IJSEM Papers in Press. Published November 10, 2014 as doi:10.1099/ijs.0.069708-0
Sphingobium subterraneum sp. nov., isolated from ground water
1
2
Jae-Chan Lee1, Song-Gun Kim3,4, Kyung-Sook Whang1,2
3 4 5 6 7 8 9 10 11 12
1
Institute of Microbial Ecology and Resources, Mokwon University, 88 Doanbuk-ro, Seo-gu,
Daejeon 302-318, Republic of Korea 2
Department of Microbial & Nano Materials, College of Science & Technology, Mokwon
University, 88 Doanbuk-ro, Seo-gu, Daejeon 302-318, Republic of Korea 3
Microbial Resource Center/KCTC, Korea Research Institute of Bioscience and
Biotechnology, Daejeon 305-806, Republic of Korea 4
University of Science and Technology, Daejeon 305-850, Republic of Korea
13 14 15 16
Running title: Sphingobium subterraneum sp. nov.
17 18
Key words: Sphingobium subterraneum sp. nov., ground water, taxonomy
19 20 21
Author for correspondence: Kyung-Sook Whang
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Tel: +82 42 829 7598
23
Fax: +82 42 829 7599
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E-mail: [email protected]
25 26 27
Category: New taxa - Proteobacteria
28 29 30 31
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain S-
32
II-13T is FJ796422. 1
33
A Gram-staining-negative, strictly aerobic, non-motile, non-spore-forming, yellow
34
colored and rod-shaped bacterium, designated S-II-13T, was isolated from ground water
35
at Daejeon in Korea. Strain S-II-13T grew between 15 and 30 °C (optimal growth at
36
28 °C), between pH 6.0 and 9.0 (optimal growth at pH 7.5) and at salinities of 0–1.5 %
37
(w/v) NaCl, growing optimally at 0.5 % (w/v) NaCl. On the basis of 16S rRNA gene
38
sequence analysis, strain S-II-13T was shown to belong to the genus Sphingobium
39
showed closest phylogenetic similarity to Rhizorhapis suberifaciens CA1 (97.0 %),
40
Sphingobium sufflavum HL-25T (96.9 %) and Sphingobium vulgare BHU1-GD12T
41
(96.6%). The major polar lipids were phosphatidylglycerol, diphosphatidyl-glycerol,
42
phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidyl coline
43
and sphingoglycolipid. The predominant ubiquinone was Q-10. The major fatty acids
44
were C18:1 ω7c, C14:0 2-OH, C16:0 and C16:1 ω7c and/or C15:0 iso 2-OH. The DNA G+C
45
content of this novel isolate was 63.5 mol%. DNA-DNA relatedness between strain S-II-
46
13T and Rhizorhapis suberifaciens LMG 17323T, Sphingobium sufflavum KCTC 23953T
47
and Sphingobium vulgare KCTC 22289T was 24, 52 and 55 %, respectively. On the basis
48
of polyphasic evidence from this study, strain S-II-13T represents a novel species of the
49
genus Sphingobium for which the name Sphingobium subterraneum sp. nov. is proposed.
50
The type strain is S-II-13T (=KACC 17606T=NBRC 109814T).
51 52
The genus Sphingobium in the family Sphingomonadaceae, belonging to the Alpha-
53
proteobacteria, was first described by Yabuuchi et al. (1990) and proposed by Takeuchi et al.
54
(2001) by dissection of the genus Sphingomonas on the basis of phylogenetic and
55
chemotaxonomic analysis. At the recently study, Sphingobium suberifaciens which was
56
reclassified from Sphingomonas suberifaciens by Chen et al. (2013), was proposed to
57
reclassify as Rhizorhapis suberifaciens gen. nov., comb. nov. by Francis et al. (2014). At the
58
time of writing the genus Sphingobium comprised 37 recognized species (Parte, 2014).
59
Sphingobium are Gram-negative, non-sporulating, strictly aerobic, non-motile or motile rods
60
and they are characterized chemotaxonomically by the presence of ubiquinone-10 and C18:1 as
61
the predominant fatty acid, C16:0 as a minor component, and C14:0 2-OH as the major hydroxyl
62
fatty acids and sphingoglycolipid and members of the genus possess DNA G + C contents of
63
between 62 and 67 mol% (Takeuchi et al. 2001).
64
Strain S-II-13T was isolated from ground water at Daejeon in Korea during studies focused 2
65
on the isolation of oligotrophic bacteria. It was isolated by diluting a water sample in sterile
66
distilled water and plating on 100-fold dilution of nutrient broth (10-2 NB) containing 1.2 %
67
(w/v) agar, followed by aerobic incubation at 28 °C for 10 days. Nutrient broth (NB) was
68
comprised of 1 % (w/v) beef extract (Junsei), 1 % (w/v) polypeptone (Junsei) and 0.5 % (w/v)
69
NaCl. The pH of the medium was adjusted to pH 7.0 with 1M NaOH. The strain was
70
subsequently purified three times by plating on R2A (Difco) medium 28 °C for 3 days and
71
maintained on the same medium. The strain was stored at –80 °C on this medium without
72
agar and supplemented with 20 % (v/v) glycerol. In order to characterize strain S-II-13T
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phenotypically, the isolate was routinely grown aerobically on R2A agar for 3 days at 28 °C
74
and pH 7.0, except where indicated otherwise.
75 76
The morphology of the isolate was observed by Gram staining and transmission electron
77
microscopy (201; Phillips) and motility was observed by phase-contrast microscopy (Eclipse
78
80i; Nikon) using cells from exponentially growing cultures. Gram staining was performed
79
by the Burke method (Murray et al., 1994). Catalase, oxidase and nitrate reduction,
80
hydrolysis of aesculin and production of indole were tested as recommended by Smibert &
81
Krieg (1994) with 0.01% (w/v) of substrate concentration. Substrate utilization profile was
82
tested in minimal media supplemented with carbon sources corresponding to API50CH
83
(bioMérieux) gallery ingredients. All compounds were sterilized by filtration and were added
84
to the media. Cell suspension was performed in sterile distilled water. Enzymic activities
85
were tested using the API ZYM kit system according to the instructions of the manufacturer
86
(bioMérieux). To determine the optimal temperature and pH for growth of the strain, broth
87
cultures in R2A broth were incubated at 0–50 °C at intervals of 5 °C plus at 4, 28 and 37 °C
88
and at pH 5–11 at intervals of 0.5 pH units. The pH values of 9 were obtained
89
by using sodium acetate/acetic acid, Tris/HCl and glycine/sodium hydroxide buffers,
90
respectively. Growth at different salt concentrations (0, 0.01, 0.03. 0.05, 0.1, 0.5, 1.0, 3.0 and
91
5.0 %, w/v) was tested in nutrient broth (10-2 NB; Difco) medium at pH 7.0. Growth was
92
monitored by turbidity at OD600 by using a spectroscopic method (model UV-1650PC;
93
Shimadzu).
