IJSEM Papers in Press. Published May 15, 2015 as doi:10.1099/ijs.0.000334
International Journal of Systematic and Evolutionary Microbiology Winogradskyella eckloniae sp. nov., a marine bacterium isolated from a brown alga Ecklonia cava --Manuscript Draft-Manuscript Number:
Winogradskyella eckloniae sp. nov., a marine bacterium isolated from a brown alga Ecklonia cava
Winogradskyella eckloniae sp. nov., a marine bacterium isolated from a brown alga Ecklonia cava
New taxa - Bacteroidetes
Duck-Chul Oh, Ph.D Jeju National Universtiy Jeju, KOREA, REPUBLIC OF
Order of Authors:
Ji-Young Kim So-Hyun Park Ga-Young Seo Young-Ju Kim Duck-Chul Oh, Ph.D
Manuscript Region of Origin:
KOREA, REPUBLIC OF
A novel bacterial strain, designated EC29T, was isolated from the brown alga Ecklonia cava collected on Jeju Island, Republic of Korea. Cells of strain EC29T were Gramnegative, aerobic, rod-shaped and motile by gliding. Growth was observed at 10-30 °C (optimum, 20-25°C), at pH 6.0-9.5 (optimum, pH 7.5) and in the presence of 1-5 % (w/v) NaCl. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain belonged to the genus Winogradskyella. Strain EC29T exhibited the highest 16S rRNA gene sequence similarities of 96.5-97.8 % to the type strains of Winogradskyella pulchriflava EM106T, Winogradskyella echinorum KMM 6211T and Winogradskyella ulvae KMM6390 T. Strain EC29T exhibited < 27% DNA-DNA relatedness with Winogradskyella pulchriflava EM106T and Winogradskyella echinorum KMM 6211T. The predominant fatty acids of strain EC29T were iso-C15:0, iso-C15:1 G, C15:0, iso-C17:0 3-OH, iso-C15:0 3-OH and anteiso C15:0. The DNA G+C content was 31.1 mol% and the major respiratory quinone was menaquinone-6 (MK-6). Based on a polyphasic study, strain EC29T is considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella eckloniae sp. nov. is proposed. The type strain is EC29T (=KCTC 32172 T = JCM 18703 T).
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Winogradskyella eckloniae sp. nov., a marine bacterium isolated from
a brown alga Ecklonia cava
Ji-Young Kim, 1, 2 So-Hyun Park,3 Ga-Young Seo,1 Young-Ju Kim,2* and Duck-Chul Oh 1*
Department of Biology, Jeju National University, Jeju 690-756, Republic of Korea
Jeju Biological Resource Co., Ltd, CTC Business Incubator, Jeju Tourism College, Jeju
690-756, Republic of Korea
Department of Aquatic Life Medicine, College of Ocean Science, Jeju 690-756, Republic of
Author for correspondence: Duck-Chul Oh; E-mail: [email protected]
; Tel : +82-64-754-
3524; Fax : +82-64-756-3541. Young-Ju Kim; E-mail: [email protected]
Running title: Winogradskyella eckloniae sp. nov.
Subject category: New taxa (Bacteroidetes)
The GenBank accession number for the 16S rRNA gene sequence of strain EC29T (=KCTC
32172 T = JCM 18703 T) is JF820845.
A novel bacterial strain, designated EC29T, was isolated from the brown alga Ecklonia
cava collected on Jeju Island, Republic of Korea. Cells of strain EC29T were Gram-negative,
aerobic, rod-shaped and motile by gliding. Growth was observed at 10-30 °C (optimum, 20-
25°C), at pH 6.0-9.5 (optimum, pH 7.5) and in the presence of 1-5 % (w/v) NaCl.
Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain
belonged to the genus Winogradskyella. Strain EC29T exhibited the highest 16S rRNA gene
sequence similarities of 96.5-97.8 % to the type strains of Winogradskyella pulchriflava
EM106T, Winogradskyella echinorum KMM 6211T and Winogradskyella ulvae KMM6390 T.
Strain EC29T exhibited < 27% DNA-DNA relatedness with Winogradskyella pulchriflava
EM106T and Winogradskyella echinorum KMM 6211T. The predominant fatty acids of strain
EC29T were iso-C15:0, iso-C15:1 G, C15:0, iso-C17:0 3-OH, iso-C15:0 3-OH and anteiso C15:0. The
DNA G+C content was 31.1 mol% and the major respiratory quinone was menaquinone-6
(MK-6). Based on a polyphasic study, strain EC29T is considered to represent a novel species
of the genus Winogradskyella, for which the name Winogradskyella eckloniae sp. nov. is
proposed. The type strain is EC29T (=KCTC 32172 T = JCM 18703 T).
