IJSEM Papers in Press. Published April 14, 2015 as doi:10.1099/ijs.0.000263
International Journal of Systematic and Evolutionary Microbiology Phaeobacterium nitratireducens gen. nov., sp. nov., a phototrophic gammaproteobacterium isolated from a sediment sample of Coringa mangrove forest, India --Manuscript Draft-Manuscript Number:
IJS-D-14-00548R1
Full Title:
Phaeobacterium nitratireducens gen. nov., sp. nov., a phototrophic gammaproteobacterium isolated from a sediment sample of Coringa mangrove forest, India
Short Title:
Phaeobacterium nitratireducens gen. nov., sp. nov.
Article Type:
Standard
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Anil Kumar Pinnaka, Ph.D Institute of Microbial Technology Chandigarh, Chandigarh INDIA
First Author:
Nupur Nupur, M.Sc
Order of Authors:
Nupur Nupur, M.Sc Naga Radha Srinivas Tanuku, Ph.D Takaichi Shinichi, Ph.D Anil Kumar Pinnaka, Ph.D
Manuscript Region of Origin:
INDIA
Abstract:
The novel, brown coloured, Gram-negative-staining, rod shaped, motile phototrophic bacterium designated strain AK40T, was isolated in pure culture from a sediment sample collected from Coringa mangrove forest. Strain AK40T, containing bacteriochlorophyll a and carotenoid of rhodopin as major photosynthetic pigments. Strain AK40T was able to grow photoheterotrophically and could utilize a number of organic substrates. It was unable to grow photoautotrophically and did not utilize sulfide and thiosulfate as electron donors. Thiamine and riboflavin were required for growth of strain AK40T. The dominant fatty acids were C12:0, C16:0, C18:1 7c and C16:1 7c and/or iso-C15:0 2OH (summed feature 3). Polar lipid content of strain AK40T was found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and eight unidentified lipids. Q-10 was the predominant respiratory quinone. The G + C content of DNA of the strain AK40T was 65.5 mol%. The 16S rRNA gene sequence comparisons indicated that the isolate represented a member of the family Chromatiaceae within the class Gammaproteobacteria. The 16S rRNA gene sequence analysis indicated that strain AK40T was closely related to Phaeochromatium fluminis with 95.2% pair-wise sequence similarity and with other species of the family, sequence similarity between 90.8 to 94.8%. Distinct morphological, physiological and genotypic differences from previously described taxa supported the classification of this isolate as a representative of a novel species in a new genus, for which the name Phaeobacterium nitratireducens gen. nov., sp. nov. is proposed. The type strain of Phaeobacterium nitratireducens is AK40T (= JCM 19219T = MTCC 11824T).
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1 1
26-03-2015
2
Phaeobacterium nitratireducens gen. nov., sp.
3
nov., a phototrophic gammaproteobacterium
4
isolated from a sediment sample of Coringa
5
mangrove forest, India
6 7
Nupur1, Naga Radha Srinivas Tanuku2, Takaichi
8
Shinichi3, and Anil Kumar Pinnaka1*
9 10
1
11
CSIR-Institute of Microbial Technology, Sector 39A,
12
Chandigarh – 160 036, INDIA
13
2
14
Centre, 176, Lawsons Bay Colony, Visakhapatnam-
15
530017, INDIA
16
3
17
Kosugi-cho, Nakahara, Kawasaki 211-0063, Japan
Microbial Type Culture Collection and Gene Bank,
CSIR-National Institute of Oceanography, Regional
Nippon Medical School, Department of Biology,
18 19
Address for correspondence*
Dr. P. Anil Kumar
20
Microbial Type Culture Collection and Gene Bank,
21
Institute of Microbial Technology (CSIR), Sector 39A,
22
Chandigarh – 160 036, INDIA
23
Email: [email protected]
24
Telephone: 00-91-172-6665170
25 26
Running title
Phaeobacterium nitratireducens gen. nov., sp. nov.
Subject category
New taxa (Gammaproteobacteria)
27 28 29 30
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
31
AK40T is HF968497.
32 33
All authors contributed equally for the present work.
2 34
The novel, brown coloured, Gram-negative-staining, rod shaped, motile phototrophic
35
purple sulphur bacterium designated strain AK40T, was isolated in pure culture from a
36
sediment sample collected from Coringa mangrove forest. Strain AK40T, containing
37
bacteriochlorophyll a and carotenoid of rhodopin as major photosynthetic pigments. Strain
38
AK40T was able to grow photoheterotrophically and could utilize a number of organic
39
substrates. It was unable to grow photoautotrophically and did not utilize sulfide and
40
thiosulfate as electron donors. Thiamine and riboflavin were required for growth of strain
41
AK40T. The dominant fatty acids were C12:0, C16:0, C18:1 7c and C16:1 7c and/or iso-C15:0
42
2OH (summed feature 3). Polar lipid content of strain AK40T was found to be
43
diphosphatidylglycerol,
44
unidentified lipids. Q-10 was the predominant respiratory quinone. The G + C content of
45
DNA of the strain AK40T was 65.5 mol%. The 16S rRNA gene sequence comparisons
46
indicated that the isolate represented a member of the family Chromatiaceae within the class
47
Gammaproteobacteria. The 16S rRNA gene sequence analysis indicated that strain AK40T
48
was closely related to Phaeochromatium fluminis with 95.2% pair-wise sequence similarity
49
and with other species of the family, sequence similarity between 90.8 to 94.8%. Based on
50
the sequence comparison data, strain AK40T was positioned distinctly outside the group
51
formed
52
Thiohalocapsa,
53
physiological and genotypic differences from previously described taxa supported the
54
classification of this isolate as a representative of a novel species in a new genus, for which
55
the name Phaeobacterium nitratireducens gen. nov., sp. nov. is proposed. The type strain of
56
Phaeobacterium nitratireducens is AK40T (= JCM 19219T = MTCC 11824T).
57
Key words: Phaeobacterium nitratireducens, Coringa mangrove forest, 16S rRNA gene
58
based phylogeny, chemotaxonomy, Gammaproteobacteria
by
the
6
phosphatidylethanolamine,
genera
Phaeochromatium,
Rhabdochromatium
and
phosphatidylglycerol
Marichromatium,
Thiorhodovibrio.
and
eight
Halochromatium,
Distinct
morphological,
3 59
Mangrove habitats are vital to humankind by virtue of their use and aesthetic value.