94 95
Susceptibility to antibiotics was tested on R2A plates using antibiotic discs containing the
96
following: amikacin, 30 μg; amoxicillin, 10 μg; ampicillin, 20 μg; bacitracin, 10 U;
97
carbenicillin, 100 μg; cefotaxime, 30 μg; cefoxitin, 30 μg; cephalexin, 30 μg; 3
98
chloramphenicol, 10 μg; ciprofloxacin, 5 μg; colistinsulphate, 10 μg; doxycycline, 30 μg;
99
erythromycin, 15 μg; gentamicin, 10 μg; kanamycin, 30 μg; lincomycin, 15 μg; methicillin, 5
100
μg; nalidixic acid, 30 μg; neomycin, 30 μg; nitrofurantoin, 300 μg; norfloxacin, 10 μg;
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novobiocin, 30 μg; nystatin, 100 μg; oxacillin, 1 μg; penicillin, 10 U; piperacillin, 75 μg;
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polymixin B, 100 U; rifampicin, 30 μg; streptomycin, 10 μg; teicoplanin, 30 μg; tetracycline,
103
30 μg; tobramycin, 10 μg; and vancomycin, 30 μg
104 105
Strain S-II-13T showed a range of phenotypic properties typical of members of the genus
106
Sphingobium (Yabuuchi et al., 1990; Takeuchi et al., 2001). It was Gram-staining-negative,
107
non-motile and strictly aerobic and, colonies were yellow in colour, round and convex with
108
entire margins when grown for 3 days at 28 °C on R2A agar. Cells were rods with a width of
109
0.6–0.7 µm and a length of 0.8–1.2 µm (Supplementary Fig. S1). Strain S-II-13T was able to
110
grow at 15–30 °C, at pH 6.0–9.0 and in 0.3–1.5 % (w/v) NaCl. Optimal growth was observed
111
at 28°C, at pH 7.5 and with 0.5 % (w/v) NaCl. Cells were catalase-positive and oxidase-
112
negative. Growth was observed on the media, nutrient agar (NA; Difco) Luria-Bertani agar
113
(LB; Difco), trypticase soy agar (TSA; Difco) and ISP2 (Difco). Other phenotypic features
114
are included in the species description and the characteristics that differentiate strain S-II-13T
115
from the related type strains of species of the genus Sphingobium are summarized in Table 1.
116 117
Genomic DNA from strain S-II-13T was prepared by using the method described by Tomaoka
118
& Komagata (1984). The 16S rRNA gene was amplified by PCR with the forward primer
119
Eubac 27F and the reverse primer 1492R (DeLong, 1992). Direct sequence determination of
120
the PCR-amplified DNA was carried out using an automated DNA sequencer (model ABI
121
3730XL; Applied Biosystems). Full sequences of the 16S rRNA gene were compiled using
122
SeqMan software (DNASTAR). The 16S rRNA gene sequence was aligned with the published
123
sequences of closely related bacteria with CLUSTAL W 2.0 software (Larkin et al., 2007). Gaps
124
at the 5' and 3' ends of the alignment were omitted in further analyses. Phylogenetic trees
125
were constructed by using three different methods: the neighbour-joining (Saitou & Nei,
126
1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971)
127
algorithms within the MEGA5 program (Tamura et al. 2011). Evolutionary distance matrices
128
for the neighbour-joining method were calculated using the algorithm of the Kimura two-
129
parameter model (Kimura, 1980). To evaluate the stability of the phylogenetic tree, a
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bootstrap analysis (1000 replications) was performed (Felsenstein, 1985). The 16S rRNA 4
131
gene sequences used for phylogenetic comparisons were obtained from GenBank and their
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strain designations and accession numbers are shown in Fig. 1.
133 134
To determine genomic relatedness, DNA–DNA hybridization was performed using the
135
modified method of Ezaki et al. (1989). Probe labelling for DNA-DNA hybridization was
136
conducted by using the nonradioactive DIG-High prime system (Roche); hybridized DNA
137
was visualized using the DIG luminescent detection kit (Roche) and the level of DNA-DNA
138
relatedness was quantified by using a densitometer (Bio-Rad). Isolation of DNA (Saito &
139
Miura, 1963) and determination of the DNA G + C content were performed by HPLC (SPD-
140
10AV; Shimadzu), as described by Mesbah et al. (1989).
141 142
The almost-complete 16S rRNA gene sequence (1459 bp) of strain S-II-13T was obtained and
143
used for initial BLAST searches in GenBank and phylogenetic analysis. The 16S rRNA gene
144
sequences of related taxa were obtained from the GenBank and EzTaxon-e servers and the
145
identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene
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sequence
147
(http://www.ezbiocloud.net/eztaxon; Kim et al., 2012). Strain S-II-13T was shown to belong
148
to the genus Sphingobium or Rhizorhapis showed closest phylogenetic similarity to
149
Rhizorhapis suberifaciens CA1T (97.0 %), Sphingobium sufflavum HL-25T (96.9 %) and
150
Sphingobium vulgare BHU1-GD12T (96.6 %) on the basis of 16S rRNA gene sequence
151
analysis. Phylogentic analysis revealed that the strain S-II-13T was located in the genus
152
Sphingobium
153
(Supplementary Fig. S2) and maximum-parsimony (Supplementary Fig. S3) resulted in
154
highly similar tree topologies forming a distinct phyletic line in the genus Sphingobium with
155
>85% bootstrap value and in maximum-parsimony and neighbor-joining (Fig. 1) also resulted
156
similar tree topology forming a separate phyletic line in the genus Rhizorhapis even though it
157
was separated from the genus Sphingobium. 16S rRNA sequence similarity with members of
158
the other species of the genus Sphingobium species was in the range from 96.9 to 94.8 %.
159
DNA–DNA hybridization values between strain S-II-13T and Rhizorhapis suberifaciens LMG
160
17323T, Sphingobium sufflavum KCTC 23953T and Sphingobium vulgare KCTC 22289T were
161
24, 52 and 55 %, respectively. All values were clearly below the threshold value of 70 %
162
DDH that is recommended for the assignment of the genomic species (Wayne et al., 1987).
