IJSEM Papers in Press. Published January 21, 2015 as doi:10.1099/ijs.0.000080
International Journal of Systematic and Evolutionary Microbiology Frigidibacter albus gen. nov., sp. nov., a novel genus of the family Rhodobacteraceae, isolated from lake water --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00100R2
Full Title:
Frigidibacter albus gen. nov., sp. nov., a novel genus of the family Rhodobacteraceae, isolated from lake water
Short Title:
Frigidibacter albus gen. nov., sp. nov.
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Aihua Li institute of microbiology,China academy of sciences Beijing, CHINA
First Author:
Aihua Li
Order of Authors:
Aihua Li Yu-Guang Zhou
Manuscript Region of Origin:
CHINA
Abstract:
Three Gram-staining-negative, strictly aerobic, non-pigmented, non-motile, rod-shaped bacterial strains SP32T, SR68 and SP95 were isolated from two water samples of a coldwater lake in Xinjiang province, China. Growth was observed at 4-25℃ and pH6.09.0, optimum growth occurred at 18-20℃ and at pH7.0-7.5. Phylogenetic analysis of 16S rRNA gene sequences revealed that these isolates belonged to the family Rhodobacteraceae, but formed a distinct evolutionary lineage from other validly published species of this family. Strain SP32T showed the highest 16S rRNA gene sequences similarity (96.7%) to Rhodobacter veldkampii ATCC35703T, and the similarities to genera Defluviimonas, Haematobacter and Pseudorhodobacter were 95.8-96.4%, 96.0-96.1% and 95.3-96.1%, respectively. The genomic DNA G+C content of strain SP32T was 67.6 mol%. The major fatty acids (>5%) were summed feature 8 (C18:1ω7c/C18:1ω6c) and C18:1ω7c 11-methyl. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, one unidentified glycolipid and one unidentified polar lipid were found to be main polar lipids. Ubiquinone-10 (Q-10) was the sole respiratory quinone. While, strain SP32T neither produced photosynthetic pigments nor contained the gene pufM, by which it differed from the phototrophic species of the family Rhodobacteraceae. Based on the distinct phenotypic, chemotaxonomic and phylogenetic properties, strain SP32T represent a novel species in a novel genus within the family Rhodobacteraceae, for which we proposed the name Frigidibacter albus gen. nov., sp. nov. The type strain of the type species is strain SP32T (=CGMCC 1.13995T = NBRC 109671T ).
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1
Frigidibacter albus gen. nov., sp. nov., a novel genus of the family
2
Rhodobacteraceae, isolated from lake water
3
Ai-Hua Li, Yu-Guang Zhou*
4
China General Microbiological Culture Collection Center and State Key Laboratory of Microbial
5
Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, P. R. China
6 7
* Corresponding author:
8
Yu-Guang Zhou
9
Tel: 86-10-6480-7355; Fax: 86-10-6480-7288
10
Email: [email protected]
11
Running title: Frigidibacter albus gen. nov., sp. nov.
12
Subject category: New Taxa; Subsection: Rhodobacteraceae
13
The GenBank accession numbers for 16S rRNA gene sequences of strains SP32T, SR68 and SP95
14
reported here are KF944301, KF944302, KF944303, respectively.
15 16
Summary:
17
Three Gram-staining-negative, strictly aerobic, non-pigmented, non-motile, rod-shaped bacterial
18
strains SP32T, SR68 and SP95 were isolated from two water samples of a coldwater lake in
19
Xinjiang province, China. Growth was observed at 4-25℃ and pH6.0-9.0, optimum growth
20
occurred at 18-20℃ and at pH7.0-7.5. Phylogenetic analysis of 16S rRNA gene sequences
21
revealed that these isolates belonged to the family Rhodobacteraceae, but formed a distinct
22
evolutionary lineage from other validly published species of this family. Strain SP32T showed the
23
highest 16S rRNA gene sequences similarity (96.7%) to Rhodobacter veldkampii ATCC35703T,
24
and the similarities to genera Defluviimonas, Haematobacter and Pseudorhodobacter were 1
25
95.8-96.4%, 96.0-96.1% and 95.3-96.1%, respectively. The genomic DNA G+C content of strain
26
SP32T was 67.6 mol%. The major fatty acids (>5%) were summed feature 8 (C18:1ω7c/C18:1ω6c)
27
and C18:1ω7c 11-methyl. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol,
28
phosphatidylcholine, one unidentified glycolipid and one unidentified polar lipid were found to be
29
main polar lipids. Ubiquinone-10 (Q-10) was the sole respiratory quinone. While, strain SP32T
30
neither produced photosynthetic pigments nor contained the gene pufM, by which it differed from
31
the phototrophic species of the family Rhodobacteraceae. Based on the distinct phenotypic,
32
chemotaxonomic and phylogenetic properties, strain SP32T represent a novel species in a novel
33
genus within the family Rhodobacteraceae, for which we proposed the name Frigidibacter albus
34
gen. nov., sp. nov. The type strain of the type species is strain SP32T (=CGMCC 1.13995T =
35
NBRC 109671T ).
36 37
The family Rhodobacteraceae (Garrity et al., 2005), which is a member of the class
38
Alphaproteobacteria, embraces approximately 104 valid genera at the time of writing. Members
39
of the family Rhodobacteraceae are physiologically and metabolically heterogenous, including
40
both phototrophic and non-phototrophic bacteria. In recent years, many novel species isolated
41
from various habitats were taxonomically proved to belong to the family Rhodobacteraceae, such
42
as Youngimonas (Hameed et al., 2014), Cribrihabitans (Chen et al., 2014), Planktomarina (Giebel
43
et al., 2013), Pelagimonas (Hahnke et al., 2013), Profundibacterium (Lai et al., 2013), Sagittula
44
(Lee et al., 2013), Epibacterium (Penesyan et al., 2013) and Falsirhodobacter (Subhash et al.,
45
2013). In this communication, we report a novel non-phototrophic species of a new genus
46
associated with the family Rhodobacteraceae, which was isolated from a coldwater lake.
47
Five samples (NO.1-5) of lake water were collected from Sayram Lake located in Xingjiang
48
province in west of China, at an altitude of 2071.9 m (44°61’N, 81°17’E). The annual mean water
49
temperature is 5℃. Bacterial strains were isolated by standard dilution plating technique and
50
incubated on PYG (5.0g bacto peptone, 0.2g yeast extract, 5.0g glucose, 3.0g beef extract, 0.5g
51
NaCl, 1.5g MgSO4·7H2O, 1000 ml sterile water, 15g agar, pH7.0) and R2A agar at 8℃ for one
52
month. All the single colonies were transferred and purified, and the purified strains were
53
preserved by lyophilization. Strains SP32T and SP95 were isolated from sample NO.1 on PYG 2
54
agar, and strain SR68 was isolated from sample NO.3 on R2A agar. These three strains were
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selected for further taxonomic research, as they might be novel strains according to the 16S rRNA
56
gene sequence analysis.
