IJSEM Papers in Press. Published May 27, 2015 as doi:10.1099/ijs.0.000357
International Journal of Systematic and Evolutionary Microbiology Bizionia arctica sp. nov., isolated from Arctic fjord seawater and emended description of the genus Bizionia --Manuscript Draft-Manuscript Number:
IJS-D-15-00254R1
Full Title:
Bizionia arctica sp. nov., isolated from Arctic fjord seawater and emended description of the genus Bizionia
Short Title:
Bizionia arctica sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Bin-Bin Xie Shandong University CHINA
First Author:
Hai Li
Order of Authors:
Hai Li Xi-Ying Zhang Chang Liu Ang Liu Qi-Long Qin Hai-Nan Su Mei Shi Bai-Cheng Zhou Xiu-Lan Chen Yu-Zhong Zhang Bin-Bin Xie
Manuscript Region of Origin:
CHINA
Abstract:
A Gram-negative, yellow-colored, aerobic, non-flagellated, non-gliding bacterial strain, designated SM1203T, was isolated from surface seawater of Kongsfjorden, Svalbard. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1203T was affiliated with the genus Bizionia in the family Flavobacteriaceae. The strain shared the highest 16S rRNA gene sequence similarity (>96 %) with type strains of Formosa spongicola (96.8 %), Bizionia paragorgiae (96.3 %), B. saleffrena (96.3 %) and B. echini (96.1 %) and 95.4~95.7 % sequence similarities with type strains of other known Bizionia species. The strain grew at 4-30 °C, and in the presence of 1.0-5.0 % (w/v) NaCl. The major fatty acids of strain SM1203T were iso-C15:0, iso-C15:1, anteiso-C15:0 and C15:0 and the main polar lipids were phosphatidylethanolamine and one unidentified lipid. The major respiratory quinone of strain SM1203T was menaquinone 6 (MK-6). The genomic DNA G+C content of strain SM1203T was 34.8 mol %. Based on the polyphasic characterization of strain SM1203T in this study, the strain represents a novel species in the genus Bizionia, for which the name Bizionia arctica sp. nov. is proposed. The type strain of Bizionia arctica is SM1203T (=CGMCC 1.12751T =JCM 30333T).
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2
Bizionia arctica sp. nov., isolated from Arctic fjord seawater
3
and emended description of the genus Bizionia
4
Hai Li1,2, Xi-Ying Zhang1,2*, Chang Liu1,2, Ang Liu1,2, Qi-Long Qin1,2, Hai-Nan Su1,2,
5
Mei Shi1,2, Bai-Cheng Zhou1,2, Xiu-Lan Chen1,2, Yu-Zhong Zhang1,2 and Bin-Bin
6
Xie1,2
7
1
8
Center, Shandong University, Jinan 250100, China
9
*Co first author: Xi-Ying Zhang contributed equally to this paper as Hai Li.
State Key Laboratory of Microbial Technology, 2Marine Biotechnology Research
10
Running title: Bizionia arctica sp. nov.
11
Subject category: New Taxa- Bacteroidetes
12
Correspondence:
13
Bin-Bin Xie
14
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100,
15
China;
16
Tel: 86-531-88364326; Fax: 86-531-88564326; E-mail: [email protected]
17
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of
18
strain SM1203T is KJ508751.
1
19 20
21
Summary
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A Gram-negative, yellow-colored, aerobic, non-flagellated, non-gliding bacterial
23
strain, designated SM1203T, was isolated from surface seawater of Kongsfjorden,
24
Svalbard. Phylogenetic analysis based on 16S rRNA gene sequences revealed that
25
strain
26
Flavobacteriaceae. The strain shared the highest 16S rRNA gene sequence similarity
27
(>96 %) with type strains of Formosa spongicola (96.8 %), Bizionia paragorgiae
28
(96.3 %), B. saleffrena (96.3 %) and B. echini (96.1 %) and 95.4~95.7 % sequence
29
similarities with type strains of other known Bizionia species. The strain grew at
30
4–30 °C, and in the presence of 1.0–5.0 % (w/v) NaCl. The major fatty acids of strain
31
SM1203T were iso-C15:0, iso-C15:1, anteiso-C15:0 and C15:0 and the main polar lipids
32
were phosphatidylethanolamine and one unidentified lipid. The major respiratory
33
quinone of strain SM1203T was menaquinone 6 (MK-6). The genomic DNA G+C
34
content of strain SM1203T was 34.8 mol %. Based on the polyphasic characterization
35
of strain SM1203T in this study, the strain represents a novel species in the genus
36
Bizionia, for which the name Bizionia arctica sp. nov. is proposed. The type strain of
37
Bizionia arctica is SM1203T (=CGMCC 1.12751T =JCM 30333T).
SM1203T
was
affiliated
with
the
38
39
40
2
genus
Bizionia
in
the
family
41
42
The genus Bizionia belonging to the family Flavobacteriaceae in the phylum
43
Bacteroidetes was first proposed by Nedashkovskaya et al. (2005) to accommodate a
44
Gram-negative, strictly aerobic, heterotrophic, non-gliding, yellow-pigmented marine
45
bacterium. This genus currently comprises nine species: Bizionia paragorgiae (type
46
species; Nedashkovskaya et al., 2005), B. algoritergicola (Bowman & Nichols, 2005),
47
B. myxarmorum (Bowman & Nichols, 2005), B. saleffrena (Bowman & Nichols,
48
2005), B. argentinensis (Bercovich et al., 2008), B. echini (Nedashkovskaya et al.,
49
2010), B. gelidisalsuginis (Bowman & Nichols, 2005), B. hallyeonensis (Yoon et al.,
50
2013) and B. psychrotolerans (Song et al., 2014). Five of them (Bizionia
51
algoritergicola, B. myxarmorum, B. saleffrena, B. gelidisalsuginis and B.
52
argentinensis) have been isolated from Antarctic marine habitats including costal
53
seawater, sea-ice brines and amphipods, while other Bizionia species have been
54
isolated from coastal seawater and invertebrates from temperate seas. During a
55
bacterial diversity study of Arctic seawater samples, a yellow-pigmented Bizionia-like
56
bacterial strain, designated SM1203T, was isolated. In this study, we report the
57
polyphasic characterization of the new isolate and propose that it represents the first
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Arctic species in the genus Bizionia.
