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 KK2020170T from surface seawater in Kojima Bay, Okayama, Japan and examined whether this strain represents a new species under the genus Flavobacterium.

Materials and methods

Isolation and cultivation

Strain KK2020170T 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 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 KK2020170T 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/GenBank). 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 KK2020170T, whole-genome 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 KK2020170T 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 KK2020170T (F. haoranii LQY-7 T (Zhang et al. 2010), F. sediminis MEBjC07310T [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 KK2020170T) was routinely grown on TSA or in TSB at 30 °C for morphological and biochemical characterization.

To observe the morphology of strain KK2020170T cells, cells were cultured on TSA at 30 °C for 72 h. Harvested cells were Gram-stained using Favor G Nissui (Nissui Pharmaceutical, Tokyo, Japan) per the supplier’s protocols, and they were visualized under a phase-contrast microscope (BX50F4; Olympus, Tokyo, Japan) at × 1000 magnification. Colony morphology was observed after incubating cells at 30 °C for 72 h. Growth phenotypes at wide ranges of temperatures (4, 10, 15, 20, 25, 30, 35, 40, and 45 °C) were tested on TSA for 72 h. Salt sensitivity was tested in TSB supplemented with different final concentrations of NaCl (0%–8% w/v, increased in 1% increments). The pH sensitivity of strain KK2020170T was tested in TSB with a series of different pH from 4.5 to 10.0 in 0.5-unit increments [pH was fine-tuned using 10 mM MES (pH 4–6) or 10 mM Tris (pH 7–10) buffers]. The existence of flexirubin-type pigments in colonies was evaluated using previously reported methods (Bernardet et al. 2002). Catalase and oxidase activities were evaluated using 3% (v/v) H2O2 and 1% (w/v) tetramethyl-p-phenylenediamine, respectively, using previously described methods (Smibert et al. 1994). Growth ability was assessed on Marine Broth 2216 agar (Becton Dickinson, Franklin Lakes, USA), nutrient agar (Oxoid, Basingstoke, UK), and MacConkey agar (Nissui Pharmaceutical, Tokyo, Japan). Casein and starch hydrolysis was examined for 7 days at 30 °C according to standard protocols (Smibert, and NR. 1994; Cowan 1965). Acid production from carbohydrates, enzyme activities, and other biochemical features of strain KK2020170T were evaluated using API 20NE and API ZYM (BioMérieux, Marcy-l'Étoile, France), both of which are widely used for phenotype-based bacterial characterization or identification purposes, following the manufacturer’s protocols. Susceptibility to the following antibiotics was tested by the disc diffusion procedure (µg/disc) on TSA for 24 h at 30 °C using Sensi-Disc (Becton Dickinson, Franklin Lakes, USA): amoxicillin (25), ampicillin (10), bacitracin (10), carbenicillin (100), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), lincomycin (2), rifampicin (5), penicillin G (10), polymyxin B (300), tetracycline (30), spectinomycin (100), streptomycin (10), and vancomycin (30). The anaerobic growth test was performed by incubating a TSA plate at 37 °C for 72 h, and oxygen was absorbed using an Anaero Pack gas system (Anaero Pack disposable, Mitsubishi Gas Chemical, Tokyo, Japan).

To compare fatty acid compositions, reference strains (F. haoranii LQY-7 T and F. sediminis MEBiC07310T) 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 KK2020170T and the reference strains (LQY-7 T and MEBiC07310T) 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 KK2020170T 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).

Results and discussion

Phylogenetic analysis

Sequence similarity calculations of 16S rRNA gene (with 1439 unambiguously aligned base pairs) suggested that strain KK2020170T was most closely related to F. haoranii LQY-7 T (98.1% similarity) (Zhang et al. 2010), followed by F. sediminis MEBiC07310T (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 KK2020170T clearly belongs to the genus Flavobacterium, surrounded by legitimately named Flavobacterium members (Fig. 1). The same phylogenetic position of strain KK2020170T 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.

Fig. 1
figure 1

Neighbor-joining tree depicting the evolutionary relationships of F. okayamense strain KK2020170T 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

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Whole-genome sequence analysis

The full genome of strain KK2020170T 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 KK2020170T from F. haoranii LQY-7 T (ASM936305v1) and F. sediminis MEBiC07310T (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 KK2020170T and F. haoranii LQY-7 T was 81.3%, versus 75.8% between strain KK2020170T and F. sediminis MEBiC07310T. 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 KK2020170T and F. haoranii LQY-7 T was calculated as 24.6%. This value was 20.0% when strain KK2020170T and F. sediminis MEBiC07310T 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 KK2020170T is a new species in Flavobacterium.

