IJSEM Papers in Press. Published May 28, 2015 as doi:10.1099/ijs.0.000360
International Journal of Systematic and Evolutionary Microbiology Oceanobacillus arenosus sp. nov., a moderate halophilic bacterium isolated from marine sand --Manuscript Draft-Manuscript Number:
IJS-D-15-00043R2
Full Title:
Oceanobacillus arenosus sp. nov., a moderate halophilic bacterium isolated from marine sand
Short Title:
Oceanobacillus arenosus sp. nov.
Article Type:
Note
Section/Category:
New taxa - Firmicutes and related organisms
Corresponding Author:
Wonyong Kim Chung-Ang University College of Medicine Seoul, KOREA, REPUBLIC OF
First Author:
Wonyong Kim
Order of Authors:
Wonyong Kim Chatuphon Siamphan Jong-Hwa Kim Ampaitip Sukhoom
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A Gram-positive, spore-forming, rod-shaped, motile, strictly aerobic bacterium, designated CAU 1183T, was isolated from a marine sand and its taxonomic position was investigated using a polyphasic approach. The bacterium grew optimally at a temperature of 30C at pH 8.5 in the presence of 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAU 1183T formed a distinct lineage within the genus Oceanobacillus and exhibited the highest similarity to Oceanobacillus chungangensis (97.6%). The strain contained MK-7 as the predominant isoprenoid quinone and anteiso-C15:0 was the major cellular fatty acid. The cell wall peptidoglycan contains meso-diaminopimelic acid. The polar lipid pattern of strain CAU 1183T consisted of diphosphatidylglycerol, phosphatidylglycerol, and unidentified lipids including two phospholipids, two glycolipids, phosphoglycolipid, and two lipids. The G+C content of the genomic DNA was 37.5 mol%. On the basis of phenotypic, chemotaxonomic, and phylogenetic data, strain CAU 1183T should be classified as a novel species in the genus Oceanobacillus, for which the name Oceanobacillus arenosus sp. nov. is proposed. The type strain is CAU 1183T (=KCTC 33037T, =CECT 8560T).
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1
Oceanobacillus arenosus sp. nov., a moderate halophilic bacterium isolated from marine
2
sand
3 4
Wonyong Kim,1 Chatuphon Siamphan,1 Jong-Hwa Kim,1 Ampaitip Sukhoom2*
5 6
1
7
of Korea
8
2
9
90112, Thailand
Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Republic
Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla,
10 11
*Correspondence: Ampaitip Sukhoom, Tel 66-074-28-8008, E-mail: [email protected]
12 13
Subject category: New taxa (Firmicutes and Related Organisms)
14 15
Running title: Oceanobacillus arenosus sp. nov.
16
Keywords: Oceanobacillus arenosus, Firmicutes; Bacillaceae, Marine sand
17 18
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
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CAU 1183T is JX233495.
20
1
21
Summary
22
A Gram-positive, spore-forming, rod-shaped, motile, strictly aerobic bacterium, designated
23
CAU 1183T, was isolated from a marine sand and its taxonomic position was investigated
24
using a polyphasic approach. The bacterium grew optimally at a temperature of 30C at pH
25
8.5 in the presence of 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene
26
sequences showed that strain CAU 1183T formed a distinct lineage within the genus
27
Oceanobacillus and exhibited the highest similarity to Oceanobacillus chungangensis
28
(97.6%). The strain contained MK-7 as the predominant isoprenoid quinone and anteiso-C15:0
29
was the major cellular fatty acid. The cell wall peptidoglycan contains meso-diaminopimelic
30
acid. The polar lipid pattern of strain CAU 1183T consisted of diphosphatidylglycerol,
31
phosphatidylglycerol, and unidentified lipids including two phospholipids, two glycolipids,
32
phosphoglycolipid, and two lipids. The G+C content of the genomic DNA was 37.5 mol%.
33
On the basis of phenotypic, chemotaxonomic, and phylogenetic data, strain CAU 1183T
34
should be classified as a novel species in the genus Oceanobacillus, for which the name
35
Oceanobacillus arenosus sp. nov. is proposed. The type strain is CAU 1183T (=KCTC
36
33037T, =CECT 8560T).
37 38
The genus Oceanobacillus, a member of the family Bacillaceae, was proposed by Lu et al.
39
(2001) with the description of O. iheyensis as the type species. The genus Oceanobacillus
40
comprises aerobic, Gram-stain-positive, motile, rod-shaped bacteria that are characterized
41
chemo-taxonomically by the presence of menaquinone-7 (MK-7) as the major isoprenoid
42
quinone and anteiso-C15:0 as the predominant cellular fatty acid (Namwong et al. 2009; Lee et
43
al. 2010; Whon et al. 2010). Currently, the genus comprises 17 recognized species with
44
validly published names: O. iheyensis (Lu et al. 2001), O. picturae (Heyrman et al. 2003), O.
45
oncorhynchi (Yumoto et al. 2005), O. chironomi (Raats and Halpern 2007), O. profundus
2
46
(Kim et al. 2007), O. caeni (Nam et al. 2008), O. kapialis (Namwong et al. 2009), O. sojae
47
(Tominaga et al. 2009), O. neutriphilus (Yang et al. 2010), O. locisalsi (Lee et al. 2010), O.
48
kimchii (Whon et al. 2010), O. chungangensis (Lee et al., 2013), O. polygoni (Hirota et al.,
49
2013), O. indicireducens (Hirota et al., 2013), O. pacificus (Yu et al., 2014), O. limi
50
(Amoozegar et al., 2014), and O. luteolus (Wu et al., 2014) (http://www.bacterio.net/).
51
Members of the genus Oceanobacillus have been isolated from various habitats such as
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marine environments (deep-sea sediment, marine solar saltern, and salt lake), mural paintings,
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freshwater fish, insect, activated sludge, food, soy sauce production equipment, and soil. In
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the course of the screening of bacteria from marine environments, a bacterial strain,
55
designated CAU 1183T, was isolated from a sand sample collected in Jeju Island
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(33º22’06.16”N, 126º33’11.11”E) in the Republic of Korea. The purpose of the present study
57
was to establish the taxonomic position of this bacterial strain by using a polyphasic approach
58
that included the determination of phenotypic, chemotaxonomic, and genotypic properties.
59 60
Strain CAU 1183T was isolated from a marine sand sample of Jeju Island according to
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Gordon & Mihm (1962) using marine agar 2216 (MA; Difco Laboratories Detroit MI, USA).
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The sand sample was crushed and diluted with sterilized saline solution so that appropriate
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dilutions could be spread on MA plates. The plates were incubated under aerobic conditions
64
at 30˚C for 7 days. A pure single colony was purified by subculturing and preserved at -80˚C
65
in marine broth 2216 (MB; Difco Laboratories Detroit MI, USA) supplemented with 25%
66
(w/v) glycerol for taxonomic analysis. Strain CAU 1183T was deposited in the Korean
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Collection for Type Cultures (KCTC; Taejon, Republic of Korea) and the Colección
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Española de Cultivos Tipo (CECT; Paterna, Spain) as KCTC 33037T and CECT 8560T,
69
respectively. The type strains of four closely related species and type strain of the type
70
species of the genus, O. iheyensis HTE831T, were used as reference strains in most analyses.