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The Arctic surface seawater sample was collected from Kongsfjorden, Svalbard
60
(78º 59′ N, 11º 40′ E) in July, 2011, during the 8th Chinese National Arctic Yellow
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River Station Scientific Expedition (summer). Approximately 0.8 liter of seawater
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sampled was vacuum-filtered through a 0.22 μm membrane filter. The membrane was
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then suspended in 50 ml of sterile artificial seawater and was vigorously shaken for 30
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min at 15 °C. The suspension obtained was directly spread onto trypticase yeast
3
65
extract (TYS) agar [0.5 % tryptone (Oxoid), 0.1 % yeast extract (Oxoid), 1.5 % agar
66
and artificial seawater] plates and incubated at 15 °C for 2–3 weeks. Artificial
67
seawater was prepared with commercial sea salts (Sigma, 3 %). Colonies showing
68
different morphologies on the plates were selected and purified by repeated streaking
69
on TYS agar plates. Strain SM1203T isolated was preserved at −80 ºC in TYS broth
70
(0.5 % tryptone, 0.1 % yeast extract and artificial seawater) containing 20 % (v/v)
71
glycerol. It was routinely cultivated on TYS agar or in TYS broth at 20 °C. Reference
72
strains used for some phenotypic tests and fatty acids analysis, including B.
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paragorgiae KCTC 12304T, B. echini KCTC 22015T, B. saleffrena CIP 108534T, B.
74
gelidisalsuginis CIP 108536T and Formosa spongicola KCTC 22662T, were also
75
routinely cultivated on TYS agar or in TYS broth at 20 °C.
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Genomic DNA of strain SM1203T for the 16S rRNA gene amplification was
77
extracted using a BioTeke (China) bacterial genomic DNA isolation kit according to
78
the manufacturer’s protocol. The 16S rRNA gene of strain SM1203T was
79
PCR-amplified using primers: 8-27F (5′-AGAGTTTGATCTTGGCTCAG-3′) and
80
1492-1510R (5′-TACGGCTACCTTGTTACGACTT-3′) (Lane, 1991) and then
81
sequenced by using an Applied Biosystems 3730 automated DNA sequencer. The
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resultant 16S rRNA gene sequence was compared with those of validly published
83
bacterial species in the EzTaxon-e database using the BLASTN program
84
(http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). Phylogenetic trees were
85
reconstructed by the neighbor-joining (Saitou & Nei, 1987), maximum likelihood
86
(Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) methods using MEGA
87
version 5 (Tamura et al., 2011), with bootstrap analyses based on 1000 replicates
88
(Felsenstein, 1985) and the Kimura’s two-parameter model (Kimura, 1980). Values of
89
pairwise sequence similarity calculated using the global alignment algorithm were 4
90
also obtained through the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et
91
al., 2012).
92
The almost-complete 16S rRNA gene sequence of strain SM1203T was
93
determined (1,442 bp) and comparative analysis of the sequence showed that strain
94
SM1203T shared the highest 16S rRNA gene sequence similarities (>96 %) with
95
Formosa spongicola (96.8 %), B. (96.3 %), B. saleffrena (96.3 %) and B. echini
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(96.1 %). Sequence similarities with other recognized Bizionia species were
97
95.4~95.7 % and with other Formosa species were 95.2~95.9 %. It was also found
98
that strain SM1203T exhibited relatively high 16S rRNA gene sequence similarities
99
(95.0~95.5 %) with respect to some species in the genera of Flavivirga, Olleya,
100
Flaviramulus, Algibacter, Winogradskyella, Gaetbulibacter and Arenitalea in the
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family Flavobacteriaceae. However, the phylogenetic trees based on 16S rRNA gene
102
sequences
103
maximum-parsimony methods all showed that strain SM1203T was grouped into the
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genus Bizionia and formed a sub-group with B. paragorgia, B. saleffrena and B.
105
gelidisalsuginis (Fig. 1 and Supplementary Figs. S1 and S2).
inferred
by
the
neighbor-joining,
maximum-likelihood
and
106
For the cellular fatty acid analysis, strain SM1203T and the reference strains: B.
107
paragorgiae KCTC 12304T, B. KCTC 22015T, and F. spongicola KCTC 22662T were
108
all cultivated in TYS broth at 20 ºC for 3 days. Cellular fatty acids were analyzed by
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GC (Hewlett Packard 6890) using the Sherlock MIS software (version 4.5 and the
110
TSBA40 database). Polar lipids were extracted as previously descried (Komagata &
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Suzuki, 1987) and further analyzed using two-dimensional TLC with appropriate
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spraying reagents including ethanolic molybdophosphoric acid [10 % (w/v), total
113
lipids], ninhydrin (aminolipids) and Zinzadze reagent (phospholipids) (Collins &
5
114
Jones, 1980). Quinones were extracted as described by Komagata & Suzuki (1987)
115
and analyzed using an LC-MS system consisting of a Dionex Ultimate 3000 HPLC
116
coupled with a Bruker Impact HD mass spectrometer. The genomic DNA G+C
117
content of strain SM1203T was determined by the thermal denaturation method
118
(Marmur & Doty, 1962) using a Beckman DU800 Spectrophotometer with
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chromosomal DNA extracted using the method of Marmur (1961).
