Phylogenetic and Genome sequence analysis and analysis
Based on the 16S rRNA gene sequence similarities, strains BT291T and BT350T were affiliated with the family Methylobacteriaceae and showed high sequence similarities with the genus Microvirga. The strain BT291T was closely related to Microvirga aerophila 5420S-12T (97.5 % 16S rRNA gene similarity) and Microvirga subterranean DSM 14364T (97.2 %). The strain BT350T was closely related to Microvirga aerophila 5420S-12T (97.6 %) and Microvirga brassicacearum CDVBN77T (96.8 %). The 16S rRNA gene sequence similarities of strains BT291T and BT350T with the closely related type strains were less than 97.5 % and 97.6 %, respectively and with other Microvirga species were less than 96.9 %. These values were around or below the 98.7 % 16S rRNA gene sequence similarity recently used as the threshold for differentiating among bacterial species (Chun et al. 2018). The remaining Microvirga species exhibited sequence similarities lower than 97.0 %. After the reconstruction of neighbor-joining, maximum-likelihood (Fig. S2) and maximum-parsimony (Fig. S3) trees strain BT350T clustered with M. brassicacearum CDVBN77T and M. flavescens c27j1T and strain BT291T clustered independently (Fig. 1). The phylogenetic analysis results clearly showed that strains BT291T and BT350T are two new species within the genus Microvirga.
The draft genome of strain BT291T was 4.77 Mb (51.2×) long and consisted of 4,473 protein-coding genes, 57 RNA genes (6 rRNA genes, 50 tRNA genes) and 8 pseudogenes. The draft genome of strain BT350T was 4,42 Mb (29.9×) long and consisted of 4,014 protein-coding genes, 51 RNA genes (4 rRNA genes, 47 tRNA genes) and 66 pseudogenes. The genome sequence of the strains BT291T and BT350T have been deposited in GenBank under the accession numbers NZ_JAFEMB000000000 and NZ_JADQDO010000000, respectively. The DNA G + C contents of strains BT291T and BT350T were 64.7 mol% and 61.9 mol%, respectively. These values were within the range of the G + C contents for the genus Microvirga as previously reported (63.5–64.3 mol%). The digital DNA-DNA hybridization values between strains BT291T and BT350T and other related type strains of genus Microvirga were less than 23.1 %, respectively (Table S1), which are below the cutoff (70 %) point (Meier- Kolthoff et al. 2013). Average nucleotide identity (ANI) values between strains BT291T and BT350T and other related type strains of genus Microvirga were less than 79.1 %, respectively (Table S1). These values are below the ANI species threshold (95–96 % ANI value) as described by Ritcher and Rossello-Mora (2009).
Chemotaxonomic characterization
The fatty acid profiles of strains BT291T and BT350T and three reference strains of genus Microvirga were presented in Table 2. The major fatty acids of strain BT291T C18:1 ω7c (58.2 %) and cyclo-C19:0 ω8c (25.7 %). Strain BT291T has smaller amounts of C16:0 (5.8 %), summed feature 2 (iso-C16:1 I / C14:0 3OH) (2.2 %) and C18:0 (1.5 %), whereas other closely related Microvirga species (M. aerophila 5420S-12T, M. subterranean DSM 14364T and M. subterranea FaiI4T) have larger amounts of corresponding fatty acids. Strain BT291T contained C19:0 10-methyl (1.1 %) and C14:0 (> 1 %), but other closely related Microvirga species (M. aerophila 5420S-12T, M. subterranean DSM 14364T and M. subterranea FaiI4T) did not contain those fatty acids.
The major fatty acid profiles of strain BT350T were C18:1 ω7c (38.5 %) and cyclo-C19:0 ω8c (27.7 %). Strain BT350T has larger amount of C16:0 (10.4 %), cyclo-C17:0 (6.4 %) and C18:0 3OH (1.8 %), whereas other closely related Microvirga species (M. aerophila 5420S-12T, M. subterranean DSM 14364T and M. subterranea FaiI4T) have smaller amounts of corresponding fatty acids. Strain BT350T did not contain C17:1 ω8c, C17:1 ω6c and C17:1 ω7c 11-methyl, but other closely related Microvirga species (M. aerophila 5420S-12T, M. subterranean DSM 14364T and M. subterranea FaiI4T) contained those fatty acids.
