IJSEM Papers in Press. Published April 15, 2015 as doi:10.1099/ijs.0.000266
International Journal of Systematic and Evolutionary Microbiology Chryseobacterium solani sp. nov. isolated from field-grown eggplant rhizosphere soil --Manuscript Draft-Manuscript Number:
IJS-D-14-00481R2
Full Title:
Chryseobacterium solani sp. nov. isolated from field-grown eggplant rhizosphere soil
Short Title:
Chryseobacterium solani sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Tae-Hoo Yi Kyung-Hee University Yongin, KOREA, REPUBLIC OF
First Author:
Juan Du
Order of Authors:
Juan Du hien T.T. Ngo KyungHwa Won Ki–Young Kim Feng–Xie Jin Tae-Hoo Yi
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
Strain THG-EP9T, a Gram-negative, aerobic, motile, rod-shaped bacterium was isolated from field-grown eggplant (Solanum melongena) rhizosphere soil in Pyeongtaek, Gyeonggi-do, Republic of Korea. Based on 16S rRNA gene sequence comparisons, strain THG-EP9T had closely similarity with Chryseobacterium ginsenosidimutans THG 15T (97.3 %), Chryseobacterium soldanellicola PSD1-4T (97.2 %), Chryseobacterium zeae JM-1085T (97.2 %) and Chryseobacterium indoltheticum LMG 4025T (96.8 %). DNA-DNA hybridization showed 5.7 % and 9.1 % DNA re-association with Chryseobacterium ginsenosidimutans KACC 14527T and Chryseobacterium soldanellicola KCTC 12382T, respectively. Chemotaxonomic data revealed that strain THG-EP9T possesses menaquinone-6 as the only respiratory quinone and iso-C15:0 (29.0 %), C16:0 (12.5 %) and iso-C17:0 3OH (11.9 %) as the major fatty acids. The polar lipid profiles consisted of phosphatidylethanolamine, unidentified aminophospholipid, two unidentified glycolipids, six unidentified aminolipids and two unidentified polar lipids. The G+C content was 35.3 mol%. These data corroborated the affiliation of strain THG-EP9T to the genus Chryseobacterium. Thus, the isolate represents a novel species, for which the name Chryseobacterium solani sp. nov. is proposed, with THG-EP9T as the type strain (= KACC 17652T = JCM 19456T).
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1
Chryseobacterium solani sp. nov. isolated from field–grown
2
eggplant rhizosphere soil
3
Juan Du 1 † , Hien T.T. NGO 1 † , KyungHwa Won1 , Ki–Young Kim1 , Feng–Xie Jin2 and Tae–
4
Hoo Yi1*
5 6
1 College
7
446–701, Republic of Korea
8
2 College
9
Ganjingzi–qu, Dalia 116034, PR China
of Life science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Gyeonggi
of Bio and Food Technology, Dalian Polytechnic University, Qinggong–yuan No. 1,
10 11
†These
authors equally contributed to this work.
12 13
* Corresponding
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Tel: +82 31 201 2609, Fax: +82 31 206 2537
15
E–mail: [email protected]
author. Tae–Hoo Yi
16 17
Subject category: New taxa in Bacteroidetes
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Running title: Chryseobacterium solani sp. nov.
19 20
The NCBI GenBank accession number for the 16S rRNA gene sequence of strain THG–EP9T
21
is KF532126.
1
22
A phylogenetic tree, cell morphology and TLC of the polar lipids are available as
23
supplementary data in the Online version of this paper.
24
2
25
ABSTRACT
26
Strain THG–EP9T , a Gram–negative, aerobic, motile, rod–shaped bacterium was isolated
27
from field–grown eggplant (Solanum melongena) rhizosphere soil in Pyeongtaek, Gyeonggi–
28
do, Republic of Korea. Based on 16S rRNA gene sequence comparisons, strain THG–EP9T
29
had closely similarity with Chryseobacterium ginsenosidimutans THG 15T (97.3 %),
30
Chryseobacterium soldanellicola PSD1–4T (97.2 %), Chryseobacterium zeae JM–1085T
31
(97.2 %) and Chryseobacterium indoltheticum LMG 4025T (96.8 %). DNA–DNA
32
hybridization showed 5.7 % and 9.1 % DNA re-association with Chryseobacterium
33
ginsenosidimutans KACC 14527T and Chryseobacterium soldanellicola KCTC 12382T ,
34
respectively. Chemotaxonomic data revealed that strain THG–EP9T possesses menaquinone–
35
6 as the only respiratory quinone and iso–C15:0 (29.0 %), C16:0 (12.5 %) and iso–C17:0 3OH
36
(11.9
37
phosphatidylethanolamine, unidentified aminophospholipid, two unidentified glycolipids, six
38
unidentified aminolipids and two unidentified polar lipids. The G+C content was 35.3 mol%.
39
These data corroborated the affiliation of strain THG–EP9T to the genus Chryseobacterium.
40
Thus, the isolate represents a novel species, for which the name Chryseobacterium solani sp.
41
nov. is proposed, with THG–EP9T as the type strain (= KACC 17652T = JCM 19456T ).
%)
as
the
major
fatty
acids.
The polar
lipid
profiles consisted
of
42 43
The genus Chryseobacterium, which belongs to the family Flavobacteriaceae, was first
44
established by Vandamme et al. (1994). Subsequently, the genus expanded rapidly to
45
encompass more than 85 species, they were isolated from various environments such as soils
46
(Venil et al., 2014), clinical (Holmes et al., 2013), fresh water (Kirk et al., 2013), sewage and
47
wastewater (Kämpfer et al., 2003), plant (Sang et al., 2013), fish (Loch et al., 2014) and meat
48
(De Beer et al., 2005). The typical characteristics of the genus Chryseobacterium include
3
49
aerobic type of metabolism, production of flexirubin–type pigments, the presence of
50
branched–chain fatty acids (iso–C15
51
phosphatidylethanolamine (PE) as a major polar lipid and menaquinone–6 (MK–6) as the
52
characteristic respiratory quinone (Bernardet et al., 2006, 2011; Im et al 2011; Kämpfer et al.,
53
2009; Park et al., 2006; Vandamme et al., 1994; Wu et al., 2013). In this study, a
54
Chryseobacterium–like bacterial strain, THG–EP9T , was characterized by a polyphasic
55
taxonomic approach.
: 0
and iso–C17
: 0
3–OH) as the major fatty acids,
56 57
Strain THG–EP9T was isolated from field–grown eggplant rhizosphere soil in Pyeongtaek,
58
Gyeonggi–do, South Korea (36° 99′ N 127° 11′ E). About one gram soil was thoroughly
59
suspended in 10 ml sterile 0.85 % NaCl (w/v; saline solution), serially diluted samples were
60
spread on nutrient agar (NA, Oxoid) and incubated at 28 °C for 3 days. Single colonies were
61
purified by transferring to new NA plates. One isolate, THG–EP9T was cultured routinely on
62
NA agar at 28°C and stored in NA broth containing glycerol suspension (25 %, w/v) at –
63
70 °C.