120
Fatty acids of strain SM1203T predominantly included (≥5 %) iso-C15:0 (25.5 %),
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iso-C15:1 (20.3 %), anteiso-C15:0 (14.2 %) and C15:0 (5.3 %) with iso-C17:0 3-OH
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(4.8 %), iso-C16:0 3-OH (3.9 %), anteiso-C15:1 (3.4 %), iso-C14:0 (3.4 %), iso-C15:0
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3-OH (2.7 %), summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH) (2.0 %), C15:0 2-OH
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(1.7 %), C17:0 2-OH (1.2 %), iso-C16:1 H (1.2 %) and an unknown acid with an
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equivalent chain-length of 13.565 (1.5 %) being minor components (>1 %), showing a
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profile essentially similar to those of B. paragorgiae KCTC 12304T and B. echini
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KCTC 22015T (Table 1). But proportions of iso-C15:0 (25.5 %) and iso-C15:1 (20.3%)
128
in strain SM1203T were significantly higher than those in B. paragorgiae KCTC
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12304T (18.3 % for iso-C15:0 and 15.6 % for iso-C15:1, respectively) while lower than
130
those in B. echini KCTC 22015T (34.2 % and 24.2 %, respectively); in addition,
131
proportion of anteiso-C15:0 (14.2 %) in strain SM1203T was apparently higher than
132
those in B. paragorgiae KCTC 12304T (2.9 %) and B. echini KCTC 22015T (8.0 %).
133
These reflected the difference among them. Major lipids of strain SM1203T were
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phosphatidylethanolamine (PE) and one unidentified lipid (L1) with moderate
135
amounts of one unidentified phospholipid (PL) and aminolipid (AL1) and minor
136
amounts of one unidentified lipid (L2) and aminophospholipid (APL) and two
137
unidentified aminolipids (AL2 and AL3) (Supplementary Fig. S3). The major
138
respiratory quinone of strain SM1203T was menaquinone 6 (MK-6). The DNA G+C 6
139
content of strain SM1203T was 34.8 mol %, within the range reported for the genus
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Bizionia (34.0−45.0 mol %).
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The temperature (4, 10−45 °C at intervals of 5 °C) and pH (4.0−9.0 in
142
increments of 0.5 pH units) ranges for growth were determined in TYS broth. For the
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pH range examination, various buffers including MES (pH 5.0−6.0), MOPS (pH
144
6.5−7.0), Tris (pH 7.5−8.5) and CHES (pH 9.0) were respectively added into the TYS
145
broth at the concentration of 50 mM. Growth at different concentrations of NaCl (0,
146
0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 %, w/v) was investigated in a medium containing 0.5 %
147
tryptone (Oxoid), 0.1 % yeast extract (Oxoid), 0.5 % MgCl2, 0.2 % MgSO4, 0.05 %
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CaCl2, 0.1 % KCl, 0.0001 % FeSO4 and distilled water supplemented with appropriate
149
amounts of NaCl.
150
Gram-staining was performed using the Hucker’s method as described in Murray
151
et al. (1994). Cell morphology and flagellation were examined using a transmission
152
electron microscopy (JEM−100CX II) with cells grown in TYS broth at 20 °C for 3
153
days. Oxidase activity was detected using commercial oxidase test strips (Merck) and
154
catalase activity was assessed by observation of the bubble production in 3 % (v/v)
155
hydrogen peroxide solution. The presence of gliding motility was checked using the
156
hanging drop technique and the ability to produce flexirubin-type pigments were
157
detected by the KOH test (Bernardet et al. 2002). DNA hydrolysis was determined on
158
DNase agar (Oxoid). The ability to hydrolyze carboxymethyl-cellulose (CMC) was
159
examined on MA containing 1% (w/v) CMC (Sigma) (Lee et al., 2012). Hydrolysis of
160
other ploymers including agar, casein, gelatin, starch and Tweens 20, 40, 60 and 80
161
was determined as described in Smibert & Krieg (1994). Susceptibility to
162
antimicrobial agents was examined using the disc-diffusion method on TYS agar
7
163
(Kobayashi et al., 2003) using Oxoid susceptibility discs impregnated with different
164
antimicrobial agents including ampicillin (10 μg/disc), carbenicillin (100 U/disc),
165
cephalexin (30 μg/disc), chloramphenicol (30 μg/disc), erythromycin (15 μg/disc),
166
gentamicin (10 μg/disc), kanamycin (30 μg/disc), neomycin (30 μg/disc), novobiocin
167
(5 μg/disc), penicillin G (10 U/disc), polymyxin B (300 U/disc), streptomycin (10
168
μg/disc), tetracycline (30 μg/disc) and vancomycin (30 μg/disc). Anaerobic growth
169
was tested in TYS broth in Hungate tubes, supplemented with potassium nitrate
170
(0.1 %, w/v), cysteine hydrochloride (0.05 %, w/v) and sodium sulfide (0.05 %, w/v).
171
Other enzymatic activities and biochemical properties were tested using API ZYM
172
and API 20NE strips (bioMérieux), following the manufacturer’s instructions with
173
inocula prepared by suspending cells in sterile artificial sea water.
174
Cells of strain SM1203T were Gram-negative, non-flagellated, non-gliding long
175
thin rods. Anaerobic growth was not observed. Other phenotypic characteristics of
176
strain SM1203T were given in the species description. Strain SM1203T could be
177
phenotypically differentiated from the closely related species by a number of
178
characteristics, including temperature and NaCl concentration ranges for growth,
179
nitrate reduction, hydrolysis of various substrates, assimilation of carbohydrates and
180
genomic DNA G+C content, as shown in Table 2. Thus, according to the phylogenetic
181
analysis of the 16S rRNA gene sequences and the results from the chemotaxonomic
182
and phenotypic characterizations, we concluded that strain SM1203T represents a
183
novel Bizionia species, for which the name Bizionia arctica sp. nov. is proposed.
184
Description of Bizionia arctica sp. nov.
