Microvirga Amygdalina sp. nov., and Microvirga Alba sp. nov., Two New Species Isolated from Soil in South Korea

Isolation and cultural conditions

Strain BT291^T was isolated from Uijeongbu city (37° 44′ 55″ N, 127° 2′ 20″ E) and strain BT350^T was isolated from Jeju island (33° 22′ 48″ N, 126° 31′ 48″ E) located in South Korea. Colonies were isolated using Reasoner's 2A (R2A) agar medium (Difco) after incubation at 25°C for 9 days. After that, the strains were routinely cultured on R2A agar at 25°C, maintained at 4°C and stored in 10 % (w/v) glycerol suspension at − 80°C. The 16S rRNA gene sequences of the purified colonies were identified using the EzBioCloud server (https://www.ezbiocloud.net/).

Morphology, physiology and biochemical analysis

The cell morphology was examined using transmission electron microscopy (JEOL, JEM1010) with negative staining. The Gram-staining reaction was performed using a kit, following the manufacturer’s instruction (bioMérieux). Catalase activity was examined with 3 % (w/v) H₂O₂ solution and oxidase activity was examined by the addition of 1 % (w/v) tetramethyl-p-phenylenediamine (Cappuccino and Sherman 2002). The growth of strains BT291^T and BT350^T was tested on Reasoner’s 2A (R2A) agar, Luria-Bertani (LB) agar, Tryptic Soy Agar (TSA), Nutrient Agar (NA) and on MacConkey (MAC) agar. Growth was tested at different temperatures (10, 15, 25 and 30°C) under various pH conditions (5 to 9, 1 pH intervals) and different NaCl concentrations (1 % to 5 % [w/v %], 1 % intervals). API 20NE and API ZYM tests were performed according to the manufacturer’s instruction (bioMérieux).

Phylogenetic analysis

The 16S rRNA genes of strains BT291^T (1,428 bp) and BT350^T (1,435 bp) were amplified and sequenced using two universal bacterial primers 27F and 1492R (Weisburg et al. 1991) using the BT291^T and BT350^T genomic DNA as a template. The sequencing was then done using the 337F, 518R, 785F, and 926R universal primers (Macrogen). To determine the taxonomic positions of strains BT291^T and BT350^T, 16S rRNA sequences of similarity searches were obtained from EzBioCloud (Yoon et al. 2017) and compared with of strains BT291^T and BT350^T using EzEditor2 server. Phylogenetic trees were reconstructed using the MEGAX program (Kumar et al. 2018) with the neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony algorithms (Fitch 1971). The stability of the tree topologies was evaluated by bootstrap analysis based on 1,000 resampling method (Felsenstein 1985). Evolutionary distances were calculated according to the Kimura two-parameter model (Kimura 1983).

Genome sequencing

The genomic DNA was extracted using a genomic DNA extraction kit according to the manufacturer’s instruction (Solgent). The sequencing libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina), and whole-genome sequencing was performed by iSeq 100. The genome sequences were assembled using SPAdes 3.10.1 (Algorithmic Biology Lab, St. Petersburg Academic University of the Russian Academy of Sciences). The whole-genome sequences of strains BT291^T and BT350^T were deposited in GenBank (www.ncbi.nlm.nih.gov/) database. The genome sequences of strains BT291^T and BT350^T were annotated by the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016). The average nucleotide identity (ANI) was calculated using the EzBioCloud (https://www.ezbiocloud.net) and the digital DNA-DNA hybridization (dDDH) was calculated using the Genome-to Genome Distance Calculator (GGDC) with the recommended formula 2 (Table 1) (Meier-Kolthoff et al. 2013).

Chemotaxonomic characteristics

For analysis of cellular fatty acid, polar lipid and quinone strains BT291^T and BT350^T were grown on R2A agar at 25°C for three days and cells were freeze-dried. Polar lipids of strains BT291^T and BT350^T were extracted as described previously. 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 (Komagata and Suzuki 1987; Minnikin et al. 1984). The fatty acids were purified by saponification, methylation and extraction procedures (Sasser 1990). The quinones were extracted using the Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) based on the previous methods (Hiraishi et al. 1996). The fatty acid methyl esters (FAME) were identified using the Sherlock Microbial Identification System V6.01 (MIS, database TSBA6, MIDI Inc).