163
The G + C content of the DNA of strain S-II-13T was 63.5 mol%, which is in the range (62.0–
similarity
and
were
separated
achieved
from
by
the
genus
5
using
the
Rhizorhapis.
EzTaxon-e
server
Maximum-likelihood
164
67.0 mol%) for members of the genus Sphingobium and different from the genus Rhizorhapis
165
(58–60 mol%) (Francis et al., 2014).
166 167
For analysis of fatty acids, strain S-II-13T was cultured on R2A at 28 °C for 3 days and the
168
closely related type strains were cultured under the same conditions. Cellular fatty acids were
169
extracted and analysed by GC (6890N; Agilent Technologies) according to the standard
170
protocol of the Sherlock Microbial Identification System (version 4.5; MIDI database
171
TSBA40 4.10). For the analysis of quinones and polar lipids, cells were harvested in the late-
172
exponential phase and freeze-dried. Isoprenoid quinones were extracted and analysed by
173
HPLC (SPD-10AV; Shimadzu), as described by Collins & Jones (1981). For polar lipid
174
analysis, the cellular lipids were extracted twice, washed and hydrolysed with 0.5 M NaOH
175
as described by Yabuuchi et al. (1990, 1999). The total lipids were separated by two-
176
dimensional TLC with a solvent system composed of chloroform/methanol/water (65:25:4,
177
by vol.) in the first direction and chloroform/methanol/acetic acid/water (80:15:12:4, by vol.)
178
in the second direction. For polyamine analysis, strain S-II-13T was grown aerobically in
179
R2A broth at 28 °C and harvested in the late-exponential phase. Polyamines were extracted
180
from l00 mg of lyophilized cells according to the methods described by Busse et al. (1997)
181
and sequentially benzoylated by the methods described by Taibi et al. (2000). Quantitative
182
analysis of polyamines was performed by using a reverse-phase HPLC equipped with a
183
photodiode array detector (234 nm) (Series 20 HPLC; Shimadzu) and Watcher 120 ODS-AP
184
column (250 × 4.6 mm internal diameter, 5 μm particle size) (Isu Industry Corp)
185 186
Major fatty acids in strain S-II-13T were C18:1 ω7c (23.1 %), C16:0 (15.5 %) and summed
187
feature 3 (12.4 %; C16:1 ω7c and/or iso-C15:0 2-OH). 2-Hydroxy fatty acids were present as
188
C14:0 2-OH (16.3 %) but 3-hydroxy fatty acid were not present. These are common
189
characteristic features of members of the genus Sphingomonas. The presence of C15:0, C19:0
190
cyclo ω8c and C16:0 2-OH distinguished strain S-II-13T from closely related type strains of the
191
genus Sphingobium and Rhizorhapis (Supplementary Table. S1). The major isoprenoid
192
quinone of isolate S-II-13T was ubiquinone 10 (Q-10), as in all known members of the family
193
Sphingomonadaceae. Polar lipids of S-II-13T comprised phosphatidylethanolamine (PE),
194
phosphatidylmonomethylethanolamine
195
diphosphatidylglycerol (DPG), phosphatidyl-choline (PC), sphingoglycolipid (SGL) and,
196
unknown aminoglycolipid (AGL) (Supplementary Fig. S4). The presence of abundant
(PME),
6
phosphatidylglycerol
(PG),
197
sphingoglycolipid in strain S-II-13T indicates that the isolate is a member of the family
198
Sphingomonadaceae. The polyamine pattern of strain S-II-13T consisted of predominantly
199
spermidine [18.9 µmol (g dry weight)-1] and trace amount of putrescine [0.1 µmol (g dry
200
weight)-1] and cadaverine [0.05 µmol (g dry weight)-1]. This polyamine pattern is in
201
agreement with the description of the genus Sphingobium (Takeuchi et al., 2001).
202 203
Strain S-II-13T shared similar chemotaxonomic characteristics with members of the genus
204
Sphingobium sensu stricto in terms of the DNA G + C content, polar lipids, major polyamine,
205
predominant ubiquinone and major fatty acids. However, strain S-II-13T could be
206
distinguished from other closely related species by some chemotaxonomic and phenotypic
207
features, such as colony color, oxidase negative, presence of C15:0, C19:0 cyclo ω8c and C16:0
208
2-OH.
209 210
Therefore, on the basis of the results of this polyphasic taxonomic study, we propose that the
211
new strain represents a novel species of the genus Sphingobium, for which the name
212
Sphingobium subterraneum sp. nov. is proposed, with the type strain S-II-13T.
213 214
Description of Sphingobium subterraneum sp. nov.
215
Sphingobium subterraneum (sub.ter.ra'ne.um. L. neut. adj. subterraneum, underground).
216 217
Cells are Gram-negative, non-motile, rods, 0.6−0.7×0.8−1.2 µm in size. Colonies are circular,
218
convex, entire, yellow in colour, and 0.5−0.9 mm in diameter on R2A agar after 3 days
219
incubation at 28 ºC. Growth also occurs on NA, LB, TSA and ISP2. Grows at 15−30 °C
220
(optimally at 28 °C) and pH 6.0−9.0 (optimally at pH 7.5) and with 0.3−1.5 % (w/v) NaCl,
221
with optimal growth at 0.5 % (w/v) NaCl. Catalase-positive and oxidase-negative. Esculin
222
and starch are hydrolysed but L-arginine, urea, L-tyrosine, xanthine and gelatin are not.
223
Nitrate is reduced to nitrite. Indole is not produced. Acid is not produced from D-glucose.