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Bizionia arctica (arc'ti.ca. L. fem. adj. arctica northern, from the Arctic)
8
186
Cells of the type strain are Gram-negative, aerobic, non-flagellated and
187
non-gliding rods, 1.8−4 μm in length and 0.3−0.5 μm in diameter. When incubated on
188
TYS agar at 20 °C for 5-7 days, colonies are yellow-pigmented, shiny, circular
189
(0.3−1.2 mm in diameter) with entire edges and convex. The type strain is oxidase-
190
negative and catalase-positive. It hydrolyzes casein, gelatin and Tweens 20, 40 and 80
191
but does not hydrolyze agar, carboxymethyl-cellulose (CMC), DNA, aesculin, starch
192
or Tween 60. The type strain grows at 4−30 °C (optimum at 20 °C), in the presence of
193
1−6 % NaCl (w/v, optimum in 2 %) and at pH 5.5−8.5 (optimum at pH 7.0). It does
194
not produce flexirubin-type pigments. In API 20NE tests, the type strain is positive for
195
arginine dihydrolase, acid production from glucose and hydrolysis of gelatin. In
196
addition, it does not utilize D-glucose, gluconate, caprate, arabinose, mannose,
197
mannitol, N-acetylglucosamine, maltose, adipate, malate, citrate and phenylacetate. In
198
the API ZYM tests, the type strain is positive for alkaline phosphatase, esterase (C4),
199
esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase,
200
trypsin, α-chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase but
201
negative for lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase,
202
α-glucosidase,
203
β-fucosidase. The type strain is susceptible to ampicillin, carbenicillin, cephalexin,
204
vancomycin,
205
chloramphenicol, but resistant to gentamicin, kanamycin, neomycin, polymyxin B and
206
streptomycin. The major fatty acids (>5 %) of the type strain are iso-C15:0, iso-C15:1,
207
anteiso-C15:0
208
phosphatidylethanolamine, two unidentified lipids, one unidentified phospholipid,
209
three unidentified aminolipids and one unidentified aminophospholipid. The major
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respiratory quinone of the type strain is menaquinone 6 (MK-6). The genomic G+C
β-glucosidase,
penicillin
and
C15:0.
G,
The
N-acetyl-β-glucosaminidase,
erythromycin,
polar
lipids
9
α-mannosidase
tetracycline,
of
the
type
novobiocin
strain
and
and
comprise
211
212
content of the type strain is 34.8 mol %. The type strain is SM1203T (=CGMCC 1.12751T =JCM 30333T), which was
213
isolated from surface seawater of Kongsfjorden, Svalbard.
214
Emended description of the genus Bizionia Nedashkovskaya et al. (2005)
215
The description is as given by Nedashkovskaya et al. (2005) with the following
216
amendments. Species in the genus Bizionia are oxidase- positive or negative.
217
Acknowledgements
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The work was supported by the China Ocean Mineral Resources R & D Association
219
(COMRA) Special Foundation (grants DY125-15-R-03 and DY125-15-T-05), the
220
National Natural Science Foundation of China (grants 31270117, 31290231,
221
31470541 and 41376153), the Hi-Tech Research and Development Program of China
222
(grants 2012AA092103, 2012AA092105 and 2014AA093509), the Fundamental
223
Research Funds of Shandong University (2014QY006) and the Chinese National
224
Arctic Yellow River Station Scientific Expedition in 2011 (2011YR06006).
225
226
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Song, E. -J., Lee, M. -H., Seo, M. -J., Yim, K. J., Hyun, D. -W., Bae, J.-W., Park,
290
S. -l., Roh, S. W. & Y. -D. Nam (2014). Bizionia psychrotolerans sp. nov., a
291
psychrophilic bacterium isolated from the intestine of a sea cucumber (Apostichopus
292
japonicus). Antonie van Leeuwenhoek 106, 837–844.
293
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011).
294
MEGA5: molecular evolutionary genetics analysis using maximum likelihood,
295
evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28,
296
2731–2739.
297
Yoon, B. -J. & Oh, D. -C. (2011). Formosa spongicola sp. nov., isolated from the 13
298
marine sponge Hymeniacidon flavia. Int J Syst Evol Microbiol, 61, 330–333.
299
Yoon, J. -H., Kang C. -H., Jung Y. -T. & Kang S. -J. (2013). Bizionia
300
hallyeonensis sp. nov., isolated from seawater in an oyster farm. Int J Syst Evol
301
Microbiol 63, 685–690.
302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331
14
332
Table 1. Cellular fatty acid compositions (%) of strain SM1203T and type strains of
333
closely related species in the genera of Bizionia and Formosa. Fatty acid 1 2 3 4 Straight-chain fatty acids C15:0 5.3 4.0 2.0 TR C16:0 TR TR TR 1.4 Branched fatty acids iso-C14:0 3.4 TR TR TR iso-C15:0 25.5 18.3 34.2 27.7 iso-C15:1 20.3 15.6 24.2 20.6 anteiso-C15:0 14.2 8.0 2.9 4.1 3.4 5.8 TR 1.4 anteiso-C15:1 iso-C16:0 TR 1.2 TR TR iso-C16:1 1.2 1.6 − TR iso-C17:1ω9c TR 2.4 2.7 3.2 Hydroxy fatty acids C15:0 2-OH 1.7 1.6 TR TR 2.7 8.4 4.8 5.9 iso-C15:0 3-OH 3.9 6.0 2.0 TR iso-C16:0 3-OH C17:0 2-OH 1.2 3.5 TR TR 4.8 6.4 11.9 13.0 iso-C17:0 3-OH Unsaturated fatty acids C15:1ω6c TR 1.7 TR TR C18:1ω9c TR − TR 1.5 2.0 4.9 2.9 6.7 Summed feature 3* Unknown 13.565§ 1.5 TR 3.0 4.4
334 335
Strains:1, SM1203T (this study); 2, B. paragorgiae KCTC 12304T (this study); 3, B.
336
echini KCTC 22015T (this study); 4, F. spongicola KCTC 22662T (this study).
337
Fatty acids amounting to 5 %) in each strain are shown in bold type.
339
−, not detected.
340
TR, trace (70 %) based on 1000
358
replicates are indicated at nodes. Bar, 0.01 substitutions per
359
nucleotide position.
17
Figure 1 Click here to download Figure: Figure 1.doc
82 100 93
Formosa algae KMM3553T (AY228461) Formosa arctica IMCC9485T (KF148059)
Formosa spongicola A2T (FJ348469) Bizionia gelidisalsuginis IC164T (AY694004) 100 99 Bizionia saleffrena HFDT (AY694005) Bizionia paragorgiae KMM 6029T (AY651070) Bizionia arctica SM1203T (KJ508751) Bizionia myxarmorum ADA-4T (AY694002) Bizionia argentinensis JUB59T (EU021217) 99 Bizionia echini KMM6177T (FJ716799) 72 Bizionia psychrotolerans PB-M7 T (KJ461691) 83 Bizionia algoritergicola APA-1T (AY694003) 84 Bizionia hallyeonensis T-y7T (JN885199) Gelidibacter mesophilus 2SM29T (AJ344133)
74
0.01
Fig.1.
