Jeotgalibacillus Auranticolor sp. nov., Isolated from Freshwater Collected from Baiyangdian Lake Lake, China

A novel Gram-positive, strictly aerobic, rod-shaped, orange-pigmented bacterial strain, designated R-1-5s-1 T , was isolated from Baiyangdian Lake, China. Strain R-1-5s-1 T grew at 15-37 ℃ (optimum 37 ℃ ) and pH 7-11 (optimum pH 8) in Luria-Bertani medium. Based on 16S rRNA gene sequence analysis, strain R-1-5s-1 T was assigned to the genus Jeotgalibacillus and showed the closest relationships with Jeotgalibacillus salarius ASL-1 T (97.69%), Jeotgalibacillus alkaliphilus JC303 T (97.29%), Jeotgalibacillus marinus DSM 1297 T (97.15%), Jeotgalibacillus campisalis SF-57 T (97.01%), and Jeotgalibacillus spp. ( ≤ 97%). The predominant polar lipids were phosphatidylglycerol and diphosphatidylglycerol; the major cellular fatty acids were iso-C 15:0 , anteiso-C 15:0 , iso-C 17:0 , and anteiso-C 17:0 ; and the major respiratory quinones were MK-7 and MK-8. The peptidoglycan type of the cell wall was A1a linked via L-lysine as the diamino acid. The G+C content was 43.6%, and the draft genome size of strain R-1-5s-1 T was 3.4 Mbp. Between strain R-1-5s-1 T and the related strain J. salarius ASL-1 T , the ANI and dDDH relatedness values were 78.9% and 20.8%, respectively. Phylogenetic, chemotaxonomic, and genotypic analyses revealed that strain R-1-5s-1 T is a novel species in the genus Jeotgalibacillus, for which the name Jeotgalibacillus auranticolor nov. is proposed. The type strain is R-1-5s-1 T


Introduction
The genus Jeotgalibacillus belongs to the family Planococcaceae of the class Bacilli, the original species of which was Jeotgalibacillus alimentarius, isolated from jeotgal in Korea by Yoon et al. (2001). The J. alimentarius strain possesses MK-7 and MK-8 as major respiratory quinones (Yoon et al., 2001). Currently, the genus Jeotgalibacillus comprises eight species with validly published names according to the List of Prokaryotic Names with Standing in Nomenclature website (http://www.bacterio.net/). These members of the genus Jeotgalibacillus have been isolated from diverse environments, including salt pan, sandy beach, seawater, and soil (Srinivas et al., 2016). We focused on a novel species, the type strain of which, R-1-5s-1 T , was isolated from water collected from Baiyangdian Lake.
Baiyangdian Lake is the largest freshwater lake and most important ecological resource bank in northern China. As an important body of freshwater, Baiyangdian Lake is important for economic development in the surrounding area (Cui et al., 2018). The composition, activity, and physiology of the microbial community in water, sediment and aquatic plant of Baiyangdian Lake has been investigated extensively, particularly of functional microorganisms (Du et al., 2018;Sun et al., 2021;Zhou et al., 2020a;Zhou et al., 2020b). To assess the community structure and species diversity of culturable bacteria in lakes, we analyzed the microbial populations in Baiyangdian Lake and identi ed a new species.

Samples and isolation
A colony of strain R-1-5s-1 T was isolated from water of Baiyangdian Lake (38.850°N 116.000°E; salinity 0.58 g/L, pH 8.48, temperature 28.08℃, oxygen content 2.28 mg/L), the largest freshwater lake in northern China. The water samples were diluted (10 -3 -10 -5 ), spread on solid Luria-Bertani (LB) medium (1 L of deionized water containing 10 g of tryptone, 10 g of yeast extract, and 15 g of agar) and incubated for 3 days on LB agar at 28℃. The strain R-1-5s-1 T was isolated from the medium and stored in 50% (v/v) glycerol at -80℃. Two related type strains, Jeotgalibacillus salarius ASL-1 T and Jeotgalibacillus alimentarius YKJ-13 T , were obtained from the Korean Collection for Type Cultures.

