IJSEM Papers in Press. Published February 25, 2015 as doi:10.1099/ijs.0.000146
International Journal of Systematic and Evolutionary Microbiology Mameliella phaeodactyli sp. nov., a member of the family Rhodobacteraceae isolated from the marine algae Phaeodactylum tricornutum --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00212R3
Full Title:
Mameliella phaeodactyli sp. nov., a member of the family Rhodobacteraceae isolated from the marine algae Phaeodactylum tricornutum
Short Title:
Mameliella phaeodactyli sp. nov.,
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Tianling Zheng Xiamen University CHINA
First Author:
Zhangran Chen
Order of Authors:
Zhangran Chen Jingyan Zhang Xueqian Lei Qiliang Lai Luxi Yang Huajun Zhang Yi Li Wei Zheng Yun Tian Zhiming Yu Hong Xu Tianling Zheng
Manuscript Region of Origin:
CHINA
Abstract:
A novel Gram-staining-negative, aerobic, rod-shaped, non-motile, yellow bacterium designated strain KD53T, was isolated from a culture of the alga Phaeodactylum tricornutum, Xiamen, Fujian Province, China. 16S rRNA gene sequence comparison showed that strain KD53T was a member of the Roseobacter clade within the family Rhodobacteraceae, forming a distinct lineage with species of the genus Mameliella. The 16S rRNA gene sequence similarities between strain KD53T and other strains were all less than 97.0 %. Strain KD53T was found to grow optimally at 28 °C, at pH 7.5-8.0 and in the presence of 3 % (w/v) NaCl. The dominant fatty acids of strain KD53T were C18:1 ω6c, C18:1 ω7c, C18:0, C16:0. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol. The DNA G+C content was 65 mol% and the major respiratory quinone was ubiquinone 10 (Q-10). On the basis of phenotypic data and phylogenetic inference, strain KD53T represents a novel strain of Mameliella, then the name Mameliella phaeodactyli sp. nov. is proposed. The type strain is KD53T (= MCCC 1K00273T = KCTC 42178T).
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1
Mameliella phaeodactyli sp. nov., a member of the family
2
Rhodobacteraceae isolated from the marine algae Phaeodactylum
3
tricornutum.
4
Zhangran Chen1†, Jingyan Zhang1, Xueqian Lei1, Qiliang Lai1,2, Luxi Yang1, Huajun
5
Zhang1, Yi Li1, Wei Zheng1, Yun Tian1, Zhiming Yu3，Hong Xu1*, Tianling Zheng1*
6
1
7
Education for Coastal and Wetland Ecosystem, School of Life Sciences, Xiamen
8
University, Xiamen 361005, China
9
2
State Key Laboratory of Marine Environment and Key lab of the Ministry of
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography,
10
State Oceanic Administration, People’s Republic of China
11
3
12
Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China
13
*Corresponding author: Tianlin Zheng. E-mail: [email protected]; Hong Xu.
14
E-mail: [email protected]; Tel: +86-592-2183217. Fax: +86-592-2184528
15
Subject category: New Taxa (Proteobacteria)
16
Running title: Mameliella phaeodactyli sp. nov.
17
Abbreviations: KCTC, Korean Collection For Type Cultures; DSMZ, German
18
Collection of Microorganisms and Cell Cultures; MCCC, Marine Culture Collection
19
of China. CGMCC, China General Microbiological Culture Collection Center; CICC,
20
China center of industrial culture collection.
Key Laboratory of Marine Ecology and Environmental Science, Institute of
21 22
The GenBank [/EMBL/DDBJ] accession number for the 16S rRNA gene sequence of 1
23
strain KD53T is KJ850205.
24 25
Abstract:
26
A novel Gram-staining-negative, aerobic, rod-shaped, non-motile, yellow bacterium
27
designated strain KD53T, was isolated from a culture of the alga Phaeodactylum
28
tricornutum, Xiamen, Fujian Province, China. 16S rRNA gene sequence comparison
29
showed that strain KD53T was a member of the Roseobacter clade within the family
30
Rhodobacteraceae, forming a distinct lineage with species of the genus Mameliella.
31
The 16S rRNA gene sequence similarities between strain KD53T and other strains
32
were all less than 97.0 %. Strain KD53T was found to grow optimally at 28 °C, at pH
33
7.5–8.0 and in the presence of 3 % (w/v) NaCl. The dominant fatty acids of strain
34
KD53T were C18:1 ω6c, C18:1 ω7c, C18:0, C16:0. The major polar lipids were
35
phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. The DNA
36
G+C content was 65 mol% and the major respiratory quinone was ubiquinone 10
37
(Q-10). On the basis of phenotypic data and phylogenetic inference, strain KD53T
38
represents a novel strain of Mameliella, then the name Mameliella phaeodactyli sp.
39
nov. is proposed. The type strain is KD53T (= MCCC 1K00273T = KCTC 42178T).
40 41
Members of the Roseobacter clade (Wagner-Döbler et al., 2003) in the order
42
Rhodobacterales of the class Alphaproteobacteria are abundant in marine
43
environments. The abundance and diverse physiological characteristics within this
44
group suggest that they have the function to degrade aromatic compounds (Buchan et 2
45
al., 2005) and participate in the biogeochemical cycles of carbon and sulfur. In this
46
study, strain KD53T, which was isolated from coastal seawater, was found to form a
47
clade with Mameliella alba CGMCC 1.7290T (Zheng et al., 2010), after comparative
48
16S rRNA gene sequence analysis. Accordingly, the aim of the present work was to
49
determine the exact taxonomic position of strain KD53T by using polyphasic
50
characterization including the determination of phenotypic properties and a detailed
51
phylogenetic analysis based on 16S rRNA gene sequences.
52 53
Strain KD53T was isolated from a culture of the alga Phaeodactylum tricornutum,
54
Xiamen, Fujian Province, China in December 2013. The samples were serially diluted
55
(10-fold dilution) by sterile seawater and 0.1 mL aliquots of each dilution were spread
56
onto marine agar 2216 (MA; Difco) followed by incubation for 3 days at 28 °C.
57
Individual colonies of distinct morphology were further purified three times and
58
stored at -80 °C in marine broth 2216 (MB; Difco) supplemented with 10 % (v/v)
59
glycerol.
60 61
The genomic DNA of strain KD53T was extracted according to the method of Ausubel
62
et al. (1995) and the 16S rRNA gene was amplified by PCR using the primer pair
63
P27F and P1492R (DeLong, 1992). Purification of the PCR product was carried out
64
using a TIANquick Midi purification kit (TIANGEN, China) and the purified PCR
65
product was cloned into vector pMD19-T and sequenced. Sequences of related taxa
66
were downloaded from the GenBank database and the EzTaxon-e server 3
67
(http://eztaxon-e.ezbiocloud.net/) (Kim et al., 2012). Phylogenetic analysis was
68
performed using MEGA version 4 (Tamura et al., 2007) after multiple alignment of
69
data by DNAMAN (version 5.1). Evolutionary distances and clustering were
70
performed by using the neighbour-joining method (Saitou & Nei, 1987). The resulting
71
tree topology was evaluated by using bootstrap analysis based on 1000 replicates.