Formosa undariae WS-MY3T (KC832835) Formosa agariphila KMM3901T (AY187688)
Supplementary figures Click here to download Supplementary Material Files: Supplymentary figures-1.2-.pdf
International Journal of Systematic and Evolutionary Microbiology Bizionia arctica sp. nov., isolated from Arctic fjord seawater and emended description of the genus Bizionia --Manuscript Draft-Manuscript Number:
IJS-D-15-00254R1
Full Title:
Bizionia arctica sp. nov., isolated from Arctic fjord seawater and emended description of the genus Bizionia
Short Title:
Bizionia arctica sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Bin-Bin Xie Shandong University CHINA
First Author:
Hai Li
Order of Authors:
Hai Li Xi-Ying Zhang Chang Liu Ang Liu Qi-Long Qin Hai-Nan Su Mei Shi Bai-Cheng Zhou Xiu-Lan Chen Yu-Zhong Zhang Bin-Bin Xie
Manuscript Region of Origin:
CHINA
Abstract:
A Gram-negative, yellow-colored, aerobic, non-flagellated, non-gliding bacterial strain, designated SM1203T, was isolated from surface seawater of Kongsfjorden, Svalbard. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1203T was affiliated with the genus Bizionia in the family Flavobacteriaceae. The strain shared the highest 16S rRNA gene sequence similarity (>96 %) with type strains of Formosa spongicola (96.8 %), Bizionia paragorgiae (96.3 %), B. saleffrena (96.3 %) and B. echini (96.1 %) and 95.4~95.7 % sequence similarities with type strains of other known Bizionia species. The strain grew at 4-30 °C, and in the presence of 1.0-5.0 % (w/v) NaCl. The major fatty acids of strain SM1203T were iso-C15:0, iso-C15:1, anteiso-C15:0 and C15:0 and the main polar lipids were phosphatidylethanolamine and one unidentified lipid. The major respiratory quinone of strain SM1203T was menaquinone 6 (MK-6). The genomic DNA G+C content of strain SM1203T was 34.8 mol %. Based on the polyphasic characterization of strain SM1203T in this study, the strain represents a novel species in the genus Bizionia, for which the name Bizionia arctica sp. nov. is proposed. The type strain of Bizionia arctica is SM1203T (=CGMCC 1.12751T =JCM 30333T).
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1
2
Bizionia arctica sp. nov., isolated from Arctic fjord seawater
3
and emended description of the genus Bizionia
4
Hai Li1,2, Xi-Ying Zhang1,2*, Chang Liu1,2, Ang Liu1,2, Qi-Long Qin1,2, Hai-Nan Su1,2,
5
Mei Shi1,2, Bai-Cheng Zhou1,2, Xiu-Lan Chen1,2, Yu-Zhong Zhang1,2 and Bin-Bin
6
Xie1,2
7
1
8
Center, Shandong University, Jinan 250100, China
9
*Co first author: Xi-Ying Zhang contributed equally to this paper as Hai Li.
State Key Laboratory of Microbial Technology, 2Marine Biotechnology Research
10
Running title: Bizionia arctica sp. nov.
11
Subject category: New Taxa- Bacteroidetes
12
Correspondence:
13
Bin-Bin Xie
14
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100,
15
China;
16
Tel: 86-531-88364326; Fax: 86-531-88564326; E-mail: [email protected]
17
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of
18
strain SM1203T is KJ508751.
1
19 20
21
Summary
22
A Gram-negative, yellow-colored, aerobic, non-flagellated, non-gliding bacterial
23
strain, designated SM1203T, was isolated from surface seawater of Kongsfjorden,
24
Svalbard. Phylogenetic analysis based on 16S rRNA gene sequences revealed that
25
strain
26
Flavobacteriaceae. The strain shared the highest 16S rRNA gene sequence similarity
27
(>96 %) with type strains of Formosa spongicola (96.8 %), Bizionia paragorgiae
28
(96.3 %), B. saleffrena (96.3 %) and B. echini (96.1 %) and 95.4~95.7 % sequence
29
similarities with type strains of other known Bizionia species. The strain grew at
30
4–30 °C, and in the presence of 1.0–5.0 % (w/v) NaCl. The major fatty acids of strain
31
SM1203T were iso-C15:0, iso-C15:1, anteiso-C15:0 and C15:0 and the main polar lipids
32
were phosphatidylethanolamine and one unidentified lipid. The major respiratory
33
quinone of strain SM1203T was menaquinone 6 (MK-6). The genomic DNA G+C
34
content of strain SM1203T was 34.8 mol %. Based on the polyphasic characterization
35
of strain SM1203T in this study, the strain represents a novel species in the genus
36
Bizionia, for which the name Bizionia arctica sp. nov. is proposed. The type strain of
37
Bizionia arctica is SM1203T (=CGMCC 1.12751T =JCM 30333T).
SM1203T
was
affiliated
with
the
38
39
40
2
genus
Bizionia
in
the
family
41
42
The genus Bizionia belonging to the family Flavobacteriaceae in the phylum
43
Bacteroidetes was first proposed by Nedashkovskaya et al. (2005) to accommodate a
44
Gram-negative, strictly aerobic, heterotrophic, non-gliding, yellow-pigmented marine
45
bacterium. This genus currently comprises nine species: Bizionia paragorgiae (type
46
species; Nedashkovskaya et al., 2005), B. algoritergicola (Bowman & Nichols, 2005),
47
B. myxarmorum (Bowman & Nichols, 2005), B. saleffrena (Bowman & Nichols,
48
2005), B. argentinensis (Bercovich et al., 2008), B. echini (Nedashkovskaya et al.,
49
2010), B. gelidisalsuginis (Bowman & Nichols, 2005), B. hallyeonensis (Yoon et al.,
50
2013) and B. psychrotolerans (Song et al., 2014). Five of them (Bizionia
51
algoritergicola, B. myxarmorum, B. saleffrena, B. gelidisalsuginis and B.
