The family Flavobacteriaceae is one of the major branches of the phylum Bacteroidota Krieg et al. 2021 (Oren and Garrity, 2021). Over the past two decades, numerous novel genera and species have been reported as new member of the family Flavobacteriaceae. However, phylogenetic positioning based on 16S rRNA gene sequence of strains within the family remained somewhat ambiguous, especially in the branch including the genera Algibacter, Hyunsoonleella, Lacinutrix, etc. The genus Seonamhaeicola, adjacent to the genus Hyunsoonleella, also belong to the family Flavobacteriaceae and was first proposed by Park et al. (2014) and its description was subsequently amended by Zhou et al. (2016). Most members of the genus Seonamhaeicola are characterized as Gram-stain-negative, rod-shaped, facultatively anaerobic, heterotrophic, non-motile, non-spore-forming, yellowish- to orange- coloured bacteria, with menaquinone-6 (MK-6) as the major respiratory quinone, and iso-C_15:0 as the major fatty acid. Members of the genus have been isolated from diverse habitats such as coral (Yoon et al., 2019), marine algae (Zhou et al., 2016; Fang et al., 2017), and sediments (Park et al., 2014; Cao et al., 2020; Zhang et al., 2020). A novel marine bacterial strain affiliated to the genus Seonamhaeicola has been isolated from a debris of Eisenia bicyclis and its taxonomic properties are reported here.

Sampling, isolation and culture conditions

During the exploration of marine bacteria, strain MEBiC01930^T (=DOKDO 020^T) was isolated from a debris of marine algae (Eisenia bicyclis) sampled at Dokdo Island, Republic of Korea (37° 14' 34'' N, 131° 52' 00'' E) in Oct. 2004. For strains isolation, a bit of algal debris (approx. 0.1 g) was grinded and serially diluted with sterilized seawater, plated on marine agar 2216 medium (MA; BD) and cultivated at 25℃ for 3 days, then, distinctive colonies were selected for pure culture. After primary isolation and purification, strain MEBiC01930^T was cultivated at 25°C on the same medium for biochemical and physiological characterization. This strain was also stored at −80°C in marine broth 2216 (MB; BD) with 20% (v/v) glycerol. For phenotypic comparisons, Seonamhaeicola aphaedonensis KCTC 32578^T, S. acroporae KCTC 62713^T, S. algicola KCTC 42396^T, S. maritimus KCTC 72528^T, S. sediminis KCTC 72085^T, and S. marinus NBRC 112333^T were purchased from KCTC (Korean Collection for Type Cultures) or NBRC (Biological Resource Center, NITE) and grown on MA at 25℃. Unless stated otherwise, strain MEBiC01930^T was cultivated at 25℃ for 2 days.

Phylogenetic and genomic analysis

Extraction of genomic DNA was carried out using a commercial DNA extraction kit (MoBio), and the 16S rRNA gene was amplified by using a bacterial primer set 27F and 1492R (Giovannoni, 1991), the detailed procedure and conditions were described in Lee et al. (2013). The 16S rRNA gene sequencing was performed using an ABI 3730xl automatic sequencer. The obtained 16S rRNA gene sequence was compared by BLAST pair-wise alignment with sequences in the EzBioCloud database (Kim et al., 2012). Phylogenetic analysis based on the almost-complete 16S rRNA gene sequence (1,407 bp) of strain MEBiC01930^T was conducted with members of the genus Seonamhaeicola and closely related genera by using MEGA11 (Tamura et al., 2021). The phylogenetic tree with 1,000 replicated bootstrap analysis and the complete deletion option was reconstructed by using the neighbor-joining method [NJ; (Saitou and Nei, 1987)] with the Kimura 2 parameter model (Kimura, 1980) as well as the maximum-likelihood [ML; (Felsenstein,1981)] and maximum-parsimony [MP; (Fitch, 1971)] methods. The sequences of Flavobacterium aquatile DSM 1132^T (AM230485) and Kordia algicida OT-1^T (AY195836) were used as outgroups (Fig. 1).