57
During the polyphasic investigation, strains were cultivated on PYG agar or broth for most of the
58
experiments. Six strains were used as reference strains in this study: Rhodobacter veldkampii
59
CGMCC 1.5006T and Rhodobacter capsulatus CGMCC 1.8920T were obtained from China
60
General Microbiological Culture Collection Center (CGMCC), Defluviimonas denitrificans DSM
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18921T from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Defluviimonas
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aestuarii JCM 18630T and Pseudorhodobacter ferrugineus JCM 20687T from Japan Collection of
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Microorgnism (JCM) and Pseudorhodobacter wandonensis KCTC 23672T were from Korean
64
Collection for Type Cultures (KCTC).
65
Phenotypic characteristics of these isolates were tested as following. Cell morphology and flagella
66
were observed using light microscope and transmission electron microscopy (JEM1400; JEOL;
67
Fig. S1). Motility was determined by the hanging-drop technique (Skerman, 1967). Gram staining
68
was performed according to the procedure of Collins et al. (1989). Growth of strains was tested on
69
tryptic soy agar, nutrient agar and marine agar 2216 (MA; BD Difco). For assessment of pH range
70
for growth, strains were cultured in PYG broth adjusted to pH4.0-10.0 using 100 mM acetate
71
buffer (for pH4.0-5.0), 100 mM phosphate buffer (pH6.0-8.0) or 100mM NaHCO3/NaCO3 buffer
72
(pH9.0-10.0) (Breznak & Costilow, 2007; Yumoto et al., 2004), respectively, and medium were
73
sterilized by filtration. The requirement and tolerance of NaCl were tested on PYG broth
74
supplemented with various NaCl concentrations (from 0-5.0%, at 0.5% intervals). Growth at 4, 8,
75
15, 20, 30, 35, 37 ℃ was observed on PYG slant after 15-days incubation. Photoautotrophic and
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photoheterotrophic growth under anaerobic condition was tested in screw-capped tubes and in agar
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deeps at 2400lx, using the DSMZ medium 27 and PYG medium, respectively. For analysis of
78
photosynthetic pigments, cells were suspended in 60% sucrose solution and the in vivo absorption
79
spectrum was measured by using a Lambda 35 UV/Vis spectrometer (Trüper & Pfennig, 1981).
80
Catalase and oxidase activity were determined by using 3% (v/v) H2O2 and Bactident Oxidase
81
strips (Merck), respectively. Hydrolysis of Tween20, Tween60, Tween80, starch, esculin,
82
CM-cellulose, casein and gelatin were assessed according to Smibert & Krieg (1994). DNase assay 3
83
was carried out on DNase test agar (Merck). Other enzyme activities were determined using the
84
commercial system API 20E, API 20NE and API ZYM (bioMérieux) following the manufacturer’s
85
instructions. Acid production from carbohydrates was tested in API 50CH strip (bioMérieux), and
86
oxidation of substrates was evaluated using the GN3 MicroPlate system (Biolog). Sensitivity to
87
antibiotics were tested on PYG plates by using antibiotic discs (Beijing Tiantan Biological
88
Products) containing ampicillin (10μg), amikacin (30μg), azithromycin (15μg), cefaclor (30μg),
89
cefazolin (30μg), cefoperazone (75μg), cefotaxine (30μg), ceftazidime (30μg), ceftriazone (30μg),
90
cefuroxime sodium (30μg), cephalotin (30μg), chloramphenicol (30μg), ciprofloxacin (5μg),
91
clarithromycin (15μg), clindamycin (2μg), doxycycline (30μg), erythromycin (15μg), fleroxacin
92
(5μg), lomefloxacin (10μg), minocycline (30μg), netilmicin (30μg), nitrofurantoin (300μg),
93
oxacillin (1μg), penicillin G (10 IU), piperacillin (100μg), rifampicin (5μg), sulfamethoxazole
94
(1.25μg), tetracycline (30μg), tobramycin (10μg) and vancomycin (30μg). The morphological,
95
biochemical and physiological properties of strain SP32T are given in Table 1 and in the species
96
description.
97
For fatty acid profile analysis, new isolates and reference strains were incubated on TSA at 18℃
98
for 3 days, and cell masses were harvested during the same exponential growth phase and were
99
subjected to saponification, methylation and extraction as described previously (Kämpfer &
100
Kroppenstedt, 1996) and subsequently analyzed by gas chromatograph (Agilent 6890). Peaks
101
were determined using MIDI (version 6) by adopting the TSBA6.0 database (Sasser, 1990).
102
Respiratory quinones of these three isolates and R. veldkampii CGMCC 1.5006T were extracted
103
according to Collins (1985) and analyzed by HPLC (Wu et al., 1989). Polar lipids were extracted
104
by the procedure of Minnikin et al. (1984) and determined by two-dimensional TLC. The
105
separated spots were confirmed by spraying 5% ethanolic molybdophosphoric acid, molybdenum
106
blue, ninhydrin and α-naphthol for total polar lipids, phospholipids, aminolipids and glycolipids,
107
respectively. The polar lipid features of R. veldkampii CGMCC 1.5006T, D. denitrificans DSM
108
18921T and P. wandonensis KCTC 23672T were used as references.
109
Genomic DNA of these strains was extracted by using a commercial Genomic DNA Rapid
110
Isolation Kit for Bacterial Cell (BioDev-Tech, China) following the instruction. Gene pufM was
111
amplified by using the primer 2F (5’-CAGATCGGGCCGATCTA-3’) and 4R (5’-CCAGACGTA 4
112
CCAGTTGTC-3’) (Uchino et al., 2002). PCR products of pufM were detected by electrophoresis.
113
16S rRNA gene sequence were amplified by using universal primer 27F and 1492R (Lane, 1991).
114
The PCR products of 16S rRNA gene were cloned using the pGEM-T Easy vector and sequenced.
115
The nearly full-length 16S rRNA gene sequence (1428bp) was obtained, and aligned with
116
available published sequences on the GenBank and EzTaxon-e server (Kim et al., 2012).
117
According to the analysis of 16S rRNA gene sequences, strains SP32T, SR68 and SP95 shared 100%
118
similarity with each other, and the 16S similarities with the phylogenetic neighbors were as
119
following: R. veldkampii ATCC 35703T (96.6%), D. aestuarii BS14T (96.3%), H. missouriensis
120
CCUG 52307T (96.3%), H. massiliensis CCUG 47968T (96.2%) and P. wandonensis WT-MW11T
121
(95.9%), respectively. 16S rRNA gene sequences of the most closely related taxa were retrieved
122
and aligned with BioEdit (Hall, 1999). Phylogenetic tree were constructed with Mega5.0 by using
123
neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and
124
maximum-parsimony (Fitch, 1971) methods. Bootstrap values were calculated based on 1000
125
replications in order to evaluate confidence levels of the nodes. The neighbor-joining phylogenetic
126
analysis of 16S rRNA gene sequences revealed that these three isolates constituted a distinct
127
phylogenetic lineage within the family Rhodobacteraceae, and clustered with the genera
128
Rhodobacter, Defluviimonas, Pseudorhodobacter and Heamobacter (Figure 1). The topologies of
129
maximum-likelihood tree and maximum-parsimony tree (Fig. S2) also supported the notion that
130
these isolates represented a novel genus which was distinct from their most closely related genera
131
of the family Rhodobacteraceae.