60
Mangroves serve as nursery, feeding and spawning grounds for commercial fin and shell
61
fishes. Mangrove habitats are found in locations where a river enters the sea and these
62
dynamic ecosystems support numerous diversified soil microbes. Mangroves are found in
63
different states of India such as: Andhra Pradesh, Andaman and Nicobar Islands, Goa,
64
Gujarat, Karnataka, Kerala, Maharashtra, Tamil Nadu and West Bengal. The significance of
65
microbes in aquatic ecosystems is recognized well by many pioneer studies (Longhurst &
66
Harrison, 1989; Wheeler & Kirchman, 1986). Microbes play different roles such as: primary
67
producers, secondary producers, consumers and decomposers. The activities of these
68
microbes (photosynthetic, lithotrophic and organotrophic) are important for maintaining
69
balance in the environment; which in turn, is very vital for the maintenance of other life
70
forms. A number of novel bacterial taxa are identified from mangrove habitats. The family
71
Chromatiaceae was proposed by Bavendamm (1924), with the type genus Chromatium and
72
later emended by Imhoff (1984). Members of the Chromatiaceae are Gram negative, ovoid,
73
rod, spherical, spiral, or vibrioid shaped, motile by flagella or non-motile, multiply by binary
74
fission, may contain gas vesicles and consist of internal photosynthetic membranes which are
75
either tubular or vescicular type. Cells in majority of the taxa contain photosynthetic
76
pigments such as: bacteriochlorophyll a or b and carotenoids of the okenone, rhodopinal, or
77
spirilloxanthin groups (Takaichi, 1999). Cultures appear in different colours like orange-
78
brown to brownish red or pink, purple-red, and purple-violet depending on the type of
79
carotenoids present. Cells grow under anoxic conditions in the presence of light
80
(photolithoautotropically) with sulfide or sulfur as electron donor and deposit sulfur granules
81
within the cells as an intermediate oxidation product and may convert to sulfate; and can use
82
hydrogen or reduced iron as an electron donor and also grow mixotrophically and
83
photoassimilate a number of organic substrates. Some taxa are also capable to grow
84
photoorganoheterotrophically. Most of the taxa are strictly anaerobic and obligately
4 85
phototrophic but others grow chemolithoautotrophically or chemoorganoheterotrophically
86
under microoxic to oxic conditions in the dark. Carbon fixation was by reductive pentose
87
phosphate cycle in all species tested so far. Several taxa can fix N2 and store elemental sulfur,
88
poly-β-hydroxybutyrate, polysaccharides and polyphosphate. Some taxa may require
89
different growth factors. Chromatiaceae members commonly dwell in the areas where anoxic
90
conditions prevail with sulfide or sulfur sources with illumination, of diverse aquatic habitats
91
and sediments like ditches, estuaries, lakes, marine habitats, moist and muddy soils, ponds,
92
rivers, salt and soda lakes and sulfur springs. The G + C content of the DNA among the
93
species varies between 45.5-70.4 mol% (Imhoff, 2005a). In the course of bacterial diversity
94
studies of mangrove samples, a bacterial strain, AK40T, was isolated from a mangrove
95
sediment sample collected near Coringa, Andhra Pradesh, India. Phylogenetic analysis based
96
on 16S rRNA gene sequence revealed that strain AK40T was closely related to the genus
97
Phaeochromatium of the family Chromatiaceae. In this study, a polyphasic approach,
98
including genotypic, phenotypic and chemotaxonomic characterizations, was used to
99
determine the taxonomic position of strain AK40T.
100 101
Strain AK40T was isolated from the sediment sample collected from Coringa
102
mangrove forest, India. The temperature and pH of the sediment sample was 30 oC and 7.5,
103
respectively.
104
photolithoheterotrophic enrichments of the sediment sample, and subsequent purification was
105
performed as previously described (Anil Kumar et al., 2008a, b) in modified Pfennig medium
106
(Pfennig & Trüper, 1992) supplemented with NaCl (2 % w/v), pyruvate (0.3 % w/v), sodium
107
thiosulfate (2 mM) and ammonium chloride (0.12 % w/v). Modified Pfennig medium was
108
used throughout the study unless mentioned otherwise. Preservation was done at -80°C in
109
modified Pfennig broth with 20% glycerol.
Anoxygenic
phototrophic
bacterium
AK40T
was
isolated
using
5 110
Colony morphology was studied after 72 hrs growth of the strain on modified Pfennig
111
medium at 30 oC under 2000 lux illumination, anaerobically. The isolated pure colony was
112
checked for cell morphology and motility by using phase contrast microscopy (Olympus,
113
USA) at 1000X magnification and also by transmission electron microscope (Jeol JEM 2100)
114
at operating voltage of 200 kV. Gram reaction was determined by using the Gram staining kit
115
from HIMEDIA as described by manufacturer’s protocol.
116
Physiological and biochemical characteristics were determined as previously
117
described (Anil Kumar et al., 2008a, b; Srinivas et al., 2006). In vivo absorption spectrum
118
was measured with a Spectronic Genesys 2 spectrophotometer in sucrose solution (Trüper &
119
Pfennig, 1981). Absorption spectrum of pigments extracted with acetone was also recorded.
120
Carotenoid composition was analysed by using C18-HPLC (Takaichi & Shimada, 1992).
121
Strains AK40T and Phaeochromatium fluminis JA418T were grown on modified
122
Pfennig medium with 2% NaCl at 30 oC under 2000 lux illumination, anaerobically for 3
123
days to prepare fatty acid methyl esters (classical methods) and were analysed using Sherlock
124
Microbial Identification System (MIDI-6890 with database TSBA50) by the protocol
125
described by the manufacturer. At the time of harvesting, the cells were at logarithmic phase
126
of growth. Freeze-dried cells were analysed for polar lipids and quinones. Cells were
127
extracted for the polar lipid analysis (Bligh & Dyer, 1959) and analyzed by 2D thin-layer
128
chromatography followed by spraying with appropriate detection reagents (5% ethanolic
129
molybdatophosphoric acid, molybdenum blue, ninhydrin and molisch reagents) (Komagata &
130
Suzuki, 1987). Genomic DNA was isolated by using the procedure of Marmur (1961) and the
131
DNA G + C content was determined from melting point (Tm) curves (Sly et al., 1986)
132
obtained by using Lambda 35; Perkin Elmer spectrophotometer equipped with Templab 2.0
133
software package.
134
For 16S rRNA gene sequencing, DNA was prepared using a bacterial DNA isolation
135
kit (Qiagen). The 16S rRNA gene was amplified by PCR using universal bacterial primers
6 136
27f (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492r (5'-TAC GGY TAC CTT GTT
137
ACG ACT T-3'). The PCR product was purified using QIA quick PCR purification kit
138
(Qiagen) and sequenced using an ABI PRISM model 3700 automatic DNA sequencer and
139
Big Dye Terminator cycle sequencing kit (Applied Biosystems). The 16S rRNA gene
140
sequence of strain AK40T was subjected to BLAST sequence similarity search (Altschul et
141
al., 1990) and EzTaxon-e server (Kim et al., 2012) to identify the nearest taxa. Based on
142
BLAST results all 16S rRNA gene sequences of type species of phototrophic members of the
143
genera belonging to the family Chromatiaceae were downloaded from the NCBI database
144
(http://www.ncbi.nlm.nih.gov) and aligned using the CLUSTAL_W program in MEGA5
145
(Tamura et al., 2011). The evolutionary history was inferred by using the maximum-
146
likelihood method (Tamura & Nei, 1993), neighbor-joining (NJ) method (Saitou & Nei,
147
1987) and maximum-parsimony method (Nei & Kumar, 2000) using the MEGA5 package
148
(Tamura et al., 2011). The percentage of trees in which the associated taxa clustered together
149
is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the
150
number of substitutions per site. All positions containing gaps and missing data were
151
eliminated. There were a total of 1355 positions in the final dataset. Evolutionary analyses
152
were conducted in MEGA5 (Tamura et al., 2011). The percentage of replicate trees in which
153
the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to
154
the branches (Felsenstein, 1985). Fast DNAML tree was constructed using ARB software
155
package (Ludwig et al., 2004).