Fig. 1. Phylogenetic tree based on nearly complete 16S rRNA gene sequences (1,407 bp) showing relationship between strain MEBiC01930^T (=DOKDO020^T) and closely related members of the family Flavobacteriaceae. The tree is based on the Kimura 2 distances model and the neighbour-joining algorithm. Bootstrap values from NJ, ML, and MP by 1,000 replicated are designated (●; recovered by three methods with >90%, ○; recovered by three methods with >70%, ▲; recovered by three methods, △; recovered by two methods) at each node. Scale bar, 0.01 nucleotide substitutions per nucleotide position.

The genome sequencing of strain MEBiC01930^T was performed using a PacBio RS II system. Sequence assembly was performed by using HGAP version 3.0. Obtained genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2016). A phylogenomic analysis was conducted with the obtained genome sequence of strain MEBiC01930^T with the closely related strains in the family Flavobacteriaceae (Table 1) using PhyloPhlAn (Segata et al., 2013). Average nucleotide identity (ANI) and Average amino acids identity (AAI) values were computed using the OrthoANIu algorithm (Yoon et al., 2017) and EzAAI (Kim et al., 2021), respectively. The genus delineation cut-off of AAI values was calculated according to Park et al. (2022).

Table 1

Genomes used in the calculation of ANI and AAI values and construction of the phylogenomic tree in this study. Two reference strains were not used in calculation of ANI and AAI values

No.	Genus	Strain	Accession No.	Size (Mb)	Contigs	Protein	RNAs (r/t/o)	Genes	G + C ratio
1	Seonamhaeicola citrea	DOKDO020	CP104270	3.65	1	3102	6/49/4	3170	32.42
2	Seonamhaeicola algicola	Gy8	VOSC00000000	3.71	52	3086	4/38/4	3144	32.32
3	Seonamhaeicola aphaedonensis	CECT 8487	QRDX00000000	3.51	54	2950	5/38/4	3010	33.72
4	Seonamhaeicola marinus	B011	VSDQ00000000	5.06	1039	4593	13/41/5	5034	33.34
5	Seonamhaeicola maritimus	1505	VRKQ00000000	4.38	35	3627	7/38/4	3687	33.29
6	Seonamhaeicola sediminis	W255	SMZJ00000000	3.54	50	2957	5/38/4	3021	33.52
7	Hyunsoonleella aestuarii	KCTC 23449	VAWB00000000	3.14	9	2805	6/36/4	2865	33.18
8	Hyunsoonleella aquatilis	SJ7	JACNMF000000000	3.93	24	3313	5/36/4	3369	38.15
9	Hyunsoonleella flava	T58	SIRT00000000	3.81	63	3299	4/42/4	3363	34.99
10	Hyunsoonleella jejuensis	DSM 21035	FOFN00000000	3.48	11	2938	5/37/4	2992	34.61
11	Hyunsoonleella pacifica	SW033	SIRS00000000	4.13	37	3493	5/46/4	3565	32.70
12	Hyunsoonleella ulvae	HU1-3	JAEPJQ000000000	4.06	23	3442	3/44/4	3501	33.89
13	Jujuia pallidilutea	DSM 21165	PVEO00000000	4.11	34	3414	5/36/4	3473	34.02
14	Aestuariivivens insulae	AH-MY3	JAKMCS000000000	4.23	33	3456	3/40/4	3525	36.59
15	Tamlana sedimentorum	JCM 19808	JTDW00000000	3.96	52	3360	3/40/4	3421	32.91
16	Hwangdonia seohaensis	HD-3	JAVSCK00000000	3.75	10	3203	3/34/4	3250	36.23
17	Yeosuana aromaticivorans	JCM 12862	BMNR00000000	3.76	34	3302	5/39/4	3374	35.37