3
71
O. chungangensis CAU 1051T, O. profundus CL-MP28T, and O. caeni S-11T were obtained
72
from the Korean Collection for Type Cultures (KCTC; Taejon, Korea).
73 74
Genomic DNA of strain CAU 1183T was extracted using the method of Marmur (1961). PCR
75
amplification of the 16S rRNA gene was performed following established procedures (Cho et
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al., 2008). The amplified 16S rRNA gene was sequenced directly using a BigDye terminator
77
v3.1 cycle sequencing kit and an automatic 3730 DNA sequencer (Applied Biosystems Life
78
Technologies Co., Carlsbad, CA, USA). Multiple alignments and calculation of 16S rRNA
79
gene sequence similarity between strain CAU 1183T and a broad selection of the closely
80
related
81
(http://www.ezbiocloud.net/eztaxon), and CLUSTAL-X 2.1 program (Larkin et al., 2007).
82
Evolutionary distance matrices were created by the neighbor-joining method defined by
83
Jukes and Cantor (1969). Phylogenetic trees were generated using the neighbour-joining
84
(Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967), and maximum-likelihood
85
(Felsenstein, 1981) algorithms in the PHYLIP package (Felsenstein, 1989). Branch support in
86
the neighbour-joining tree was evaluated by the bootstrap resampling method based on 1,000
87
replicates (Felsenstein, 1985). The extent of DNA–DNA relatedness between CAU 1183T
88
and the most closely related phylogenetic neighbour, Oceanobacillus chungangensis CAU
89
1051T was determined using the fluorometric microplate method (Ezaki et al., 1989), as
90
modified by Goris et al. (1998). The mol% G+C content of the genomic DNA was examined
91
by HPLC, according to the method of Tamaoka and Komagata (1984).
species
were
carried
out
by
using
the
EzTaxon
-
EzBioCloud.net
92 93
Strain CAU 1183T was cultivated on MA at 30˚C for the investigation of morphological,
94
physiological and biochemical characteristics, except for spore formation that was assessed
95
on nutrient sporulation medium (Nicholson & Setlow, 1990). Cell morphology was examined
4
96
under a DM 1000 light microscope (Leica Microsystems AG, Wetzlar, Germany) using cells
97
from an exponentially growing culture. Gram reaction was performed using the bioMérieux
98
Gram staining kit (bioMérieux SA, Marcy l'Etoile, France). Motility was assessed on an MB
99
culture using the hanging-drop method (Bowman et al., 2000). After 5 days of growth, spore
100
formation was determined by staining with malachite green as described previously by Conn
101
et al. (1957). Growth on MA at 4, 10, 20, 30, 37, 45, and 55C was evaluated in an aerobic
102
incubator (model MIR-253; Sanyo Electric Co. Ltd, Wood Dale, IL, USA) and an anaerobic
103
chamber (model Bactron; Sheldon Manufacturing, Inc., Cornelius, OR, USA) by measuring
104
the turbidity of the broth after 72 hrs. The optimal growth pH was investigated by culturing at
105
30C in MB that was adjusted to pH 4.5–10.0 (at intervals of 0.5 pH unit) using sodium
106
acetate/acetic acid and Na2CO3 buffers, and values were confirmed after autoclaving. Growth
107
in the absence of NaCl and in the presence of 0–15.0% (w/v) NaCl was explored by culturing
108
in MB prepared according to the Difco formula except that NaCl was excluded and that
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0.45% (w/v) MaCl2.6H2O and 0.06% (w/v) KCl was added.
110 111
Oxidase activity was assessed from the oxidation of 0.1% (w/v) tetramethyl-p-
112
phenylenediamine (Cappuccino & Sherman, 2002). Catalase activity was tested by bubble
113
production in 3% (v/v) H2O2 solution. Hydrolysis of casein, starch and urea were evaluated
114
according to the methods of Lányí (1987) and Smibert & Krieg (1994). Acid production from
115
carbohydrates, enzyme activity, and other physiological and biochemical features were tested
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using the API 50CH and API 20E systems at 30˚C (bioMérieux). API 20E strips were read
117
after 24 hrs and API 50CH strips after 24 hrs and 48 hrs.
118 119
For fatty acid analysis, the cell mass of strain O. iheyensis HTE831T, O. chungangensis CAU
120
1051T, O. profundus CL-MP28T, and O. caeni S-11T was harvested from TSA (Difco) after
5
121
cultivation for 3 days at 30C. The physiological ages of the biomasses were standardized by
122
observing growth development during incubation of the three different cultures and choose
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the moment of harvesting according to the standard MIDI method (Sherlock Microbial
124
Identification System version 6.1, Newark, DE, USA). Cellular fatty acid methyl esters
125
(FAMEs) were obtained according to the method of Minnikin et al. (1980) and separated in a
126
6890N gas chromatograph fitted with a 7683 autosampler (Agilent Technologies Inc., Santa
127
Clara, CA, USA). Peaks were identified using the Microbial Identification software package
128
(MOORE library ver. 5.0; MIDI database TSBA6). Peptidoglycan was analyzed as described
129
by Schleifer & Seidl (1985). The polar lipids of strain CAU 1183T were identified using two-
130
dimensional thin-layer chromatography (TLC) by the method of Minnikin et al. (1984). The
131
plate was sprayed with 10% ethanolic molybdatophosphoric acid (for the total lipids),
132
molybdenum blue (for phospholipids), ninhydrin (for aminolipids) and α-naphthol/sulphuric
133
acid reagent (for glycolipids). Menaquinones were determined on HPLC using an isocratic
134
solvent system [methanol/isopropyl ether (3:1, v/v)] and the solvent flow (1 ml/min) (Hiraishi
135
et al., 1984).
136 137
The nearly complete 16S rRNA gene sequence of strain CAU 1183T (1,547 bp) was
138
determined and compared to the reference sequences for other bacterial species in the public
139
database. Phylogenetic analysis indicated that the novel isolate fell into the genus
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Oceanobacillus. The neighbour-joining tree is shown in Fig. 1. Pairwise analysis indicated
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that the most closely related strains were O. chungangensis CAU 1051T (16S rRNA gene
142
similarity, 97.6%), O. profundus CL-MP28T (similarity, 96.5%), O. polygoni SA9T
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(similarity, 96.3%), and O. caeni S-11T (similarity, 95.7%). The DNA-DNA relatedness
144
between CAU 1183T and the most closely related strain, O. chungangensis CAU 1051T was
145
44.2% when CAU 1183T was used as a probe and 43.6% when O. chungangensis CAU 1051T
6
146
was used as a probe. These value is well below the 70% cut-off point recommended by
147
Wayne et al. (1987) for the delineation of genomic species, supporting the proposal that strain
148
CAU 1183T represents a separate species. The DNA G+C content of the strain CAU1183T
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was 37.5 mol%, which was in accordance with the previously reported values of the genus
150
Oceanobacillus; 33.6 mol% (O. caeni S-11T; Nam et al., 2008) – 40.6 mol% (O. polygoni
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SA9T; Hirota et al., 2013).