132
The DNA G+C contents were assessed by HPLC (Mesbah et al., 1989), and DNA of Lambda
133
phage (Sigma) (49.8 mol %) and R. veldkampii CGMCC1.5006T were used as reference.
134
As list in Table S1, the predominant fatty acids of strain SP32T was mainly summed feature 8
135
(C18:1ω7c/ C18:1ω6c; 82.1%), which was identical with other genera of the family
136
Rhodobacteraceae. Strain SP32T distinguished from its neighbor genera Rhodobacter and
137
Pseudorhodobacter by containing C18:1ω7c 11-methyl (5%).
138
The major polar lipids of strain SP32T were phosphatidylglycerol (PG), phosphatidylethanolamine
139
(PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), one unidentified glycolipid (GL) 5
140
and one unidentified polar lipid (L4). Besides, there are 6 unidentified polar lipids (L1, 2, 3, 4, 5,
141
6, 7) present in strain SP32T with trace amount (Fig. S3). As the polar lipids profiles of strains
142
SR68 and SP95 was identical with strain SP32T, results of strains SR68 and SP95 were not shown.
143
The presence of one unidentified glycolipid (GL) and several unidentified polar lipids were the
144
key feature of strains SP32T, SP95 and SR68 that distinguished them from their closest
145
phylogenetic neighbors, R. veldkampii CGMCC 1.5006T, D. denitrificans DSM 18921T and P.
146
ferrugineus JCM 20687T.
147
Ubiquinone-10 was the sole respiratory quinone, which in line with members of the family
148
Rhodobacteraceae. The genomic DNA G+C content of strains SP32T, SR68 and SP95 were 67.6,
149
67.9, 67.8 mol %, respectively.
150
According to the phenotypic and physiological characteristics comparison list in Table 1, strain
151
SP32T differed from the genus Rhodobacter by it phenotypic characteristics and cultivation
152
conditions. Photoautotrophic and photoheterotrophic growth under anaerobic condition were not
153
observed for strain SP32T. No absorption maxima at 377, 590, 803 and 860 nm were detected,
154
which confirmed that they did not contain any photosynthetic pigments. In addition, strain SP32T
155
did not contain the photosynthetic gene pufM (Fig. S4). Besides, strain SP32T distinguished from
156
genera Defluviimonas, Pseudorhodobacter and Haematobacter by its polar lipids features and the
157
fatty acids profiles. Therefore, on the basis of these distinctive phenotypic, physiological,
158
biochemical and phylogenetic characteristics, strain SP32T represents a novel species in a novel
159
genus within the family Rhodobacteraceae, which is proposed as Frigidibacter albus gen. nov., sp.
160
nov. As the genomic, physiological and chemical taxonomic characteristics of strains SP95 and
161
SR68 are identical with strain SP32T, they are also proposed as strains of Frigidibacter albus gen.
162
nov., sp. nov.
163
Description of Frigidibacter gen. nov
164
Frigidibacter (Fri.gi.di.bac'ter. L. adj. frigidus cold; N.L. masc. n. bacter, a rod; N.L. masc. n.
165
Frigidibacter, a rod from a cold environment)
166
Cells are Gram-staining negative, short rods, and non-spore forming. Positive for catalase and
167
oxidase. Photoautotrophic and photoheterotrophic growth are not present. Voges-proskauer test 6
168
and urease are positive. Q-10 is the sole respiratory quinone. The predominant polar lipids are
169
phosphatidyglycerol, phosphatidylethanolamine, phosphatidylcholine, one unidentified glycolipid
170
and one unidentified polar lipid. The main fatty acid are summed feature 8 (C18:1ω7c/ C18:1ω6c)
171
and C18:1ω7c 11-methyl. The G+C content of genomic DNA is 67-68 mol%. The type species is
172
Frigidibacter albus SP32T.
173
Description of Frigidibacter albus sp. nov.
174
Frigidibacter albus (al'bus. L. masc. adj. albus white).
175
Cells are Gram-staining-negative, non-pigmented, non-motile, strictly aerobic, short-rods with
176
0.6-0.8μm in width and 1.0-2.2μm in length. Colonies are creamy, convex, round, opaque with
177
smooth edges and 2-4mm in diameter after 4-days incubation on PYG agar at 20℃. Growth
178
occurs at 4-25℃ and pH6.0-9.0 (with optimum at 18-20℃ and pH7.0-7.5). The range of NaCl
179
tolerance is 0-4% (with optimum 0-1.5%). Grow on R2A, NA, but not on Marine 2216 agar.
