Microbulbifer okhotskensis sp. nov., isolated from a deep bottom sediment of the Okhotsk Sea

A Gram-negative, aerobic, non-motile bacterium КMM 9862T was isolated from a deep bottom sediment sample obtained from the Okhotsk Sea, Russia. Based on the 16S rRNA gene and whole genome sequences analyses the novel strain КMM 9862T fell into the genus Microbulbifer (class Gammaproteobacteria) sharing the highest 16S rRNA gene sequence similarities of 97.4% to Microbulbifer echini AM134T and Microbulbifer epialgicus F-104T, 97.3% to Microbulbifer pacificus SPO729T, 97.1% to Microbulbifer variabilis ATCC 700307T, and similarity values of < 97.1% to other recognized Microbulbifer species. The average nucleotide identity and digital DNA–DNA hybridization values between strain КMM 9862T and M. variabilis ATCC 700307T and M. thermotolerans DSM 19189T were 80.34 and 77.72%, and 20.2 and 19.0%, respectively. Strain КMM 9862T contained Q-8 as the predominant ubiquinone and C16:0, C16:1ω7c, C12:0, and C10:0 3-OH as the major fatty acids. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified aminophospholipid, an unidentified aminolipid, two unidentified phospholipids, phosphatidic acid, and an unidentified lipid. The DNA G+C content of 49.8% was calculated from the genome sequence. On the basis of the phylogenetic evidence and distinctive phenotypic characteristics, the marine bacterium KMM 9862T is proposed to be classified as a novel species Microbulbifer okhotskensis sp. nov. The type strain of the species is strain KMM 9862T (= KACC 22804T).


Introduction
The genus Microbulbifer was created by Gonzalez et al. (1997) with the description of the type species Microbulbifer hydrolyticus and emended by Tang et al. (2008). The genus Microbulbifer belongs to the family Microbulbiferaceae (Spring et al. 2015) and contains currently 25 species with validly published names as listed at https:// lpsn. dsmz. de/ genus/ micro bulbi fer. Bacteria of the genus Microbulbifer are widely distributed in marine or saline environments being recovered from diverse sources, including solar saltern (Yoon et al. 2007), deep-sea sediment (Miyazaki et al. 2008), saline soil (Tang et al. 2008), Pacific marine algae (Nishijima et al. 2009), mangroves (Baba et al. 2011;Vashist et al. 2013), coastal soil (Kämpfer et al. 2012), marine sediment (Zhang et al. 2012;Xiong et al. 2019), intertidal sediment and marine sponge specimen (Jeong et al. 2013), rhizosphere of a halophytic plant (Camacho et al. 2016), a purple sea urchin (Lee et al. 2017), coastal sand (Huang et al. 2020). Several Microbulbifer members have been reported to be polysaccharide-degrading bacteria (Miyazaki et al. 2008;Baba et al. 2011;Vashist et al. 2013;Huang et al. 2020). In the present study the taxonomic position of a Gram-negative, aerobic, non-motile bacterium KMM 9862 T , isolated from a deep bottom sediment sampled from the Okhotsk Sea, Russia, was characterized. On the basis of combined phylogenetic analyses and phenotypic properties, a novel species, Microbulbifer okhotskensis sp. nov., is described.

Bacterial strains
Strain KMM 9862 T was isolated from a deep bottom sediment sample obtained at a depth of 46.2 m from the Okhotsk Sea, Russia, during the expedition of R/V Academician Oparin, in September 2020. The novel bacterium was cultivated aerobically on marine agar 2216 (MA) or in marine broth (MB) 2216 (BD Difco) at 28 °C and stored at − 70 °C in MB 2216 supplemented with 30% (v/v) glycerol. The type strains of Microbulbifer echini KACC 18258 T was kindly provided by the Korean Agricultural Culture Collection (KACC), Agricultural Microbiology Division, National Academy of Agricultural Science, Korea, and Microbulbifer thermotolerans DSM 19189 T was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, Braunschweig, Germany, to be used in the phenotypic and lipids analyses.