152 153
The detailed phenotypic characteristics are presented in Table 1 and the new species
154
description. Strain CAU 1183T was Gram-stain-positive, central ovoid spore-forming (Suppl.
155
Fig. S1), strictly aerobic, and motile. Colonies on MA were cream colored, entire, circular,
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and convex with diamsporeeters of 1.2–2.0 mm after 3 days of cultivation at 30˚C. No
157
colonies were formed under anaerobic growth condition. Cell are rod-shaped approximately
158
0.5–1.0 µm in diameter and 1.5–4.3 µm in length. Growth occurs at 20–37C (optimum,
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30C) and at pH 5.5-9.0 (optimum, 8.5), and at 0–8% (w/v) NaCl (optimum, 2%). Phenotypic
160
characteristics data of strain CAU 1183T differed from its closest relatives, O. chungangensis
161
KCTC 33035T (Lee et al., 2013) and the members in the genus Oceanobacillus (Table 1).
162
The growth temperature of CAU 1183T could be distinguished from O. profundus (15-42C),
163
O. polygoni (5–48C), and O. iheyensis (15–42C). In addition, the pH range and NaCl
164
tolerance of strain CAU 1183T differed from O. polygoni (pH 7.0–12.0 / 3–12% NaCl), and O.
165
iheyensis (pH 6.5–10.0 / 0–21% NaCl). Strain CAU 1183T differed from its closest relatives,
166
O. profundus CL-MP28T (Kim et al., 2007), O. polygoni SA9T (Hirota et al., 2013) and O.
167
caeni S-11T (Nam et al., 2008), and the type strain O. iheyensis (Lu et al., 2002) by its acid
168
production from amidon and glycogen. In addition, strain CAU 1183T differed from O.
169
iheyensis HTE831T (Lu et al., 2002) by its acid production from D-trehalose. Overall, the
170
chemotaxonomic characteristic was in agreement with previously published data for the 7
171
genus Oceanobacillus. The predominant isoprenoid quinone detected in strain CAU 1183T
172
was menaquinone 7 (MK-7), in line with all members of the genus Oceanobacillus, which is
173
consistent with data for the type species of the genus, O. iheyensis HTE831T (Lu et al., 2001).
174
The strain contained saturated and branched-chain fatty acids (Table 2). The peptidoglycan of
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strain CAU 1183T contained meso-diaminopimelic acid as the diagnostic cell-wall diamino
176
acid, which was present in the strains of recognized Oceanobacillus species. The major fatty
177
acids (>10%) were anteiso-C15:0 (50.4%), C16:0 (11.5%), anteiso-C17:0 (10.3%), iso-C15:0
178
(10.3%). The following fatty acids (>1%) C16:0 (8.5%), iso-C14:0 (5.0%), iso-C17:0 (2.4%), and
179
C14:0 (1.4%) were also present. This fatty acid profile was almost same as that of closest
180
relatives, although there were differences in the amounts and presence of some fatty acids.
181
Anteiso-C15:0 was major compound in strain CAU 1183T and the closely related other species
182
of the genus Oceanobacillus; O. chungangensis CAU 1051T, O. profundus KCTC 13625T
183
and O. caeni KCTC 13061T and O. polygoni SA9T, except O. iheyensis KCTC 3954T. Strain
184
1183T contained diphosphatidylglycerol and phosphatidylglycerol as the major polar lipids
185
identified as in O. iheyensis KCTC 3954T. In addition, cells of this novel isolate contained
186
unidentified lipids: two phospholipids, two glycolipids, one phosphoglycolipid, and two
187
lipids (Suppl. Fig. S2). Diphosphatidylglycerol and phosphatidylglycerol were also detected
188
as the major polar lipids in O. luteolus WM-1T, O. limi H9BT, O. chungangensis CAU 1051T,
189
O. sojae JCM 15792T, O. kapialis SSK2-2T. Phosphatidylethanolamine was detected in O.
190
pacificus XH204T, O. polygoni SA9T, O. indicireducens A21T, but this polar lipid was not
191
detected in strain CAU 1183T.
192 193
In conclusion, it is evident from the phenotypic, chemotaxonomic and genotypic data that
194
strain CAU 1183T represents a novel species of the genus Oceanobacillus, for which the
195
name Oceanobacillus arenosus sp. nov. is proposed.
8
196 197
Description of Oceanobacillus arenosus sp. nov.
198
Oceanobacillus sp. nov. (a.re.no'sus. L. masc. adj. arenosus sandy).
199 200
Gram-positive, obligate aerobic, motile rod with optimal growth that occurs at 30C (range of
201
20–37C), pH 8.5 (pH 5.5–9.01), and 2% (w/v) sea salt (0–8%). Forms central endospores.
202
Catalase and oxidase are positive. Esculin and gelatin are hydrolysed. Starch, urea and citrate
203
are negative. Acid is produced from amidon, amygdalin, D-fructose, D-galactose, D-glucose,
204
D-maltose, D-mannitol, D-melibiose, D-lactose, D-raffinose, D-sorbitol, D-trehalose, D-
205
xylose, glycogen and salicin. The major isoprenoid quinone is MK-7. The cell wall
206
peptidoglycan contains meso-diaminopimelic acid. The polar lipid pattern consists of
207
diphosphatidylglycerol,
208
unidentified glycolipids, one unidentified phosphoglycolipid and two unidentified lipids. The
209
major cellular fatty acid is anteiso-C15:0. The DNA G+C content is 37.5 mol%. The type
210
strain CAU 1183T (=KCTC 33037T, =CECT 8560T), isolated from a marine sand collected
211
from Jeju Island in the Republic of Korea.
phosphatidylglycerol,
two
unidentified
phospholipids,
two
212 213
Acknowledgment
214
This work was supported by the project on survey of indigenous species of Korea of the
215
National Institute of Biological Resources (NIBR) under the Ministry of Environment (MOE).
216 217
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& Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid
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quinones and polar lipids. J Microbiol Methods 2:233–241.
315
13
316
Nam, J.-H., Bae. W. & Lee, D.-H. (2008). Oceanobacillus caeni sp. nov., isolated from a
317
Bacillus-dominated wastewater treatment system in Korea. Int J Syst Evol Microbiol 58,
318
1109–1113.
319 320
Namwong, S., Tanasupawat, S., Lee, K. C. & Lee, J.-S. (2011). Oceanobacillus kapialis sp.
321
nov., from fermented shrimp paste in Thailand. Int J Syst Evol Microbiol, 59, 2254–2259.
322
Nicholson, W. L. & Setlow, P. (1990). Sporulation, germination and outgrowth. In
323
Molecular Biological Methods for Bacillus, pp. 391–450. Edited by C. R. Harwood & S. M.
