IJSEM Papers in Press. Published May 21, 2015 as doi:10.1099/ijs.0.000350
International Journal of Systematic and Evolutionary Microbiology Winogradskyella crassostreae sp. nov., isolated from oyster --Manuscript Draft-Manuscript Number:
IJS-D-15-00255R1
Full Title:
Winogradskyella crassostreae sp. nov., isolated from oyster
Short Title:
Winogradskyella crassostreae sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Jung-Hoon Yoon Sungkyunkwan University Suwon, KOREA, REPUBLIC OF
First Author:
Sooyeon Park
Order of Authors:
Sooyeon Park Ji-Min Park Sung-Min Won Jung-Hoon Yoon
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A Gram-stain-negative, non-flagellated, non-gliding, aerobic and rod-shaped bacterium, designated TYO-19T, was isolated from an oyster collected from the South Sea in South Korea, and subjected to a polyphasic taxonomic approach. Strain TYO19T grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 1.0-2.0 % (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that strain TYO-19T belonged to the genus Winogradskyella, clustering coherently with the type strain of Winogradskyella epiphytica. Strain TYO-19T exhibited 16S rRNA gene sequence similarity values of 99.7 % to W. epiphytica KMM 3906T and 94.2-96.9 % to the type strains of the other Winogradskyella species. Strain TYO-19T contained MK-6 as the predominant menaquinone and iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and anteiso-C15:0 as the major fatty acids. The major polar lipids detected in strain TYO19T were phosphatidylethanolamine and one unidentified lipid. The DNA G+C content was 39.0 mol% and its mean DNA-DNA relatedness with the type strain of W. epiphytica was 59±4.3 %. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain TYO-19T is separated from recognized Winogradskyella species. On the basis of the data presented, strain TYO-19T is considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella crassostreae sp. nov. is proposed. The type strain is TYO-19T (= KCTC 42462T = NBRC 110924T).
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1
Winogradskyella crassostreae sp. nov., isolated from oyster
2 3
Sooyeon Park, Ji-Min Park, Sung-Min Won and Jung-Hoon Yoon
4 5
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu,
6
Suwon, South Korea
7 8 9
Running title: Winogradskyella crassostreae sp. nov.
10 11 12
Subject category: New taxa - Bacteroidetes
13 14 15 16 17 18 19 20
Author for correspondence: Prof. Jung-Hoon Yoon Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, South Korea Tel : +82-31-290-7800 Fax : +82-31-290-7882 e-mail : [email protected]
21 22 23 24 25 26 27
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TYO19T is KP981392. Two supplementary figures are available with the online version of this paper.
1
28
(Abstract)
29
A Gram-stain-negative, non-flagellated, non-gliding, aerobic and rod-shaped bacterium,
30
designated TYO-19T, was isolated from an oyster collected from the South Sea in South Korea,
31
and subjected to a polyphasic taxonomic approach. Strain TYO-19T grew optimally at 30 °C, at
32
pH 7.0-8.0 and in the presence of 1.0-2.0 % (w/v) NaCl. The phylogenetic trees based on 16S
33
rRNA gene sequences showed that strain TYO-19T belonged to the genus Winogradskyella,
34
clustering coherently with the type strain of Winogradskyella epiphytica. Strain TYO-19T
35
exhibited 16S rRNA gene sequence similarity values of 99.7 % to W. epiphytica KMM 3906T
36
and 94.2-96.9 % to the type strains of the other Winogradskyella species. Strain TYO-19T
37
contained MK-6 as the predominant menaquinone and iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and
38
anteiso-C15:0 as the major fatty acids. The major polar lipids detected in strain TYO-19T were
39
phosphatidylethanolamine and one unidentified lipid. The DNA G+C content was 39.0 mol%
40
and its mean DNA-DNA relatedness with the type strain of W. epiphytica was 59±4.3 %.
41
Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness,
42
revealed that strain TYO-19T is separated from recognized Winogradskyella species. On the
43
basis of the data presented, strain TYO-19T is considered to represent a novel species of the
44
genus Winogradskyella, for which the name Winogradskyella crassostreae sp. nov. is proposed.
45
The type strain is TYO-19T (= KCTC 42462T = NBRC 110924T).
46 47 2
48
The genus Winogradskyella, a member of family Flavobacteriaceae of the phylum
49
Bacteroidetes, was proposed by Nedashkovskaya et al. (2005) with the descriptions of three
50
novel species, Winogradskyella thalassocola (the type species of the genus), Winogradskyella
51
epiphytica and Winogradskyella eximia. At the time of writing, the genus comprises 20 species
52
with validly published names (http://www.bacterio.net/uw/winogradskyella.html; Euzéby, 1997;
53
Kim & Oh, 2012; Oren & Garrity, 2015). All members of the genus Winogradskyella have been
54
isolated from a variety of marine environments and marine organisms (Lau et al., 2005;
55
Nedashkovskaya et al., 2005, 2009, 2012; Pinhassi et al. 2009; Romanenko et al., 2009;
56
Ivanova et al., 2010; Yoon & Lee, 2012; Begum et al., 2013; Kang et al., 2013; Park & Yoon,
57
2013; Park et al., 2014). During a screening of novel marine bacteria from an oyster collected
58
from the South Sea, South Korea, many novel bacterial strains have been isolated and
59
characterized taxonomically. One of these bacterial strains, designated TYO-19T, which is most
60
phylogenetically affiliated to the genus Winogradskyella, is described in this study. The aim of
61
the present work was to determine the exact taxonomic position of strain TYO-19T by using a
62
polyphasic taxonomic characterization including phenotypic, phylogenetic and genetic analyses.
63 64 65
An oyster was collected from the South Sea at Tongyeong, South Korea, and used as the source
66
for the isolation of bacterial strains. Strain TYO-19T was isolated by the standard dilution
67
plating technique at 25 °C on marine agar 2216 (MA; Becton Dickinson) and cultivated
3
68
routinely at 30 °C on MA. Winogradskyella epiphytica KCTC 12220T and Winogradskyella
69
thalassocola KCTC 12221T, which were used as reference strains for fatty acid and polar lipid
70
analyses and DNA-DNA hybridization, were obtained from the Korean Collection for Type
71
Cultures (KCTC; South Korea). The cell morphology, Gram reaction, pH range for growth,
72
anaerobic growth, requirement for Mg2+ ions, hydrolysis of gelatin and urea and susceptibility to
73
antibiotics were determined as described by Park et al. (2014). Gliding motility was investigated
74
as described by Bowman (2000). Growth at 4, 10, 15, 20, 25, 30, 37 and 40 °C was measured on
75
MA to measure the optimal temperature and temperature range for growth. Growth at various
76
concentrations of NaCl (0, 0.5 and 1.0-10.0 %, at increments of 1.0 %) was investigated by
77
supplementing appropriate concentrations of NaCl in marine broth 2216 (MB) prepared
78
according to the formula of the Becton Dickinson medium except that NaCl was excluded.
