Vibrio japonicus sp. nov., a novel member of the Nereis clade in the genus Vibrio isolated from the coast of Japan

A novel Vibrio strain, JCM 31412T, was isolated from seawater collected from the Inland Sea (Setonaikai), Japan, and characterized as a Gram-negative, oxidase-positive, catalase-negative, facultatively anaerobic, motile, ovoid-shaped bacterium with one polar flagellum. Based on 16S rDNA gene identity, strain JCM 31412T showed a close relationship with type strains of Vibrio brasiliensis (LMG 20546T, 98.2% identity), V. harveyi (NBRC 15634T, 98.2%), V. caribbeanicus (ATCC BAA-2122T, 97.8%) and V. proteolyticus (NBRC 13287T, 97.8%). The G+C content of strain JCM 31412T DNA was 46.8%. Multi-locus sequence analysis (MLSA) of eight loci (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA; 5535bp) further clustered strain JCM 31412T in the Nereis clade, genus Vibrio. Phenotypically, strain JCM 31412T differed from the closest related Vibrio species in its utilization of melibiose and raffinose, and its lack of casein and gelatin hydrolysis. It was further differentiated based on its fatty acid composition, specifically properties of C12:03OH and summed features, which were significantly different from those of V. brasiliensis, V. nigripulchritudo and V. caribbeanicus type strains. Overall, the results of DNA-DNA hybridization, and physiological and biochemical analysis differentiated strain JCM 31412T from other described species of the genus Vibrio. Based on these polyphasic taxonomic findings, it was therefore concluded that JCM 31412T was a novel Vibrio species, for which the name Vibrio japonicus sp. nov. was proposed, with JCM 31412T (= LMG 29636T = ATCC TSD-62T) as the type strain.


Phenotypic characterization
Strain JCM 31412 T was subjected to the following phenotypic tests: cell morphology and mobility; Gram staining; Voges-Proskaur test; oxidase and catalase activity; oxidation/fermentation; hydrolysis of gelatin, esculin and casein; and DNA decomposition. Catalase activity was determined by bubble formation in 3% H 2 O 2 solution, and oxidase activity using cytochrome oxidase paper (Nissui, Tokyo). DNA decomposition (DNase production) was tested by using the toluidine blue-DNA agar containing 1.0% Bacto peptone (Difco), 1.0% yeast extract (Difco), 0.2% deoxyribonucleic acid (from salmon testes), 0.01% toluidine blue, 2.0% agar (Difco) and 3.0% NaCl (pH 7.6). Growth at various temperatures (4-40˚C), pH (4)(5)(6)(7)(8)(9)(10)(11)(12), NaCl concentrations (0-10%) using tryptic soy broth (Difco) with 1.5% NaCl (for temperature and pH) at 28˚C (for pH and NaCl) with shaking, and on thiosulfate-citrate-bile sucrose (TCBS) agar (Nippon Beckton Dickinson) was also examined. Additional biochemical characterization was performed using standardized API 20E, API 20NE, API ZYM and API 50CH identification systems (bioMérieux) with incubation at 28˚C according to the manufacturers' instructions, except that sterile 1.5% (w/v) NaCl was used to prepare the inocula. Strain JCM 31412 T was phenotypically characterized in comparison with the following closely related Vibrio species obtained from bacterial culture collections: V. brasiliensis LMG 20546 T [15], V. nigripulchritudo LMG 03896 T [16] and V. caribbeanicus ATCC BAA-2122 T [17] (Table 1). Antibiotic sensitivity was established using a disc susceptibility assay as described by the Clinical and Laboratory Standard Institute (CLSI), document M45-A2 (2010) [18]. Sensitivity to the vibriostatic agent O/129 (2, 4-diamino-6, 7-diisopropylpteridine) was determined using Oxoid discs containing 10 and 150μg O/129 per disc. The inhibition zone of each antibiotic was measured using strains grown on Müller-Hinton agar (Difco) supplemented with 1.5% (w/v) NaCl for 16-18h at 35˚C. For negatively stained transmission electric micrograph observations of strain JCM 31412 T cells, cells from an overnight culture in tryptic soy broth (Difco) with 1.5% NaCl at 28˚C with shaking were harvested by centrifugation and washed with sterile saline, and were then fixed for 30 min at 4˚C in 2% glutaraldehyde in 0.1M phosphate buffer (pH 7). A droplet of cell suspension was placed on carbon-film with a 400 Cu grid for two minutes. Excess liquid was removed using filter paper. The cells on the grid were negatively stained with 2% (w/v) uranyl acetate (Cerac, USA) for two minutes after drying the excess liquid at room temperature. Samples were observed by transmission electron microscopy (TEM) with H-7600 (HITACHI, Tokyo) at 100 kV. Fatty acid analysis Cellular fatty acid methyl esters were obtained from strain JCM 31412 T , Vibrio brasiliensis LMG 20546 T , V. nigripulchritudo LMG 03896 T and V. caribbeanicus ATCC BAA-2122 T by saponification, methylation and extraction after growth for 24h at 28˚C on trypticase soy agar containing 1.5% NaCl (w/v). The cellular fatty acids were extracted according to the protocol of the Sherlock Microbial Identification System [19] and subsequently identified by gas chromatography. Individual fatty acids were identified using the Microbial Identification software package (Sherlock version 6.0) based on the TSBA6 calculation method and TSBA6 library databases [20]. Fatty acid methyl esters were analyzed by gas chromatography with flame-ionization detection (GC-FID) using rapid Microbial Identification System software (RBTR20; MIDI Inc.) to identify the relative amounts of each fatty acid. For comparison, the three closest species based on 16S rDNA analysis were also examined in parallel.