324
Cutting. Chichester: Wiley.
325 326
Raats, D. & Halpern, M. (2007). Oceanobacillus chironomi sp. nov., a halotolerant and
327
facultatively alkaliphilic species isolated from a chironomid egg mass. Int J Syst Evol
328
Microbiol 57, 255–259.
329 330
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing
331
phylogenetic trees. Mol Biol Evol 4, 406–425.
332 333
Schleifer, K. H. & Seidl, P. H. (1985). Chemical composition and structure of murein. In
334
Chemical Methods in Bacterial Systematics, pp. 201-219. Edited by M. Goodfellow & D. E.
335
Minnikin. London: Academic Press.
336 337
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General
338
and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A.
339
Wood, N. R. Krieg. Washington, DC: American Society for Microbiology.
340
14
341
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reverse-
342
phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
343 344
Tominaga, T., An, S. Y., Oyaizu, H. & Yokota, A. (2009). Oceanobacillussoja sp. nov.
345
isolated from soy sauce production equipment in Japan. J Gen Appl Microbiol 55, 225–232.
346 347
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O.,
348
Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors.
349
(1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee
350
on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
351 352
Whon, T. W., Jung, M.-J., Roh, S. W., Nam, Y.-D., Park, E.-J., Shin, K.-S. & Bae, J.-W.
353
(2010). Oceanobacillus kimchii sp. nov. isolated from a traditional Korean fermented food. J
354
Microbiol 48, 862–866.
355 356
Wu, M., Yang, G., Yu, Z., Zhuang, L., Jin, Y. & Zhou, S. (2014). Oceanobacillus luteolus
357
sp. nov., isolated from soil. Int J Syst Evol Microbiol 64, 1495–1500.
358 359
Yang, J.-Y., Huo, Y.-Y., Xu, X.-W., Meng, F.-X., Wu, M. & Wang, C.-S. (2010).
360
Oceanobacillus neutriphilus sp. nov., isolated from activated sludge in a bioreactor. Int J Syst
361
Evol Microbiol 60, 2409–2414.
362 363
Yu, C., Yu, S., Zhang, Z., Li, Z. & Zhang, X.-H. (2014). Oceanobacillus pacificus sp. nov.,
364
isolated from a deep-sea sediment. Int J Syst Evol Microbiol, 64, 1278–1283.
365
15
366
Yumoto, I., Hirota, K., Nodasaka, Y. & Nakajima, K. (2005). Oceanobacillus
367
oncorhynchi sp. nov., a halotolerant obligate alkaliphile isolated from the skin of a rainbow
368
trout (Oncorhynchus mykiss), and emended description of the genus Oceanobacillus. Int J
369
Syst Evol Microbiol 55, 1521–1524.
16
370
Table 1. Phenotypic properties of strain CAU 1183T the type strains of the closely related
371
Oceanobacillus species. Strains: 1, CAU 1183T (data from this study); 2, O. chungangensis
372
CAU 1051T; 3, O. profundus CL-MP28T; 4, O. caeni S-11T; 5, O. polygoni SA9T; 6, O.
373
iheyensis HTE831T. All strain indicated sporangial morphology as ellipsoidal. +, positive; -,
374
negative; w, weakly positive Characteristic
1
2
3
4
5
Spore formation
Central
Terminal
Terminalb
Centralc
Centrald
Temperature range (˚C) Optimum
20–37 30
20–37a 30a
15–42b 35b
20–45c 35c
5–48d 35d
Terminal or subterminale 15–42e 30e
pH range Optimum
5.5–9.0 8.5
4.5–10.0a 5a
6.5–9.5b 8b
6.0–9.0c 7c
7.0–12.0d 9d
6.5–10.0e 8.3e
0–8 2
0–10a 0a
0–14b 2b
0–10c 3.5c
3–12d 3d
0–21e 3e
Hydrolysis of : Casein Gelatin Esculin
+ +
+
+ + +
-
+ +
+ + +
Acid production from : Amygdalin Glycerol D-Galactose
+ +
+ +
+ + +
+ + -
+ -
+ + -
D-Mannose D-Melezitose D-Melibiose D-Raffinose D-Ribose D-Sorbitol D-Trehalose L-Arabinose
+ + + + -
+ + + + +
+ + + + + -
+ + + + -
+ + + + -
+ + + +
L-Rhamnose DNA G+C content (mol%)
-
-
-
+
-
-
37.5
36.3
40.2
33.6
40.6
35.8
NaCl tolerance (%) Optimum
6
375
*Data taken from: a, Lee et al., 2013; b, Kim et al., 2007; c, Nam et al., 2008; d, Hirota et al.,
376
2013; e, Lu et al., 2002.
377
17
378
Table 2. Cellular fatty acid compositions (%) of strain CAU 1183T the type strains of the
379
most closely related Oceanobacillus species. Strains: 1, CAU 1183T; 2, O. chungangensis
380
CAU 1051T; 3, O. profundus CL-MP28T; 4, O. caeni S-11T; 5, O. polygoni SA9T (data from
381
Hirota et al., 2013); 6, O. iheyensis HTE831T; All data from this study except O. polygoni
382
SA9T uses data from Hirota et al. (2013). Only those fatty acids amounting to >1.0% in all
383
strains are shown, -, not detected, ND, data not shown Fatty acids
1
2
3
4
5
6
C14:0
1.41
1.5
-
-
2.0
4.1
C16:0
11.5
16.4
4.9
6.7
9.0
9.9
anteiso-C15:0
50.4
44.2
53.3
56.7
60.3
32.0
anteiso-C17:0
10.3
13.3
18.0
16.9
8.6
4.2
iso-C14:0
5.0
4.8
3.7
2.6
3.5
6.8
iso-C15:0
10.3
9.6
8.5
8.0
8.3
33.5
iso-C16:0
8.5
6.6
8.6
5.4
3.5
6.1
iso-C17:0
2.4
2.5
1.9
1.8
1.5
2.3
Saturated
Branched-chain
384 385
18
386
Legend figure
387 388
Fig. 1 Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene
389
sequences showing the relationships between strain CAU 1183T and the type strains of
390
Oceanobacillus species. Dots indicate that the corresponding nodes were also recovered in
391
the trees generated with the maximum-likelihood and least squares algorithms. The numbers
392
at the nodes indicate levels of bootstrap support based on a neighbour-joining analysis of
393
1,000 resampled datasets; only values > 70% are given. Bar, 0.01 substitutions per nucleotide
394
position. Macrococcus hajekii CCM 4809T (AY119685) is used as an outgroup organism.
