Characteristics of Planococcus antioxidans sp. nov., an antioxidant‐producing strain isolated from the desert soil in the Qinghai–Tibetan Plateau

Abstract Strain Y74T was an isolate from the sandy soil in the town of Huatugou, Qinghai–Tibet Plateau, China. An analysis of this strain's phenotypic, chemotaxonomic, and genomic characteristics established the relationship of the isolate with the genus Planococcus. Strain Y74T was able to grow between 4 and 42°C (with an optimum temperature of 28°C) at pH values of 6–8.5 and in 0%–7% (w/v) NaCl. The dominant quinones were MK‐8 and MK‐7. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and an unknown phospholipid. The majority of the fatty acid content was anteiso‐C15:0 (28.8%) followed by C16:1 ω7c alcohol (20.9%) and iso‐C14:0 (13.4%). The 16S rRNA gene sequence similarity analysis demonstrated a stable branch formed by strain Y74T and Planococcus halotolerans SCU63T (99.66%). The digital DNA–DNA hybridization between these two strains was 57.2%. The G + C content in the DNA of Y74T was 44.5 mol%. In addition, the morphological, physiological, and chemotaxonomic pattern clearly differentiated the isolates from their known relatives. In conclusion, the strain Y74T (=JCM 32826T = CICC24461T) represents a novel member of the genus Planococcus, for which the name Planococcus antioxidans sp. nov. is proposed. Strain Y74T was found to have potent antioxidant activity via its hydrogen peroxide tolerance and its 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activity. The DPPH radical‐scavenging activity was determined to be 40.2 ± 0.7%. The genomic analysis indicated that six peroxidases genes, one superoxide dismutase gene, and one dprA (DNA‐protecting protein) are present in the genome of Y74T.


| INTRODUC TI ON
The accumulation of free radicals in living organisms can lead to many diseases, such as cancer and neurodegenerative diseases (Fischer & Maier, 2015;Lin & Beal, 2006). Thus, it may be possible to reduce and prevent these chronic diseases by decreasing the presence of free radicals and increasing the intake of antioxidants (Bonda et al., 2010;Fischer & Maier, 2015). Microorganisms are an abundant source of bioactive metabolites (Berdy, 2005;Velho-Pereira, Parvatkar, & Furtado, 2015). Therefore, in order to prevent the toxic effects of free radicals, potent natural antioxidants have been an important target for researchers. Recently, exploring new taxa for new antioxidants has been one of the effective strategies employed in this search.
The Qinghai-Tibet Plateau is the highest plateau in the world, where the average altitude is above 4,500 m (Zhang et al., 2019).
Because of the stressful conditions, such as low air temperatures, high UV radiation, and low atmospheric oxygen content, the organisms have had to adapt to survive on this plateau Zhang, Tang, et al., 2016). This environment is a potential source of genetic diversity and is an ideal place to search for antioxidant-producing microbes (Zhang, Wu, et al., 2016).
According to our research, a new Planococcus species strain, Y74 T , was isolated from the desert soil in the Qinghai-Tibetan Plateau, China. Strain Y74 T demonstrated a strong antioxidant activity, which has potential antioxidant applications.

| Bacteria isolation
The desert soil samples were obtained from the town of Huatugou, Qinghai province, China. Strains Y74 T was isolated with modified 216 L agar medium (per liter distilled water: 1.0 g sodium acetate, 10.0 g tryptone, 2.0 g yeast extract, 0.5 g sodium citrate, 0.2 g ammonium nitrate, 0.5 g nutrient broth medium, 20.0 g agar, pH 7.6) and incubated for 7 days at 20°C, after which it was preserved at −80°C in 20% (v/v) glycerol (Wang, Wang, & Shao, 2010).

| Genome sequencing and analysis
Genomic DNA was extracted with a bacterial genomic DNA extraction kit (Omega Bio-tek, Inc.), according to the manufacturer's instructions, and the sequence was determined by the Illumina HiSeq 2000. The reads from the sequencing were assembled de novo using the Velvet 1.2.10 program. The genomes of the type strains that were similar to Y74 T were retrieved from GenBank. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were used to assess the degree of similarity of each pair. The ANI was calculated with the JSpeciesWS (Richter, Rossello-Mora, Glockner, & Peplies, 2016). The ANI could be divided into ANIb and ANIm, depending on the BLASTN (Basic Local Alignment Search Tool) algorithm or the MUMMER ultra-rapid aligning tool. The dDDH was computed by an online tool, GGDC 2.0: the results of this computation were obtained using the recommended formula 2 (Meier-Kolthoff, Auch, Klenk, & Goker, 2013). The genome of strain Y74 T was annotated using IMG Annotation Pipeline v.5.0.3 . The G + C content of the DNA of strain Y74 T was deduced from the genomic data. Y74 T horizontal gene transfer analysis by the method of Bertelli, Laird, and Williams (2017).