64 65
For the sequencing of the 16S rRNA gene, genomic DNA was achieved using a Solgent
66
genomic DNA extraction kit (Korea) and the 16S rRNA gene was amplified according to the
67
methods of Weisburg et al. (1991). The 16S rRNA gene sequencing was performed by
68
Solgent Co. Ltd (Korea), sequences of related taxa were obtained from the EzTaxon–e server
69
(http://www.ezbiocloud.net/eztaxon; Kim et al., 2012) and GenBank database. Multiple
70
alignments were performed via program CLUSTAL_X (Thompson et al., 1997), followed
71
with gap editions in the BioEdit program (Hall, 1999). The Kimura two–parameter model
72
(Kimura, 1983) was used to calculate the evolutionary distances. The neighbor–joining
73
method (Saitou & Nei, 1987) and the maximum–likelihood method were used to construct
4
74
phylogenetic trees as implemented in MEGA 4 and MEGA 5.2 respectively (Kumar et al.,
75
2008). The bootstrap values were calculated based on 1,000 replications (Felsenstein, 1985).
76 77
The 16S rRNA gene sequence of strain THG–EP9T (1,424 bp) was analyzed. Sequence
78
similarity indicated that the close phylogenetic neighbors of strain THG–EP9T were
79
Chryseobacterium ginsenosidimutans THG 15T (97.3 %), Chryseobacterium soldanellicola
80
PSD1–4T (97.2 %), Chryseobacterium zeae JM–1085T (97.2 %) and Chryseobacterium
81
indoltheticum LMG 4025T (96.8 %). This relationship between strain THG–EP9T and other
82
recognized species of the genus Chryseobacterium was evidenced in the phylogenetic tree
83
(Figure 1 and Supplementary Fig. S1).
84 85
Gram reaction was determined by using bioMérieux Gram stain kit according to the
86
manufacturer’s instructions. Cell morphology was observed by transmission electron
87
microscope (Model JEM1010; JEOL) at × 11,000 magnification, using cells grown for 24h at
88
28 °C on NA. Motility was tested in sulfide–indole–motility medium (SIM; Difco).
89
Anaerobic growth was performed in nutrient broth (NB) supplemented with thioglycollate
90
[0.1 % (w/v), Sigma] with 5 days incubation, for the air was substituted with nitrogen gas.
91
Growth at different temperatures (4, 10, 18, 25, 28, 30, 35, 37 and 42 °C) was assessed after 7
92
days of incubation on NA. Salt tolerance was tested in NB containing 0–5 % (w/v) NaCl (at
93
intervals of 0.5 %), growth at various pH conditions (pH 4–10, at intervals of 0.5 pH units)
94
was performed in NB. For the pH experiments, two different buffers were used (final
95
concentration, 100 mM): acetate buffer was used for pH 4.0–6.5 and phosphate buffer was
96
used for pH 7.0–10.0. Growth was evaluated by monitoring the OD600 after 5 days of
97
incubation. Production of flexirubin–type pigments was determined by the color shift to red,
98
purple or brown when colonies were flooding with aqueous 20 % KOH solution (Fautz &
5
99
Reichenbach, 1980). Methyl red and Voges–Proskauer reactions were tested in Clark–Lubs’
100
medium. Oxidase and catalase activity were tested with 1 % (w/v) N, N, N′, N′–tetramethyl–
101
1,4–phenylenediamine reagent and 3 % (v/v) H2 O2 , respectively. Tests for degradation of
102
esculin [0.1 % (w/v) esculin and 0.05% (w/v) ferric citric acid, (Sigma)], casein [2.0 % (w/v)
103
skim milk, Oxoid], starch [1.0 % (w/v), Difco], DNA (DNase agar, Oxoid), Tween 20 [1.0 %
104
(w/v), Sigma], Tween 80 [1.0 % (w/v), Sigma], L–tyrosine [0.5 % (w/v), Sigma], chitin [1.0 %
105
(w/v), Sigma], and CM–cellulose [1.0 % (w/v), Sigma] were determined after 5 days of
106
incubation at 28 °C. Tests were also made for growth on R2A agar (Difco), marine agar (MA,
107
Oxoid), tryptone soya agar (TSA, Oxoid) and MacConkey agar (Oxoid). In addition, API
108
20NE, ID 32 GN and API ZYM tests (bioMérieux, France) were carried out to evaluate
109
carbon–source utilization and enzyme activities on the bases of the instructions of the
110
manufacturer. The strains of species Chryseobacterium ginsenosidimutans KACC 14527T , C.
111
soldanellicola KCTC 12382T , C. indoltheticum KCTC 2920T were included as references for
112
the investigation of the biochemical tests using the same laboratory conditions.
113 114
Strain THG–EP9T was Gram–negative, motile by gliding, aerobic, rod–shaped (1.50–1.90 ×
115
0.55–0.60 μm) and no flagella (Supplementary Fig. S2, available in IJSEM Online). The
116
isolate grew well on NA, TSA, MA and R2A agar, but not on MacConkey agar. Methyl red
117
test was weakly positive; Voges–Proskauer test was negative. Strain THG–EP9T produced
118
flexirubin–type pigments (the reversible color shifted from yellow to red). Growth occurred
119
at 4–35 °C, pH 6.0–10.0 and 0–1.5 % (w/v) NaCl. Physiological and biochemical
120
characteristics of strain THG–EP9T are summarized in the species description and a
121
comparison of strain THG–EP9T and related type strains is given in Table 1.
122
6
123
In order to determine G+C content and to perform DNA–DNA hybridization, genomic DNA
124
of strain THG–EP9T was prepared as described previously (Moore et al., 1995). The G+C
125
content was analyzed as described by Mesbah et al. (1989) using reverse–phase HPLC
126
system. DNA–DNA hybridization was performed fluorometrically, according to the method
127
developed by Ezaki et al. (1989), with photobiotin–labelled probes in microplate wells.
128
DNA–DNA hybridization experiments were performed between strain THG–EP9T and
129
closely related type strains of genus Chryseobacterium at 29.0 °C.
130 131 132
The DNA G+C content of strain THG–EP9T was 35.3 mol%, which conform to the expected
133
range of G+C contents for the genus Chryseobacterium (Kook et al., 2014). The DNA–DNA
134
relatedness values between strain THG–EP9T and related strains were below 10 %
135
(Chryseobacterium ginsenosidimutans KACC 14527T , 5.7 % and Chryseobacterium
136
soldanellicola KCTC 12382T , 9.1 %). These very low DNA relatedness values suggested that
137
THG–EP9T as a novel species of genus Chryseobacterium (Tindall et al., 2010).
138 139
For the cellular fatty acid analysis, cells of strain THG–EP9T and reference strains were
140
harvested from NA plates after incubation for 2 days at 28 ºC. The cellular fatty acid profiles
141
were prepared according to the protocol of Sherlock Microbial Identification System (MIDI)
142
and identified with GC (Hewlett Packard 6890) using Sherlock Aerobic Bacterial Database
143
(TSBA60) (Sasser, 1990).For the extraction of polar lipids and isoprenoid quinones of strain
144
THG–EP9T and polar lipids for type strain of the closest related species, C.
145
ginsenosidimutans KACC 14527T , cells were cultured in NB for 3 days and freeze-dried after
146
harvesting. The polar lipids of strain THG–EP9T and C. ginsenosidimutans KACC 14527T
147
were extracted (Minnikin et al., 1977; 1984) and detected using 2–dimensional thin–layer
7
148
chromatography (Tindall, 1990). TLC plates were sprayed with reagents: 5 %
149
molybdophosphoric acid was used for detecting total polar lipids, 0.2 % ninhydrin for amino
150
lipids, molybdenum blue reagent for phospholipids and α–naphthol sulphuric acid reagent for
151
glycolipids. Isoprenoid quinones analysis was performed by RP–HPLC Waters 2690 Alliance
152
system [solvent; methanol/2–propanol (7:5, v/v), flow rate; 1.0 ml/min] as previously
153
described (Collins & Jones, 1981; Hiraishi et al., 1996; Tamaoka et al., 1983).