180
Photoautotrophic and photoheterotrophic growth under anaerobic condition do not occur for strain
181
SP32T. Photosynthetic pigments and the photosynthetic gene pufM are absent. Urease-, Catalase-
182
and oxidase- positive. H2S and indole production are negative. The Voges-Proskauer test is
183
positive. Do not reduce nitrate to nitrite. Negative for DNase activity. Esculin is hydrolyzed, but
184
gelatin, starch, casein, CM-cellulose, Tween20, Tween60 and Tween80 are not. In the test of API
185
20E, API 20NE and API ZYM, strain SP32T are positive for alkaline phosphatase esterase (C4),
186
esterase
187
naphthol-AS-BI-phosphohydrolase and α-glucosidase, weakly positive for cystine arylamidase,
188
α-chymotrypsin, acid phosphatase, β-glucosidase and N-acetyl-β-glucosaminidase; but negative
189
for arginine dihydrolase, β-galactosidase, lysine decarboxylase, ornithine decarboxylase,
190
tryptophane
191
β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. In API 50CH
192
detection, acid is produced from L-arabinose, D-arabitol, D-cellobiose, esculin, D-fucose,
193
D-lactose, maltose, D-turanose, D-xylose; weakly from L-arabitol, D-fructose, gluconate,
194
5-ketogluconate, and salicin; but not from N-acetyl-glucosamine, D-adonitol, amygdalin,
195
D-arabinose, arbutin, dulcitol, erythritol, L-fucose, D-galactose, D-gentiobiose, D-glucose,
196
glycerol, glycogen, glycol, inositol, 2-ketogluconate, D-mannose, D-melezitose, melibiose,
lipase
(C8),
deaminase,
lipase
arginine
(C14),
leucine
dihydrolase,
7
arylamidase,
trypsin,
valine
α-galactosidase,
arylamidase,
β-galactosidase,
197
methyl-α-D-glucopyranoside,
methyl-α-D-mannopyranoside,
methyl-β-D-xylopyranoside,
198
D-raffinose, L-rhamnose, D-ribose, sorbitol, L-sorbose, starch, sucrose, synanthrin, D-tagatose,
199
D-turanose, trehalose, xylitol and L-xylose. In the GN3 MicroPlate, the following substrates are
200
oxidized by strain SP32T: D-maltose, D-trehalose, D-cellobiose, sucrose, α-D-glucose, D-frucose,
201
D-galactose, D-sorbitol, D-arabitol, D-mannitol, glycerol, D-aspartic acid, glycyl-L-proline,
202
L-alanin, L-aspartic acid, L-glutamic acid, L-serine, pectin, D-gluconic acid, mucic acid, quinic
203
acid, D-saccharic acid, p-hydroxy-phenylacetic acid, methyl pyruvate, L-lactic acid, citric acid,
204
α-keto-glutaric acid, D-malic acid, L-malic acid, bromo-succinic acid, γ-amino-butrytic acid, α-
205
hydroxy-butyric acid, β-hydroxyl-D, L-butyric acid, α-keto-butyric acid, acetoacetic acid,
206
propionic acid, acetic acid, turanose (weak), formic acid (weak); the other substrates in the GN3
207
MicroPlate are not oxidized. Strain SP32T is resistant to amikacin (30μg), clindamycin (2μg);
208
weakly resistant to clarithromycin (15μg), erythromycin (15μg), nitrofurantoin (300μg), oxacillin
209
(1μg); but sensitive to amikacin (30μg), ampicillin (10μg), azithromycin (15μg), cefaclor (30μg),
210
cefazolin (30μg), cefoperazone (75μg), cefotaxine (30μg), ceftazidime (30μg), ceftriazone (30μg),
211
cefurosimc sodium (30μg), cephalotin (30μg), chloramphenicol (30μg), ciprofloxacin (5μg),
212
doxycycline (30μg), fleroxacin (5μg), lomefloxacin (10μg), minocycline (30μg), netilmicin
213
(30μg), penicillin G (10 IU), piperacillin (100μg), rifampin (5μg), tetracycline (30μg), tobramycin
214
(10μg) and vancomycin (30μg). The major fatty acids are summed feature 8 (C18:1ω7c/ C18:1ω6c;
215
82.1%) and C18:1ω7c 11-methyl (5%). Q-10 is the sole ubiquinone. The predominant polar lipids
216
are phosphatidyglycerol, phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol,
217
one unidentified glycolipid and one unidentified polar lipid. The DNA G+C content of strain
218
SP32T is 67.6 mol %.
219
The type strain SP32T (=CGMCC 1.13995T =NBRC 109671T) was isolated from water sample of a
220
coldwater lake in the west of China.
221 222
ACKNOWLEDGEMENTS
223
We appreciate JCM, DSMZ and KCTC for supporting reference strains for this study. We thank
224
Dr. Jing-Nan Liang for TEM observing. This work was supported by the National Science and
225
Technology Foundation Project (2012FY111600). 8
226
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Lai, P. Y., Miao, L., Lee, O. O., Liu, L. L., Zhou, X. J., Xu, Y., Al-Suwailem, A. & Qian, P. Y.
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(2013). Profundibacterium mesophilum gen. nov., sp. nov., a novel member in the family
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Rhodobacteraceae isolated from deep-sea sediment in the Red Sea, Saudi Arabia. Int J Syst Evol
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Microbiol 63, 1007-1012.
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Systematics, pp. 115-175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
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Lee, D. H., Cho, S. J., Kim, S. M. & Lee, S. B. (2013). Sagittula marina sp. nov., isolated from
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seawater and emended description of the genus Sagittula. Int J Syst Evol Microbiol 63, 2101-2107.
290
Lee, M.H., Lee, S.Y., Jung, Y.T., Park S. & Yoon J.H. (2013) Pseudorhodobacter wandonensis
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sp. nov., isolated from wood falls, and emended description of the genus Pseudorhodobacter. Int J
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Syst Evol Microbiol 63, 1479–1484.
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Math, R. K., Jin, H. M., Jeong, S. H. & Jeon, C. O. (2013). Defluviimonas aestuarii sp. nov., a
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marine bacterium isolated from a tidal flat, and emended description of the genus Defluviimonas
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Foesel et al. 2011.Int J Syst Evol Microbiol 63, 2895–2900.
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C
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Schmid, M., Tindall, B. J., Klenk, H.-P. & Camacho, M. (2013). Chryseobacterium hispalense
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sp. nov., a plant-growth-promoting bacterium isolated from a rainwater pond in an olive plant
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nursery, and emended descriptions of Chryseobacterium defluvii, Chryseobacterium indologenes,
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Chryseobacterium wanjuense and Chryseobacterium gregarium. Int J Syst Evol Microbiol 63,
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4386–4395.
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Penesyan, A., Breider, S., Schumann, P., Tindall, B. J., Egan, S. & Brinkhoff, T. (2013).
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Epibacterium ulvae gen. nov., sp. nov., epibiotic bacteria isolated from the surface of a marine alga.
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Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing
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phylogenetic trees. Mol Biol Evol 4, 406–425.
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Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI
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Technical Note 101. Newark, DE: MIDI Inc.
315
Shalem Raj, P., Ramaprasad, E.V.V., Vaseef, S., Sasikala, Ch. & Ramana, Ch.V.
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(2013). Rhodobacter viridis sp. nov., a phototrophic bacterium isolated from mud of a stream. Int J
317
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318
Skerman, V. B. D. (1967). A Guide to the Identification of the Genera of Bacteria, 2nd edn.
319
Baltimore: Williams & Wilkins.
320
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and
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Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R.
322
Krieg. Washington, DC: American Society for Microbiology.
323
Subhash, Y., Tushar, L., Sasikala, Ch. & Ramana, Ch. V. (2013). Falsirhodobacter halotolerans
324
gen. nov., sp. nov., isolated from dry soils of a solar saltern. Int J Syst Evol Microbiol 63,
325
2132-2137.
326
Trüper, H. G. & Pfennig, N. (1981). Isolation of members of the families Chromatiaceae and
327
Chlorobiaceae. In The Prokaryotes: a Handbook on Habitats, Isolation, and Identification of
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Bacteria, pp. 279–289. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. G. Schlegel.
329
Berlin: Springer.
330
Uchino, Y., Hamada, T. & Yokota, A. (2002). Proposal of Pseudorhodobacter ferrugineus gen.
331
nov., comb. nov., for a non-photosynthetic marine bacterium, Agrobacterium ferrugineum, related
332
to the genus Rhodobacter. J Gen Appl Microbiol, 48, 309–319.