| Phylogenetic analysis
The closely related type strains of Y74 T were obtained by comparing their 16S rRNA gene sequences, retrieved from the genome in the EzTaxon-e database (Kim et al., 2012). Phylogenetic trees based on the 16S rRNA gene sequences were generated utilizing neighbor joining (Saitou & Nei, 1987), maximum parsimony (Tamura et al., 2011), and maximum likelihood (Felsenstein, 1981) algorithms in MEGA X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018). The sequences were aligned with ClustalW (Larkin et al., 2007). The remaining parameters followed the model of Jukes and Cantor (Jukes & Cantor, 1969), and the bootstrap value was 1,000 re-samplings (Felsenstein, 1985). A phylogenetic tree based on 25 housekeeping genes nucleotide sequences was generated using neighbor joining algorithms. The parameters were as same as for the phylogenetic tree based on 16S rRNA gene sequences. The sequences of 25 housekeeping genes were obtained from genomes of 19 type strain of genus Planococcus, Planomicrobium, and 1 outgroup strain (Lysinibacillus sphaericus IAM 13420 T ) after annotated using Rapid Annotations using Subsystems Technology (RAST) (Brettin et al., 2015). The sequences of 25 housekeeping genes were concatenated in the following order: CTP
Gram staining was tested using the Solarbio Gram staining kit.
Oxidase activity was detected with 1% (w/v) tetramethyl-p-phenylenediamine. Starch and gelatin hydrolysis, nitrate reduction, catalase activity, methyl red, and Voges-Proskauer tests were performed according to the description of Kurup and Schmitt (1973). A carbohydrate utilization test was performed as described previously . Additional enzyme activities were detected by API ZYM systems.

| Chemotaxonomic analysis
For the chemotaxonomic analysis, cells were collected by centrifugation from strains cultured at 28°C in TSB medium (per liter distilled water: 17.0 g tryptone, 3.0 g soy peptone, 2.5 g D-glucose, 5.0 g sodium chloride, 2.5 g monopotassium phosphate, pH 7.3) for 3 days and then washed twice with distilled water. The cell-wall peptidoglycan was analyzed by the method of Schleifer and Kandler (1972). The whole-cell sugars were analyzed by the methods of Lechevalier and Lechevalier (1970). The quinones and the polar lipids were analyzed by the method of Collins et al. and HPLC (Collins, Pirouz, Goodfellow, & Minnikin, 1977;Kroppenstedt, 1982) and by the method of Minnikin et al. (1984), respectively. The methylation, extraction, and analysis of the fatty acids were based on the methods of Sasser (1990) and identified in the TSBA 6.0 database of the Sherlock Microbial identification (MIDI) system (Kämpfer & Kroppenstedt, 1996).

| Antioxidant activity analysis
The effect of hydrogen peroxide on the growth of strain Y74 T was tested as follows: an inoculum of 100 μl of strain Y74 T within the exponential growth phase (OD600 = 0.6) was mixed with 50 ml LB medium containing 0, 1, and 5 mM H 2 O 2 and then incubated at 30°C for 48 hr. The cell concentration was monitored by spectrophotometer (absorbance at 600 nm). All experiments were performed in triplicate. The growth fitting curves of strain Y74 T were drawn with Origin 2018 (logistics nonlinear fitting).
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity was tested in steps. First, an inoculum of 1 ml of strain Y74 T within the exponential growth phase (OD600 = 1.0) was centrifuged at 5,300 g for 10 min, after which the supernatant was discarded, and the precipitate was resuspended with 500 μl PBS. This process was repeated three times. The resuspended precipitate was then mixed with 500 μl 0.4 mmol/L DPPH•ethanol (the control group used an equal volume of distilled water), after which the mixture was allowed to react in at low-light area for 30 min at room temperature and subsequently centrifuged at 5,300 g for 10 min. The absorbance of the supernatant was measured with a spectrophotometer at 517 nm. The DPPH free radical-scavenging rate was calculated as follows: scavenging activity (%) = [1 − (As − Ab)/Ac] × 100%, where Ab is the absorbance of the blank group, Ac is the absorbance of the control group, and As is the absorbance of the sample set.