79
Catalase and oxidase activities were determined as described by Lányí (1987). Hydrolysis of
80
casein, starch, hypoxanthine, L-tyrosine and xanthine was investigated on MA using the
81
substrate concentrations described by Barrow & Feltham (1993). Hydrolysis of aesculin and
82
Tweens 20, 40, 60 and 80 and nitrate reduction were investigated as described previously
83
(Lányí, 1987) with the modification that artificial seawater was used for the preparation of
84
media. Hydrolysis of DNA was investigated by using DNase test agar with methyl green
85
(Becton Dickinson), with the modification that artificial seawater was used for the preparation
86
of media. Hyrolysis of carboxymethylcellulose (CMC; Sigma) was tested on basal medium agar
87
[12 g gellan gum, 1 g yeast extract, 0.5 g NH4Cl, 50 ml 1M Tris/HCl (pH 7.4) and 5 g low
4
88
melting agarose l-1 artificial seawater] containing 0.5 % (w/v) of CMC. Hyrolysis of CMC was
89
revealed by flooding the agar with a 0.1 % (w/v) Congo red aqueous solution. The artificial
90
seawater contained (l-1 distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2·6H2O, 5.94 g
91
MgSO4·7H2O and 1.3 g CaCl2·2H2O (Bruns et al., 2001). The presence of flexirubin-type
92
pigments was investigated as described previously (Reichenbach, 1992; Bernardet et al., 2002).
93
Acid production from carbohydrates was tested as described by Leifson (1963). Enzyme
94
activities were determined, after incubation for 8 h at 30 ºC, by using the API ZYM system
95
(bioMérieux); the strip was inoculated with cells suspended in artificial seawater from which
96
CaCl2 was excluded to avoid the formation of precipitates. For in vivo and in vitro pigment-
97
absorption spectrum analyses, strain TYO-19T was cultivated aerobically in the dark at 30 C in
98
MB. The pigment absorption spectrum was analysed as described by Rainey et al. (2003). The
99
culture was washed twice using centrifugation with a MOPS buffer (MOPS/NaOH, 0.01 M;
100
KCl, 0.1 M; MgCl2, 0.001 M; pH 7.5) and disrupted by means of sonication (VC505; Sonics &
101
Materials, Inc.). After removal of cell debris by centrifugation, the absorption spectrum of the
102
supernatant was examined on 300-950 nm using the Eon Microplate spectrophotometer (Biotek).
103
The culture was centrifuged, and pigments from the cells were also extracted using
104
acetone/methanol (7:2, v/v). The absorption spectrum of the pigments was examined on the Eon
105
Microplate spectrophotometer (Biotek). Morphological, cultural, physiological and biochemical
106
characteristics of strain TYO-19T are given in the species description and in Table 1 or Fig. S1
107
(available in the online Supplementary Material). Sonicated in vivo extracts and
5
108
acetone/methanol extracts of strain TYO-19T showed no absorption maximum characteristic to
109
carotenoids.
110 111
Cell biomass of strain TYO-19T for DNA extraction and for the analyses of isoprenoid quinones
112
and polar lipids was obtained from cultures grown for 2 days in MB at 30 °C. Chromosomal
113
DNA was extracted and purified as described previously (Yoon et al., 1996), with the exception
114
that RNase T1 was used in combination with RNase A to minimize the contamination with
115
RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998)
116
using two universal primers, 9F (5’-GAGTTTGATCCTGGCTCAG-3’) and 1512R (5’-
117
ACGGTTACCTTGTTACGACTT-3’). Sequencing of the amplified 16S rRNA gene and
118
phylogenetic analysis were performed as described by Yoon et al. (2003). The almost-complete
119
16S rRNA gene sequence of strain TYO-19T comprising 1481 nucleotides, representing
120
approximately 95 % of the E. coli 16S rRNA gene sequence, was determined in this study. In
121
the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain TYO-19T
122
fell within the clade comprising Winogradskyella species, particularly clustering coherently
123
with the type strain of W. epiphytica with a bootstrap resampling value of 100 % (Fig. 1). The
124
relationship between strain TYO-19T and W. epiphytica KMM 3906T was also found in the trees
125
constructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain
126
TYO-19T exhibited 16S rRNA gene sequence similarity values of 99.7 % to W. epiphytica
127
KMM 3906T and of 94.2-96.9 % to the type strains of the other Winogradskyella species.
6
128 129
DNA-DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989)
130
using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed
131
with five replications for each sample. The highest and lowest values obtained for each sample
132
were excluded and the mean of the remaining three values was quoted as DNA-DNA
133
relatedness value. The DNAs of strain TYO-19T and W. epiphytica KCTC 12220T were used
134
individually as labelled DNA probes for reciprocal hybridization. Mean DNA-DNA relatedness
135
value between strain TYO-19T and W. epiphytica KCTC 12220T was 59±4.3%, which is below
136
the value of 70 % that is commonly accepted to define a new species (Wayne et al., 1987). The
137
DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the
138
modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-
139
phase HPLC equipped with a YMC ODS-A (2504.6 mm) column. The nucleotides were eluted
140
by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40:1, v/v), using a flow rate of 1
141
ml min-1 at room temperature and detected by UV absorbance at 270 nm. The DNA G+C
142
content of strain TYO-19T was 39.0 mol%, a value higher than those of the type strains of W.
143
epiphytica and the other Winogradskyella species (Table 1; Kim & Oh, 2012; Yoon & Lee,
144
2012; Begum et al., 2013; Kang et al., 2013; Kim et al., 2013; Park et al., 2013, 2014).
145 146
Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and
147
analyzed using reversed-phase HPLC and a YMC ODS-A (250×4.6 mm) column. The
7
148
isoprenoid quinones were eluted by a mixture of methanol/isopropanol (2:1, v/v) using a flow
149
rate of 1 ml min-1 at room temperature and detected by UV absorbance at 270 nm. The
150
predominant isoprenoid quinone detected in strain TYO-19T was menaquinone-6 (MK-6) in line
151
with the genus Winogradskyella (Nedashkovskaya et al., 2005, 2012; Begum et al., 2013) and
152
all other members of the family Flavobacteriaceae (Bernardet, 2011). For cellular fatty acid
153
analysis, cell masses of strain TYO-19T were harvested from MA plates after cultivation for 2, 3
154
and 5 days at 25 ºC and cell masses of W. epiphytica KCTC 12220T and W. thalassocola KCTC
155
12221T were harvested from MA plates after cultivation for 3 days at 25 ºC. Fatty acids were
156
saponified, methylated and extracted using the standard MIDI protocol (Sherlock Microbial
157
Identification System, version 6.2B). The fatty acids were analysed by GC (Hewlett Packard
158
6890) and identified using the TSBA6 database of the Microbial Identification System (Sasser,
159
1990). The cellular fatty acid profiles of strain TYO-19T and the type strains of W. epiphytica
160
and W. thalassocola are compared in Table 2. The major fatty acids (> 10 % of the total fatty
161
acids) detected in strain TYO-19T were iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and anteiso-C15:0.