Morphology, Physiology and Biochemical analysis

Strains BT291^T and BT350^T were Gram-staining-negative bacteria and they showed rod-shaped morphology (Fig. S1). Colonies of strains BT291^T and BT350^T were circular, convex and smooth after incubation for three days at 25°C. Colonies color of strains BT291^T and BT350^T were light-pink and white, respectively. Cells of strain BT291^T could survive at 10 to 30°C (optimum 25°C) and pH 6.0–9.0 (optimum 8.0) in R2A medium and the cells of strain BT350^T could survive at 10 to 30°C (optimum 25°C) and pH 5.0–9.0 (optimum 8.0) in R2A medium. Differential features between the new strains and reference strains were provided in Table 1. In addition, the negative reaction of strains BT291^T and BT350^T on API kits were given as supplementary tables (Table S2 and S3).

Phylogenetic and Genome sequence analysis and analysis

Based on the 16S rRNA gene sequence similarities, strains BT291^T and BT350^T were affiliated with the family Methylobacteriaceae and showed high sequence similarities with the genus Microvirga. The strain BT291^T was closely related to Microvirga aerophila 5420S-12^T (97.5 % 16S rRNA gene similarity) and Microvirga subterranean DSM 14364^T (97.2 %). The strain BT350^T was closely related to Microvirga aerophila 5420S-12^T (97.6 %) and Microvirga brassicacearum CDVBN77^T (96.8 %). The 16S rRNA gene sequence similarities of strains BT291^T and BT350^T 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 BT350^T clustered with M. brassicacearum CDVBN77^T and M. flavescens c27j1^T and strain BT291^T clustered independently (Fig. 1). The phylogenetic analysis results clearly showed that strains BT291^T and BT350^T are two new species within the genus Microvirga.

The draft genome of strain BT291^T 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 BT350^T 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 BT291^T and BT350^T have been deposited in GenBank under the accession numbers NZ_JAFEMB000000000 and NZ_JADQDO010000000, respectively. The DNA G + C contents of strains BT291^T and BT350^T 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 BT291^T and BT350^T 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 BT291^T and BT350^T 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 BT291^T and BT350^T and three reference strains of genus Microvirga were presented in Table 2. The major fatty acids of strain BT291^T C_18:1 ω7c (58.2 %) and cyclo-C_19:0 ω8c (25.7 %). Strain BT291^T has smaller amounts of C_16:0 (5.8 %), summed feature 2 (iso-C_16:1 I / C_14:0 3OH) (2.2 %) and C_18:0 (1.5 %), whereas other closely related Microvirga species (M. aerophila 5420S-12^T, M. subterranean DSM 14364^T and M. subterranea FaiI4^T) have larger amounts of corresponding fatty acids. Strain BT291^T contained C_19:0 10-methyl (1.1 %) and C_14:0 (> 1 %), but other closely related Microvirga species (M. aerophila 5420S-12^T, M. subterranean DSM 14364^T and M. subterranea FaiI4^T) did not contain those fatty acids.

The major fatty acid profiles of strain BT350^T were C_18:1 ω7c (38.5 %) and cyclo-C_19:0 ω8c (27.7 %). Strain BT350^T has larger amount of C_16:0 (10.4 %), cyclo-C_17:0 (6.4 %) and C_18:0 3OH (1.8 %), whereas other closely related Microvirga species (M. aerophila 5420S-12^T, M. subterranean DSM 14364^T and M. subterranea FaiI4^T) have smaller amounts of corresponding fatty acids. Strain BT350^T did not contain C_17:1 ω8c, C_17:1 ω6c and C_17:1 ω7c 11-methyl, but other closely related Microvirga species (M. aerophila 5420S-12^T, M. subterranean DSM 14364^T and M. subterranea FaiI4^T) contained those fatty acids.

The polar lipids of strain BT291^T 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 BT350^T 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 BT291^T and BT350^T was Q-10. These results supported that chemotaxonomic characteristic of strains BT291^T and BT350^T are similar to those of the other species in the genus Microvirga. Based on phenotypic, phylogenetic and biochemical features, it is concluded that strains BT291^T and BT350^T 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 C_18:1 ω7c (58.2 %) and cyclo-C_19:0 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 BT291^T 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 BT291^T is MT795755 (1,422 bp). The type strain BT291^T (= KCTC 72368^T = NBRC 114845^T) 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 C_18:1 ω7c (38.5 %) and cyclo-C_19:0 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 BT350^T 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 BT350^T is MT795757 (1,416 bp). The type strain BT350^T (= KCTC 72385^T = NBRC 114848^T) was isolated from a soil sample collected in Jeju island (33° 22′ 48″ N, 126° 31′ 48″ E), South Korea.