Microvirga Jeongseonensis Sp. Nov., New Species Isolated From Soil in South Korea

A novel Gram-stain-negative, aerobic, rod-shaped, convex, and light pink-colored strain BT688 T was isolated from a soil sample collected in Jeongseon city, South Korea. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain BT688 T belongs to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alpha Proteobacteria, phylum Proteobacteria). The 16S rRNA gene sequence similarity between strain BT688 T and Microvirga aerilata 5420S-16 T was 98.5%. Strain BT688 T had Q-10 as a major respiratory quinone and the major polar lipids of strain BT688 T was diphosphatidilglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). The major cellular fatty acids of strain BT688 T were C 18:1 ω7c (76.0%) and summed feature 3 (9.6%). Based on the polyphasic characteristics, strain BT688 T can be suggested as a novel bacterial species within the genus Microvirga and the proposed name is Microvirga jeongseonensis. The type strain of Microvirga jeongseonensis is BT688 T (= KCTC XXXX T =NBRC 114857 T ).

In this study, strain BT688 T was newly isolated from a soil sample collected in Jeongseon city, South Korea, and characterized. Phylogenetic analysis, phenotypic, genotypic, and chemotaxonomic characterization were performed to determine the taxonomic position of strain BT688 T . The results suggest that strain BT688 T represents a novel species of the genus Microvirga, for which the name Microvirga jeongseonensis sp. nov. is proposed.

Materials And Methods
Bacteria isolation and culture conditions Strain BT688 T was isolated from Jeongseon city (37° 22' 38.1"N, 128° 47' 17.0" E) located in South Korea. A single colony was isolated using Reasoner's 2A (R2A) agar medium (Difco) after incubation at 25°C for 10 days. Then the strain was routinely cultured on R2A agar at 25°C, maintained at 4°C, and stored in 20% (w/v) glycerol suspension at − 80°C, respectively.

Phylogenetic analysis
The 16S rRNA gene of strain BT688 T was ampli ed using two universal bacterial primers 27F and 1492R (Weisburg et al. 1991). The 16S rRNA gene sequence was identi ed using the EzBioCloud server (https://www.ezbiocloud.net/). The sequence was analyzed using the 337F, 518R, 785F and 926R universal primers (Macrogen, Korea). To determine the taxonomic position of strain BT688 T , 16S rRNA sequences of closely related species were obtained from EzBioCloud (Yoon et al. 2017) and compared with that of strain BT688 T using EzEditor2 program. Phylogenetic trees were reconstructed using the MEGAX program (Kumar et al. 2018) with the neighbor-joining (Saitou and Nei 1987), maximumlikelihood (Felsenstein 1981), and maximum-parsimony algorithms (Fitch 1971). The stability of the tree topologies was evaluated by bootstrap analysis based on the 1,000 resampling method (Felsenstein 1985). Evolutionary distances were calculated according to the Kimura two-parameter model (Kimura 1983).

Whole-genome sequence analysis
The genomic DNA of strain BT688 T was extracted using a genomic DNA extraction kit according to the manufacturer's instruction (Solgent, Korea). The sequencing libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina) and whole-genome sequencing was performed using iSeq 100. The partial genome sequences were assembled by using the SPAdes algorism (ver.

Chemotaxonomic characteristics
To analyze the composition of cellular fatty acid, polar lipid, and quinone strain BT688 T was grown on R2A agar at 25°C for 3 days and collected cells were freeze-dried. Polar lipids of strain BT688 T were extracted as described by Minnikin et al. (1984). The total lipids, glycolipids, phosphatidylcholine, and amino lipid groups were separated using two-dimensional thin-layer chromatography (TLC) and detected by using proper detection reagents (Komagata and Suzuki 1987). Fatty acids were puri ed by saponi cation, methylation, and extraction procedures (Sasser 1990). Lipoquinones were extracted using the Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) based on previous methods (Hiraishi et al. 1996). The fatty acid methyl esters (FAME) were identi ed using the Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc).

Results And Discussion
Morphology, Physiology and Biochemical analysis Strain BT688 T was Gram-negative and had rod-shaped morphology (Fig. S1). Colonies of strain BT688 T were circular, convex, smooth, and light pink color after incubation for three days at 25°C. Cells of strain BT688 T could grow at 10 to 30°C (optimum 25°C) and pH 6.0-9.0 (optimum 7.0) on R2A agar. Different features between the newly isolated strain and reference strains were provided in Table 1. The negative reactions of strain BT688 T on API kits were shown in the supplementary table (Table S1).

Phylogenetic and genome sequence analysis
The length of the 16S rRNA gene of strain BT688 T was 1,428 bp. Based on the 16S rRNA gene sequence similarities, strain BT688 T was a liated with the family Methylobacteriaceae and showed high sequence similarities with the genus Microvirga. The strain BT688 T was similar to Microvirga aerilata 5420S-16 T (98.5% of 16S rRNA gene similarity) and Microvirga makkahensis SV1470 T (98.2% of 16S rRNA gene similarity). These values were around or below the 98.7 % 16S rRNA gene sequence similarity recently used as the threshold for bacterial species classi cation (Chun et al. 2018). Analysis of neighbor-joining (Fig. 1), maximum-likelihood (Fig. S2), and maximum-parsimony (Fig. S3) trees clearly showed that strain BT688 T belongs to the genus Microvirga.
The draft genome length of strain BT688 T was 6.62 Mb (29.6×) and consisted of 6,356 protein-coding genes and 55 RNA genes including 2 rRNA genes and 53 tRNA genes. The genome sequence of strain BT688 T was deposited in GenBank under the accession number NZ_JACXAB000000000. The DNA G + C contents of strain BT688 T was 62.2 mol%. which value was within the range of the G + C contents of the genus Microvirga (63.5-64.3 mol%). The average nucleotide identity (ANI) value between strain BT688 T and the most closely related type strain 5420S-16 T (genus Microvirga, Microvirga aerilata) was 83.3%. This value is below the ANI species threshold (95-96 % ANI value) as described by Ritcher and Rossello-Mora (2009).
The polar lipids of strain BT688 T consisted of one diphosphatidilglycerol (DPG), one phosphatidylglycerol (PG), one phosphatidylethanolamine (PE), one phosphatidylcholine (PC), one amino lipid (AL), one aminophospholipid (APL), one phospholipid (PL) and two unknown lipids (L) (Fig. S4). The major respiratory quinone of strain BT688 T was Q-10. The results of the chemotaxonomic analysis indicated that strain BT688 T is similar to those of the other species in the genus Microvirga. Based on phenotypic, phylogenetic, and biochemical characteristics, it can be concluded that strain BT688 T represents a novel species of the genus Microvirga, for which the name Microvirga jeongseonensis is proposed.