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:
IJSEM-D-14-00411R1
Full Title:
Winogradskyella eckloniae sp. nov., a marine bacterium isolated from a brown alga Ecklonia cava
Short Title:
Winogradskyella eckloniae sp. nov., a marine bacterium isolated from a brown alga Ecklonia cava
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Duck-Chul Oh, Ph.D Jeju National Universtiy Jeju, KOREA, REPUBLIC OF
First Author:
Ji-Young Kim
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
Abstract:
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
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a brown alga Ecklonia cava
3 4
Ji-Young Kim, 1, 2 So-Hyun Park,3 Ga-Young Seo,1 Young-Ju Kim,2* and Duck-Chul Oh 1*
5 6
1
Department of Biology, Jeju National University, Jeju 690-756, Republic of Korea
7
2
Jeju Biological Resource Co., Ltd, CTC Business Incubator, Jeju Tourism College, Jeju
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690-756, Republic of Korea
9
3
10
Department of Aquatic Life Medicine, College of Ocean Science, Jeju 690-756, Republic of
Korea
11 12
Author for correspondence: Duck-Chul Oh; E-mail:
[email protected]; Tel : +82-64-754-
13
3524; Fax : +82-64-756-3541. Young-Ju Kim; E-mail: jym1006@ jejunu.ac.kr
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Running title: Winogradskyella eckloniae sp. nov.
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Subject category: New taxa (Bacteroidetes)
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The GenBank accession number for the 16S rRNA gene sequence of strain EC29T (=KCTC
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32172 T = JCM 18703 T) is JF820845.
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Abstract
22 23
A novel bacterial strain, designated EC29T, was isolated from the brown alga Ecklonia
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cava collected on Jeju Island, Republic of Korea. Cells of strain EC29T were Gram-negative,
25
aerobic, rod-shaped and motile by gliding. Growth was observed at 10-30 °C (optimum, 20-
26
25°C), at pH 6.0-9.5 (optimum, pH 7.5) and in the presence of 1-5 % (w/v) NaCl.
27
Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain
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belonged to the genus Winogradskyella. Strain EC29T exhibited the highest 16S rRNA gene
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sequence similarities of 96.5-97.8 % to the type strains of Winogradskyella pulchriflava
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EM106T, Winogradskyella echinorum KMM 6211T and Winogradskyella ulvae KMM6390 T.
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Strain EC29T exhibited < 27% DNA-DNA relatedness with Winogradskyella pulchriflava
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EM106T and Winogradskyella echinorum KMM 6211T. The predominant fatty acids of strain
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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
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DNA G+C content was 31.1 mol% and the major respiratory quinone was menaquinone-6
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(MK-6). Based on a polyphasic study, strain EC29T is considered to represent a novel species
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of the genus Winogradskyella, for which the name Winogradskyella eckloniae sp. nov. is
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proposed. The type strain is EC29T (=KCTC 32172 T = JCM 18703 T).
38 39
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The genus Winogradskyella was first reported by Nedashkovskaya et al. (2005) and belongs
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to the family Flavobacteriaceae (Bernardet et al., 2002; Bernardet, 2011) within the phylum
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Bacteroidetes. At the time of writing, the genus comprised 20 species with validly published
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names (http://www.bacterio.cict.fr/). Members of the genus Winogradskyella have been
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isolated from various marine environments, such as marine animals (Lau et al., 2005;
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Nedashkovskaya et al., 2009; Ivanova et al., 2010), marine sediments (Romanenko et al.,
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2009; Begum et al., 2013; Kang et al., 2013; Kim et al., 2013; Park et al., 2014), seawater
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(Pinhassi et al., 2009; Kim & Nedashkovskaya, 2010; Yoon et al., 2011; Yoon & Lee, 2012;
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Lee et al., 2012, 2013) and seaweeds (Nedashkovskaya et al., 2005; Nedashkovskaya et al.,
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2012; Park & Yoon, 2013).
50 51
During our investigation of the biodiversity of marine bacteria from seaweed, strain EC29T
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was isolated from brown alga and its taxonomic position was investigated using a polyphasic
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approach. The brown alga sample of Ecklonia cava was collected from the coast of Jeju
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Island, Republic of Korea. The seaweed sample was homogenized with sterile seawater, and
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the homogenate was spread on marine agar 2216 (MA; Difco) using a dilution-plating
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technique. After incubation for 7 days at 25 °C, the purified colonies were maintained in a
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glycerol solution (20 %, w/v) at -70 °C. For phenotypic comparison and DNA-DNA
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hydridization experiments, Winogradskyella pulchriflava KCTC 23858T, Winogradskyella
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echinorum KCTC 22026T and Winogradskyella ulvae KCTC 23626T were used as reference
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strains in parallel tests.