156 157
Cells of strain AK40T were Gram-negative-staining, rod-shaped, brown pigmented,
158
motile and multiplied by binary fission. Cells were 1.0–4.0 µm in long and 0.9–1.3 µm in
159
wide (Fig. 1). Colonies were 0.5-1.5 mm in diameter, circular, smooth, brown, opaque,
160
convex and raised with entire margin on modified Pfennig medium. Strain AK40T grow
161
photoorganoheterotrophically (optimum light intensity is 2000 lux). Photolithoautotrophic
7 162
growth [anaerobic, light (2000 lux), Na2S/Na2S2O3 (0.5 mM), NaHCO3 (0.1%, w/v)],
163
chemolithoautotrophic growth [aerobic, dark, Na2S2O3 (0.5 mM), NaHCO3 (0.1%, w/v)],
164
chemoorganoheterotrophic growth [aerobic, dark with glucose/pyruvate (0.3%, w/v)] and
165
fermentative growth [anaerobic, dark with glucose/pyruvate (0.3%, w/v)] were not detected.
166
Salt (NaCl) was not required for growth, but tolerated up to 7% (w/v, NaCl), the optimum
167
NaCl concentration being 1.5–3% (w/v). The pH range for growth of strain AK40T was 5–9
168
with the optimum at 7.5. The temperature range was from 22 to 37 oC and the optimum
169
temperature for growth was 30 oC. Growth factors such as thiamine and riboflavin were
170
required for growth. The colour of photosynthetically grown cell suspensions was brown. The
171
whole-cell absorption spectrum (Supplementary Fig. S2a) of strain AK40T in sucrose solution
172
gave maxima at 376, 488, 592, 702, 800, 836 nm, confirming the presence of
173
bacteriochlorophyll a and carotenoids. The absorption spectrum of acetone extracted
174
pigments (Supplementary Fig. S2b) gave maxima at 446, 476, 506 nm, indicating the
175
presence of carotenoids. The carotenoid composition of strain AK40T, as determined by using
176
C18-HPLC analysis, was lycopene (5%), rhodopin (71%), anhydrorhodovibrin (15%),
177
rhodovibrin (3%) and spirilloxanthin (6%) (Supplementary Fig. S4). They were identified
178
based on absorption spectra in the C18-HPLC eluent of methanol, and the retention times on
179
the C18-HPLC (Takaichi & Shimada, 1992). Other physiological characteristics are given in
180
the species description, Tables 1 and 2.
181
The cellular fatty acid composition of the strain AK40T showed a profile of eleven
182
fatty acids of C12:0 (5.6%), C14:0 (0.6%), C16:0 (22.5%), antesio-C16:0 (1.1%), antesio-C17:0
183
(1.0%), C18:0 (0.7%), C18:1 2OH (1.9%), C18:1 7c (32.0%), C18:3 6,9,12c (2.8%), C12:0
184
aldehyde/iso-C16:1I/C14:03OH (summed feature 2) (0.9%), and C16:1 7c/iso-C15:0 2OH
185
(summed feature 3) (30.9%). Unsaturated fatty acids constituted 65.7%. Major polar lipids
186
consisted
187
phosphatidylglycerol (PG) and eight unidentified lipids (L) (Supplementary Fig. S3). Q-10
of
diphosphatidylglycerol
(DPG),
phosphatidylethanolamine
(PE),
8 188
was the predominant respiratory quinone. The G + C content of DNA of strain AK40T was
189
65.5 mol% (Tm).
190
For phylogenetic assessment of strain AK40T, 1464 bp of 16S rRNA gene sequence
191
was amplified and sequenced. The sequence was compared with the database of type strains
192
with validly published prokaryotic names [Available online http://eztaxon-e.ezbiocloud.net/
193
(Kim et al., 2012)], which revealed its closeness to Phaeochromatium fluminis of the class
194
Gammaproteobacteria. Based on pair-wise sequence similarity, strain AK40T was closely
195
related to Phaeochromatium fluminis with 95.2% and to other members of the family
196
Chromatiaceae with 90.8 to 94.8% sequence similarities. Phylogenetic tree based on
197
maximum-likelihood method showed the clustering of strain AK40T with Phaeochromatium
198
fluminis and a clade comprising Marichromatium gracile, Halochromatium salexigens,
199
Thiohalocapsa halophila, Rhabdochromatium marinum and Thiorhodovibrio winogradskyi
200
(Fig. 2). In neighbour joining phylogenetic tree, strain AK40T clustered with all six taxa
201
together (Supplementary Fig. S1). In ARB fast DNAML tree, strain AK40T clustered with all
202
six taxa together (Supplementary Fig. S5).
203
Based on blast result strain AK40T was compared with Phaeochromatium fluminis
204
JA418T, which is the nearest neighbor and a number of differences were observed (Table 1).
205
For instance, strain AK40T differed from the Phaeochromatium fluminis JA418T with respect
206
to cell size, sulfur granules per cell, colour of cell suspension, NaCl range, temperature
207
growth range, pH growth range and optimum, growth modes, vitamin requirement, sulfide
208
tolerance, carbon substate utilization, nitrogen and sulfur sources, major fatty acids, polar
209
lipids and DNA G + C content (mol%) (Table 1). Based on the phylogenetic analysis a
210
comparison was made between the characteristics of strain AK40T and all the genera of the
211
family Chromatiaceae, Allochromatium, Halochromatium, Isochromatium, Lamprocystis,
212
Marichromatium, Nitrosococcus, Phaeochromatium, Rhabdochromatium, Rheinheimera,
213
Thermochromatium, Thioalkalicoccus, Thiobaca, Thiocapsa, Thiococcus, Thiocystis,
9 214
Thiodictyon, Thioflavicoccus, Thiohalocapsa, Thiolamprovum, Thiopedia, Thiophaeococcus,
215
Thiorhodococcus, Thiorhodovibrio and Thiospirillum (Table 2) and a number of differences
216
were observed. Thus, the cumulative differences that strain AK40T exhibited with the above
217
closely related type strains unambiguously supported the creation of a new genus
218
Phaeobacterium, and a new type species of this genus, for which the name Phaeobacterium
219
nitratireducens sp. nov., was proposed.
220 221
Description of Phaeobacterium gen. nov.
222
Phaeobacterium (Phae.o.bac.te'ri.um. Gr. adj. phaeos brown; L. neut. n. bacterium a stick,
223
staff, rod; N.L. neut. n. (nominative in apposition) phaeobacterium the brown rod).
224
Cells are Gram negative-staining, rod shaped, oxidase positive, catalase negative,
225
brown pigmented, motile and anaerobic. Cells grow photoorganoheterotrophically. The major
226
fatty acids are C12:0, C16:0, C18:1 7c and C16:1 7c/iso-C15:0 2OH (summed in feature 3). The
227
predominant
228
phosphatidylglycerol and eight unidentified lipids. Q-10 is the predominant respiratory
229
quinone. The DNA G + C content is 65.5 mol%. The genus is a member of the family
230
Chromatiaceae of the class Gammaproteobacteria in the phylum Proteobacteria. The type
231
species is Phaeobacterium nitratireducens.
polar
lipids
are
diphosphatidylglycerol,
phosphatidylethanolamine,
232 233
Description of Phaeobacterium nitratireducens sp. nov.
234
Phaeobacterium nitratireducens (ni.tra.ti.re.du′cens. N.L. n. nitras -atis, nitrate; L. part. adj.
235
reducens, leading back, bringing back and in chemistry converting to a different oxidation
236
state; N.L. part. adj. nitratireducens, reducing nitrate).
237
The species exhibits the following properties in addition to those given in the genus
238
description. Cells are 1.0–4.0 µm in long and 0.9-1.3 µm in wide, and they occur singly.
