Sphingomonas Negativus sp. nov., and Sphingomonas Gyeonggiense sp. nov., Bacteria Isolated From Soil in South Korea

Two novel Gram-staining-negative bacterial strains BT553 T and BT552 T were isolated from soil collected in Gyeonggi province, Korea. Phylogenetic analysis using 16S rRNA gene sequences revealed that the strains BT553 T and BT552 T both belong to a distinct lineage in the genus Sphingomonas (family Sphingomonadaceae, order Sphingomonadales, class Alphaproteobacteria). Strain BT553 T was closely related to Sphingomonas melonis DAPP-PG 224 T (98.1 % 16S rRNA gene similarity) and Sphingomonas aquatilis JSS7 T (98.1%). Strain BT553 T was closely related to Sphingomonas melonis DAPP-PG 224 T (98.2 %) and Sphingomonas aquatilis JSS7 T (98.1%). The genome size of strain BT553 T was 3,941,714 bp. Bacterial growth was observed at 10°C–30°C (optimum 25°C), pH 5.0–9.0 (optimum pH 7.0) in R2A agar and the presence of up to 2% NaCl. The genome size of strain BT552 T was 4,035,561 bp. Bacterial growth was observed at 10°C–30°C (optimum 25°C), pH 5.0–9.0 (optimum pH 7.0) in R2A agar and in the presence up to 2% NaCl. The major cellular fatty acids of strains BT553 T and BT552 T were Summed Feature 3 and (16:1 ω 6c / 16:1 ω 7c), Summed Feature 8 (18:1 ω 7c / 18:1 ω 6c), and 14:0. In addition, their predominant respiratory quinone was Q-10. The major polar lipids of strain BT553 T was identied to be (DPG), (PE), phosphatidylglycerol (PG), (PC), and sphingoglycolipid (SL). The major polar lipids of strain BT552 T was identied to be diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), phospholipid (PL), and sphingoglycolipid (SL). Based on the biochemical, chemotaxonomic, and phylogenetic analyses, strains BT553 T and BT552 T are novel bacterial species within the genus Sphingomonas. The type strain of Sphingomonas negativus is BT553 T (= KCTC 82095 T = NBRC XXXX T ) and the type strain of Sphingomonas gyeonggiense is BT552 T (= KCTC 82094 T = NBRC XXXX T ).


Introduction
The genus Sphingomonas, a member of the family Sphingomonadaceae in the phylum Proteobacteria, was rst described by Holmes et al. (1977) and later was modi ed by Yabuuchi et al. (1990). At the time of writing (May 2021), the genus Sphingomonas comprised 161 species with validly published and correct names (https://lpsn.dsmz.de/genus/sphingomonas). The species in this genus are characterized as obligately aerobic, Gram-negative, non-spore forming, straight or slightly curved rods or ovoid cells, and motile of nonmotile. Mostly, colonies are lemon-yellow colored, and they produce catalase (Weisburg et al. 1991). They produce acids from pentoses, hexoses, and disaccharides by oxidation, but not from polyalcohols or inulin. They use ubiquinone 10 as a respiratory quinone. Cellular lipid contains two major fatty acids including cis-octadecenoic and 2-hydroxymyristic and it also contains sphingoglycolipid (SGL). The genus probably distributes widely in various environments and some species are opportunistic pathogen to humans. Usually, the nucleotide position 1290 (in Escherichia coli) is deleted in 16S rRNA and G + C contents of DNA are 64-66 mol% (Yabuuchi et al.1990).
During the soil sample screening, we found a strain designated as HKS19 that showed similar characteristics to the genus Sphingomonas. The results of our polyphasic taxonomic analysis were described in this paper.

Strain isolation
Two strains BT553 T and BT552 T were isolated from soil collected in Gyeonggi province, Korea. The soil was stored in a sterile plastic bottle at 4°C as soon as it was collected and then transported to laboratory. Soil (g) was added to 10 mL sterilized seawater, shaken at 25°C for 2 h, and then diluted by tenfold; 100 µL of the diluent was spread on R2A agar (Difco) and incubated at 25°C for 7 days. The colonies are puri ed and identi ed by 16S rRNA gene sequences using the EzBioCloud server (https://www.ezbiocloud.net/).

Morphology, physiology, and biochemical analysis
Morphological properties were observed using light microscopy (B1 series; Motic) and scanning electron microscopy (JEOL, JEM1010). The Gram-staining-reaction was performed using a commercial kit (bioMérieux, France), following the manufacturer's instructions. Oxidase and catalase activities were determined using 3% (w/v) H 2 O 2 solution and 1% (w/v) tetramethyl-p-phenylenediamine, respectively (Cappuccino and Sherman 2002). Growth of the strains were tested at 25°C under various pH conditions (4 to 10, 0.5 pH intervals) and various NaCl concentrations (0 to 5% [w/v%], 1% intervals). The growth on various media was tested on R2A agar plates in Luria-Bertani (LB) agar, nutrient agar (NA), MacConkey (MAC) agar, and trypticase soy agar (TSA) mediums (all purchased from Difco) and were observed for three days at different temperatures (4°C, 10°C, 25°C, 30°C). The biochemical and physiological tests were performed using API 20NE kits, and enzymic activities were tested using an API ZYM kit (bioMérieux) following the manufacturer's instructions.

Genome sequencing
The genomic DNA of strains BT553 T and BT552 T were extracted using a genomic DNA extraction kit (Solgent). The DNA sequencing library was prepared by the Nextera DNA Flex Library Prep Kit (Illumina).
Whole-genome sequencing was done using iSeq 100 (150 bp paired-end). The sequence was assembled using SPAdes 3.

Phylogenetic analysis
The 16S rRNA genes of strains BT553 T and BT552 T were ampli ed by 27F and 1492R universal bacterial primers and the sequencing was performed using the 337F, 518R, 785F and 926R universal primers (Macrogen).
The taxonomic positions of strains BT553 T and BT552 T were determined using 16S rRNA sequences of related taxa, obtained from EzBioCloud and compared using the EzEditor2 program (Jeon et al. 2014). The phylogenetic tree was constructed using the MEGAX program (Kumar et al. 2018) with the neighborjoining (Saitou and Nei 1987), maximum-likelihood, and maximum-parsimony algorithms (Ziheng 1995).
The evolutionary distances were calculated according to the Kimura two-parameter model (Kimura 1983). A bootstrap analysis was conducted with 1,000 replicates (Felsenstein 1985).

Chemotaxonomic characteristics
The polar lipids were extracted according to Minnikin et al. (1984) and identi ed by two-dimensional TLC.
The separated spots were detected with detection reagents sprayed onto the lipids (Komagata and Suzuki 1987). The quinones were extracted and puri ed using Sep-Pak Vac cartridges (Waters) and were analyzed by high-performance lipid chromatography (HPLC) according to the method of Hiraishi et al. (1996). For cellular fatty acid analysis, strains BT553 T and BT552 T were grown on R2A agar plates for 3 days at 25°C, and 300 mg of cells were harvested and freeze-dried. Cellular fatty acids were extracted, methylated and analyzed according to the standard protocol of the Sherlock Microbial Identi cation System (version 6.0; MIDIdatabase TSBA6) (Sasser 1990).