Phenotypic, physiological and biochemical properties

Unless otherwise stated, the physiological and morphological characterization of the strain was conducted according to methods described by Yang et al. (2006). Transmission electron micrographs were taken using a LIBRA120 (Carl-Zeiss) electron microscope with fixed cells that were negatively stained with 2% phosphotungstic acid at pH 7.0. Additionally, scanning electron micrographs (SEM) were taken using a VEGAII LSU (Tescan) after dehydrated with series of ethanol. The range and optimal growth temperatures were determined in MB at 12 different temperatures (10, 16, 20, 23, 26, 29, 32, 36, 39, 42, 46 and 50°C) in a temperature gradient incubator (TVS126MA; Adaventec) for up to 3 days. The tolerance range for NaCl was tested in modified ZoBell 2216 broth (ZoBell, 1941) prepared with distilled water and supplemented with the NaCl (Sigma; 0, 0.5, 1, 2, 3, 3.5, 4, 6, 10, 15, and 20%, w/v) for 1 week. The tolerance range for pH was determined (pH 4, 5, 6, 6.5, 7, 7.5, 8, 9, and 10) in MB with the pH adjusted by 1 N HCl or 1 N NaOH solutions and using 10 mM MES (pH 4–6), 10 mM HEPES (pH 6–8) or 10 mM AMPSO (pH 8–10) as biological buffers for 1 week. The tolerance ranges for NaCl and pH were also determined in a temperature gradient incubator and the OD₆₀₀ was monitor at 10 min intervals. The bacterial suspension used to inoculate into API 20E, 20NE, API ZYM kit (bioMérieux) and a Microlog GN2 system (Biolog) was prepared in a 2% sea salt (Sigma) solution. API and Microlog panels were recorded after incubation at 25°C for 2 days. To confirm anaerobic growth, cells were inoculated with 0.3% sodium nitrate and 0.5% yeast extract in the serum vial capped with aluminum seal, purged with deoxygenated nitrogen gas until color of indicator (final 0.2 mg/L of resazurin) was disappeared, and cultivated at 25°C for 5 days and observing growth.

Chemotaxonomic analysis

The cellular fatty acid profiles of strain MEBiC01930^T and reference strains were determined using the MIDI/Hewlett Packard Microbial Identification System (MIS) (Sasser, 1990) with Sherlock version 6.2 and the TSBA6 database according to the manufacturer’s instructions at KCCM (Korea Culture Center of Microorganisms). Briefly, strains were streaked on marine agar to produce four segments, and the third segment was sampled when growth was observed at the fourth segment. Then, the samples were saponified, methylated, extracted, purified, and analyzed by gas chromatograph. Polar lipids were extracted using a chloroform/methanol system and separated by two-dimensional TLC using silica gel 60 F₂₅₄ aluminum-backed thin-layer plates (Merck) (Minnikin et al., 1984). The detailed procedure is described in Yang et al. (2013). After 2-dimensional development, each component was visualized using the following reagents; all lipids -10 % (w/v) molybdatophosphoric acid, free amino groups -0.2% ninhydrin solution (Consden and Gordon, 1948), phosphorus - Zinzadze reagent (Dittmer and Lester, 1964), sugar groups - α- naphthol reagent (Jacin and Mishkin, 1965), and phosphatidylcholine (PC) - Dragendorff’s reagent. The major respiratory quinone in strain MEBiC01930^T was determined by HPLC analysis according to the method described by Collins (1985).

Nucleotide sequence accession numbers

The GenBank/DDBJ/EMBL accession number for the 16S rRNA gene sequence of strain MEBiC01930^T is DQ191181 and that of the genome sequence is CP104270.

Isolation and 16S rRNA gene sequence phylogeny of strain MEBiC01930^T

After purification of the strain using standard agar plating method, a yellow-coloured, circular, convex, and opaque colony that was butyrous and entire edges was selected and designated as strain MEBiC01930^T (=DOKDO020^T). Strain MEBiC01930^T was found to be closely related to Seonamhaeicola maritimus 1505^T and S. algicola Gy8^T with 97.84% and 97.71% 16S rRNA gene sequence similarity, respectively, and all other type species showing lower than 97% similarity. The 16S rRNA gene sequence similarity values were lower than the cut-off value (98.65%) of the novel species suggested by Kim et al. (2014). The NJ tree revealed that strain MEBiC01930^T formed a coherent clade with members of the genus Seonamhaeicola but phylogenetic position pf species within the genus was not stable (Fig. 1). This result implied that strain MEBiC01930^T could be a separate species in the genus Seonamhaeicola but genome based phylogenetic analysis was required for determining exact position.

