Hymenobacter Humicola sp. nov., Isolated from Soil in South Korea.

Two novel bacterial strains, designated as BT186 T and BT505, were isolated from a soil sample collected in South Korea and characterized. Both strains were Gram-stain-negative, rod-shaped, aerobic, circular, convex, and had red-colored colonies. 16S rRNA sequence analysis indicated that strains BT186 T and BT505 belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia, phylum Bacteroidetes, kingdom Bacteria). Both strains were closely related to Hymenobacter norwichensis DSM 15439 T (98.3% 16S rRNA gene similarity), Hymenobacter aquaticus JCM 31653 T (96.8%), and Hymenobacter perfusus LMG26000 T (96.5%). The strain BT186 T was found to have the MK-7 as the major respiratory quinone. The major polar lipid of strain BT186 T was identied to be phosphatidylethanolamine (PE). The major cellular fatty acid proles of strain BT186 T were C 16:1 ω5c (24.3%), iso-C 15:0 (20.3%) and summed feature 3 (C 16:1 ω6c / C 16:1 ω7c) (19.9%). Characterization based on polyphasic analysis indicated that strains BT186 T and BT505 represent novel species of the genus Hymenobacter and the name Hymenobacter humicola sp. nov. is proposed. The type strain of Hymenobacter humicola is BT186 T (= KCTC 72338 T = NBRC 114968 T ). All strains were positive for alkaline phosphatase and leucine arylamidase. All data were negative for β-glucuronidase, α-fucosidase, L-malate, α-mannosidase and lipase (C14).

In the present study, we conducted phylogenetic analysis, phenotypic, genotypic, and chemotaxonomic characterization to determine the taxonomic position of strains BT186 T and BT505. The results suggested that strains BT186 T and BT505 represent novel species of the genus Hymenobacter, for which the name Hymenobacter humicola sp. nov. is proposed.

Strain isolation
Two novel strains designated as sBT186 T and BT505, respectively, were isolated from a soil sample collected at Jungangro (35° 59′ 0″ N, 126° 43′ 0″ E) located in Gunsan City, South Korea. After one week of incubation at 25°C on Reasoner's 2A (R2A) agar medium (Difco), single colonies were picked and subcultured using the same medium at least two times to obtain pure colonies. The new bacterial cultures were routinely subcultured on R2A agar at 25 °C, maintained at 4 °C, and stored in 10% (w/v) glycerol suspension at −80 °C before use.

Morphology, physiology, and biochemical analysis
Cell morphologies of new isolates were observed using transmission electron microscopy (JEOL, JEM1010) using the negative staining method. The Gram-staining reaction was performed using a kit, following the manufacturer's instructions (bioMérieux). Catalase activity was determined by adding 3% (w/v) H 2 O 2 solution and oxidase activity was examined using 1% (w/v) tetramethyl-p-phenylenediamine diamine. (Cappuccino and Sherman 2002

Phylogenetic analysis
For phylogenetic analysis, the 16S rRNA genes were ampli ed using two universal primers 9F and 1512R (Weisburg et al. 1991). The PCR products were puri ed and sequenced using universal primers 337F, 518R, 785F, and 926R (Macrogen). The 16S rRNA gene sequences were identi ed by EzBioCloud server (https://www.ezbiocloud.net/). To determine the taxonomic positions of strains BT186 T and BT505, the 16S rRNA sequences of related taxa of the genus Hymenobacter were obtained from EzBioCloud (Yoon et al. 2017) and compared with those of strains BT186 T and BT505 using the EzEditor2 program (Jeon et al. 2014). Phylogenetic trees were constructed using the MEGAX program (Kumar et al. 2016) with the neighbor-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981), and maximum-parsimony (MP) algorithms (Fitch 1971). The stability of the tree topologies was evaluated by bootstrap analysis based on the 1,000 resampling method (Felsenstein 1985). Evolutionary distances were calculated according to the Kimura two-parameter model (Kimura 1983).

Genome sequencing
The genomic DNA of strain BT186 T was extracted using a genomic DNA extraction kit according to the manufacturer's instruction (Solgent). Then, the DNA library was prepared using the Nextera DNA Flex Library Prep Kit (Illumina) according to the manufacturer's protocol. Whole-genome sequencing (WGS) was performed using iSeq 100 and assembled by using the SPAdes software version 3.10.1 (Algorithmic Biology Lab, St. Petersburg Academic University of the Russian Academy of Sciences). The wholegenome sequence of strain BT186 T was 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). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values based on whole-genome sequences were calculated by using the EzBioCloud (https://www.ezbiocloud.net) and Genome-to Genome Distance Calculator (GGDC), respectively, with the recommended formula 2 (Table S1) (Meier-Kolthoff et al. 2013).