Physiology and chemotaxonomy

Cells of strain KK2020170T were Gram-stain-negative rods (0.5–0.6 × 1.0–9.5 μm, Fig. S2A). Gliding motility was not observed. Strain KK2020170T colonies grown on TSA plate were yellow, circular, and smooth with low convex (Fig. S2B). In addition to aerobic growth, weak anaerobic growth was observed. Flexirubin-type pigments were not detected. Cells were susceptible to amoxicillin, ampicillin, carbenicillin, chloramphenicol, erythromycin, lincomycin, penicillin G, polymyxin B, rifampicin, spectinomycin, tetracycline, and vancomycin but resistant to bacitracin, gentamicin, kanamycin, and streptomycin. The physiological and biochemical properties used to differentiate strain KK2020170T and closely connected Flavobacterium species are summarized in Table 1. Briefly, strain KK2020170T; unlike F. haoranii LQY-7 T, does not hydrolyze aesculin, gelatin, and starch. Conversely, strain KK2020170T, unlike F. haoranii LQY-7 T, hydrolyzes casein. Urease and α-chymotrypsin activities were observed for F. haoranii LQY-7 T but not for strain KK2020170T.

Table 1 Differential phenotypic attributes of F. okayamense strain KK2020170T and closely related Flavobacterium type strains
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The major fatty acids (> 10%) of strain KK2020170T were iso-C15:0 (53.6%), iso-C15:1 G (12.9%), and iso-C17:03-OH (12.2%). In Table 2, the cellular fatty acid profile of strain KK2020170T is paralleled with those of F. haoranii LQY-7 T, F. sediminis MEBiC07310T, F. indicum GPTSA 100-9 T, and F. urocaniciphilum YIT 12746 T. The fatty acid profile of strain KK2020170T resembled that of F. haoranii LQY-7 T, in which the major detected components were iso-C15:0, iso-C15:1 G, and iso-C17:0 3-OH (Table 2). The fatty acid composition of strain KK2020170T was distinguishable from those of F. haoranii LQY-7 T and F. sediminis MEBiC07310T in that iso-C14:0 and C17:1ω6c were not detected in strain KK2020170T. Menaquinone 6 (MK-6) was detected as the dominant (99.9%) respiratory quinone in strain KK2020170T, similar to the findings in other members of the family Flavobacteriaceae. Strain KK2020170T exhibited a complex polar lipid profile consisting of one phosphatidylethanolamine as the dominant element, three aminolipids, one lyso-phosphatidyl-ethanolamine, and four unidentified lipids (Fig. S3).

Table 2 Cellular fatty acid proportions (%) of F. okayamense strain KK2020170T and closely related Flavobacterium type strains
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Based on comparative phylogenetic analysis using 16S rRNA gene sequences, strain KK2020170T was suggested to belong to the genus Flavobacterium. Subsequent whole-genome shotgun sequencing showed that strain KK2020170T, F. haoranii LQY-7 T, and F. sediminis MEBiC07310T are closely related but clearly distinct from each other at the species level. Physiological and chemotaxonomic characterization revealed that strain KK2020170T had typical properties as a member of the genus Flavobacterium. For example, strain KK2020170T 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 MEBiC07310T. However, some characteristics of strain KK2020170T 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 KK2020170T 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).

Cells are Gram-stain-negative, facultatively anaerobic, non-motile, and rod-shaped (0.5–0.6 × 1.0–9.5 μm). Flexirubin-type pigment adsorption by a colony is not detected. Colonies on TSA (after cultivation for 72 h at 30 °C) are yellow, circular, and smooth with low convex. Optimal growth occurs at 30 °C (range 15–40 °C), at pH 7.0 (range, pH 6.5–8.5), and in the presence of 1% (w/v) NaCl (range 1–4%). Cells hydrolyze casein but not starch and tyrosine. Growth occurs on Marine Broth 2216 agar and nutrient agar but not on MacConkey agar. Oxidase and catalase activity is present. In API 20NE tests, hydrolysis of aesculin and gelatin; reduction of nitrate to nitrite; glucose fermentation; indole production; arginine dihydrolase, β-galactosidase, and urease activities; and assimilation of arabinose, mannose, maltose, potassium gluconate, N-acetylglucosamine, glucose, arabinose, mannitol, caprate, adipate, malate, citrate, and phenyl acetate are negative. In the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase activities are present, but other enzyme activities are absent. The major cellular fatty acids are iso-C15:0, iso-C15:1 G, and iso-C17:0 3-OH. The most abundant isoprenoid quinone is MK-6. The major polar lipid is phosphatidylethanolamine.

The type strain, KK2020170T (= ATCC TSD-280 T = NBRC 115344 T), was isolated from surface seawater in Kojima Bay (34° 60′ N 133° 99′ E), Okayama, Japan. The genomic DNA G + C content of the type strain is 31.1 mol%.