| Phylogenetic analysis
The entirety of the 16S rRNA gene sequences was extracted from the genome of strain Y74 T (1,512 bp, KU601236). The genome of strain Y74 T was deposited at DDBJ/EMBL/GenBank with the accession number RCWH00000000.
Compared with the EzTaxon database, type strain Planococcus Planococcus maitriensis S1 T (98.23%), and Planomicrobium mcmeekinii S23F2 T (98.16%) were found to show high degrees of similarity with strain Y74 T (Kim et al., 2012). The three 16S rRNA gene phylogenetic trees indicated that Y74 T and Planococcus halotolerans SCU63 T formed a stable clade (Figure 1). However, many adjacent clades were not stable. Therefore, a more stable phylogenetic tree was constructed, which was based on 25 concatenated housekeeping genes of strain Y74 T and their related type strains ( Figure 2). According to this phylogenetic tree, strain Y74 T should be a member of the genus Planococcus. The exact position of Planomicrobium oekanokoites IFO12536 T could not exactly be resolved within the analysis. The dDDH between strain Y74 T and Planococcus halotolerans SCU63 T , Planomicrobium okeanokoites IFO 12536 T , or Planococcus maitriensis S1 T were 57.2%, 30.5%, and 19.1%, respectively. The ANIb values between strain Y74 T and Planococcus halotolerans SCU63 T , Planomicrobium okeanokoites F I G U R E 1 Neighbor joining phylogenetic tree, based on nearly complete 16S rRNA gene sequences, showing the relationships among strain Y74 T and their related species. Numbers at nodes are bootstrap values based on 1,000 re-samplings (only values above 50% are shown). Asterisks and hash marks indicate that the clades were also recovered in the maximum parsimony and maximum likelihood trees F I G U R E 2 Neighbor joining phylogenetic tree based on 25 concatenated housekeeping genes of strain Y74 T and their similar related type strains. Numbers at nodes are bootstrap values based on 1,000 re-samplings (only values above 50% are shown) IFO 12536 T , or Planococcus maitriensis S1 T were 94.15%, 85.43%, and 72.19%, respectively, with ANIm values of 94.66%, 87.45%, and 83.50%, respectively (Table 1). These values were below the species demarcation threshold in prokaryotic species, the generally accepted species boundary for ANI and dDDH values were 95 ~ 96% and 70%, respectively (Chun et al., 2018;Kim, Oh, Park, & Chun, 2014;Meier-Kolthoff et al., 2013). For all of these reasons, Planococcus antioxidans sp. nov. Y74 T was designated as a novel species in the genus Planococcus.

| Morphological and physiological characteristics
Strain Y74 T was determined to be Gram-positive, whose cellular shape was cocci, short rods, or rods, and whose colony color was all white (Figure 3). The growth temperature range of Y74 T was 4-42°C (optimum temperature 30°C) with a pH range of 6-8.5 and a NaCl tolerance of up to 7% (w/v) ( Table 2). Strain Y74 T had a wide range of growth temperatures and a high salt tolerance, which was similar to other closely related type strains of Planococcus or Planomicrobium Jung et al., 2009).  (Table 2). However, Planococcus halotolerans SCU63 T was able to utilize sucrose but could not utilize D-sorbitol or melibiose .
There were some distinctions with other reference type strains.
Strain Y74 T showed positivity for catalase and for gelatin hydrolysis and negativity for oxidase, nitrate reduction, methyl red, and the Voges-Proskauer tests ( Table 2). The result of the API ZYM test showed that strain Y74 T was weakly positive for weak positive for α-glucosidase, cystine arylamidase, esterase lipase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, and valine arylamidase.