333
Wang, D., Liu, H.-L., Zheng, S.-X. & Wang G.-J. (2014). Paenirhodobacter enshiensis gen. nov.,
334
sp. nov., a non-photosynthetic bacterium isolated from soil, and emended descriptions of the
335
genera Rhodobacter and Haematobacter. Int J Syst Evol Microbiol 64, 551–558.
336
Wu, C., Lu, X., Qin, M., Wang, Y. & Ruan, J. (1989). Analysis of menaquinone compound in
337
microbial cells by HPLC. Microbiology [English translation of Microbiology (Beijing)] 16,
338
176–178.
339
Yumoto, I., Hirota, K., Yamaga, S., Nodasaka, Y., Kawasaki, T., Matsuyama, H. & Nakajima,
340
K. (2004). Bacillus asahii sp. nov., a novel bacterium isolated from soil with the ability to 12
341
deodorize the bad smell generated from short-chain fatty acids. Int J Syst Evol Microbiol 54,
342
1997–2001.
343
344
Figure Legend
345
Fig.1. Neighbour-joining tree based on 16S rRNA gene sequences, showing the phylogenetic
346
relationship of strains SP32T, SR68 and SP95 and related genera of the family Rhodobacteraceae.
347
Rhodospirillum rubrum ATCC11170T was used as an outgroup. Only bootstrap values (percentages
348
of 1000 replications) of >50% are shown at branching points. Filled circles indicate nodes also
349
recovered in the trees drew by maximum-likehood and maximum-parsimony methods, and open
350
circles indicate nodes recovered by maximum-likehood tree. Bar, 0.01 substitutions per nucleotide
351
position.
352
13
Differential characteristics of strains SP32T, SR68, SP95 and type strains of the
353
Table1.
354
closely related genera of the family Rhodobacteraceae.
355
Strains: 1: SP32T; 2: strain SR68; 3: strain SP95; 4: Rhodobacter veldkampii CGMCC1.5006T; 5:
356
Rhodobacter capsulatus CGMCC1.8920T; 6: Defluviimonas aestuarii JCM 18630T; 7:
357
Defluviimonas denitrificans DSM18921T; 8: Pseudorhodobacter ferrugineus NBRC 20687T; 9:
358
Pseudorhodobacter wandonensis KCTC 23672T; 10: Haematobacter massiliensis CCUG 47968T;
359
11: Haematobacter missouriensis CCUG 52307T. Data are from this study unless otherwise
360
indicated. +, positive; -, negative; w, weak growth; ND, not done; tr, trace (
International Journal of Systematic and Evolutionary Microbiology Frigidibacter albus gen. nov., sp. nov., a novel genus of the family Rhodobacteraceae, isolated from lake water --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00100R2
Full Title:
Frigidibacter albus gen. nov., sp. nov., a novel genus of the family Rhodobacteraceae, isolated from lake water
Short Title:
Frigidibacter albus gen. nov., sp. nov.
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Aihua Li institute of microbiology,China academy of sciences Beijing, CHINA
First Author:
Aihua Li
Order of Authors:
Aihua Li Yu-Guang Zhou
Manuscript Region of Origin:
CHINA
Abstract:
Three Gram-staining-negative, strictly aerobic, non-pigmented, non-motile, rod-shaped bacterial strains SP32T, SR68 and SP95 were isolated from two water samples of a coldwater lake in Xinjiang province, China. Growth was observed at 4-25℃ and pH6.09.0, optimum growth occurred at 18-20℃ and at pH7.0-7.5. Phylogenetic analysis of 16S rRNA gene sequences revealed that these isolates belonged to the family Rhodobacteraceae, but formed a distinct evolutionary lineage from other validly published species of this family. Strain SP32T showed the highest 16S rRNA gene sequences similarity (96.7%) to Rhodobacter veldkampii ATCC35703T, and the similarities to genera Defluviimonas, Haematobacter and Pseudorhodobacter were 95.8-96.4%, 96.0-96.1% and 95.3-96.1%, respectively. The genomic DNA G+C content of strain SP32T was 67.6 mol%. The major fatty acids (>5%) were summed feature 8 (C18:1ω7c/C18:1ω6c) and C18:1ω7c 11-methyl. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, one unidentified glycolipid and one unidentified polar lipid were found to be main polar lipids. Ubiquinone-10 (Q-10) was the sole respiratory quinone. While, strain SP32T neither produced photosynthetic pigments nor contained the gene pufM, by which it differed from the phototrophic species of the family Rhodobacteraceae. Based on the distinct phenotypic, chemotaxonomic and phylogenetic properties, strain SP32T represent a novel species in a novel genus within the family Rhodobacteraceae, for which we proposed the name Frigidibacter albus gen. nov., sp. nov. The type strain of the type species is strain SP32T (=CGMCC 1.13995T = NBRC 109671T ).
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1
Frigidibacter albus gen. nov., sp. nov., a novel genus of the family
2
Rhodobacteraceae, isolated from lake water
3
Ai-Hua Li, Yu-Guang Zhou*
4
China General Microbiological Culture Collection Center and State Key Laboratory of Microbial
5
Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, P. R. China
6 7
* Corresponding author:
8
Yu-Guang Zhou
9
Tel: 86-10-6480-7355; Fax: 86-10-6480-7288
10
Email: [email protected]
11
Running title: Frigidibacter albus gen. nov., sp. nov.
12
Subject category: New Taxa; Subsection: Rhodobacteraceae
13
The GenBank accession numbers for 16S rRNA gene sequences of strains SP32T, SR68 and SP95
14
reported here are KF944301, KF944302, KF944303, respectively.
15 16
Summary:
17
Three Gram-staining-negative, strictly aerobic, non-pigmented, non-motile, rod-shaped bacterial
18
strains SP32T, SR68 and SP95 were isolated from two water samples of a coldwater lake in
19
Xinjiang province, China. Growth was observed at 4-25℃ and pH6.0-9.0, optimum growth
20
occurred at 18-20℃ and at pH7.0-7.5. Phylogenetic analysis of 16S rRNA gene sequences
21
revealed that these isolates belonged to the family Rhodobacteraceae, but formed a distinct
22
evolutionary lineage from other validly published species of this family. Strain SP32T showed the
23
highest 16S rRNA gene sequences similarity (96.7%) to Rhodobacter veldkampii ATCC35703T,
24
and the similarities to genera Defluviimonas, Haematobacter and Pseudorhodobacter were 1
25
95.8-96.4%, 96.0-96.1% and 95.3-96.1%, respectively. The genomic DNA G+C content of strain
26
SP32T was 67.6 mol%. The major fatty acids (>5%) were summed feature 8 (C18:1ω7c/C18:1ω6c)
27
and C18:1ω7c 11-methyl. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol,
28
phosphatidylcholine, one unidentified glycolipid and one unidentified polar lipid were found to be
29
main polar lipids. Ubiquinone-10 (Q-10) was the sole respiratory quinone. While, strain SP32T
30
neither produced photosynthetic pigments nor contained the gene pufM, by which it differed from
31
the phototrophic species of the family Rhodobacteraceae. Based on the distinct phenotypic,
32
chemotaxonomic and phylogenetic properties, strain SP32T represent a novel species in a novel
33
genus within the family Rhodobacteraceae, for which we proposed the name Frigidibacter albus
34
gen. nov., sp. nov. The type strain of the type species is strain SP32T (=CGMCC 1.13995T =
35
NBRC 109671T ).