239
Internal photosynthetic membranes are of vesicular type. Colonies grown for 3 days at 30 oC
10 240
on modified Pfennig medium are circular, 0.5-1.5 mm in diameter, smooth, brown, opaque
241
and raised with entire margin. The in vivo absorption spectrum of intact cells in sucrose
242
exhibits maxima at 376, 488, 592, 702, 800, 836 nm. The absorption spectrum in acetone
243
extracted pigments gives absorption maxima at 446, 476, 506 nm. Major photosynthetic
244
pigments are bacteriochlorophyll a and rhodopin. The type strain is mesophilic (range 22–37
245
o
246
optimum 7.5) and does not require NaCl for growth but can tolerate concentrations of NaCl
247
up to 7% w/v with optimum growth between 1.5-3% NaCl. Growth occurs under anaerobic
248
conditions in the light (photoorganoheterotrophy). Photoautotrophic and chemoautotrophic
249
growth is not possible in the presence of sulfide and thiosulfate as electron donor and
250
NaHCO3 as carbon source. Chemoorganoheterotrophic growth is not possible in presence of
251
pyruvate as carbon source under aerobic in dark conditions and fermentative growth is also
252
not
253
Photoorganoheterotrophy with various organic compounds is the preferred growth mode.
254
Substrates that are utilized as carbon source/electron donors under photoheterotrophic
255
condition include acetate, fumarate, glycolate, malate, propionate, pyruvate, succinate,
256
valerate and yeast extract but not butyrate, citrate, crotonate, fructose, glucose, glycerol,
257
methanol, sucrose, casamino acids and trehalose. Ammonium chloride, glutamine, nitrate and
258
yeast extract are utilized as nitrogen source but not nitrite. Sulfate (2 mM), thiosulfate (2
259
mM) and sulfide (1.0 mM) are utilized as sulfur sources under photoheterotrophic condition
260
but not sulfur. Ferrous iron cannot act as electron donor. Thiamine and riboflavin are required
261
for growth.
262
The type strain AK40T (= JCM 19219T = MTCC 11824T) was isolated from a sediment
263
sample collected from Coringa mangrove forest, Andhra Pradesh, India.
C, optimum 30 oC), can grow both in acidic and alkaline conditions (pH range 5.0–9.0,
observed
under
264 265
Acknowledgements
anaerobic
dark
conditions
in
the
presence
of
pyruvate.
11 266
We thank Council of Scientific and Industrial Research (CSIR), Directors of IMTECH,
267
Chandigarh and NIO, Goa and Department of Biotechnology, Government of India for
268
encouragement and financial assistance. We would like to thank Mr. Deepak for his help in
269
sequencing 16S rRNA gene and Dr. Subash and Mr. Anil Theophilus for help in transmission
270
electron microscopy. TNRS is thankful to project PSC0105 for providing facilities and
271
funding respectively. IMTECH communication number is 0158/2013.
272 273 274 275 276 277 278 279 280
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18 447 448 449 450 451 452
Figure Legends:
453
Fig. 1. Electron micrograph of negatively stained cells of strain AK40T. Bar, 0.5 µm.
454
Fig. 2. Maximum-likelihood tree based on 16S rRNA gene sequences representing the
455
phylogenetic relationships between the strain AK40T and the type species of the genera
456
belongs to phototrophic members of the family Chromatiaceae. Numbers at the nodes are
457
bootstrap values >50%. Escherichia coli ATCC 11775T (X80725) was taken as the out group
458
organism. Bar, 0.02 substitutions per nucleotide position.
459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485
19 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501
Table 1. Phenotypic features that distinguish strain AK40T with the closely related type strain Phaeochromatium fluminis JA418T Data for strains AK40T and Phaeochromatium fluminis JA418T are from the present study. Both strains were rod-shaped, Gram-negative-staining and motile, optimally grew at 30 °C and 1.5-3 % NaCl (w/v), utilize acetate, fumarate, propionate, pyruvate, and succinate as carbon sources; ammonium chloride, glutamine as nitrogen sources; sulfate, sulfide and thiosulfate as sulfur sources. Both strains cannot grow under aerobic condition, does not utilize glucose, glycerol and citrate as carbon sources. +, Substrate utilized/present; (+), weak positive; −, substrate not utilized/absent; PLA, photolithoautotrophic; PLH, photolithoheterotrophic; POH, photoorganoheterotrophic; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; AL, unidentified aminolipid; L, unidentified lipid. Organic substrate utilization was tested during photoheterotrophic growth. For testing carbon sources, minimal media supplemented with respective carbon sources (5mM) were used. For Nitrogen sources, NH4Cl was replaced by different nitrogen sources with final concentration 0.12% (w/v). For sulfur sources, MgSO4.7H2O was replaced by respective sulfur sources like sodium thiosulfate, sodium sulfide of concentration (2mM) was added in media.
Characteristic Cell diameter (μm) Sulfur granules per cell (n) NaCl range (%) pH range pH optimum Temperature range (°C) Growth modes Colour of cell suspension Carotenoids Vitamin requirement Other growth factor requirement Sulfide tolerance (mM) Carbon/electron donors Oxoglutarate Methanol Fructose Valerate Malate Sucrose Nitrogen sources Nitrate Chemotaxonomy Major fatty acids
Phaeobacterium nitratireducens AK40T 2-4 x 0.9-1.3 No 0-7 5-9 7.5 22-37 PLH, POH Brown Spirilloxanthin group Thiamine and riboflavin None 1
Phaeochromatium fluminis JA418T 4-6 x 0.4-0.5 1-3 0.5-6.5 6.5-10 8-8.5 25-30 PLH, PLA, POH Reddish-brown Spirilloxanthin group None Yeast extract 4
+ (+) -
(+) (+) + (+)
+
-
C12:0, C16:0, C18:1 7c and C16:1 7c/iso-C15:0 2OH Polar lipids DPG, PE, PG, L1-8 DNA G + C content (mol%) 65.5 502 *Data taken from Shivali et al., 2012.
C16 : 0, C16 : 1ω7c/ C16 : 1 ω6c and C18 : 1 ω7c *DPG, PE, PG, AL1-2 *71.4
20 503 504 505 506 507 508 509 510 511
Table 2. Phenotypic features that distinguish strain AK40T with the type species of the genera, Chromatiaceae 1. Strain AK40T; 2. Allochromatium (Imhoff, 2005b); 3. Halochromatium (Imhoff & Caumette, 2005a); 4. Isochromatium (Imhoff, 2005c); 5. Lamprocystis (Imhoff, 2001); 6. Marichromatium (Imhoff, 2005d); 7. Nitrosococcus (Koops & Roser, 2005); 8. Phaeochromatium (Sucharita et al., 2010 & Shivali et al., 2012); 9. Rhabdochromatium (Imhoff, 2005e), 10. Rheinheimera (Brettar et al., 2002); 11. Thermochromatium (Imhoff & Madigan, 2005); 12. Thioalkalicoccus (Imhoff, 2005f); 13. Thiobaca (Rees et al., 2002); 14. Thiocapsa (Imhoff & Caumette, 2005b); 15. Thiococcus (Imhoff, 2005g); 16. Thiocystis (Imhoff, 2005h); 17. Thiodictyon (Imhoff, 2005i); 18. Thioflavicoccus (Imhoff & Pfennig, 2001); 19.Thiohalocapsa (Imhoff & Caumette, 2005c); 20. Thiolamprovum (Guyoneaud et al., 1998); 21. Thiopedia (Imhoff, 2005j); 22. Thiophaeococcus (Anil Kumar et al., 2008b); 23. Thiorhodococcus (Guyoneaud et al., 1997); 24. Thiorhodovibrio (Overmann et al., 1992); 25. Thiospirillum (Imhoff, 2005k).
