IJSEM Papers in Press. Published May 11, 2015 as doi:10.1099/ijs.0.000321
International Journal of Systematic and Evolutionary Microbiology Phaeodactylibacter luteus sp. nov., isolated from the oleaginous microalga Picochlorum sp. --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00410R2
Full Title:
Phaeodactylibacter luteus sp. nov., isolated from the oleaginous microalga Picochlorum sp.
Short Title:
Phaeodactylibacter luteus sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Tianling Zheng Xiamen University CHINA
First Author:
Xueqian Lei
Order of Authors:
Xueqian Lei Yi Li Guanghua Wang Yao Chen Qiliang Lai Zhangran Chen Jingyan Zhang Pingping Liao Hong Zhu Wei Zheng Tianling Zheng
Manuscript Region of Origin:
CHINA
Abstract:
A Gram-staining-negative, orange-pigmented, non-motile, aerobic bacterial strain, designated GYP20T, was isolated from a culture of the alga Picochlorum sp., a promising feedstock for biodiesel production, which was isolated from the India Ocean. Growth was observed at temperatures from 20 to 37 °C, at salinities from 0 to 3% and at pH from 5 to 9. Mg2+ and Ca2+ ions are required for growth. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain was a member of the genus Phaeodactylibacter, which belongs to the family Saprospiraceae. Strain GYP20T was most closely related to Phaeodactylibacter xiamenensis KD52T (95.5% sequence similarity). The dominant fatty acids were iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH and Summed Feature 3. The predominant respiratory quinone was menaquinone-7 (MK-7). The polar lipids of strain GYP20T were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified glycolipid, two unidentified phospholipid and three unidentified aminolipid. According to its morphology, physiology, fatty acid composition and 16S rRNA sequence data, the novel strain most appropriately belongs to the genus Phaeodactylibacter, but can readily be distinguished from known Phaeodactylibacter xiamenensis GYP20T. The name Phaeodactylibacter luteus sp. nov. is proposed (type strain GYP20T =MCCC 1F01222T =KCTC 42180T).
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1 2 3
Phaeodactylibacter luteus sp. nov., isolated from the oleaginous microalga Picochlorum sp.
4
Xueqian Lei1†, Yi Li1†, Guanghua Wang2, Yao Chen1, Qiliang Lai3, Zhangran Chen1,
5
Jingyan Zhang1, Pingping Liao1, Hong Zhu1, Wei Zheng1, Tianling Zheng1*
6
1
7
Xiamen University, Xiamen 361005, China
8
2
9
Guangdong Key Laboratory of Marine Materia Medica (LMMM-GD), South China
10
Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301,
11
China
12
3
13
State Oceanic Administration, People’ s Republic of China
Key Laboratory of MOE for Coast and Wetland Ecosystems, School of Life Sciences,
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB-CAS),
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography,
14 15
†
Co-first author: These authors contributed equally to this work.
16
*
17
+86-592-2184528; E-mail:
[email protected];
18
Subjective category: Bacteroidetes
19
Running title: Phaeodactylibacter luteus sp. nov.
20
Abbreviations: KCTC, Korean Collection For Type Cultures; MCCC, Marine
21
Culture Collection of China.
22
The GenBank [/EMBL/DDBJ] accession number for the 16S rRNA gene sequence of
23
strain GYP20T is KM235292.
24
Four supplementary figures are available with the online Supplementary Material.
Corresponding
author:
Tianling
Zheng.
Tel:
+86-592-2183217.
Fax:
25
Abstract:
26
A Gram-staining-negative, orange-pigmented, non-motile, aerobic bacterial strain,
27
designated GYP20T, was isolated from a culture of the alga Picochlorum sp., a
28
promising feedstock for biodiesel production, which was isolated from the India
29
Ocean. Growth was observed at temperatures from 20 to 37 °C, at salinities from 0 to
30
3% and at pH from 5 to 9. Mg2+ and Ca2+ ions were required for growth. Phylogenetic
31
analysis based on 16S rRNA gene sequencing revealed that the strain was a member
32
of the genus Phaeodactylibacter, which belongs to the family Saprospiraceae. Strain
33
GYP20T was most closely related to Phaeodactylibacter xiamenensis KD52T (95.5%
34
sequence similarity). The major fatty acids were iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH
35
and Summed Feature 3. The predominant respiratory quinone was menaquinone-7
36
(MK-7). The polar lipids of strain GYP20T were found to consist of
37
diphosphatidylglycerol,
38
unidentified glycolipid, two unidentified phospholipid and three unidentified
39
aminolipid. According to its morphology, physiology, fatty acid composition and 16S
40
rRNA sequence data, the novel strain most appropriately belongs to the genus
41
Phaeodactylibacter, but can readily be distinguished from known Phaeodactylibacter
42
xiamenensis GYP20T. The name Phaeodactylibacter luteus sp. nov. is proposed for
43
strain GYP20T (=MCCC 1F01222T =KCTC 42180T).
