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Maribellus comscasis sp. nov., isolated from the deep-sea cold
seep 1
Rikuan Zheng1,2,3,4, Chaomin Sun1,2,4* 2
1CAS Key Laboratory of Experimental Marine Biology & Center
of Deep Sea 3
Research, Institute of Oceanology, Chinese Academy of Sciences,
Qingdao, China 4
2Laboratory for Marine Biology and Biotechnology, Qingdao
National Laboratory 5
for Marine Science and Technology, Qingdao, China 6
3College of Earth Science, University of Chinese Academy of
Sciences, Beijing, 7
China 8
4Center of Ocean Mega-Science, Chinese Academy of Sciences,
Qingdao, China 9
10
* Corresponding author 11
Chaomin Sun Tel.: +86 532 82898857; fax: +86 532 82898857.
12
E-mail address: [email protected] 13
14
Category: New taxon in Bacteroidetes 15
Running title: A novel Bacteroidetes bacterium 16
17
The NCBI GenBank accession numbers for the 16S rRNA gene
sequence and 18
whole-genome sequence (WGS) of strain WC007T are MN096653 and
CP046401, 19
respectively. 20
21
22
23
24
25
26
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ABSTRACT 27
A facultatively anaerobic, Gram-stain-negative, non-motile,
curved rod-shaped 28
bacterium, designated WC007T, was isolated from the deep-sea
cold seep, P. R. 29
China. Strain WC007T was found to grow at temperatures from 28
to 37 °C (optimum, 30
30 °C), at pH values between pH 6.0 and 8.0 (optimum, pH 7.0)
and in 0-5.0% (w/v) 31
NaCl (optimum, 1.0%). The major fatty acids (>10.0%) were
iso-C15:0, C16:0, summed 32
feature 3 and summed feature 8. The major isoprenoid quinone was
MK-7. 33
Predominant polar lipids were phosphatidylethanolamine, one
unidentified 34
phospholipid, one unidentified aminolipid and one unidentified
lipid. The G+C 35
content of the genomic DNA was 38.38%. The average nucleotide
identity (ANIb and 36
ANIm), amino acid identity (AAI), the tetranucleotide signatures
(Tetra) and in silico 37
DNA-DNA hybridization (isDDH) similarities between the genome
sequences of 38
isolate WC007T and Maribellus luteus XSD2T were 70.11%, 84.94%,
71.0%, 0.92022 39
and 20.40%, respectively, indicating that strain WC007T was
distinguished from M. 40
luteus. Phylogenetic analysis based on 16S rRNA gene sequences
placed strain 41
WC007T within the genus Maribellus and showed the highest
similarity to strain 42
XSD2T (95.70%). In combination of the results of phylogenetic
analysis and 43
phenotypic and chemotaxonomic data, strain WC007T was considered
to represent a 44
novel species of the genus Maribellus, for which the name
Maribellus comscasis sp. 45
nov. is proposed. The type strain is WC007T (=KCTC 25169T = MCCC
1K04777T). 46
The available of the genome sequence of strain WC007T would be
helpful in 47
understanding the degradation mechanism of difficult-to-degrade
polysaccharides. 48
49
50
Keywords 51
Maribellus comscasis, 16S rRNA gene sequence, polysaccharides,
deep-sea cold seep 52
53
54
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The family Prolixibacteraceae was originally described and
classified under the order 55
Bacteroidales [1], class Bacteroidia, phylum Bacteroidetes, and
the genus 56
Prolixibacter is the type genus [2]. After that, the families
Prolixibacteraceae, 57
Marinilabiliaceae and Marinifilaceae were transferred to the
order Marinilabiliales in 58
2016 [3]. At the time of writing, 11 genera including Aquipluma,
Meniscus, 59
Puteibacter, Sunxiuqinia, Prolixibacter, Mangrovibacterium,
Draconibacterium, 60
Mariniphaga, Tangfeifania, Roseimarinus and Maribellus had been
identified within 61
the family Prolixibacteraceae [4-6]. Members of the family
Prolixibacteraceae were 62
isolated from various habitats, such as coastal sediment [7, 8],
crude oil [9], mangrove 63
sediment [1], freshwater lake [5], and marine sediments [10].