DNA isolation and genotypic analysis
Genomic DNA was extracted and purified using a commercial QIAamp DNA Blood Maxi Kit (QIAGEN) following the manufacturer's protocol. The base sequence of complete 16S rDNA was determined according to the method of Nakagawa et al. [21]. Sequences were analyzed with an ABI PRISM 3130 xl genetic analyzer system (Applied Biosystems, CA, USA). A homology search was carried out using the BLASTN (https://blast.ncbi.nlm.nih. gov) and the 16S rDNA base sequences of other known related strains were retrieved from GenBank/EMBL/DDBJ. Sequences were aligned using CLUSTAL W [22] and phylogenetic trees reconstructed using neighbor-joining algorithms (MEGA version 5.05) [23,24]. The stability of the grouping was estimated by bootstrap analysis with 1000 replications. Multi locus sequence analysis (MLSA) of eight concatenated housekeeping gene sequences encoding topoisomerase I (topA), cell division protein (ftsZ), glyceraldehydes-3-phosphate dehydrogenase (gapA), DNA gyrase B subunit (gyrB), actin-like cytoskeleton protein (mreB), uridylate kinase (pyrH), recombination repair protein (recA) and RNA polymerase alphasubunit (rpoA) was carried out as described previously [25,26]. PCR amplicons were edited and assembled into consensus sequences using CLUSTAL W [22] (accession numbers are listed in S1 Table). DNA-DNA hybridization experiments using photobiotin-labelled DNA was performed at 44˚C according to the method of Suzuki et al. [27]. The G+C content of the DNA was determined by high-performance liquid chromatography (HPLC) [

Morphological description
Cells of strain JCM 31412 T were found to be a motile but swarming, ovoid and variable in size, ranging from 0.95-1.3 μm long and 0.6-0.9 μm wide. A sheathed single polar flagellum approximately 5.9 μm long was observed in TEM observations (Fig 1). Short rod bodies formed in older cultures. No endospores were observed. Colonies on ZoBell 2216E agar supplemented with 2.0% NaCl (w/v) were Gram-negative, cream colored, non-pigmented, nonluminescent, circular, smooth and convex, and 1-2 mm in diameter after incubation at 27˚C for 24h.

Phenotypic analysis
Phenotypic traits and characteristics of strain JCM 31412 T and closely related Vibrio species are listed in Table 1. Strain JCM 31412 T showed distinct phenotypic features of the genus Vibrio that were Gram-negative, oxidase positive, grown on TCBS agar and were facultative anaerobes [29]. It was further identified as a facultative anaerobe capable of both fermentative and respiratory metabolism. Furthermore, it required salt (0.5-9.0%), and was capable of growth from 10 to 37˚C and at pH 7.0-12.0. Vigorous growth also occurred on ZoBell marine agar 2216E (Difco) and tryptic soy agar supplemented with 2.0% NaCl (w/v) at 25-37˚C for 12 h. Optimal growth was observed at 35˚C with 2.0% NaCl (w/v) at pH 8.0. Growth on TCBS agar (Nippon Beckton Dickinson) was also observed in the form of a yellow, convex, round colony, about 1 mm in diameter, after 72 h incubation at 28˚C. Strain JCM 31412 T was positive for