19
Figure Click here to download Figure: Fig. 1_IJS-D-15-00043R2.ppt
Virgibacillus litoralis JSM 089168T (FJ425909) ●100 ●
83
100●
Virgibacillus salinus XH-22T (FM205010)
Virgibacillus carmonensis LMG 20964T (AJ316302) Virgibacillus halodenitrificans DSM 10037T (AY543169)
●
Oceanobacillus picturae LMG 19492T (AJ315060) ●
100 Oceanobacillus manasiensis YD3-56T (FJ386518) ● 97
●
Oceanobacillus kapialis SSK2-2T (AB366005)
Ornithinibacillus contaminans CCUG 53201T (FN597064) ● ●
Paucisalibacillus globulus B22T (AM114102) ● 79
Ornithinibacillus bavariensis WSBC 24001T (Y13066)
● ● 78
Ornithinibacillus californiensis MB-9T (AF326365) Ornithinibacillus scapharcae TW25T (AEWH01000025)
‘ Oceanobacillus massiliensis N'DiopT (HQ586877)’ Oceanobacillus polygoni SA9T (AB750685)
●100
Oceanobacillus profundus CL-MP28T (DQ386635)
Oceanobacillus chungangensisC AU1051T (JX233492)
●100
Oceanobacillus arenosus CAU 1183T (JX233495) Oceanobacillus caeni S-11T (AB275883)
Oceanobacillus limi H9BT (HQ433455) Oceanobacillus iheyensis HTE831T (BA000028)
●100
Oceanobacillus kimchii X50T (GU784860)
100
●
●98
●
Oceanobacillus neutriphilus A1gT (EU709018) 89
●100
Oceanobacillus sojae Y27T (AB473561)
Oceanobacillus oncorhynchi subsp. incaldanensis 20AGT (AJ640134)
● 96
Oceanobacillus oncorhynchi subsp. oncorhynchi R-2T (AB188089)
● 100
Oceanobacillus chironomi LMG 23627T (DQ298074)
Oceanobacillus indicireducensi A21T (AB623011) Macrococcus hajekii CCM 4809T (AY119685) 0.01
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary Fig. IJS-D-15-00043R2.pdf
International Journal of Systematic and Evolutionary Microbiology Oceanobacillus arenosus sp. nov., a moderate halophilic bacterium isolated from marine sand --Manuscript Draft-Manuscript Number:
IJS-D-15-00043R2
Full Title:
Oceanobacillus arenosus sp. nov., a moderate halophilic bacterium isolated from marine sand
Short Title:
Oceanobacillus arenosus sp. nov.
Article Type:
Note
Section/Category:
New taxa - Firmicutes and related organisms
Corresponding Author:
Wonyong Kim Chung-Ang University College of Medicine Seoul, KOREA, REPUBLIC OF
First Author:
Wonyong Kim
Order of Authors:
Wonyong Kim Chatuphon Siamphan Jong-Hwa Kim Ampaitip Sukhoom
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A Gram-positive, spore-forming, rod-shaped, motile, strictly aerobic bacterium, designated CAU 1183T, was isolated from a marine sand and its taxonomic position was investigated using a polyphasic approach. The bacterium grew optimally at a temperature of 30C at pH 8.5 in the presence of 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAU 1183T formed a distinct lineage within the genus Oceanobacillus and exhibited the highest similarity to Oceanobacillus chungangensis (97.6%). The strain contained MK-7 as the predominant isoprenoid quinone and anteiso-C15:0 was the major cellular fatty acid. The cell wall peptidoglycan contains meso-diaminopimelic acid. The polar lipid pattern of strain CAU 1183T consisted of diphosphatidylglycerol, phosphatidylglycerol, and unidentified lipids including two phospholipids, two glycolipids, phosphoglycolipid, and two lipids. The G+C content of the genomic DNA was 37.5 mol%. On the basis of phenotypic, chemotaxonomic, and phylogenetic data, strain CAU 1183T should be classified as a novel species in the genus Oceanobacillus, for which the name Oceanobacillus arenosus sp. nov. is proposed. The type strain is CAU 1183T (=KCTC 33037T, =CECT 8560T).
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Manuscript_IJS-D-15-00043R2.doc
1
Oceanobacillus arenosus sp. nov., a moderate halophilic bacterium isolated from marine
2
sand
3 4
Wonyong Kim,1 Chatuphon Siamphan,1 Jong-Hwa Kim,1 Ampaitip Sukhoom2*
5 6
1
7
of Korea
8
2
9
90112, Thailand
Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Republic
Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla,
10 11
*Correspondence: Ampaitip Sukhoom, Tel 66-074-28-8008, E-mail: [email protected]
12 13
Subject category: New taxa (Firmicutes and Related Organisms)
14 15
Running title: Oceanobacillus arenosus sp. nov.
16
Keywords: Oceanobacillus arenosus, Firmicutes; Bacillaceae, Marine sand
17 18
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
19
CAU 1183T is JX233495.
20
1
21
Summary
22
A Gram-positive, spore-forming, rod-shaped, motile, strictly aerobic bacterium, designated
23
CAU 1183T, was isolated from a marine sand and its taxonomic position was investigated
24
using a polyphasic approach. The bacterium grew optimally at a temperature of 30C at pH
25
8.5 in the presence of 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene
26
sequences showed that strain CAU 1183T formed a distinct lineage within the genus
27
Oceanobacillus and exhibited the highest similarity to Oceanobacillus chungangensis
28
(97.6%). The strain contained MK-7 as the predominant isoprenoid quinone and anteiso-C15:0
29
was the major cellular fatty acid. The cell wall peptidoglycan contains meso-diaminopimelic
30
acid. The polar lipid pattern of strain CAU 1183T consisted of diphosphatidylglycerol,
31
phosphatidylglycerol, and unidentified lipids including two phospholipids, two glycolipids,
32
phosphoglycolipid, and two lipids. The G+C content of the genomic DNA was 37.5 mol%.
33
On the basis of phenotypic, chemotaxonomic, and phylogenetic data, strain CAU 1183T
34
should be classified as a novel species in the genus Oceanobacillus, for which the name
35
Oceanobacillus arenosus sp. nov. is proposed. The type strain is CAU 1183T (=KCTC
36
33037T, =CECT 8560T).
37 38
The genus Oceanobacillus, a member of the family Bacillaceae, was proposed by Lu et al.
39
(2001) with the description of O. iheyensis as the type species. The genus Oceanobacillus
40
comprises aerobic, Gram-stain-positive, motile, rod-shaped bacteria that are characterized
41
chemo-taxonomically by the presence of menaquinone-7 (MK-7) as the major isoprenoid
42
quinone and anteiso-C15:0 as the predominant cellular fatty acid (Namwong et al. 2009; Lee et
43
al. 2010; Whon et al. 2010). Currently, the genus comprises 17 recognized species with
44
validly published names: O. iheyensis (Lu et al. 2001), O. picturae (Heyrman et al. 2003), O.
45
oncorhynchi (Yumoto et al. 2005), O. chironomi (Raats and Halpern 2007), O. profundus
2
46
(Kim et al. 2007), O. caeni (Nam et al. 2008), O. kapialis (Namwong et al. 2009), O. sojae
47
(Tominaga et al. 2009), O. neutriphilus (Yang et al. 2010), O. locisalsi (Lee et al. 2010), O.