The new genus 64
Maribellus within the family Prolixibacteraceae was proposed
recently [4], and 65
Maribellus luteus XSD2T, isolated from coastal seawater, was the
only strain in the 66
genus Maribellus. The predominant respiratory quinone of strain
XSD2T was MK-7, 67
which is the most frequently identified respiratory quinone in
bacteria belonging to 68
the family Prolixibacteraceae [4]. 69
The phylum Bacteroidetes, specialized on polysaccharide
degradation, was the 70
most abundant group of bacteria in the ocean after
Proteobacteria and Cyanobacteria 71
[11]. Marine Bacteroidetes were commonly assumed to have a key
role in degrading 72
phytoplankton polysaccharides [12], which had a great number and
diversity of 73
carbohydrate-active enzymes (CAZymes) [13]. The CAZymes are
categorized into 74
families of glycoside hydrolases (GHs), glycoside transferases
(GTs), 75
carbohydrate-binding modules (CBMs), carbohydrate esterases
(CEs), polysaccharide 76
lyases (PLs), sulfatases (targeting sulfated polysaccharides)
plus a range of auxiliary 77
enzymes [12, 14, 15]. And the components of Bacteroidetes for
the degradation of 78
polysaccharides was often encoded in distinct polysaccharide
utilization loci (PULs) 79
[16], which were strictly regulated by the gene clusters that
encode CAZymes and 80
protein ensembles required for the degradation of complicated
carbohydrates. Besides 81
the various substrate-specific CAZymes genes, Bacteroidetes PULs
also contain genes 82
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encoding SusCD-like proteins that are extracellular lipoproteins
and integral 83
membrane beta-barrels termed TonB-dependent transporters [17].
In addition, PULs 84
of marine Bacteroidetes are frequently found many sulfatases
[15, 18-21], because 85
abundant polysaccharides from marine algae were often sulfated.
So far most 86
functional studies of Bacteroides polysaccharide degradation
have been conducted in 87
human gut Bacteroides [22], however, only a few studies have
investigated 88
polysaccharide degradation by marine Bacteroides, especially the
deep-sea variety 89
[23]. 90
In this study, a facultatively anaerobic strain WC007T,
belonging to the 91
Maribellus genus, was isolated from deep-sea sediment at a depth
of 1,146 m in the 92
cold seep (22º 06' 58.598'' N 119º 17' 07.322'' E) as described
previously [24], P. R. 93
China. Strain WC007T was isolated from an enrichment medium
containing (per liter 94
of seawater): 1.0 g NaHCO3, 1.0 g CH3COONa, 1.0 g NH4Cl, 0.5 g
KH2PO4, 0.2 g 95
MgSO4.7H2O, 1.0 g polysaccharide (cellulose, pectin and xylan),
1.0 mL 0.1 % (w/v) 96
resazurin, 0.7 g cysteine hydrochloride (pH 7.0) at atmospheric
pressure and the 97
medium was prepared anaerobically as previously described [25].
The cultures were 98
repeatedly purified by using the Hungate roll-tube method in the
medium containing 99
1.5% (w/v) agar. After incubation for 7 days, several colonies
were picked by 100
sterilized bamboo skewers and then harvested and cultured in the
liquid medium. The 101
process of isolation was repeated several times until the
isolates were deemed to be 102
axenic. The purity of this isolate was confirmed routinely by
transmission electron 103
microscopy (TEM) and by repeated sequencing of the 16S rRNA
gene. Then the 104
single colony was transferred to a new medium (ORG) for further
culture. The ORG 105
medium contained (L−1): 1.0 g NaHCO3, 1.0 g NH4Cl, 1.0 g
CH3COONa, 0.2 g 106
MgSO4.7H2O, 0.5 g KH2PO4, 1.0 g yeast extract, 1.0 g peptone,
0.7 g cysteine 107
hydrochloride, 1 mL 0.1% (w/v) resazurin; the pH was adjusted to
7.0 [26]. The 108
isolate was cultured in ORG broth and maintained at -80 °C as a
suspension in ORG 109
supplemented with glycerol (20 %, v/v). 110
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Genomic DNA was extracted from strain WC007T cultured for 7 d at