The genus Winogradskyella was first reported by Nedashkovskaya et al. (2005) and belongs
to the family Flavobacteriaceae (Bernardet et al., 2002; Bernardet, 2011) within the phylum
Bacteroidetes. At the time of writing, the genus comprised 20 species with validly published
names (http://www.bacterio.cict.fr/). Members of the genus Winogradskyella have been
isolated from various marine environments, such as marine animals (Lau et al., 2005;
Nedashkovskaya et al., 2009; Ivanova et al., 2010), marine sediments (Romanenko et al.,
2009; Begum et al., 2013; Kang et al., 2013; Kim et al., 2013; Park et al., 2014), seawater
(Pinhassi et al., 2009; Kim & Nedashkovskaya, 2010; Yoon et al., 2011; Yoon & Lee, 2012;
Lee et al., 2012, 2013) and seaweeds (Nedashkovskaya et al., 2005; Nedashkovskaya et al.,
2012; Park & Yoon, 2013).
During our investigation of the biodiversity of marine bacteria from seaweed, strain EC29T
was isolated from brown alga and its taxonomic position was investigated using a polyphasic
approach. The brown alga sample of Ecklonia cava was collected from the coast of Jeju
Island, Republic of Korea. The seaweed sample was homogenized with sterile seawater, and
the homogenate was spread on marine agar 2216 (MA; Difco) using a dilution-plating
technique. After incubation for 7 days at 25 °C, the purified colonies were maintained in a
glycerol solution (20 %, w/v) at -70 °C. For phenotypic comparison and DNA-DNA
hydridization experiments, Winogradskyella pulchriflava KCTC 23858T, Winogradskyella
echinorum KCTC 22026T and Winogradskyella ulvae KCTC 23626T were used as reference
strains in parallel tests.
The 16S rRNA gene was amplified from genomic DNA by PCR using the universal
primers of bacterial 27F and 1522R (Weisburg et al., 1991). The resultant products were
cloned using the TOPO cloning kit (Invitrogen) and the sequencing of the cloned 16S rRNA
gene was subject to a service by Genotech (Korea). A full sequence of the 16S rRNA gene
was generated using SeqMan software (DNASTAR) and compared with the corresponding
e.ezbioclould.net/; Kim et al., 2012). Multiple alignments were performed using CLUSTAL
X version 1.83 program (Thompson et al., 1997) and gaps (at 5`- and 3`ends) were excluded
by using the BioEdit program (Hall, 1999). Phylogenetic trees were reconstructed using
MEGA program (version 6.0; Tamura et al., 2013) including the neighbor-joining (Saitou &
Nei, 1987), maximum likelihood (Felsenstein, 1981) and maximum parsimony (Kluge and
Farris, 1969) algorithms. The reliability of tree topology was evaluated by bootstrap analysis
(Felsenstein, 1985) based on 1,000 replicates. DNA–DNA hybridization was tested to
determine the relationship between strains EC29T and the type strain W. pulchriflava KCTC
23858T and W. echinorum KCTC 22026T according to the measured photobiotin-labelled
DNA probes using the microplate hybridization method (Ezaki et al., 1989; Christensen et al.,
The cell morphology of strain EC29T was observed by light microscopy (ECLIPS 80I; Nikon)
and scanning electron microscopy (SIE-3000M; SEC), with cells grown on MA for 3 days at
25 °C. Cell motility was investigated by the development of turbidity throughout a tube of
semi-solid media (Bowman, 2000). The Gram reaction was determined using the Gram stain
kit (BD) according to the manufacturer’s instructions. The temperature (4, 10, 15, 20, 25, 30,
35, 37, 40, and 45 °C) and pH (4.0 - 10.0, at intervals of 0.5 pH unit) range for growth were
examined on MA. The requirement and tolerance of various NaCl concentrations were
adjusted to 0-10 % (w/v, at interval of 1.0 %) using Zobell's marine agar medium (Zobell,
1941; 5.0 g Bacto peptone, 1.0 g yeast extract, 0.1 g ferric citrate, 5.94 g MgSO4·7H2O, 4.53g
MgCl2·6H2O, 0.64g KCl, 1.3g CaCl2 and 15.0 g Bacto agar in 1 liter distilled water, pH 7.0).