48
kimchii (Whon et al. 2010), O. chungangensis (Lee et al., 2013), O. polygoni (Hirota et al.,
49
2013), O. indicireducens (Hirota et al., 2013), O. pacificus (Yu et al., 2014), O. limi
50
(Amoozegar et al., 2014), and O. luteolus (Wu et al., 2014) (http://www.bacterio.net/).
51
Members of the genus Oceanobacillus have been isolated from various habitats such as
52
marine environments (deep-sea sediment, marine solar saltern, and salt lake), mural paintings,
53
freshwater fish, insect, activated sludge, food, soy sauce production equipment, and soil. In
54
the course of the screening of bacteria from marine environments, a bacterial strain,
55
designated CAU 1183T, was isolated from a sand sample collected in Jeju Island
56
(33º22’06.16”N, 126º33’11.11”E) in the Republic of Korea. The purpose of the present study
57
was to establish the taxonomic position of this bacterial strain by using a polyphasic approach
58
that included the determination of phenotypic, chemotaxonomic, and genotypic properties.
59 60
Strain CAU 1183T was isolated from a marine sand sample of Jeju Island according to
61
Gordon & Mihm (1962) using marine agar 2216 (MA; Difco Laboratories Detroit MI, USA).
62
The sand sample was crushed and diluted with sterilized saline solution so that appropriate
63
dilutions could be spread on MA plates. The plates were incubated under aerobic conditions
64
at 30˚C for 7 days. A pure single colony was purified by subculturing and preserved at -80˚C
65
in marine broth 2216 (MB; Difco Laboratories Detroit MI, USA) supplemented with 25%
66
(w/v) glycerol for taxonomic analysis. Strain CAU 1183T was deposited in the Korean
67
Collection for Type Cultures (KCTC; Taejon, Republic of Korea) and the Colección
68
Española de Cultivos Tipo (CECT; Paterna, Spain) as KCTC 33037T and CECT 8560T,
69
respectively. The type strains of four closely related species and type strain of the type
70
species of the genus, O. iheyensis HTE831T, were used as reference strains in most analyses.
3
71
O. chungangensis CAU 1051T, O. profundus CL-MP28T, and O. caeni S-11T were obtained
72
from the Korean Collection for Type Cultures (KCTC; Taejon, Korea).
73 74
Genomic DNA of strain CAU 1183T was extracted using the method of Marmur (1961). PCR
75
amplification of the 16S rRNA gene was performed following established procedures (Cho et
76
al., 2008). The amplified 16S rRNA gene was sequenced directly using a BigDye terminator
77
v3.1 cycle sequencing kit and an automatic 3730 DNA sequencer (Applied Biosystems Life
78
Technologies Co., Carlsbad, CA, USA). Multiple alignments and calculation of 16S rRNA
79
gene sequence similarity between strain CAU 1183T and a broad selection of the closely
80
related
81
(http://www.ezbiocloud.net/eztaxon), and CLUSTAL-X 2.1 program (Larkin et al., 2007).
82
Evolutionary distance matrices were created by the neighbor-joining method defined by
83
Jukes and Cantor (1969). Phylogenetic trees were generated using the neighbour-joining
84
(Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967), and maximum-likelihood
85
(Felsenstein, 1981) algorithms in the PHYLIP package (Felsenstein, 1989). Branch support in
86
the neighbour-joining tree was evaluated by the bootstrap resampling method based on 1,000
87
replicates (Felsenstein, 1985). The extent of DNA–DNA relatedness between CAU 1183T
88
and the most closely related phylogenetic neighbour, Oceanobacillus chungangensis CAU
89
1051T was determined using the fluorometric microplate method (Ezaki et al., 1989), as
90
modified by Goris et al. (1998). The mol% G+C content of the genomic DNA was examined
91
by HPLC, according to the method of Tamaoka and Komagata (1984).
species
were
carried
out
by
using
the
EzTaxon
-
EzBioCloud.net
92 93
Strain CAU 1183T was cultivated on MA at 30˚C for the investigation of morphological,
94
physiological and biochemical characteristics, except for spore formation that was assessed
95
on nutrient sporulation medium (Nicholson & Setlow, 1990). Cell morphology was examined
4
96
under a DM 1000 light microscope (Leica Microsystems AG, Wetzlar, Germany) using cells
97
from an exponentially growing culture. Gram reaction was performed using the bioMérieux
98
Gram staining kit (bioMérieux SA, Marcy l'Etoile, France). Motility was assessed on an MB
99
culture using the hanging-drop method (Bowman et al., 2000). After 5 days of growth, spore
100
formation was determined by staining with malachite green as described previously by Conn
101
et al. (1957). Growth on MA at 4, 10, 20, 30, 37, 45, and 55C was evaluated in an aerobic
102
incubator (model MIR-253; Sanyo Electric Co. Ltd, Wood Dale, IL, USA) and an anaerobic
103
chamber (model Bactron; Sheldon Manufacturing, Inc., Cornelius, OR, USA) by measuring
104
the turbidity of the broth after 72 hrs. The optimal growth pH was investigated by culturing at
105
30C in MB that was adjusted to pH 4.5–10.0 (at intervals of 0.5 pH unit) using sodium
106
acetate/acetic acid and Na2CO3 buffers, and values were confirmed after autoclaving. Growth
107
in the absence of NaCl and in the presence of 0–15.0% (w/v) NaCl was explored by culturing
108
in MB prepared according to the Difco formula except that NaCl was excluded and that
109
0.45% (w/v) MaCl2.6H2O and 0.06% (w/v) KCl was added.
110 111
Oxidase activity was assessed from the oxidation of 0.1% (w/v) tetramethyl-p-
112
phenylenediamine (Cappuccino & Sherman, 2002). Catalase activity was tested by bubble
113
production in 3% (v/v) H2O2 solution. Hydrolysis of casein, starch and urea were evaluated
114
according to the methods of Lányí (1987) and Smibert & Krieg (1994). Acid production from
115
carbohydrates, enzyme activity, and other physiological and biochemical features were tested
116
using the API 50CH and API 20E systems at 30˚C (bioMérieux). API 20E strips were read
117
after 24 hrs and API 50CH strips after 24 hrs and 48 hrs.
118 119
For fatty acid analysis, the cell mass of strain O. iheyensis HTE831T, O. chungangensis CAU
120
1051T, O. profundus CL-MP28T, and O. caeni S-11T was harvested from TSA (Difco) after
5
121
cultivation for 3 days at 30C. The physiological ages of the biomasses were standardized by
122
observing growth development during incubation of the three different cultures and choose
123
the moment of harvesting according to the standard MIDI method (Sherlock Microbial
124
Identification System version 6.1, Newark, DE, USA). Cellular fatty acid methyl esters
125
(FAMEs) were obtained according to the method of Minnikin et al. (1980) and separated in a
126
6890N gas chromatograph fitted with a 7683 autosampler (Agilent Technologies Inc., Santa
127
Clara, CA, USA). Peaks were identified using the Microbial Identification software package
128
(MOORE library ver. 5.0; MIDI database TSBA6). Peptidoglycan was analyzed as described
129
by Schleifer & Seidl (1985). The polar lipids of strain CAU 1183T were identified using two-
130
dimensional thin-layer chromatography (TLC) by the method of Minnikin et al. (1984). The
131
plate was sprayed with 10% ethanolic molybdatophosphoric acid (for the total lipids),
132
molybdenum blue (for phospholipids), ninhydrin (for aminolipids) and α-naphthol/sulphuric
133
acid reagent (for glycolipids). Menaquinones were determined on HPLC using an isocratic
134
solvent system [methanol/isopropyl ether (3:1, v/v)] and the solvent flow (1 ml/min) (Hiraishi
135
et al., 1984).