30 °C using 111
a bacteria genomic DNA kit (Takara, Japan). The whole genome
sequencing (WGS) 112
of strain WC007T was performed on the nanopore sequencing
technology platform 113
with the Oxford Nanopore MinION (Oxford, UK) and Illumina MiSeq
sequencing 114
platform (San Diego, USA). The experimental process was
implemented in 115
accordance with the standard protocol provided by Oxford
Nanopore Technologies 116
(Oxford, UK). And the library building includes the following
four steps: (1) High 117
quality genomic DNA was extracted, and then Nanodrop, Qubit and
0.35 % agarose 118
gel electrophoresis were used for purity, concentration and
integrity inspection, 119
respectively; (2) The large fragments of DNA were recovered by
the BluePippin 120
automatic nucleic acid recovery system; (3) Library construction
was conducted by 121
using the SQK-LSK109 connection Kit (Japan), which including DNA
damage repair 122
and terminal repair, magnetic bead purification, ligation of
sequencing adapters and 123
magnetic bead purification. (4) After the library construction,
computer sequencing 124
will be performed and Canu V1.5 software was used to assemble
the filtered subreads 125
[27]. Finally, Pilon software was used to correct the assembled
genome with 126
second-generation data to obtain the final genome with higher
accuracy [28]. 127
The whole genome of strain WC007T had been deposited at GenBank
under the 128
accession number CP046401. The genome size of strain WC007T was
7,811,310 bp 129
with a DNA G+C content of 38.38%. The number of contigs was 1,
the total of N50 130
was 7,811,310 and the sequencing depth was 50.0×. Annotation of
the genome of 131
strain WC007T consisted of 6,176 coding sequences that included
54 RNA genes (6 132
rRNA genes, 45 tRNA genes and 3 other ncRNAs). And we checked
the authenticity 133
of the genome using the QUAST-5.0.2 software. The results showed
that the genome 134
assembly quality of WC007T was high. In the genome of WC007T,
genes encoding 135
SusC, SusD, CAZymes and sulfatase were ubiquitously distributed,
strongly 136
indicating it possesses potentials for polysaccharide
degradation. As measures of 137
relatedness between strain WC007T and closely related strains,
the genome 138
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relatedness values were calculated by several approaches:
Average Nucleotide 139
Identity (ANI) based on the MUMMER ultra-rapid aligning tool
(ANIm), ANI based 140
on the BLASTN algorithm (ANIb), the tetranucleotide signatures
(Tetra), and in silico 141
DNA–DNA similarity. ANIm, ANIb, and Tetra frequencies were
calculated, using 142
JSpecies WS (http://jspecies.ribohost.com/jspeciesws/) [29]. The
recommended 143
species criterion cut-offs were used; 95% for the ANIb and ANIm
and 0.99 for the 144
Tetra signature [30]. The amino acid identity (AAI) values were
calculated by 145
AAI-profiler (http://ekhidna2.biocenter.helsinki.fi/AAI/) [31].
The in silico 146
DNA-DNA similarity (isDDH) values were calculated by the
Genome-to-Genome 147
Distance Calculator (GGDC) (http://ggdc.dsmz.de/) [32]. The
isDDH results were 148
based on the recommended formula 2, which is independent of
genome size and, thus, 149
is robust when using whole-genome sequences. 150
From ANI calculations, it could be observed that the comparison
between strain 151
WC007T and three closely relatives were noticeably lower (ANIb:
70.11-70.51% and 152
ANIm: 83.71-84.94%) than the 95% cut-off proposed for bacterial
species delineation. 153
The AAI values of 71.0-71.7% between strain WC007T and closely
relatives were 154
obtained, which were also far below the 95% cut-off value
generally recommended 155
for species differentiation. The Tetra values were also lower
(0.92022-0.9496) than 156
the 0.99 cut-off proposed for bacterial species differentiation.