Whole genome analysis of strain MEBiC01930^T

The genome of strain MEBiC01930^T was assembled into 1 contig with a size of 3.65 Mbp (3652651 bp). Calculated coverage was 4437×, and considering the self-mapping coverage and 16S rRNA gene sequences, genome was not contaminated by materials from other organisms. Briefly, the genome contains 3,102 protein-coding genes (CDSs) in addition to the 2 set of rRNAs, 49 tRNAs, 4 ncRNAs and 9 pseudogenes. The DNA G + C content is estimated to be 32.4 mol% (Table 1). Details on the genome contents can be found in Oh et al. (2024) and they reported that the strain possesses a set of alginate lyases such as guluronate-specific alginate lyases, mannuronate-specific alginate lyases, and an oligo-alginate lyase, but was not agarose, carragenase and cellulae. The 2 set of 16S rRNA genes encoded in the genome were identical but shows 1 bp difference against that by Sanger method. One or two copies of rRNA gene cluster are common in the closely related strains (Table 1). The copy number of the rRNA operon has been thought to be related to growth rate but no direct relationship was shown in nature. However, it can be related to the availability of limited nutrients (Valdivia-Anistro et al., 2016).

The phylogenomic tree inferred by PhyloPhlan showed that strain MEBiC01930^T was well-clustered with the members of the genus Seonamhaeicola and this result demonstrated that strain MEBiC01930^T is a member of the genus Seonamhaeicola (Fig. 2). The ANI used only for species delineation but AAI values widely used not only species but genus delineation (Park et al., 2022). The ANI values calculated were lower than 86.61% and demonstrated that strain MEBiC01930^T is a novel species (Table 2). In case of the AAI values, higher than 76.44% within the genus Seonamhaeicola. Yang et al. (2024) reported that AAI value for genus delineation at adjacent groups of the family Flavobacteriaceae ranged between 76.0–78.7% and the above value (76.44%) is in this range.

Table 2

ANI and AAI values among 15 species in the family Flavobacteriaceae closely related with the genus Seonamhaeicola.

Species numbers are as designated in the Table 1. Species numbers affiliated with the same genus designated with the same color and species reported as the same genus but showed discrepancy in ANI and/or AAI values are indicated in red color numbering column or raw. The ANI/AAI values were presented in a gradient from blue (low) to red (high).

ANI	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
AAI
1		76.44	79.09	77.25	81.17	79.30	74.72	72.95	73.29	73.58	72.91	72.78	74.04	73.34	72.88
2	76.60		77.04	79.57	77.45	76.97	73.35	72.33	72.62	73.50	72.10	72.42	72.91	72.61	72.69
3	77.64	76.53		77.03	80.14	89.16	74.62	74.66	74.52	75.2	74.32	74.88	73.8	74.38	72.23
4	76.97	78.5	76.23		77.71	76.79	73.17	71.86	72.02	72.77	71.96	72.38	71.79	71.69	70.39
5	80.11	76.70	78.27	76.64		80.46	75.37	73.43	73.54	74.04	73.16	72.95	73.29	73.59	71.59
6	77.93	76.74	86.61	76.52	78.45		74.82	74.64	74.72	75.14	74.47	75.03	74.11	74.28	72.28
7	75.10	74.45	74.49	74.28	75.63	74.67		74.77	75.17	75.43	74.34	74.34	74.80	75.44	72.34
8	73.22	72.42	73.55	72.4	73.41	73.73	73.32		79.01	78.75	77.68	77.95	77.34	73.19	70.9
9	74.25	73.60	74.23	73.62	74.18	74.48	74.65	75.44		83.14	82.95	82.40	81.50	73.42	71.58
10	74.28	74.65	74.74	73.87	74.7	74.98	75.08	75.32	78.73		81.01	81.18	82.78	73.91	72.17
11	74.68	74.68	74.97	74.26	74.69	75.08	74.84	75.02	79.66	78.16		88.66	79.82	72.77	70.63
12	74.05	74.20	74.93	73.7	74.39	75.05	74.44	75.06	79.20	77.88	86.19		80.02	72.65	70.71
13	74.67	75.19	74.19	74.13	74.5	74.55	74.82	74.82	78.19	79.44	77.96	77.73		73.32	72.26
14	73.73	74.02	74.04	73.25	73.87	74.02	74.18	72.91	73.61	73.61	73.58	73.27	74.06		71.75
15	74.48	75.44	73.94	73.83	74.3	73.81	74.35	72.19	73.80	73.75	73.95	73.57	74.46	73.92

