Characterization of Gymnodinialimonas Ceratoperidinii gen. nov.,sp. nov., a New Bacterium Isolated From Rare Marine Dino agellate Ceratoperidinium Margale i

Yue Jiang KRIBB: Korea Research Institute of Bioscience and Biotechnology Yuxin Peng KRIBB: Korea Research Institute of Bioscience and Biotechnology Hyeon Ho Shin KIOST: Korea Institute of Ocean Science and Technology Hyun Jung Kim KRIBB: Korea Research Institute of Bioscience and Biotechnology Ki-Hyun Kim KRIBB: Korea Research Institute of Bioscience and Biotechnology Lingmin Jiang KRIBB: Korea Research Institute of Bioscience and Biotechnology Jiyoung Lee KRIBB: Korea Research Institute of Bioscience and Biotechnology Zhun Li (  lizhun@kribb.re.kr ) Korea Research Institute of Bioscience and Biotechnology https://orcid.org/0000-0001-8961-9966


Introduction
Dino agellates play a major role in the dynamics of the Earth's oceans and climate (Moustafa et al. 2010). Vitamins are an important growth factor for the majority of dino agellates (Tang et al. 2010). Marine Rhodobacteraceae are major vitamin suppliers for dino agellates (Sanudo-Wilhelmy et al. 2014). The family Rhodobacteraceae (Liang et al. 2021) belongs to the order Rhodobacterales within the class Alphaproteobacteria. Members are Gram-stain-negative, callus or rod-shaped and non-spore-forming bacteria. The major respiratory quinone is ubiquinone-10 (Q-10) and the major cellular fatty acid is unsaturated fatty acid C 18 : 1 ω7c. And Rhodobacteraceae strains usually contain phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) as the main polar lipids. Members of the family Rhodobacteraceae are found in various saline environmental habitats, such as sea water, sea grass, marine sediment and hypersaline lake.
In the present study, we describe strain J12C1-MA-4 T , which was isolated from rare dino agellate C. margale i during a study on the relationship between bacteria and marine dino agellate. Polyphasic taxonomic analyses revealed that the strain J12C1-MA-4 T should be proposed as a novel species of a new genus in the family Rhodobacteraceae, named as Gymnodinialimonas ceratoperidinii gen. nov.,sp. nov.

Materials And Methods
Isolation and culture conditions The bacterial strain was isolated from a liquid culture of the dino agellate Ceratoperidinium margale i (J12C1), which was collected from the East China Sea (31• 59′ 53′′ N, 127• 41′ 59′′ E) ( Figure 1). The dino agellate was isolated from seawater and the pure dino agellate was cultured in F/2 medium for 30 days. Collect 100 microliters from the seed culture sample and spread on MA agar (MA, Marine agar 2216) and then cultured at 25°C. After 3 days, single colonies were transferred onto a new MA agar plate under the same conditions. A round, yellow, smooth, strain named J12C1-MA-4 T was stored at -80°C in a suspension containing 10% (w/v) skimmed milk, and was selected for polyphasic sorting, and incubated as usual with MA at 25°C. Jannaschia helgolandensis KCTC 12191 T ; Pseudooctadecabacter jejudonensis KCTC 32525 T ; Loktanella acticola KCTC 52837 T ; Loktanella ponticola KCTC 42133 T ; Alterinioella_nitratireducens KCTC 72738 T were obtained from KCTC as closely related strains in order to further investigate the characteristics of strain J12C1-MA-4 T . DNA extraction, PCR and 16S rRNA-based phylogenetic analysis Bacteria grown was used as a template on MA agar plates for 3 days. The DNA of strain F J12C1-MA-4 T was extracted with a spin columns ltration method (QIAamp DNA mini kit). The 16S rRNA gene of strain J12C1-MA-4 T was ampli ed by polymerase chain reaction (PCR) using two bacterial speci c universal primers 27F and 1492R and then the puri ed PCR product was sequenced by Biofact using primers 518F and 805R. Sequences were viewed and assembled using Geneious Prime (https://www.geneious.com). The almost complete 16S rRNA gene sequence (1402 bp) was submitted to the GeneBank (https://www.ncbi.nlm.nih.gov/nuccore/MZ457320). Subsequently, the obtained 16S rRNA gene sequence was compared to the type strain sequences available on EzBioCloud server (http://www.ezbiocloud.net) (Yoon et al. 2017a). Phylogenetic analyses using the neighbor-joining, maximum-likelihood and maximumparsimony algorithms were performed as described by Jiang et al. (Jiang et al. 2021 . AAI values between of complete genome of J12C1-MA-4 T and each reference genome were calculated using an online AAI calculation tool available at the webserver (http://enveomics.ce.gatech.edu/) (Luo et al. 2014). The up-to-date bacterial core genome (UBCG), consisting of 92 core genes, was used to construct a genome-wide phylogenetic tree after collecting the whole genome sequences of all closely related strains in NCBI GenBank as described by Na et al. (Na et al. 2018).

