Laetantesicola Maris Gen. Nov., Sp. Nov. Isolated From the Sea of Japan Shallow Sediments.


 A Gram-negative, non-motile bacterium КММ 3653T was isolated from a sediment sample from the Sea of Japan seashore, Russia. On the basis of the 16S rRNA gene sequence analysis the strain КММ 3653T was positioned within the family Rhodobacteraceae (class Alphaproteobacteria) forming a distinct lineage with the highest gene sequence similarities to the members of the genera Pacificibacter (95.2-94.7%) and Nioella (95.1-94.5%), respectively. According to the phylogenomic tree based on 400 conserved protein sequences, strain КММ 3653T was placed in the cluster comprising Vannielia litorea, Nioella nitratireducens, Litoreibacter albidus and Pseudoruegeria aquimaris as a separate lineage adjacent to Vannielia litorea KCTC 32083T. The average nucleotide identity values between strain КММ 3653T and Vannielia litorea KCTC 32083T, Nioella nitratireducens KCTC 32417T, Litoreibacter albidus KMM 3851T, and Pseudoruegeria aquimaris CECT 7680T were 71.1, 70.3, 69.6, and 71.0%, respectively. Strain КММ 3653T contained Q-10 as the predominant ubiquinone and C18:1ω7c as the major fatty acid followed by C16:0. The polar lipids were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid, two unidentified aminolipids, and five unidentified lipids. The DNA G+C content of 61.8 % was calculated from the genome sequence. Based on the phylogenetic evidence and distinctive phenotypic characteristics, strain КММ 3653T is proposed to be classified as a novel genus and species Laetantesicola maris gen. nov., sp. nov. The type strain of Laetantesicola maris gen. nov., sp. nov. is КММ 3653T (=KCTC 82575T).


Introduction
The Roseobacter clade (family Rhodobacteraceae, class Alphaproteobacteria) comprises a large group of bacterial genera (Garrity et al. 2005;Brinkhoff et al. 2008) which have been reported to be wide-spread microorganisms in marine environments, being isolated from seawater, sediments, polar sea ice, microbial mats, seaweeds and animals (Buchan et al. 2005). A recent phylogenomic study of Wirth and Whitman (2018) demonstrated the insu ciency of the 16S rRNA gene sequence analysis for phylogeny of the Roseobacter group in the family Rhodobacteraceae, whereas phylogeny based on a set of housekeeping core gene sequences is more reliable. As a result, applying this approach a number of genera and species of Alphaproteobacteria were reclassi ed and proposed as novel or recognized taxa (Wirth and Whitman 2018; Hördt et al. 2020). During a survey of microorganisms capable of dwelling shallow sediments of the Sea of Japan the Gram-negative, non-motile bacterium designated КММ 3653 T was found and investigated by using phenotypic and molecular methods and results obtained are reported in the present study. Based on the phylogenetic analyses data and distinctive phenotypic characteristics, a novel genus and species Laetantesicola maris gen. nov., sp. nov. is described to accommodate the strain КММ 3653 T (=KCTC 82575 T ).

