Flavobacterium okayamense sp. nov. isolated from surface seawater

Strain KK2020170T, a Gram-stain negative, yellow colony-forming bacterium, was isolated from surface seawater sampled in Kojima Bay, Okayama, Japan. Phylogenetic analysis based on the 16S rRNA gene revealed that strain KK2020170T belongs to the genus Flavobacterium, with Flavobacterium haoranii LQY-7T (98.1% similarity) being its closest relative, followed by Flavobacterium sediminis MEBiC07310T (96.9%) and Flavobacterium urocaniciphilum YIT 12746T (96.0%). Whole-genome shotgun sequencing showed that strain KK2020170T, when paralleled with F. haoranii LQY-7 T, had 81.3% average nucleotide identity, and 24.6% in silico DNA–DNA hybridization values, respectively. The DNA G + C content of strain KK2020170T was 31.1 mol%. The most abundant fatty acids (> 10%) of strain KK2020170T were iso-C15: 0, iso-C17: 0 3-OH and iso-C15: 1 G. The dominant respiratory quinone of the strain was menaquinone MK-6. Based on the phylogenetic and phenotypic analysis results, we propose that strain KK2020170T represents a novel species, for which the name Flavobacterium okayamense sp. nov. has been proposed. The type strain is KK2020170T (= ATCC TSD-280 T = NBRC 115344 T). Supplementary Information The online version contains supplementary material available at 10.1007/s00203-023-03682-x.


Introduction
The genus Flavobacterium, a member of the family Flavobacteriaceae within the phylum Bacteroidota, was proposed by Bergey et al. (Bergey et al. 1923;Zhang et al. 2019) and subsequently revised by Bernardet et al. (1996).This genus contains at least 314 species with legitimately published names at the time of writing (https:// lpsn.dsmz.de/, April 2023).Flavobacterium species have been recognized in a wide variety of biotopes, including soil (Máté et al. 2022;Hou et al. 2022), river water (Watanabe et al. 2022), plants (Seo et al. 2022), seawater (Sun et al. 2022), sites in Antarctica (Králová et al. 2021), and chinstrap penguins (Irgang et al. 2023).Physiologically, cells of the genus Flavobacterium are typically Gram-stain-negative, rod-shaped, aerobic, and heterotrophic, and they yield yellow or orange colonies (McBride 2014).This study described the isolation of strain KK2020170 T from surface seawater in Kojima Bay, Okayama, Japan and examined whether this strain represents a new species under the genus Flavobacterium.

Isolation and cultivation
Strain KK2020170 T was isolated from surface seawater in Kojima Bay (34° 6048′ N 133° 9856′ E) in Okayama, Japan via a traditional dilution-plate method on trypticase soy agar (TSA) (BBL, Becton Dickinson, Franklin Lakes, USA).Sampling was performed in late October 2020.The sampled seawater had a temperature of 22 °C, NaCl concentration of 1.15% (w/w), and pH of 7.79.A yellow colony on the TSA, which had been incubated for 72 h at 30 °C, was picked up and single-colony isolation processes were repeated three 346 Page 2 of 7 times to establish a pure culture.The isolated strain was routinely grown at 30 °C on TSA or in trypticase soy broth (TSB) (BBL, Becton Dickinson, Franklin Lakes, USA).To preserve the strain, an overnight TSB culture was mixed well with 20% (final v/v) glycerol and preserved at −80 °C as a glycerol stock.

Phylogeny
To examine the 16S rRNA gene, genomic DNA was isolated from the cell pellet of a 1-ml overnight liquid culture of strain KK2020170 T using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) per the manufacturer's instructions.The 16S rRNA gene was directly PCR-amplified from the template (genomic DNA) using a universal primer set for bacteria (27F and 1492R) (Lane 1991;Kitahara et al. 2012).The resulting PCR product, which was well separated as a single band (approximately 1500 bp) by agarose-gel electrophoresis, was excised from the gel and purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).The amplicon was directly sequenced using an ABI 3130XL automatic sequencer (Applied Biosystems, Waltham, USA) according to the supplier's procedure.A similarity search of the sequenced 16S rRNA gene was performed using international nucleotide sequence databases (DDBJ/ENA/Gen-Bank).Phylogenetic trees were built by the neighbor-joining (NJ) and maximum-likelihood (ML) algorithms (Felsenstein 1981;Fitch 1971;Saitou, and Nei 1987) using MEGA v.7.0 (Kumar et al. 2016).Each topology of the two phylogenetic trees was validated by 1000 random bootstrap replicates.

