Morphology, physiology and biochemical analysis
Strains 172403-2T and BT310T were isolated from soil collected in the Pyeongchang and Uijeongbu city, respectively. The colonies of strain 172403-2T on R2A agar medium were observed to be red, convex and circular after 72-hour incubation at 25°C, whereas strain BT310T formed orange color colonies. Both strains were Gram-staining-negative, non-flagellated and short rods (Fig. 1). Strain 172403-2T could grow at 10-30°C, pH 5.0-9.0 without NaCl. Strain BT310T 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. Strain 172403-2T could be differentiated from strain BT310T based on several characteristics, such as growth at 3% of NaCl. Strain 172403-2T could be differentiated from P. chitinilyticus 17gy-14 T on the basis of arginine dihydrolase, α-chymotrypsin, α-fucosidase, β-galactosidase (ONPG), α-glucosidase (starch hydrolysis), β-glucosidase, α-mannosidase enzyme activities and assimilation of D-glucose, D-maltose, D-mannose, D-mannitol and N-acetyl-D-glucosamine. Strain BT310T could be differentiated from P. populi HLY7-15 T on the basis of acid phosphatase, α-chymotrypsin, cystine arylamidase, esterase (C8), naphtol-AS-BI-phosphohydrolase enzyme activities and assimilation of D-glucose and D-mannose. (Table 1).
Genome sequencing and Phylogenetic analysis
The genome size of strain 172403-2T 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 172403-2T was submitted to the GenBank/EMBL/DDBJ under the accession number NZ_JADQDR000000000. The genome size of strain BT310T 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 BT310T was submitted to the GenBank/EMBL/DDBJ under the accession number NZ_JAELXU010000000.
Based on 16S rRNA gene sequence similarity, strains 172403-2T and BT310T were affiliated with the family Hymenobacteraceae and showed high sequence similarities with the genus Pontibacter. Strain 172403-2T was most closely related to P. chitinilyticus 17gy-14 T (95.7% 16S rRNA gene sequence similarity) and strain BT310T 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 172403-2T and BT310T belong to the genus Pontibacter and represent two novel species. The phylogenomic genomic tree (Fig. S1) based on UBCGs supported the 16S rRNA genes based phylogenetic tree.
The genome of the strains 172403-2T and BT310T contain genes associated with nitrogen metabolism, including denitrification. Both the strains 172403-2T and BT310T contain the key enzymes such as cytochrome nitrite reductase (EC:1.7.2.1) and Nitric-oxide reductase (EC 1.7.99.7) involved in the denitrification process (Xu at al. 2014, Philippon et al. 2021). Furthermore, genes associated with menaquinone biosynthesis are also identified in the genome, which is an essential component of the electron transfer pathway in prokaryotes (Dairi 2012).
Chemotaxonomic characterization
The total cellular fatty acids of strains 172403-2T and BT310T and their most related species were shown in Table 2. The predominant fatty acids of strain 172403-2T were iso-C15:0 (28.7%) and summed feature 4 (iso-C17:1 I / anteiso-C17:1 B) (22.1%). The fatty acid profile of strain 172403-2T 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-C15:0, iso-C16:03OH, iso-C17:0 3OH and small amount of C14:1 ω5c, iso-C15:1F (Table 2). The predominant fatty acids of strain BT310T were iso-C15:0 (29.5%) and summed feature 4 (iso-C17:1 I / anteiso-C17:1 B) (20.9%). The fatty acid profile of strain BT310T 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-C15:1 G, iso-C16:1 H, C16:1 ω5c, C17:1 ω6c and small amount of iso-C17:0 3OH (Table 3). The major polar lipid of strains 172403-2T and BT310T was phosphatidylethanolamine (PE). The total polar lipids profile of strain 172403-2T showed phosphatidylethanolamine (PE), one aminolipid, one aminophospholipid, two glycolipids and two unknown polar lipids (Fig. S2). The total polar lipids profile of strain BT310T showed phosphatidylethanolamine (PE), one aminolipid, one aminophospholipid, one glycolipid, two phospholipids, and six unknown polar lipids (Fig. S3). The major respiratory quinone of strains 172403-2T and BT310T was MK-7, which is common in the species of the genus Pontibacter.