72 73
A nearly full-length 16S rRNA gene sequence (1371 bp, GenBank [/EMBL/DDBJ]
74
accession number KJ850205) of strain KD53T was determined. Preliminary
75
comparison of the 16S rRNA gene sequence of strain KD53T with other sequences
76
indicated that the new isolate showed 96.71% similarity to Ponticoccus litoralis DSM
77
18986T (GenBank no. EF211829). Phylogenetic analysis of the 16S rRNA sequence
78
of strain KD53T indicated that it belongs to the family Rhodobacteraceae (Fig. 1, Fig.
79
S2 and S3). In the phylogenetic tree based on the neighbour-joining algorithm, strain
80
KD53T joined a phylogenetic clade comprising M. alba CGMCC 1.7290T, with which
81
it exhibited the 16S rRNA gene sequence similarity (95.6%). The relatively low levels
82
of 16S rRNA gene sequence similarity to its most closely related strain suggested that
83
strain KD53T may represent a novel species of the genus Mameliella.
84 85
Cell morphology and motility were observed by using transmission electron
86
microscopy (model JEM-2100HC; JEOL) and phase-contrast light microscopy (model
87
50i; Nikon), with cells from the early exponential phase grown on MA at 28 °C.
88
Spore production was determined as Gonzalez et al described (1997). The presence of 4
89
flexirubin-type pigments was assessed using the bathochromic shift test with 20%
90
(w/v) KOH, as described by Bernardet (2002). Colony morphology was examined
91
from cultures grown on MA for 6 days. Gliding motility was investigated as described
92
by Bowman (2000). The Gram reaction was determined by using the bioMérieux
93
Gram stain kit according to the manufacturer’s instructions. Anaerobic growth was
94
assessed on MA that was autoclaved and cooled to room temperature under nitrogen
95
atmosphere (99.999 % purity). Triplicate cultures were grown in 50 ml anaerobic
96
serum bottles sealed with thick butyl rubber stoppers and aluminum caps, and
97
incubated statically in the dark at 28 °C for 21 days. Growth in MB was tested at 4, 10,
98
16, 20, 28, 30, 37 and 42 °C and at pH 3.0-10.0 (at 1 pH unit intervals during 3.0-7.0
99
and 0.5 pH unit intervals during 7.0-10.0). The pH of MB was adjusted prior to
100
sterilization using the following buffers: citric acid/sodium citrate (pH 3.0–6.0),
101
Na2HPO4/citric acid (pH 7.0–8.0) and Lysine/NaOH (pH 9.0–10.0). Verification of
102
the pH values after autoclaving revealed only minor changes (Su et al., 2013). The
103
NaCl concentration range and optimum for growth were determined in NaCl-free MB
104
(tryptone 5.0 g L-1, yeast extract 1.0 g L-1, 1L distilled water, pH 7.6 ~7.8),
105
supplemented with 0–7% (at 1% intervals) and 9.0–13.0% (at 2% intervals) NaCl
106
(w/v). Catalase and oxidase activities were assessed by addition of 3% Hydrogen
107
peroxide to exponential-phase colonies and by using oxidase reagent (bioMérieux),
108
respectively. Hydrolysis of starch, chitin, tyrosine, casein, gelatin, urea and Tweens 20,
109
40, 60 and 80 was tested using MA supplemented with 0.5 % (w/v) of starch and 1 %
110
(w/v) of the other substrates. Results were examined twice after growth on agar plates 5
111
for 3 and 5 days. The above mentioned tests were carried out for strain KD53T and the
112
four reference strains. Biochemical tests were carried out using API 20NE, API 20E
113
and API ZYM strips (bioMérieux) according to the manufacturer’s instructions,
114
except that the NaCl concentration in all tests was adjusted to 3.0%. Susceptibility to
115
antibiotics was tested on MA for 4 days by using filter-paper discs (OXOID)
116
containing various antibiotics (Lányí, 1987, Smibert & Krieg, 1994) and the results
117
showed that strain KD53T was susceptible to cefazolin, ampicillin, carbenicillin,
118
ciprofloxacin, vibramycin, erythromycin, kanamycin, minomycin, norfloxacin,
119
ofloxacin, oxacillin, penicillin G, polymyxin B, piperacillin, streptomycin, tetracycline,
120
vancomycin, novobiocin, trimethoprim, neomycin; Resistant to sulphafurazole,
121
metronidazole, clindamycin and chloramphenicol. All above-mentioned tests were
122
incubated at 28 °C. All strains were tested using MA or MB except Antarctobacter
123
heliothermus DSM 11445T) (grown on R2A agar). The novel isolate displayed the
124
characteristics found in many members of the family Rhodobacteraceae, e.g.
125
rod-shaped, non-motile bacterium (Hwang et al., 2008; Zheng et al., 2010). Other
126
phenotypic properties of strain KD53T are given in the species description and in
127
Table 1.
128 129
To determine the DNA G+C content, genomic DNA was extracted from cells cultured
130
on MA for 3 days at 28 °C and analysed by reverse-phase HPLC (Tamaoka &
131
Komagata, 1984). The DNA G+C content of the new isolate KD53T was 65 mol%,
132
which is within the range reported for the members of the family Rhodobacteraceae 6
133
(Zheng et al., 2010; Labrenz et al., 1998., Hwang et al., 2008).
134 135
For cellular fatty acid analysis, the fatty acids of strain KD53T and the four reference
136
strains except Antarctobacter heliothermus DSM 11445 (grown on R2A agar) grown
137
on MA at 28 °C for 4 days were saponified, methylated and extracted using the
138
standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B).
139
The fatty acids were analysed by GC (Agilent Technologies 6850) and identified by
140
using the TSBA6 database of the Microbial Identification System (Sasser, 1990). The
141
five strains had similar growth rates at 28 °C and the same physiological age at the
142
time they were harvested. The dominant fatty acids of strain KD53T were identified as
143
summed feature 8 (comprising C18:1 ω6c and/or C18:1 ω7c, 75.3%), C18:0 (9.3%), C16:0
144
(5.6%), which accounted for 90.2% of the total. The fatty acid profile of the strain
145
KD53T was observed to be in good agreement with those of members of family
146
Rhodobacteraceae (Gonzalez et al., 1997; Labrenz et al., 1998; Hwang et al., 2008;
147
Zheng et al., 2010) but it can be differentiated from other species by the percentage of
148
C18:0
149
phosphatidylglycerol were the major polar lipids detected in strain KD53 (Fig. S4,
150
available in the online Supplementary Material), along with small amounts of
151
unidentified
152
Phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol have also
153
been reported to be the major constituents of the polar lipids in the P. litoralis DSM
154
18986T, although unidentified phospholipid was not the major polar lipid (Hwang &
(Table
2).
Phosphatidylcholine,
phospholipids,
unidentified
7
phosphatidylethanolamine
aminolipids,
unknown
and
lipids.
155
Cho, 2008).
156 157
The major respiratory quinone of the strain KD53T was analyzed at the CICC on an
158
LDC Analytical (thermo separation products) HPLC fitted with a reverse phase
159
column (Macherey–Nagel, 2 mm × 125 mm, 3 μm, RP18) using Methanol:Heptane
160
9:1 (v/v) as the eluent. The major respiratory ubiquinone present in strain KD53T was
161
Q-10, in line with Rhodobacteraceae species (Zheng et al., 2010;. Hwang et al.,
162
2008).
163 164
On the basis of morphological, physiological and chemotaxonomic characteristics, as
165
well as phylogenetic inference (Tables 1, 2 and Fig.1), strain KD53T should be
166
assigned to a novel species of in the genus Mameliella for which the name Mameliella
167
phaeodactyli sp. nov. is proposed.