154 155
The major cellular fatty acids (> 10.0 %) of strain THG–EP9T were iso–C15:0 (29.0 %), C16:0
156
(12.5 %) and iso–C17:0 3OH (11.9 %), which are consistent with the genus Chryseobacterium.
157
A comparison of fatty acid profiles between strain THG–EP9T and selected closely strains is
158
shown in Table 2. The polar lipid profiles of strain THG–EP9T and C. ginsenosidimutans
159
KACC 14527T are shown in Supplementary Fig. S3. A spot was detected with
160
molybdophosphoric acid, ninhydrin and
161
phosphatidylethanolamine (PE), which had shown similar retention on strain C.
162
ginsenosidimutans KACC 14527T
163
Chryseobacterium; eight lipids were observed to be ninhydrin positive, one of them were
164
molybdenum blue positive which were believed to be aminophospholipid (APL), the rest six
165
molybdenum blue negative spots were aminolipids (AL1–6); two spots, appeared as α–
166
naphthol sulphuric acid positive, were glycolipids (GL1–2). The polar lipid profiles of strain
167
THG–EP9T was clearly distinguishable from that of C. ginsenosidimutans KACC 14527T by
168
presence of two unidentified aminolipids (AL4–6), and absence of unidentified lipid (L3).
169
Strain THG–EP9T contained MK–6 as the single respiratory quinone, which is typical for the
170
genus Chryseobacterium (Wu et al., 2013).
molybdenum blue,
is in
was
believed to be
line with all other members of genus
171
8
172
On the basis of 16S rRNA gene sequence, fatty acid and polar lipid profiles, G+C content and
173
quinone system, strain THG–EP9T is suggested to belong to the genus Chryseobacterium as a
174
novel species, for which the name Chryseobacterium solani sp. nov. is proposed.
175 176
Description of Chryseobacterium solani sp. nov.
177
Chryseobacterium solani (so.la’ni. N.L. gen. neut. n. solani of the eggplant).
178
Cells are aerobic, Gram–negative, motile and rod–shaped (1.50–1.90 × 0.55–0.60 μm).
179
Colonies are yellowish, translucent and shiny on NA. Growth occurs at 4–35 °C (optimum
180
25–30 °C), at pH 6.0–10.0 (optimum 6.0–8.5) and at 0–1.5 % (w/v) NaCl. Grows well on NA,
181
TSA MA and R2A agar, but do not on MacConkey agar. Strain THG–EP9T produces
182
flexirubin–type pigments. Catalase and oxidase activity are positive. Weakly positive for
183
Methyl red test; Negative for Voges–Proskauer test. Esculin, casein, starch, DNA, Tween 20,
184
Tween 80, CM–cellulose CM cellulose and L–tyrosine are hydrolysed but chitin is not.
185
According to the API ZYM tests, positive results are obtained from alkaline phosphatase,
186
esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase,
187
cystine arylamidase, trypsin, α–chymotrypsin, acid phosphatase, α–glucosidase, β–
188
glucosidase, α–fucosidase, α–mannosidase and naphtol–AS–BI–phosphohydrolase; weakly
189
positive result is obtained from β–galactosidase; negative results are obtained from α–
190
galactosidase, β–glucuronidase and N–acetyl–β–glucosaminidase. In API 20NE tests, the
191
result of D–maltose, adipic acid, D–mannitol and gelatin hydrolysis, β–glucosidase, arginine
192
dihydrolase, and urease activity and indole production are positive; the assimilation for malic
193
acid and citric acid are weakly positive. In ID32 GN tests, glycogen, salicin, L–proline, D–
194
mannitol, D–glucose, L–rhamnose and D–maltose are assimilated; L–histidine, D–sucrose, D–
195
melibiose,
L–arabinose,
L–fucose,
D–sorbitol,
9
capric acid, valerate, citric acid, 2–
196
ketogluconate, 3–hydroxy–butyrate, 4–hydroxy–benzoate, N–acetyl–glucosamine, D–ribose,
197
itaconate, suberate, malonate, acetate, lactate,
198
benzoate and 5–ketogluconate are not assimilated; propionate is weakly assimilated. The
199
major fatty acids (> 10 %) are iso–C15:0 , C16:0 and iso–C17:0 3OH. The only isoprenoid
200
quinone is MK–6. The G+C content of genomic DNA is 35.3 mol%. The composition of
201
polar lipids is phosphatidylethanolamine, unidentified aminophospholipid, six unidentified
202
aminolipids, two unidentified glycolipids and two unidentified lipids.
L–alanin, L–serine,
inositol, 3–hydroxyl–
203 204
The type strain, THG–EP9T (= KACC 17652T = JCM 19456T ) was isolated from field–grown
205
eggplant rhizosphere soil in Gyeonggi–do, Republic of Korea.
206 207
10
208
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RE, Moore DD, Seidman JG, Smith JA & Struhl K. New York: Wiley.
282
Park, M. S., Jung, S. R., Lee, K. H., Lee, M. S., Do, J. O., Kim, S. B. & Bae, K. S. (2006).
283
Chryseobacterium soldanellicola sp. nov. and Chryseobacterium taeanense sp. nov.,
284
isolated from roots of sand–dune plants.Int J Syst Evol Microbiol 56, 433–438
285
Saitou, N. & Nei, M. (1987). The neighbor–joining method: a new method for reconstructing
286
phylogenetic trees. Mol Bio Evol 4, 406–425.
287
Sang, M. K., Kim, H. S., Myung, I. S., Ryu, C. M., Kim, B. S. and Kim, K. D. (2013).
288
Chryseobacterium kwangjuense sp. nov., isolated from pepper (Capsicum annuum L.)
289
root. Int. J. Syst. Evol. Microbiol., 63, 2835-2840.
290 291
Sasser, M. (1990). Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. MIDI Technical Note 101. Newark, DE: MIDI Inc.
292
Tamaoka, J., Katayama–Fujiruma, A. & Kuraishi, H. (1983). Analysis of bacterial
293
menaquinone mixtures by high performance liquid chromatography. J Appl Bacieriol
294
54, 31–36.
295
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997).
296
The CLUSTAL_X windows interface: flexible strategies for multiple sequence
297
alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
298 299
Tindall, B. J. (1990). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66, 199–202.
300
Tindall, B. J., Rosselló-Mora, R., Busse, H. J., Ludwig, W., & Kämpfer, P. (2010). Notes
301
on the characterization of prokaryote strains for taxonomic purposes.International .
302
Int J Syst Bacteriol 60, 249-266.
14
303
Vandamme, P., Bernardet, J. F., Segers, P., Kersters, K. & Holmes, B. (1994). New
304
perspectives
in
the
classification
of
the
Flavobacteria–description
of
305
Chryseobacterium gen. nov., Bergeyella gen. nov., and Empedobacter nom rev. Int J
306
Syst Bacteriol 44, 827–831.
307
Venil, C. K., Nordin, N., Zakaria, Z. A. & Ahmad, W. A. (2014). Chryseobacterium
308
artocarpi sp. nov., isolated from the rhizosphere soil of Artocarpus integer. Int. J.