162
The fatty acid profiles of strain TYO-19T from the three different growth phases were found to
163
be similar. The fatty acid profile of strain TYO-19T was similar with those of the type strains of
164
W. epiphytica KCTC 12220T and W. thalassocola KCTC 12221T, although there were
165
differences in the proportions of some fatty acids (Table 2). Polar lipids were extracted
166
according to the procedures described by Minnikin et al. (1984), and separated by two-
167
dimensional TLC using chloroform/methanol/water (65:25:3.8, by vol.) for the first dimension
8
168
and chloroform/methanol/acetic acid/water (40:7.5:6:1.8, by vol.) for the second dimension as
169
described by Embley & Wait (1994). Individual polar lipids were identified by spraying the
170
plates with 10 % ethanolic molybdophosphoric acid, molybdenum blue, ninhydrin and -
171
naphthol reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987) and with Dragendorff’s
172
reagent
173
phosphatidylethanolamine and one unidentified lipid; minor amounts of seven unidentified
174
lipids, two unidentified aminophospholipids and one unidentified aminolipid were also present
175
(Fig. S2). The polar lipid profile of strain TYO-19T was similar with those of W. epiphytica
176
KCTC 12220T and W. thalassocola KCTC 12221T in that phosphatidylethanolamine is the only
177
major phospholipid identified and one unidentified lipid is a major polar lipid, but
178
distinguishable from those of W. epiphytica KCTC 12220T and W. thalassocola KCTC 12221T
179
by the absence of major or significant amounts of some polar lipids, particularly unidentified
180
phospholipid (PL) in W. epiphytica KCTC 12220T and one unidentified lipid (L11) and one
181
unidentified glycolipid (GL) in W. thalassocola KCTC 12221T (Fig. S2).
(Sigma).
The
major
polar
lipids
detected
in
strain
TYO-19T
were
182 183
The results obtained from the phylogenetic and chemotaxonomic analyses are sufficient to
184
assign strain TYO-19T as a member of the genus Winogradskyella. Strain TYO-19T was
185
distinguished from the type strains of W. epiphytica and W. thalassocola by differences in
186
several phenotypic characteristics, including gliding motility, optimal temperature for growth,
187
hydrolysis of some substrates, acid production from some substrates, susceptibility to some
9
188
antibiotics, activity of some enzymes and polar lipid profiles (Table 1). These differences, in
189
combination with phylogenetic and genetic distinctiveness of strain TYO-19T, suggest that the
190
novel strain is separated from other species of the genus Winogradskyella (Wayne et al., 1987;
191
Stackebrandt & Goebel, 1994). On the basis of the phenotypic, chemotaxonomic, phylogenetic
192
and genetic data, strain TYO-19T is considered to represent a novel species of the genus
193
Winogradskyella, for which the name Winogradskyella crassostreae sp. nov. is proposed.
194 195 196
Description of Winogradskyella crassostreae sp. nov.
197 198
Winogradskyella crassostreae (crass.os’tre.ae. N.L. gen. n. crassostreae of Crassostrea, named
199
after the generic name of the Pacific oyster Crassostrea gigas, from which the type strain was
200
isolated).
201 202
Cells are Gram-stain-negative, non-flagellated, non-gliding and rod-shaped, approximately 0.2-
203
0.4 µm in width and 0.4->10.0 µm in length; a few cells greater than 10 m in length are
204
observed. Colonies on MA are circular, slightly convex, smooth, glistening, pale yellow in
205
colour and 1.0-1.5 mm in diameter after incubation for 3 days at 30 °C. Optimal temperature for
206
growth is 30 °C; growth occurs at 4 and 37 °C, but not at 40 °C. Optimal pH for growth is 7.0-
207
8.0; growth occurs at pH 5.5, but not at pH 5.0. Growth occurs with 0.5-7.0 % (w/v) NaCl
10
208
(optimum, 1.0-2.0 %). Mg2+ ions are not required for growth. Growth does not occur under
209
anaerobic conditions on MA and on MA supplemented with nitrate. Nitrate is not reduced.
210
Catalase- and oxidase-positive. Carotenoids are not produced. Flexirubin-type pigments are not
211
produced. H2S is not produced. Casein, gelatin and Tweens 20, 40, 60 and 80 are hydrolysed,
212
but aesculin, DNA, CMC, hypoxanthine, starch, L-tyrosine, urea and xanthine are not. Acid is
213
produced from maltose and weakly from D-glucose, but not from L-arabinose, D-cellobiose, D-
214
fructose, D-galactose, lactose, D-mannose, D-melezitose, melibiose, D-raffinose, L-rhamnose,
215
D-ribose, sucrose, D-trehalose, D-xylose, myo-inositol, D-mannitol and D-sorbitol. In assays
216
with API ZYM system, activity of alkaline phosphatase, esterase (C4), esterase lipase (C8),
217
leucine
218
phosphohydrolase is present, but activity of lipase (C14), cystine arylamidase, trypsin, α-
219
chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase,
220
N-acetyl- -glucosaminidase, α-mannosidase and α-fucosidase is absent. Susceptible to
221
chloramphenicol, lincomycin, novobiocin, oleandomycin, penicillin G and tetracycline, but not
222
to ampicillin, carbenicillin, cephalotin, gentamicin, kanamycin, neomycin, polymyxin B and
223
streptomycin. The predominant menaquinone is MK-6. The major fatty acids (> 10 % of the
224
total fatty acids) are iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and anteiso-C15:0. The major polar
225
lipids are phosphatidylethanolamine and one unidentified lipid. The DNA G+C content of the
226
type strain is 39.0 mol%.
227
The type strain, TYO-19T (= KCTC 42462T = NBRC 110924T), was isolated from an oyster
arylamidase,
valine
arylamidase,
11
acid
phosphatase
and
naphthol-AS-BI-
228
(Crassostrea gigas) collected from the South Sea at Tongyeong, South Korea.
229 230 231
Acknowledgements
232 233
This work was supported by the project on survey of indigenous species of Korea of the
234
National Institute of Biological Resources (NIBR) under the Ministry of Environment (MOE)
235
and the Program for Collection, Management and Utilization of Biological Resources (grant
236
NRF-2013M3A9A5075953) from the Ministry of Science, ICT & Future Planning (MSIP) of
237
the Republic of Korea.
238 239 240
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Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A.
310
& Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid
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quinones and polar lipids. J Microbiol Methods 2, 233-241.
312 313
Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Snauwaert, C., Vancanneyt, M., Swings, J.,
314
Kim, K.-O., Lysenko, A. M., Rohde, M., Frolova, G. M., Mikhailov, V. V. & Bae, K. S.
315
(2005). Winogradskyella thalassocola gen. nov., sp. nov., Winogradskyella epiphytica sp. nov.
316
and Winogradskyella eximia sp. nov., marine bacteria of the family Flavobacteriaceae. Int J
317
Syst Evol Microbiol 55, 49-55.
318 319
Nedashkovskaya, O. I., Vancanneyt, M., Kim, S. B. & Zhukova, N. V. (2009).
320
Winogradskyella echinorum sp. nov., a marine bacterium of the family Flavobacteriaceae
321
isolated from the sea urchin Strongylocentrotus intermedius. Int J Syst Evol Microbiol 59, 1465-
322
1468.
323 324
Nedashkovskaya, O. I., Kukhlevskiy, A. D. & Zhukova, N. V. (2012). Winogradskyella ulvae
325
sp. nov., a novel epiphyte of a Pacific seaweed and emended descriptions of the genus
326
Winogradskyella
327
Winogradskyella exilis and Winogradskyella eximia. Int J Syst Evol Microbiol 62, 1450-1456.
and
Winogradskyella
thalassocola,
16
Winogradskyella
echinorum,
328 329
Oren, A. & Garrity, G.-M. (2015). Winogradskyella jejuensis sp. nov. In List of new names
330
and new combinations previously effectively, but not validly, published, Validation List no. 161.
331
Int J Syst Evol Microbiol 65, 1-4.
332 333
Park, S. & Yoon, J.-H. (2013). Winogradskyella undariae sp. nov., a member of the family
334
Flavobacteriaceae isolated from a brown algae reservoir. Antonie van Leeuwenhoek 104, 619-
335
626.