168 169
Description of Mameliella phaeodactyli sp. nov.
170
Mameliella
171
Phaeodactylum tricornutum).
phaeodactyli
(phae.o.dac'ty.li.
N.L.
gen.
n.
phaeodactyli
of
172 173
Cells are gram staining negative, aerobic, non-spore-forming, non-flagella, rod shaped,
174
with cells 2.2-2.9 μm in length and 0.5-1.0 μm (Fig. S1, available in the online
175
Supplementary Material) in width. On MA medium plates, strain KD53T forms yellow,
176
circular, small, translucent and smooth colonies after 3 days incubation at 28 °C. The 8
177
diameter of the colony is 0.5-2 mm. Strain KD53T is found to grow at 16-37 °C
178
(optimum 28 °C), in 1-6% NaCl (optimum 3%), at pH 6.0-9.5 (optimum 7.5-8.0).
179
Strain KD53T cannot grow under anaerobic conditions or in the absence of NaCl. It
180
can hydrolyze tyrosine and urea, but not Tweens 20, 40, 60, 80, casein, starch, chitin.
181
In the API 20E test, positive for ß-galactosidase, arginine dihydrolase, urease,
182
tryptophane deaminase, gelatinase, production of acetoin, utilization of glucose,
183
saccharose, amygdalin and arabinose; negative for lysine decarboxylase, ornithine
184
decarboxylase activities, citrate utilization, production of H2S and indole, utilization
185
of mannitol, inositol, sorbitol, rhamnose and melibiose. In the API 20NE test, positive
186
for nitrate reduction, arginine dihydrolase, urease, aesculin hydrolysis, gelatin
187
hydrolysis,
188
N-acetyl-glucosamine, D-maltose, potassium gluconate; Weakly positive for
189
utilization of L-arabinose, D-mannose, adipic acid, malic acid and trisodium citrate;
190
negative for indole production, D-glucose fermentation, utilization of capric acid and
191
phenylacetic acid. According to the API ZYM test, positive for alkaline phosphatase,
192
esterase (C4), esterase lipase (C8), lipase (C14), leucine aminopeptidase, valine
193
aminopeptidase, cystine aminopeptidase, trypsin, β-glucosidase, α-mannosidase;
194
negative for α-chymotrypsin, acid phosphatase, naphtol-AS-Bl-phosphoamidase,
195
α-galactosidase,
196
N-acetyl-β-glucosaminidase, α-fucosidase. The predominant fatty acids (>5% of the
197
total) are Summed Feature 8 (comprising C18:1 ω6c and/or C18:1 ω7c), C18:0, C16:0. The
198
complete fatty acid composition is given in Table 2. The DNA G+C content of the
ß-galactosidase,
utilization
β-galactosidase,
of
D-glucose,
β-glucuronidase,
9
D-mannitol,
α-glucosidase,
199
new isolate KD53T is 65 mol%. The major respiratory quinone is Q-10 and the major
200
polar
201
phosphatidylglycerol. Other phenotypic characteristics are given in Table 1.
lipids
are
phosphatidylcholine,
phosphatidylethanolamine
and
202 203
The type strain, KD53T (= MCCC 1K00273T = KCTC 42178T), is isolated from a
204
culture of marine alga Phaeodactylum tricornutum, Xiamen, China.
205 206
Emended description of the genus Mameliella
207
This emended description of the genus Mameliella is based on the description of
208
Mameliella alba published by Zheng et al. (2010) and on this study.
209 210
Cells are gram staining negative, aerobic, rod shaped, non-flagella and non-motile.
211
Positive for oxidase activities but negative for hydrolyse of chitin, Tween 20, 40, 60
212
and 80. The predominant fatty acids (>5% of the total) are Summed Feature 8
213
(comprising C18:1 ω6c and/or C18:1 ω7c), C18:0 and C16:0. The major respiratory
214
quinone is Q-10. The DNA G+C content is approximately 64 mol%.
215 216
Acknowledgements：
217
This work was supported by the Joint project of National Natural Science Foundation
218
of China (NSFC) and Shandong province, Marine ecology and environmental
219
sciences (U1406403), public science and technology research funds projects of ocean
220
(201305016) and the National Nature Science Foundation of China (41376119, 10
221
41476095).
222 223
References
224
Ausubel, F., Brent, R., Kingston, R. E., Moore, D., Seidman, J. & Smith, J. K.
225
Struhl, K.(editors) (1995). Short Protocols in Molecular Biology: a Compendium of
226
Methods from Current Protocols in Molecular Biology, 3rd edn. New York: Wiley.
227
Bernardet, J. F., Nakagawa, Y. & Holmes, B. (2002). Proposed minimal standards
228
for describing new taxa of the family Flavobacteriaceae and emended description of
229
the family. Int J Syst Evol Micr .52. 1049-1070.
230
Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov., isolated from
231
the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell
232
and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst
233
Evol Micr. 50, 1861-1868.
234
Buchan, A., González, J. M. & Moran, M. A. (2005). Overview of the marine
235
Roseobacter lineage. Appl Environ Microb 71, 5665-5677.
236
DeLong, E. F. (1992). Archaea in coastal marine environments. P Natl Acad Sci USA.
237
89, 5685-5689.
238
González, J., Mayer, F., Moran, M., Hodson, R., Whitman, W. (1997). Sagittula
239
stellata gen. nov., sp. nov., a lignin-transforming bacterium from a coastal
240
environment. Int J Syst Bacteriol, 47(3), 773-780.
241
Hwang, C. Y. & Cho, B. C. (2008). Ponticoccus litoralis gen. nov., sp. nov., a marine
242
bacterium in the family Rhodobacteraceae. Int J Syst Evol Micr 58, 1332-1338. 11
243
Kates, M. (1986). Lipid extraction procedures in Techniques of lipidology: Isolation,
244
Analysis and Identification of Lipids. Edited by M. Kates. Elsevier, Amsterdam,
245
100-111.
246
Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., Jeon, Y.
247
S., Lee, J.-H. & Yi, H. (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene
248
sequence database with phylotypes that represent uncultured species. Int J Syst Evol
249
Micr 62, 716-721.
250
Labrenz, M., Collins, M.D; Lawson, P.A., Tindall, B.J., Braker, G& Hirsch. P.
251
(1998). Antarctobacter heliothermus gem nov,, sp. now, a budding bacterium from
252
hypersaline and heliothermal Ekho Lake. Int J Syst Bacteriol. 48, 1363-1372.
253
Lányí, B. (1987). Classical and Rapid Identification Methods for Medically Important.
254
Method Microbiol 19, 1.
255
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for
256
reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.
257
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty
258
acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
259
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for
260
General and Molecular Bacteriology. pp. 607-654. Edited by P. Gerhardt, R. G. E.
261
Murray. W. A. Wood & N. R. Krieg. Washington, DC: American Society for
262
Microbiology.
263
Su, J., Zhou, Y., Lai, Q., Li, X., Dong, P., Yang, X., Zhang, B., Zhang, J., Zheng,
264
X., Tian, Y & Zheng, T. (2013). Sinobacterium caligoides gen. nov., sp. nov., a new 12
265
member of the family Oceanospirillaceae isolated from the South China Sea, and
266
emended description of Amphritea japonica. Int J Syst Evol Micr 63, 2095-2100.
267
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by
268
reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett. 25,
269
125-128.