309
Syst. Evol. Microbiol., 64, 3153-3159.
310 311
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.
312
Wu, Y. F., Wu, Q. L. & Liu Sh. J. (2013). Chryseobacterium taihuense sp. nov., isolated
313
from a eutrophic lake, and emended descriptions of the genus Chryseobacterium,
314
Chryseobacterium taiwanense, Chryseobacterium jejuense and Chryseobacterium
315
indoltheticum. Int J Syst Evol Microbiol 63, 913–919.
316
15
317
Table 1. Phenotypic characte ristics of Chryseobacterium solani THG–EP9 T and related
318
type strains of species of genus Chryseobacterium.
319
Strains: 1. Chryseobacterium solani THG–EP9T ; 2. Chryseobacterium ginsenosidimutans
320
KACC 14527T ; 3. Chryseobacterium soldanellicola KCTC 12382T ; 4. Chryseobacterium
321
zeae JM–1085T and 5. Chryseobacterium indoltheticum KCTC 2920T . For strains 1–3 and 5,
322
all data (except for DNA G+C content; taken from Im et al., 2011; Park et al., 2006 and
323
Vandamme et al., 1994) was carried out in this study; for stain 4, data were taken from
324
Kämpfer et al. (2013). All strains are oxidase and catalase positive, aerobic, growth on NA,
325
R2A and TSA and non–growth on MacConkey agar, positive for hydrolysis of esculin;
326
negative for nitrate reduction, acidification of glucose, capric acid, gluconate and
327
phenylacetic acid. The following compounds are not utilised as a sole source of carbon for all
328
species: D–melibiose, D–sorbitol, capric acid, citric acid, L–histidine, 4–hydroxy–benzoate, N–
329
acetyl–glucosamine,
330
hydroxyl–benzoate. In API ZYM tests, strains 1–3 and 5 are all positive for alkaline
331
phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine
332
arylamidase, acid phosphatase, naphtol–AS–BI–phosphohydrolase and α–glucosidase;
333
negative for α–Galactosidase, β–Glucuronidase. (NA): not available; (+): positive; (w):
334
weakly positive; (–): negative.
D–ribose,
itaconate, suberate, lactate, L–alanin, L–serine, inositol, 3–
Characteristic Temperature range for growth (°C) pH range for growth Growth with 2% NaCl (w/v) Gliding motility Growth on MA Indole production Assimilation of Adipic acid Salicin Acetate
1 4–35 6.0–10.0 – + + +
2 10–35 5.5–10.0 – – – +
3 4–37 5.0–7.0 + + + +
4 8–36 NA + – NA –
5 4–35 5.0–8.0 + – – +
+ + –
– – +
W – –
– + –
W – +
16
Propionate L–Arabinose D–Mannose D–Maltose D–Mannitol D–Sucrose D–Glucose Hydrolysis of Starch DNA Casein CMC L–Tyrosine Tween 80 Enzyme activities Urease Arginine dihydrolase a–Fucosidase N–Acetyl–β–glucosaminidase α–Mannosidase Trypsin Cystine arylamidase DNA G+C content (mol%)
W – – + + – –
W + + + – + +
– + + + + + +
– W – + – + W
– – + – – – +
+ + + + + +
+ + + + – +
+ + + + + –
– – – – – NA
+ + + + + +
+ + + – + + + 35.3
– – – + – – – 35.7
– – – – – + + 28.8
– – NA NA NA NA NA NA
– – – + – + + 33.8
335
17
336
Table 2. Cellular fatty acid profiles of strain THG–EP9 T and phylogenetically related
337
species of the genus Chryseobacterium.
338
1. Chryseobacterium solani THG–EP9T ; 2. Chryseobacterium ginsenosidimutans KACC
339
14527T ; 3. Chryseobacterium soldanellicola
340
indoltheticum KCTC 2920T . All data were carried out in this study. Cells were cultured under
341
same condition. Fatty acids of less than 0.5 % in all strains were not listed; (ND): not
342
detected; Tr: traces (< 1.0 %). Fatty acid
KCTC 12382T ; 4. Chryseobacterium
1
2
3
4
12.5 6.2
5.5 3.3
7.7 4.4
5.8 2.1
2.0
Tr
1.9
Tr
Tr 29.0 2.3 4.3 2.7 1.7 1.2 11.9 7.4 8.4
Tr 34.1 3.8 2.3 1.3 Tr 1.4 16.5 13.2 11.7
1.6 28.4 3.5 4.7 Tr 1.3 Tr 14.4 7.5 16.6
ND 25.0 1.9 6.5 1.9 2.3 Tr 9.5 22.7 12.9
Saturated C16:0 C18:0
Unsaturated C16:0 3OH
Branched–chain iso–C13:0 iso–C15:0 iso–C15:0 3OH anteiso–C15:0 iso–C16:0 iso–C16:0 3OH iso–C17:0 iso–C17:0 3OH iso–C17:1 ω9c * Summed Feature 3 343 344
*Summed features represent groups of two or three fatty acids that could not be separated by
345
GLC with the MIDI system. Summed feature 3 contained C16:1 ω7c and/or C16:1 ω6c.
346 347
18
348
Figure legends
349
Fig. 1. Neighbour–joining phylogenetic tree based on 16S rRNA gene sequences showing
350
the relationships of Chryseobacterium solani THG–EP9 T with related Chryseobacterium
351
species.
352
Bootstrap values (expressed as percentage of 1,000 replications) over 70% are shown at the branching
353
points. Bergeyella zoohelcum ATCC 43767 T (AGYA01000006) was used as outgroup. Bar, 0.005
354
substitutions per nucleotide position.
355 356
Suppleme ntary Fig. S1. Maximum–likelihood tree based on 16S rRNA gene sequences
357
showing the relationships between strain THG–EP9T and other related type species.
358
Numbers at nodes represent percentages of bootstrap support based on a maximum–likelihood
359
analysis of 1,000 resampled datasets.
360 361
Suppleme ntary Fig. S2. Trans mission electron micrograph of cells of Chryseobacterium
362
solani THG–EP9 T.
363
The detection was performed after negative staining with uranyl acetate. Bar, 0.5µm.
364 365
Suppleme ntary Fig. S3. Two–dimensional thin–layer chromatography of polar lipids of
366
strain THG–EP9T.
367
a and b: Total lipids were detected by spraying with 5% molybdatophosphoric acid for strain THG–
368
EP9T and C. ginsenosidimutans KACC 14527T , respectively; c: Aminolipids detected by spraying
369
with 0.2% ninhydrin; d: Phospholipids detected by spraying with molybdenum blue; e: Glycolipids
370
revealed by α–naphthol–sulphuric acid. Abbreviations: phosphatidylethanolamine (PE), unidentified
371
glycolipid (GL), unidentified aminophospholipids (APL), unidentified phospholipid (PL), unidentified
372
aminolipid (AL) and unidentified lipid (L).