Materials And Methods
Bacterial strains Strain KMM 3653 T was isolated from a sandy sediment sample collected from the Sea of Japan seashore, Russia, in July 2002 as described in a previous paper (Romanenko et al. 2004 and growth at different salinities (0-12% NaCl), temperatures (4-45 °C) and pH values (4.5-10.5) were carried out using arti cial sea water (ASW) as described in previous papers (Romanenko et al. 2011b(Romanenko et al. , 2013. The arti cial sea water (ASW) contained (per liter of distilled water): 24 g NaCl, 4.9 g MgCl 2 , 2.0 g MgSO 4 , 0.5 g CaCl 2 , 1.0 g KCl, 0.01 g FeSO 4 . Acid production from carbohydrates was examined using the oxidation/fermentation medium of Leifson (1963). Biochemical tests were performed using API 20E, API 20NE, API ID32 GN, and API ZYM test kits (bioMérieux, France) as described by the manufacturer except the cultures were suspended in ASW. Antibiotic susceptibility was examined using commercial . Two-dimensional thin layer chromatography of polar lipids was carried out on Silica gel 60 F 254 (10 x 10 cm, Merck, Germany) using chloroform-methanol-water (65:25:4, v/v) for the rst direction, and chloroform-methanol-acetic acid-water (80:12:15:4, v/v) for the second one (Collins and Shah 1984) and spraying with speci c reagents (Collins et al. 1980). Respiratory lipoquinones were analyzed by reversed-phase high performance thin-layer chromatography as described by Mitchell and Fallon (1990). Fatty acid methyl esters (FAMEs) were prepared according to the procedure of the Microbial Identi cation System (MIDI) (Sasser 1990). The analysis of FAMEs was performed using the GC-2010 chromatograph (Shimadzu, Kyoto, Japan) equipped with capillary columns (30 m x 0.25 mm I.D.), one coated with Supecowax-10 and the other with SPB-5. Identi cation of FAMEs was accomplished by equivalent chain length values and comparing the retention times of the samples to those of standards. In addition, FAMEs were analyzed using a GLC-MS Shimadzu GC-MS model QP2020 (column Shimadzu SH-Rtx-5MS, the temperature program from 160 о С to 250 о С, at a rate of 2 о С/min). Production of bacteriochlorophyll a (Bchl a) was spectrophotometrically tested in methanolic extracts of cells grown on MA and MB in the dark as described by Lafay et al. (1995).

16S rRNA gene sequence and phylogenetic analysis
Genomic DNA of the strain КММ 3653 T was extracted using a commercial genomic DNA extraction kit (Fermentas, EU) following the manufacturer's instruction. The universal bacterial primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1522R (5'-AAGGAGGTGATCCAGCCGCA-3') (Edwards et al. 1989) were used for ampli cation of the 16S rRNA gene. The 16S rRNA gene was PCR-ampli ed and sequenced as described in a previous paper (Romanenko et al. 2019). The 16S rRNA gene sequence obtained for strain КММ 3653 T was compared with those of the closest relatives using the BLAST (http://www.ncbi.nlm.nih.gov/blast/) and EzBioCloud service (Yoon et al. 2017). Phylogenetic analysis was conducted using Molecular Evolutionary Genetics Analysis (MEGA X) (Kumar et al. 2018). Phylogenetic trees were constructed by the neighbor-joining and the maximum-likelihood and the distances were calculated according to the Kimura two-parameter model (Kimura 1980). The robustness of phylogenetic trees was estimated by the bootstrap analysis of 1000 replicates.

Whole-genome sequencing and genome-based phylogenetic analysis
The genomic DNA was obtained from the strain КММ 3653 T using the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland). The quantity and quality of the genomic DNA was measured using DNA gel electrophoresis and the Qubit 3.0 Fluorometer (Thermo Fisher Scienti c, USA). Preparation of the DNA sequencing library was carried out using Nextera DNA Flex kits (Illumina, San Diego, CA, USA) and whole-genome sequencing was performed subsequently using paired-end runs on an Illumina MiSeq platform with a 150-bp read length. The reads were trimmed using Trimmomatic (Bolger et al. 2014) and their quality assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

Results And Discussion
16S rRNA genesequencing and genome-based phylogenetic analysis Comparative 16S rRNA gene sequence analysis showed that the novel strain КММ 3653 T belongs to the family Rhodobacteraceae (class Alphaproteobacteria) and its closest phylogenetic neighbours were found to be members of the genera Paci cibacter (95.2-94.7% gene sequence similarity) and Nioella (95.1-94.5%), respectively. Different treeing algorithms (neighbor-joining, Fig. 1, and maximum- Table S1. The phylogenomic analysis data evidence that the strain КММ 3653 T does not belong to any of recognized genera and could be classi ed as an individual genus and species of the family Rhodobacteraceae.