Finally, the isDDH 157
values were significantly lower (18.40-20.40%) than the 70%
cut-off value generally 158
recommended for species differentiation. The related genome
comparison data were 159
listed in the Table S1. 160
Genomic DNA extraction and PCR amplification of the 16S rRNA
gene of strain 161
WC007T were carried out as described by Hetharua et al [33]. The
PCR product was 162
cloned into the vector pMD-19T (TaKaRa, Japan), sequenced and
then compared to 163
the 16S rRNA gene sequence extracted from the genome. The
results exhibited 99.9 % 164
sequence similarity. The whole full-length 16S rRNA gene
sequence (1,516 bp) of 165
WC007T was obtained from the genome, which had been deposited in
the GenBank 166
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database (accession number MN096653). Moreover, the 16S rRNA
gene sequences of 167
related taxa were obtained from NCBI (www.ncbi.nlm.nih.gov/). A
sequence 168
similarity calculation using the NCBI server indicated the
closest relatives of strain 169
WC007T were Maribellus luteus XSD2T (95.70%), Mariniphaga
sediminis SY21T 170
(93.44%) and Draconibacterium sediminis MCCC 1A00734T (92.99%).
Phylogenetic 171
analysis was performed using the software MEGA version 6.0 [34].
Phylogenetic 172
trees were constructed by the neighbor-joining algorithm [35],
maximum Likelihood 173
[36] and minimum-evolution methods [37]. The numbers above or
below the branches 174
are bootstrap values based on 1,000 replicates. Phylogenetic
analyses based on the 175
16S rRNA and genome sequences showed strain WC007T belonged to
the genus 176
Maribellus and formed an independent phyletic line with the type
strain Maribellus 177
luteus XSD2T (Fig. 1 and Fig. S1). Therefore, we propose strain
WC007T to be 178
classified as the type strain of a novel species in the genus
Maribellus, for which the 179
name Maribellus comscasis sp. nov. is proposed. On the basis of
the phylogenetic 180
results, Maribellus luteus XSD2T (=MCCC 1H00347T) was selected
as the closest 181
recognized neighbor of strain WC007T and was used as a reference
strain in most of 182
the subsequent phenotypic tests. 183
To observe the morphological characteristics of M. comscasis
WC007T, cells were 184
examined using transmission electronic microscopy (TEM) (HT7700;
Hitachi, Japan) 185
with a JEOL JEM 12000 EX (equipped with a field emission gun) at
100 kV. The 186
cells suspension of M. comscasis WC007T was washed with Milli-Q
water and 187
centrifuged at 5,000 g for 5 min. Subsequently, the sample was
taken by immersing 188
copper grids coated with a carbon film for 20 min in the
bacterial suspensions and 189
washed for 10 min in distilled water and dried for 20 min at
room temperature [38]. 190
Strain WC007T was Gram-stain-negative and showed a curved
rod-shaped, 191
1.0-3.5×0.5-0.8 µm in size, which had no flagellum as indicated
by TEM (Fig. 2). 192
For phenotypic characteristics comparison, the temperature, pH
and NaCl 193
concentration ranges for the growth of strain WC007T were
determined in duplicate 194
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experiments using the ORG medium as described above. Water baths
and 195
thermostatic incubators were used to incubate bacterial cultures
from 4 to 80 °C. The 196
pH of the culture medium was adjusted by 6 M HCl for low pH and
10% NaHCO3 197
(w/v) for high pH. The pH range for growth was tested from pH
3.0 to pH 11.0 (initial 198
pH at 30 °C) with increments of 0.5 pH units. Salt tolerance was
determined by 199
directly weighing NaCl (0-100 g L−1) into the Hungate tubes
before packaging the 200
autotrophic medium. Catalase activity was determined by
observation of bubble 201
production after the application of 3% (v/v) hydrogen peroxide
solution. Oxidase 202
activity was evaluated by the oxidation of 1% (w/v) tetramethyl
p-phenylenediamine. 203
Substrate utilization was tested at atmospheric pressure in
duplicate, using Hungate 204
tubes containing basal medium contained (L−1): 1.0 g NaHCO3, 1.0
g NH4Cl, 1.0 g 205
CH3COONa, 0.2 g MgSO4.7H2O, 0.5 g KH2PO4, 0.7 g cysteine
hydrochloride, 1 mL 206
0.1% (w/v) resazurin. Single substrate (including cellulose,
pectin, xylan, glucose, 207
acetate, maltose, butyrate, fructose, glycine, ethanol, formate,
lactate, sucrose, sorbitol, 208
D-mannose) was added from sterile filtered stock solutions to
the final concentration 209
at 20 mM, respectively. Cell culture without adding any other
substrates was used as a 210
control. The pH was adjusted to 7.0 with NaOH. The cultures were
incubated at 30 °C 211
for 7 days and then determined by spectrophotometry at 600 nm.
