Microvirga Terrestris sp. nov., and Microvirga Arvi sp. nov., Two Novel Species Isolated From Soil in South Korea


 Two novel Gram-stain-negative, aerobic, rod shaped bacterial strains BT290T and BT689T were isolated from soil collected in South Korea. Colony morphologies of both strains were circular and convex while the colors of BT290T and BT689T were light-pink and white, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that BT290T and BT689T belong to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alphaproteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rRNA gene sequence similarity between two strains was 97.9 %. Both strains had the similar quinone system, with ubiquinone 10 (Q-10) as the major respiratory quinone. The major polar lipids of strains BT290T and BT689T were phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC) and phosphatydilglycerol (PG). The major cellular fatty acids of strain BT290T were C18:1 ω7c (58.2 %) and C16:0 (17.7 %), while those of strain BT689T were C18:1 ω7c (61.8 %) and C16:0 (10.8 %).On the bases of polyphasic analysis (phylogenetic, chemotaxonomic and biochemical), strains BT290T and BT689T can be suggested as novel bacterial species within the genus Microvirga and the proposed names are Microvirga terrestris and Microvirga arvi, respectively. The type strain of Microvirga terrestris is BT290T (= KCTC 72367T=NBRC 114844T) and the type strain of Microvirga arvi is BT689T (= KACC 22016T = NBRC 114858T), respectively.

In this study, two strains BT290 T and BT689 T were newly isolated from soil samples collected in South Korea. A phylogenetic analysis was conducted based on the 16S rRNA gene sequences and phenotypic, genotypic, and chemotaxonomic characteristics were performed to determine the taxonomic position of strains BT290 T and BT689 T . The results suggested that strains BT290 T and BT689 T represent two novel species of the genus Microvirga, for which the name Microvirga terrestris sp. nov. and Microvirga arvi sp. nov. are proposed, respectively.

Materials And Methods
Isolation and culture conditions of bacteria Two soil samples were obtained in South Korea to isolate new strains. The strain BT290 T was isolated from Jeongseon province (37° 22′ 45″ N, 128° 39′ 53″ E) and strain BT689 T was isolated from Uijeongbu city (37° 44′ 55″ N, 127° 2′ 20″ E). Colonies were isolated using Reasoner's 2A (R2A) agar medium (Difco) after incubation at 25°C for 10 days and single colonies puri cation were performed under the same condition. The strains were routinely sub-cultured on R2A agar at 25°C, maintained at 4°C and stock solutions were stored in 10 % (w/v) glycerol suspension at −80°C before use.

Morphology, physiology and biochemical analysis
The cell morphologies of strains BT290 T and BT689 T were examined using a transmission electron microscopy (JEOL, JEM1010) after grown on R2A agar for 3 days at 25°C, using negative staining method. The Gram-staining was performed using a commercialized kit, following the manufacturer's instruction (bioMérieux). Catalase activity was examined with 3 % (w/v) hydrogen peroxide and oxidase activity was examined by addition of 1 % (w/v) tetramethyl-p-phenylenediamine [10].

Phylogenetic analysis
The 16S rRNA genes of strains BT290 T (1,417 bp) and BT689 T (1,451 bp) were ampli ed by PCR using two universal bacterial primers 27F and 1492R [11] using the BT290 T and BT689 T genomic DNA as templates. Then, sequencing was performed using four universal primers including 337F, 518R, 785F, and 926R (Macrogen). To determine the taxonomic positions of strains BT290 T and BT689 T , similar 16S rRNA sequences were obtained from EzBioCloud [12] and compared with those of strains BT290 T and BT689 T using EzEditor2 server. Phylogenetic trees were constructed using the MEGAX program [13] with the neighbor-joining [14], maximum-likelihood [15] and maximum-parsimony algorithms [16]. The stability of the tree topologies was calculated based on 1,000 replications [17] and evolutionary distances were calculated using Kimura's two-parameter model [18].

