Genome sequence analysis
The draft genome of strain BT290T was 4.20 Mb (38.6×) long and consisted of 3,867 protein-coding genes, 50 RNA genes (5 rRNA genes, 45 tRNA genes), and 41 pseudogenes. The draft genome of strain BT689T was 6.10 Mb (29.9×) long and consisted of 4,014 protein-coding genes, 51 RNA genes (4 rRNA genes, 44 tRNA genes), and 66 pseudogenes. The genome sequences of strains BT290T and BT689T were deposited in GenBank database (accession numbers: NZ_JADQDN000000000 for BT290T and NZ_JAFEMD000000000 for BT689T, respectively). The DNA G+C contents of strains BT290T and BT689T were 62.3 mol% and 62.4 mol%, respectively. These values were within the range of the G+C contents for the genus Microvirga those previously reported (61.1 - 65.1 mol %) (Zhang e al. 2019), again supported that two strains belong to genus Microvirga. The digital DNA-DNA hybridization values between strains BT290T and BT689T and other related type strains of genus Microvirga were less than 26.7% and 28.4%, respectively (Table S3), which are below the cutoff (70%) point (Meier-Kolthoff et al. 2013). Average nucleotide identity (ANI) values between strains BT290T and BT689T and other related type strains of genus Microvirga were less than 82.9% and 83.9%, respectively (Table S3). These values are below the ANI species threshold (95 - 96% ANI value) as described by Ritcher and Rossello-Mora (Richter et al. 2009).
Chemotaxonomic characterization
Fatty acid profiles of strains BT290T and BT689T and three reference strains of genus Microvirga were presented in Table 2. The major fatty acids of strain BT290T were C18:1 ω7c (58.2%) and C16:0 (17.7%). Interestingly, strain BT290T has large amounts of C18:0 (5.7%), cyclo-C19:0 ω8c (2.2%), C14:0 (1.6%) and C18:1 ω7c 11-methyl (1.2%), whereas the most closely related species M. aerilata 5420S-16T has smaller amounts of corresponding fatty acids. In addition, strain BT290T did not contain C15:1 ω8c while most closely related species M. aerilata 5420S-16T contained that fatty acid. The polar lipids of strain BT290T consisted of phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC), phosphatydilglycerol (PG), aminolipid (AL), and four unknown lipid (L) (Fig. S3). The major fatty acid profiles of strain BT689T were C18:1 ω7c (61.8%) and C16:0 (10.8%). Strain BT689T has large amounts of cyclo-C19:0 ω8c (8.2%), C18:0 (6.6%), C18:1 ω7c 11-methyl (2.3%), C14:0 (1.3%) and C18:0 3OH (1.0%), whereas the most closely related species M. aerilata 5420S-16T has smaller amounts of corresponding fatty acids. Strain BT689T contained C17:0, cyclo-C17:0 and C18:1 ω9c, but most closely related species M. aerilata 5420S-16T did not contain those fatty acids. The polar lipids of strain BT689T consisted of phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatydilglycerol (PG), phosphatidylcholine (PC), aminolipid (AL) and nine unknown lipid (L) (Fig. S4).
The dominant respiratory quinone of strains BT290T and BT689T was ubiquinone 10 (Q-10).
Regarding the chemotaxonomic characteristics of BT290T and BT689T, both strains could be differentiated from other Microvirga species. Based on phenotypic, phylogenetic and biochemical features, it is concluded that strains BT290T and BT689T represent two novel species of the genus Microvirga, for which the name Microvirga terrestris and Microvirga arvi, respectively, are proposed.
Description of Microvirga terrestris sp. nov.
Microvirga terrestris (ter.res'tris. L. fem. adj. terrestris of the earth).
Cells are Gram-stain-negative, aerobic, rod-shaped, 0.5 - 1.4 µm in diameter and about 1.3 - 2.4 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 R2A agar and MacConkey (MAC) agar (weakly) but not on Luria-Bertani agar (LB), Tryptic Soy Agar (TSA) and Nutrient Agar (NA). Cells are positive for oxidase and catalase activity. The major respiratory quinone of strain BT290T is ubiquinone 10 (Q-10). The dominant cellular fatty acids are C16:0 and C18:1 ω7c. The major polar lipids are phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC) and phosphatydilglycerol (PG). Positive for D-glucose (API 20NE). Positive for acid phosphatase (API ZYM). The whole-genome sequence of strain BT290T has been deposited in GenBank under the accession number NZ_JADQDN000000000 (6.10 Mb). The genome-based G+C content is 62.3 mol %. The GenBank accession number for the 16S rRNA gene sequence of strain BT290T is MT795754 (1,417 bp). The type strain BT290T (= KCTC 72367T=NBRC 114844T) was isolated from a soil sample collected in Jeongseon province (37° 22′ 45″ N, 128° 39′ 53″ E), South Korea.
Description of Microvirga arvi sp. nov.
Microvirga alba (ar'vi. L. gen. n. arvi of a field).
Cells are Gram-stain-negative, aerobic, rod-shaped, 0.6 - 0.7 µm in diameter and about 1.1 - 2.0 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 6.0 - 9.0 (optimum pH 8.0) on R2A agar. Cells grow on R2A agar, Luria-Bertani agar (LB) (weakly) and MacConkey (MAC) (weakly) agar but not on Tryptic Soy Agar (TSA) and Nutrient Agar (NA). Cells are positive for oxidase and catalase activity. The major respiratory quinone of strain is ubiquinone 10 (Q-10). The dominant cellular fatty acids are C18:1 ω7c and C16:0. The major polar lipids are phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC) and phosphatydilglycerol (PG). Positive for nitrate reduction to NO2 (weakly), nitrate reduction to N2, D-glucose and l-malate (weakly) (API 20NE). Positive for alkaline phosphatase and acid phosphatase (API ZYM).
The whole genome sequence of strain BT689T has been deposited in GenBank under the accession number NZ_JAFEMD000000000 (4,42 Mb). The genome-based G+C content is 62.4 mol%. The GenBank accession number for the 16S rRNA gene sequence of strain BT689T is MT795749 (1,451 bp). The type strain BT689T (= KACC 22016T = NBRC 114858T) was isolated from a soil sample collected in Uijeongbu city (37° 44′ 55″ N, 127° 2′ 20″ E), South Korea.