16S rDNA genotypic analysis
After analyzing and assembling the 1477-bp 16S rDNA gene sequence of strain JCM 31412 T , the consensus sequence was used to query the GenBank database of NCBI using BLAST to identify strains with the highest sequence identity. Sequence searches demonstrated that strain JCM 31412 T belongs to the genus Vibrio. Analysis (query coverage 100%) further revealed that type strains of V. brasiliensis (LMG 20546 T ), V. harveyi (NBRC 13287 T ), V. proteolyticus (NBRC 13287 T ) and V. carribeanicus (ATCC BAA-2122 T ) were most closely related, sharing 98.2, 98.2, 97.8 and 97.8% 16S rDNA gene sequence identity, respectively. Moreover, a neighbor-joining tree derived from 16S rDNA gene sequences of 24 different Vibrio species including strain JCM 31412 T included novel strain JCM 31412 T in a cluster with V. brasiliensis LMG 20546 T , V. nigripulchritudo ATCC 27043 T and V. caribbeanicus N384 T (Fig 2).

MLSA
Vibrios are comprised of closely related species and are therefore difficult to identify [30]. MLSA of housekeeping genes has proven to be an accurate tool for delineation of microorganisms [31], as well as phylogenetic studies of the genus Vibrio [25]. To further optimize the taxonomic resolution, a neighbor-joining tree was therefore constructed from concatenated sequences of eight housekeeping genes (topA, ftsZ, gapA, gyrB, mreB, pyrH, recA and rpoA) as recommended by Sawabe et al. [32]. As a result, MLSA clustered strain JCM 31412 T in the Nereis clade along with V. nereis LMG 3895 T and V. xuii LMG 21346 T , with high bootstrap values. Based on 16S rDNA gene sequence identity, the closest related neighbors were found to be type strains of V. brasiliensis (Orientalis clade), V. nigripulchritudo (Nigripulchritudo clade) and V. carribeanicus (Pectenicida clade). Phylogenic analysis of sequences of protein coding genes therefore revealed substantial differences from the phylogenic tree obtained based on 16S rDNA gene sequences due to low interspecies resolution with the latter [26,[33][34][35]. Gen-Bank/EMBL/DDBJ accession numbers of the 16S rDNA, topA, ftsZ, gapA, gyrB, mreB, pyrH, recA and rpoA gene sequences of strain JCM 31412 T , and all other gene sequences used in this study are listed in the S1 Table. DNA-DNA hybridization analysis The phylogenetic tree based on 16S rDNA gene sequences of strain JCM 31412 T and related species showed that strain JCM 31412 T formed its closest phylogenetic cluster with type strains of V. brasiliensis, V. nigripulchritudo and V. caribbeanicus but with low bootstrap support (Fig  2). In contrast, MLSA revealed that strain JCM 31412 T formed a distinct cluster in the Nereis clade with type strains of V. nereis and V. xuii, with high bootstrap values (Fig 3). DNA-DNA hybridization experiments were therefore performed between novel strain JCM 31412 T and established strains grouped in the Nereis clade as well as closest phylogenetic neighbors based on 16S rDNA gene sequences. DNA of strain JCM 31412 T showed relatively low DNA-DNA relatedness with V. brasiliensis LMG 20546 T [15], V. nigripulchritudo LMG 03896 T [16], V. caribbeanicus ATCC BAA-2122 T [17], V. nereis LMG 3895 T [36] and V. xuii LMG 21346 T [15] (36.4±1.6, 24.8±1.8, 24.5±3.0, 36.4±2.6 and 32.7±3.4%, respectively), significantly below the recommended cut-off threshold of 70% DNA-DNA hybridization for the identification of bacterial species (Table 3) [37]. These results further support the suggestion that strain JCM 31412 T represents a species distinct from either of its nearest neighbors based on MLSA, as well as from type strains of V. nereis and V. xuii.

G+C content analysis
The mean G+C content of strain JCM 31412 T was calculated as 46.8 mol%, which falls within the range of 38-51 mol% previously found for other members of the genus Vibrio [38].

Nucleotide sequence accession numbers
All sequence files are available on the GenBank nucleotide database with accession numbers for the 16S rDNA gene, topA, ftsZ, gapA, gyrB, mreB, pyrH, recA and rpoA sequences, respectively. Other relevant data are provided in S1 Table.

Conclusion
This study characterized strain JCM 31412 T both phenotypically and genotypically, supporting its description as a novel and previously uncharacterized Vibrio species. Strain JCM 31412 T formed a stable group in the Nereis clade, and could be differentiated from closely related species based on phylogenetic analysis, DNA-DNA relatedness and phenotypic characterization. Overall, these data confirm that strain JCM 31412 T is a novel species belonging to the genus Vibrio, for which the name Vibrio japonicus sp. nov. is proposed.
Supporting information S1