Characteristics Cell shape Cell size (µm)
1 R 2-4
2 R 1-4
3 RO 2-4
5 SO 2-3.5
6 R 1-1.7
+ -
4 R 3.54.5 + -
Motility Slime capsule Gas vesicles Aggregate formation
+ -
+ -
Internal photosynthetic Membranes Colour of cell suspension Bacteriochlorophyll Carotenoid group
V
+ + + B
+ Spc
V
V
V
V
V
Rb
Ob
Pr
Pv
a RN
a RA
a SP
PvPuv a RA
pH optimum
7.5
6.5-7.6
Temperature range (oC) Salt requirement Growth mode(s)
2237 POH
25-35
7.27.6 20-35
7.07.3 20-30
PLA,
+ PLA, CLA, COH
6.57.6 2530 + PLA
Sulfate assimilation Vitamin B12 requirement DNA G+C content (mol%)
-
+ ±
+ ±
65.5
55.166.3
64.666.3
512 513 514 515 516 517 518
7 SE 1.52.5 + NA NA Si pi EI
8 R 0.40.5 + NA NA -
9 R 1.51.7 + NA NA -
10 RC 0.51.5 + NA NA -
11 R 1-2 + NA -
12 SO 1.31.8 -
V
V
-
V
T
Br Pur a SP Ok 6.57.6 25-37
NA
Db
BOB
NA
NA
a RN
a LY
DblLbl NA
NA
7.0
NA
7.0
NA
6.57.5 NA
4-30
PLA, CLA
+ PLA, CLA, COH
+ CLH
+ PLH
2035 + PLA
+ +
NA NA
+ -
NA NA
NA -
62.262.8
63.464.1
68.970.4
50.5
68-69
a RA
13 R 1.6
14 S 1.2-3
15 S 1.2-1.5
16 R, SO 2.5-3
17 R 1.5-2
+ NA Si/ Pi V
± tet
-
+ + Irr
V
T
Yb,O b b THS
Br
P, Rr
a LY
48-50
8.89.2 25
COH
PLA
+ PLA
+ NA
NA
+ -
NA -
60.4
47.848.9
60-63
63.664.8
a SP
19 S 1.52.5 + -
20 SO 2
21 SO 2-2.5
22 S 2-2.5
23 SO 1-2.0
NA + Irr
18 Co 0.8 -1 + -
25 RS 2.5-4
+ Si,Pi,irr
24 VS 1.21.6 + NA NA -
+ Plt
+ Rec
+ -
V
V
T
V
V
V
V
V
V
V
Ob
Puv
Puv
Pur
P, Rr
a SP
b THS
a RA
a RA
YbOb b THS
Pur
S-B
Bo
P
Y-Ob
a Ok
a SP
a OK
a LY
a SP
a SP
6.57.5 20-30
7.4-7.6
7.37.5 20
7.5
7.0-7.2
20-35
30-35
7.07.4 14-37
a LY, RN 7.0
NA
7.3
6.5 -7.5
2530 PL H, PO H NA
20-35
20-35
6.5 7.6 25-35
6.77.3 20-30
6.57.5 20-30
-§ PLA, POH, CLA, COH + -
± PLA
PLA, CLA, COH
PLA
+ PLA
+ PLA, POH, CLA, COH +
PLA, CLA
PLA
+ PLA
PLA CLA
+ PLA
PLA
-
± -
NA NA
+ -
-
-
-
-
+ -
+ +
63
63.366.3
69.469.9
61.367.9
65.366.3
66.5
65.966.6
64.566.5
62.563.5
68.5
66.8-67.0
61.0
45.5
37
Co, coccoid; R, rod; S, sphere; RO, rod or ovoid to spherical; SO, spherical to oval; RC, Rod to coccoid; SE, spherical to ellipsoidal; VS,vibroid to spirilloid, RS, rod to spiral; B, branching; Irr, Irregular; Plt, platelet; Rec, rectangular; Spc, Sin pairs clumps; Si, sin; pi, pairs; tet, tetrad; V, vesicular; T, Tubular; EI- extensive intracytoplasmic membrane systems arranged centrally in the protoplasm; Ob, orange brownish; Pv, pinkishviolet; Dbl, dark blue; Lbl, light blue; Yb, yellowish brown; Puv, purple-violet; Yb, yellow-beige; Pr, pinkish-red; Pur, purple-red; S-B, sandy brown-Baker’s chocolate; Bo, brown-orange; Rb, reddish-brown; Db, dark brown; Br, brownish; BOB, beige to orange-brown; P, pinkish; Rr, rose-red; RA, rhodopinal; SP, spirilloxanthin; RN, rhodopin; RA, rhodopinal; THS, tetrahydrospirilloxanthin; Ok, okenone; LY, lycopenal; §, marine strains may tolerate low concentrations of NaCl; PLA, photolithoautotrophic; PLH, photolithoheterotrophic; POH, photoorganoheterotrophic; CLA, chemolithoautotrophic; CLH, chemolithoheterotrophic; COH, chemoorganoheterotrophic; +, present; −, absent; ±, variable; NA, no data available.
+ NA NA -
20-25
Figure 1 Click here to download Figure: Figure 1.tiff
Figure 2 Click here to download Figure: Figure 2.pptx
Thiophaeococcus mangrovi JA304T (AM748925) Thiorhodococcus minor CE2203T (Y11316) Thiocapsa roseopersicina 1711T (AF113000)
Thiobaca trueperi BCHT (AJ404006) Lamprocystis roseopersicina DSM 229T (AJ006063)
50
Thiodictyon elegans DSM 232T (EF999973) Chromatium okenii DSM 169T (AJ223234)
59
Thiocystis violacea DSMZ 207T (Y11315) Thermochromatium tepidum DSM 3771T (M59150)
86
Allochromatium vinosum DSM 180T (FM178268)
Isochromatium buderi DSM 176T (AJ224430) Thiolamprovum pedioforme DSM 3802T (Y12297) Thioalkalicoccus limnaeus A26T (AJ277023)
64
Thioflavicoccus mobilis 8320 (AJ010125)
Phaeobacterium nitratireducens AK40T (HF968497) Phaeochromatium fluminis JA418T (FM210274) Marichromatium gracile BN 5210T (X93473)
Halochromatium salexigens 6310T (X98597)
95
Thiohalocapsa halophila 4270T (AJ002796)
53
87
Rhabdochromatium marinum DSM 5261T (X84316) Thiorhodovibrio winogradskyi MBIC2776 (AB016986) Escherichia coli ATCC 11775T (X80725)
0.02
Fig. 2
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International Journal of Systematic and Evolutionary Microbiology Phaeobacterium nitratireducens gen. nov., sp. nov., a phototrophic gammaproteobacterium isolated from a sediment sample of Coringa mangrove forest, India --Manuscript Draft-Manuscript Number:
IJS-D-14-00548R1
Full Title:
Phaeobacterium nitratireducens gen. nov., sp. nov., a phototrophic gammaproteobacterium isolated from a sediment sample of Coringa mangrove forest, India
Short Title:
Phaeobacterium nitratireducens gen. nov., sp. nov.