19
Figure Click here to download Figure: figure 1 EP9.pdf
84 94 86
0.005
Chryseobacterium indoltheticum LMG 4025T (AY468448) Chryseobacterium scophthalmum LMG 13028T (AJ271009) Chryseobacterium greenlandense UMB 34T (932652) 100 Chryseobacterium aquaticum 10-46T (AM748690) Chryseobacterium piscicola VQ-6316sT (EU869190) Chryseobacterium soli JS6-6T (EF591302) Chryseobacterium ginsenosidimutans THG 15T (GU138380) Chryseobacterium solani THG-EP9T (KF532126) Chryseobacterium soldanellicola PSD1-4T (AY883415) 73 Chryseobacterium geocarposphaerae 91A-561T (HG738132) Chryseobacterium gwangjuense THG-A18T (JN196134) Chryseobacterium gregarium DSM 19109T (AM773820) 92 Chryseobacterium camelliae THG C4-1T (JX843771) Chryseobacterium daeguense K105T (EF076759) Chryseobacterium aahli T68T (AM773820) 100 Chryseobacterium yeoncheonense DCY67T (JX141782) Chryseobacterium massiliae 90BT (AF531766) Chryseobacterium elymi RHA3-1T (DQ673671) Chryseobacterium taihuense THMBM1T (IQ283114) Chryseobacterium zeae JM-1085T (HG738135) Chryseobacterium hungaricum CHB-20pT (EF685359) Chryseobacterium hominis NF802T (AM261868) Chryseobacterium viscerum 687B-08T (FR871426) Chryseobacterium oncorhynchi 701B-08 (FN674441) Chryseobacterium jejuense JS17-8T (EF591303) Chryseobacterium vietnamense GIMN1.005T (HM212415) Bergeyella zoohelcum ATCC 43767 (AGYA01000006) Figure 1, Du et al.
Click here to download Supplementary Material Files: Supplementary file-EP9.pdf
International Journal of Systematic and Evolutionary Microbiology Chryseobacterium solani sp. nov. isolated from field-grown eggplant rhizosphere soil --Manuscript Draft-Manuscript Number:
IJS-D-14-00481R2
Full Title:
Chryseobacterium solani sp. nov. isolated from field-grown eggplant rhizosphere soil
Short Title:
Chryseobacterium solani sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Tae-Hoo Yi Kyung-Hee University Yongin, KOREA, REPUBLIC OF
First Author:
Juan Du
Order of Authors:
Juan Du hien T.T. Ngo KyungHwa Won Ki–Young Kim Feng–Xie Jin Tae-Hoo Yi
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
Strain THG-EP9T, a Gram-negative, aerobic, motile, rod-shaped bacterium was isolated from field-grown eggplant (Solanum melongena) rhizosphere soil in Pyeongtaek, Gyeonggi-do, Republic of Korea. Based on 16S rRNA gene sequence comparisons, strain THG-EP9T had closely similarity with Chryseobacterium ginsenosidimutans THG 15T (97.3 %), Chryseobacterium soldanellicola PSD1-4T (97.2 %), Chryseobacterium zeae JM-1085T (97.2 %) and Chryseobacterium indoltheticum LMG 4025T (96.8 %). DNA-DNA hybridization showed 5.7 % and 9.1 % DNA re-association with Chryseobacterium ginsenosidimutans KACC 14527T and Chryseobacterium soldanellicola KCTC 12382T, respectively. Chemotaxonomic data revealed that strain THG-EP9T possesses menaquinone-6 as the only respiratory quinone and iso-C15:0 (29.0 %), C16:0 (12.5 %) and iso-C17:0 3OH (11.9 %) as the major fatty acids. The polar lipid profiles consisted of phosphatidylethanolamine, unidentified aminophospholipid, two unidentified glycolipids, six unidentified aminolipids and two unidentified polar lipids. The G+C content was 35.3 mol%. These data corroborated the affiliation of strain THG-EP9T to the genus Chryseobacterium. Thus, the isolate represents a novel species, for which the name Chryseobacterium solani sp. nov. is proposed, with THG-EP9T as the type strain (= KACC 17652T = JCM 19456T).
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): MR THG-EP9-11.04.2015-Submission.docx
1
Chryseobacterium solani sp. nov. isolated from field–grown
2
eggplant rhizosphere soil
3
Juan Du 1 † , Hien T.T. NGO 1 † , KyungHwa Won1 , Ki–Young Kim1 , Feng–Xie Jin2 and Tae–
4
Hoo Yi1*
5 6
1 College
7
446–701, Republic of Korea
8
2 College
9
Ganjingzi–qu, Dalia 116034, PR China
of Life science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Gyeonggi
of Bio and Food Technology, Dalian Polytechnic University, Qinggong–yuan No. 1,
10 11
†These
authors equally contributed to this work.
12 13
* Corresponding
14
Tel: +82 31 201 2609, Fax: +82 31 206 2537
15
E–mail: [email protected]
author. Tae–Hoo Yi
16 17
Subject category: New taxa in Bacteroidetes
18
Running title: Chryseobacterium solani sp. nov.
19 20
The NCBI GenBank accession number for the 16S rRNA gene sequence of strain THG–EP9T
21
is KF532126.
1
22
A phylogenetic tree, cell morphology and TLC of the polar lipids are available as
23
supplementary data in the Online version of this paper.
24
2
25
ABSTRACT
26
Strain THG–EP9T , a Gram–negative, aerobic, motile, rod–shaped bacterium was isolated
27
from field–grown eggplant (Solanum melongena) rhizosphere soil in Pyeongtaek, Gyeonggi–
28
do, Republic of Korea. Based on 16S rRNA gene sequence comparisons, strain THG–EP9T
29
had closely similarity with Chryseobacterium ginsenosidimutans THG 15T (97.3 %),
30
Chryseobacterium soldanellicola PSD1–4T (97.2 %), Chryseobacterium zeae JM–1085T
31
(97.2 %) and Chryseobacterium indoltheticum LMG 4025T (96.8 %). DNA–DNA
32
hybridization showed 5.7 % and 9.1 % DNA re-association with Chryseobacterium
33
ginsenosidimutans KACC 14527T and Chryseobacterium soldanellicola KCTC 12382T ,
34
respectively. Chemotaxonomic data revealed that strain THG–EP9T possesses menaquinone–
35
6 as the only respiratory quinone and iso–C15:0 (29.0 %), C16:0 (12.5 %) and iso–C17:0 3OH
36
(11.9
37
phosphatidylethanolamine, unidentified aminophospholipid, two unidentified glycolipids, six
38
unidentified aminolipids and two unidentified polar lipids. The G+C content was 35.3 mol%.
39
These data corroborated the affiliation of strain THG–EP9T to the genus Chryseobacterium.
40
Thus, the isolate represents a novel species, for which the name Chryseobacterium solani sp.
41
nov. is proposed, with THG–EP9T as the type strain (= KACC 17652T = JCM 19456T ).
%)
as
the
major
fatty
acids.
The polar
lipid
profiles consisted
of
42 43
The genus Chryseobacterium, which belongs to the family Flavobacteriaceae, was first
44
established by Vandamme et al. (1994). Subsequently, the genus expanded rapidly to
45
encompass more than 85 species, they were isolated from various environments such as soils
46
(Venil et al., 2014), clinical (Holmes et al., 2013), fresh water (Kirk et al., 2013), sewage and
47
wastewater (Kämpfer et al., 2003), plant (Sang et al., 2013), fish (Loch et al., 2014) and meat
48
(De Beer et al., 2005). The typical characteristics of the genus Chryseobacterium include
3
49
aerobic type of metabolism, production of flexirubin–type pigments, the presence of
50
branched–chain fatty acids (iso–C15
51
phosphatidylethanolamine (PE) as a major polar lipid and menaquinone–6 (MK–6) as the
52
characteristic respiratory quinone (Bernardet et al., 2006, 2011; Im et al 2011; Kämpfer et al.,
53
2009; Park et al., 2006; Vandamme et al., 1994; Wu et al., 2013). In this study, a
54
Chryseobacterium–like bacterial strain, THG–EP9T , was characterized by a polyphasic
55
taxonomic approach.