224
Utilizes D-ribose, L-xylose, D-adonitol, methyl-β-D-xylopyranoside, D-galactose, D-fructose,
225
D-mannose,
226
mannopyranoside, methyl-α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin,
227
esculin, L-salicin, D-cellobiose, D-lactose, D-melibiose, D-sucrose, inulin, D-melezitose, D-
228
raffinose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose,
L-rhamnose,
dulicitol,
inositol,
7
D-mannitol,
D-sorbitol,
methyl-α-D-
229
L-fucose, D-arabitol, L-arabitol and potassium gluconate. Acid is produced from amygdalin,
230
arbutin,
231
acetylglucosamine, D-ribose, L-salicin, starch, D-sucrose and D-trehalose. Enzyme activity is
232
observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine
233
arylamidase, valine arylamidase and crystine arylamidase, but not for trypsin,
D-cellobiose,
esculin,
D-fructose,
glycerol,
glycogen,
D-maltose,
N-
236
αchymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, βgalactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosamininase, αmannosidase and α-fucosidase (API ZYM). Susceptible to teicoplanin, amikacin, ampicillin,
237
nalidixic acid, kanamycin, gentamicin, neomycin, vancomycin, erythromycin, oleandomycin,
238
amoxicillin, spiramycin, rifampicin, nystatin, tetracycline, roxithromycin, sphingomyelin,
239
apramycin, hygromycin B, capreomycin, sisomycin, gramicidin S, chloramphenicol and
240
chloramphenicol, but resistant to lincomycin, streptomycin, penicillin, polymixin B,
241
bacitracin, cycloheximide, salinomycin, amphotericin, phosphomycin and streptomycin. The
242
major fatty acids are C18:1 ω7c, C14:0 2-OH, C16:0 and C16:1 ω7c and/or C15:0 iso 2-OH. Main
243
hydroxy-fatty
244
phosphatidylmonomethylethanolamine,
245
phosphatidyl-choline, sphingoglycolipid and unknown aminoglycolipid. Major polyamine is
246
spermidine. The predominant isoprenoid quinone is ubiquinone Q-10.
234 235
acid
is
C14:0 2-OH.
Polar
lipids
are
phosphatidylethanolamine,
phosphatidylglycerol,
diphosphatidylglycerol,
247 248
The type strain, S-II-13T (=KACC 17606T=NBRC 109814T), was isolated from ground
249
water at Daejeon in Korea. The DNA G + C content of the type strain is 63.5 mol%.
250 251 252 253
Acknowledgements
254 255
This work was supported by a grant from the Regional SubGenBank Support Program of
256
Rural Development Administration, Republic of Korea
257 258 259 260 261 8
262 263
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Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General
321
and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A.
322
Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
323
Taibi, G., Schiavo, M. R., Gueli, M. C., Rindina, P. C., Muratore, R. & Nicotra, C. M. A.
324
(2000). Rapid and simultaneous high-performance liquid chromatography assay of
325
polyamines and monoacetylpolyamines in biological specimens. J Chromatogr B Biomed Sci
326
Appl 745, 431–437.
327
Takeuchi, M., Hamana, K. & Hiraishi, A. (2001). Proposal of the genus Sphingomonas
328
sensu stricto and three new genera, Sphingobium, Sphingobium and Sphingopyxis, on the
329
basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51, 1405–
330
1417.
331
Tamura K., Peterson D., Peterson N., Stecher G., Nei M., and Kumar S. (2011). MEGA5:
332
molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance,
333
and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
334
Tomaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reverse-
335
phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
336
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O.,
337
Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors
338
(1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee
339
on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
340
Yabuuchi, E., Kosako, Y., Naka, T., Suzuki, S. & Yano, I. (1999). Proposal of 11
341
Sphingomonas suberifaciens (van Bruggen, Jochimsen and Brown 1990) comb. nov.,
342
Sphingomonas natatoria (Sly 1985) comb. nov., Sphingomonas ursincola (Yurkov et al.,
343
1997) comb. nov., and emendation of the genus Sphingomonas. Microbiol Immunol 43, 339–
344
349.
345
Yabuuchi, E., Yano, I., Oyaizu, H., Hashimoto, Y., Ezaki, T. & Yamamoto, H. (1990).
346
Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas
347
parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp.
348
nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas.
349
Microbiol Immunol 34, 99–119.
350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 12
373
Figure Legend
374 375
Fig. 1. Rooted neighbor-joining tree based on 16S rRNA gene sequences showing the
376
phylogenetic position of strain S-II-13T and related bacteria in the genus Sphingobium and
377
Rhizorhapis. Bootstrap values, expressed as a percentage of 1000 replications, are given at
378
branching points when >50%. Sphingomonas paucimobilis ATCC 2983T was used as an
379
outgroup. Bar, 0.01 subtitutions per nucleotide position.
380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 13
406
Table 1. Phenotypic characteristics for differentiating strain S-II-13T from the related type
407
strains of species the genus Sphingobium and Rhizorhapis
408 409 410 411 412 413 414 415 416 417
Strains: 1, S-II-13T; 2, Rhizorhapis suberifaciens LMG 17323T; 3, Sphingobium boeckii DSM 25079T; 4, Sphingobium sufflavum KCTC 23953T; 5, Sphingobium vulgare KCTC 22289T, 6, Sphingobium xanthum LMG 12560T. All data were obtained from this study, except where indicated otherwise. All strains are Gram-stainingnegative, aerobic rods and positive for catalase. All strains share the following characteristics: positive for alkaline phosphate and negative for indole production, arginine dihydrolase, α-galactosidase and N-acetyl-βglucosamininase activities hydrolysis esculin but not gelatin, L-arginine and urea; no assimilation of D,Larabinose and caprate; no acid production from D-adonitol, L-rhamnose, D-mannitol and D-raffinose. All strains are susceptible to kanamycin (30 ㎍), gentamicin (10 ㎍), vancomycin (30 ㎍) and erythromycin (15 ㎍). +, Positive; −, negative; w, weakly positive; S, sensitive; R, resistant. Characteristic
1
2
3
4
5
6
Yellow
White
Creamy yellow
Light yellow
Yellow
Bright yellow
−
+
+
+
+
+
−
−
+
−
+
+
+
−
−
−
−
+
D-Glucose
−
+
−
+
+
+
N-Acetyl- D-glucosamine
+
−
+
+
−
−
D-Galactose
+
−
+
+
−
+
D-Mannose
+
−
+
+
−
+
Rhamnose
+
−
−*
+
−
+
D-Melibiose
+
−
+
+
−
+*
Colony color Oxidase Growth at 37
℃
Hydrolysis of: Starch Assimilation of:
Glycogen
+
−
+
+
−
+
Adipate
−
−
−
+
+
+
Citrate
−
−
+
+
−
+
Malate
−
−
−
+
+*
+
D-Fructose
+
−
−
−
+
+
D-Maltose
+
+
−
−
+
+
D-Sucrose
+
−
−
−
−
+
D-Trehalose
+
−
+
−
+
+
Glycerol
+
−
−
+
+
−
D-Ribose
+
−
−
+*
+
−
D-Cellobiose
+
−
+
−
−
+
Esterase (C4)
+
−
w
+
+
−
Valine arylamidase
+
−
−
+
−
+
α-glucosidase
−
+
−
−
−
−
β-galactosidase
−
+
+
+
−
−
R
Acid production from:
Enzyme activities:
Susceptibility to: Penicillin (12 U)
R
S
S
S
R
Nystatin (50
S
R
R
S
S
S
R
S
S
R
R
R
S
R
R
S
S
㎍)
Polymixin B (50 Tetracycline (30
418 419
㎍) ㎍)
DNA G+C contents (mol%) 63.5 58.2-59.5a† 64.6 b 63.8c 66.8d * Data that were different from the results obtained in previous studies † Data obtained from: a, Francis et al. (2014); b, Chen et al. (2013); c, Sheu et al. (2013); d, Baek et al. (2010)
14
S 64.7a
Sphingobium subterraneum sp. nov., isolated from ground water
1
2
Jae-Chan Lee1, Song-Gun Kim3,4, Kyung-Sook Whang1,2
3 4 5 6 7 8 9 10 11 12
1
Institute of Microbial Ecology and Resources, Mokwon University, 88 Doanbuk-ro, Seo-gu,
Daejeon 302-318, Republic of Korea 2
Department of Microbial & Nano Materials, College of Science & Technology, Mokwon
University, 88 Doanbuk-ro, Seo-gu, Daejeon 302-318, Republic of Korea 3
Microbial Resource Center/KCTC, Korea Research Institute of Bioscience and
Biotechnology, Daejeon 305-806, Republic of Korea 4
University of Science and Technology, Daejeon 305-850, Republic of Korea
13 14 15 16
Running title: Sphingobium subterraneum sp. nov.