36 37
The family Rhodobacteraceae (Garrity et al., 2005), which is a member of the class
38
Alphaproteobacteria, embraces approximately 104 valid genera at the time of writing. Members
39
of the family Rhodobacteraceae are physiologically and metabolically heterogenous, including
40
both phototrophic and non-phototrophic bacteria. In recent years, many novel species isolated
41
from various habitats were taxonomically proved to belong to the family Rhodobacteraceae, such
42
as Youngimonas (Hameed et al., 2014), Cribrihabitans (Chen et al., 2014), Planktomarina (Giebel
43
et al., 2013), Pelagimonas (Hahnke et al., 2013), Profundibacterium (Lai et al., 2013), Sagittula
44
(Lee et al., 2013), Epibacterium (Penesyan et al., 2013) and Falsirhodobacter (Subhash et al.,
45
2013). In this communication, we report a novel non-phototrophic species of a new genus
46
associated with the family Rhodobacteraceae, which was isolated from a coldwater lake.
47
Five samples (NO.1-5) of lake water were collected from Sayram Lake located in Xingjiang
48
province in west of China, at an altitude of 2071.9 m (44°61’N, 81°17’E). The annual mean water
49
temperature is 5℃. Bacterial strains were isolated by standard dilution plating technique and
50
incubated on PYG (5.0g bacto peptone, 0.2g yeast extract, 5.0g glucose, 3.0g beef extract, 0.5g
51
NaCl, 1.5g MgSO4·7H2O, 1000 ml sterile water, 15g agar, pH7.0) and R2A agar at 8℃ for one
52
month. All the single colonies were transferred and purified, and the purified strains were
53
preserved by lyophilization. Strains SP32T and SP95 were isolated from sample NO.1 on PYG 2
54
agar, and strain SR68 was isolated from sample NO.3 on R2A agar. These three strains were
55
selected for further taxonomic research, as they might be novel strains according to the 16S rRNA
56
gene sequence analysis.
57
During the polyphasic investigation, strains were cultivated on PYG agar or broth for most of the
58
experiments. Six strains were used as reference strains in this study: Rhodobacter veldkampii
59
CGMCC 1.5006T and Rhodobacter capsulatus CGMCC 1.8920T were obtained from China
60
General Microbiological Culture Collection Center (CGMCC), Defluviimonas denitrificans DSM
61
18921T from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Defluviimonas
62
aestuarii JCM 18630T and Pseudorhodobacter ferrugineus JCM 20687T from Japan Collection of
63
Microorgnism (JCM) and Pseudorhodobacter wandonensis KCTC 23672T were from Korean
64
Collection for Type Cultures (KCTC).
65
Phenotypic characteristics of these isolates were tested as following. Cell morphology and flagella
66
were observed using light microscope and transmission electron microscopy (JEM1400; JEOL;
67
Fig. S1). Motility was determined by the hanging-drop technique (Skerman, 1967). Gram staining
68
was performed according to the procedure of Collins et al. (1989). Growth of strains was tested on
69
tryptic soy agar, nutrient agar and marine agar 2216 (MA; BD Difco). For assessment of pH range
70
for growth, strains were cultured in PYG broth adjusted to pH4.0-10.0 using 100 mM acetate
71
buffer (for pH4.0-5.0), 100 mM phosphate buffer (pH6.0-8.0) or 100mM NaHCO3/NaCO3 buffer
72
(pH9.0-10.0) (Breznak & Costilow, 2007; Yumoto et al., 2004), respectively, and medium were
73
sterilized by filtration. The requirement and tolerance of NaCl were tested on PYG broth
74
supplemented with various NaCl concentrations (from 0-5.0%, at 0.5% intervals). Growth at 4, 8,
75
15, 20, 30, 35, 37 ℃ was observed on PYG slant after 15-days incubation. Photoautotrophic and
76
photoheterotrophic growth under anaerobic condition was tested in screw-capped tubes and in agar
77
deeps at 2400lx, using the DSMZ medium 27 and PYG medium, respectively. For analysis of
78
photosynthetic pigments, cells were suspended in 60% sucrose solution and the in vivo absorption
79
spectrum was measured by using a Lambda 35 UV/Vis spectrometer (Trüper & Pfennig, 1981).
80
Catalase and oxidase activity were determined by using 3% (v/v) H2O2 and Bactident Oxidase
81
strips (Merck), respectively. Hydrolysis of Tween20, Tween60, Tween80, starch, esculin,
82
CM-cellulose, casein and gelatin were assessed according to Smibert & Krieg (1994). DNase assay 3
83
was carried out on DNase test agar (Merck). Other enzyme activities were determined using the
84
commercial system API 20E, API 20NE and API ZYM (bioMérieux) following the manufacturer’s
85
instructions. Acid production from carbohydrates was tested in API 50CH strip (bioMérieux), and
86
oxidation of substrates was evaluated using the GN3 MicroPlate system (Biolog). Sensitivity to
87
antibiotics were tested on PYG plates by using antibiotic discs (Beijing Tiantan Biological
88
Products) containing ampicillin (10μg), amikacin (30μg), azithromycin (15μg), cefaclor (30μg),
89
cefazolin (30μg), cefoperazone (75μg), cefotaxine (30μg), ceftazidime (30μg), ceftriazone (30μg),
90
cefuroxime sodium (30μg), cephalotin (30μg), chloramphenicol (30μg), ciprofloxacin (5μg),
91
clarithromycin (15μg), clindamycin (2μg), doxycycline (30μg), erythromycin (15μg), fleroxacin
92
(5μg), lomefloxacin (10μg), minocycline (30μg), netilmicin (30μg), nitrofurantoin (300μg),
93
oxacillin (1μg), penicillin G (10 IU), piperacillin (100μg), rifampicin (5μg), sulfamethoxazole
94
(1.25μg), tetracycline (30μg), tobramycin (10μg) and vancomycin (30μg). The morphological,
95
biochemical and physiological properties of strain SP32T are given in Table 1 and in the species
96
description.
97
For fatty acid profile analysis, new isolates and reference strains were incubated on TSA at 18℃
98
for 3 days, and cell masses were harvested during the same exponential growth phase and were
99
subjected to saponification, methylation and extraction as described previously (Kämpfer &
100
Kroppenstedt, 1996) and subsequently analyzed by gas chromatograph (Agilent 6890). Peaks
101
were determined using MIDI (version 6) by adopting the TSBA6.0 database (Sasser, 1990).