52
argentinensis) have been isolated from Antarctic marine habitats including costal
53
seawater, sea-ice brines and amphipods, while other Bizionia species have been
54
isolated from coastal seawater and invertebrates from temperate seas. During a
55
bacterial diversity study of Arctic seawater samples, a yellow-pigmented Bizionia-like
56
bacterial strain, designated SM1203T, was isolated. In this study, we report the
57
polyphasic characterization of the new isolate and propose that it represents the first
58
Arctic species in the genus Bizionia.
59
The Arctic surface seawater sample was collected from Kongsfjorden, Svalbard
60
(78º 59′ N, 11º 40′ E) in July, 2011, during the 8th Chinese National Arctic Yellow
61
River Station Scientific Expedition (summer). Approximately 0.8 liter of seawater
62
sampled was vacuum-filtered through a 0.22 μm membrane filter. The membrane was
63
then suspended in 50 ml of sterile artificial seawater and was vigorously shaken for 30
64
min at 15 °C. The suspension obtained was directly spread onto trypticase yeast
3
65
extract (TYS) agar [0.5 % tryptone (Oxoid), 0.1 % yeast extract (Oxoid), 1.5 % agar
66
and artificial seawater] plates and incubated at 15 °C for 2–3 weeks. Artificial
67
seawater was prepared with commercial sea salts (Sigma, 3 %). Colonies showing
68
different morphologies on the plates were selected and purified by repeated streaking
69
on TYS agar plates. Strain SM1203T isolated was preserved at −80 ºC in TYS broth
70
(0.5 % tryptone, 0.1 % yeast extract and artificial seawater) containing 20 % (v/v)
71
glycerol. It was routinely cultivated on TYS agar or in TYS broth at 20 °C. Reference
72
strains used for some phenotypic tests and fatty acids analysis, including B.
73
paragorgiae KCTC 12304T, B. echini KCTC 22015T, B. saleffrena CIP 108534T, B.
74
gelidisalsuginis CIP 108536T and Formosa spongicola KCTC 22662T, were also
75
routinely cultivated on TYS agar or in TYS broth at 20 °C.
76
Genomic DNA of strain SM1203T for the 16S rRNA gene amplification was
77
extracted using a BioTeke (China) bacterial genomic DNA isolation kit according to
78
the manufacturer’s protocol. The 16S rRNA gene of strain SM1203T was
79
PCR-amplified using primers: 8-27F (5′-AGAGTTTGATCTTGGCTCAG-3′) and
80
1492-1510R (5′-TACGGCTACCTTGTTACGACTT-3′) (Lane, 1991) and then
81
sequenced by using an Applied Biosystems 3730 automated DNA sequencer. The
82
resultant 16S rRNA gene sequence was compared with those of validly published
83
bacterial species in the EzTaxon-e database using the BLASTN program
84
(http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). Phylogenetic trees were
85
reconstructed by the neighbor-joining (Saitou & Nei, 1987), maximum likelihood
86
(Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) methods using MEGA
87
version 5 (Tamura et al., 2011), with bootstrap analyses based on 1000 replicates
88
(Felsenstein, 1985) and the Kimura’s two-parameter model (Kimura, 1980). Values of
89
pairwise sequence similarity calculated using the global alignment algorithm were 4
90
also obtained through the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et
91
al., 2012).
92
The almost-complete 16S rRNA gene sequence of strain SM1203T was
93
determined (1,442 bp) and comparative analysis of the sequence showed that strain
94
SM1203T shared the highest 16S rRNA gene sequence similarities (>96 %) with
95
Formosa spongicola (96.8 %), B. (96.3 %), B. saleffrena (96.3 %) and B. echini
96
(96.1 %). Sequence similarities with other recognized Bizionia species were
97
95.4~95.7 % and with other Formosa species were 95.2~95.9 %. It was also found
98
that strain SM1203T exhibited relatively high 16S rRNA gene sequence similarities
99
(95.0~95.5 %) with respect to some species in the genera of Flavivirga, Olleya,
100
Flaviramulus, Algibacter, Winogradskyella, Gaetbulibacter and Arenitalea in the
101
family Flavobacteriaceae. However, the phylogenetic trees based on 16S rRNA gene
102
sequences
103
maximum-parsimony methods all showed that strain SM1203T was grouped into the
104
genus Bizionia and formed a sub-group with B. paragorgia, B. saleffrena and B.
105
gelidisalsuginis (Fig. 1 and Supplementary Figs. S1 and S2).
inferred
by
the
neighbor-joining,
maximum-likelihood
and
106
For the cellular fatty acid analysis, strain SM1203T and the reference strains: B.
107
paragorgiae KCTC 12304T, B. KCTC 22015T, and F. spongicola KCTC 22662T were
108
all cultivated in TYS broth at 20 ºC for 3 days. Cellular fatty acids were analyzed by
109
GC (Hewlett Packard 6890) using the Sherlock MIS software (version 4.5 and the
110
TSBA40 database). Polar lipids were extracted as previously descried (Komagata &
111
Suzuki, 1987) and further analyzed using two-dimensional TLC with appropriate
112
spraying reagents including ethanolic molybdophosphoric acid [10 % (w/v), total
113
lipids], ninhydrin (aminolipids) and Zinzadze reagent (phospholipids) (Collins &
5
114
Jones, 1980). Quinones were extracted as described by Komagata & Suzuki (1987)
115
and analyzed using an LC-MS system consisting of a Dionex Ultimate 3000 HPLC
116
coupled with a Bruker Impact HD mass spectrometer. The genomic DNA G+C
117
content of strain SM1203T was determined by the thermal denaturation method
118
(Marmur & Doty, 1962) using a Beckman DU800 Spectrophotometer with
119
chromosomal DNA extracted using the method of Marmur (1961).
