tetramethyl-p-phenylenediamine and 3% (w/v) H2O2 solution, respectively (Cappuccino and Sherman 2002). Bacterial growth was tested at 25 °C under various pH conditions (4 to 10, 0.5 pH intervals, pH 4.0 ~ 5.5: acetic acid buffer, pH 6.0 ~ 8.0: potassium phosphate buffer, pH 8.5 ~ 10.0: Glycine—NaOH buffer) and different NaCl concentrations (0 to 5% [w/v%], 1% intervals). Bacterial growth on R2A agar plate was observed for 3 days at different temperatures (4, 10, 15, 18, 25 and 30 °C). The bacteria were grown on R2A agar plates, Luria–Bertani (LB) agar, nutrient agar (NA), MacConkey (MAC) agar, and trypticase soy agar (TSA) (all purchased from Difco, New Jersey, United States) and were observed for three days at different temperatures (4 °C, 10 °C, 25 °C, 30 °C). Biochemical and physiological tests were performed using API 20NE kits, and enzymic activities were tested using an API ZYM kit (bioMérieux) following the manufacturer’s instructions. Two reference strains, H. soli PB17T and H. terrae DG7AT were obtained from the Korean Collection for Type Cultures and were cultured under the same conditions for comparative analysis.

Genome sequencing

Genomic DNA was extracted using a genomic DNA extraction kit (Solgent, Daejeon, Korea).

The DNA sequencing library was prepared by the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, United States), and whole-genome sequencing was accomplished using iSeq 100 (150 bp paired-end). The genome sequence was assembled using SPAdes 3.10.1 (Algorithmic Biology Lab, St. Petersburg Academic University of the Russian Academy of Sciences). The whole-genome sequences of strains BT442T and BT584 were deposited in the GenBank (www.ncbi.nlm.nih.gov/) database. The genome sequences of strains BT442T and BT584 were annotated using the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016).

Phylogenetic analysis

The 16S rRNA genes of strains BT442T and BT584 were amplified by 27F and 1492R universal bacterial primers (Macrogen) using BT442T and BT584 genomic DNA as a template. Then, sequencing was performed using the 337F, 518R, 785F and 926R universal primers (Macrogen). To determine the taxonomic positions of strains BT442T and BT584, 16S rRNA sequences of related taxa were obtained from EzBioCloud and compared with those of BT442T and BT584 using the EzEditor2 program (Jeon et al. MEGA). A phylogenetic tree was constructed using the MEGAX program (Kumar et al. 2018) with the neighbor-joining (Saitou and Nei 1987), maximum-likelihood and maximum-parsimony algorithms (Ziheng 1995). Evolutionary distances were calculated according to the Kimura two-parameter model (Kimura 1983). A bootstrap analysis was conducted with 1,000 replicates (Felsenstein 1985). DNA sequence similarities among the strains BT442T, BT584, and the closest type strain were evaluated using average nucleotide identity (ANI) analysis and in silico DNA-DNA hybridization. ANI values were calculated using the Orthologous Average Nucleotide Identity Tool version 0.98 (Lee et al. 2016) and digital DNA-DNA hybridization was performed using Genome-to Genome Distance Calculator (GGDC) with the recommended formula 2 (Meier-Kolthoff et al. 2013).

Chemotaxonomic characteristics

Polar lipids were extracted from the cells according to a previously described method (Minnikin et al. 1984) and separated by two-dimensional thin-layer chromatography (TLC). Spots were detected with detection reagents sprayed onto the lipids (Komagata and Suzuki 1987). Respiratory quinones were extracted using Sep-Pak Vac cartridges (Waters, Massachusetts, USA) and were analyzed by high-performance lipid chromatography (HPLC) based on the method of Hiraishi et al. (1996). For cellular fatty acid analysis, strains BT442T and BT584 were grown on R2A agar plates for 3 days at 25 °C, and 300 mg of cells were harvested and freeze-dried. Cellular fatty acids were purified by saponification, methylation, and extraction procedures (Sasser 1990), then identified using the Sherlock Microbial Identification System V6.01 (MIS, database TSBA6, MIDI Inc., Newark, USA).

Results and discussion

Morphology, physiology, and biochemical analysis

Strains BT442T and BT584 were isolated from dry soil collected in Busan and Guri, Korea. Strains BT442T and BT584 were Gram negative and rod shaped (Fig. S1). Colonies of strain BT442T and BT584 were convex, smooth, circular, and pink in color after incubation for three days at 25 °C. Cells could grow at 10–30 °C (optimum 25 °C) and pH 6.0–8.0 (optimum 6.0) in the R2A medium. Different features between two novel strains and reference strains are provided in Table 1. Features from API 20NE and API ZYM test were listed in Table S1.

Table 1 Different characteristics of Hymenobacter negativus sp. nov., and closely related species
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Genome sequence analysis and phylogenetic analysis

The draft genome of strain BT442T consisted of 5,143,362 bp with a coverage of 20-fold. A total of 4,202 protein-coding genes and 44 RNA genes (3 rRNA genes, 41 tRNA genes) were predicted by NCBI Prokaryotic Annotation Pipeline (PGAP) analysis. The genome sequence of the strain BT442T was deposited in GenBank under accession number NZ_JAEDAE010000000. The draft genome of strain BT584 consisted of 5,156,552 bp with a coverage of 55-fold. A total of 4209 protein-coding genes and 43 RNA genes (3 rRNA genes, 40 tRNA genes) were predicted by NCBI Prokaryotic Annotation Pipeline (PGAP) analysis. The genome sequence of the strain BT584 has been deposited in GenBank under accession number NZ_JAEDAD000000000.

