Pontibacter rubellus sp. nov., and Pontibacter situs sp. nov. bacteria isolated from soil

Gram-stain-negative, aerobic, non-agellated strains 172403-2 T and BT310 T were isolated from the soil collected in Pyeongchang city and city, Korea. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strains 172403-2 T and BT310 T formed a distinct lineage within the family Hymenobacteraceae (order Chitinophagales, class Chitinophagia) and were most closely related to members of the genus Pontibacter, Pontibacter chitinilyticus 17gy-14 T (95.7%), and Pontibacter populi HLY7-15 T (97.1% 16S rRNA gene sequence similarity) respectively. The optimal growth of strains 172403-2 T and BT310 T occurred at pH 7.0, in the absence of NaCl, and 25°C and 30°C, respectively. The predominant cellular fatty acids were iso-C 15:0 and summed feature 4 (iso-C 17:1 I / anteiso-C 17:1 B). The major respiratory quinone of the two strains was MK-7. The major polar lipid of the two strains was phosphatidylethanolamine. Biochemical, chemotaxonomic and phylogenetic analyses indicated that strains 172403-2 T and BT310 T represent novel bacterial species within the genus Pontibacter, for which the names Pontibacter rubellus and Pontibacter situs are proposed. The type strains of Pontibacter rubellus and Pontibacter situs are 172403-2 T and BT310 T , respectively. N-acetyl-D-glucosamine, adipate and L-malate. protease (gelatin hydrolysis), D-mannitol, gluconate, caprate, citrate and phenyl acetate. In API ZYM test, strain


Introduction
The genus Pontibacter is a member of the family Hymenobacteraceae in the phylum Bacteroidetes. The family Hymenobacteraceae contains six genera (http://www.bacterio.net) and Pontibacter is one of the largest genera. The genus Pontibacter was rst proposed by Nedashkovskaya et al. (2005) with P. actiniarum as a type species. At the time of writing, the genus Pontibacter is comprised of 38 species (http://www.bacterio.net/Pontibacter.html).
The species of Pontibacter have been isolated mainly from soil samples (Srinivasan et

Materials And Methods
Organism and culture conditions Strains 172403-2 T and BT310 T were isolated from a soil sample collected in Uijeongbu city, South Korea.
Soil (1 g) was added to 10 mL if sterile normal saline and shaken at 37 °C for 1h, and then serially diluted. 100 μL of the diluent was spread on Reasoner's 2A (R2A, Difco) agar and incubated at 25°C; after 3 days, various colonies were selected and puri ed. Strains 172403-2 T and BT310 T were stored at -80°C in 20% (v/v) glycerol with R2A broth. The 16S rRNA gene sequences of the strains were compared with the closely related sequences including Pontibacter populi HLY7-15 T and Pontibacter amylolyticus 9-2 T using the EzBioCloud (https://www.ezbiocloud.net).

Morphology, physiology and biochemical analysis
Cell morphology was observed by transmission electron microscopy (JEOL, JEM1010) after 3 days of incubation on R2A at 30 °C. Gram-staining reaction was performed according to the standard Gram reaction kit (bioMérieux). The growth temperature range was tested at 4, 10, 15, 25, 30, 35, 37, 42 and 45°C . The salt tolerance was measured in R2A supplemented with various concentrations of NaCl (1-10% at intervals of 1%, w/v). The pH range was measured in R2A from pH 4.0 to 10.0 with an interval of 0.5 units using R2A broth at 25°C. Oxidase activities of strains 172403-2 T and BT310 T were tested using 1% (w/v) tetramethyl-p-phenylene diamine diamine (Smibert and Krieg 1981) and catalase activities were tested by measuring bubble production after applying 3% (v/v) hydrogen peroxide solution (Cappuccino and Sherman 2002). Growth on the different mediums was observed on R2A agar, nutrient agar (NA, BD Difco), tryptic soy agar (TSA, BD Difco), MacConkey agar (BD Difco) and lysogeny broth (LB, BD Difco).
API 20NE (bioMérieux) was used to determine the utilization and fermentation of various carbon sources and API ZYM (bioMérieux) was used to determine the enzymic activities of the strains according to the manufacturer's instructions.

Phylogenetic analysis and Genome sequencing
The genomic DNA of strains 172403-2 T and BT310 T were extracted using a genomic DNA extraction kit (Qiagen). The 16S rRNA gene was Ampli ed using a standard PCR method with a universal bacterial primer set 27F and 1492R (Weisburg et al. 1991). The ampli ed 16S rRNA gene was sequenced by Macrogen (Korea) with the 518F, 785F, 800R and 926R universal primers. To determine the taxonomic positions of strains 172403-2 T and BT310 T , 16S rRNA gene sequences of closely related taxa were obtained from EzBioCloud (http://ezbiocloud.net) and EzEditor2 program was used for the alignment of the sequences. The phylogenetic tree was constructed using the MEGAX program (Kumar et al. 2018) and neighbor-joining (NJ) algorithm (Saitou and Nei 1987). A bootstrap analysis with 1,000 replicates was conducted (Felsenstein 1985). added automatically x errors, x frameshifts, and back ll gaps (Overbeek et al. 2014). The genomebased phylogenetic tree was reconstructed using the UBCG set pipeline (Na et al. 2018), with which we used a concatenated sequence dataset of 92 single-copy bacterial core genes (www.ezbiocloud.net/tools/ubcg).