17 18
Key words: Sphingobium subterraneum sp. nov., ground water, taxonomy
19 20 21
Author for correspondence: Kyung-Sook Whang
22
Tel: +82 42 829 7598
23
Fax: +82 42 829 7599
24
E-mail: [email protected]
25 26 27
Category: New taxa - Proteobacteria
28 29 30 31
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain S-
32
II-13T is FJ796422. 1
33
A Gram-staining-negative, strictly aerobic, non-motile, non-spore-forming, yellow
34
colored and rod-shaped bacterium, designated S-II-13T, was isolated from ground water
35
at Daejeon in Korea. Strain S-II-13T grew between 15 and 30 °C (optimal growth at
36
28 °C), between pH 6.0 and 9.0 (optimal growth at pH 7.5) and at salinities of 0–1.5 %
37
(w/v) NaCl, growing optimally at 0.5 % (w/v) NaCl. On the basis of 16S rRNA gene
38
sequence analysis, strain S-II-13T was shown to belong to the genus Sphingobium
39
showed closest phylogenetic similarity to Rhizorhapis suberifaciens CA1 (97.0 %),
40
Sphingobium sufflavum HL-25T (96.9 %) and Sphingobium vulgare BHU1-GD12T
41
(96.6%). The major polar lipids were phosphatidylglycerol, diphosphatidyl-glycerol,
42
phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidyl coline
43
and sphingoglycolipid. The predominant ubiquinone was Q-10. The major fatty acids
44
were C18:1 ω7c, C14:0 2-OH, C16:0 and C16:1 ω7c and/or C15:0 iso 2-OH. The DNA G+C
45
content of this novel isolate was 63.5 mol%. DNA-DNA relatedness between strain S-II-
46
13T and Rhizorhapis suberifaciens LMG 17323T, Sphingobium sufflavum KCTC 23953T
47
and Sphingobium vulgare KCTC 22289T was 24, 52 and 55 %, respectively. On the basis
48
of polyphasic evidence from this study, strain S-II-13T represents a novel species of the
49
genus Sphingobium for which the name Sphingobium subterraneum sp. nov. is proposed.
50
The type strain is S-II-13T (=KACC 17606T=NBRC 109814T).
51 52
The genus Sphingobium in the family Sphingomonadaceae, belonging to the Alpha-
53
proteobacteria, was first described by Yabuuchi et al. (1990) and proposed by Takeuchi et al.
54
(2001) by dissection of the genus Sphingomonas on the basis of phylogenetic and
55
chemotaxonomic analysis. At the recently study, Sphingobium suberifaciens which was
56
reclassified from Sphingomonas suberifaciens by Chen et al. (2013), was proposed to
57
reclassify as Rhizorhapis suberifaciens gen. nov., comb. nov. by Francis et al. (2014). At the
58
time of writing the genus Sphingobium comprised 37 recognized species (Parte, 2014).
59
Sphingobium are Gram-negative, non-sporulating, strictly aerobic, non-motile or motile rods
60
and they are characterized chemotaxonomically by the presence of ubiquinone-10 and C18:1 as
61
the predominant fatty acid, C16:0 as a minor component, and C14:0 2-OH as the major hydroxyl
62
fatty acids and sphingoglycolipid and members of the genus possess DNA G + C contents of
63
between 62 and 67 mol% (Takeuchi et al. 2001).
64
Strain S-II-13T was isolated from ground water at Daejeon in Korea during studies focused 2
65
on the isolation of oligotrophic bacteria. It was isolated by diluting a water sample in sterile
66
distilled water and plating on 100-fold dilution of nutrient broth (10-2 NB) containing 1.2 %
67
(w/v) agar, followed by aerobic incubation at 28 °C for 10 days. Nutrient broth (NB) was
68
comprised of 1 % (w/v) beef extract (Junsei), 1 % (w/v) polypeptone (Junsei) and 0.5 % (w/v)
69
NaCl. The pH of the medium was adjusted to pH 7.0 with 1M NaOH. The strain was
70
subsequently purified three times by plating on R2A (Difco) medium 28 °C for 3 days and
71
maintained on the same medium. The strain was stored at –80 °C on this medium without
72
agar and supplemented with 20 % (v/v) glycerol. In order to characterize strain S-II-13T
73
phenotypically, the isolate was routinely grown aerobically on R2A agar for 3 days at 28 °C
74
and pH 7.0, except where indicated otherwise.