Morphological, physiological and chemotaxonomic characterization
Its morphological features were examined by using a scanning electron microscope (JEOL Ltd, Tokyo, Japan), samples were prepared as described by Li et al. (Li et al. 2019). To explore the optimal medium for J12C1-MA-4 T , a variety of media were selected here for testing, such as Marine agar 2216 (MA, Difco), Tryptic Soy agar (TSA, Difco), Luria-Bertani agar (LB, Difco), Potato Dextrose agar (PD, Difco), Nutrient agar (NA, beef extract 3.0 g/L, peptone 5.0 g/L, and agar 15.0 g/L). The temperature range for growth was determined in MA medium at 25°C for 3 days at different temperatures of 4, 10, 15, 20, 25, 30, 35, 37, 40, 45, 50 and 60°C. Growth at different pH values (pH 3.0-12.0 in 1.0 unit intervals) (adjusted with 1N HCl and 1N NaOH, 1.0 pH unit interval) was measured in modi ed MA broth with 10 mM acetate or 10 mM Tris-HCl buffer described by Jiang et al. (Jiang et al. 2020). NaCl tolerance was tested in DW containing MA broth (NaCl concentrations of 0-12%). The pH and NaCl tolerance were monitored by measuring the absorbance of OD600 using a DU 700 UV-Vis spectrophotometer (Beckman Coulter). Motility was assessed on soft agar (0.4%; w/v) test tubes. Gram stain test using Gram stain kit (Difco). The bubbles produced after the addition of 3% (v/v) hydrogen peroxide solution determined the activity of catalase, which was determined by the appearance of a purple color change in the colonies after the addition of an oxidase reagent (bioMérieux). Other biochemical characteristics were tested using API 20NE, API 50CH and API ZYM (bioMérieux) according to the manufacturer's instructions.
The appropriate liquid medium was prepared, and strain J12C1-MA-4 T and closely related strains were cultured in a shaker incubator, shaken for 3 days, and then the bacteria were harvested by centrifugation at 6000 rpm, lyophilized, and polar lipids and quinones were extracted with 100 mg of lyophilized cells. For the analysis of chemosynthetic properties, methods including polar lipids (Minnikin et al. 1984) and quinones (Collins et al. 1980) were used. Polar lipid spots were separated by two-dimensional thin-layer chromatography and spray dyes including 0.2% ninhydrin (Sigma-Aldrich), γ-naphthol, molybdenum blue (Sigma-Aldrich), 4% phosphomolybdic acid and Dragendorff solution for the identi cation of aminocontaining lipids, glycolipids, phospholipids, total lipids and quaternary nitrogen-containing lipids, respectively. Cellular fatty acid methyl esters (FAMEs) were obtained from cells by saponi cation, methylation and extraction according to the protocol of Sherlock Microbial Identi cation System (MIDI, USA). Cellular FAMEs were separated by gas chromatography (6890) and identi ed and quanti ed using Sherlock Microbial Identi cation System software (Sasser 2006 Phylogenetic analyses using the neighbor-joining, maximum-likelihood and maximum-parsimony algorithms were performed as described by Jiang et al. (Jiang et al. 2021). NJ, ML, and ME algorithms obtained similar results ( Figure 2). Based on 16S rRNA phylogenetic analysis, strain J12C1-MA-4 T is located on a separate branch in the family Rhodobacteraceae, next to the genus Loktanella, Planktomarina, Thalassobius, Pseudooctadecabacter and Octadecabacter.

Taxonomic conclusion
In summary, the phylogenetic and chemotaxonomic analysis suggest that strain J12C1-MA-4 T belongs to the family Rhodobacteraceae. However, 16S rRNA similarity (below 96.9%), ANI, AAI, fatty acid composition, dDDH values as well as some physiological characteristics (such as API 20NE, API 50CH, and API ZYM test), show that strain J12C1-MA-4 T was different with the closely related strains. Thus, strain J12C1-MA-4 T represents a new species in the new genus of the family Rhodobacteraceae, and the name of Gymnodinialimonas ceratoperidinii gen. nov., sp. nov. is proposed.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. J12C1MA4supplementarymaterial.docx