Genome sequencing and analysis
Genomic DNA was extracted using a genomic DNA extraction kit according to the manufacturer's instruction (Solgent). Sequencing libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina) and whole-genome sequencing was performed by iSeq 100 system (Illumina). The obtained genome sequences were assembled using a SPAdes 3.10.1 (Algorithmic Biology Lab, St. Petersburg Academic University of the Russian Academy of Sciences). Whole-genome sequences of strains BT290 T and BT689 T were deposited in GenBank (www.ncbi.nlm.nih.gov/) database, respectively. The genome sequences of strains BT290 T and BT689 T were annotated by the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (PGAP) [19]. The average nucleotide identity (ANI) was calculated using the EzBioCloud (https://www.ezbiocloud.net) and digital DNA-DNA hybridization (dDDH) values were calculated using the Genome-to Genome Distance Calculator (GGDC) with the recommended formula 2 (Table 1) [20].

Chemotaxonomic characteristics
To analyze the cellular composition of polar lipid, fatty acid, and quinone of strains BT290 T and BT689 T , both strains were grown on R2A agar at 25°C for three days and then cells were freeze-dried. Polar lipids were extracted as described previously [21]. 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 [22,23]. The fatty acids were puri ed by saponi cation, methylation and extraction procedures and analyzed by Sherlock Microbial Identi cation System V6.01 (MIS, database TSBA6, MIDI Inc., Newark, DE, USA) [24]. The quinones of strains BT290 T and BT689 T were extracted using the Sep-Pak Vac cartridges (Waters) and analyzed by high-performance lipid chromatography (HPLC) based on the previous methods [25]. 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 Strains BT290 T and BT689 T were Gram-staining-negative bacteria and they showed rod-shaped morphology ( Fig. 1). Colonies of strains BT290 T and BT689 T were circular, convex, smooth, light-pink and white colored after incubation for three days at 25°C. Cells of strains BT290 T and BT689 T could survive at 10 to 30°C (optimum 25°C) and pH 6.0 -9.0 (optimum 8.0) on R2A medium. Distinct features of new strains and reference strains were presented in Table 1. The negative reaction of strains BT290 T and BT689 T by API analysis were given as supplementary tables (Table S1 and S2, respectively). . The results of neighbor-joining tree (Fig. 2), maximumlikelihood tree (Fig. S1) and maximum-parsimony tree (Fig. S2) showed that strains BT290 T and BT689 T were clustered with M. soli R491 T and M. occulans ATCC BAA-817 T , respectively, at >70 % bootstrap support (Fig. 2). The phylogenetic analysis results clearly showed that strains BT290 T and BT689 T are two new species within the genus Microvirga.

Genome sequence analysis
The  (Table S3), which are below the cutoff (70 %) point [20]. Average nucleotide identity (ANI) values between strains BT290 T and BT689 T 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 [27].

Chemotaxonomic characterization
Fatty acid pro les of strains BT290 T and BT689 T and three reference strains of genus Microvirga were presented in Table 2. The major fatty acids of strain BT290 T were C 18:1 ω7c (58.2 %) and C 16:0 (17.7 %).
The dominant respiratory quinone of strains BT290 T and BT689 T was ubiquinone 10 (Q-10).
Regarding the chemotaxonomic characteristics of BT290 T and BT689 T , both strains could be differentiated from other Microvirga species. Moreover, based on phenotypic, phylogenetic and biochemical features, it is concluded that strains BT290 T and BT689 T 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.
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 The whole genome sequence of strain BT689 T 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 BT689 T is MT795749 (1,451 bp). The type strain BT689 T (= KACC 22016 T = NBRC 114858 T ) was isolated from a soil sample collected in Uijeongbu city (37° 44′ 55″ N, 127° 2′ 20″ E), South Korea. Figure 1 Transmission electron micrograph of strains BT290T (a) and BT689T (b) from cultures grown on R2A agar for three days at 25 °C. Bars are 500 nm.

Figure 2
Neighbor-joining phylogenetic tree reconstructed from a comparative analysis of 16S rRNA gene sequences showing the relationships of strains BT290T and BT689T with closely related validly published species. Bootstrap values (based on 1,000 replications) greater than 70 % based on neighborjoining method is shown at the internodes. Circles indicate that the corresponding nodes were also recovered in the maximum-parsimony tree. Triangles indicate that the corresponding nodes were recovered in the maximum-likelihood tree. Phyllobacterium loti S658T was used as an outgroup. Bar, 0.01