75 76
The morphology of the isolate was observed by Gram staining and transmission electron
77
microscopy (201; Phillips) and motility was observed by phase-contrast microscopy (Eclipse
78
80i; Nikon) using cells from exponentially growing cultures. Gram staining was performed
79
by the Burke method (Murray et al., 1994). Catalase, oxidase and nitrate reduction,
80
hydrolysis of aesculin and production of indole were tested as recommended by Smibert &
81
Krieg (1994) with 0.01% (w/v) of substrate concentration. Substrate utilization profile was
82
tested in minimal media supplemented with carbon sources corresponding to API50CH
83
(bioMérieux) gallery ingredients. All compounds were sterilized by filtration and were added
84
to the media. Cell suspension was performed in sterile distilled water. Enzymic activities
85
were tested using the API ZYM kit system according to the instructions of the manufacturer
86
(bioMérieux). To determine the optimal temperature and pH for growth of the strain, broth
87
cultures in R2A broth were incubated at 0–50 °C at intervals of 5 °C plus at 4, 28 and 37 °C
88
and at pH 5–11 at intervals of 0.5 pH units. The pH values of 9 were obtained
89
by using sodium acetate/acetic acid, Tris/HCl and glycine/sodium hydroxide buffers,
90
respectively. Growth at different salt concentrations (0, 0.01, 0.03. 0.05, 0.1, 0.5, 1.0, 3.0 and
91
5.0 %, w/v) was tested in nutrient broth (10-2 NB; Difco) medium at pH 7.0. Growth was
92
monitored by turbidity at OD600 by using a spectroscopic method (model UV-1650PC;
93
Shimadzu).
94 95
Susceptibility to antibiotics was tested on R2A plates using antibiotic discs containing the
96
following: amikacin, 30 μg; amoxicillin, 10 μg; ampicillin, 20 μg; bacitracin, 10 U;
97
carbenicillin, 100 μg; cefotaxime, 30 μg; cefoxitin, 30 μg; cephalexin, 30 μg; 3
98
chloramphenicol, 10 μg; ciprofloxacin, 5 μg; colistinsulphate, 10 μg; doxycycline, 30 μg;
99
erythromycin, 15 μg; gentamicin, 10 μg; kanamycin, 30 μg; lincomycin, 15 μg; methicillin, 5
100
μg; nalidixic acid, 30 μg; neomycin, 30 μg; nitrofurantoin, 300 μg; norfloxacin, 10 μg;
101
novobiocin, 30 μg; nystatin, 100 μg; oxacillin, 1 μg; penicillin, 10 U; piperacillin, 75 μg;
102
polymixin B, 100 U; rifampicin, 30 μg; streptomycin, 10 μg; teicoplanin, 30 μg; tetracycline,
103
30 μg; tobramycin, 10 μg; and vancomycin, 30 μg
104 105
Strain S-II-13T showed a range of phenotypic properties typical of members of the genus
106
Sphingobium (Yabuuchi et al., 1990; Takeuchi et al., 2001). It was Gram-staining-negative,
107
non-motile and strictly aerobic and, colonies were yellow in colour, round and convex with
108
entire margins when grown for 3 days at 28 °C on R2A agar. Cells were rods with a width of
109
0.6–0.7 µm and a length of 0.8–1.2 µm (Supplementary Fig. S1). Strain S-II-13T was able to
110
grow at 15–30 °C, at pH 6.0–9.0 and in 0.3–1.5 % (w/v) NaCl. Optimal growth was observed
111
at 28°C, at pH 7.5 and with 0.5 % (w/v) NaCl. Cells were catalase-positive and oxidase-
112
negative. Growth was observed on the media, nutrient agar (NA; Difco) Luria-Bertani agar
113
(LB; Difco), trypticase soy agar (TSA; Difco) and ISP2 (Difco). Other phenotypic features
114
are included in the species description and the characteristics that differentiate strain S-II-13T
115
from the related type strains of species of the genus Sphingobium are summarized in Table 1.
116 117
Genomic DNA from strain S-II-13T was prepared by using the method described by Tomaoka
118
& Komagata (1984). The 16S rRNA gene was amplified by PCR with the forward primer
119
Eubac 27F and the reverse primer 1492R (DeLong, 1992). Direct sequence determination of
120
the PCR-amplified DNA was carried out using an automated DNA sequencer (model ABI
121
3730XL; Applied Biosystems). Full sequences of the 16S rRNA gene were compiled using
122
SeqMan software (DNASTAR). The 16S rRNA gene sequence was aligned with the published
123
sequences of closely related bacteria with CLUSTAL W 2.0 software (Larkin et al., 2007). Gaps
124
at the 5' and 3' ends of the alignment were omitted in further analyses. Phylogenetic trees
125
were constructed by using three different methods: the neighbour-joining (Saitou & Nei,
126
1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971)
127
algorithms within the MEGA5 program (Tamura et al. 2011). Evolutionary distance matrices
128
for the neighbour-joining method were calculated using the algorithm of the Kimura two-
129
parameter model (Kimura, 1980). To evaluate the stability of the phylogenetic tree, a
130
bootstrap analysis (1000 replications) was performed (Felsenstein, 1985). The 16S rRNA 4
131
gene sequences used for phylogenetic comparisons were obtained from GenBank and their
132
strain designations and accession numbers are shown in Fig. 1.
133 134
To determine genomic relatedness, DNA–DNA hybridization was performed using the
135
modified method of Ezaki et al. (1989). Probe labelling for DNA-DNA hybridization was
136
conducted by using the nonradioactive DIG-High prime system (Roche); hybridized DNA
137
was visualized using the DIG luminescent detection kit (Roche) and the level of DNA-DNA
138
relatedness was quantified by using a densitometer (Bio-Rad). Isolation of DNA (Saito &
139
Miura, 1963) and determination of the DNA G + C content were performed by HPLC (SPD-
140
10AV; Shimadzu), as described by Mesbah et al. (1989).
141 142
The almost-complete 16S rRNA gene sequence (1459 bp) of strain S-II-13T was obtained and
143
used for initial BLAST searches in GenBank and phylogenetic analysis. The 16S rRNA gene
144
sequences of related taxa were obtained from the GenBank and EzTaxon-e servers and the
145
identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene
146
sequence
147
(http://www.ezbiocloud.net/eztaxon; Kim et al., 2012). Strain S-II-13T was shown to belong
148
to the genus Sphingobium or Rhizorhapis showed closest phylogenetic similarity to
149
Rhizorhapis suberifaciens CA1T (97.0 %), Sphingobium sufflavum HL-25T (96.9 %) and
150
Sphingobium vulgare BHU1-GD12T (96.6 %) on the basis of 16S rRNA gene sequence
151
analysis. Phylogentic analysis revealed that the strain S-II-13T was located in the genus
152
Sphingobium
153
(Supplementary Fig. S2) and maximum-parsimony (Supplementary Fig. S3) resulted in
154
highly similar tree topologies forming a distinct phyletic line in the genus Sphingobium with
155
>85% bootstrap value and in maximum-parsimony and neighbor-joining (Fig. 1) also resulted
156
similar tree topology forming a separate phyletic line in the genus Rhizorhapis even though it
157
was separated from the genus Sphingobium. 16S rRNA sequence similarity with members of
158
the other species of the genus Sphingobium species was in the range from 96.9 to 94.8 %.