Phenotypic characteristics
Cell morphology and size were observed using a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) and SU1080 scanning electron microscope (Hitachi, Tokyo, Japan) after 24 h of incubation at 37°C on LB agar. The temperature range for growth was examined in modi ed LB broth at 4,15,20,25,30,37, and 45°C (Gordon RE et al., 1974). The pH range for growth was evaluated in modi ed LB broth at pH 4-13 (at intervals of 1.0 pH unit) The range of NaCl concentrations for growth was examined in modi ed LB medium containing 0-15 % (w/v) NaCl (at intervals of 1 %). Gram staining was carried out using a Gram stain kit (Solarbio, Beijing, China). Cell motility was tested on 0.5% agar plates, as described by Xu et al. (2013). Oxidase activity was evaluated using an oxidase reagent (bioMérieux, Marcy-l'Étoile, France), and catalase activity was examined by adding 5% (v/v) H 2 O 2 to colonies. Growth under anoxic conditions was evaluated in LB broth in an anaerobic tube after incubation for 1 week at 37°C. Starch hydrolysis was tested in LB agar as a modi ed basal medium (Gordon et al., 1974). A physiological analysis of strain R-1-5s-1 T was performed using the API 20NE (test assimilation), API 50CH (test enzyme activities), and API ZYM (test acid production) systems following the manufacturer's instructions (bioMérieux) with incubation at 37°C. The Biolog-Gen -Microplate system was used to evaluate the strain's carbon metabolic capability at 37°C.

Chemotaxonomic characterization
The cell wall was prepared, and the peptidoglycan structure was determined as described previously (Schleifer and Kandler, 1972). The cellular fatty acid properties of strain R-1-5s-1 T , Azospirillum doebereinerae GSF71 T , and Azospirillum lipoferum59b T were evaluated in parallel using a 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA), and analyzed against the TSBA v. 6.0 database (Liu et al., 2016). The polar lipid and respiratory quinone properties of strain R-1-5s-1 T , J. salarius R-1-5s-1 T , and ASL-1 T were examined in parallel. Polar lipids were extracted and separated as described previously with modi cations and two-dimensional thin-layer chromatography (Kamekura 2020; Tindall et al., 2007). Respiratory quinones were extracted and puri ed as described previously (Collins 1985) and analyzed by high-performance liquid chromatography (Kroppenstedt 1986;Hu et al., 2004).
The strain was orange and rod-shaped. Strain R-1-5s-1 T had peritrichous agella under transmission electron microscopy ( Fig. S1a-b). Strain R-1-5s-1 T formed slightly raised colonies on LB agar and produced pigment. The cells were Gram-positive and were motile peritrichous agella (Fig. S1b). The API 20NE, API ZYM, and API 50CH test results are shown in Table 1. Strain R-1-5s-1 T differed from J. salarius ASL-1 T based on its ability to assimilate D-glucose, D-mannitol, and D-maltose; inability to hydrolyze gelatin; presence of α-galactosidase and naphthol-AS-BI-phosphohydrolase activity; lack of cystine arylamidase activity; and ability to use D-galactose, glycogen, D-mannitol, and D-melibiose to produce acid (Table 1).

Conclusion
The phylogenetic, morphologic, and chemotaxonomic results show that strain R-1-5s-1 T is a liated with genus Jeotgalibacillus. However, the strain can be distinguished from other Jeotgalibacillus species by several phenotypic differences, such as the temperature and pH range; gelatin; use of D-glucose, Dmannitol, D-maltose, D-galactose, glycogen, and gelatin; and fatty acid composition (Table 2). Based on these ndings, strain R-1-5s-1 T represents a novel species of the genus Jeotgalibacillus, for which the name Jeotgalibacillus auranticolor sp. nov. is proposed.

Abbreviations
showing the strain R-1-5s-1 T belong to genus Jeotgalibacillus and the reference strain was J. salarius ASL-1 T . GenBank accession numbers are given behind strains. Bootstrap values above 50% based on 1000 replicates are shown at branch nodes. Bar, 0.01 substitutions per nucleotide.

Figure 3
Phylogenetic tree using the maximum-parsimony method based on the 16S rRNA genes sequence, showing the strain R-1-5s-1 T belong to genus Jeotgalibacillus and the reference strain was J. salarius ASL-1 T . GenBank accession numbers are given behind strains. Bootstrap values above 50% based on 1000 replicates are shown at branch nodes. Bar, 10 substitutions per nucleotide.

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