For each substrate 212
was repeated three times. All tested substrates were listed in
the species description. 213
The detailed physiological characteristics that differentiate
the Maribellus luteus 214
XSD2T were listed as Table 1. Strain WC007T required NaCl for
growth and growth 215
was observed at 0-5.0% NaCl (optimum: 1.0% NaCl). And growth
occurred at 216
28-37 °C (optimum, 30 °C) and at pH 6.0-8.0 (optimum, pH 7.0).
In addition, the 217
positive activities of acetate, maltose, fructose, ethanol,
formate, lactate, sorbitol and 218
D-mannose in strain WC007T distinguished those in strain XSD2T.
The differential 219
phenotypic characteristics between strain WC007T and the closely
related type strain 220
XSD2T are shown in the Table 1. Overall, the phenotypic
characterization supports 221
that strain WC007T represents a novel species of the genus
Maribellus. 222
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For chemotaxonomic analysis, cells of WC007T were cultured and
collected under 223
the same conditions unless stated otherwise, with the closely
related type strains were 224
grown on the ORG solid medium for 7 d at 30 °C under the same
condition. Cells of 225
WC007T and the closely related type strain were harvested from
cultures at the 226
mid-exponential phase of growth and freeze-dried. Cellular fatty
acids were extracted 227
and determined from dried cells by using GC (model 7890A,
Agilent, USA) 228
according to the protocol of the Sherlock Microbial
Identification System [39]. Polar 229
lipids were extracted and determined as described by Tindall et
al [40]. 230
The predominant respiratory quinone of strain WC007T was MK-7,
which is the 231
most frequently identified respiratory quinone in bacteria
belonging to the family 232
Prolixibacteraceae [4]. The major cellular fatty acids (>10.0
%) in strain WC007T 233
were iso-C15:0, C16:0, summed feature 3 and summed feature 8.
The amount of 234
iso-C15:0 in strain WC007T (14.07%) was higher than that found
in strain XSD2T 235
(1.45%), while the amount of C18:0 and C18:1ω9c in strain WC007T
(2.39%, 7.62%) 236
was lower than that found in strain XSD2T (17.33%, 16.57%),
respectively. The major 237
polar lipids in strain WC007T were phosphatidylethanolamine, one
unidentified 238
phospholipid, one unidentified aminolipid and one unidentified
lipid (Fig. S2). Strain 239
WC007T was apparently different from the type strain XSD2T by
the presence of one 240
unidentified phospholipid. 241
In summary, phylogenetic analysis of strain WC007T based on 16S
rRNA gene 242
sequence similarities confirmed the distinctness of strain
WC007T from the closely 243
related strain XSD2T. Moreover, based on a polyphasic taxonomic
approach and 244
several phenotypic characteristics, strain WC007T distinguished
from Maribellus 245
luteus XSD2T, the only recognized species of the genus
Maribellus. Therefore, we 246
propose that strain WC007T was classified as the type strain of
a novel species in the 247
genus Maribellus, for which the name Maribellus comscasis sp.
nov. is proposed. 248
Description of Maribellus comscasis sp. nov. 249
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Maribellus comscasis (com.sca'sis. L. gen. pl. n. comscasis from
the Center for Ocean 250
Mega-Science, Chinese Academy of Sciences). 251
Cells are Gram-stain-negative curve-shaped, 2.0-6.0 µm in length
and 0.5-0.8 µm 252
in width. Facultative anaerobic and oxidase-positive. The
temperature range for 253
growth is 28-37 °C with an optimum at 30 °C. Growing at pH
values of 6.0-8.0 254
(optimum, pH 7.0). Growth occurs at NaCl concentrations between
0.0-5.0% with 255
optimum growth at 1.0% NaCl. By analyzing the hydrolysis of
polysaccharides, the 256
growth is promoted significantly by cellulose, pectin and xylan.