336 337
Park, S., Park, J.-M., Won, S.-M., Bae, K. S. & Yoon, J.-H. (2014). Winogradskyella
338
wandonensis sp. nov., isolated from a tidal flat. Int J Syst Evol Microbiol 64, 1520-1525.
339 340
Pinhassi, J., Nedashkovskaya, O. I., Hagström, Å. & Vancanneyt, M. (2009).
341
Winogradskyella rapida sp. nov., isolated from protein-enriched seawater. Int J Syst Evol
342
Microbiol 59, 2180-2184.
343 344
Rainey, F. A., Silva, J., Nobre, M. F., Silva, M. T. & da Costa, M. S. (2003). Porphyrobacter
345
cryptus sp. nov., a novel slightly thermophilic, aerobic, bacteriochlorophyll a-containing species.
346
Int J Syst Evol Microbiol 53, 35-41.
347 17
348
Reichenbach, H. (1992). The order Cytophagales. In The Prokaryotes. A Handbook on the
349
Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, 2nd edn, pp. 3631-
350
3675. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New
351
York: Springer.
352 353
Romanenko, L. A., Tanaka, N., Frolova, G. M. & Mikhailov, V. V. (2009). Winogradskyella
354
arenosi sp. nov., a member of the family Flavobacteriaceae isolated from marine sediments
355
from the Sea of Japan. Int J Syst Evol Microbiol 59, 1443-1446.
356 357
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids.
358
MIDI Technical Note 101. Newark, DE: MIDI. Inc.
359 360
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA
361
reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology.
362
Int J Syst Bacteriol 44, 846-849.
363 364
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reverse-
365
phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125-128.
366 367
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky,
18
368
M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987).
369
International Committee on Systematic Bacteriology. Report of the ad hoc committee on
370
reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463-464.
371 372
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. &
373
Park, Y.-H. (1996). Identification of Saccharomonospora strains by the use of genomic DNA
374
fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502-505.
375 376
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific phylogenetic analysis of
377
the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48,
378
187-194.
379 380
Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp. nov., isolated from
381
jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449-454.
382 383
Yoon, J.-H. & Lee, S.-Y. (2012). Winogradskyella multivorans sp. nov., a polysaccharide-
384
degrading bacterium isolated from seawater of an oyster farm. Antonie van Leeuwenhoek 102,
385
231-238.
386 387 19
388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403
Table 1. Differential characteristics of strain TYO-19T and the type strains of Winogradskyella epiphytica and Winogradskyella thalassocola. Strains: 1, TYO-19T; 2, W. epiphytica KCTC 12220T; 3, W. thalassocola KCTC 12221T. Data of reference strains taken from Yoon & Lee (2012) unless indicated otherwise. ＋, positive reaction; , negative reaction; w, weakly positive reaction. All strains are positive for the followings: activity of catalase and oxidase; hydrolysis of casein, gelatin and Tweens 20, 40, 60 and 80; acid production from maltose; susceptibility to chloramphenicol, lincomycin and oleandomycin; and activity of alkaline phosphatase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and [esterase (C4), esterase lipase (C8) and acid phosphatase] (weak for W. thalassocola KCTC 12221T). All strains are negative for the followings: anaerobic growth; Gram-staining; nitrate reduction; H2S production; production of flexirubin-type pigments; hydrolysis of hypoxanthine, xanthine, starch and urea; acid production from L-arabinose, D-fructose, D-galactose, lactose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, D-trehalose, D-xylose, myo-inositol, D-mannitol and D-sorbitol; susceptibility to ampicillin, gentamicin, kanamycin, neomycin, polymyxin B and streptomycin; and activity of trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, αglucosidase, β-glucosidase, N-acetyl--glucosaminidase, α-mannosidase and α-fucosidase.
Characteristic Gliding motility Optimal growth temperature (°C) Hydrolysis of Agar Aesculin L-Tyrosine Acid production from D-Cellobiose D-Glucose D-Mannose Sucrose Susceptibility to Carbenicillin Cephalothin Novobiocin Penicillin G Tetracycline Enzyme activity (API ZYM) Lipase (C14) Valine arylamidase Cystine arylamidase DNA G+C content (mol%)
404
1 30
2 +* 23-25*
3 +* 21-23*
+* +
+* + +
w
+ + + +
+ + +
+ + +
+ +
+ 39.0
+ + + 35.2*
34.6*
*Data taken from Nedashkovskaya et al. (2005).
405 406 407 408 409 20
410 411 412 413 414 415 416
Table 2. Cellular fatty acid compositions (%) of strain TYO-19T and the type strains of Winogradskyella epiphytica and Winogradskyella thalassocola. Strains: 1, TYO-19T (2 days); 2, TYO-19T (3 days); 3, TYO-19T (5 days); 4, W. epiphytica KCTC 12220T; 5, W. thalassocola KCTC 12221T. Data obtained from this study. Fatty acids that represented < 1.0 % in all columns were omitted. Fatty acids that represented > 10.0 % were indicated as bold. TR, Traces (< 1.0 %); －, Not detected.
Fatty acid Branched iso-C14:0 iso-C15:0 iso-C15:1 G* anteiso-C15:0 anteiso-C15:1 A* iso-C16:0 iso-C16:1 H* Hydroxy C15:0 2-OH C15:0 3-OH C17:0 2-OH iso-C13:0 3-OH iso-C15:0 3-OH iso-C16:0 3-OH iso-C17:0 3-OH Unsaturated C15:1 ω6c C18:1 ω5c iso-C17:1 ω9c anteiso-C17:1 ω9c Summed feature 3†
417 418
1
2
3
4
5
TR 11.7 17.6 10.6 6.2 2.4 1.8
TR 12.8 16.5 11.0 6.8 1.8 1.8
1.1 10.8 13.3 14.0 6.1 2.4 1.5
TR 11.2 11.0 11.6 4.8 3.0 1.7
1.4 19.6 16.9 11.0 2.9 1.1 2.5
2.2 － 5.7 1.1 4.0 7.1 13.1
2.7 － 6.2 － 4.3 4.8 13.3
3.4 － 7.7 TR 4.6 6.2 13.7
2.4 － 10.1 TR 3.7 8.8 14.3
1.4 1.4 1.8 － 10.6 9.6 7.9
－ 1.4 3.2 TR 7.7
－ 1.1 4.2 TR 8.9
－ TR 2.3 1.2 6.9
－ 1.6 4.3 TR 6.4
2.4 1.2 3.0 － 2.5
*Double bond position indicated by a capital letter is unknown. †Summed feature 3 contained C 16:1 ω7c and/or C16:1 ω6c.
419 420 421 422 423 424 425 426 21
427
Legend to Figure
428 429
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the
430
positions of Winogradskyella crassostreae TYO-19T, the type strains of other Winogradskyella
431
species and representatives of some other related taxa. Only bootstrap values (expressed as
432
percentages of 1000 replications) greater than 50 % are shown at branching points. Filled circles
433
indicate that the corresponding nodes were also recovered in the trees generated with the
434
maximum-likelihood and maximum-parsimony algorithms. Capnocytophaga ochracea ATCC
435
27872T (GenBank accession number, U41350) was used as an outgroup. Scale bar, 0.01
436
substitutions per nucleotide position.