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The 16S rRNA gene was amplified from genomic DNA by PCR using the universal
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primers of bacterial 27F and 1522R (Weisburg et al., 1991). The resultant products were
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cloned using the TOPO cloning kit (Invitrogen) and the sequencing of the cloned 16S rRNA
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gene was subject to a service by Genotech (Korea). A full sequence of the 16S rRNA gene
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was generated using SeqMan software (DNASTAR) and compared with the corresponding
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sequences
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(http://blast.ncbi.nlm,nih.gov/Blast.cgi)
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e.ezbioclould.net/; Kim et al., 2012). Multiple alignments were performed using CLUSTAL
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X version 1.83 program (Thompson et al., 1997) and gaps (at 5`- and 3`ends) were excluded
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by using the BioEdit program (Hall, 1999). Phylogenetic trees were reconstructed using
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MEGA program (version 6.0; Tamura et al., 2013) including the neighbor-joining (Saitou &
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Nei, 1987), maximum likelihood (Felsenstein, 1981) and maximum parsimony (Kluge and
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Farris, 1969) algorithms. The reliability of tree topology was evaluated by bootstrap analysis
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(Felsenstein, 1985) based on 1,000 replicates. DNA–DNA hybridization was tested to
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determine the relationship between strains EC29T and the type strain W. pulchriflava KCTC
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23858T and W. echinorum KCTC 22026T according to the measured photobiotin-labelled
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DNA probes using the microplate hybridization method (Ezaki et al., 1989; Christensen et al.,
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2000).
of
related
taxa
obtained and
from EzTaxon-e
the
GenBank server
database
(http://eztaxon-
80 81
The cell morphology of strain EC29T was observed by light microscopy (ECLIPS 80I; Nikon)
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and scanning electron microscopy (SIE-3000M; SEC), with cells grown on MA for 3 days at
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25 °C. Cell motility was investigated by the development of turbidity throughout a tube of
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semi-solid media (Bowman, 2000). The Gram reaction was determined using the Gram stain
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kit (BD) according to the manufacturer’s instructions. The temperature (4, 10, 15, 20, 25, 30,
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35, 37, 40, and 45 °C) and pH (4.0 - 10.0, at intervals of 0.5 pH unit) range for growth were
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examined on MA. The requirement and tolerance of various NaCl concentrations were
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adjusted to 0-10 % (w/v, at interval of 1.0 %) using Zobell's marine agar medium (Zobell,
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1941; 5.0 g Bacto peptone, 1.0 g yeast extract, 0.1 g ferric citrate, 5.94 g MgSO4·7H2O, 4.53g
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MgCl2·6H2O, 0.64g KCl, 1.3g CaCl2 and 15.0 g Bacto agar in 1 liter distilled water, pH 7.0).
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Anaerobic growth was investigated after incubation for 1 month on MA in the AnaeroPack
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system (Oxoid).
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Hydrolysis of casein, carboxymethylcellulose, starch and Tween 20, 40, 60, and 80 were
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carried out using MA, supplemented with each substance at a concentration as described
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previously (Smibert & Krieg, 1994). Hydrolysis of DNA was tested using DNase test agar
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(Difco) prepared with artificial seawater. Catalase and oxidase activities were examined in a
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3 % solution (v/v) of hydrogen peroxide and 1 % (w/v) tetramethyl p-phenylenediamine,
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respectively. Other physiological and biochemical properties were determined with the API
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20E, API 20NE and API ZYM (bioMérieux) according to the manufacturer`s instructions.
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Susceptibility to antibiotic was investigated using filter-paper discs (BBL) as described by
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Kim & Oh (2012).
102 103
For the analysis of cellular fatty acids, the cell mass of strain EC29T and reference strains
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were harvested on MA after incubation for 3 days at 25 °C. The identification of fatty acid
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methyl esters was performed using the Sherlock Microbial Identification System (MIDI;
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version 6.1) and the TSBA6 database (Sasser, 1990). The DNA G+C content of strain EC29T
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was determined by extracting genomic DNA according to the methods described by Marmur
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(1961) and the thermal denaturation method (Tm) with Escherichia coli K-12 as a reference
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strain (Marmur & Doty, 1962). Analysis of the respiratory quinones was performed using a
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service of the Korean Culture Center of Microorganisms (KCCM). Polar lipids were
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separated by two-dimensional thin-layer chromatography (TLC) and analyzed according to
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the methods described by Minnikin et al. (1984). For the detection of polar lipids,
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molybdophosphoric acid, ninhydrin, Zinzade and α-naphthol reagents were applied and
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through co-migration with authentic standards (Minnikin et al., 1984; Komagata and Suzuki,
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1987).