270
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular
271
evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24,
272
1596-1599.
273
Wagner-Döbler, I., Rheims, H., Felske, A., Pukall, R. & Tindall, B. J. (2003).
274
Jannaschia helgolandensis gen. nov., sp. nov., a novel abundant member of the
275
marine Roseobacter clade from the North Sea. Int J Syst Evol Micr 53, 731-738.
276
Zheng, Q., Chen, C., Yan, X.-J., Wang, Y.-N., Zeng, Y.-H., Hao, L.-K., He, W.-H.
277
& Jiao, N.-Z. (2010). Mameliella alba gen. nov., sp. nov., a marine bacterium of the
278
Roseobacter clade in the order Rhodobacterales. Int J Syst Evol Micr 60, 953-957.
279 280 281 282 283 284 285 286 13
287
Figure Legends
288
Fig. 1. Neighbour-joining tree showing the phylogenetic positions of Mameliella
289
phaeodactyli KD53T and related genera of family Rhodobacteraceae, based on 16S
290
rRNA gene sequences. Filled circles indicate nodes that were also recovered in
291
maximum-likelihood and minimum evolution trees based on the same sequences.
292
Bootstrap values (expressed as percentages of 1000 replications) are shown at branch
293
points. Bar, 0.005 nucleotide substitution rate (Knuc) units. Oceanibaculum indicum
294
P24T (EU656113) was used as outgroup.
295 296 297 298 299 300 301 302 303 304 305 306 307 308 14
309 310
Table 1. Differential characteristics between strain KD53T and M. alba CGMCC
311
1.7290T.
312
Strains species: 1, KD53T. 2, M. alba CGMCC 1.7290T. All data were from this study
313
except where otherwise indicated. Data of catalase, oxidase, API 20NE, API 20E and
314
API ZYM for these two strains were done at the same time in this study. Two strains
315
are positive for oxidase activities and hydrolyse of gelatin but negative for hydrolyse
316
of chitin, Tween 20, 40, 60 and 80. In the API ZYM, they are positive for alkaline
317
phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), trypsin, leucine
318
aminopeptidase, valine aminopeptidase, α-mannosidase, cystine aminopeptidase and
319
β-glucosidase
320
naphtol-AS-Bl-phosphoamidase. In the API 20E, they are positive for ß-galactosidase,
321
arginine dihydrolase, urease, gelatinase, acetoin production and acid production from
322
amygdalin, saccharose and arabinose while negative for lysine decarboxylase,
323
ornithine decarboxylase, citrate utilization, H2S production and acid production from
324
rhamnose, mannitol, melibiose, inositol and sorbitol. In the API 20NE strip, they are
325
positive for β-glucosidase, gelatin hydrolysis, urease, β-galactosidase, arginine
326
dihydrolase and utilization of N-acetyl-glucosamine, trisodium citrate, malic acid,
327
adipic acid, L-arabinose, D-glucose and D-mannitol while negative for denitrification
328
and utilization of D-maltose, potassium gluconate, and capric acid. All strains are
329
susceptible to cefobid, neomycin, novobiocin, vancomycin, tetracycline, streptomycin,
330
piperacillin, polymyxin B, penicillin G, oxacillin, ofloxacin, norfloxacin, minomycin,
while
negative
for
α-chymotrypsin,
15
acid
phosphatase
and
331
kanamycin, erythromycin, vibramycin, ciprofloxacin, carbenicillin, ampicillin,
332
trimethoprim and cefazolin while resistant to metronidazole, clindamycin and
333
sulphafurazole. +, positive; －, negative; W, weakly positive. Characteristic
1
2
Colony colour
Yellow
White
Catalase
-
+
Growth temperature (°C)
16-37
20-37
Optimal salinity (%)
3.0
2-3
pH
6-9.5
6.0-9.0
-
+
-
+
-
+
-
+
-
+
-
+
Tryptophane deaminase
+
-
Indole production
-
+
Reduction of nitrate
+
-
Indole production
-
+
API
ZYM
α-Galactosidase β-Galactosidase β-Glucuronidase α-Glucosidase N-Acetyl-β-Glucosaminidase α-Fucosidase API 20E
API 20NE
16
D-Glucose fermentation
-
+
D-Mannose
w
-
Phenylacetic acid
-
+
-
+
Starch
-
+
Casein
-
w
Tyrosine
+
-
DNA G+C content (mol%)
65
63.7*
Susceptibility to: Chloramphenicol Degradation of
334
*data from Zheng et al., (2010).
335 336 337 338
Table 2. Cellular fatty acid contents of strain KD53T and closely related members of
339
the family Rhodobacteraceae.
340
Strains species: 1, KD53T. 2, M. alba CGMCC 1.7290T. 3, P. litoralis DSM 18986T. 4,
341
A. heliothermus DSM 11445T; 5, S. stellata DSM 11524T All data are from this
342
study. Results are presented as percentages of the total fatty acids. Fatty acids less
343
than 1% are marked as tr (trace); ND, Not detected. Fatty acid C12:1 3-OH C12:0 3-OH C16:0
1 2.9 tr 5.6
2 4.0 tr 5.6
3 5.1 1.4 4.1 17
4 2.6 ND 2.4
5 3.9 3.7 5.7
C17:0 C17:1ω7c C18:0 C18:1ω7c 11-methyl C19:0 cyclo ω8c Summed feature 8*
tr tr 9.3 3.4 tr 75.3
tr 1.3 7.4 3.9 tr 76.0
ND tr 2.6 6.6 ND 78.4
ND tr 1.1 4.5 tr 81.8
tr ND 9.0 3.0 1.2 69.1
344
*
345
separated by GLC with the MIDI system. Summed Feature 8: C18:1ω6c and/or
346
C18:1ω7c.
