Variovorax terrae sp. nov. Isolated from Soil with Potential Antioxidant Activity

A white-pigmented, non-motile, gram-negative, and rod-shaped bacterium, designated CYS-02T, was isolated from soil sampled at Suwon, Gyeonggi-do, Republic of Korea. Cells were strictly aerobic, grew optimally at 20-28ºC and hydrolyzed Tween 40. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain CYS-02T formed a lineage within the family Comamonadaceae and clustered as members of the genus Variovorax. The closest members were Variovorax guangxiensis DSM 27352T (98.6% sequence similarity), Variovorax paradoxus NBRC 15149T (98.5%), and Variovorax gossypii JM-310T (98.3%). The principal respiratory quinone was Q-8 and the major polar lipids contain phosphatidylethanolamine (PE), phosphatidylethanolamine (PG), and diphosphatidylglycerol (DPG). The predominant cellular fatty acids were C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The DNA GC content was 67.7 mol%. The ANI and dDDH values between strain CYS-02T and the closest members in the genus Variovorax were ≤ 79.0 and 22.4%, respectively, and the AAI and POCP values between CYS-02T and the other related species in the family Comamonadaceae were > 70% and > 50%, respectively. The genome of strain CYS-02T showed a putative terpene biosynthetic cluster responsible for antioxidant activity which was supported by DPPH radical scavenging activity test. Based on genomic, phenotypic and chemotaxonomic analyses, strain CYS-02T was classified into a novel species in the genus Variovorax, for which the name Variovorax terrae sp. nov., has been proposed. The type strain is CYS-02T (= KACC 22656T = NBRC 00115645T).


Phylogenetic Analysis
The 16S rRNA gene of strain CYS-02 T was extracted using the InstaGene Matrix Kit (Bio-Rad, USA) as per the manufacturer's instructions. Isolation of 16S rRNA gene was determined by PCR using primers 27F and 1492R [15]. Sequencing was carried out by using an Applied Biosystems 3770XL DNA Analyser with a BigDye Terminator cycle sequencing Kit v.3.1 (Applied Biosystems, USA). Near-complete sequences of 16S rRNA genes (1457 bp) were assembled with SeqMan software (DNASTAR Inc.). The closest phylogenetic neighbors were identified using the EzBioCloud database [16]. All the 16S rRNA gene sequences of the closest phylogenetic members were retrieved from the NCBI GenBank database and aligned using SILVA alignment [17]. Phylogenetic trees were reconstructed using MEGA X software [18].

Chemotaxonomic Analysis
Fatty acids of strain CYS-02 T and reference strains were harvested from same culture condition (at 28°C for 4 days) during the late log phase. Fatty acids were extracted by the standard MIDI protocol (Sherlock Microbial Identification System, version 6.0B), analyzed with a gas chromatograph (HP 6890 Series GC System; Hewlett Packard, Country), and identified using the TSBA6 database of the Microbial Identification System [22]. Polar lipids and isoprenoid quinones were extracted from freeze-dried cells according to procedures described by Minnikin et al. [23]. Appropriate detection reagents were used to identify the spots [24].

Genomic and Genotypic Characterization
Whole genome-based approaches were used to confirm the taxonomic status of novel strains. For wholegenome sequencing, genomic DNA was extracted using DNeasy Blood and Tissue kits (Qiagen, Germany). Whole-genome shotgun sequencing of strain CYS-02 T was performed by Macrogen (Republic of Korea) using the Illumina HiSeq platform and assembled by SPAdes [25]. The authenticity of the genome assembly was checked by comparing 16S rRNA gene sequence using NCBI Align Sequences Nucleotide BLAST tool [26] and the potential contamination was checked by ContEst16S algorithm [27]. After analysis, the genome sequence was annotated utilizing the NCBI Prokaryotic Genome Annotation Pipeline [28] and Rapid Annotation using Subsystem Technology (RAST) server [29]. The anti-SMASH server was utilized to identify the biosynthetic gene clusters (BGCs) for various secondary metabolites [30]. Genome-based relatedness between strain CYS-02 T and closely related strains was observed based on Average Nucleotide Identity (ANI) utilizing the OrthoANIu algorithm [31]. DNA-DNA hybridization (DDH) was calculated in silico by the Genome-to-Genome Distance Calculator (GGDC 2.1) utilizing the blast method [32]. The average amino acid identity (AAI) utilizing Prodigal (Hyatt, D et al., 2010), MMseqs2 [33] and the EzAAI [34] were compared among strain CYS-02 T , closely related strains, and those of the other related species in the family Comamonadaceae. To evaluate the percentatage of conserved proteins (POCP) analysis, open reading frames (ORFs) were performed with the Prodigal tool and reciprocal Blastp was conducted as described by Qin et al. [35].