phosphatidylethanolamine,
phosphatidylglycerol,
four
44 45
Main text:
46
Members of the family Saprospiraceae, are considered to be important members of
47
the bacterial community involved in ecophysiological activities in a variety of natural
48
environments (Yoon et al., 2011). The family Saprospiraceae consists of the genera
49
Saprospira (Reichenbach et al., 1989), Haliscomenobacter (Van Veen et al., 1973),
50
Lewinella (Sly et al., 1998), ‘Portibacter’ (Yoon et al., 2011), Aureispira (Hosoya, et
51
al., 2006), ‘Rubidimonas’ (Yoon et al., 2012) and Phaeodactylibacter (Chen et al.,
52
2014), and they are found in various habitats. Comparative 16S rRNA gene sequence
53
analysis indicated that strain GYP20T formed a clade within the genus
54
Phaeodactylibacter. The genus Phaeodactylibacter was first proposed by Chen et al.
55
(2014). At present, this genus includes one species with validly published names:
56
Phaeodactylibacter xiamenensis KD52T. Accordingly, the aim of the present work
57
was to determine the exact taxonomic position of strain GYP20T by using polyphasic
58
characterization including the determination of phenotypic properties and a detailed
59
phylogenetic analysis based on 16S rRNA gene sequences.
60
Strain GYP20T was isolated from a culture of the alga Picochlorum sp., a promising
61
feedstock for biodiesel production, which was isolated from the India Ocean (Yang et
62
al., 2014). The samples were serially diluted (10-fold dilution) by sterile seawater and
63
0.1 mL aliquots of each dilution were spread onto marine agar 2216 (MA; Difco)
64
followed by incubation for 7 days at 28 °C. Individual colonies of distinct
65
morphology were further purified three times and stored at -80 °C in marine broth
66
2216 (MB; Difco) supplemented with 10% (v/v) glycerol.
67
Genomic DNA was extracted according to the method of Ausubel et al. (1995). The
68
16S rRNA gene sequence was amplified by PCR using primers 27F and 1492R
69
(DeLong, 1992). Purification of the PCR product was carried out according to the
70
protocol of the TIANquick midi purification kit (TIANGEN, China). The purified
71
DNA was cloned into vector pMD19-T and sequenced. Sequences of related taxa
72
were
73
(http://eztaxon-e.ezbiocloud.net/) (Kim et al., 2012). Phylogenetic analysis was
74
performed using MEGA version 5.0 (Tamura et al., 2011) on the basis of
75
neighbour-joining (Saitou & Nei, 1987), minimum evolution (Rzhetsky & Nei, 1993)
76
and maximum likelihood (Felsenstein 1981) algorithms, with bootstrap resampling of
77
1,000 replications.
78
A nearly full-length 16S rRNA gene sequence (1484 bp) of strain GYP20T was
79
determined. Phylogenetic analysis of the strain GYP20T based on the 16S rRNA gene
80
sequence indicated that this strain belonged to the family Saprospiraceae, forming a
81
robust clade within the genus Phaeodactylibacter (Fig. 1, Fig. S2 and Fig. S3,
82
available in the online Supplementary Material). The closest related species was P.
83
xiamenensis KD52T (95.5%). Strain GYP20T formed a separate phylogenetic clade
84
with P. xiamenensis KD52T.
85
Cell morphology and motility were observed by using transmission electron
86
microscopy (model JEM-2100HC; JEOL) and phase-contrast light microscopy (model
87
50i; Nikon), with cells from the early exponential phase grown on MA at 28 °C.