Based on phenotypic, phylogenetic and biochemical characteristics, we concluded that strain 172403-2T and strain BT310T 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 172403-2T and BT310T are MW237669 and MT795756, respectively.
Description of Pontibacter rubellus sp. nov.
Pontibacter rubellus (ru.bel'lus. L. masc. adj. rubellus reddish).
The cells are Gram-stain-negative, non-flagellated and short rod-shaped. Colonies on R2A agar are convex, circular and red colored after 72 hours of growth at 25°C. Cells are around 0.4 µm wide and 0.5 µm long. Growth occurs at 10-30°C and pH 5.0-9.0 (optimum 7.0). Cells grow well on R2A agar, TSA, LB and NA but not on MAC agar. Oxidase and catalase activities are positive. In API 20NE test, strain 172403-2T was positive for β-glucosidase (esculin hydrolysis), protease (gelatin hydrolysis), and β-galactosidase (PNPG). But negative for nitrate reduction, arginine dihydrolase, urease, D-glucose, L-arabinose, D-mannitol, D-maltose, gluconate, D-mannose, production of indole, production of acid from glucose, N-acetyl-D-glucosamine, caprate, adipate L-malate, citrate, and phenyl acetate. In API ZYM test, strain 172403-2T was positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, and N–acetyl-β-glucosaminidase. weakly positive for esterase (C4), esterase (C8), cystine arylamidase, and α-galactosidase. But negative for lipase (C14), trypsin, α-chymotrypsin, β-galactosidase (ONPG) and β-glucuronidase, α-glucosidase (starch hydrolysis), β-glucosidase, α-mannosidase. and α-fucosidase. The major respiratory quinone is MK-7. The dominant cellular fatty acids are iso-C15:0 and summed feature 4 (iso-C17:1 I / anteiso-C17:1 B). The major polar lipid is phosphatidylethanolamine (PE).
The type strain for Pontibacter rubellus, 172403-2T (=KCTC 62072T = NBRC XXXXT) was isolated from soil in Korea. The GenBank accession number for 16S rRNA gene sequence of strain 172403-2T is MW237669.
Description of Pontibacter situs sp. nov.
Pontibacter situs (si.tus. L. masc. adj. situs soil).
The cells are Gram-stain-negative, non-flagellated and short rod-shaped. Colonies on R2A agar are convex, circular and red colored after 72 hours of growth at 25°C. Cells are around 0.9-1.1 µm wide and 2.2-2.4 µm long. Growth occurs at 10-30°C and pH 5.0-9.0. Cells grow well on R2A agar, TSA, LB and NA but not on MAC agar. Oxidase activity is negative and catalase activity is positive. In API 20NE test, strain BT310T was positive for β-glucosidase (esculin hydrolysis). But negative for nitrate reduction, production of indole, production of acid from glucose, arginine dihydrolase, urease, β-galactosidase (PNPG), D-maltose, D-glucose, L-arabinose, D-mannose, N-acetyl-D-glucosamine, adipate and L-malate. protease (gelatin hydrolysis), D-mannitol, gluconate, caprate, citrate and phenyl acetate. In API ZYM test, strain BT310T was positive for alkaline phosphatase, esterase (C4), esterase (C8), leucine arylamidase, valine arylamidase, and trypsin. But negative for lipase (C14), cystine arylamidase, α-chymotrypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase (ONPG), β-glucuronidase, α-glucosidase (starch hydrolysis), β-glucosidase, N–acetyl-β-glucosaminidase and α-mannosidase and α-fucosidase. The major respiratory quinone is MK-7. The dominant cellular fatty acids are summed feature 4 (iso-C17:1 I / anteiso-C17:1 B) and iso-C15:0. The major polar lipid is phosphatidylethanolamine (PE).
The type strain for Pontibacter situs, BT310T (=KCTC 72363T = NBRC 114378T) was isolated from soil in Korea. The GenBank accession number for 16S rRNA gene sequence of strain BT310T is MT795756.