Summed Features represent groups of two or three fatty acids that could not be
347 348 349 350 351 352 353
18
Figure Click here to download Figure: Fig.1.pptx 100
Pelagibaca abyssi JLT2014T (JX878396) Pelagibaca bermudensis HTCC2601T (AATQ01000039)
70
Thalassococcus halodurans UST050418-052T (DQ397336) Thalassococcus lentus YCS-24T (JX090308)
77
Roseivivax lentus S5-5T (FJ875966) Thalassobius maritimus GSW-M6T (HM748766) Tropicibacter naphthalenivorans C02T (AB302370) Tropicibacter phthalicicus KU27E1T (AB636139)
86
Ponticoccus litoralis CL-GR66T (EF211829) Sagittula stellata E-37T (AAYA01000003) 93
Antarctobacter heliothermus EL-219T (Y11552)
72 50
Mameliella phaeodactyli KD53T (KJ850205) 93
Mameliella alba JLT354-WT (EU734592) Primorskyibacter sedentarius KMM 9018T (AB550558)
69 73 60 71
Pelagicola litorisediminis D1-W8T (KC708867) Leisingera methylohalidivorans DSM 14336T (CP006773) Seohaeicola saemankumensis SD-15T (EU221274) Epibacterium ulvae U95T (JN935021) Pseudoruegeria aquimaris SW-255T (DQ675021)
70 75
Shimia marina CL-TA03T (AY962292) Oceanicola batsensis HTCC2597T (AAMO01000005) Maritimibacter alkaliphilus HTCC2654T (AAMT01000002) Boseongicola aestuarii BS-W15T (KF977837) Dinoroseobacter shibae DFL 12T (CP000830) Oceanibaculum indicum P24T (EU656113)
0.005
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary.pdf
International Journal of Systematic and Evolutionary Microbiology Mameliella phaeodactyli sp. nov., a member of the family Rhodobacteraceae isolated from the marine algae Phaeodactylum tricornutum --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00212R3
Full Title:
Mameliella phaeodactyli sp. nov., a member of the family Rhodobacteraceae isolated from the marine algae Phaeodactylum tricornutum
Short Title:
Mameliella phaeodactyli sp. nov.,
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Tianling Zheng Xiamen University CHINA
First Author:
Zhangran Chen
Order of Authors:
Zhangran Chen Jingyan Zhang Xueqian Lei Qiliang Lai Luxi Yang Huajun Zhang Yi Li Wei Zheng Yun Tian Zhiming Yu Hong Xu Tianling Zheng
Manuscript Region of Origin:
CHINA
Abstract:
A novel Gram-staining-negative, aerobic, rod-shaped, non-motile, yellow bacterium designated strain KD53T, was isolated from a culture of the alga Phaeodactylum tricornutum, Xiamen, Fujian Province, China. 16S rRNA gene sequence comparison showed that strain KD53T was a member of the Roseobacter clade within the family Rhodobacteraceae, forming a distinct lineage with species of the genus Mameliella. The 16S rRNA gene sequence similarities between strain KD53T and other strains were all less than 97.0 %. Strain KD53T was found to grow optimally at 28 °C, at pH 7.5-8.0 and in the presence of 3 % (w/v) NaCl. The dominant fatty acids of strain KD53T were C18:1 ω6c, C18:1 ω7c, C18:0, C16:0. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol. The DNA G+C content was 65 mol% and the major respiratory quinone was ubiquinone 10 (Q-10). On the basis of phenotypic data and phylogenetic inference, strain KD53T represents a novel strain of Mameliella, then the name Mameliella phaeodactyli sp. nov. is proposed. The type strain is KD53T (= MCCC 1K00273T = KCTC 42178T).
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): manuscript.doc
1
Mameliella phaeodactyli sp. nov., a member of the family
2
Rhodobacteraceae isolated from the marine algae Phaeodactylum
3
tricornutum.
4
Zhangran Chen1†, Jingyan Zhang1, Xueqian Lei1, Qiliang Lai1,2, Luxi Yang1, Huajun
5
Zhang1, Yi Li1, Wei Zheng1, Yun Tian1, Zhiming Yu3，Hong Xu1*, Tianling Zheng1*
6
1
7
Education for Coastal and Wetland Ecosystem, School of Life Sciences, Xiamen
8
University, Xiamen 361005, China
9
2
State Key Laboratory of Marine Environment and Key lab of the Ministry of
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography,
10
State Oceanic Administration, People’s Republic of China
11
3
12
Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China
13
*Corresponding author: Tianlin Zheng. E-mail: [email protected]; Hong Xu.
14
E-mail: [email protected]; Tel: +86-592-2183217. Fax: +86-592-2184528
15
Subject category: New Taxa (Proteobacteria)
16
Running title: Mameliella phaeodactyli sp. nov.
17
Abbreviations: KCTC, Korean Collection For Type Cultures; DSMZ, German
18
Collection of Microorganisms and Cell Cultures; MCCC, Marine Culture Collection
19
of China. CGMCC, China General Microbiological Culture Collection Center; CICC,
20
China center of industrial culture collection.
Key Laboratory of Marine Ecology and Environmental Science, Institute of
21 22
The GenBank [/EMBL/DDBJ] accession number for the 16S rRNA gene sequence of 1
23
strain KD53T is KJ850205.
24 25
Abstract:
26
A novel Gram-staining-negative, aerobic, rod-shaped, non-motile, yellow bacterium
27
designated strain KD53T, was isolated from a culture of the alga Phaeodactylum
28
tricornutum, Xiamen, Fujian Province, China. 16S rRNA gene sequence comparison
29
showed that strain KD53T was a member of the Roseobacter clade within the family
30
Rhodobacteraceae, forming a distinct lineage with species of the genus Mameliella.
31
The 16S rRNA gene sequence similarities between strain KD53T and other strains
32
were all less than 97.0 %. Strain KD53T was found to grow optimally at 28 °C, at pH
33
7.5–8.0 and in the presence of 3 % (w/v) NaCl. The dominant fatty acids of strain
34
KD53T were C18:1 ω6c, C18:1 ω7c, C18:0, C16:0. The major polar lipids were
35
phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. The DNA
36
G+C content was 65 mol% and the major respiratory quinone was ubiquinone 10
37
(Q-10). On the basis of phenotypic data and phylogenetic inference, strain KD53T
38
represents a novel strain of Mameliella, then the name Mameliella phaeodactyli sp.
39
nov. is proposed. The type strain is KD53T (= MCCC 1K00273T = KCTC 42178T).
40 41
Members of the Roseobacter clade (Wagner-Döbler et al., 2003) in the order
42
Rhodobacterales of the class Alphaproteobacteria are abundant in marine
43
environments. The abundance and diverse physiological characteristics within this
44
group suggest that they have the function to degrade aromatic compounds (Buchan et 2
45
al., 2005) and participate in the biogeochemical cycles of carbon and sulfur. In this
46
study, strain KD53T, which was isolated from coastal seawater, was found to form a
47
clade with Mameliella alba CGMCC 1.7290T (Zheng et al., 2010), after comparative
48
16S rRNA gene sequence analysis. Accordingly, the aim of the present work was to
49
determine the exact taxonomic position of strain KD53T by using polyphasic
50
characterization including the determination of phenotypic properties and a detailed
51
phylogenetic analysis based on 16S rRNA gene sequences.
52 53
Strain KD53T was isolated from a culture of the alga Phaeodactylum tricornutum,
54
Xiamen, Fujian Province, China in December 2013. The samples were serially diluted
55
(10-fold dilution) by sterile seawater and 0.1 mL aliquots of each dilution were spread
56
onto marine agar 2216 (MA; Difco) followed by incubation for 3 days at 28 °C.
57
Individual colonies of distinct morphology were further purified three times and
58
stored at -80 °C in marine broth 2216 (MB; Difco) supplemented with 10 % (v/v)
59
glycerol.
60 61
The genomic DNA of strain KD53T was extracted according to the method of Ausubel
62
et al. (1995) and the 16S rRNA gene was amplified by PCR using the primer pair
63
P27F and P1492R (DeLong, 1992). Purification of the PCR product was carried out
64
using a TIANquick Midi purification kit (TIANGEN, China) and the purified PCR
65
product was cloned into vector pMD19-T and sequenced. Sequences of related taxa
66
were downloaded from the GenBank database and the EzTaxon-e server 3
67
(http://eztaxon-e.ezbiocloud.net/) (Kim et al., 2012). Phylogenetic analysis was
68
performed using MEGA version 4 (Tamura et al., 2007) after multiple alignment of
69
data by DNAMAN (version 5.1). Evolutionary distances and clustering were
70
performed by using the neighbour-joining method (Saitou & Nei, 1987). The resulting
71
tree topology was evaluated by using bootstrap analysis based on 1000 replicates.