The polar lipids of strain BT291T consisted of a phosphatidylethanolamine (PE), an unknown diphosphatydilglycerol (DPG), unknown phosphatidylcholine (PC), an unknown phosphatidylglycerol (PG), an unknown aminolipid (AL), an unknown aminophospholipid (APL), an unknown phospholipid (PL), an unknown glycolipid (GL) and two unknown lipids (L) (Fig. S4). In contrast, strain BT350T consisted of a phosphatidylethanolamine (PE), an unknown diphosphatydilglycerol (DPG), an unknown phosphatidylglycerol (PG), unknown phosphatidylcholine (PC), an unknown amino lipid (AL) and an unknown lipid (L) (Fig. S5). The dominant respiratory quinone of strains BT291T and BT350T was Q-10. These results supported that chemotaxonomic characteristic of strains BT291T and BT350T are similar to those of the other species in the genus Microvirga. Based on phenotypic, phylogenetic and biochemical features, it is concluded that strains BT291T and BT350T represent two novel species of the genus Microvirga, for which the name Microvirga amygdalina and Microvirga alba are proposed.
Description of Microvirga amygdalina sp. nov.
Microvirga amygdalina (a.myg.da.li’na L. fem. adj. amygdalina of almonds).
Cells are Gram-stain-negative, aerobic, rod-shaped, 0.6–1.3 µm in diameter and about 1.6–2.7 µm in length, non spore forming and non-motile. Colonies are irregular, convex and light-pink-colored on Reasoner's 2A (R2A) agar plates after growth for three days at 25°C. Growth is observed at temperatures ranging from 10 to 30°C (optimum 25°C). The pH range for growth is 6.0–9.0 (optimum pH 8.0) on R2A agar. Normal cell growth occurs at 10–30°C (optimum 25°C) and pH 6.0–9.0 (optimum 8.0). Cells grow on Reasoner’s 2A agar (R2A), Luria-Bertani agar (LB), Tryptic Soy Agar (TSA), Nutrient Agar (NA) and Macconkey (MAC) agar (weakly). Cells are positive for oxidase and catalase activity. The major respiratory quinone is Q-10. The dominant cellular fatty acids are C18:1 ω7c (58.2 %) and cyclo-C19:0 ω8c (25.7 %). The major polar lipids are phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC), phosphatidylglycerol (PG). Positive for nitrate reduction, arginine dihydrolase and urease (API 20NE). Positive for esterase (C4) and acid phosphatase (API ZYM). The whole-genome sequence of strain BT291T has been deposited in GenBank under the accession number NZ_JAFEMB000000000 (4.77 Mb). The genome-based G + C content is 64.7 mol%. The GenBank accession number for the 16S rRNA gene sequence of strain BT291T is MT795755 (1,422 bp). The type strain BT291T (= KCTC 72368T = NBRC 114845T) was isolated from a soil sample collected in Uijeongbu city (37° 44′ 55″ N, 127° 2′ 20″ E), South Korea.
Description of Microvirga alba sp. nov.
Microvirga alba (al'ba. L. fem. adj. alba white).
Cells are Gram-stain-negative, aerobic, rod-shaped, 0.4–0.9 µm in diameter and about 0.5–1.2 µm in length, non spore forming and non-motile. Colonies are irregular, convex and white colored on Reasoner's 2A (R2A) agar plates after growth for three days at 25°C. Growth is observed at temperatures ranging from 10 to 30°C (optimum 25°C). The pH range for growth is 5.0–9.0 (optimum pH 8.0) on R2A agar. Cells grow on Reasoner’s 2A agar (R2A), Tryptic Soy Agar (TSA) and Nutrient Agar (NA) but not on Luria-Bertani agar (LB) and Macconkey (MAC) agar. Cells are positive for oxidase and catalase activity. The major respiratory quinone is Q-10. The dominant cellular fatty acids are C18:1 ω7c (38.5 %) and cyclo-C19:0 ω8c (27.7 %). The major polar lipids are phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC) and phosphatydilglycerol (PG). Weakly positive for trisodium citrate (API 20NE). Positive for alkaline phosphatase and esterase (C4) (API ZYM).
The whole-genome sequence of strain BT350T has been deposited in GenBank under the accession number NZ_JADQDO010000000 (4,42 Mb). The genome-based G + C content is 61.9 mol%. The GenBank accession number for the 16S rRNA gene sequence of strain BT350T is MT795757 (1,416 bp). The type strain BT350T (= KCTC 72385T = NBRC 114848T) was isolated from a soil sample collected in Jeju island (33° 22′ 48″ N, 126° 31′ 48″ E), South Korea.