Phenotypic characterization
Gram-staining, oxidase and catalase reactions, and motility (the hanging drop method) were determined as described by Gerhardt et al. (1994). The morphology of cells grown in MB and negatively stained with a 1% phosphotungstic acid on carbon-coated 200-mesh copper grids was examined by electronic transmission microscopy [Libra 120 FE (Carl Zeiss), provided by the Far Eastern Centre of electronic microscopy, Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences]. Hydrolysis of starch, casein, gelatin, Tween 80, DNA, L-tyrosine, chitin, nitrate reduction (sulfanilic acid/α-naphthylamine test), and growth at different salinities (0-12% NaCl), temperatures (5-45 °C), and pH values (4.0-10.5) were carried out using artificial sea water (ASW) as described in a previous paper (Romanenko et al. 2013). The artificial sea water (ASW) contained (per liter of distilled water): 30 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 . 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.

Chemotaxonomic characterization
Strain KMM 9862 T and related type strains, M. thermotolerans DSM 19189 T and M.echini KACC 18258 T were grown on MA 2216 at 28 °C. Lipids were extracted using the method of Folch et al. (1957). Two-dimensional thin layer chromatography of polar lipids was carried out on Silica gel 60 F 254 (10 × 10 cm, Merck, Germany) using chloroform-methanol-water (65:25:4, v/v) for the first direction, and chloroform-methanol-acetic acid-water (80:12:15:4, v/v) for the second one (Collins and Shah 1984) and spraying with specific 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 Identification System (MIDI) (Sasser 1990). The analysis of FAMEs was performed using the GC-2010 chromatograph (Shimadzu, Kyoto, Japan) equipped with capillary columns (30 m × 0.25 mm I.D.), one coated with Supecowax-10 and the other with SPB-5. Identification 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 °C to 250 °C, at a rate of 2 °C/min).

16S rRNA gene sequence and phylogenetic analysis
Genomic DNA of the strain КMM 9862 T was extracted using a commercial genomic DNA extraction kit (Fermentas, EU) following the manufacturer's instruction. The universal bacterial primers 8F (5ʹ-AGA GTT TGA TCC TGG CTC AG-3ʹ) and 1522R (5ʹ-AAG GAG GTG ATC CAG CCG CA-3ʹ) (Edwards et al. 1989) were used for amplification of the 16S rRNA gene. The 16S rRNA gene was PCR-amplified and sequenced as described in a previous paper (Romanenko et al. 2019). The 16S rRNA gene sequence of the strain КMM 9862 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 methods 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 КMM 9862 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 Scientific, USA). Preparation of the DNA sequencing library was carried out using Nextera DNA Flex kits (Illumina, San Diego, Page 3 of 7 548 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 version 0.39 (Bolger et al. 2014) and their quality assessed using FastQC version 0.11.8 (https:// www. bioin forma tics. babra ham. ac. uk/ proje cts/ fastqc/). Filtered reads were assembled into contigs with SPAdes version 3.15.3 (Bankevich et al. 2012) and genome metrics were calculated with the help of QUAST version 5.0.2 (Gurevich et al. 2013). The draft genome assembly was annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016). Comparisons of the Average Nucleotide Identity (ANI) and in silico DNA-DNA hybridization (dDDH) values of the strain КMM 9862 T and its closest neighbors were performed with the online server ANI/AAI-Matrix (Rodriguez-R and Konstantinidis 2016), and TYGS platform (Meier-Kolthoff and Göker 2019), respectively. The phylogenomic analysis was performed using PhyloPhlAn 3.0 software based on a set of 400 conserved bacterial protein sequences (Asnicar et al. 2020).  (Fig. 1). The 16S rRNA gene sequence similarities were not exceeding the threshold value of 98.7%-98.6% recommended by Stackebrandt and Ebers (2006) and Kim et al. (2014) for the species discrimination  (Fig. 2). The ANI between strain КMM 9862 T and M. variabilis ATCC 700307 T and M. thermotolerans DSM 19189 T were 80.34 and 77.72%, respectively, which are below the threshold ANI values of 95-96% for delineating bacterial species (Richter and Rossello-Mora 2009). The estimated dDDH values between strain КMM 9862 T and M. variabilis ATCC 700307 T and M. thermotolerans DSM 19189 T were 20.2 and 19.0%, respectively, which are lower the dDDH value of 70% accepted as the threshold value for bacterial species discrimination (Goris et al. 2007;Chun et al. 2018). The genomic and phylogenetic analyses data evidence strain KMM 9862 T could be classified as an individual species of the genus Microbulbifer.