Fig. 2. Phylogenomic tree inferred by PhyloPhlan showing relationship between strain MEBiC01930^T and the members of the genus Seonamhaeicola.

Phenotypic, physiological and biochemical analysis of strain MEBiC01930^T

Cell images obtain by TEM and SEM were rod shaped (0.3–0.5 × 1.6–2.1 mm) without flagella (Fig. 3), and this result is well matched with previously reported species of the genus Seonamhaeicola (Zhang et al., 2020). Staining the cell wall of strain was resulted as Gram-negative. Growth observed at temperature between 20.0 and 32.2°C (optimum 26–29°C), pH between 6.5 and 8.5 (optimum pH 7.5) and in the presence of 0–4% (w/v, optimum 2%) NaCl. No growth was observed at 18°C and 35°C, with 5% NaCl, and at pH 6.0 and 9.0 (Table 3). Anaerobic growth was observed by reducing nitrate to nitrite. Enzyme activities and utilization of carbon and energy sources, as measured using commercial kits, are summarized in Table 3 and in the description of the species and it can be briefly summarized as follows; strain MEBiC01930^T was able to utilize a variety of substrates such as carbohydrates, short chain organic acids, and amino acids. Compared with the previously reported species, basic physiological properties were similar. However, cell size of strain MEBiC01930^T was smaller than others and growth ranges for temperature, NaCl, and pH revealed to be lower than previously reported species (Table 3).

Table 3

Differential phenotypic characteristics of strain MEBiC01930^T and the species of the genus Seonamhaeicola

Strains: 1, MEBiC01930T (this study); 2, S. marinus NBRC 112333^T (data from this study and Fang et al. (2017)); 3, S. maritims KCTC 72528^T (data from this study and Cao et al. (2020)); 4, S. acroporae KCTC 62713^T (data from this study and Yoon et al. (2019)); 5, S. sediminis KCTC 72085^T (data from this study and Zhang et al. (2020)); 6, S. aphaedonesis KCTC 32578^T (data from this study and Park et al. [2014]); 7, S. algicola KCTC 42396^T (data from this study and Zhou et al. [2016]).; +, Positive reaction; -, negative reaction; w, weakly positive; nd, not determinted. FAn; facultative anaerobic, SA; Strictly aerobic, PE; phosphatidyl ethanolamine, PL; phospholipid, AL; aminolipid, APL; aminophospholipid, AGL; aminoglycolipid, L; polar lipid. All strains were positive for enzyme activity of Alkaline phosphatase and negative for Lipase (C₁₄), phosphohydrolase α-galactosidase, β-glucuronidase, α- mannosidase, and α-fucosidase.