159
DNA–DNA hybridization values between strain S-II-13T and Rhizorhapis suberifaciens LMG
160
17323T, Sphingobium sufflavum KCTC 23953T and Sphingobium vulgare KCTC 22289T were
161
24, 52 and 55 %, respectively. All values were clearly below the threshold value of 70 %
162
DDH that is recommended for the assignment of the genomic species (Wayne et al., 1987).
163
The G + C content of the DNA of strain S-II-13T was 63.5 mol%, which is in the range (62.0–
similarity
and
were
separated
achieved
from
by
the
genus
5
using
the
Rhizorhapis.
EzTaxon-e
server
Maximum-likelihood
164
67.0 mol%) for members of the genus Sphingobium and different from the genus Rhizorhapis
165
(58–60 mol%) (Francis et al., 2014).
166 167
For analysis of fatty acids, strain S-II-13T was cultured on R2A at 28 °C for 3 days and the
168
closely related type strains were cultured under the same conditions. Cellular fatty acids were
169
extracted and analysed by GC (6890N; Agilent Technologies) according to the standard
170
protocol of the Sherlock Microbial Identification System (version 4.5; MIDI database
171
TSBA40 4.10). For the analysis of quinones and polar lipids, cells were harvested in the late-
172
exponential phase and freeze-dried. Isoprenoid quinones were extracted and analysed by
173
HPLC (SPD-10AV; Shimadzu), as described by Collins & Jones (1981). For polar lipid
174
analysis, the cellular lipids were extracted twice, washed and hydrolysed with 0.5 M NaOH
175
as described by Yabuuchi et al. (1990, 1999). The total lipids were separated by two-
176
dimensional TLC with a solvent system composed of chloroform/methanol/water (65:25:4,
177
by vol.) in the first direction and chloroform/methanol/acetic acid/water (80:15:12:4, by vol.)
178
in the second direction. For polyamine analysis, strain S-II-13T was grown aerobically in
179
R2A broth at 28 °C and harvested in the late-exponential phase. Polyamines were extracted
180
from l00 mg of lyophilized cells according to the methods described by Busse et al. (1997)
181
and sequentially benzoylated by the methods described by Taibi et al. (2000). Quantitative
182
analysis of polyamines was performed by using a reverse-phase HPLC equipped with a
183
photodiode array detector (234 nm) (Series 20 HPLC; Shimadzu) and Watcher 120 ODS-AP
184
column (250 × 4.6 mm internal diameter, 5 μm particle size) (Isu Industry Corp)
185 186
Major fatty acids in strain S-II-13T were C18:1 ω7c (23.1 %), C16:0 (15.5 %) and summed
187
feature 3 (12.4 %; C16:1 ω7c and/or iso-C15:0 2-OH). 2-Hydroxy fatty acids were present as
188
C14:0 2-OH (16.3 %) but 3-hydroxy fatty acid were not present. These are common
189
characteristic features of members of the genus Sphingomonas. The presence of C15:0, C19:0
190
cyclo ω8c and C16:0 2-OH distinguished strain S-II-13T from closely related type strains of the
191
genus Sphingobium and Rhizorhapis (Supplementary Table. S1). The major isoprenoid
192
quinone of isolate S-II-13T was ubiquinone 10 (Q-10), as in all known members of the family
193
Sphingomonadaceae. Polar lipids of S-II-13T comprised phosphatidylethanolamine (PE),
194
phosphatidylmonomethylethanolamine
195
diphosphatidylglycerol (DPG), phosphatidyl-choline (PC), sphingoglycolipid (SGL) and,
196
unknown aminoglycolipid (AGL) (Supplementary Fig. S4). The presence of abundant
(PME),
6
phosphatidylglycerol
(PG),
197
sphingoglycolipid in strain S-II-13T indicates that the isolate is a member of the family
198
Sphingomonadaceae. The polyamine pattern of strain S-II-13T consisted of predominantly
199
spermidine [18.9 µmol (g dry weight)-1] and trace amount of putrescine [0.1 µmol (g dry
200
weight)-1] and cadaverine [0.05 µmol (g dry weight)-1]. This polyamine pattern is in
201
agreement with the description of the genus Sphingobium (Takeuchi et al., 2001).
202 203
Strain S-II-13T shared similar chemotaxonomic characteristics with members of the genus
204
Sphingobium sensu stricto in terms of the DNA G + C content, polar lipids, major polyamine,
205
predominant ubiquinone and major fatty acids. However, strain S-II-13T could be
206
distinguished from other closely related species by some chemotaxonomic and phenotypic
207
features, such as colony color, oxidase negative, presence of C15:0, C19:0 cyclo ω8c and C16:0
208
2-OH.
209 210
Therefore, on the basis of the results of this polyphasic taxonomic study, we propose that the
211
new strain represents a novel species of the genus Sphingobium, for which the name
212
Sphingobium subterraneum sp. nov. is proposed, with the type strain S-II-13T.
213 214
Description of Sphingobium subterraneum sp. nov.
215
Sphingobium subterraneum (sub.ter.ra'ne.um. L. neut. adj. subterraneum, underground).
216 217
Cells are Gram-negative, non-motile, rods, 0.6−0.7×0.8−1.2 µm in size. Colonies are circular,
218
convex, entire, yellow in colour, and 0.5−0.9 mm in diameter on R2A agar after 3 days
219
incubation at 28 ºC. Growth also occurs on NA, LB, TSA and ISP2. Grows at 15−30 °C
220
(optimally at 28 °C) and pH 6.0−9.0 (optimally at pH 7.5) and with 0.3−1.5 % (w/v) NaCl,
221
with optimal growth at 0.5 % (w/v) NaCl. Catalase-positive and oxidase-negative. Esculin
222
and starch are hydrolysed but L-arginine, urea, L-tyrosine, xanthine and gelatin are not.
223
Nitrate is reduced to nitrite. Indole is not produced. Acid is not produced from D-glucose.