120
Fatty acids of strain SM1203T predominantly included (≥5 %) iso-C15:0 (25.5 %),
121
iso-C15:1 (20.3 %), anteiso-C15:0 (14.2 %) and C15:0 (5.3 %) with iso-C17:0 3-OH
122
(4.8 %), iso-C16:0 3-OH (3.9 %), anteiso-C15:1 (3.4 %), iso-C14:0 (3.4 %), iso-C15:0
123
3-OH (2.7 %), summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH) (2.0 %), C15:0 2-OH
124
(1.7 %), C17:0 2-OH (1.2 %), iso-C16:1 H (1.2 %) and an unknown acid with an
125
equivalent chain-length of 13.565 (1.5 %) being minor components (>1 %), showing a
126
profile essentially similar to those of B. paragorgiae KCTC 12304T and B. echini
127
KCTC 22015T (Table 1). But proportions of iso-C15:0 (25.5 %) and iso-C15:1 (20.3%)
128
in strain SM1203T were significantly higher than those in B. paragorgiae KCTC
129
12304T (18.3 % for iso-C15:0 and 15.6 % for iso-C15:1, respectively) while lower than
130
those in B. echini KCTC 22015T (34.2 % and 24.2 %, respectively); in addition,
131
proportion of anteiso-C15:0 (14.2 %) in strain SM1203T was apparently higher than
132
those in B. paragorgiae KCTC 12304T (2.9 %) and B. echini KCTC 22015T (8.0 %).
133
These reflected the difference among them. Major lipids of strain SM1203T were
134
phosphatidylethanolamine (PE) and one unidentified lipid (L1) with moderate
135
amounts of one unidentified phospholipid (PL) and aminolipid (AL1) and minor
136
amounts of one unidentified lipid (L2) and aminophospholipid (APL) and two
137
unidentified aminolipids (AL2 and AL3) (Supplementary Fig. S3). The major
138
respiratory quinone of strain SM1203T was menaquinone 6 (MK-6). The DNA G+C 6
139
content of strain SM1203T was 34.8 mol %, within the range reported for the genus
140
Bizionia (34.0−45.0 mol %).
141
The temperature (4, 10−45 °C at intervals of 5 °C) and pH (4.0−9.0 in
142
increments of 0.5 pH units) ranges for growth were determined in TYS broth. For the
143
pH range examination, various buffers including MES (pH 5.0−6.0), MOPS (pH
144
6.5−7.0), Tris (pH 7.5−8.5) and CHES (pH 9.0) were respectively added into the TYS
145
broth at the concentration of 50 mM. Growth at different concentrations of NaCl (0,
146
0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 %, w/v) was investigated in a medium containing 0.5 %
147
tryptone (Oxoid), 0.1 % yeast extract (Oxoid), 0.5 % MgCl2, 0.2 % MgSO4, 0.05 %
148
CaCl2, 0.1 % KCl, 0.0001 % FeSO4 and distilled water supplemented with appropriate
149
amounts of NaCl.
150
Gram-staining was performed using the Hucker’s method as described in Murray
151
et al. (1994). Cell morphology and flagellation were examined using a transmission
152
electron microscopy (JEM−100CX II) with cells grown in TYS broth at 20 °C for 3
153
days. Oxidase activity was detected using commercial oxidase test strips (Merck) and
154
catalase activity was assessed by observation of the bubble production in 3 % (v/v)
155
hydrogen peroxide solution. The presence of gliding motility was checked using the
156
hanging drop technique and the ability to produce flexirubin-type pigments were
157
detected by the KOH test (Bernardet et al. 2002). DNA hydrolysis was determined on
158
DNase agar (Oxoid). The ability to hydrolyze carboxymethyl-cellulose (CMC) was
159
examined on MA containing 1% (w/v) CMC (Sigma) (Lee et al., 2012). Hydrolysis of
160
other ploymers including agar, casein, gelatin, starch and Tweens 20, 40, 60 and 80
161
was determined as described in Smibert & Krieg (1994). Susceptibility to
162
antimicrobial agents was examined using the disc-diffusion method on TYS agar
7
163
(Kobayashi et al., 2003) using Oxoid susceptibility discs impregnated with different
164
antimicrobial agents including ampicillin (10 μg/disc), carbenicillin (100 U/disc),
165
cephalexin (30 μg/disc), chloramphenicol (30 μg/disc), erythromycin (15 μg/disc),
166
gentamicin (10 μg/disc), kanamycin (30 μg/disc), neomycin (30 μg/disc), novobiocin
167
(5 μg/disc), penicillin G (10 U/disc), polymyxin B (300 U/disc), streptomycin (10
168
μg/disc), tetracycline (30 μg/disc) and vancomycin (30 μg/disc). Anaerobic growth
169
was tested in TYS broth in Hungate tubes, supplemented with potassium nitrate
170
(0.1 %, w/v), cysteine hydrochloride (0.05 %, w/v) and sodium sulfide (0.05 %, w/v).
171
Other enzymatic activities and biochemical properties were tested using API ZYM
172
and API 20NE strips (bioMérieux), following the manufacturer’s instructions with
173
inocula prepared by suspending cells in sterile artificial sea water.
174
Cells of strain SM1203T were Gram-negative, non-flagellated, non-gliding long
175
thin rods. Anaerobic growth was not observed. Other phenotypic characteristics of
176
strain SM1203T were given in the species description. Strain SM1203T could be
177
phenotypically differentiated from the closely related species by a number of
178
characteristics, including temperature and NaCl concentration ranges for growth,
179
nitrate reduction, hydrolysis of various substrates, assimilation of carbohydrates and
180
genomic DNA G+C content, as shown in Table 2. Thus, according to the phylogenetic
181
analysis of the 16S rRNA gene sequences and the results from the chemotaxonomic
182
and phenotypic characterizations, we concluded that strain SM1203T represents a
183
novel Bizionia species, for which the name Bizionia arctica sp. nov. is proposed.
184
Description of Bizionia arctica sp. nov.