: 0
and iso–C17
: 0
3–OH) as the major fatty acids,
56 57
Strain THG–EP9T was isolated from field–grown eggplant rhizosphere soil in Pyeongtaek,
58
Gyeonggi–do, South Korea (36° 99′ N 127° 11′ E). About one gram soil was thoroughly
59
suspended in 10 ml sterile 0.85 % NaCl (w/v; saline solution), serially diluted samples were
60
spread on nutrient agar (NA, Oxoid) and incubated at 28 °C for 3 days. Single colonies were
61
purified by transferring to new NA plates. One isolate, THG–EP9T was cultured routinely on
62
NA agar at 28°C and stored in NA broth containing glycerol suspension (25 %, w/v) at –
63
70 °C.
64 65
For the sequencing of the 16S rRNA gene, genomic DNA was achieved using a Solgent
66
genomic DNA extraction kit (Korea) and the 16S rRNA gene was amplified according to the
67
methods of Weisburg et al. (1991). The 16S rRNA gene sequencing was performed by
68
Solgent Co. Ltd (Korea), sequences of related taxa were obtained from the EzTaxon–e server
69
(http://www.ezbiocloud.net/eztaxon; Kim et al., 2012) and GenBank database. Multiple
70
alignments were performed via program CLUSTAL_X (Thompson et al., 1997), followed
71
with gap editions in the BioEdit program (Hall, 1999). The Kimura two–parameter model
72
(Kimura, 1983) was used to calculate the evolutionary distances. The neighbor–joining
73
method (Saitou & Nei, 1987) and the maximum–likelihood method were used to construct
4
74
phylogenetic trees as implemented in MEGA 4 and MEGA 5.2 respectively (Kumar et al.,
75
2008). The bootstrap values were calculated based on 1,000 replications (Felsenstein, 1985).
76 77
The 16S rRNA gene sequence of strain THG–EP9T (1,424 bp) was analyzed. Sequence
78
similarity indicated that the close phylogenetic neighbors of strain THG–EP9T were
79
Chryseobacterium ginsenosidimutans THG 15T (97.3 %), Chryseobacterium soldanellicola
80
PSD1–4T (97.2 %), Chryseobacterium zeae JM–1085T (97.2 %) and Chryseobacterium
81
indoltheticum LMG 4025T (96.8 %). This relationship between strain THG–EP9T and other
82
recognized species of the genus Chryseobacterium was evidenced in the phylogenetic tree
83
(Figure 1 and Supplementary Fig. S1).
84 85
Gram reaction was determined by using bioMérieux Gram stain kit according to the
86
manufacturer’s instructions. Cell morphology was observed by transmission electron
87
microscope (Model JEM1010; JEOL) at × 11,000 magnification, using cells grown for 24h at
88
28 °C on NA. Motility was tested in sulfide–indole–motility medium (SIM; Difco).
89
Anaerobic growth was performed in nutrient broth (NB) supplemented with thioglycollate
90
[0.1 % (w/v), Sigma] with 5 days incubation, for the air was substituted with nitrogen gas.
91
Growth at different temperatures (4, 10, 18, 25, 28, 30, 35, 37 and 42 °C) was assessed after 7
92
days of incubation on NA. Salt tolerance was tested in NB containing 0–5 % (w/v) NaCl (at
93
intervals of 0.5 %), growth at various pH conditions (pH 4–10, at intervals of 0.5 pH units)
94
was performed in NB. For the pH experiments, two different buffers were used (final
95
concentration, 100 mM): acetate buffer was used for pH 4.0–6.5 and phosphate buffer was
96
used for pH 7.0–10.0. Growth was evaluated by monitoring the OD600 after 5 days of
97
incubation. Production of flexirubin–type pigments was determined by the color shift to red,
98
purple or brown when colonies were flooding with aqueous 20 % KOH solution (Fautz &
5
99
Reichenbach, 1980). Methyl red and Voges–Proskauer reactions were tested in Clark–Lubs’
100
medium. Oxidase and catalase activity were tested with 1 % (w/v) N, N, N′, N′–tetramethyl–
101
1,4–phenylenediamine reagent and 3 % (v/v) H2 O2 , respectively. Tests for degradation of
102
esculin [0.1 % (w/v) esculin and 0.05% (w/v) ferric citric acid, (Sigma)], casein [2.0 % (w/v)
103
skim milk, Oxoid], starch [1.0 % (w/v), Difco], DNA (DNase agar, Oxoid), Tween 20 [1.0 %
104
(w/v), Sigma], Tween 80 [1.0 % (w/v), Sigma], L–tyrosine [0.5 % (w/v), Sigma], chitin [1.0 %
105
(w/v), Sigma], and CM–cellulose [1.0 % (w/v), Sigma] were determined after 5 days of
106
incubation at 28 °C. Tests were also made for growth on R2A agar (Difco), marine agar (MA,
107
Oxoid), tryptone soya agar (TSA, Oxoid) and MacConkey agar (Oxoid). In addition, API
108
20NE, ID 32 GN and API ZYM tests (bioMérieux, France) were carried out to evaluate
109
carbon–source utilization and enzyme activities on the bases of the instructions of the
110
manufacturer. The strains of species Chryseobacterium ginsenosidimutans KACC 14527T , C.
111
soldanellicola KCTC 12382T , C. indoltheticum KCTC 2920T were included as references for
112
the investigation of the biochemical tests using the same laboratory conditions.
113 114
Strain THG–EP9T was Gram–negative, motile by gliding, aerobic, rod–shaped (1.50–1.90 ×
115
0.55–0.60 μm) and no flagella (Supplementary Fig. S2, available in IJSEM Online). The
116
isolate grew well on NA, TSA, MA and R2A agar, but not on MacConkey agar. Methyl red
117
test was weakly positive; Voges–Proskauer test was negative. Strain THG–EP9T produced
118
flexirubin–type pigments (the reversible color shifted from yellow to red). Growth occurred
119
at 4–35 °C, pH 6.0–10.0 and 0–1.5 % (w/v) NaCl. Physiological and biochemical
120
characteristics of strain THG–EP9T are summarized in the species description and a
121
comparison of strain THG–EP9T and related type strains is given in Table 1.
122
6
123
In order to determine G+C content and to perform DNA–DNA hybridization, genomic DNA
124
of strain THG–EP9T was prepared as described previously (Moore et al., 1995). The G+C
125
content was analyzed as described by Mesbah et al. (1989) using reverse–phase HPLC
126
system. DNA–DNA hybridization was performed fluorometrically, according to the method
127
developed by Ezaki et al. (1989), with photobiotin–labelled probes in microplate wells.
128
DNA–DNA hybridization experiments were performed between strain THG–EP9T and
129
closely related type strains of genus Chryseobacterium at 29.0 °C.
130 131 132
The DNA G+C content of strain THG–EP9T was 35.3 mol%, which conform to the expected
133
range of G+C contents for the genus Chryseobacterium (Kook et al., 2014). The DNA–DNA
134
relatedness values between strain THG–EP9T and related strains were below 10 %
135
(Chryseobacterium ginsenosidimutans KACC 14527T , 5.7 % and Chryseobacterium
136
soldanellicola KCTC 12382T , 9.1 %). These very low DNA relatedness values suggested that
137
THG–EP9T as a novel species of genus Chryseobacterium (Tindall et al., 2010).