| Chemotaxonomic characteristics
The whole-cell hydrolysate of Y74 T contained ribose. The peptidoglycan type was L-Lys-D-Glu. The dominant quinone compounds of Y74 T were MK-8 (76%) and MK-7 (24%) ( Table 2). Table 2 shows that the predominant isoprenoid quinone compound of the type strains of Planococcus and Planomicrobium were all MK-8 and MK-7.
C, cocci; SR, short rods; R, rods; +, positive; −, negative; W, weak positive; N, not determined. a The range of the growth temperatures was not reported. b The lower limit was the minimum value that the strain could grow in, and the upper limit was the maximum value of the optimal growth range.
c The optimal growth range. some extreme environments, such as the Arctic permafrost or the Antarctic (Mykytczuk et al., 2012;Reddy et al., 2002). Therefore, these strains have strong stress-resistant abilities generally. Previous research found a new antioxidant in Planococcus (Shindo & Misawa, 2014). The species of Planococcus genus had the potential for antioxidant applications, but few studies were emphasized on this property.

| Genome properties
The draft genome of strain Y74 T was 3,672,033 bp (Table 4).
The G + C content of the DNA of Y74 T was 44.5 mol%. A total of 3,831 genes were detected in strain Y74 T , 3,668 of which were F I G U R E 5 Characteristics and position of the predicted genomic islands found in the draft genome sequence of strain Y74 T . The genomic islands show that several horizontal gene transfer events have occurred in strain Y74 T . The red in the circles represents the prediction from integrating three different methods (IslandPath-DIMOB, SIGI-HMM, and IslandPick); the orange represents the prediction result using IslandPath-DIMOB; the dark blue represents the prediction result using SIGI-HMM protein-coding genes. There were 967 protein-coding genes containing enzymes. The number of protein-coding genes with a function prediction was 2,913. The number of protein-coding genes with COGs was 3,006, which accounted for 78.47% of all genes. The genome of strain Y74 T contained 106 RNA genes, including 67 tRNA genes, 10 5S rRNA genes, 7 16S rRNA genes, and 12 23S rRNA genes ( Table 4). The copy number of the rRNA operons in prokaryotic organisms is generally thought to be related to growth rates (Klappenbach, Dunbar, & Schmidt, 2000). It could be suggested that strain Y74 T could grow rapidly at lower temperatures. Multiple copy numbers of key genes could increase the radiation resistance of the bacteria as well (Slade & Radman, 2011). There are six peroxidase genes in the genome of Y74 T (Table A1), including two glutathione peroxidases, one catalase family peroxidase, one heme-dependent peroxidase, one thioredoxin-dependent thiol peroxidase, and one thiol peroxidase. Peroxidases are enzymes that catalyze the oxidation of substrates by hydrogen peroxide as an electron acceptor (Welinder, 1992). This property may explain why the strain can grow in a medium containing hydrogen peroxide. Genes with antioxidant abilities, DNA-protecting protein (DprA), and superoxide dismutase were also found in the genome of Y74 T .
In addition to the core proteins and other orthologs presenting in the organism, some nonortholog proteins were found in the strain of Y74 T . Therefore, the gene cluster of strain Y74 T was analyzed by the method of Bertelli et al. (2017). The results showed that multiple horizontal gene transfer events were found in the Y74 T genome.
There were 28 gene islands be found in the genome, which contained 395 genes ranging from 4,000 to 700,000 bp, including 162 unclear functional genes of them were annotated as hypothetical proteins; 16 genes were predicted to be recombinase and phage-associated proteins ( Figure 5, Table A2). Based on the gene function analysis on gene island, most of the genes were involved in metabolism, signal transduction, and DNA repair.

| CON CLUS IONS
According to an analysis of phenotypic, phylogenetic, and chemotaxonomic characteristics, strain Y74 T was determined to be a new member within the genus Planococcus. Therefore, it was named as Planococcus antioxidans sp. nov. Y74 T . Strain Y74 T was found to have potent antioxidant activity via its hydrogen peroxide tolerance and its DPPH radical-scavenging activity.
The type strain, Y74 T (=JCM 32826 T = CICC24461 T ), was isolated from the sandy soil in the town of Huatugou, Qinghai province, China. The G + C content of the DNA of strain Y74 T is 44.5 mol%.

ACK N OWLED G M ENTS
The research was funded by the Bureau of International Cooperation, Chinese Academy of Sciences (131B62KYSB20160014), and the National Natural Science Foundation of China (No. 41801045, 31570498), CAS "Light of West China" Program.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are provided in full in the results section of this paper apart from the DNA sequences. The 16S rRNA gene sequence of strain