185
Bizionia arctica (arc'ti.ca. L. fem. adj. arctica northern, from the Arctic)
8
186
Cells of the type strain are Gram-negative, aerobic, non-flagellated and
187
non-gliding rods, 1.8−4 μm in length and 0.3−0.5 μm in diameter. When incubated on
188
TYS agar at 20 °C for 5-7 days, colonies are yellow-pigmented, shiny, circular
189
(0.3−1.2 mm in diameter) with entire edges and convex. The type strain is oxidase-
190
negative and catalase-positive. It hydrolyzes casein, gelatin and Tweens 20, 40 and 80
191
but does not hydrolyze agar, carboxymethyl-cellulose (CMC), DNA, aesculin, starch
192
or Tween 60. The type strain grows at 4−30 °C (optimum at 20 °C), in the presence of
193
1−6 % NaCl (w/v, optimum in 2 %) and at pH 5.5−8.5 (optimum at pH 7.0). It does
194
not produce flexirubin-type pigments. In API 20NE tests, the type strain is positive for
195
arginine dihydrolase, acid production from glucose and hydrolysis of gelatin. In
196
addition, it does not utilize D-glucose, gluconate, caprate, arabinose, mannose,
197
mannitol, N-acetylglucosamine, maltose, adipate, malate, citrate and phenylacetate. In
198
the API ZYM tests, the type strain is positive for alkaline phosphatase, esterase (C4),
199
esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase,
200
trypsin, α-chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase but
201
negative for lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase,
202
α-glucosidase,
203
β-fucosidase. The type strain is susceptible to ampicillin, carbenicillin, cephalexin,
204
vancomycin,
205
chloramphenicol, but resistant to gentamicin, kanamycin, neomycin, polymyxin B and
206
streptomycin. The major fatty acids (>5 %) of the type strain are iso-C15:0, iso-C15:1,
207
anteiso-C15:0
208
phosphatidylethanolamine, two unidentified lipids, one unidentified phospholipid,
209
three unidentified aminolipids and one unidentified aminophospholipid. The major
210
respiratory quinone of the type strain is menaquinone 6 (MK-6). The genomic G+C
β-glucosidase,
penicillin
and
C15:0.
G,
The
N-acetyl-β-glucosaminidase,
erythromycin,
polar
lipids
9
α-mannosidase
tetracycline,
of
the
type
novobiocin
strain
and
and
comprise
211
212
content of the type strain is 34.8 mol %. The type strain is SM1203T (=CGMCC 1.12751T =JCM 30333T), which was
213
isolated from surface seawater of Kongsfjorden, Svalbard.
214
Emended description of the genus Bizionia Nedashkovskaya et al. (2005)
215
The description is as given by Nedashkovskaya et al. (2005) with the following
216
amendments. Species in the genus Bizionia are oxidase- positive or negative.
217
Acknowledgements
218
The work was supported by the China Ocean Mineral Resources R & D Association
219
(COMRA) Special Foundation (grants DY125-15-R-03 and DY125-15-T-05), the
220
National Natural Science Foundation of China (grants 31270117, 31290231,
221
31470541 and 41376153), the Hi-Tech Research and Development Program of China
222
(grants 2012AA092103, 2012AA092105 and 2014AA093509), the Fundamental
223
Research Funds of Shandong University (2014QY006) and the Chinese National
224
Arctic Yellow River Station Scientific Expedition in 2011 (2011YR06006).
225
226
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227
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Bernardet, J.-F., Nakagawa, Y. & Holmes, B. (2002). Proposed minimal standards 10
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Bowman, J. P. & Nichols, D. S. (2005). Novel members of the family
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Table 1. Cellular fatty acid compositions (%) of strain SM1203T and type strains of
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closely related species in the genera of Bizionia and Formosa. Fatty acid 1 2 3 4 Straight-chain fatty acids C15:0 5.3 4.0 2.0 TR C16:0 TR TR TR 1.4 Branched fatty acids iso-C14:0 3.4 TR TR TR iso-C15:0 25.5 18.3 34.2 27.7 iso-C15:1 20.3 15.6 24.2 20.6 anteiso-C15:0 14.2 8.0 2.9 4.1 3.4 5.8 TR 1.4 anteiso-C15:1 iso-C16:0 TR 1.2 TR TR iso-C16:1 1.2 1.6 − TR iso-C17:1ω9c TR 2.4 2.7 3.2 Hydroxy fatty acids C15:0 2-OH 1.7 1.6 TR TR 2.7 8.4 4.8 5.9 iso-C15:0 3-OH 3.9 6.0 2.0 TR iso-C16:0 3-OH C17:0 2-OH 1.2 3.5 TR TR 4.8 6.4 11.9 13.0 iso-C17:0 3-OH Unsaturated fatty acids C15:1ω6c TR 1.7 TR TR C18:1ω9c TR − TR 1.5 2.0 4.9 2.9 6.7 Summed feature 3* Unknown 13.565§ 1.5 TR 3.0 4.4
334 335
Strains:1, SM1203T (this study); 2, B. paragorgiae KCTC 12304T (this study); 3, B.
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echini KCTC 22015T (this study); 4, F. spongicola KCTC 22662T (this study).
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Fatty acids amounting to 5 %) in each strain are shown in bold type.
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−, not detected.
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TR, trace (70 %) based on 1000
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replicates are indicated at nodes. Bar, 0.01 substitutions per
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nucleotide position.
17
Figure 1 Click here to download Figure: Figure 1.doc
82 100 93
Formosa algae KMM3553T (AY228461) Formosa arctica IMCC9485T (KF148059)
Formosa spongicola A2T (FJ348469) Bizionia gelidisalsuginis IC164T (AY694004) 100 99 Bizionia saleffrena HFDT (AY694005) Bizionia paragorgiae KMM 6029T (AY651070) Bizionia arctica SM1203T (KJ508751) Bizionia myxarmorum ADA-4T (AY694002) Bizionia argentinensis JUB59T (EU021217) 99 Bizionia echini KMM6177T (FJ716799) 72 Bizionia psychrotolerans PB-M7 T (KJ461691) 83 Bizionia algoritergicola APA-1T (AY694003) 84 Bizionia hallyeonensis T-y7T (JN885199) Gelidibacter mesophilus 2SM29T (AJ344133)
74
0.01
Fig.1.
Formosa undariae WS-MY3T (KC832835) Formosa agariphila KMM3901T (AY187688)
Supplementary figures Click here to download Supplementary Material Files: Supplymentary figures-1.2-.pdf