Characteristic	1	2	3	4	5	6	7
Cell size (μm)	0.3–0.5 × 1.6–2.1	0.5–0.7 × 2.0–8.0a	0.5–0.7 × 2.0–11.0b	0.3–0.4c	0.3–0.8 × 1.0–4.0d	0.2–0.7 × 0.9–8.5e	0.3–0.5 ×4.0–12.0f
Respiration mode	FAn	FAna	FAnb	SAc	ndd	nde	FAnf
Growth range of:
Temperature (°C)†	20–32 (26–29)	10–40 (28)a	10–35(28)b	10–40 (37)c	15–37 (28)d	10–37 (25)e	10–40 (33–37)f
pH†	6.5–8.5(7.5)	6–8.5(6.5–7.5)a	6.5–9 (7.5)b	6.5–8.5 (7)c	6.0–8.5(7.5)d	6–8 (7)e	6–8 (7–7.5)f
NaCl (%, w/v)†	0–4 (2)	1–5 (3)a	1–4 (2)b	1–5 (3)c	1–5 (3)d	1–5 (2)e	0–7 (2.2)f
Nitrate reduction	+	+ a	+ b	– c	+ d	– e	– f
Motility	–	– a	– b	– c	+ d	– e	+ f
Hydrolysis of :
Agar	–	+ a	– b	– c	– d	– e	+ e
DNase	–	+ a	– b	+ c	+ d	– e	– e
Gelatin	–	+ a	nd b	– c	nd d	– e	+ e
Strach	–	– a	+ b	– c	– d	+ e	+ e
Tween 80	–	– a	– b	– c	– d	+ e	– e
Acid production from
esculin	+	+	–	+	–	–	–
sucrose	+	+	w	–	–	+	–
5-keto-gluconate	+	+	+	+	+	–	–
Glycerol	–	–	+	–	–	–	–
D-arabinose	–	w	–	–	–	–	+
D-xylose	–	–	w	–	+	+	+
N-acethyl-glucosamine	–	–	–	–	+	+	–
cellobiose	–	–	–	–	+	–	+
Enzyme activities
Catalase, Leucine arylamidase	+	–	+	+	+	+	+
Alkaline phosphatase	+	+	+	+	+	+	–
Esterase (C4)	+	–	+	+	–	–	+
Esterase Lipase (C8)	+	w	+	–	–	–	+
Valine arylamidase	–	–	+	+	+	–	+
Trypsin	–	+	–	–	–	–	+
Major polar lipids	PE, 3L, 2AL	PE, PL, AL, APL 5L a	PE, 6AL, 4L b	PE, 2AL, 2L c	PE, AL, AGL, 2L d	PE, PL, AL, 3L e	PE, PL, AL, AGL, GL, 2L f
DNA G + C content (mol%)	35.4	33.9 a	33.3 b	34.3 c	33.5 d	34.8 e	35.3 f

^† Optimum value in the parentheses.

* Data from: a, Fang et al. (2017); b, Cao et al. (2020); c, Yoon et al. (2019); d, Zhang et al. (2020); e, Park et al. (2014); f, Zhou et al. (2016).

Fig. 3. Transmission electron micrograph images of strain MEBiC01930^T (A) and scanning electron micrograph images of strain MEBiC01930^T (B) cultivated on MA medium at 25°C for 48 h.

The dominant fatty acids of strain MEBiC01930^T were determined to be iso-C_15:1 G (17.2%), iso-C_15:0 (14.8%), iso-C_15:0 3-OH (9.8%), and iso-C_17:0 3-OH (12.2%) and summed feature 3 (C_16:1 ω6c and/or C_16:1 ω7c; 4.0%) (Table 4) and were not quite different from other species in the genus Seonamhaeicola (Table 4). The predominant polar lipids of strain MEBiC01930^T were phosphatidylethanolamine (PE), two unidentified aminolipids (ALs), and additional three unidentified lipids (Ls) (Fig. 4). PE as the major polar lipid is consistent with previously reported results for the genus Seonamhaeicola. The major respiratory quinone of strain MEBiC01930^T was determined to be MK-6 by HPLC analysis according to Collins (1985). The DNA G + C content deduced from genome sequence was 32.4 mol%.