102
Respiratory quinones of these three isolates and R. veldkampii CGMCC 1.5006T were extracted
103
according to Collins (1985) and analyzed by HPLC (Wu et al., 1989). Polar lipids were extracted
104
by the procedure of Minnikin et al. (1984) and determined by two-dimensional TLC. The
105
separated spots were confirmed by spraying 5% ethanolic molybdophosphoric acid, molybdenum
106
blue, ninhydrin and α-naphthol for total polar lipids, phospholipids, aminolipids and glycolipids,
107
respectively. The polar lipid features of R. veldkampii CGMCC 1.5006T, D. denitrificans DSM
108
18921T and P. wandonensis KCTC 23672T were used as references.
109
Genomic DNA of these strains was extracted by using a commercial Genomic DNA Rapid
110
Isolation Kit for Bacterial Cell (BioDev-Tech, China) following the instruction. Gene pufM was
111
amplified by using the primer 2F (5’-CAGATCGGGCCGATCTA-3’) and 4R (5’-CCAGACGTA 4
112
CCAGTTGTC-3’) (Uchino et al., 2002). PCR products of pufM were detected by electrophoresis.
113
16S rRNA gene sequence were amplified by using universal primer 27F and 1492R (Lane, 1991).
114
The PCR products of 16S rRNA gene were cloned using the pGEM-T Easy vector and sequenced.
115
The nearly full-length 16S rRNA gene sequence (1428bp) was obtained, and aligned with
116
available published sequences on the GenBank and EzTaxon-e server (Kim et al., 2012).
117
According to the analysis of 16S rRNA gene sequences, strains SP32T, SR68 and SP95 shared 100%
118
similarity with each other, and the 16S similarities with the phylogenetic neighbors were as
119
following: R. veldkampii ATCC 35703T (96.6%), D. aestuarii BS14T (96.3%), H. missouriensis
120
CCUG 52307T (96.3%), H. massiliensis CCUG 47968T (96.2%) and P. wandonensis WT-MW11T
121
(95.9%), respectively. 16S rRNA gene sequences of the most closely related taxa were retrieved
122
and aligned with BioEdit (Hall, 1999). Phylogenetic tree were constructed with Mega5.0 by using
123
neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and
124
maximum-parsimony (Fitch, 1971) methods. Bootstrap values were calculated based on 1000
125
replications in order to evaluate confidence levels of the nodes. The neighbor-joining phylogenetic
126
analysis of 16S rRNA gene sequences revealed that these three isolates constituted a distinct
127
phylogenetic lineage within the family Rhodobacteraceae, and clustered with the genera
128
Rhodobacter, Defluviimonas, Pseudorhodobacter and Heamobacter (Figure 1). The topologies of
129
maximum-likelihood tree and maximum-parsimony tree (Fig. S2) also supported the notion that
130
these isolates represented a novel genus which was distinct from their most closely related genera
131
of the family Rhodobacteraceae.
132
The DNA G+C contents were assessed by HPLC (Mesbah et al., 1989), and DNA of Lambda
133
phage (Sigma) (49.8 mol %) and R. veldkampii CGMCC1.5006T were used as reference.
134
As list in Table S1, the predominant fatty acids of strain SP32T was mainly summed feature 8
135
(C18:1ω7c/ C18:1ω6c; 82.1%), which was identical with other genera of the family
136
Rhodobacteraceae. Strain SP32T distinguished from its neighbor genera Rhodobacter and
137
Pseudorhodobacter by containing C18:1ω7c 11-methyl (5%).
138
The major polar lipids of strain SP32T were phosphatidylglycerol (PG), phosphatidylethanolamine
139
(PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), one unidentified glycolipid (GL) 5
140
and one unidentified polar lipid (L4). Besides, there are 6 unidentified polar lipids (L1, 2, 3, 4, 5,
141
6, 7) present in strain SP32T with trace amount (Fig. S3). As the polar lipids profiles of strains
142
SR68 and SP95 was identical with strain SP32T, results of strains SR68 and SP95 were not shown.
143
The presence of one unidentified glycolipid (GL) and several unidentified polar lipids were the
144
key feature of strains SP32T, SP95 and SR68 that distinguished them from their closest
145
phylogenetic neighbors, R. veldkampii CGMCC 1.5006T, D. denitrificans DSM 18921T and P.
146
ferrugineus JCM 20687T.
147
Ubiquinone-10 was the sole respiratory quinone, which in line with members of the family
148
Rhodobacteraceae. The genomic DNA G+C content of strains SP32T, SR68 and SP95 were 67.6,
149
67.9, 67.8 mol %, respectively.
150
According to the phenotypic and physiological characteristics comparison list in Table 1, strain
151
SP32T differed from the genus Rhodobacter by it phenotypic characteristics and cultivation
152
conditions. Photoautotrophic and photoheterotrophic growth under anaerobic condition were not
153
observed for strain SP32T. No absorption maxima at 377, 590, 803 and 860 nm were detected,
154
which confirmed that they did not contain any photosynthetic pigments. In addition, strain SP32T
155
did not contain the photosynthetic gene pufM (Fig. S4). Besides, strain SP32T distinguished from
156
genera Defluviimonas, Pseudorhodobacter and Haematobacter by its polar lipids features and the
157
fatty acids profiles. Therefore, on the basis of these distinctive phenotypic, physiological,
158
biochemical and phylogenetic characteristics, strain SP32T represents a novel species in a novel
159
genus within the family Rhodobacteraceae, which is proposed as Frigidibacter albus gen. nov., sp.
160
nov. As the genomic, physiological and chemical taxonomic characteristics of strains SP95 and
161
SR68 are identical with strain SP32T, they are also proposed as strains of Frigidibacter albus gen.
162
nov., sp. nov.
163
Description of Frigidibacter gen. nov
164
Frigidibacter (Fri.gi.di.bac'ter. L. adj. frigidus cold; N.L. masc. n. bacter, a rod; N.L. masc. n.
165
Frigidibacter, a rod from a cold environment)
166
Cells are Gram-staining negative, short rods, and non-spore forming. Positive for catalase and
167
oxidase. Photoautotrophic and photoheterotrophic growth are not present. Voges-proskauer test 6
168
and urease are positive. Q-10 is the sole respiratory quinone. The predominant polar lipids are
169
phosphatidyglycerol, phosphatidylethanolamine, phosphatidylcholine, one unidentified glycolipid
170
and one unidentified polar lipid. The main fatty acid are summed feature 8 (C18:1ω7c/ C18:1ω6c)
171
and C18:1ω7c 11-methyl. The G+C content of genomic DNA is 67-68 mol%. The type species is
172
Frigidibacter albus SP32T.
173
Description of Frigidibacter albus sp. nov.
174
Frigidibacter albus (al'bus. L. masc. adj. albus white).