138 139
For the cellular fatty acid analysis, cells of strain THG–EP9T and reference strains were
140
harvested from NA plates after incubation for 2 days at 28 ºC. The cellular fatty acid profiles
141
were prepared according to the protocol of Sherlock Microbial Identification System (MIDI)
142
and identified with GC (Hewlett Packard 6890) using Sherlock Aerobic Bacterial Database
143
(TSBA60) (Sasser, 1990).For the extraction of polar lipids and isoprenoid quinones of strain
144
THG–EP9T and polar lipids for type strain of the closest related species, C.
145
ginsenosidimutans KACC 14527T , cells were cultured in NB for 3 days and freeze-dried after
146
harvesting. The polar lipids of strain THG–EP9T and C. ginsenosidimutans KACC 14527T
147
were extracted (Minnikin et al., 1977; 1984) and detected using 2–dimensional thin–layer
7
148
chromatography (Tindall, 1990). TLC plates were sprayed with reagents: 5 %
149
molybdophosphoric acid was used for detecting total polar lipids, 0.2 % ninhydrin for amino
150
lipids, molybdenum blue reagent for phospholipids and α–naphthol sulphuric acid reagent for
151
glycolipids. Isoprenoid quinones analysis was performed by RP–HPLC Waters 2690 Alliance
152
system [solvent; methanol/2–propanol (7:5, v/v), flow rate; 1.0 ml/min] as previously
153
described (Collins & Jones, 1981; Hiraishi et al., 1996; Tamaoka et al., 1983).
154 155
The major cellular fatty acids (> 10.0 %) of strain THG–EP9T were iso–C15:0 (29.0 %), C16:0
156
(12.5 %) and iso–C17:0 3OH (11.9 %), which are consistent with the genus Chryseobacterium.
157
A comparison of fatty acid profiles between strain THG–EP9T and selected closely strains is
158
shown in Table 2. The polar lipid profiles of strain THG–EP9T and C. ginsenosidimutans
159
KACC 14527T are shown in Supplementary Fig. S3. A spot was detected with
160
molybdophosphoric acid, ninhydrin and
161
phosphatidylethanolamine (PE), which had shown similar retention on strain C.
162
ginsenosidimutans KACC 14527T
163
Chryseobacterium; eight lipids were observed to be ninhydrin positive, one of them were
164
molybdenum blue positive which were believed to be aminophospholipid (APL), the rest six
165
molybdenum blue negative spots were aminolipids (AL1–6); two spots, appeared as α–
166
naphthol sulphuric acid positive, were glycolipids (GL1–2). The polar lipid profiles of strain
167
THG–EP9T was clearly distinguishable from that of C. ginsenosidimutans KACC 14527T by
168
presence of two unidentified aminolipids (AL4–6), and absence of unidentified lipid (L3).
169
Strain THG–EP9T contained MK–6 as the single respiratory quinone, which is typical for the
170
genus Chryseobacterium (Wu et al., 2013).
molybdenum blue,
is in
was
believed to be
line with all other members of genus
171
8
172
On the basis of 16S rRNA gene sequence, fatty acid and polar lipid profiles, G+C content and
173
quinone system, strain THG–EP9T is suggested to belong to the genus Chryseobacterium as a
174
novel species, for which the name Chryseobacterium solani sp. nov. is proposed.
175 176
Description of Chryseobacterium solani sp. nov.
177
Chryseobacterium solani (so.la’ni. N.L. gen. neut. n. solani of the eggplant).
178
Cells are aerobic, Gram–negative, motile and rod–shaped (1.50–1.90 × 0.55–0.60 μm).
179
Colonies are yellowish, translucent and shiny on NA. Growth occurs at 4–35 °C (optimum
180
25–30 °C), at pH 6.0–10.0 (optimum 6.0–8.5) and at 0–1.5 % (w/v) NaCl. Grows well on NA,
181
TSA MA and R2A agar, but do not on MacConkey agar. Strain THG–EP9T produces
182
flexirubin–type pigments. Catalase and oxidase activity are positive. Weakly positive for
183
Methyl red test; Negative for Voges–Proskauer test. Esculin, casein, starch, DNA, Tween 20,
184
Tween 80, CM–cellulose CM cellulose and L–tyrosine are hydrolysed but chitin is not.
185
According to the API ZYM tests, positive results are obtained from alkaline phosphatase,
186
esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase,
187
cystine arylamidase, trypsin, α–chymotrypsin, acid phosphatase, α–glucosidase, β–
188
glucosidase, α–fucosidase, α–mannosidase and naphtol–AS–BI–phosphohydrolase; weakly
189
positive result is obtained from β–galactosidase; negative results are obtained from α–
190
galactosidase, β–glucuronidase and N–acetyl–β–glucosaminidase. In API 20NE tests, the
191
result of D–maltose, adipic acid, D–mannitol and gelatin hydrolysis, β–glucosidase, arginine
192
dihydrolase, and urease activity and indole production are positive; the assimilation for malic
193
acid and citric acid are weakly positive. In ID32 GN tests, glycogen, salicin, L–proline, D–
194
mannitol, D–glucose, L–rhamnose and D–maltose are assimilated; L–histidine, D–sucrose, D–
195
melibiose,
L–arabinose,
L–fucose,
D–sorbitol,
9
capric acid, valerate, citric acid, 2–
196
ketogluconate, 3–hydroxy–butyrate, 4–hydroxy–benzoate, N–acetyl–glucosamine, D–ribose,
197
itaconate, suberate, malonate, acetate, lactate,
198
benzoate and 5–ketogluconate are not assimilated; propionate is weakly assimilated. The
199
major fatty acids (> 10 %) are iso–C15:0 , C16:0 and iso–C17:0 3OH. The only isoprenoid
200
quinone is MK–6. The G+C content of genomic DNA is 35.3 mol%. The composition of
201
polar lipids is phosphatidylethanolamine, unidentified aminophospholipid, six unidentified
202
aminolipids, two unidentified glycolipids and two unidentified lipids.
L–alanin, L–serine,
inositol, 3–hydroxyl–
203 204
The type strain, THG–EP9T (= KACC 17652T = JCM 19456T ) was isolated from field–grown
205
eggplant rhizosphere soil in Gyeonggi–do, Republic of Korea.
206 207
10
208
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209
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310 311
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312
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313
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314
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315
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316
15
317
Table 1. Phenotypic characte ristics of Chryseobacterium solani THG–EP9 T and related
318
type strains of species of genus Chryseobacterium.
319
Strains: 1. Chryseobacterium solani THG–EP9T ; 2. Chryseobacterium ginsenosidimutans
320
KACC 14527T ; 3. Chryseobacterium soldanellicola KCTC 12382T ; 4. Chryseobacterium
321
zeae JM–1085T and 5. Chryseobacterium indoltheticum KCTC 2920T . For strains 1–3 and 5,
322
all data (except for DNA G+C content; taken from Im et al., 2011; Park et al., 2006 and
323
Vandamme et al., 1994) was carried out in this study; for stain 4, data were taken from
324
Kämpfer et al. (2013). All strains are oxidase and catalase positive, aerobic, growth on NA,
325
R2A and TSA and non–growth on MacConkey agar, positive for hydrolysis of esculin;
326
negative for nitrate reduction, acidification of glucose, capric acid, gluconate and
327
phenylacetic acid. The following compounds are not utilised as a sole source of carbon for all
328
species: D–melibiose, D–sorbitol, capric acid, citric acid, L–histidine, 4–hydroxy–benzoate, N–
329
acetyl–glucosamine,
330
hydroxyl–benzoate. In API ZYM tests, strains 1–3 and 5 are all positive for alkaline
331
phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine
332
arylamidase, acid phosphatase, naphtol–AS–BI–phosphohydrolase and α–glucosidase;
333
negative for α–Galactosidase, β–Glucuronidase. (NA): not available; (+): positive; (w):
334
weakly positive; (–): negative.