72 73
A nearly full-length 16S rRNA gene sequence (1371 bp, GenBank [/EMBL/DDBJ]
74
accession number KJ850205) of strain KD53T was determined. Preliminary
75
comparison of the 16S rRNA gene sequence of strain KD53T with other sequences
76
indicated that the new isolate showed 96.71% similarity to Ponticoccus litoralis DSM
77
18986T (GenBank no. EF211829). Phylogenetic analysis of the 16S rRNA sequence
78
of strain KD53T indicated that it belongs to the family Rhodobacteraceae (Fig. 1, Fig.
79
S2 and S3). In the phylogenetic tree based on the neighbour-joining algorithm, strain
80
KD53T joined a phylogenetic clade comprising M. alba CGMCC 1.7290T, with which
81
it exhibited the 16S rRNA gene sequence similarity (95.6%). The relatively low levels
82
of 16S rRNA gene sequence similarity to its most closely related strain suggested that
83
strain KD53T may represent a novel species of the genus Mameliella.
84 85
Cell morphology and motility were observed by using transmission electron
86
microscopy (model JEM-2100HC; JEOL) and phase-contrast light microscopy (model
87
50i; Nikon), with cells from the early exponential phase grown on MA at 28 °C.
88
Spore production was determined as Gonzalez et al described (1997). The presence of 4
89
flexirubin-type pigments was assessed using the bathochromic shift test with 20%
90
(w/v) KOH, as described by Bernardet (2002). Colony morphology was examined
91
from cultures grown on MA for 6 days. Gliding motility was investigated as described
92
by Bowman (2000). The Gram reaction was determined by using the bioMérieux
93
Gram stain kit according to the manufacturer’s instructions. Anaerobic growth was
94
assessed on MA that was autoclaved and cooled to room temperature under nitrogen
95
atmosphere (99.999 % purity). Triplicate cultures were grown in 50 ml anaerobic
96
serum bottles sealed with thick butyl rubber stoppers and aluminum caps, and
97
incubated statically in the dark at 28 °C for 21 days. Growth in MB was tested at 4, 10,
98
16, 20, 28, 30, 37 and 42 °C and at pH 3.0-10.0 (at 1 pH unit intervals during 3.0-7.0
99
and 0.5 pH unit intervals during 7.0-10.0). The pH of MB was adjusted prior to
100
sterilization using the following buffers: citric acid/sodium citrate (pH 3.0–6.0),
101
Na2HPO4/citric acid (pH 7.0–8.0) and Lysine/NaOH (pH 9.0–10.0). Verification of
102
the pH values after autoclaving revealed only minor changes (Su et al., 2013). The
103
NaCl concentration range and optimum for growth were determined in NaCl-free MB
104
(tryptone 5.0 g L-1, yeast extract 1.0 g L-1, 1L distilled water, pH 7.6 ~7.8),
105
supplemented with 0–7% (at 1% intervals) and 9.0–13.0% (at 2% intervals) NaCl
106
(w/v). Catalase and oxidase activities were assessed by addition of 3% Hydrogen
107
peroxide to exponential-phase colonies and by using oxidase reagent (bioMérieux),
108
respectively. Hydrolysis of starch, chitin, tyrosine, casein, gelatin, urea and Tweens 20,
109
40, 60 and 80 was tested using MA supplemented with 0.5 % (w/v) of starch and 1 %
110
(w/v) of the other substrates. Results were examined twice after growth on agar plates 5
111
for 3 and 5 days. The above mentioned tests were carried out for strain KD53T and the
112
four reference strains. Biochemical tests were carried out using API 20NE, API 20E
113
and API ZYM strips (bioMérieux) according to the manufacturer’s instructions,
114
except that the NaCl concentration in all tests was adjusted to 3.0%. Susceptibility to
115
antibiotics was tested on MA for 4 days by using filter-paper discs (OXOID)
116
containing various antibiotics (Lányí, 1987, Smibert & Krieg, 1994) and the results
117
showed that strain KD53T was susceptible to cefazolin, ampicillin, carbenicillin,
118
ciprofloxacin, vibramycin, erythromycin, kanamycin, minomycin, norfloxacin,
119
ofloxacin, oxacillin, penicillin G, polymyxin B, piperacillin, streptomycin, tetracycline,
120
vancomycin, novobiocin, trimethoprim, neomycin; Resistant to sulphafurazole,
121
metronidazole, clindamycin and chloramphenicol. All above-mentioned tests were
122
incubated at 28 °C. All strains were tested using MA or MB except Antarctobacter
123
heliothermus DSM 11445T) (grown on R2A agar). The novel isolate displayed the
124
characteristics found in many members of the family Rhodobacteraceae, e.g.
125
rod-shaped, non-motile bacterium (Hwang et al., 2008; Zheng et al., 2010). Other
126
phenotypic properties of strain KD53T are given in the species description and in
127
Table 1.
128 129
To determine the DNA G+C content, genomic DNA was extracted from cells cultured
130
on MA for 3 days at 28 °C and analysed by reverse-phase HPLC (Tamaoka &
131
Komagata, 1984). The DNA G+C content of the new isolate KD53T was 65 mol%,
132
which is within the range reported for the members of the family Rhodobacteraceae 6
133
(Zheng et al., 2010; Labrenz et al., 1998., Hwang et al., 2008).
134 135
For cellular fatty acid analysis, the fatty acids of strain KD53T and the four reference
136
strains except Antarctobacter heliothermus DSM 11445 (grown on R2A agar) grown
137
on MA at 28 °C for 4 days were saponified, methylated and extracted using the
138
standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B).
139
The fatty acids were analysed by GC (Agilent Technologies 6850) and identified by
140
using the TSBA6 database of the Microbial Identification System (Sasser, 1990). The
141
five strains had similar growth rates at 28 °C and the same physiological age at the
142
time they were harvested. The dominant fatty acids of strain KD53T were identified as
143
summed feature 8 (comprising C18:1 ω6c and/or C18:1 ω7c, 75.3%), C18:0 (9.3%), C16:0
144
(5.6%), which accounted for 90.2% of the total. The fatty acid profile of the strain
145
KD53T was observed to be in good agreement with those of members of family
146
Rhodobacteraceae (Gonzalez et al., 1997; Labrenz et al., 1998; Hwang et al., 2008;
147
Zheng et al., 2010) but it can be differentiated from other species by the percentage of
148
C18:0
149
phosphatidylglycerol were the major polar lipids detected in strain KD53 (Fig. S4,
150
available in the online Supplementary Material), along with small amounts of
151
unidentified
152
Phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol have also
153
been reported to be the major constituents of the polar lipids in the P. litoralis DSM
154
18986T, although unidentified phospholipid was not the major polar lipid (Hwang &
(Table
2).
Phosphatidylcholine,
phospholipids,
unidentified
7
phosphatidylethanolamine
aminolipids,
unknown
and
lipids.
155
Cho, 2008).
156 157
The major respiratory quinone of the strain KD53T was analyzed at the CICC on an
158
LDC Analytical (thermo separation products) HPLC fitted with a reverse phase
159
column (Macherey–Nagel, 2 mm × 125 mm, 3 μm, RP18) using Methanol:Heptane
160
9:1 (v/v) as the eluent. The major respiratory ubiquinone present in strain KD53T was
161
Q-10, in line with Rhodobacteraceae species (Zheng et al., 2010;. Hwang et al.,
162
2008).
163 164
On the basis of morphological, physiological and chemotaxonomic characteristics, as
165
well as phylogenetic inference (Tables 1, 2 and Fig.1), strain KD53T should be
166
assigned to a novel species of in the genus Mameliella for which the name Mameliella
167
phaeodactyli sp. nov. is proposed.