From the sole carbon 257
source utilization test, growth is stimulated by acetate,
maltose, fructose, lactate, 258
sorbitol and D-mannose. Weak growth occurs with ethanol and
formate. The major 259
polar lipids are phosphatidylethanolamine, one unidentified
phospholipid, one 260
unidentified aminolipid, one unidentified lipid. Containing
significant proportions 261
(>10%) of the cellular fatty acids iso-C15:0, C16:0, summed
feature 3 (containing 262
C16:1ω7c and/or C16:1ω6c) and summed feature 8 (containing
C18:1ω7c and/or 263
C18:1ω6c). 264
The type strain, WC007T (=KCTC 25169T =MCCC 1K04777T), was
isolated from 265
the sediment of deep-sea cold seep, P.R. China. The DNA G+C
content of the type 266
strain is 38.38%. 267
268
Funding information 269
This work was funded by the National Key R and D Program of
China (Grant No. 270
2018YFC0310800), China Ocean Mineral Resources R&D
Association Grant (Grant 271
No. DY135-B2-14), Strategic Priority Research Program of the
Chinese Academy of 272
Sciences (Grant No. XDA22050301), the Taishan Young Scholar
Program of 273
Shandong Province (tsqn20161051), and Qingdao Innovation
Leadership Program 274
(Grant No. 18-1-2-7-zhc) for Chaomin Sun. 275
Conflicts of interest 276
The authors have no conflict of interest. 277
(which was not certified by peer review) is the author/funder.
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395
396
397
398
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Table 1. Differential physiological characteristics of the novel
strain WC007T and its 399
closest related type strain Maribellus luteus XSD2T. Strains: 1,
WC007T (all data from 400
this study); 2, Maribellus luteus XSD2T (all data from this
study except DNA G+C 401
content and polar lipids). +, Positive result or growth; -,
negative result or no growth. 402
Characteristic 1 2
Cell length (µm)
Temperature range
for growth (°C) Optimum
pH range for growth
Optimum
NaCl range for growth (%)
Optimum
Oxidase activity
Hydrolysis of:
Cellulose
Pectin
Xylan
Utilization as a sole carbon source:
Acetate
Maltose
Fructose
Ethanol
Formate
Lactate
Sorbitol
D-mannose
Major menaquinone(s)
Polar lipids
Major fatty acids (>10 %)
DNA G+C content (mol%)
Isolation source
2.0-6.0
28-37
30
6.0-8.0
7.0
0-5
1
+
+
+
+
+
+
+
-
-
+
+
+
MK-7
PE, PL, AL, L
iso-C15:0, C16:0, summed
feature 3, summed
feature 8
38.38
deep-sea sediments
2.0-9.0
20–40
28
6.0-8.5
7.0
1-5
2
-
-
-
-
-
-
-
+
+
-
-
-
MK-7
PE, AL, 3L
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15
C16:0, C18:1ω9c,
summed feature 8,
C18:0
44.1
surface seawater
*Summed features are groups of two or three fatty acids that
could not be separated 403
by GLC using the MIDI system. Summed feature 8 contains C18:1ω7c
and/or C18:1ω6c. 404
Summed feature 8 contains C16:1ω7c/C16:1ω6c and/or
C16:1ω6c/C16:1ω7c. 405
Table 2. Percentages of fatty acids useful for distinguishing
WC007T from its closest 406
relative Maribellus luteus XSD2T. Strains: 1, WC007T (all data
from this study); 2, 407
Maribellus luteus XSD2T (all data from this study). 408
Fatty acid Percentage (w/v) of total fatty acids
1 2
Saturated:
C18:0
Branched:
2.39
17.33
iso-C15:0
C18:1ω9c
14.07
7.62
1.45
16.57
409
410
411
412
413
414
415
416
417
418
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16
419
420
421
422
423
424
425
426
427
428
Figure legends 429
Fig. 1. Neighbor-joining tree based on 16S rRNA gene sequences
showing the 430
position of the novel strain WC007T among bacterial members of
the family 431
Prolixibacteraceae and other closely related families. The
filled circles indicate 432
branches of the tree that were also formed using the
maximum-likelihood and 433
minimum-evolution methods. Numbers at branching points are
bootstrap values 434
(expressed as percentages of 1000 replications). The access
number of each 16S 435
rRNA is indicated after the strain’s name. The sequence of
Agarivorans albus LMG 436
21761T is used as an outgroup. Bar, 0.05 substitutions per
nucleotide position. 437
Fig. 2. Transmission electron microscopy (TEM) observation of a
negatively stained 438
culture of strain WC007T. Bar is 2 μm. 439
440
441
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