Based on its sequence similarity to the 16S rRNA gene, strain BT442T was affiliated with the family Hymenobacteraceae, and it showed high sequence similarities with the genus Hymenobacter. 16S rRNA sequence from PCR results was identical with that of whole genome. The similarity of the 16S rRNA gene sequence between the BT442T and BT584 strains was 99.23%, which indicated that they represent nearly identical species. The novel isolate BT442T was related to Hymenobacter soli PB17T (98.0% 16S rRNA gene similarity) and Hymenobacter terrae DG7AT (97.6%). According to Chun et al (2018), BT442T could be regarded a new species as its 16S rRNA similarity was 98.7% or less. The OrthoANI value between strains BT422T and the closest type strain Hymenobacter terrae DG7AT was 81.74%, and the OrthoANI values between strain BT584 and H. terrae DG7AT was 81.73%. The ANI and dDDH values between strain BT422T and H. terrae DG7AT was 80.40% and 17.0%, respectively. The ANI and dDDH values between strain BT584 and H. terrae DG7AT was 80.38% and 17.0%, respectively. In the neighbor-joining, maximum-likelihood and maximum parsimony tree, strain BT442T formed an independent cluster (Fig. 1). The phylogenetic analysis result clearly showed that strain BT442T is a novel species within the genus Hymenobacter.

Fig. 1
figure 1

Neighbor-joining phylogenetic tree reconstructed from a comparative analysis of 16S rRNA gene sequences showing the relationships of strains BT442T and BT584 with closely related validly published species. Bootstrap values (> 70%) based on the neighbor-joining method are shown at the branch nodes. Circles indicate that the corresponding nodes were also recovered in maximum-likelihood and maximum-parsimony trees. Bar, 0.020 substitutions per nucleotide position. Chitinophaga hutchinsonii NBRC 15051 T was used as the outgroup. The compact triangles represent other species of genus Hymenobacter

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Chemotaxonomic characterization

The fatty acid profiles of strains BT442Tand BT584 were compared with those of the closely related three species of the genus Hymenobacter (Table 2). The major fatty acids of strains BT442T and BT584 were C15:0, anteiso-C15:0, C16:1 ω5c; summed feature 3 (C16:1 ω7c/ C16:1 ω6c); and summed feature 4 (iso-C17:1 I / anteiso-C17:1 B). The fatty acid profiles of strains BT442T and BT584 were similar to those of the two most closely related Hymenobacter species.

Table 2 Cellular fatty acid profiles of Hymenobacter negativus sp. nov., and closely related species
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The polar lipids of strain BT442T consisted of phosphatidylethanolamine, phospholipid, glycolipid, two aminolipids, four aminophospholipids, and two unknown polar lipids (Fig. S2). Polar lipids of strain BT584 consisted of one phosphatidylethanolamine, one phospholipid, one glycolipid, two aminolipids, two aminophospholipids, and two unknown lipids (Fig. S3). The dominant respiratory quinone of strain BT442T was MK-7.

The morphological, biochemical, and chemotaxonomic characters of strains BT442T and BT584 were consistent with descriptions of the genus Hymenobacter. On the basis of the phylogenetic differences between strain BT442T and species of the genus Hymenobacter, a novel species, Hymenobacter negativus sp. nov., has been proposed, with BT442T as the strain type.

Description of Hymenobacter negativus sp. nov

Hymenobacter negativus (ne.g.,a.ti'vus. L. masc. adj. negativus, negative, because of the Gram-negative staining reaction).

Cells are Gram negative and rod shaped. Colonies on R2A agar are irregular, convex, and pink in color after the three-day incubation at 25 °C. The cell size of strain BT442T is approximately 1.2–1.5 µm in diameter and approximately 5.3–5.8 µm in length. Growth occurs at 10–30 °C (optimum 25 °C) and pH 6.0–9.0 (optimum 6.0). Cells grow on R2A agar, TSA, and NA but not on LB agar and Macconkey agar. Cells are oxidase activity negative and catalase activity positive and do not assimilate D-mannitol. The major respiratory quinone is MK-7. The dominant cellular fatty acids are C15:0, anteiso-C15:0, C16:1 ω5c and summed feature 3 (C16:1 ω7c/ C16:1 ω6c), summed feature 4 (iso-C17:1 I / anteiso-C17:1 B). The major polar lipids are phosphatidylethanolamine (PE), aminophospholipid (APL), and aminolipid (AL). The whole genome sequences of strains BT442T and BT584 have been deposited in GenBank under accession numbers NZ_JAEDAE000000000 and NZ_JAEDAD000000000, respectively. The GenBank accession number for the 16S rRNA gene sequence of strains BT442T and BT584 are MT815535 and MT893355, respectively. The strain type BT442T (= KCTC 72902 T = NBRC XXXXT) was isolated from a soil sample collected in Korea.