136 137
The nearly complete 16S rRNA gene sequence of strain CAU 1183T (1,547 bp) was
138
determined and compared to the reference sequences for other bacterial species in the public
139
database. Phylogenetic analysis indicated that the novel isolate fell into the genus
140
Oceanobacillus. The neighbour-joining tree is shown in Fig. 1. Pairwise analysis indicated
141
that the most closely related strains were O. chungangensis CAU 1051T (16S rRNA gene
142
similarity, 97.6%), O. profundus CL-MP28T (similarity, 96.5%), O. polygoni SA9T
143
(similarity, 96.3%), and O. caeni S-11T (similarity, 95.7%). The DNA-DNA relatedness
144
between CAU 1183T and the most closely related strain, O. chungangensis CAU 1051T was
145
44.2% when CAU 1183T was used as a probe and 43.6% when O. chungangensis CAU 1051T
6
146
was used as a probe. These value is well below the 70% cut-off point recommended by
147
Wayne et al. (1987) for the delineation of genomic species, supporting the proposal that strain
148
CAU 1183T represents a separate species. The DNA G+C content of the strain CAU1183T
149
was 37.5 mol%, which was in accordance with the previously reported values of the genus
150
Oceanobacillus; 33.6 mol% (O. caeni S-11T; Nam et al., 2008) – 40.6 mol% (O. polygoni
151
SA9T; Hirota et al., 2013).
152 153
The detailed phenotypic characteristics are presented in Table 1 and the new species
154
description. Strain CAU 1183T was Gram-stain-positive, central ovoid spore-forming (Suppl.
155
Fig. S1), strictly aerobic, and motile. Colonies on MA were cream colored, entire, circular,
156
and convex with diamsporeeters of 1.2–2.0 mm after 3 days of cultivation at 30˚C. No
157
colonies were formed under anaerobic growth condition. Cell are rod-shaped approximately
158
0.5–1.0 µm in diameter and 1.5–4.3 µm in length. Growth occurs at 20–37C (optimum,
159
30C) and at pH 5.5-9.0 (optimum, 8.5), and at 0–8% (w/v) NaCl (optimum, 2%). Phenotypic
160
characteristics data of strain CAU 1183T differed from its closest relatives, O. chungangensis
161
KCTC 33035T (Lee et al., 2013) and the members in the genus Oceanobacillus (Table 1).
162
The growth temperature of CAU 1183T could be distinguished from O. profundus (15-42C),
163
O. polygoni (5–48C), and O. iheyensis (15–42C). In addition, the pH range and NaCl
164
tolerance of strain CAU 1183T differed from O. polygoni (pH 7.0–12.0 / 3–12% NaCl), and O.
165
iheyensis (pH 6.5–10.0 / 0–21% NaCl). Strain CAU 1183T differed from its closest relatives,
166
O. profundus CL-MP28T (Kim et al., 2007), O. polygoni SA9T (Hirota et al., 2013) and O.
167
caeni S-11T (Nam et al., 2008), and the type strain O. iheyensis (Lu et al., 2002) by its acid
168
production from amidon and glycogen. In addition, strain CAU 1183T differed from O.
169
iheyensis HTE831T (Lu et al., 2002) by its acid production from D-trehalose. Overall, the
170
chemotaxonomic characteristic was in agreement with previously published data for the 7
171
genus Oceanobacillus. The predominant isoprenoid quinone detected in strain CAU 1183T
172
was menaquinone 7 (MK-7), in line with all members of the genus Oceanobacillus, which is
173
consistent with data for the type species of the genus, O. iheyensis HTE831T (Lu et al., 2001).
174
The strain contained saturated and branched-chain fatty acids (Table 2). The peptidoglycan of
175
strain CAU 1183T contained meso-diaminopimelic acid as the diagnostic cell-wall diamino
176
acid, which was present in the strains of recognized Oceanobacillus species. The major fatty
177
acids (>10%) were anteiso-C15:0 (50.4%), C16:0 (11.5%), anteiso-C17:0 (10.3%), iso-C15:0
178
(10.3%). The following fatty acids (>1%) C16:0 (8.5%), iso-C14:0 (5.0%), iso-C17:0 (2.4%), and
179
C14:0 (1.4%) were also present. This fatty acid profile was almost same as that of closest
180
relatives, although there were differences in the amounts and presence of some fatty acids.
181
Anteiso-C15:0 was major compound in strain CAU 1183T and the closely related other species
182
of the genus Oceanobacillus; O. chungangensis CAU 1051T, O. profundus KCTC 13625T
183
and O. caeni KCTC 13061T and O. polygoni SA9T, except O. iheyensis KCTC 3954T. Strain
184
1183T contained diphosphatidylglycerol and phosphatidylglycerol as the major polar lipids
185
identified as in O. iheyensis KCTC 3954T. In addition, cells of this novel isolate contained
186
unidentified lipids: two phospholipids, two glycolipids, one phosphoglycolipid, and two
187
lipids (Suppl. Fig. S2). Diphosphatidylglycerol and phosphatidylglycerol were also detected
188
as the major polar lipids in O. luteolus WM-1T, O. limi H9BT, O. chungangensis CAU 1051T,
189
O. sojae JCM 15792T, O. kapialis SSK2-2T. Phosphatidylethanolamine was detected in O.
190
pacificus XH204T, O. polygoni SA9T, O. indicireducens A21T, but this polar lipid was not
191
detected in strain CAU 1183T.
192 193
In conclusion, it is evident from the phenotypic, chemotaxonomic and genotypic data that
194
strain CAU 1183T represents a novel species of the genus Oceanobacillus, for which the
195
name Oceanobacillus arenosus sp. nov. is proposed.
8
196 197
Description of Oceanobacillus arenosus sp. nov.
198
Oceanobacillus sp. nov. (a.re.no'sus. L. masc. adj. arenosus sandy).
199 200
Gram-positive, obligate aerobic, motile rod with optimal growth that occurs at 30C (range of
201
20–37C), pH 8.5 (pH 5.5–9.01), and 2% (w/v) sea salt (0–8%). Forms central endospores.