22
Figure 1 Click here to download Figure: Fig. 1.ppt
Winogradskyella undariae WS-MY5T (KC261665) 66.3
Winogradskyella pacifica KMM 6019T (GQ181061) Winogradskyella psychrotolerans RS-3T (FN377721)
Winogradskyella thalassocola KMM 3907T (AY521223) Winogradskyella rapida SCB 36T (U64013) Winogradskyella arenosi R 60T (AB438962)
100
Winogradskyella lutea A73T (FJ919968) 100
Winogradskyella crassostreae TYO-19T (KP981392) Winogradskyella epiphytica KMM 3906T (AY521224) Winogradskyella multivorans T-Y1T (JQ354979)
Winogradskyella damuponensis F081-2T (HQ336488) 61.3
79.1
Winogradskyella eximia KMM 3944T (AY521225) Winogradskyella pulchriflava EM106T (JN896598)
97.0
Winogradskyella echinorum KMM 6211T (EU727254)
79.6
Winogradskyella ulvae KMM 6390T (HQ456127) 71.9
98.4
Winogradskyella aquimaris DPG-24T (HM368527) Winogradskyella poriferorum UST030701-295T (AY848823)
92.6 62.7
Winogradskyella exilis 022-2-26T (FJ595484) Winogradskyella jejuensis CP32T (JF820844)
98.1
Winogradskyella wandonensis WD-2-2T (KF768343) 97.7
Winogradskyella litorisediminis DPS-8T (JQ432561) Postechiella marina M091T (HQ336487) 100
65.3
Bizionia hallyeonensis T-y7T (JN885199) Bizionia echini KMM 6177T (FJ716799) Bizionia paragorgiae KMM 6029T (AY651070) Geojedonia litorea YCS-16T (JX994295) Psychroserpens burtonensis ACAM 188T (U62913)
100
Psychroserpens damuponensis F051-1T (HQ336490) Gaetbulibacter marinus IMCC1914T (EF108219)
83.6
Gaetbulibacter saemankumensis SMK-12T (AY883937) Mariniflexile gromovii KMM 6038T (DQ312294)
99.0
Mariniflexile jejuense SSK2-3T (JQ739457)
Capnocytophaga ochracea ATCC 27872T (U41350) 0.01
Fig. 1
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary materials.pdf
International Journal of Systematic and Evolutionary Microbiology Winogradskyella crassostreae sp. nov., isolated from oyster --Manuscript Draft-Manuscript Number:
IJS-D-15-00255R1
Full Title:
Winogradskyella crassostreae sp. nov., isolated from oyster
Short Title:
Winogradskyella crassostreae sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Jung-Hoon Yoon Sungkyunkwan University Suwon, KOREA, REPUBLIC OF
First Author:
Sooyeon Park
Order of Authors:
Sooyeon Park Ji-Min Park Sung-Min Won Jung-Hoon Yoon
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A Gram-stain-negative, non-flagellated, non-gliding, aerobic and rod-shaped bacterium, designated TYO-19T, was isolated from an oyster collected from the South Sea in South Korea, and subjected to a polyphasic taxonomic approach. Strain TYO19T grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 1.0-2.0 % (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that strain TYO-19T belonged to the genus Winogradskyella, clustering coherently with the type strain of Winogradskyella epiphytica. Strain TYO-19T exhibited 16S rRNA gene sequence similarity values of 99.7 % to W. epiphytica KMM 3906T and 94.2-96.9 % to the type strains of the other Winogradskyella species. Strain TYO-19T contained MK-6 as the predominant menaquinone and iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and anteiso-C15:0 as the major fatty acids. The major polar lipids detected in strain TYO19T were phosphatidylethanolamine and one unidentified lipid. The DNA G+C content was 39.0 mol% and its mean DNA-DNA relatedness with the type strain of W. epiphytica was 59±4.3 %. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain TYO-19T is separated from recognized Winogradskyella species. On the basis of the data presented, strain TYO-19T is considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella crassostreae sp. nov. is proposed. The type strain is TYO-19T (= KCTC 42462T = NBRC 110924T).
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): IJS-D-15-00255(revised).doc
1
Winogradskyella crassostreae sp. nov., isolated from oyster
2 3
Sooyeon Park, Ji-Min Park, Sung-Min Won and Jung-Hoon Yoon
4 5
Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu,
6
Suwon, South Korea
7 8 9
Running title: Winogradskyella crassostreae sp. nov.
10 11 12
Subject category: New taxa - Bacteroidetes
13 14 15 16 17 18 19 20
Author for correspondence: Prof. Jung-Hoon Yoon Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, South Korea Tel : +82-31-290-7800 Fax : +82-31-290-7882 e-mail : [email protected]
21 22 23 24 25 26 27
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TYO19T is KP981392. Two supplementary figures are available with the online version of this paper.
1
28
(Abstract)
29
A Gram-stain-negative, non-flagellated, non-gliding, aerobic and rod-shaped bacterium,
30
designated TYO-19T, was isolated from an oyster collected from the South Sea in South Korea,
31
and subjected to a polyphasic taxonomic approach. Strain TYO-19T grew optimally at 30 °C, at
32
pH 7.0-8.0 and in the presence of 1.0-2.0 % (w/v) NaCl. The phylogenetic trees based on 16S
33
rRNA gene sequences showed that strain TYO-19T belonged to the genus Winogradskyella,
34
clustering coherently with the type strain of Winogradskyella epiphytica. Strain TYO-19T
35
exhibited 16S rRNA gene sequence similarity values of 99.7 % to W. epiphytica KMM 3906T
36
and 94.2-96.9 % to the type strains of the other Winogradskyella species. Strain TYO-19T
37
contained MK-6 as the predominant menaquinone and iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and
38
anteiso-C15:0 as the major fatty acids. The major polar lipids detected in strain TYO-19T were
39
phosphatidylethanolamine and one unidentified lipid. The DNA G+C content was 39.0 mol%
40
and its mean DNA-DNA relatedness with the type strain of W. epiphytica was 59±4.3 %.
41
Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness,
42
revealed that strain TYO-19T is separated from recognized Winogradskyella species. On the
43
basis of the data presented, strain TYO-19T is considered to represent a novel species of the
44
genus Winogradskyella, for which the name Winogradskyella crassostreae sp. nov. is proposed.
45
The type strain is TYO-19T (= KCTC 42462T = NBRC 110924T).
46 47 2
48
The genus Winogradskyella, a member of family Flavobacteriaceae of the phylum
49
Bacteroidetes, was proposed by Nedashkovskaya et al. (2005) with the descriptions of three
50
novel species, Winogradskyella thalassocola (the type species of the genus), Winogradskyella
51
epiphytica and Winogradskyella eximia. At the time of writing, the genus comprises 20 species
52
with validly published names (http://www.bacterio.net/uw/winogradskyella.html; Euzéby, 1997;
53
Kim & Oh, 2012; Oren & Garrity, 2015). All members of the genus Winogradskyella have been
54
isolated from a variety of marine environments and marine organisms (Lau et al., 2005;
55
Nedashkovskaya et al., 2005, 2009, 2012; Pinhassi et al. 2009; Romanenko et al., 2009;
56
Ivanova et al., 2010; Yoon & Lee, 2012; Begum et al., 2013; Kang et al., 2013; Park & Yoon,
57
2013; Park et al., 2014). During a screening of novel marine bacteria from an oyster collected
58
from the South Sea, South Korea, many novel bacterial strains have been isolated and
59
characterized taxonomically. One of these bacterial strains, designated TYO-19T, which is most
60
phylogenetically affiliated to the genus Winogradskyella, is described in this study. The aim of
61
the present work was to determine the exact taxonomic position of strain TYO-19T by using a
62
polyphasic taxonomic characterization including phenotypic, phylogenetic and genetic analyses.