Morphological, physiological, and chemotaxonomic characteristics
Morphological, physiological, biochemical, and chemotaxonomic characteristics of strain КMM 9862 T are given in Table 1, Table S1, Table S2, Supplementary Figure S1, Supplementary Figure S2, and in the species description. Strain КMM 9862 T was rod-shaped bacteria capable of producing extracellular material (Supplementary Figure S1). The novel bacterium КMM 9862 T was able to grow in the narrow salinity (1-4%) and temperature (7-35 °C) ranges and not able to assimilate carbon sources in the 32ID GN tests (Table S1). Strain КMM 9862 T contained ubiquinone Q-8 as the major respiratory quinone and C 16:0 , C 16:1 ω7c, C 12:0 , and C 10:0 3-OH as the major fatty acids (Table S2). Strain КMM 9862 T was close in its fatty acid profile to those of related type strains M. echini KACC 18258 T and M. variabilis ATCC 700307 T (Nishijima et al. 2009) although strain КMM 9862 T differed in content of C 12:0 and C 10:0 3-OH. M. thermotolerans DSM 19189 T contained significant amounts of iso-C 15:0 and iso-C 17:1 and lesser amounts of C 16:0 and C 16:1 ω7c (Table S2). The polar lipids of the strain КMM 9862 T comprised phosphatidylglycerol (PG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), an unidentified aminophospholipid (APL), an unidentified aminolipid (AL), two unidentified phospholipids (PL1, PL2), phosphatidic acid (PA), and an unidentified lipid (L) (Fig. S2). The polar lipid profile of strain КMM 9862 T was most similar to that of related type strain M. thermotolerans DSM 19189 T , but one unidentified aminolipid  (Fig. S2). The DNA G+C content of 49.8% was calculated from the genome sequence of the strain КMM 9862 T . The value obtained for the strain КMM 9862 T was close to those of 48.1-49.7% as reported for M. variabilis and M. epialgicus strains (Nishijima et al. 2009), whereas the DNA G+C means found for other recognized Microbulbifer species were significantly higher and ranged from 55 up to 64% (Miyazaki et al. 2008;Tang et al. 2008). Chemotaxonomic characteristics found for the strain KMM 9862 T , including ubiquinone Q-8, the predominance of C 16:0 , C 16:1 ω7c, C 12:0 and C 10:0 3-OH, the major polar lipid components of PE, PG, DPG, APL, and the DNA G+C content, are corroborated with those previously described for some of Microbulbifer species (Nishijima et al. 2009;Kämpfer et al. 2012;Zhang et al. 2012;Lee et al. 2017) and supported its assignment to this genus.
The phylogenetic and genetic distinctions obtained were supported by phenotypic differences of the novel isolate КMM 9862 T in its growth temperature and salinity ranges, enzyme activity 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 КMM 9862 T as a novel species, Microbulbifer okhotskensis sp. nov.
The type strain of the species is strain KMM 9862 T (= KACC 22804 T ), isolated from a bottom sediments sample, collected from the Okhotsk Sea, Russia.