168 169
Description of Mameliella phaeodactyli sp. nov.
170
Mameliella
171
Phaeodactylum tricornutum).
phaeodactyli
(phae.o.dac'ty.li.
N.L.
gen.
n.
phaeodactyli
of
172 173
Cells are gram staining negative, aerobic, non-spore-forming, non-flagella, rod shaped,
174
with cells 2.2-2.9 μm in length and 0.5-1.0 μm (Fig. S1, available in the online
175
Supplementary Material) in width. On MA medium plates, strain KD53T forms yellow,
176
circular, small, translucent and smooth colonies after 3 days incubation at 28 °C. The 8
177
diameter of the colony is 0.5-2 mm. Strain KD53T is found to grow at 16-37 °C
178
(optimum 28 °C), in 1-6% NaCl (optimum 3%), at pH 6.0-9.5 (optimum 7.5-8.0).
179
Strain KD53T cannot grow under anaerobic conditions or in the absence of NaCl. It
180
can hydrolyze tyrosine and urea, but not Tweens 20, 40, 60, 80, casein, starch, chitin.
181
In the API 20E test, positive for ß-galactosidase, arginine dihydrolase, urease,
182
tryptophane deaminase, gelatinase, production of acetoin, utilization of glucose,
183
saccharose, amygdalin and arabinose; negative for lysine decarboxylase, ornithine
184
decarboxylase activities, citrate utilization, production of H2S and indole, utilization
185
of mannitol, inositol, sorbitol, rhamnose and melibiose. In the API 20NE test, positive
186
for nitrate reduction, arginine dihydrolase, urease, aesculin hydrolysis, gelatin
187
hydrolysis,
188
N-acetyl-glucosamine, D-maltose, potassium gluconate; Weakly positive for
189
utilization of L-arabinose, D-mannose, adipic acid, malic acid and trisodium citrate;
190
negative for indole production, D-glucose fermentation, utilization of capric acid and
191
phenylacetic acid. According to the API ZYM test, positive for alkaline phosphatase,
192
esterase (C4), esterase lipase (C8), lipase (C14), leucine aminopeptidase, valine
193
aminopeptidase, cystine aminopeptidase, trypsin, β-glucosidase, α-mannosidase;
194
negative for α-chymotrypsin, acid phosphatase, naphtol-AS-Bl-phosphoamidase,
195
α-galactosidase,
196
N-acetyl-β-glucosaminidase, α-fucosidase. The predominant fatty acids (>5% of the
197
total) are Summed Feature 8 (comprising C18:1 ω6c and/or C18:1 ω7c), C18:0, C16:0. The
198
complete fatty acid composition is given in Table 2. The DNA G+C content of the
ß-galactosidase,
utilization
β-galactosidase,
of
D-glucose,
β-glucuronidase,
9
D-mannitol,
α-glucosidase,
199
new isolate KD53T is 65 mol%. The major respiratory quinone is Q-10 and the major
200
polar
201
phosphatidylglycerol. Other phenotypic characteristics are given in Table 1.
lipids
are
phosphatidylcholine,
phosphatidylethanolamine
and
202 203
The type strain, KD53T (= MCCC 1K00273T = KCTC 42178T), is isolated from a
204
culture of marine alga Phaeodactylum tricornutum, Xiamen, China.
205 206
Emended description of the genus Mameliella
207
This emended description of the genus Mameliella is based on the description of
208
Mameliella alba published by Zheng et al. (2010) and on this study.
209 210
Cells are gram staining negative, aerobic, rod shaped, non-flagella and non-motile.
211
Positive for oxidase activities but negative for hydrolyse of chitin, Tween 20, 40, 60
212
and 80. The predominant fatty acids (>5% of the total) are Summed Feature 8
213
(comprising C18:1 ω6c and/or C18:1 ω7c), C18:0 and C16:0. The major respiratory
214
quinone is Q-10. The DNA G+C content is approximately 64 mol%.
215 216
Acknowledgements：
217
This work was supported by the Joint project of National Natural Science Foundation
218
of China (NSFC) and Shandong province, Marine ecology and environmental
219
sciences (U1406403), public science and technology research funds projects of ocean
220
(201305016) and the National Nature Science Foundation of China (41376119, 10
221
41476095).
222 223
References
224
Ausubel, F., Brent, R., Kingston, R. E., Moore, D., Seidman, J. & Smith, J. K.
225
Struhl, K.(editors) (1995). Short Protocols in Molecular Biology: a Compendium of
226
Methods from Current Protocols in Molecular Biology, 3rd edn. New York: Wiley.
227
Bernardet, J. F., Nakagawa, Y. & Holmes, B. (2002). Proposed minimal standards
228
for describing new taxa of the family Flavobacteriaceae and emended description of
229
the family. Int J Syst Evol Micr .52. 1049-1070.
230
Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov., isolated from
231
the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell
232
and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst
233
Evol Micr. 50, 1861-1868.
234
Buchan, A., González, J. M. & Moran, M. A. (2005). Overview of the marine
235
Roseobacter lineage. Appl Environ Microb 71, 5665-5677.
236
DeLong, E. F. (1992). Archaea in coastal marine environments. P Natl Acad Sci USA.
237
89, 5685-5689.
238
González, J., Mayer, F., Moran, M., Hodson, R., Whitman, W. (1997). Sagittula
239
stellata gen. nov., sp. nov., a lignin-transforming bacterium from a coastal
240
environment. Int J Syst Bacteriol, 47(3), 773-780.
241
Hwang, C. Y. & Cho, B. C. (2008). Ponticoccus litoralis gen. nov., sp. nov., a marine
242
bacterium in the family Rhodobacteraceae. Int J Syst Evol Micr 58, 1332-1338. 11
243
Kates, M. (1986). Lipid extraction procedures in Techniques of lipidology: Isolation,
244
Analysis and Identification of Lipids. Edited by M. Kates. Elsevier, Amsterdam,
245
100-111.
246
Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., Jeon, Y.
247
S., Lee, J.-H. & Yi, H. (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene
248
sequence database with phylotypes that represent uncultured species. Int J Syst Evol
249
Micr 62, 716-721.
250
Labrenz, M., Collins, M.D; Lawson, P.A., Tindall, B.J., Braker, G& Hirsch. P.
251
(1998). Antarctobacter heliothermus gem nov,, sp. now, a budding bacterium from
252
hypersaline and heliothermal Ekho Lake. Int J Syst Bacteriol. 48, 1363-1372.
253
Lányí, B. (1987). Classical and Rapid Identification Methods for Medically Important.
254
Method Microbiol 19, 1.
255
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for
256
reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.
257
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty
258
acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
259
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for
260
General and Molecular Bacteriology. pp. 607-654. Edited by P. Gerhardt, R. G. E.
261
Murray. W. A. Wood & N. R. Krieg. Washington, DC: American Society for
262
Microbiology.
263
Su, J., Zhou, Y., Lai, Q., Li, X., Dong, P., Yang, X., Zhang, B., Zhang, J., Zheng,
264
X., Tian, Y & Zheng, T. (2013). Sinobacterium caligoides gen. nov., sp. nov., a new 12
265
member of the family Oceanospirillaceae isolated from the South China Sea, and
266
emended description of Amphritea japonica. Int J Syst Evol Micr 63, 2095-2100.
267
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by
268
reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett. 25,
269
125-128.
270
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular
271
evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24,
272
1596-1599.