63 64 65
An oyster was collected from the South Sea at Tongyeong, South Korea, and used as the source
66
for the isolation of bacterial strains. Strain TYO-19T was isolated by the standard dilution
67
plating technique at 25 °C on marine agar 2216 (MA; Becton Dickinson) and cultivated
3
68
routinely at 30 °C on MA. Winogradskyella epiphytica KCTC 12220T and Winogradskyella
69
thalassocola KCTC 12221T, which were used as reference strains for fatty acid and polar lipid
70
analyses and DNA-DNA hybridization, were obtained from the Korean Collection for Type
71
Cultures (KCTC; South Korea). The cell morphology, Gram reaction, pH range for growth,
72
anaerobic growth, requirement for Mg2+ ions, hydrolysis of gelatin and urea and susceptibility to
73
antibiotics were determined as described by Park et al. (2014). Gliding motility was investigated
74
as described by Bowman (2000). Growth at 4, 10, 15, 20, 25, 30, 37 and 40 °C was measured on
75
MA to measure the optimal temperature and temperature range for growth. Growth at various
76
concentrations of NaCl (0, 0.5 and 1.0-10.0 %, at increments of 1.0 %) was investigated by
77
supplementing appropriate concentrations of NaCl in marine broth 2216 (MB) prepared
78
according to the formula of the Becton Dickinson medium except that NaCl was excluded.
79
Catalase and oxidase activities were determined as described by Lányí (1987). Hydrolysis of
80
casein, starch, hypoxanthine, L-tyrosine and xanthine was investigated on MA using the
81
substrate concentrations described by Barrow & Feltham (1993). Hydrolysis of aesculin and
82
Tweens 20, 40, 60 and 80 and nitrate reduction were investigated as described previously
83
(Lányí, 1987) with the modification that artificial seawater was used for the preparation of
84
media. Hydrolysis of DNA was investigated by using DNase test agar with methyl green
85
(Becton Dickinson), with the modification that artificial seawater was used for the preparation
86
of media. Hyrolysis of carboxymethylcellulose (CMC; Sigma) was tested on basal medium agar
87
[12 g gellan gum, 1 g yeast extract, 0.5 g NH4Cl, 50 ml 1M Tris/HCl (pH 7.4) and 5 g low
4
88
melting agarose l-1 artificial seawater] containing 0.5 % (w/v) of CMC. Hyrolysis of CMC was
89
revealed by flooding the agar with a 0.1 % (w/v) Congo red aqueous solution. The artificial
90
seawater contained (l-1 distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2·6H2O, 5.94 g
91
MgSO4·7H2O and 1.3 g CaCl2·2H2O (Bruns et al., 2001). The presence of flexirubin-type
92
pigments was investigated as described previously (Reichenbach, 1992; Bernardet et al., 2002).
93
Acid production from carbohydrates was tested as described by Leifson (1963). Enzyme
94
activities were determined, after incubation for 8 h at 30 ºC, by using the API ZYM system
95
(bioMérieux); the strip was inoculated with cells suspended in artificial seawater from which
96
CaCl2 was excluded to avoid the formation of precipitates. For in vivo and in vitro pigment-
97
absorption spectrum analyses, strain TYO-19T was cultivated aerobically in the dark at 30 C in
98
MB. The pigment absorption spectrum was analysed as described by Rainey et al. (2003). The
99
culture was washed twice using centrifugation with a MOPS buffer (MOPS/NaOH, 0.01 M;
100
KCl, 0.1 M; MgCl2, 0.001 M; pH 7.5) and disrupted by means of sonication (VC505; Sonics &
101
Materials, Inc.). After removal of cell debris by centrifugation, the absorption spectrum of the
102
supernatant was examined on 300-950 nm using the Eon Microplate spectrophotometer (Biotek).
103
The culture was centrifuged, and pigments from the cells were also extracted using
104
acetone/methanol (7:2, v/v). The absorption spectrum of the pigments was examined on the Eon
105
Microplate spectrophotometer (Biotek). Morphological, cultural, physiological and biochemical
106
characteristics of strain TYO-19T are given in the species description and in Table 1 or Fig. S1
107
(available in the online Supplementary Material). Sonicated in vivo extracts and
5
108
acetone/methanol extracts of strain TYO-19T showed no absorption maximum characteristic to
109
carotenoids.
110 111
Cell biomass of strain TYO-19T for DNA extraction and for the analyses of isoprenoid quinones
112
and polar lipids was obtained from cultures grown for 2 days in MB at 30 °C. Chromosomal
113
DNA was extracted and purified as described previously (Yoon et al., 1996), with the exception
114
that RNase T1 was used in combination with RNase A to minimize the contamination with
115
RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998)
116
using two universal primers, 9F (5’-GAGTTTGATCCTGGCTCAG-3’) and 1512R (5’-
117
ACGGTTACCTTGTTACGACTT-3’). Sequencing of the amplified 16S rRNA gene and
118
phylogenetic analysis were performed as described by Yoon et al. (2003). The almost-complete
119
16S rRNA gene sequence of strain TYO-19T comprising 1481 nucleotides, representing
120
approximately 95 % of the E. coli 16S rRNA gene sequence, was determined in this study. In
121
the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain TYO-19T
122
fell within the clade comprising Winogradskyella species, particularly clustering coherently
123
with the type strain of W. epiphytica with a bootstrap resampling value of 100 % (Fig. 1). The
124
relationship between strain TYO-19T and W. epiphytica KMM 3906T was also found in the trees
125
constructed using the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain
126
TYO-19T exhibited 16S rRNA gene sequence similarity values of 99.7 % to W. epiphytica
127
KMM 3906T and of 94.2-96.9 % to the type strains of the other Winogradskyella species.
6
128 129
DNA-DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989)
130
using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed
131
with five replications for each sample. The highest and lowest values obtained for each sample
132
were excluded and the mean of the remaining three values was quoted as DNA-DNA
133
relatedness value. The DNAs of strain TYO-19T and W. epiphytica KCTC 12220T were used
134
individually as labelled DNA probes for reciprocal hybridization. Mean DNA-DNA relatedness
135
value between strain TYO-19T and W. epiphytica KCTC 12220T was 59±4.3%, which is below
136
the value of 70 % that is commonly accepted to define a new species (Wayne et al., 1987). The
137
DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the
138
modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-
139
phase HPLC equipped with a YMC ODS-A (2504.6 mm) column. The nucleotides were eluted
140
by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40:1, v/v), using a flow rate of 1
141
ml min-1 at room temperature and detected by UV absorbance at 270 nm. The DNA G+C
142
content of strain TYO-19T was 39.0 mol%, a value higher than those of the type strains of W.
143
epiphytica and the other Winogradskyella species (Table 1; Kim & Oh, 2012; Yoon & Lee,
144
2012; Begum et al., 2013; Kang et al., 2013; Kim et al., 2013; Park et al., 2013, 2014).