Anaerobic growth was investigated after incubation for 1 month on MA in the AnaeroPack
Hydrolysis of casein, carboxymethylcellulose, starch and Tween 20, 40, 60, and 80 were
carried out using MA, supplemented with each substance at a concentration as described
previously (Smibert & Krieg, 1994). Hydrolysis of DNA was tested using DNase test agar
(Difco) prepared with artificial seawater. Catalase and oxidase activities were examined in a
3 % solution (v/v) of hydrogen peroxide and 1 % (w/v) tetramethyl p-phenylenediamine,
respectively. Other physiological and biochemical properties were determined with the API
20E, API 20NE and API ZYM (bioMérieux) according to the manufacturer`s instructions.
Susceptibility to antibiotic was investigated using filter-paper discs (BBL) as described by
Kim & Oh (2012).
For the analysis of cellular fatty acids, the cell mass of strain EC29T and reference strains
were harvested on MA after incubation for 3 days at 25 °C. The identification of fatty acid
methyl esters was performed using the Sherlock Microbial Identification System (MIDI;
version 6.1) and the TSBA6 database (Sasser, 1990). The DNA G+C content of strain EC29T
was determined by extracting genomic DNA according to the methods described by Marmur
(1961) and the thermal denaturation method (Tm) with Escherichia coli K-12 as a reference
strain (Marmur & Doty, 1962). Analysis of the respiratory quinones was performed using a
service of the Korean Culture Center of Microorganisms (KCCM). Polar lipids were
separated by two-dimensional thin-layer chromatography (TLC) and analyzed according to
the methods described by Minnikin et al. (1984). For the detection of polar lipids,
molybdophosphoric acid, ninhydrin, Zinzade and α-naphthol reagents were applied and
through co-migration with authentic standards (Minnikin et al., 1984; Komagata and Suzuki,
The nearly complete 16S rRNA gene sequence of strain EC29T was a continuous stretch of
1,442 bp and was GenBank accession number JF820845. Phylogenetic analysis established
that strain EC29T belonged to the genus Winogradskyella, forming a well separated branch
with type strains of W. pulchriflava EM106T (97.8 %), W. echinorum KMM 6211T (97.5 %),
W. ulvae KMM 6390T (96.5 %), W. eximia KMM 3994T (96.2%) and W. lutea A73T (96.1 %)
in the neighbor-joining phylogenetic tree (Fig. 1). Sequence similarities of < 93.8 % was
observed with the other species of the genus Winogradskyella. Although the bootstrap value
for the node between the isolate and closely related type strains was relatively low (56 %),
the topologies of the phylogenetic trees built using the maximum-likelihood and maximum-
parsimony algorithms supported the finding that strain EC29T formed lineage separated from
members of the genus Winogradskyella (Fig S1). The levels of DNA–DNA relatedness
between strain EC29T and the type strains W. pulchriflava KCTC 23858T and W. echinorum
KCTC 22026T was 26.8 ± 0.7 and 24.9 ± 1.3 %, respectively. These values confirmed the
assignment of the novel isolate EC29T to a separate species according to the cut-off value of
70 % proposed Wayne et al. (1987) for the definition of members of a species.
Cells of strain EC29T are Gram-negative, aerobic, motile by gliding and rod-shaped, able to
grow at temperatures of 10-30 °C (optimum 25 °C), at pH 6.0-9.5 (optimum pH 7.5) and in
the presence of 1-5 % NaCl. Colonies are circular, have a rough surface and are yellow-
colored when grown on MA at 25 °C. Other phenotypic and biochemical characteristics of
the strain EC29T are presented in Table 1 and the related Winogradskyella species description.
The predominant cellular fatty acids of the strain EC29T (> 5 % of the total fatty acids) were
iso-C15:0 (24.9 %), iso-C15:1 G (23.5 %), C15:0 (9.1 %), iso-C17:0 3-OH (8.8 %), iso-C15:0 3-OH
(7.1 %), and anteiso C15:0 (5.6 %). This fatty acid profile was similar to those of closely related
members within the genus Winogradskyella, with difference in the respective proportions of
some fatty acids (Table 2). The predominant respiratory quinone found in the strain EC29T
was menaquinone-6 (MK-6), which is a characteristic quinone system of the family
Flavobacteriaceae (Bernardet and Nakagawa, 2006).
was 31.1 mol%, this supports their affiliation with the genus Wingradskyella, members of
which have DNA G+C content between 30.1 and 36.4 mol%. The polar lipid profiles of strain
EC29T were phosphatidylethanolamine (PE), unknown aminolipids (AL1-AL2), and
unknown lipids (L1-L5) (Fig. S2). The presence of phosphatidylethanolamine and aminolipid
was consistent with results of analyses of the genus Winogradskyella (Nedashkovskaya et al.,
2012; Kim et al., 2012; Kim et al., 2013; Park et al., 2014).