Table 4

Fatty acids compositions (%) of strain MEBiC01930^T and the species of the genus Seonamhaeicola

Taxa: 1, MEBiC1930^T; 2, S. marinus NBRC 112333^T; 3, S. maritims KCTC 72528^T; 4, S. acroporae KCTC 62713^T; 5, S. sediminis KCTC 72085^T; 6, S. aphaedonesis KCTC 32578^T; 7, S. algicola KCTC 42396^T. Characters are scored as: -, not detected: tr, Trace amount (<1%); Lower than 1% in all strains are not recorded; Bold, >10%. All data were obtained in the present study.

Fatty acid	1	2	3	4	5	6	7
Saturated
C10:0	3.0	1.0	1.4	-	tr	-	-
Iso-C14:0	1.1	tr	1.1	tr	tr	tr	tr
C12:0	5.2	2.3	3.3	-	1.0	tr	-
Iso-C14:0	-	1.1	tr	tr	tr	1.8	2.1
C14:0	tr	1.1	tr	tr	tr	tr	tr
iso-C15:1 G	17.2	13.6	18.6	24.5	21.3	19.3	12.0
anteios-C15:1	tr	tr	tr	1.7	4.5	2.7	tr
iso-C15:0	14.8	20.2	18.2	18.1	10.4	17.1	21.0
Anteiso-C15:0	4.6	1.8	2.9	5.5	8.6	9.0	5.2
Iso-C16:0 anteios	tr	1.4	tr	tr	1.0	1.6	5.0
C16:0	3.4	7.8	3.1	3.3	4.9	3.2	3.6
C17:0	-	tr	-	-	1.4	-	-
Unsaturated
C15:1 ω5c	2.1	1.8	1.7	3.0	1.6	3.7	2.9
C15:1 ω6c	tr	tr	tr	-	tr	-	1.1
C15:1 ω8c	-	-	-	4.4	3.1	-	-
C17:1 ω6c	tr	tr	tr	-	tr	-	1.3
C18:1 ω9c	1.0	tr	tr	tr	-	tr	tr
Hydroxy
C10:0 3-OH	3.7	tr	1.1	-	-	-	-
iso-C15:0 3-OH	9.8	4.8	12.1	9.7	6.7	7.5	4.8
C15:0 2-OH	-	1.7	2.2	1.5	4.3	2.8	1.6
iso-C16:0 3-OH	1.4	4.4	2.4	2.7	1	6.5	11.0
C16:0 3-OH	tr	2.9	tr	tr	-	-	1.0
iso-C17:0 3-OH	12.2	14.3	13.2	11.0	4.7	8.6	11.0
C17:0 2-OH	3.0	1.3	1.8	2.6	2.9	3.4	2.8
C17:0 3-OH	tr	1.6	tr	tr	-	tr	1.0
Summed feature 3	4.0	5.5	6.1	4.3	8.8	8.2	5
Summed feature 9	1.4	tr	tr	-	-	-	tr

* Summed Features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 3 contained one or more of the following fatty acids: C_16:1ω7c and/or C_16:1ω6c and Summed feature 9 contained one or more of the following fatty acids: C_18:1ω7c and/or C_18:1ω6c.

Fig. 4. Two-dimensional TLC after staining with molybdatophosphoric acid showing the total polar lipid profiles of strain MEBiC01930^T cultivated on MA medium at 25°C for 48 h.

Taxonomic Conclusion

Strain MEBiC01930^T could be classified into the genus Seonamhaeicola on the basis of the phylogenetic and phylogenomic trees (Figs. 1 and 2), fatty acids profile (Table 4), types of respiratory quinone, DNA G + C ratio, reduction of nitrate, enzyme activities determined by API ZYM kit (Table 3) etc. However, the isolate could be distinguished from other type strains by the existence of some short-chain fatty acids (Table 4), range and optimum of pH and NaCl for growth (Table 3), and the assimilation of some carbon sources such as gluconate and malate (Table 3). Additionally, ANI and AAI values of strain MEBiC01930^T against members of the genus Seonamhaeicola were lower than the species delineation range (Kim et al., 2014) (Table 2). On the basis of this polyphasic taxonomical evidence, it was concluded that strain MEBiC01930^T is a novel species in the genus Seonamhaeicola and proposed it as a name Seonamhaeicola citrea sp. nov.