145 146
Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and
147
analyzed using reversed-phase HPLC and a YMC ODS-A (250×4.6 mm) column. The
7
148
isoprenoid quinones were eluted by a mixture of methanol/isopropanol (2:1, v/v) using a flow
149
rate of 1 ml min-1 at room temperature and detected by UV absorbance at 270 nm. The
150
predominant isoprenoid quinone detected in strain TYO-19T was menaquinone-6 (MK-6) in line
151
with the genus Winogradskyella (Nedashkovskaya et al., 2005, 2012; Begum et al., 2013) and
152
all other members of the family Flavobacteriaceae (Bernardet, 2011). For cellular fatty acid
153
analysis, cell masses of strain TYO-19T were harvested from MA plates after cultivation for 2, 3
154
and 5 days at 25 ºC and cell masses of W. epiphytica KCTC 12220T and W. thalassocola KCTC
155
12221T were harvested from MA plates after cultivation for 3 days at 25 ºC. Fatty acids were
156
saponified, methylated and extracted using the standard MIDI protocol (Sherlock Microbial
157
Identification System, version 6.2B). The fatty acids were analysed by GC (Hewlett Packard
158
6890) and identified using the TSBA6 database of the Microbial Identification System (Sasser,
159
1990). The cellular fatty acid profiles of strain TYO-19T and the type strains of W. epiphytica
160
and W. thalassocola are compared in Table 2. The major fatty acids (> 10 % of the total fatty
161
acids) detected in strain TYO-19T were iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and anteiso-C15:0.
162
The fatty acid profiles of strain TYO-19T from the three different growth phases were found to
163
be similar. The fatty acid profile of strain TYO-19T was similar with those of the type strains of
164
W. epiphytica KCTC 12220T and W. thalassocola KCTC 12221T, although there were
165
differences in the proportions of some fatty acids (Table 2). Polar lipids were extracted
166
according to the procedures described by Minnikin et al. (1984), and separated by two-
167
dimensional TLC using chloroform/methanol/water (65:25:3.8, by vol.) for the first dimension
8
168
and chloroform/methanol/acetic acid/water (40:7.5:6:1.8, by vol.) for the second dimension as
169
described by Embley & Wait (1994). Individual polar lipids were identified by spraying the
170
plates with 10 % ethanolic molybdophosphoric acid, molybdenum blue, ninhydrin and -
171
naphthol reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987) and with Dragendorff’s
172
reagent
173
phosphatidylethanolamine and one unidentified lipid; minor amounts of seven unidentified
174
lipids, two unidentified aminophospholipids and one unidentified aminolipid were also present
175
(Fig. S2). The polar lipid profile of strain TYO-19T was similar with those of W. epiphytica
176
KCTC 12220T and W. thalassocola KCTC 12221T in that phosphatidylethanolamine is the only
177
major phospholipid identified and one unidentified lipid is a major polar lipid, but
178
distinguishable from those of W. epiphytica KCTC 12220T and W. thalassocola KCTC 12221T
179
by the absence of major or significant amounts of some polar lipids, particularly unidentified
180
phospholipid (PL) in W. epiphytica KCTC 12220T and one unidentified lipid (L11) and one
181
unidentified glycolipid (GL) in W. thalassocola KCTC 12221T (Fig. S2).
(Sigma).
The
major
polar
lipids
detected
in
strain
TYO-19T
were
182 183
The results obtained from the phylogenetic and chemotaxonomic analyses are sufficient to
184
assign strain TYO-19T as a member of the genus Winogradskyella. Strain TYO-19T was
185
distinguished from the type strains of W. epiphytica and W. thalassocola by differences in
186
several phenotypic characteristics, including gliding motility, optimal temperature for growth,
187
hydrolysis of some substrates, acid production from some substrates, susceptibility to some
9
188
antibiotics, activity of some enzymes and polar lipid profiles (Table 1). These differences, in
189
combination with phylogenetic and genetic distinctiveness of strain TYO-19T, suggest that the
190
novel strain is separated from other species of the genus Winogradskyella (Wayne et al., 1987;
191
Stackebrandt & Goebel, 1994). On the basis of the phenotypic, chemotaxonomic, phylogenetic
192
and genetic data, strain TYO-19T is considered to represent a novel species of the genus
193
Winogradskyella, for which the name Winogradskyella crassostreae sp. nov. is proposed.
194 195 196
Description of Winogradskyella crassostreae sp. nov.
197 198
Winogradskyella crassostreae (crass.os’tre.ae. N.L. gen. n. crassostreae of Crassostrea, named
199
after the generic name of the Pacific oyster Crassostrea gigas, from which the type strain was
200
isolated).
201 202
Cells are Gram-stain-negative, non-flagellated, non-gliding and rod-shaped, approximately 0.2-
203
0.4 µm in width and 0.4->10.0 µm in length; a few cells greater than 10 m in length are
204
observed. Colonies on MA are circular, slightly convex, smooth, glistening, pale yellow in
205
colour and 1.0-1.5 mm in diameter after incubation for 3 days at 30 °C. Optimal temperature for
206
growth is 30 °C; growth occurs at 4 and 37 °C, but not at 40 °C. Optimal pH for growth is 7.0-
207
8.0; growth occurs at pH 5.5, but not at pH 5.0. Growth occurs with 0.5-7.0 % (w/v) NaCl
10
208
(optimum, 1.0-2.0 %). Mg2+ ions are not required for growth. Growth does not occur under
209
anaerobic conditions on MA and on MA supplemented with nitrate. Nitrate is not reduced.
210
Catalase- and oxidase-positive. Carotenoids are not produced. Flexirubin-type pigments are not
211
produced. H2S is not produced. Casein, gelatin and Tweens 20, 40, 60 and 80 are hydrolysed,
212
but aesculin, DNA, CMC, hypoxanthine, starch, L-tyrosine, urea and xanthine are not. Acid is
213
produced from maltose and weakly from D-glucose, but not from L-arabinose, D-cellobiose, D-
214
fructose, D-galactose, lactose, D-mannose, D-melezitose, melibiose, D-raffinose, L-rhamnose,
215
D-ribose, sucrose, D-trehalose, D-xylose, myo-inositol, D-mannitol and D-sorbitol. In assays
216
with API ZYM system, activity of alkaline phosphatase, esterase (C4), esterase lipase (C8),
217
leucine
218
phosphohydrolase is present, but activity of lipase (C14), cystine arylamidase, trypsin, α-
219
chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase,
220
N-acetyl- -glucosaminidase, α-mannosidase and α-fucosidase is absent. Susceptible to
221
chloramphenicol, lincomycin, novobiocin, oleandomycin, penicillin G and tetracycline, but not
222
to ampicillin, carbenicillin, cephalotin, gentamicin, kanamycin, neomycin, polymyxin B and
223
streptomycin. The predominant menaquinone is MK-6. The major fatty acids (> 10 % of the
224
total fatty acids) are iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and anteiso-C15:0. The major polar
225
lipids are phosphatidylethanolamine and one unidentified lipid. The DNA G+C content of the
226
type strain is 39.0 mol%.
227
The type strain, TYO-19T (= KCTC 42462T = NBRC 110924T), was isolated from an oyster
arylamidase,
valine
arylamidase,
11
acid
phosphatase
and
naphthol-AS-BI-
228
(Crassostrea gigas) collected from the South Sea at Tongyeong, South Korea.
229 230 231
Acknowledgements
232 233
This work was supported by the project on survey of indigenous species of Korea of the
234
National Institute of Biological Resources (NIBR) under the Ministry of Environment (MOE)
235
and the Program for Collection, Management and Utilization of Biological Resources (grant
236
NRF-2013M3A9A5075953) from the Ministry of Science, ICT & Future Planning (MSIP) of
237
the Republic of Korea.