88
Colony morphology was examined from cultures grown on MA for 2 days. The
89
presence of flexirubin-type pigments was assessed using the bathochromic shift test
obtained
from
the
GenBank
database
and
EzTaxon-e
server
90
with 20% KOH, as described by Bernardet et al. (2002). The Gram reaction was
91
determined by using the bioMérieux Gram stain kit according to the manufacturer’s
92
instructions. Gliding motility was investigated as described by Bowman (2000).
93
Anaerobic growth was examined according to the protocol of Li et al. (2013).
94
Triplicate cultures were grown in 50 ml anaerobic serum bottles sealed with thick
95
butyl rubber stoppers and aluminum caps, and incubated statically in the dark at 30 °C
96
for 21 days. The optimal growth temperature was determined over the range 0–45 °C
97
(0, 4, 15, 20, 25, 28, 30, 32, 35, 37, 38, 39, 40, 42 and 45°C) in MB with triplicate and
98
measured by spectrophotometer method with OD600 every 12 h in total 3 days. The
99
specific ion requirements and NaCl tolerance were performed as described by Sohn et
100
al. (2004). Specific ion requirements and NaCl tolerance were tested by using
101
NaCl-free 2216 medium (Tryptone 5.0 g/L, yeast extract 1.0 g/L, 1 L distilled water,
102
pH 7.6-7.8) that was supplemented with NaCl or combinatorial artificial sea salts. The
103
strain showed requirement for cations, namely Na+, Mg2+ and Ca2+, as no growth was
104
observed in medium that had not been supplemented with Na+, Mg2+ and Ca2+ ions.
105
The pH range for growth was determined in MB that was adjusted to pH 3.0-12.0 (at
106
1 pH unit intervals), as previously described (Lei et al., 2014). Verification of the pH
107
values after autoclaving revealed only minor changes (Su et al., 2013). The catalase
108
activity was performed by addition of 3% (v/v) hydrogen peroxide to
109
exponential-phase colonies, and oxidase reaction was tested by using oxidase reagent
110
(bioMérieux). Hydrolysis of starch, chitin, tyrosine, casein, gelatin, urea and Tweens
111
20, 40, 60 and 80 was tested using MA supplemented with 0.5% (w/v) of starch and 1%
112
(w/v) of the other substrates. Results were examined twice after growth on agar plates
113
for 3 and 5 days. Further biochemical test of strain GYP20T and P. xiamenensis
114
KD52T were conducted using the API 20NE and API 20E test kits (bioMérieux) at
115
30 °C for 3 days and the API ZYM test kit (bioMérieux) at 30 °C for 24 h according
116
to the manufacturer’s instructions. All the commercial kits were inoculated with
117
bacterial suspensions prepared in 3% (w/v) NaCl. The physiological and biochemical
118
characteristics of strain GYP20T are shown in the species description and in Table 1.
119
Fatty acids of strain GYP20T and P. xiamenensis KD52T in whole cells grown on MA
120
at 28 °C for 48 h were extracted, saponified and esterified using the standard protocol
121
of MIDI (Sherlock Microbial Identification System, version 6.0B). The two strains
122
had similar growth rates and had same physiological age after growth on MA plates at
123
28 °C for 48 h. The fatty acids were analysed by GC (Agilent Technologies 6850) and
124
identified by using the TSBA 6.0 database of the Microbial Identification System
125
(Sasser, 1990). P. xiamenensis KD52T was tested under the same condition in this
126
study with strain GYP20T. As showed in Table 2, the major fatty acids of GYP20T
127
were Summed Feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c, 23.5%), iso-C15:0
128
(13.3%), iso-C17:0 3-OH (13.1%), iso-C15:1 G (12.0%), which accounted for 61.9%.
129
Compared with P. xiamenensis KD52T, strain GYP20T possessed a lower content of
130
iso-C15:0 and a higher content of Summed Feature 3 (comprising C16:1 ω7c and/or C16:1
131
ω6c, 23.5%).
132
The G+C content of the chromosomal DNA was determined according to the methods
133
(Mesbah & Whitman, 1989) using a reverse-phase HPLC. The DNA G+C contents of
134
the new isolate GYP20T was 53 mol%, which was similar to reference strain P.
135
xiamenensis KD52T.
136
The major respiratory quinone of the strain GYP20T was determined to be
137
menaquinone-7 (MK-7), which was carried out by the Identification Service of the
138
DSMZ, Braunschweig, Germany. This trait was in accordance with the properties of
139
the genus Phaeodactylibacter.