Article Type:
Standard
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Anil Kumar Pinnaka, Ph.D Institute of Microbial Technology Chandigarh, Chandigarh INDIA
First Author:
Nupur Nupur, M.Sc
Order of Authors:
Nupur Nupur, M.Sc Naga Radha Srinivas Tanuku, Ph.D Takaichi Shinichi, Ph.D Anil Kumar Pinnaka, Ph.D
Manuscript Region of Origin:
INDIA
Abstract:
The novel, brown coloured, Gram-negative-staining, rod shaped, motile phototrophic bacterium designated strain AK40T, was isolated in pure culture from a sediment sample collected from Coringa mangrove forest. Strain AK40T, containing bacteriochlorophyll a and carotenoid of rhodopin as major photosynthetic pigments. Strain AK40T was able to grow photoheterotrophically and could utilize a number of organic substrates. It was unable to grow photoautotrophically and did not utilize sulfide and thiosulfate as electron donors. Thiamine and riboflavin were required for growth of strain AK40T. The dominant fatty acids were C12:0, C16:0, C18:1 7c and C16:1 7c and/or iso-C15:0 2OH (summed feature 3). Polar lipid content of strain AK40T was found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and eight unidentified lipids. Q-10 was the predominant respiratory quinone. The G + C content of DNA of the strain AK40T was 65.5 mol%. The 16S rRNA gene sequence comparisons indicated that the isolate represented a member of the family Chromatiaceae within the class Gammaproteobacteria. The 16S rRNA gene sequence analysis indicated that strain AK40T was closely related to Phaeochromatium fluminis with 95.2% pair-wise sequence similarity and with other species of the family, sequence similarity between 90.8 to 94.8%. Distinct morphological, physiological and genotypic differences from previously described taxa supported the classification of this isolate as a representative of a novel species in a new genus, for which the name Phaeobacterium nitratireducens gen. nov., sp. nov. is proposed. The type strain of Phaeobacterium nitratireducens is AK40T (= JCM 19219T = MTCC 11824T).
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): PAPER-PHAEOBACTERIUM NITRATIREDUCENS 24.03
1 1
26-03-2015
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Phaeobacterium nitratireducens gen. nov., sp.
3
nov., a phototrophic gammaproteobacterium
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isolated from a sediment sample of Coringa
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mangrove forest, India
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Nupur1, Naga Radha Srinivas Tanuku2, Takaichi
8
Shinichi3, and Anil Kumar Pinnaka1*
9 10
1
11
CSIR-Institute of Microbial Technology, Sector 39A,
12
Chandigarh – 160 036, INDIA
13
2
14
Centre, 176, Lawsons Bay Colony, Visakhapatnam-
15
530017, INDIA
16
3
17
Kosugi-cho, Nakahara, Kawasaki 211-0063, Japan
Microbial Type Culture Collection and Gene Bank,
CSIR-National Institute of Oceanography, Regional
Nippon Medical School, Department of Biology,
18 19
Address for correspondence*
Dr. P. Anil Kumar
20
Microbial Type Culture Collection and Gene Bank,
21
Institute of Microbial Technology (CSIR), Sector 39A,
22
Chandigarh – 160 036, INDIA
23
Email: [email protected]
24
Telephone: 00-91-172-6665170
25 26
Running title
Phaeobacterium nitratireducens gen. nov., sp. nov.
Subject category
New taxa (Gammaproteobacteria)
27 28 29 30
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
31
AK40T is HF968497.
32 33
All authors contributed equally for the present work.
2 34
The novel, brown coloured, Gram-negative-staining, rod shaped, motile phototrophic
35
purple sulphur bacterium designated strain AK40T, was isolated in pure culture from a
36
sediment sample collected from Coringa mangrove forest. Strain AK40T, containing
37
bacteriochlorophyll a and carotenoid of rhodopin as major photosynthetic pigments. Strain
38
AK40T was able to grow photoheterotrophically and could utilize a number of organic
39
substrates. It was unable to grow photoautotrophically and did not utilize sulfide and
40
thiosulfate as electron donors. Thiamine and riboflavin were required for growth of strain
41
AK40T. The dominant fatty acids were C12:0, C16:0, C18:1 7c and C16:1 7c and/or iso-C15:0
42
2OH (summed feature 3). Polar lipid content of strain AK40T was found to be
43
diphosphatidylglycerol,
44
unidentified lipids. Q-10 was the predominant respiratory quinone. The G + C content of
45
DNA of the strain AK40T was 65.5 mol%. The 16S rRNA gene sequence comparisons
46
indicated that the isolate represented a member of the family Chromatiaceae within the class
47
Gammaproteobacteria. The 16S rRNA gene sequence analysis indicated that strain AK40T
48
was closely related to Phaeochromatium fluminis with 95.2% pair-wise sequence similarity
49
and with other species of the family, sequence similarity between 90.8 to 94.8%. Based on
50
the sequence comparison data, strain AK40T was positioned distinctly outside the group
51
formed
52
Thiohalocapsa,
53
physiological and genotypic differences from previously described taxa supported the
54
classification of this isolate as a representative of a novel species in a new genus, for which
55
the name Phaeobacterium nitratireducens gen. nov., sp. nov. is proposed. The type strain of
56
Phaeobacterium nitratireducens is AK40T (= JCM 19219T = MTCC 11824T).
57
Key words: Phaeobacterium nitratireducens, Coringa mangrove forest, 16S rRNA gene
58
based phylogeny, chemotaxonomy, Gammaproteobacteria
by
the
6
phosphatidylethanolamine,
genera
Phaeochromatium,
Rhabdochromatium
and
phosphatidylglycerol
Marichromatium,
Thiorhodovibrio.
and
eight
Halochromatium,
Distinct
morphological,
3 59
Mangrove habitats are vital to humankind by virtue of their use and aesthetic value.
60
Mangroves serve as nursery, feeding and spawning grounds for commercial fin and shell
61
fishes. Mangrove habitats are found in locations where a river enters the sea and these
62
dynamic ecosystems support numerous diversified soil microbes. Mangroves are found in
63
different states of India such as: Andhra Pradesh, Andaman and Nicobar Islands, Goa,
64
Gujarat, Karnataka, Kerala, Maharashtra, Tamil Nadu and West Bengal. The significance of
65
microbes in aquatic ecosystems is recognized well by many pioneer studies (Longhurst &
66
Harrison, 1989; Wheeler & Kirchman, 1986). Microbes play different roles such as: primary
67
producers, secondary producers, consumers and decomposers. The activities of these
68
microbes (photosynthetic, lithotrophic and organotrophic) are important for maintaining
69
balance in the environment; which in turn, is very vital for the maintenance of other life
70
forms. A number of novel bacterial taxa are identified from mangrove habitats. The family
71
Chromatiaceae was proposed by Bavendamm (1924), with the type genus Chromatium and
72
later emended by Imhoff (1984). Members of the Chromatiaceae are Gram negative, ovoid,
73
rod, spherical, spiral, or vibrioid shaped, motile by flagella or non-motile, multiply by binary
74
fission, may contain gas vesicles and consist of internal photosynthetic membranes which are
75
either tubular or vescicular type. Cells in majority of the taxa contain photosynthetic
76
pigments such as: bacteriochlorophyll a or b and carotenoids of the okenone, rhodopinal, or
77
spirilloxanthin groups (Takaichi, 1999). Cultures appear in different colours like orange-
78
brown to brownish red or pink, purple-red, and purple-violet depending on the type of
79
carotenoids present. Cells grow under anoxic conditions in the presence of light
80
(photolithoautotropically) with sulfide or sulfur as electron donor and deposit sulfur granules
81
within the cells as an intermediate oxidation product and may convert to sulfate; and can use
82
hydrogen or reduced iron as an electron donor and also grow mixotrophically and
83
photoassimilate a number of organic substrates. Some taxa are also capable to grow
84
photoorganoheterotrophically. Most of the taxa are strictly anaerobic and obligately
4 85
phototrophic but others grow chemolithoautotrophically or chemoorganoheterotrophically
86
under microoxic to oxic conditions in the dark. Carbon fixation was by reductive pentose
87
phosphate cycle in all species tested so far. Several taxa can fix N2 and store elemental sulfur,
88
poly-β-hydroxybutyrate, polysaccharides and polyphosphate. Some taxa may require
89
different growth factors. Chromatiaceae members commonly dwell in the areas where anoxic
90
conditions prevail with sulfide or sulfur sources with illumination, of diverse aquatic habitats
91
and sediments like ditches, estuaries, lakes, marine habitats, moist and muddy soils, ponds,
92
rivers, salt and soda lakes and sulfur springs. The G + C content of the DNA among the
93
species varies between 45.5-70.4 mol% (Imhoff, 2005a). In the course of bacterial diversity
94
studies of mangrove samples, a bacterial strain, AK40T, was isolated from a mangrove
95
sediment sample collected near Coringa, Andhra Pradesh, India. Phylogenetic analysis based
96
on 16S rRNA gene sequence revealed that strain AK40T was closely related to the genus
97
Phaeochromatium of the family Chromatiaceae. In this study, a polyphasic approach,
98
including genotypic, phenotypic and chemotaxonomic characterizations, was used to
99
determine the taxonomic position of strain AK40T.