D–ribose,
itaconate, suberate, lactate, L–alanin, L–serine, inositol, 3–
Characteristic Temperature range for growth (°C) pH range for growth Growth with 2% NaCl (w/v) Gliding motility Growth on MA Indole production Assimilation of Adipic acid Salicin Acetate
1 4–35 6.0–10.0 – + + +
2 10–35 5.5–10.0 – – – +
3 4–37 5.0–7.0 + + + +
4 8–36 NA + – NA –
5 4–35 5.0–8.0 + – – +
+ + –
– – +
W – –
– + –
W – +
16
Propionate L–Arabinose D–Mannose D–Maltose D–Mannitol D–Sucrose D–Glucose Hydrolysis of Starch DNA Casein CMC L–Tyrosine Tween 80 Enzyme activities Urease Arginine dihydrolase a–Fucosidase N–Acetyl–β–glucosaminidase α–Mannosidase Trypsin Cystine arylamidase DNA G+C content (mol%)
W – – + + – –
W + + + – + +
– + + + + + +
– W – + – + W
– – + – – – +
+ + + + + +
+ + + + – +
+ + + + + –
– – – – – NA
+ + + + + +
+ + + – + + + 35.3
– – – + – – – 35.7
– – – – – + + 28.8
– – NA NA NA NA NA NA
– – – + – + + 33.8
335
17
336
Table 2. Cellular fatty acid profiles of strain THG–EP9 T and phylogenetically related
337
species of the genus Chryseobacterium.
338
1. Chryseobacterium solani THG–EP9T ; 2. Chryseobacterium ginsenosidimutans KACC
339
14527T ; 3. Chryseobacterium soldanellicola
340
indoltheticum KCTC 2920T . All data were carried out in this study. Cells were cultured under
341
same condition. Fatty acids of less than 0.5 % in all strains were not listed; (ND): not
342
detected; Tr: traces (< 1.0 %). Fatty acid
KCTC 12382T ; 4. Chryseobacterium
1
2
3
4
12.5 6.2
5.5 3.3
7.7 4.4
5.8 2.1
2.0
Tr
1.9
Tr
Tr 29.0 2.3 4.3 2.7 1.7 1.2 11.9 7.4 8.4
Tr 34.1 3.8 2.3 1.3 Tr 1.4 16.5 13.2 11.7
1.6 28.4 3.5 4.7 Tr 1.3 Tr 14.4 7.5 16.6
ND 25.0 1.9 6.5 1.9 2.3 Tr 9.5 22.7 12.9
Saturated C16:0 C18:0
Unsaturated C16:0 3OH
Branched–chain iso–C13:0 iso–C15:0 iso–C15:0 3OH anteiso–C15:0 iso–C16:0 iso–C16:0 3OH iso–C17:0 iso–C17:0 3OH iso–C17:1 ω9c * Summed Feature 3 343 344
*Summed features represent groups of two or three fatty acids that could not be separated by
345
GLC with the MIDI system. Summed feature 3 contained C16:1 ω7c and/or C16:1 ω6c.
346 347
18
348
Figure legends
349
Fig. 1. Neighbour–joining phylogenetic tree based on 16S rRNA gene sequences showing
350
the relationships of Chryseobacterium solani THG–EP9 T with related Chryseobacterium
351
species.
352
Bootstrap values (expressed as percentage of 1,000 replications) over 70% are shown at the branching
353
points. Bergeyella zoohelcum ATCC 43767 T (AGYA01000006) was used as outgroup. Bar, 0.005
354
substitutions per nucleotide position.
355 356
Suppleme ntary Fig. S1. Maximum–likelihood tree based on 16S rRNA gene sequences
357
showing the relationships between strain THG–EP9T and other related type species.
358
Numbers at nodes represent percentages of bootstrap support based on a maximum–likelihood
359
analysis of 1,000 resampled datasets.
360 361
Suppleme ntary Fig. S2. Trans mission electron micrograph of cells of Chryseobacterium
362
solani THG–EP9 T.
363
The detection was performed after negative staining with uranyl acetate. Bar, 0.5µm.
364 365
Suppleme ntary Fig. S3. Two–dimensional thin–layer chromatography of polar lipids of
366
strain THG–EP9T.
367
a and b: Total lipids were detected by spraying with 5% molybdatophosphoric acid for strain THG–
368
EP9T and C. ginsenosidimutans KACC 14527T , respectively; c: Aminolipids detected by spraying
369
with 0.2% ninhydrin; d: Phospholipids detected by spraying with molybdenum blue; e: Glycolipids
370
revealed by α–naphthol–sulphuric acid. Abbreviations: phosphatidylethanolamine (PE), unidentified
371
glycolipid (GL), unidentified aminophospholipids (APL), unidentified phospholipid (PL), unidentified
372
aminolipid (AL) and unidentified lipid (L).
19
Figure Click here to download Figure: figure 1 EP9.pdf
84 94 86
0.005
Chryseobacterium indoltheticum LMG 4025T (AY468448) Chryseobacterium scophthalmum LMG 13028T (AJ271009) Chryseobacterium greenlandense UMB 34T (932652) 100 Chryseobacterium aquaticum 10-46T (AM748690) Chryseobacterium piscicola VQ-6316sT (EU869190) Chryseobacterium soli JS6-6T (EF591302) Chryseobacterium ginsenosidimutans THG 15T (GU138380) Chryseobacterium solani THG-EP9T (KF532126) Chryseobacterium soldanellicola PSD1-4T (AY883415) 73 Chryseobacterium geocarposphaerae 91A-561T (HG738132) Chryseobacterium gwangjuense THG-A18T (JN196134) Chryseobacterium gregarium DSM 19109T (AM773820) 92 Chryseobacterium camelliae THG C4-1T (JX843771) Chryseobacterium daeguense K105T (EF076759) Chryseobacterium aahli T68T (AM773820) 100 Chryseobacterium yeoncheonense DCY67T (JX141782) Chryseobacterium massiliae 90BT (AF531766) Chryseobacterium elymi RHA3-1T (DQ673671) Chryseobacterium taihuense THMBM1T (IQ283114) Chryseobacterium zeae JM-1085T (HG738135) Chryseobacterium hungaricum CHB-20pT (EF685359) Chryseobacterium hominis NF802T (AM261868) Chryseobacterium viscerum 687B-08T (FR871426) Chryseobacterium oncorhynchi 701B-08 (FN674441) Chryseobacterium jejuense JS17-8T (EF591303) Chryseobacterium vietnamense GIMN1.005T (HM212415) Bergeyella zoohelcum ATCC 43767 (AGYA01000006) Figure 1, Du et al.
Click here to download Supplementary Material Files: Supplementary file-EP9.pdf