116 117
The nearly complete 16S rRNA gene sequence of strain EC29T was a continuous stretch of
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1,442 bp and was GenBank accession number JF820845. Phylogenetic analysis established
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that strain EC29T belonged to the genus Winogradskyella, forming a well separated branch
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with type strains of W. pulchriflava EM106T (97.8 %), W. echinorum KMM 6211T (97.5 %),
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W. ulvae KMM 6390T (96.5 %), W. eximia KMM 3994T (96.2%) and W. lutea A73T (96.1 %)
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in the neighbor-joining phylogenetic tree (Fig. 1). Sequence similarities of < 93.8 % was
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observed with the other species of the genus Winogradskyella. Although the bootstrap value
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for the node between the isolate and closely related type strains was relatively low (56 %),
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the topologies of the phylogenetic trees built using the maximum-likelihood and maximum-
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parsimony algorithms supported the finding that strain EC29T formed lineage separated from
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members of the genus Winogradskyella (Fig S1). The levels of DNA–DNA relatedness
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between strain EC29T and the type strains W. pulchriflava KCTC 23858T and W. echinorum
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KCTC 22026T was 26.8 ± 0.7 and 24.9 ± 1.3 %, respectively. These values confirmed the
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assignment of the novel isolate EC29T to a separate species according to the cut-off value of
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70 % proposed Wayne et al. (1987) for the definition of members of a species.
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Cells of strain EC29T are Gram-negative, aerobic, motile by gliding and rod-shaped, able to
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grow at temperatures of 10-30 °C (optimum 25 °C), at pH 6.0-9.5 (optimum pH 7.5) and in
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the presence of 1-5 % NaCl. Colonies are circular, have a rough surface and are yellow-
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colored when grown on MA at 25 °C. Other phenotypic and biochemical characteristics of
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the strain EC29T are presented in Table 1 and the related Winogradskyella species description.
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The predominant cellular fatty acids of the strain EC29T (> 5 % of the total fatty acids) were
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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
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(7.1 %), and anteiso C15:0 (5.6 %). This fatty acid profile was similar to those of closely related
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members within the genus Winogradskyella, with difference in the respective proportions of
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some fatty acids (Table 2). The predominant respiratory quinone found in the strain EC29T
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was menaquinone-6 (MK-6), which is a characteristic quinone system of the family
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Flavobacteriaceae (Bernardet and Nakagawa, 2006).
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was 31.1 mol%, this supports their affiliation with the genus Wingradskyella, members of
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which have DNA G+C content between 30.1 and 36.4 mol%. The polar lipid profiles of strain
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EC29T were phosphatidylethanolamine (PE), unknown aminolipids (AL1-AL2), and
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unknown lipids (L1-L5) (Fig. S2). The presence of phosphatidylethanolamine and aminolipid
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was consistent with results of analyses of the genus Winogradskyella (Nedashkovskaya et al.,
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2012; Kim et al., 2012; Kim et al., 2013; Park et al., 2014).
The G+C content of the strain EC29T
151 152
The genetic distinctiveness and differential phenotypic characteristics of strain EC29T are
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sufficient to show that this strain is separate from species of the genus Winogradskyella. Based on
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data from the present polyphasic taxonomic study, we considered that strain EC29T represent a
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novel species of the genus Winogradskyella, for which the name Winogradskyella eckloniae sp.
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nov. is proposed.
157 158
Description of Winogradskyella eckloniae sp. nov.
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Winogradskyella eckloniae (eck.lo.ni`ae. N.L. fem. n. Ecklonia scientific genus name of a
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marine alga; N.L. gen. n. eckloniae of Ecklonia, referring to the isolation of the type strain
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from Ecklonia kurome).
162 163
Cells are Gram-negative, aerobic, motile by gliding and rod-shaped (0.2–0.3 µm wide and
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0.7–1.2 µm long). After incubation for 3 days at 25 °C, colonies on MA are circular to rough
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surface, yellow-colored and 1-1.5 mm in diameter. Growth is observed at 10-30 °C (optimum,
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20-25 °C) and pH 6.0-9.5 (optimum, pH 7.5). Growth occurs in the presence of 1-5 % NaCl,
167
but not below 1 % or above 5 % NaCl. Anaerobic growth is not observed after 4 weeks at
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25 °C on MA. Catalase- and oxidase- positive. Hydrolyses aesculin, gelatin, starch and Tween
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40, but does not hydrolyse casein, cellulose, Tweens 20, 60, and 80. Nitrate is not reduced.