273
Wagner-Döbler, I., Rheims, H., Felske, A., Pukall, R. & Tindall, B. J. (2003).
274
Jannaschia helgolandensis gen. nov., sp. nov., a novel abundant member of the
275
marine Roseobacter clade from the North Sea. Int J Syst Evol Micr 53, 731-738.
276
Zheng, Q., Chen, C., Yan, X.-J., Wang, Y.-N., Zeng, Y.-H., Hao, L.-K., He, W.-H.
277
& Jiao, N.-Z. (2010). Mameliella alba gen. nov., sp. nov., a marine bacterium of the
278
Roseobacter clade in the order Rhodobacterales. Int J Syst Evol Micr 60, 953-957.
279 280 281 282 283 284 285 286 13
287
Figure Legends
288
Fig. 1. Neighbour-joining tree showing the phylogenetic positions of Mameliella
289
phaeodactyli KD53T and related genera of family Rhodobacteraceae, based on 16S
290
rRNA gene sequences. Filled circles indicate nodes that were also recovered in
291
maximum-likelihood and minimum evolution trees based on the same sequences.
292
Bootstrap values (expressed as percentages of 1000 replications) are shown at branch
293
points. Bar, 0.005 nucleotide substitution rate (Knuc) units. Oceanibaculum indicum
294
P24T (EU656113) was used as outgroup.
295 296 297 298 299 300 301 302 303 304 305 306 307 308 14
309 310
Table 1. Differential characteristics between strain KD53T and M. alba CGMCC
311
1.7290T.
312
Strains species: 1, KD53T. 2, M. alba CGMCC 1.7290T. All data were from this study
313
except where otherwise indicated. Data of catalase, oxidase, API 20NE, API 20E and
314
API ZYM for these two strains were done at the same time in this study. Two strains
315
are positive for oxidase activities and hydrolyse of gelatin but negative for hydrolyse
316
of chitin, Tween 20, 40, 60 and 80. In the API ZYM, they are positive for alkaline
317
phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), trypsin, leucine
318
aminopeptidase, valine aminopeptidase, α-mannosidase, cystine aminopeptidase and
319
β-glucosidase
320
naphtol-AS-Bl-phosphoamidase. In the API 20E, they are positive for ß-galactosidase,
321
arginine dihydrolase, urease, gelatinase, acetoin production and acid production from
322
amygdalin, saccharose and arabinose while negative for lysine decarboxylase,
323
ornithine decarboxylase, citrate utilization, H2S production and acid production from
324
rhamnose, mannitol, melibiose, inositol and sorbitol. In the API 20NE strip, they are
325
positive for β-glucosidase, gelatin hydrolysis, urease, β-galactosidase, arginine
326
dihydrolase and utilization of N-acetyl-glucosamine, trisodium citrate, malic acid,
327
adipic acid, L-arabinose, D-glucose and D-mannitol while negative for denitrification
328
and utilization of D-maltose, potassium gluconate, and capric acid. All strains are
329
susceptible to cefobid, neomycin, novobiocin, vancomycin, tetracycline, streptomycin,
330
piperacillin, polymyxin B, penicillin G, oxacillin, ofloxacin, norfloxacin, minomycin,
while
negative
for
α-chymotrypsin,
15
acid
phosphatase
and
331
kanamycin, erythromycin, vibramycin, ciprofloxacin, carbenicillin, ampicillin,
332
trimethoprim and cefazolin while resistant to metronidazole, clindamycin and
333
sulphafurazole. +, positive; －, negative; W, weakly positive. Characteristic
1
2
Colony colour
Yellow
White
Catalase
-
+
Growth temperature (°C)
16-37
20-37
Optimal salinity (%)
3.0
2-3
pH
6-9.5
6.0-9.0
-
+
-
+
-
+
-
+
-
+
-
+
Tryptophane deaminase
+
-
Indole production
-
+
Reduction of nitrate
+
-
Indole production
-
+
API
ZYM
α-Galactosidase β-Galactosidase β-Glucuronidase α-Glucosidase N-Acetyl-β-Glucosaminidase α-Fucosidase API 20E
API 20NE
16
D-Glucose fermentation
-
+
D-Mannose
w
-
Phenylacetic acid
-
+
-
+
Starch
-
+
Casein
-
w
Tyrosine
+
-
DNA G+C content (mol%)
65
63.7*
Susceptibility to: Chloramphenicol Degradation of
334
*data from Zheng et al., (2010).
335 336 337 338
Table 2. Cellular fatty acid contents of strain KD53T and closely related members of
339
the family Rhodobacteraceae.
340
Strains species: 1, KD53T. 2, M. alba CGMCC 1.7290T. 3, P. litoralis DSM 18986T. 4,
341
A. heliothermus DSM 11445T; 5, S. stellata DSM 11524T All data are from this
342
study. Results are presented as percentages of the total fatty acids. Fatty acids less
343
than 1% are marked as tr (trace); ND, Not detected. Fatty acid C12:1 3-OH C12:0 3-OH C16:0
1 2.9 tr 5.6
2 4.0 tr 5.6
3 5.1 1.4 4.1 17
4 2.6 ND 2.4
5 3.9 3.7 5.7
C17:0 C17:1ω7c C18:0 C18:1ω7c 11-methyl C19:0 cyclo ω8c Summed feature 8*
tr tr 9.3 3.4 tr 75.3
tr 1.3 7.4 3.9 tr 76.0
ND tr 2.6 6.6 ND 78.4
ND tr 1.1 4.5 tr 81.8
tr ND 9.0 3.0 1.2 69.1
344
*
345
separated by GLC with the MIDI system. Summed Feature 8: C18:1ω6c and/or
346
C18:1ω7c.
Summed Features represent groups of two or three fatty acids that could not be
347 348 349 350 351 352 353
18
Figure Click here to download Figure: Fig.1.pptx 100
Pelagibaca abyssi JLT2014T (JX878396) Pelagibaca bermudensis HTCC2601T (AATQ01000039)
70
Thalassococcus halodurans UST050418-052T (DQ397336) Thalassococcus lentus YCS-24T (JX090308)
77
Roseivivax lentus S5-5T (FJ875966) Thalassobius maritimus GSW-M6T (HM748766) Tropicibacter naphthalenivorans C02T (AB302370) Tropicibacter phthalicicus KU27E1T (AB636139)
86
Ponticoccus litoralis CL-GR66T (EF211829) Sagittula stellata E-37T (AAYA01000003) 93
Antarctobacter heliothermus EL-219T (Y11552)
72 50
Mameliella phaeodactyli KD53T (KJ850205) 93
Mameliella alba JLT354-WT (EU734592) Primorskyibacter sedentarius KMM 9018T (AB550558)
69 73 60 71
Pelagicola litorisediminis D1-W8T (KC708867) Leisingera methylohalidivorans DSM 14336T (CP006773) Seohaeicola saemankumensis SD-15T (EU221274) Epibacterium ulvae U95T (JN935021) Pseudoruegeria aquimaris SW-255T (DQ675021)
70 75
Shimia marina CL-TA03T (AY962292) Oceanicola batsensis HTCC2597T (AAMO01000005) Maritimibacter alkaliphilus HTCC2654T (AAMT01000002) Boseongicola aestuarii BS-W15T (KF977837) Dinoroseobacter shibae DFL 12T (CP000830) Oceanibaculum indicum P24T (EU656113)
0.005
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary.pdf