238 239 240
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384
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386 387 19
388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403
Table 1. Differential characteristics of strain TYO-19T and the type strains of Winogradskyella epiphytica and Winogradskyella thalassocola. Strains: 1, TYO-19T; 2, W. epiphytica KCTC 12220T; 3, W. thalassocola KCTC 12221T. Data of reference strains taken from Yoon & Lee (2012) unless indicated otherwise. ＋, positive reaction; , negative reaction; w, weakly positive reaction. All strains are positive for the followings: activity of catalase and oxidase; hydrolysis of casein, gelatin and Tweens 20, 40, 60 and 80; acid production from maltose; susceptibility to chloramphenicol, lincomycin and oleandomycin; and activity of alkaline phosphatase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and [esterase (C4), esterase lipase (C8) and acid phosphatase] (weak for W. thalassocola KCTC 12221T). All strains are negative for the followings: anaerobic growth; Gram-staining; nitrate reduction; H2S production; production of flexirubin-type pigments; hydrolysis of hypoxanthine, xanthine, starch and urea; acid production from L-arabinose, D-fructose, D-galactose, lactose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, D-trehalose, D-xylose, myo-inositol, D-mannitol and D-sorbitol; susceptibility to ampicillin, gentamicin, kanamycin, neomycin, polymyxin B and streptomycin; and activity of trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, αglucosidase, β-glucosidase, N-acetyl--glucosaminidase, α-mannosidase and α-fucosidase.
Characteristic Gliding motility Optimal growth temperature (°C) Hydrolysis of Agar Aesculin L-Tyrosine Acid production from D-Cellobiose D-Glucose D-Mannose Sucrose Susceptibility to Carbenicillin Cephalothin Novobiocin Penicillin G Tetracycline Enzyme activity (API ZYM) Lipase (C14) Valine arylamidase Cystine arylamidase DNA G+C content (mol%)
404
1 30
2 +* 23-25*
3 +* 21-23*
+* +
+* + +
w
+ + + +
+ + +
+ + +
+ +
+ 39.0
+ + + 35.2*
34.6*
*Data taken from Nedashkovskaya et al. (2005).
405 406 407 408 409 20
410 411 412 413 414 415 416
Table 2. Cellular fatty acid compositions (%) of strain TYO-19T and the type strains of Winogradskyella epiphytica and Winogradskyella thalassocola. Strains: 1, TYO-19T (2 days); 2, TYO-19T (3 days); 3, TYO-19T (5 days); 4, W. epiphytica KCTC 12220T; 5, W. thalassocola KCTC 12221T. Data obtained from this study. Fatty acids that represented < 1.0 % in all columns were omitted. Fatty acids that represented > 10.0 % were indicated as bold. TR, Traces (< 1.0 %); －, Not detected.
Fatty acid Branched iso-C14:0 iso-C15:0 iso-C15:1 G* anteiso-C15:0 anteiso-C15:1 A* iso-C16:0 iso-C16:1 H* Hydroxy C15:0 2-OH C15:0 3-OH C17:0 2-OH iso-C13:0 3-OH iso-C15:0 3-OH iso-C16:0 3-OH iso-C17:0 3-OH Unsaturated C15:1 ω6c C18:1 ω5c iso-C17:1 ω9c anteiso-C17:1 ω9c Summed feature 3†
417 418
1
2
3
4
5
TR 11.7 17.6 10.6 6.2 2.4 1.8
TR 12.8 16.5 11.0 6.8 1.8 1.8
1.1 10.8 13.3 14.0 6.1 2.4 1.5
TR 11.2 11.0 11.6 4.8 3.0 1.7
1.4 19.6 16.9 11.0 2.9 1.1 2.5
2.2 － 5.7 1.1 4.0 7.1 13.1
2.7 － 6.2 － 4.3 4.8 13.3
3.4 － 7.7 TR 4.6 6.2 13.7
2.4 － 10.1 TR 3.7 8.8 14.3
1.4 1.4 1.8 － 10.6 9.6 7.9
－ 1.4 3.2 TR 7.7
－ 1.1 4.2 TR 8.9
－ TR 2.3 1.2 6.9
－ 1.6 4.3 TR 6.4
2.4 1.2 3.0 － 2.5
*Double bond position indicated by a capital letter is unknown. †Summed feature 3 contained C 16:1 ω7c and/or C16:1 ω6c.
419 420 421 422 423 424 425 426 21
427
Legend to Figure
428 429
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the
430
positions of Winogradskyella crassostreae TYO-19T, the type strains of other Winogradskyella
431
species and representatives of some other related taxa. Only bootstrap values (expressed as
432
percentages of 1000 replications) greater than 50 % are shown at branching points. Filled circles
433
indicate that the corresponding nodes were also recovered in the trees generated with the
434
maximum-likelihood and maximum-parsimony algorithms. Capnocytophaga ochracea ATCC
435
27872T (GenBank accession number, U41350) was used as an outgroup. Scale bar, 0.01
436
substitutions per nucleotide position.
22
Figure 1 Click here to download Figure: Fig. 1.ppt
Winogradskyella undariae WS-MY5T (KC261665) 66.3
Winogradskyella pacifica KMM 6019T (GQ181061) Winogradskyella psychrotolerans RS-3T (FN377721)
Winogradskyella thalassocola KMM 3907T (AY521223) Winogradskyella rapida SCB 36T (U64013) Winogradskyella arenosi R 60T (AB438962)
100
Winogradskyella lutea A73T (FJ919968) 100
Winogradskyella crassostreae TYO-19T (KP981392) Winogradskyella epiphytica KMM 3906T (AY521224) Winogradskyella multivorans T-Y1T (JQ354979)
Winogradskyella damuponensis F081-2T (HQ336488) 61.3
79.1
Winogradskyella eximia KMM 3944T (AY521225) Winogradskyella pulchriflava EM106T (JN896598)
97.0
Winogradskyella echinorum KMM 6211T (EU727254)
79.6
Winogradskyella ulvae KMM 6390T (HQ456127) 71.9
98.4
Winogradskyella aquimaris DPG-24T (HM368527) Winogradskyella poriferorum UST030701-295T (AY848823)
92.6 62.7
Winogradskyella exilis 022-2-26T (FJ595484) Winogradskyella jejuensis CP32T (JF820844)
98.1
Winogradskyella wandonensis WD-2-2T (KF768343) 97.7
Winogradskyella litorisediminis DPS-8T (JQ432561) Postechiella marina M091T (HQ336487) 100
65.3
Bizionia hallyeonensis T-y7T (JN885199) Bizionia echini KMM 6177T (FJ716799) Bizionia paragorgiae KMM 6029T (AY651070) Geojedonia litorea YCS-16T (JX994295) Psychroserpens burtonensis ACAM 188T (U62913)
100
Psychroserpens damuponensis F051-1T (HQ336490) Gaetbulibacter marinus IMCC1914T (EF108219)
83.6
Gaetbulibacter saemankumensis SMK-12T (AY883937) Mariniflexile gromovii KMM 6038T (DQ312294)
99.0
Mariniflexile jejuense SSK2-3T (JQ739457)
Capnocytophaga ochracea ATCC 27872T (U41350) 0.01
Fig. 1
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary materials.pdf