140
Polar lipids of strain GYP20T were extracted using a chloroform / methanol system
141
and analysed using one- and two-dimensional TLC, as described previously (Kates,
142
1986). Merck silica gel 60 F254 aluminium-backed thin-layer plates were used in
143
TLC analysis. The plate dotted with sample was subjected to two-dimensional
144
development, with the first solvent of chloroform-methanol-water (65:25:4, by vol.)
145
followed by second solvent of chloroform-methanol-acetic acid-water (85:12:15:4, by
146
vol.). The TLC plates were sprayed with sulfuric acid/ethanol (1: 2, v/v) followed by
147
heating at 150 °C for 3 min to detect phospholipids and glycolipids. The TLC plates
148
were also visualized by treating the plates with 10 % (w/v) molybdatophosphoric acid
149
followed by heating at 150 °C for 5 min. The polar lipids of strain GYP20T were
150
found
151
phosphatidylglycerol, four unidentified glycolipid, two unidentified phospholipid and
152
three
153
phosphatidylethanolamine, phosphatidylglycerol, unidentified glycolipid were found
154
in strain GYP20T. (Fig. S4, available in the online Supplementary Material).
to
consist
unidentified
of
diphosphatidylglycerol,
aminolipid.
Compared
with
phosphatidylethanolamine,
the
strain
KD52T,
155 156
On the basis of morphological, physiological and chemotaxonomic characteristics,
157
together with data from 16S rRNA gene sequences comparison described above,
158
strain GYP20T should be assigned to a novel species within the genus
159
Phaeodactylibacter, for which a name Phaeodactylibacter luteus sp. nov. is proposed.
160 161
Description of Phaeodactylibacter luteus sp. nov.
162
Phaeodactylibacter luteus (lu'te.us. L.masc. adj. luteus, orange-colored, referring to
163
the color of the colony).
164
Cells are Gram-staining-negative, rod-shaped, non-motile, non-gliding rods, 5.0-11.2
165
μm in length and 0.5-0.6 μm in diameter, as show in Fig. S1 (available in the online
166
Supplementary Material). Growth is visible after 48 h of incubation on MA at 28 °C.
167
Colonies on MA are orange and circular with regular, smooth edges that are 1-2 mm
168
in diameter after 48 h incubation at 28 °C, opaque and raised in the centre. Growth
169
occurs at 20-37 ºC (optimum 28 ºC). Growth occurs at pH 5–9 (optimum pH 7).
170
Growth occurs at NaCl concentrations of 0-3% (w/v), with optimal growth at 2%
171
(w/v). No growth was observed in medium that has not been supplemented with Na+,
172
Mg2+ and Ca2+ ions. Growth does not occur under anaerobic conditions on MB.
173
Flexirubin-type pigments are not produced. Positive for catalase, oxidase, reduction of
174
nitrate and hydrolysis of Tweens 20, 40 and 60 and tyrosine. Negative for hydrolysis
175
of casein, chitin, starch, Tween 80, urea and gelatin. In API 20NE strips positive for
176
reduction of nitrate to nitrite, denitrification, β-glucosidase (aesculin hydrolysis),
177
β-galactosidase; negative for indole production, arginine dihydrolase, urease activity,
178
gelatin hydrolysis, D-glucose fermentation and utilization of D-glucose, L-arabinose,
179
D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate,
180
capric acid, adipic acid, malic acid and trisodium citrate phenylacetic acid. According
181
to API 20E strips, positive for ß-galactosidase, tryptophan deaminase and acetoin
182
production; negative for arginine dihydrolase, lysine decarboxylase, ornithine
183
decarboxylase, citrate utilization, H2S production, urease activity, indole production,
184
gelatinase and acid production from glucose, mannitol, inositol, sorbitol, rhamnose,
185
saccharose, melibiose, amygdalin and arabinose. In the test of API ZYM, positive for
186
alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine aminopeptidase,
187
valine aminopeptidase, cystine aminopeptidase, trypsin, α-chymotrypsin, acid
188
phosphatase, naphtol-AS-Bl-phosphoamidase and β-glucosidase; weakly positive for
189
lipase (C14), β-galactosidase and α-glucosidase; negative for α-galactosidase,
190
β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase or α-fucosidase. The
191
major fatty acids are iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH and Summed Feature 3. The
192
DNA G+C contents of the type strain is 53 mol%. The predominant respiratory
193
quinone is menaquinone-7 (MK-7). The major polar lipids are diphosphatidylglycerol,
194
phosphatidylethanolamine, phosphatidylglycerol, four unidentified glycolipid, two
195
unidentified phospholipid and three unidentified aminolipid.