Description of Seonamhaeicola citrea sp. nov.

Seonamhaeicola citrea (ci. tre′a. N.L. fem. adj. citrea lemon-yellow coloured)

Gram-stain-negative, oxidase and catalase positive, facultatively anaerobic, non-motile, and rod-shaped cells are 0.3–0.5 × 1.6–2.1 μm in size. The ranges and optimal conditions of temperature, pH and NaCl for growth are 20.0–32°C (optimum, 26–29°C), pH 6.5–8.5 (optimum, pH 7.5) and 0–4% (w/v; optimum, 2%), respectively. Nitrate is reduced to nitrite. Urease, β-galactosidase and β-glucosidase activities are present. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities are not detected in API20E and 20NE kits. When assayed with the API ZYM system, alkaline phosphatase, esterase (C₄), esterase lipase (C₈), leucine arylamidase, acid phosphatase, naphtol-AS-BI phosphohydrolase, β-glucosidase, α-glucosidase and N-acetyl-β-glucosaminidase activities are present. Lipase (C₁₄), valine arylamidase, trypsin, α-chymotrypsin, cystine arylamidase, α-galactosidase, β-galactosidase, β-glucuronidase, α-mannosidase and α-fucosidase activities are absent. In the API 50 CHB/E test, acid is produced from esculin, sucrose and 5-keto-gluconate, but not from others. Major fatty acids (>5.0%) are C_12:0, iso-C_15:1 G, iso-C_15:0, iso-C_15:0 3-OH and iso-C_17:0 3-OH when grown at 25°C. The major respiratory quinone is MK-6. The polar lipids present are phosphatidylethanolamine, two unidentified aminolipids and three unidentified lipids. The DNA G + C content is 32.4 mol%.

The type strain MEBiC01930^T (=KCCM 42330^T =JCM 14619^T) was isolated from a debris of marine alga Eisenia bicyclis collected from Dokdo Island, Republic of Korea. The GenBank/DDBJ/EMBL accession number for the 16S rRNA gene is DQ191181 and that of the genome is CP104270.

그람 음성의 통성 혐기성의 이동성이 없는 간균 (1.6 × 2.1 μm – 0.3 × 0.5 μm) MEBiC01930^T (= DOKDO020^T)은 2004년 10월 우리나라 독도(131°52'00''E, 37°14'34''N)에서 채취된 대황 잔해에서 분리되었다. 16S rRNA 유전자 서열 분석 결과 MEBiC01930^T 균주는 Seonamhaeicola 속 균주들과 높은 유사성(최고 97.84%)을 보였다. MEBiC01930^T 균주는 섭씨 20–32도 (최적 26–29도), pH 6.5–8.5 (최적 pH 7.5) 및 0–4% (최적 2%) NaCl 농도 조건에서 성장이 관찰되었다. 주요 세포 지방산은 iso-C_15:1 G (17.2%), iso-C_15:0 (14.8%), iso-C_17:0 3-OH (12.2%), iso-C_15:0 3-OH (9.8%), C_12:0 (5.2%)였다. 균주의 유전체는 3.65 Mbp 크기에 3,170개의 유전자를 지니는 것으로 확인되었으며 DNA G + C 함량은 33.6 mol%이다. 유전체 정보 분석 결과 균주는 alginate lyase 종류를 여러 개 가지고 있음을 확인하였다. 주요 호흡기 퀴논은 MK-6이다. 이와 같은 다상분류 결과에 기초하여 균주 MEBiC01930^T는 Seonamhaeicola 속의 새로운 종으로 분류되어야 함을 제안하며 이를 Seonamhaeicola citrea sp. nov.로 제안하였다. 표준균주는 MEBiC01930^T (= KCCM 42330^T = JCM 14619^T)이다.

This work was supported by the KIOST in-house program (PEA0212) and the management of Marine Fishery Bioresources Center (2024) funded by the National Marine Biodiversity Institute of Korea (MABIK).

The authors declare that they have no conflict of interests.