175
Cells are Gram-staining-negative, non-pigmented, non-motile, strictly aerobic, short-rods with
176
0.6-0.8μm in width and 1.0-2.2μm in length. Colonies are creamy, convex, round, opaque with
177
smooth edges and 2-4mm in diameter after 4-days incubation on PYG agar at 20℃. Growth
178
occurs at 4-25℃ and pH6.0-9.0 (with optimum at 18-20℃ and pH7.0-7.5). The range of NaCl
179
tolerance is 0-4% (with optimum 0-1.5%). Grow on R2A, NA, but not on Marine 2216 agar.
180
Photoautotrophic and photoheterotrophic growth under anaerobic condition do not occur for strain
181
SP32T. Photosynthetic pigments and the photosynthetic gene pufM are absent. Urease-, Catalase-
182
and oxidase- positive. H2S and indole production are negative. The Voges-Proskauer test is
183
positive. Do not reduce nitrate to nitrite. Negative for DNase activity. Esculin is hydrolyzed, but
184
gelatin, starch, casein, CM-cellulose, Tween20, Tween60 and Tween80 are not. In the test of API
185
20E, API 20NE and API ZYM, strain SP32T are positive for alkaline phosphatase esterase (C4),
186
esterase
187
naphthol-AS-BI-phosphohydrolase and α-glucosidase, weakly positive for cystine arylamidase,
188
α-chymotrypsin, acid phosphatase, β-glucosidase and N-acetyl-β-glucosaminidase; but negative
189
for arginine dihydrolase, β-galactosidase, lysine decarboxylase, ornithine decarboxylase,
190
tryptophane
191
β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. In API 50CH
192
detection, acid is produced from L-arabinose, D-arabitol, D-cellobiose, esculin, D-fucose,
193
D-lactose, maltose, D-turanose, D-xylose; weakly from L-arabitol, D-fructose, gluconate,
194
5-ketogluconate, and salicin; but not from N-acetyl-glucosamine, D-adonitol, amygdalin,
195
D-arabinose, arbutin, dulcitol, erythritol, L-fucose, D-galactose, D-gentiobiose, D-glucose,
196
glycerol, glycogen, glycol, inositol, 2-ketogluconate, D-mannose, D-melezitose, melibiose,
lipase
(C8),
deaminase,
lipase
arginine
(C14),
leucine
dihydrolase,
7
arylamidase,
trypsin,
valine
α-galactosidase,
arylamidase,
β-galactosidase,
197
methyl-α-D-glucopyranoside,
methyl-α-D-mannopyranoside,
methyl-β-D-xylopyranoside,
198
D-raffinose, L-rhamnose, D-ribose, sorbitol, L-sorbose, starch, sucrose, synanthrin, D-tagatose,
199
D-turanose, trehalose, xylitol and L-xylose. In the GN3 MicroPlate, the following substrates are
200
oxidized by strain SP32T: D-maltose, D-trehalose, D-cellobiose, sucrose, α-D-glucose, D-frucose,
201
D-galactose, D-sorbitol, D-arabitol, D-mannitol, glycerol, D-aspartic acid, glycyl-L-proline,
202
L-alanin, L-aspartic acid, L-glutamic acid, L-serine, pectin, D-gluconic acid, mucic acid, quinic
203
acid, D-saccharic acid, p-hydroxy-phenylacetic acid, methyl pyruvate, L-lactic acid, citric acid,
204
α-keto-glutaric acid, D-malic acid, L-malic acid, bromo-succinic acid, γ-amino-butrytic acid, α-
205
hydroxy-butyric acid, β-hydroxyl-D, L-butyric acid, α-keto-butyric acid, acetoacetic acid,
206
propionic acid, acetic acid, turanose (weak), formic acid (weak); the other substrates in the GN3
207
MicroPlate are not oxidized. Strain SP32T is resistant to amikacin (30μg), clindamycin (2μg);
208
weakly resistant to clarithromycin (15μg), erythromycin (15μg), nitrofurantoin (300μg), oxacillin
209
(1μg); but sensitive to amikacin (30μg), ampicillin (10μg), azithromycin (15μg), cefaclor (30μg),
210
cefazolin (30μg), cefoperazone (75μg), cefotaxine (30μg), ceftazidime (30μg), ceftriazone (30μg),
211
cefurosimc sodium (30μg), cephalotin (30μg), chloramphenicol (30μg), ciprofloxacin (5μg),
212
doxycycline (30μg), fleroxacin (5μg), lomefloxacin (10μg), minocycline (30μg), netilmicin
213
(30μg), penicillin G (10 IU), piperacillin (100μg), rifampin (5μg), tetracycline (30μg), tobramycin
214
(10μg) and vancomycin (30μg). The major fatty acids are summed feature 8 (C18:1ω7c/ C18:1ω6c;
215
82.1%) and C18:1ω7c 11-methyl (5%). Q-10 is the sole ubiquinone. The predominant polar lipids
216
are phosphatidyglycerol, phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol,
217
one unidentified glycolipid and one unidentified polar lipid. The DNA G+C content of strain
218
SP32T is 67.6 mol %.
219
The type strain SP32T (=CGMCC 1.13995T =NBRC 109671T) was isolated from water sample of a
220
coldwater lake in the west of China.
221 222
ACKNOWLEDGEMENTS
223
We appreciate JCM, DSMZ and KCTC for supporting reference strains for this study. We thank
224
Dr. Jing-Nan Liang for TEM observing. This work was supported by the National Science and
225
Technology Foundation Project (2012FY111600). 8
226
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227
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343
344
Figure Legend
345
Fig.1. Neighbour-joining tree based on 16S rRNA gene sequences, showing the phylogenetic
346
relationship of strains SP32T, SR68 and SP95 and related genera of the family Rhodobacteraceae.
347
Rhodospirillum rubrum ATCC11170T was used as an outgroup. Only bootstrap values (percentages
348
of 1000 replications) of >50% are shown at branching points. Filled circles indicate nodes also
349
recovered in the trees drew by maximum-likehood and maximum-parsimony methods, and open
350
circles indicate nodes recovered by maximum-likehood tree. Bar, 0.01 substitutions per nucleotide
351
position.
352
13
Differential characteristics of strains SP32T, SR68, SP95 and type strains of the
353
Table1.
354
closely related genera of the family Rhodobacteraceae.
355
Strains: 1: SP32T; 2: strain SR68; 3: strain SP95; 4: Rhodobacter veldkampii CGMCC1.5006T; 5:
356
Rhodobacter capsulatus CGMCC1.8920T; 6: Defluviimonas aestuarii JCM 18630T; 7:
357
Defluviimonas denitrificans DSM18921T; 8: Pseudorhodobacter ferrugineus NBRC 20687T; 9:
358
Pseudorhodobacter wandonensis KCTC 23672T; 10: Haematobacter massiliensis CCUG 47968T;
359
11: Haematobacter missouriensis CCUG 52307T. Data are from this study unless otherwise
360
indicated. +, positive; -, negative; w, weak growth; ND, not done; tr, trace (