224
Utilizes D-ribose, L-xylose, D-adonitol, methyl-β-D-xylopyranoside, D-galactose, D-fructose,
225
D-mannose,
226
mannopyranoside, methyl-α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin,
227
esculin, L-salicin, D-cellobiose, D-lactose, D-melibiose, D-sucrose, inulin, D-melezitose, D-
228
raffinose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose,
L-rhamnose,
dulicitol,
inositol,
7
D-mannitol,
D-sorbitol,
methyl-α-D-
229
L-fucose, D-arabitol, L-arabitol and potassium gluconate. Acid is produced from amygdalin,
230
arbutin,
231
acetylglucosamine, D-ribose, L-salicin, starch, D-sucrose and D-trehalose. Enzyme activity is
232
observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine
233
arylamidase, valine arylamidase and crystine arylamidase, but not for trypsin,
D-cellobiose,
esculin,
D-fructose,
glycerol,
glycogen,
D-maltose,
N-
236
αchymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, βgalactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosamininase, αmannosidase and α-fucosidase (API ZYM). Susceptible to teicoplanin, amikacin, ampicillin,
237
nalidixic acid, kanamycin, gentamicin, neomycin, vancomycin, erythromycin, oleandomycin,
238
amoxicillin, spiramycin, rifampicin, nystatin, tetracycline, roxithromycin, sphingomyelin,
239
apramycin, hygromycin B, capreomycin, sisomycin, gramicidin S, chloramphenicol and
240
chloramphenicol, but resistant to lincomycin, streptomycin, penicillin, polymixin B,
241
bacitracin, cycloheximide, salinomycin, amphotericin, phosphomycin and streptomycin. The
242
major fatty acids are C18:1 ω7c, C14:0 2-OH, C16:0 and C16:1 ω7c and/or C15:0 iso 2-OH. Main
243
hydroxy-fatty
244
phosphatidylmonomethylethanolamine,
245
phosphatidyl-choline, sphingoglycolipid and unknown aminoglycolipid. Major polyamine is
246
spermidine. The predominant isoprenoid quinone is ubiquinone Q-10.
234 235
acid
is
C14:0 2-OH.
Polar
lipids
are
phosphatidylethanolamine,
phosphatidylglycerol,
diphosphatidylglycerol,
247 248
The type strain, S-II-13T (=KACC 17606T=NBRC 109814T), was isolated from ground
249
water at Daejeon in Korea. The DNA G + C content of the type strain is 63.5 mol%.
250 251 252 253
Acknowledgements
254 255
This work was supported by a grant from the Regional SubGenBank Support Program of
256
Rural Development Administration, Republic of Korea
257 258 259 260 261 8
262 263
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340
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342
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343
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347
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348
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349
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350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 12
373
Figure Legend
374 375
Fig. 1. Rooted neighbor-joining tree based on 16S rRNA gene sequences showing the
376
phylogenetic position of strain S-II-13T and related bacteria in the genus Sphingobium and
377
Rhizorhapis. Bootstrap values, expressed as a percentage of 1000 replications, are given at
378
branching points when >50%. Sphingomonas paucimobilis ATCC 2983T was used as an
379
outgroup. Bar, 0.01 subtitutions per nucleotide position.
380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 13
406
Table 1. Phenotypic characteristics for differentiating strain S-II-13T from the related type
407
strains of species the genus Sphingobium and Rhizorhapis
408 409 410 411 412 413 414 415 416 417
Strains: 1, S-II-13T; 2, Rhizorhapis suberifaciens LMG 17323T; 3, Sphingobium boeckii DSM 25079T; 4, Sphingobium sufflavum KCTC 23953T; 5, Sphingobium vulgare KCTC 22289T, 6, Sphingobium xanthum LMG 12560T. All data were obtained from this study, except where indicated otherwise. All strains are Gram-stainingnegative, aerobic rods and positive for catalase. All strains share the following characteristics: positive for alkaline phosphate and negative for indole production, arginine dihydrolase, α-galactosidase and N-acetyl-βglucosamininase activities hydrolysis esculin but not gelatin, L-arginine and urea; no assimilation of D,Larabinose and caprate; no acid production from D-adonitol, L-rhamnose, D-mannitol and D-raffinose. All strains are susceptible to kanamycin (30 ㎍), gentamicin (10 ㎍), vancomycin (30 ㎍) and erythromycin (15 ㎍). +, Positive; −, negative; w, weakly positive; S, sensitive; R, resistant. Characteristic
1
2
3
4
5
6
Yellow
White
Creamy yellow
Light yellow
Yellow
Bright yellow
−
+
+
+
+
+
−
−
+
−
+
+
+
−
−
−
−
+
D-Glucose
−
+
−
+
+
+
N-Acetyl- D-glucosamine
+
−
+
+
−
−
D-Galactose
+
−
+
+
−
+
D-Mannose
+
−
+
+
−
+
Rhamnose
+
−
−*
+
−
+
D-Melibiose
+
−
+
+
−
+*
Colony color Oxidase Growth at 37
℃
Hydrolysis of: Starch Assimilation of:
Glycogen
+
−
+
+
−
+
Adipate
−
−
−
+
+
+
Citrate
−
−
+
+
−
+
Malate
−
−
−
+
+*
+
D-Fructose
+
−
−
−
+
+
D-Maltose
+
+
−
−
+
+
D-Sucrose
+
−
−
−
−
+
D-Trehalose
+
−
+
−
+
+
Glycerol
+
−
−
+
+
−
D-Ribose
+
−
−
+*
+
−
D-Cellobiose
+
−
+
−
−
+
Esterase (C4)
+
−
w
+
+
−
Valine arylamidase
+
−
−
+
−
+
α-glucosidase
−
+
−
−
−
−
β-galactosidase
−
+
+
+
−
−
R
Acid production from:
Enzyme activities:
Susceptibility to: Penicillin (12 U)
R
S
S
S
R
Nystatin (50
S
R
R
S
S
S
R
S
S
R
R
R
S
R
R
S
S
㎍)
Polymixin B (50 Tetracycline (30
418 419
㎍) ㎍)
DNA G+C contents (mol%) 63.5 58.2-59.5a† 64.6 b 63.8c 66.8d * Data that were different from the results obtained in previous studies † Data obtained from: a, Francis et al. (2014); b, Chen et al. (2013); c, Sheu et al. (2013); d, Baek et al. (2010)
14
S 64.7a