196
The type strain, GYP20T (= MCCC 1F01222T =KCTC 42180T) was isolated from a
197
culture of Picochlorum sp. collected from the India Ocean.
198 199
Acknowledgements
200
This work was supported by the National Nature Science Foundation of China
201
(41376119, 40930847), Public Science and Technology Research Funds Projects of
202
Ocean (201305016). We also thank Prof. I. J. Hodgkiss of The University of Hong
203
Kong for help with English.
204 205
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280
281
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284
285
286 287
Figure Legends
288 289
Fig. 1. Neighbour-joining tree showing the phylogenetic positions of strain GYP20T
290
and representatives of some other related taxa, based on 16S rRNA gene sequences.
291
Filled circles indicate nodes that were also recovered in maximum-likelihood, and
292
minimum evolution trees based on the same sequences. Bootstrap values (expressed
293
as percentages of 1000 replications) are shown at branch points. Only bootstrap
294
values >70% are showed. Taibaiella smilacinae KCTC 32316T (KC571459) was used
295
as an outgroup. Bar, 0.02 nucleotide substitution rate (Knuc) units.
296
297
298
299
300
301
302
303
304 305 306
Table 1. Differential characteristics between strain GYP20T and P. xiamenensis
307
KD52T.
308
Strains: 1, GYP20T; 2, P. xiamenensis KD52T. Data of catalase, oxidase, API 20NE, API 20E and
309
API ZYM for two strains were done at the same time in this study. Two strains are positive for
310
oxidase activities, Tweens 20, 40 and 60; negative for Tween 80, starch, chitin, casein, gelatin,
311
urea, flexirubin-type pigments. In the API 20NE strip, two strains are positive for reduction of
312
nitrate, β-glucosidase (aesculin hydrolysis) and β-galactosidase; negative for indole production,
313
arginine dihydrolase, urease activity, gelatin hydrolysis and utilization of D-glucose, L-arabinose,
314
D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid,
315
adipic acid, malic acid, trisodium citrate and phenylacetic acid. In the test of API 20E, two strains
316
are positive for β-galactosidase, acetoin production; negative for citrate utilization, H2S
317
production, indole production and acid production from glucose, mannitol, inositol, sorbitol,
318
rhamnose, saccharose, melibiose, amygdalin and arabinose. In the API ZYM strip, two strains are
319
positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine aminopeptidase,
320
valine aminopeptidase, cystine aminopeptidase, trypsin, α-chymotrypsin, acid phosphatase,
321
naphtol-AS-Bl-phosphoamidase and β-glucosidase; weakly positive for lipase (C14); negative for
322
α-mannosidase. +, Positive; w, weakly positive; -, negative. All data from this study except growth
323
temperature, salinity range, growth pH and the DNA G+C contents of P. xiamenensis KD52T,
324
which are from Chen et al. (2014).
Characteristic
1
2
Colony colour
Orange
Reddish orange
NaCl concentration (optimum) (%, w/v)
0-3 (2)
1-9 (2.5)
20-37(28)
20-37(28)
pH range (optimal)
5-9 (7)
5-8.5 (6)
Catalase activities
+
-
+
-
Denitrification
+
-
D-Glucose fermentation
-
w
Arginine dihydrolase
-
+
Lysine decarboxylase
-
+
Ornithine decarboxylase
-
+
Urease activity
-
+
Tryptophane deaminase
+
-
Gelatinase
-
+
α-Galactosidase
-
+
β-Galactosidase
w
+
β-Glucuronidase
-
+
Growth temperature (optimum) (°C)
Hydrolysis of: Tyrosine API 20NE
API 20E
API ZYM
α-Glucosidase
w
+
N-acetyl-β-glucosaminidase
-
+
α-Fucosidase
-
+
53
51
DNA G+C content (mol%) 325
Table 2. Cellular fatty acid content of strain GYP20T and P. xiamenensis KD52T.
326
Strains: 1, GYP20T; 2, P. xiamenensis KD52T. All data were generated in this study. Values are
327
percentages of total fatty acids; tr, traces amount (