Genome features
To obtain further DNA sequence information to strengthen the phylogenetic position of strain KK2020170 T , wholegenome shotgun sequencing was performed using a MiSeq instrument (Illumina, San Diego, USA) and GridION X5 system (Oxford Nanopore Technologies, Oxford, UK).Although MiSeq can read short DNA fragments with high accuracy, GridION can read longer (thus structurally collect) DNA fragments.Combining the information of both techniques is effective for determining the complete genome of a bacterium in terms of ensuring both sequence accuracy and high structural integrity of the DNA (Miyazaki et al. 2020;Yu et al. 2019).The sequencing and data analysis methods used in this study are concordant to the specified basic standards for genome data for prokaryotic taxonomy (Chun et al. 2018).

Physiology and chemotaxonomy
The physiological and chemotaxonomic properties of strain KK2020170 T were examined with reference to the minimal requirements for describing new species in Flavobacterium (Bernardet et al. 2002;Jung et al. 2017;Yang et al. 2016).Other experiments conducted in studies describing the closest relatives of strain KK2020170 T (F.haoranii LQY-7 T (Zhang et al. 2010), F. sediminis MEBjC07310 T [Bae et al. 2018), F. indicum GPTSA100-9 T (Saha and Chakrabarti 2006) and F. urocaniciphilum YIT 12746 T (Fujii et al. 2014)] were also performed.Unless otherwise noted, the isolate (strain KK2020170 T ) was routinely grown on TSA or in TSB at 30 °C for morphological and biochemical characterization.
To compare fatty acid compositions, reference strains (F.haoranii LQY-7 T and F. sediminis MEBiC07310 T ) were procured from the Japan Collection of Microorganisms and Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, respectively.
The fatty acids of strain KK2020170 T and the reference strains (LQY-7 T and MEBiC07310 T ) were prepared per the standard MIDI protocol (Sasser 1990) by culturing on TSA at 28 °C for 24 h.The fatty acid methyl esters were examined by gas chromatography (7890A, Agilent Technologies, Santa Clara, USA) and identified using the TSBA6 database of the Microbial Identification System (Sherlock, v. 6.0).Isoprenoid quinones were extracted using a method described by Bligh and Dyer (Bligh and Dyer 1959;Tamaoka et al. 1983) and analyzed using an ACQUITY UPLC H-Class system with a PDA detector (Waters, Milford, USA) using a reversed-phase BEH C18, 2.1 (I.D.) × 150 mm, 1.7 μm column (Waters).The polar lipids of strain KK2020170 T grown on TSA for 24 h at 28 °C were extracted and tracked by two-dimensional thin-layer chromatography as previously described (Minnikin et al. 1979).

Phylogenetic analysis
Sequence similarity calculations of 16S rRNA gene (with 1439 unambiguously aligned base pairs) suggested that strain KK2020170 T was most closely related to F. haoranii LQY-7 T (98.1% similarity) (Zhang et al. 2010), followed by F. sediminis MEBiC07310 T (96.9% similarity) (Bae et al. 2018) and F. urocaniciphilum YIT 12746 T (96.0%) (Fujii et al. 2014).All other type strains had lower than 96.0%similarity.The NJ tree constructed from 16S rRNA gene sequences revealed that, strain KK2020170 T clearly belongs to the genus Flavobacterium, surrounded by legitimately named Flavobacterium members (Fig. 1).The same phylogenetic position of strain KK2020170 T was reproducibly observed in the ML-reconstructed phylogenetic tree (Fig. S1, available in the online supplementary materials).In general, it is empirically known that when a bacterial strain's 16S rRNA gene sequence has lower than 98.7% similarity to the closest type strain, the strain can possibly be classified as a novel species (Chun et al. 2018).Our 16S rRNA gene-based data suggested that it is possible that the strain has a unique phylogenetic position at species level within the genus Flavobacterium.