The G+C content of the strain EC29T
The genetic distinctiveness and differential phenotypic characteristics of strain EC29T are
sufficient to show that this strain is separate from species of the genus Winogradskyella. Based on
data from the present polyphasic taxonomic study, we considered that strain EC29T represent a
novel species of the genus Winogradskyella, for which the name Winogradskyella eckloniae sp.
nov. is proposed.
Description of Winogradskyella eckloniae sp. nov.
Winogradskyella eckloniae (eck.lo.ni`ae. N.L. fem. n. Ecklonia scientific genus name of a
marine alga; N.L. gen. n. eckloniae of Ecklonia, referring to the isolation of the type strain
from Ecklonia kurome).
Cells are Gram-negative, aerobic, motile by gliding and rod-shaped (0.2–0.3 µm wide and
0.7–1.2 µm long). After incubation for 3 days at 25 °C, colonies on MA are circular to rough
surface, yellow-colored and 1-1.5 mm in diameter. Growth is observed at 10-30 °C (optimum,
20-25 °C) and pH 6.0-9.5 (optimum, pH 7.5). Growth occurs in the presence of 1-5 % NaCl,
but not below 1 % or above 5 % NaCl. Anaerobic growth is not observed after 4 weeks at
25 °C on MA. Catalase- and oxidase- positive. Hydrolyses aesculin, gelatin, starch and Tween
40, but does not hydrolyse casein, cellulose, Tweens 20, 60, and 80. Nitrate is not reduced.
Acetoin, indole and H2S are not produced. In the API ZYM system, positive for alkaline
phosphatase, esterase (C4), esterase lipase (C8), cystine-, valine-, and leucine arylamidases,
α-chymotrypsin, trypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, and
negative for lipase (C14), α–galactosidase, β–galactosidase, α-glucosidase, β-glucosidase, β-
glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Susceptible to
ampicillin (1µg), cephalothin (30µg), chloramphenicol (30µg), erythromycin (15µg),
licomycin (2µg), novobiocin (30µg), penicillin G (10IU), and tetracycline (30µg), but
resistant to gentamycin (10µg), kanamycin (30µg), neomycin (30µg), polymyxin B (300IU),
and streptomycin (10µg). The predominant cellular fatty acids are iso-C15:0, iso-C15:1 G, C15:0,
iso-C17:0 3-OH, iso-C15:0 3-OH and anteiso C15:0. The major respiratory quinone is
menaquinone-6. The major polar lipids are phosphatidylethanolamine, two unknown
aminolipids, and five unknown lipids. The DNA G+C content of the type strain is 31.1 mol%.
The type strain is EC29T (= KCTC 23835T = JCM 18703T), which was isolated from brown
alga Ecklonia cava collected along the coast of Jeju Island, Republic of Korea.
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Table 1. Differential phenotypic characteristics of strain EC29T and the closely related
type strains of genus Winogradskyella
Strains: 1, W. eckloniae EC29T; 2, W. pulchriflava KCTC 23858 T; 3, W. echinorum KCTC
22026T; 4. W. ulvae KCTC 23626 T. All data are from this study. +, Positive; w, weakly
positive; -, negative. All strains are positive for the following: catalase and oxidase activities;
hydrolysis of aesculin and gelatin; and activities of acid phosphatase, alkaline phosphatase,
esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, and cystein
arylamidase; susceptibility of cephalothin, chloramphenicol, erythromycin, lincomycin and
novobiosin. All strains are negative for the following: hydrolysis of cellulose, urea and
Tween 20; activities of lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase, α-
glucosidase, β-glucosidase, N-acetyl- β-glucosaminidase, α-mannosidase and α-fucosidase;
susceptibility of gentamicin, neomycin, polymyxin B and streptomycin. Characteristic
NaCl (%) Fermentation of Glucose Hydrolysis of:
Enzyme activities (API ZYM): Trypsin
DNA G+C content (mol%)* 358
* DNA G+C content data from: a, Kim et al.,(2013); b, Nedashkovskaya et al., (2009); c,
Nedashkovskaya et al., (2012).
† Different results were reported by Kim et al., (2013) and Nedashkovskaya et al., (2009).
Table 2. Cellular fatty acid profiles of strain EC29T and the closely related type strains
of genus Winogradskyella
Strains: 1, W. eckloniae EC29T; 2, W. pulchriflava KCTC 23858 T; 3, W. echinorum KCTC
22026T; 4, W. ulvae KCTC 23626 T. All data were obtained in this study from cultures on MA
at 25°C for 3 days. Fatty acids founds in amounts