202
Catalase and oxidase are positive. Esculin and gelatin are hydrolysed. Starch, urea and citrate
203
are negative. Acid is produced from amidon, amygdalin, D-fructose, D-galactose, D-glucose,
204
D-maltose, D-mannitol, D-melibiose, D-lactose, D-raffinose, D-sorbitol, D-trehalose, D-
205
xylose, glycogen and salicin. The major isoprenoid quinone is MK-7. The cell wall
206
peptidoglycan contains meso-diaminopimelic acid. The polar lipid pattern consists of
207
diphosphatidylglycerol,
208
unidentified glycolipids, one unidentified phosphoglycolipid and two unidentified lipids. The
209
major cellular fatty acid is anteiso-C15:0. The DNA G+C content is 37.5 mol%. The type
210
strain CAU 1183T (=KCTC 33037T, =CECT 8560T), isolated from a marine sand collected
211
from Jeju Island in the Republic of Korea.
phosphatidylglycerol,
two
unidentified
phospholipids,
two
212 213
Acknowledgment
214
This work was supported by the project on survey of indigenous species of Korea of the
215
National Institute of Biological Resources (NIBR) under the Ministry of Environment (MOE).
216 217
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16
370
Table 1. Phenotypic properties of strain CAU 1183T the type strains of the closely related
371
Oceanobacillus species. Strains: 1, CAU 1183T (data from this study); 2, O. chungangensis
372
CAU 1051T; 3, O. profundus CL-MP28T; 4, O. caeni S-11T; 5, O. polygoni SA9T; 6, O.
373
iheyensis HTE831T. All strain indicated sporangial morphology as ellipsoidal. +, positive; -,
374
negative; w, weakly positive Characteristic
1
2
3
4
5
Spore formation
Central
Terminal
Terminalb
Centralc
Centrald
Temperature range (˚C) Optimum
20–37 30
20–37a 30a
15–42b 35b
20–45c 35c
5–48d 35d
Terminal or subterminale 15–42e 30e
pH range Optimum
5.5–9.0 8.5
4.5–10.0a 5a
6.5–9.5b 8b
6.0–9.0c 7c
7.0–12.0d 9d
6.5–10.0e 8.3e
0–8 2
0–10a 0a
0–14b 2b
0–10c 3.5c
3–12d 3d
0–21e 3e
Hydrolysis of : Casein Gelatin Esculin
+ +
+
+ + +
-
+ +
+ + +
Acid production from : Amygdalin Glycerol D-Galactose
+ +
+ +
+ + +
+ + -
+ -
+ + -
D-Mannose D-Melezitose D-Melibiose D-Raffinose D-Ribose D-Sorbitol D-Trehalose L-Arabinose
+ + + + -
+ + + + +
+ + + + + -
+ + + + -
+ + + + -
+ + + +
L-Rhamnose DNA G+C content (mol%)
-
-
-
+
-
-
37.5
36.3
40.2
33.6
40.6
35.8
NaCl tolerance (%) Optimum
6
375
*Data taken from: a, Lee et al., 2013; b, Kim et al., 2007; c, Nam et al., 2008; d, Hirota et al.,
376
2013; e, Lu et al., 2002.
377
17
378
Table 2. Cellular fatty acid compositions (%) of strain CAU 1183T the type strains of the
379
most closely related Oceanobacillus species. Strains: 1, CAU 1183T; 2, O. chungangensis
380
CAU 1051T; 3, O. profundus CL-MP28T; 4, O. caeni S-11T; 5, O. polygoni SA9T (data from
381
Hirota et al., 2013); 6, O. iheyensis HTE831T; All data from this study except O. polygoni
382
SA9T uses data from Hirota et al. (2013). Only those fatty acids amounting to >1.0% in all
383
strains are shown, -, not detected, ND, data not shown Fatty acids
1
2
3
4
5
6
C14:0
1.41
1.5
-
-
2.0
4.1
C16:0
11.5
16.4
4.9
6.7
9.0
9.9
anteiso-C15:0
50.4
44.2
53.3
56.7
60.3
32.0
anteiso-C17:0
10.3
13.3
18.0
16.9
8.6
4.2
iso-C14:0
5.0
4.8
3.7
2.6
3.5
6.8
iso-C15:0
10.3
9.6
8.5
8.0
8.3
33.5
iso-C16:0
8.5
6.6
8.6
5.4
3.5
6.1
iso-C17:0
2.4
2.5
1.9
1.8
1.5
2.3
Saturated
Branched-chain
384 385
18
386
Legend figure
387 388
Fig. 1 Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene
389
sequences showing the relationships between strain CAU 1183T and the type strains of
390
Oceanobacillus species. Dots indicate that the corresponding nodes were also recovered in
391
the trees generated with the maximum-likelihood and least squares algorithms. The numbers
392
at the nodes indicate levels of bootstrap support based on a neighbour-joining analysis of
393
1,000 resampled datasets; only values > 70% are given. Bar, 0.01 substitutions per nucleotide
394
position. Macrococcus hajekii CCM 4809T (AY119685) is used as an outgroup organism.
19
Figure Click here to download Figure: Fig. 1_IJS-D-15-00043R2.ppt
Virgibacillus litoralis JSM 089168T (FJ425909) ●100 ●
83
100●
Virgibacillus salinus XH-22T (FM205010)
Virgibacillus carmonensis LMG 20964T (AJ316302) Virgibacillus halodenitrificans DSM 10037T (AY543169)
●
Oceanobacillus picturae LMG 19492T (AJ315060) ●
100 Oceanobacillus manasiensis YD3-56T (FJ386518) ● 97
●
Oceanobacillus kapialis SSK2-2T (AB366005)
Ornithinibacillus contaminans CCUG 53201T (FN597064) ● ●
Paucisalibacillus globulus B22T (AM114102) ● 79
Ornithinibacillus bavariensis WSBC 24001T (Y13066)
● ● 78
Ornithinibacillus californiensis MB-9T (AF326365) Ornithinibacillus scapharcae TW25T (AEWH01000025)
‘ Oceanobacillus massiliensis N'DiopT (HQ586877)’ Oceanobacillus polygoni SA9T (AB750685)
●100
Oceanobacillus profundus CL-MP28T (DQ386635)
Oceanobacillus chungangensisC AU1051T (JX233492)
●100
Oceanobacillus arenosus CAU 1183T (JX233495) Oceanobacillus caeni S-11T (AB275883)
Oceanobacillus limi H9BT (HQ433455) Oceanobacillus iheyensis HTE831T (BA000028)
●100
Oceanobacillus kimchii X50T (GU784860)
100
●
●98
●
Oceanobacillus neutriphilus A1gT (EU709018) 89
●100
Oceanobacillus sojae Y27T (AB473561)
Oceanobacillus oncorhynchi subsp. incaldanensis 20AGT (AJ640134)
● 96
Oceanobacillus oncorhynchi subsp. oncorhynchi R-2T (AB188089)
● 100
Oceanobacillus chironomi LMG 23627T (DQ298074)
Oceanobacillus indicireducensi A21T (AB623011) Macrococcus hajekii CCM 4809T (AY119685) 0.01
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary Fig. IJS-D-15-00043R2.pdf