Chemotaxonomic characterization
To analyze the polar lipid, fatty acid, and lipoquinone components of strain BT186 T , cells were grown on R2A agar at 25 °C for three days. After then, cells were harvested and freeze-dried. The total lipids, glycolipids, phosphatidylcholine, and amino groups were separated using two-dimensional thin-layer chromatography (TLC). The polar lipid spots were detected by spraying the proper detection reagents as previously described (Komagata and Suzuki 1987;Minnikin et al. 1984). The cellular fatty acids methyl esters (FAME) of strain BT186 T were analyzed using the Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc) according to the protocol described by Sasser (1990). The respiratory quinone was extracted using the Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) follow the previous methods (Hiraishi et al. 1996).

Results And Discussion
Morphology, physiology, and biochemical characteristics Cells of strains BT186 T and BT505 were rod-shaped, Gram-stain-negative, aerobic, and non-motile.
Colonies of strains BT186 T and BT505 were convex, smooth, circular, and red-colored. Cells of strains BT186 T and BT505 could survive at 10-30°C (optimum 25°C) and pH 6.0-8.0 (optimum 6.0) on R2A agar plate. Distinct features between the newly isolated strains and reference strains were provided in Table 1.
The negative reactions of strain BT186 T on API kits were given as supplementary tables (Table S2).

Phylogenetic analysis and whole-genome sequence analysis
Based on the 16S rRNA gene sequence similarities, strains BT186 T and BT505 were a liated with the family Hymenobacteraceae and showed high sequence similarities with the genus Hymenobacter. The level of 16S rRNA gene sequence similarity between the strains BT186 T and BT505 was 100%, indicating that they represent an identical species. Both strains were closely related to H. norwichensis DSM 15439 T (98.3% 16S rRNA sequence similarity), H. aquaticus JCM 31653 T (96.8%), and H. perfusus LMG26000 T (96.5%). The 16S rRNA gene sequence similarities of strains BT186 T and BT505 with the closely related type strains were less than 98.3% and with other Hymenobacter species were less than 96.4%. These values were below 98.7% of which value is recently used as the threshold for differentiating among bacterial species (Chun et al. 2018). The other Hymenobacter species exhibited sequence similarities lower than 97.0%. According to the phylogenetic tree based on the neighbor-joining, maximum-likelihood ( Fig. S1), and maximum-parsimony (Fig. S2) algorithm, strains BT186 T and BT505 were placed within the genus Hymenobacter (Fig. 1).
The draft genome of strain BT186 T was 6,019,942 bp (24.7×) and consisted of 4,860 protein-coding genes, 54 RNA genes (7 rRNA genes, 44 tRNA genes), and 25 pseudogenes. The genome sequence of strain BT186 T has been deposited in GenBank under the accession numbers NZ_JAFLQZ000000000. The DNA G+C content of strain BT186 T was 57.5 mol%. This value was within the range of the G+C contents for the genus Hymenobacter (55-71 mol%) (Feng et al. 2019). The digital DNA-DNA hybridization values between strain BT186 T and other related type strains of genus Hymenobacter were less than 30.2% (Table S1), which are below the cutoff (70%) point (Meier-Kolthoff et al. 2013). Average nucleotide identity (ANI) values between strain BT186 T and other related type strains of genus Hymenobacter were less than 85.6%, respectively (Table S1). These values are below the ANI species threshold (95 -96% ANI value) as described by Richter and Rossello-Mora (2009).

Chemotaxonomic characterization
The fatty acid pro les of strain BT186 T and four reference strains were presented in Table 2 Description of Hymenobacterhumicola sp. nov.
The whole-genome sequence of strain BT186 T has been deposited in GenBank under the accession number NZ_JAFLQZ000000000 (6,019,942 bp). The genome-based G+C content is 57.5 mol%. The GenBank accession number for the 16S rRNA gene sequence of strain BT186 T is MW876235 (1,389 bp).

Declarations
Triangles indicate that the corresponding nodes were also recovered in the maximum-likelihood trees (Fig.  S2). Bar, 0.020 substitutions per nucleotide position. Cytophaga hutchinsonii NBRC 15051T were used as outgroup.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Supplementarydata.docx