Whole-genome sequence analysis
The full genome of strain KK2020170 T was identified as a single-circular chromosome of 2,781,077 bp containing 2574 protein-coding genes, 9 rRNAs, and 51 tRNA genes (Table S1).The G + C content of the genome was 31.1 mol%, which was within the reported range for Flavobacterium strains (30 mol%-41 mol%) (Bernardet et al. 1923).To distinguish strain KK2020170 T from F. haoranii LQY-7 T (ASM936305v1) and F. sediminis MEBiC07310 T (ASM314838v1), two independent in silico comparative approaches were applied.First, pyani v.0.2.10 was used to compute the average nucleotide identity (ANI) (Pritchard et al. 2016), and the ANI between strain KK2020170 T and F. haoranii LQY-7 T was 81.3%, versus 75.8% between strain KK2020170 T and F. sediminis MEBiC07310 T .When a bacterial strain, as a comparison to the closest type strain, has an ANI lower than the standard cutoff (95-96%), the strain has a high possibility of being classified as a new species.Next, in silico DNA-DNA hybridization (dDDH) using Genome-to-Genome Distance Calculator v.2.1 (http:// ggdc.dsmz.de/) (Meier-Kolthoff et al. 2013) was applied.The mean genome-to-genome distance (in silico DDH) between strain KK2020170 T and F. haoranii LQY-7 T was calculated as 24.6%.This value was 20.0% when strain KK2020170 T and F. sediminis MEBiC07310 T were compared.Both values were significantly below the conventional threshold (70%), which is used to distinguish two bacterial strains from each other at the species level.These genomic study results strongly suggest that strain KK2020170 T is a new species in Flavobacterium.
Based on comparative phylogenetic analysis using 16S rRNA gene sequences, strain KK2020170 T was suggested to belong to the genus Flavobacterium.Subsequent whole-genome shotgun sequencing showed that strain KK2020170 T , F. haoranii LQY-7 T , and F. sediminis MEBiC07310 T are closely related but clearly distinct from each other at the species level.Physiological and chemotaxonomic characterization revealed that strain KK2020170 T Fig. 1 Neighbor-joining tree depicting the evolutionary relationships of F. okayamense strain KK2020170 T and 37 adjacent type strains in the genus Flavobacterium based on 16S rRNA gene sequences.Bootstrap values (given as percentages of 1000 replicates) with > 50% values are displayed.Flavobacterium aquatile is the type species in the genus Flavobacterium.Myroides odoratus NBRC 14945 T (AB517709) was employed as an outgroup.The bar represents a Knuc distance of 0.01 had typical properties as a member of the genus Flavobacterium.For example, strain KK2020170 T contains MK-6 as the predominant respiratory quinone, forms yellow colonies, shows similar growth phenotypes (i.e., ranges of pH, salt and temperature for growth), and possesses a similar major fatty acid composition as F. haoranii LQY-7 T and F. sediminis MEBiC07310 T .However, some characteristics of strain KK2020170 T clearly differed from those of its closest neighbor F. haoranii LQY-7 T , such as the ability to hydrolyze casein; inability to hydrolyze aesculin, gelatin, and starch; minor fatty acid composition; and some biochemical characteristics.We, therefore, conclude that strain KK2020170 T represents a distinct species within the genus Flavobacterium, for which the name Flavobacterium okayamense sp.nov. is proposed.

Description of Flavobacterium okayamense sp. nov.
Flavobacterium okayamense (o.ka.ya.men'se.N.L. neut.adj.okayamense referring to Okayama in Japan, where the type of strain was isolated).