100 101
Strain AK40T was isolated from the sediment sample collected from Coringa
102
mangrove forest, India. The temperature and pH of the sediment sample was 30 oC and 7.5,
103
respectively.
104
photolithoheterotrophic enrichments of the sediment sample, and subsequent purification was
105
performed as previously described (Anil Kumar et al., 2008a, b) in modified Pfennig medium
106
(Pfennig & Trüper, 1992) supplemented with NaCl (2 % w/v), pyruvate (0.3 % w/v), sodium
107
thiosulfate (2 mM) and ammonium chloride (0.12 % w/v). Modified Pfennig medium was
108
used throughout the study unless mentioned otherwise. Preservation was done at -80°C in
109
modified Pfennig broth with 20% glycerol.
Anoxygenic
phototrophic
bacterium
AK40T
was
isolated
using
5 110
Colony morphology was studied after 72 hrs growth of the strain on modified Pfennig
111
medium at 30 oC under 2000 lux illumination, anaerobically. The isolated pure colony was
112
checked for cell morphology and motility by using phase contrast microscopy (Olympus,
113
USA) at 1000X magnification and also by transmission electron microscope (Jeol JEM 2100)
114
at operating voltage of 200 kV. Gram reaction was determined by using the Gram staining kit
115
from HIMEDIA as described by manufacturer’s protocol.
116
Physiological and biochemical characteristics were determined as previously
117
described (Anil Kumar et al., 2008a, b; Srinivas et al., 2006). In vivo absorption spectrum
118
was measured with a Spectronic Genesys 2 spectrophotometer in sucrose solution (Trüper &
119
Pfennig, 1981). Absorption spectrum of pigments extracted with acetone was also recorded.
120
Carotenoid composition was analysed by using C18-HPLC (Takaichi & Shimada, 1992).
121
Strains AK40T and Phaeochromatium fluminis JA418T were grown on modified
122
Pfennig medium with 2% NaCl at 30 oC under 2000 lux illumination, anaerobically for 3
123
days to prepare fatty acid methyl esters (classical methods) and were analysed using Sherlock
124
Microbial Identification System (MIDI-6890 with database TSBA50) by the protocol
125
described by the manufacturer. At the time of harvesting, the cells were at logarithmic phase
126
of growth. Freeze-dried cells were analysed for polar lipids and quinones. Cells were
127
extracted for the polar lipid analysis (Bligh & Dyer, 1959) and analyzed by 2D thin-layer
128
chromatography followed by spraying with appropriate detection reagents (5% ethanolic
129
molybdatophosphoric acid, molybdenum blue, ninhydrin and molisch reagents) (Komagata &
130
Suzuki, 1987). Genomic DNA was isolated by using the procedure of Marmur (1961) and the
131
DNA G + C content was determined from melting point (Tm) curves (Sly et al., 1986)
132
obtained by using Lambda 35; Perkin Elmer spectrophotometer equipped with Templab 2.0
133
software package.
134
For 16S rRNA gene sequencing, DNA was prepared using a bacterial DNA isolation
135
kit (Qiagen). The 16S rRNA gene was amplified by PCR using universal bacterial primers
6 136
27f (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492r (5'-TAC GGY TAC CTT GTT
137
ACG ACT T-3'). The PCR product was purified using QIA quick PCR purification kit
138
(Qiagen) and sequenced using an ABI PRISM model 3700 automatic DNA sequencer and
139
Big Dye Terminator cycle sequencing kit (Applied Biosystems). The 16S rRNA gene
140
sequence of strain AK40T was subjected to BLAST sequence similarity search (Altschul et
141
al., 1990) and EzTaxon-e server (Kim et al., 2012) to identify the nearest taxa. Based on
142
BLAST results all 16S rRNA gene sequences of type species of phototrophic members of the
143
genera belonging to the family Chromatiaceae were downloaded from the NCBI database
144
(http://www.ncbi.nlm.nih.gov) and aligned using the CLUSTAL_W program in MEGA5
145
(Tamura et al., 2011). The evolutionary history was inferred by using the maximum-
146
likelihood method (Tamura & Nei, 1993), neighbor-joining (NJ) method (Saitou & Nei,
147
1987) and maximum-parsimony method (Nei & Kumar, 2000) using the MEGA5 package
148
(Tamura et al., 2011). The percentage of trees in which the associated taxa clustered together
149
is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the
150
number of substitutions per site. All positions containing gaps and missing data were
151
eliminated. There were a total of 1355 positions in the final dataset. Evolutionary analyses
152
were conducted in MEGA5 (Tamura et al., 2011). The percentage of replicate trees in which
153
the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to
154
the branches (Felsenstein, 1985). Fast DNAML tree was constructed using ARB software
155
package (Ludwig et al., 2004).
156 157
Cells of strain AK40T were Gram-negative-staining, rod-shaped, brown pigmented,
158
motile and multiplied by binary fission. Cells were 1.0–4.0 µm in long and 0.9–1.3 µm in
159
wide (Fig. 1). Colonies were 0.5-1.5 mm in diameter, circular, smooth, brown, opaque,
160
convex and raised with entire margin on modified Pfennig medium. Strain AK40T grow
161
photoorganoheterotrophically (optimum light intensity is 2000 lux). Photolithoautotrophic
7 162
growth [anaerobic, light (2000 lux), Na2S/Na2S2O3 (0.5 mM), NaHCO3 (0.1%, w/v)],
163
chemolithoautotrophic growth [aerobic, dark, Na2S2O3 (0.5 mM), NaHCO3 (0.1%, w/v)],
164
chemoorganoheterotrophic growth [aerobic, dark with glucose/pyruvate (0.3%, w/v)] and
165
fermentative growth [anaerobic, dark with glucose/pyruvate (0.3%, w/v)] were not detected.