Chemotaxonomic characteristics
The polar lipids of strains 172403-2 T and BT310 T were extracted (Minnikin et al. 1984) and examined using two-dimensional thin-layer chromatography (TLC). The separated polar lipids were identi ed by spraying several reagents as described by Komagata and Suzuki (1987). Lipoquinones were extracted with Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) method (Hiraishi et al. 1996). For cellular fatty acids analysis, strains 172403-2 T and BT310 T were incubated on R2A agar for 3 days at 25°C. The cellular fatty acids were puri ed by saponi cation, methylation and extraction procedures as previously described (Sasser 1990). The fatty acid methyl esters (FAME) were identi ed using the Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc., Newark, DE, USA).

Result And Discussion
Morphology, physiology and biochemical analysis Strains 172403-2 T and BT310 T were isolated from soil collected in the Pyeongchang and Uijeongbu city, respectively. The colonies of strain 172403-2 T on R2A agar medium were observed to be red, convex and circular after 72-hour incubation at 25°C, whereas strain BT310 T formed orange color colonies. Both strains were Gram-staining-negative, non-agellated and short rods (Fig. 1). Strain 172403-2 T could grow at 10-30°C, pH 5.0-9.0 without NaCl. Strain BT310 T could grow at 10-30°C, pH 5.0-9.0 and with 3% of NaCl. The differences between the novel isolates and the reference strains were provided in Table 1.

Genome sequencing and Phylogenetic analysis
The genome size of strain 172403-2 T was 5,025,066 bp (genome coverage of 29.7X) and consisted of 4,192 coding sequences (CDSs) and 38 tRNA genes. DNA G+C content was 48.6 mol%. The obtained genome of strain 172403-2 T was submitted to the GenBank/EMBL/DDBJ under the accession number NZ_JADQDR000000000. The genome size of strain BT310 T was 4,294,440 bp (genome coverage of 45.8X) and consisted of 3,618 coding sequences (CDSs), and 38 tRNA genes. DNA G+C content was 45.2 mol%. The obtained genome of strain BT310 T was submitted to the GenBank/EMBL/DDBJ under the accession number NZ_JAELXU010000000.
Based on 16S rRNA gene sequence similarity, strains 172403-2 T and BT310 T were a liated with the family Hymenobacteraceae and showed high sequence similarities with the genus Pontibacter. Strain 172403-2 T was most closely related to P. chitinilyticus 17gy-14 T (95.7% 16S rRNA gene sequence similarity) and strain BT310 T was most closely related to P. populi HLY7-15 T (97.0% 16S rRNA gene sequence similarity). After the construction of neighbor-joining phylogenetic tree (Fig. 2), the strains each formed an independent cluster, which clearly showed that strains 172403-2 T and BT310 T belong to the genus Pontibacter and represent two novel species. The phylogenomic genomic tree (

Chemotaxonomic characterization
The total cellular fatty acids of strains 172403-2 T and BT310 T and their most related species were shown in Table 2. The predominant fatty acids of strain 172403-2 T were iso-C 15:0 (28.7%) and summed feature 4 (iso-C 17:1 I / anteiso-C 17:1 B) (22.1%). The fatty acid pro le of strain 172403-2 T was similar to those of the most closely related type strain but can be differentiated from closely related specie based on relatively high amounts of iso-C 15:0 , iso-C 16:0 3OH, iso-C 17:0 3OH and small amount of C 14:1 ω5c, iso-C 15:1 F ( Table   2). The predominant fatty acids of strain BT310 T were iso-C 15:0 (29.5%) and summed feature 4 (iso-C 17:1 I / anteiso-C 17:1 B) (20.9%). The fatty acid pro le of strain BT310 T was similar to those of the most closely related type strain but can be differentiated from closely related species based on relatively high amounts of iso-C 15:1 G, iso-C 16:1 H, C 16:1 ω5c, C 17:1 ω6c and small amount of iso-C 17:0 3OH ( Table 3). The major polar lipid of strains 172403-2 T and BT310 T was phosphatidylethanolamine (PE). The total polar lipids pro le of strain 172403-2 T showed phosphatidylethanolamine (PE), one aminolipid, one aminophospholipid, two glycolipids and two unknown polar lipids (Fig. S2). The total polar lipids pro le of strain BT310 T showed phosphatidylethanolamine (PE), one aminolipid, one aminophospholipid, one glycolipid, two phospholipids, and six unknown polar lipids (Fig. S3). The major respiratory quinone of strains 172403-2 T and BT310 T was MK-7, which is common in the species of the genus Pontibacter.
Based on phenotypic, phylogenetic and biochemical characteristics, we concluded that strain 172403-2 T and strain BT310 T represent two novel species in the genus Pontibacter, for which the name Pontibacter rubellus and Pontibacter situs are proposed. The NCBI accession numbers for 16S rRNA sequences of the strains 172403-2 T and BT310 T are MW237669 and MT795756, respectively.
The type strain for Pontibacter rubellus, 172403-2 T (=KCTC 62072 T = NBRC XXXX T ) was isolated from soil in Korea. The GenBank accession number for 16S rRNA gene sequence of strain 172403-2 T is MW237669.
Description of Pontibacter situs sp. nov.