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Acetoin, indole and H2S are not produced. In the API ZYM system, positive for alkaline
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phosphatase, esterase (C4), esterase lipase (C8), cystine-, valine-, and leucine arylamidases,
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α-chymotrypsin, trypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase, and
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negative for lipase (C14), α–galactosidase, β–galactosidase, α-glucosidase, β-glucosidase, β-
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glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Susceptible to
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ampicillin (1µg), cephalothin (30µg), chloramphenicol (30µg), erythromycin (15µg),
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licomycin (2µg), novobiocin (30µg), penicillin G (10IU), and tetracycline (30µg), but
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resistant to gentamycin (10µg), kanamycin (30µg), neomycin (30µg), polymyxin B (300IU),
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and streptomycin (10µg). The predominant cellular fatty acids are iso-C15:0, iso-C15:1 G, C15:0,
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iso-C17:0 3-OH, iso-C15:0 3-OH and anteiso C15:0. The major respiratory quinone is
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menaquinone-6. The major polar lipids are phosphatidylethanolamine, two unknown
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aminolipids, and five unknown lipids. The DNA G+C content of the type strain is 31.1 mol%.
182 183
The type strain is EC29T (= KCTC 23835T = JCM 18703T), which was isolated from brown
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alga Ecklonia cava collected along the coast of Jeju Island, Republic of Korea.
185
186
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312 313
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325
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326 327
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333
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334
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337
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338 339
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340
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341
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342 343
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344
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345
346
Table 1. Differential phenotypic characteristics of strain EC29T and the closely related
347
type strains of genus Winogradskyella
348
Strains: 1, W. eckloniae EC29T; 2, W. pulchriflava KCTC 23858 T; 3, W. echinorum KCTC
349
22026T; 4. W. ulvae KCTC 23626 T. All data are from this study. +, Positive; w, weakly
350
positive; -, negative. All strains are positive for the following: catalase and oxidase activities;
351
hydrolysis of aesculin and gelatin; and activities of acid phosphatase, alkaline phosphatase,
352
esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, and cystein
353
arylamidase; susceptibility of cephalothin, chloramphenicol, erythromycin, lincomycin and
354
novobiosin. All strains are negative for the following: hydrolysis of cellulose, urea and
355
Tween 20; activities of lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase, α-
356
glucosidase, β-glucosidase, N-acetyl- β-glucosaminidase, α-mannosidase and α-fucosidase;
357
susceptibility of gentamicin, neomycin, polymyxin B and streptomycin. Characteristic
1
2
3
4
Anarobic
-
-
-
+
37 °C
-
-
+
+
1-5
0.5-5.0
1.0-6.0
0.5-4.0
-
-
-
+
DNA
-
+
+
+
Starch
+
-
-
+
Casein
-
+
+
+
Tween 40
+
+
-
+
Tween 80
-
+
-
+
D-Glucose
-
-†
-†
+
D-mannose
-
-†
-†
+
w
-
+
-
Growth at/with
NaCl (%) Fermentation of Glucose Hydrolysis of:
Utilization of:
Enzyme activities (API ZYM): Trypsin
α-chymotrypsin
+
+
+
-
Naphtol-AS-BI-phosphohydrolase
-
+
+
+
Ampicillin
+
+
-
+
Kanamycin
-
+
-
-
Penicillin
+
+
-
+
Tetracyline
+
+
-
+
31.1
33.3 a
33.6 b
34.2 c
Susceptibility to:
DNA G+C content (mol%)* 358
* DNA G+C content data from: a, Kim et al.,(2013); b, Nedashkovskaya et al., (2009); c,
359
Nedashkovskaya et al., (2012).
360
† Different results were reported by Kim et al., (2013) and Nedashkovskaya et al., (2009).
361
362
Table 2. Cellular fatty acid profiles of strain EC29T and the closely related type strains
363
of genus Winogradskyella
364
Strains: 1, W. eckloniae EC29T; 2, W. pulchriflava KCTC 23858 T; 3, W. echinorum KCTC
365
22026T; 4, W. ulvae KCTC 23626 T. All data were obtained in this study from cultures on MA
366
at 25°C for 3 days. Fatty acids founds in amounts