Morphological, physiological and chemotaxonomic characteristics
Morphological, physiological, biochemical and chemotaxonomic characteristics of strain КММ 3653 T are given in Table 1, Table 2 Figure S2). The novel bacterium КММ 3653 T was not able to hydrolyse a number of polymeric substrates (Table 1) and assimilate most carbon sources in API 32GN, API 20E and API 20NE tests (Table S2). Strain КММ 3653 T contained ubiquinone Q-10 as the major respiratory quinone and C 18:1 ω7c as the major fatty acid followed by C 16:0 (Table 2). Fatty acid pro les found in all related type strains were similar in a large proportion of C 18:1 ω7c that is characteristic for the members of the family Rhodobacteraceae although strain КММ 3653 T differed in content of some fatty acids, such as C 14:1 3-ОН, C 12:0 3-ОН, C 16:0 2-ОН or C 19:0 cyclo ( Table 2).
The polar lipids of the strain КММ 3653 T were found to be phosphatidylcholine (PC), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), an unidenti ed phospholipid (PL), two unidenti ed aminolipids (AL) and ve unidenti ed lipids (L) (Fig. S3). The polar lipids pro le of the strain КММ 3653 T was similar to those of Paci cibacter species (Romanenko 2020 (Table 1). In the present study PE was present in V.litorea KCTC 32083 T , but was not found in N. nitratireducens KCTC 32417 T and N. aestuarii JCM 30752 T (Fig. S3). The DNA G+C content of 61.8 % was calculated from the genome sequence. The value obtained for the strain КММ 3653 T is close to some extent to those of Nioella and Litoreibacter members but signi cantly differed from those of Paci cibacter and Vannielia ( Table 1). The phylogenetic relationships observed on the basis of 16S rRNA gene and whole genome sequences, and genetic distinctness as revealed by ANI and dDDH analyses were supported by phenotypic differences of the novel isolate КММ 3653 T in its growth temperature and salinity ranges, and substrate hydrolysis.
Differential phenotypic characteristics are indicated in Table 1. Based on the combined phylogenetic evidence, phenotypic and biochemical characteristics, it is proposed to classify strain КММ 3653 T as a novel genus and species, Laetantesicola maris gen. nov., sp. nov., with the type strain of the type species КММ 3653 T (=KCTC 82575 T ).
Gram-negative, aerobic, oxidase-and catalase positive, ovoid or rod-shaped bacteria enlarged at one pole due to the cell division by budding. Chemoorganoheterotrophic. Sodium ions are essential for growth. The predominant isoprenoid quinone is Q-10. Polar lipids include phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, two unidenti ed aminolipids, an unidenti ed phospholipid, and ve unidenti ed lipids. The major fatty acid is C 18:1 ω7c followed by C 16:0 . Isolated from the marine environments. On the basis of the 16S rRNA gene sequence analysis the genus represents a separate branch within the family Rhodobacteraceae of the class Alphaproteobacteria. The type species of the genus is Laetantesicola maris.
Description of Laetantesicola maris sp. nov.
In addition to properties given in the genus description the species is characterized as follows: cells are ovoid or rod-shaped 0.9-1.4 μm in diameter and 1.0-2.7 μm in length. Cells are capable producing of capsular material. Non-motile. Whitish-pigmented, smooth and shiny colonies with the regular edges of 2-3 mm in diameter are produced on MA. Growth occurs at 7-36 °C (optimum, 28-30 °C) and in the presence of 1-5% (w/v) NaCl (optimum, 2-3% NaCl). The pH range for growth is 6.0-10.0 with an optimum of 7.0-8.0. Negative for hydrolysis of gelatin, casein, tyrosine, starch, Tweens 20, 40, 80, chitin, xanthine and hypoxanthine, and H 2 S production. Positive for DNA and urea hydrolysis. Negative for acid production from D-glucose, D-fructose, maltose, lactose, D-galactose, cellobiose, sucrose, D-xylose, Dra nose, and L-arabinose. According to the API 20NE, positive for aesculin hydrolysis and PNPG test. In the API 20E tests positive for urease production and oxidation of L-rhamnose, D-sucrose (weakly), and amygdalin. In the API ZYM tests positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, and naphthol-AS-BI-phosphohydrolase. Susceptible to (content per disc): ampicillin The type strain of the species is strain KMM 3653 T (=KCTC 82575 T ) isolated from a shallow sediment sample collected from the Sea of Japan, Russia.

Declarations
Con ict of interest The authors declare that they have no con ict of interest.
Ethical statement This article does not contain any studies with human participants or animals. Tween