Antioxidant Activities
Screening test for antioxidant activities was achieved by DPPH (2,2-diphenyl-1-picrylhydrazyl) inhibition assay, which was performed using the method described by Dahal et al. [36]. The inhibitor was prepared by centrifuging the well-grown bacterial culture in R2A medium at 4 o C. Ascorbic acid (Vitamin C) was taken as positive control. The assay was performed in 96-well plates and incubated at 37 o C for 30 min. Absorption at 516 nm was evaluated by spectrophotometer (SpectraMax 340PC384 Microplate Reader; Molecular Devices Co., USA). Each treatment was triplicated and the percentage of radical scavenging activities was calculated using following formula: where: ODexp, absorbance of the experimental sample; ODcon, absorbance of control; ODstd, absorbance of standard; and ODbln, absorbance of the blank.

Phylogenetic Analysis
On the basis of 16S rRNA gene sequence comparisons, strain CYS-02 T showed the highest similarities with V. guangxiensis DSM 27352 T (98.6% sequence similarity). Sequence similarities between strain CYS-02 T and other validly described Variovorax species were below 98.5%. Neighbor-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) trees with reasonable bootstrap values confirmed the identification of strain CYS-02 T as a novel species in the genus Variovorax. Furthermore, strain CYS-02 T was well clustered within the members of the genus Variovorax and formed a separate lineage with the other closest members (Figs. 1, S1 and S2).

Morphological, Physiological and Biochemical Analysis
The colonies of strain CYS-02 T were rod-shaped (Fig. S3), gram-negative, strictly aerobic, non-motile, and nonsporulating. Strain CYS-02 T hydrolyzed Tween 80 but was unable to hydrolyze Tween 40 and DNA. Enzyme activity of acid phosphatase was positive for CYS-02 T but negative for reference strains ( Table 1). Assimilation of D-maltose was positive for strain CYS-02 T but negative for reference strains (Table 1). Similarly, D-mannitol, potassium gluconate and malic acid were negative for strain CYS-02 T while positive for the references (Table 1). Additional physiological and biochemical differential characteristics are presented in Table 1 along with the closest members of the genus Variovorax.

Genomic and Genotypic Characterization
The DNA GC content of strain CYS-02 T was 67.7 mol%, falling within the range for Variovorax species. The ANI threshold for species demarcation is recommended at 95-96% [37], and the Average Nucleotide Identity (ANI) values between strain CYS-02 T and its phylogenetically closest neighbors available with full genome sequences were ≤ 79.0% (Table S1). The dDDH values of ≤ 22.4% were much lower than the 70% species threshold recommended for species delineation [32] (Table S1), but the values for AAI and POCP were higher than 70% and 50%, respectively, indicating it to be located within the boundary of a genus [35,38] (Table S2).
The genome of strain CYS-02 T is 4,934,485 bp and consists of eight scaffolds with genome coverage of 157.0× (Table S3). The RAST analysis showed the presence of 308 subsystems and 4 secondary metabolisms consisting of four plant hormones (Fig. S5). The different genomic features of the novel isolate and phylogenetically closet members of the genus Variovorax based on the RAST result are listed in Table S3. The general genome features are given in Table S4. The anti-SMASH analyses of BGCs observed strain CYS-02 T as containing 3 putative BGCs responsible for secondary metabolites such as betalactone, terpene and hserlactone (Table S5).

Antioxidant Activities
Screening for DPPH radical scavenging activity showed the culture supernatant of strain CYS-02 T possessed antioxidant property. DPPH radical scavenging activity for strain CYS-02 T was 44% (Fig. S6). The annotation and Table 1

. Phenotypic characteristics of strain CYS-02 T and closely related type strains of the genus Variovorax.
C h a r a c t e r i s t i c 1 2 3 4 Maximum growth temperature (°C) 30 35 a 40 a 37 b Salt tolerance at 1% (w/v) + -a + a + b pH range 6.0-8.0 5.0-9.0 a 6.0-9.0 a 4.5-9.0 b Hydrolysis of Tween 40 The data were obtained from the previous work [12]. b The data were obtained from the previous work [13]. *The DNA GC contents were calculated from the whole genome sequences in this study.
analysis of secondary metabolite biosynthesis genes by using antiSMASH [30] revealed that strain CYS-02 T contained terpene (Tables S4). This gene cluster could play important roles in antioxidant activity and its compound(s) can be used for cosmetical, pharmacological, and possible therapeutic purposes [39]. However, further investigation is needed to depict the exact bioactive chemical and pathway for antioxidant activity. In this study, Variovorax terrae CYS-02 T was similar in major fatty acid composition, predominant ubiquinone, GC content range, and morphological analysis with other Variovorax type strains closely related to CYS-02 T . Although it was distinguished from its phylogenetic neighbors due to strain CYS-02 T having differences based on the phylogenetic tree, polar lipids, biochemical, physiological features, and low ANI and dDDH values, it could belong to the same genus according to AAI and POCP values. In conclusion, based on genomic, chemotaxonomic, phenotypic and phylogenetic analyses, strain CYS-02 T represents a novel species in the genus Variovorax, and the name Variovorax terrae sp. nov. is proposed.
The type strain is CYS-02 T (= KACC 22656 T = NBRC 00115645 T ), isolated from mountain soil of Suwon in South Korea. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the whole genome sequence of strain CYS-02 T are MZ573240 and JALGBI000000000, respectively.