166
Salt (NaCl) was not required for growth, but tolerated up to 7% (w/v, NaCl), the optimum
167
NaCl concentration being 1.5–3% (w/v). The pH range for growth of strain AK40T was 5–9
168
with the optimum at 7.5. The temperature range was from 22 to 37 oC and the optimum
169
temperature for growth was 30 oC. Growth factors such as thiamine and riboflavin were
170
required for growth. The colour of photosynthetically grown cell suspensions was brown. The
171
whole-cell absorption spectrum (Supplementary Fig. S2a) of strain AK40T in sucrose solution
172
gave maxima at 376, 488, 592, 702, 800, 836 nm, confirming the presence of
173
bacteriochlorophyll a and carotenoids. The absorption spectrum of acetone extracted
174
pigments (Supplementary Fig. S2b) gave maxima at 446, 476, 506 nm, indicating the
175
presence of carotenoids. The carotenoid composition of strain AK40T, as determined by using
176
C18-HPLC analysis, was lycopene (5%), rhodopin (71%), anhydrorhodovibrin (15%),
177
rhodovibrin (3%) and spirilloxanthin (6%) (Supplementary Fig. S4). They were identified
178
based on absorption spectra in the C18-HPLC eluent of methanol, and the retention times on
179
the C18-HPLC (Takaichi & Shimada, 1992). Other physiological characteristics are given in
180
the species description, Tables 1 and 2.
181
The cellular fatty acid composition of the strain AK40T showed a profile of eleven
182
fatty acids of C12:0 (5.6%), C14:0 (0.6%), C16:0 (22.5%), antesio-C16:0 (1.1%), antesio-C17:0
183
(1.0%), C18:0 (0.7%), C18:1 2OH (1.9%), C18:1 7c (32.0%), C18:3 6,9,12c (2.8%), C12:0
184
aldehyde/iso-C16:1I/C14:03OH (summed feature 2) (0.9%), and C16:1 7c/iso-C15:0 2OH
185
(summed feature 3) (30.9%). Unsaturated fatty acids constituted 65.7%. Major polar lipids
186
consisted
187
phosphatidylglycerol (PG) and eight unidentified lipids (L) (Supplementary Fig. S3). Q-10
of
diphosphatidylglycerol
(DPG),
phosphatidylethanolamine
(PE),
8 188
was the predominant respiratory quinone. The G + C content of DNA of strain AK40T was
189
65.5 mol% (Tm).
190
For phylogenetic assessment of strain AK40T, 1464 bp of 16S rRNA gene sequence
191
was amplified and sequenced. The sequence was compared with the database of type strains
192
with validly published prokaryotic names [Available online http://eztaxon-e.ezbiocloud.net/
193
(Kim et al., 2012)], which revealed its closeness to Phaeochromatium fluminis of the class
194
Gammaproteobacteria. Based on pair-wise sequence similarity, strain AK40T was closely
195
related to Phaeochromatium fluminis with 95.2% and to other members of the family
196
Chromatiaceae with 90.8 to 94.8% sequence similarities. Phylogenetic tree based on
197
maximum-likelihood method showed the clustering of strain AK40T with Phaeochromatium
198
fluminis and a clade comprising Marichromatium gracile, Halochromatium salexigens,
199
Thiohalocapsa halophila, Rhabdochromatium marinum and Thiorhodovibrio winogradskyi
200
(Fig. 2). In neighbour joining phylogenetic tree, strain AK40T clustered with all six taxa
201
together (Supplementary Fig. S1). In ARB fast DNAML tree, strain AK40T clustered with all
202
six taxa together (Supplementary Fig. S5).
203
Based on blast result strain AK40T was compared with Phaeochromatium fluminis
204
JA418T, which is the nearest neighbor and a number of differences were observed (Table 1).
205
For instance, strain AK40T differed from the Phaeochromatium fluminis JA418T with respect
206
to cell size, sulfur granules per cell, colour of cell suspension, NaCl range, temperature
207
growth range, pH growth range and optimum, growth modes, vitamin requirement, sulfide
208
tolerance, carbon substate utilization, nitrogen and sulfur sources, major fatty acids, polar
209
lipids and DNA G + C content (mol%) (Table 1). Based on the phylogenetic analysis a
210
comparison was made between the characteristics of strain AK40T and all the genera of the
211
family Chromatiaceae, Allochromatium, Halochromatium, Isochromatium, Lamprocystis,
212
Marichromatium, Nitrosococcus, Phaeochromatium, Rhabdochromatium, Rheinheimera,
213
Thermochromatium, Thioalkalicoccus, Thiobaca, Thiocapsa, Thiococcus, Thiocystis,
9 214
Thiodictyon, Thioflavicoccus, Thiohalocapsa, Thiolamprovum, Thiopedia, Thiophaeococcus,
215
Thiorhodococcus, Thiorhodovibrio and Thiospirillum (Table 2) and a number of differences
216
were observed. Thus, the cumulative differences that strain AK40T exhibited with the above
217
closely related type strains unambiguously supported the creation of a new genus
218
Phaeobacterium, and a new type species of this genus, for which the name Phaeobacterium
219
nitratireducens sp. nov., was proposed.
220 221
Description of Phaeobacterium gen. nov.
222
Phaeobacterium (Phae.o.bac.te'ri.um. Gr. adj. phaeos brown; L. neut. n. bacterium a stick,
223
staff, rod; N.L. neut. n. (nominative in apposition) phaeobacterium the brown rod).
224
Cells are Gram negative-staining, rod shaped, oxidase positive, catalase negative,
225
brown pigmented, motile and anaerobic. Cells grow photoorganoheterotrophically. The major
226
fatty acids are C12:0, C16:0, C18:1 7c and C16:1 7c/iso-C15:0 2OH (summed in feature 3). The
227
predominant
228
phosphatidylglycerol and eight unidentified lipids. Q-10 is the predominant respiratory
229
quinone. The DNA G + C content is 65.5 mol%. The genus is a member of the family
230
Chromatiaceae of the class Gammaproteobacteria in the phylum Proteobacteria. The type
231
species is Phaeobacterium nitratireducens.
polar
lipids
are
diphosphatidylglycerol,
phosphatidylethanolamine,
232 233
Description of Phaeobacterium nitratireducens sp. nov.
234
Phaeobacterium nitratireducens (ni.tra.ti.re.du′cens. N.L. n. nitras -atis, nitrate; L. part. adj.
235
reducens, leading back, bringing back and in chemistry converting to a different oxidation
236
state; N.L. part. adj. nitratireducens, reducing nitrate).
237
The species exhibits the following properties in addition to those given in the genus
238
description. Cells are 1.0–4.0 µm in long and 0.9-1.3 µm in wide, and they occur singly.
239
Internal photosynthetic membranes are of vesicular type. Colonies grown for 3 days at 30 oC
10 240
on modified Pfennig medium are circular, 0.5-1.5 mm in diameter, smooth, brown, opaque
241
and raised with entire margin. The in vivo absorption spectrum of intact cells in sucrose
242
exhibits maxima at 376, 488, 592, 702, 800, 836 nm. The absorption spectrum in acetone
243
extracted pigments gives absorption maxima at 446, 476, 506 nm. Major photosynthetic
244
pigments are bacteriochlorophyll a and rhodopin. The type strain is mesophilic (range 22–37
245
o
246
optimum 7.5) and does not require NaCl for growth but can tolerate concentrations of NaCl
247
up to 7% w/v with optimum growth between 1.5-3% NaCl. Growth occurs under anaerobic
248
conditions in the light (photoorganoheterotrophy). Photoautotrophic and chemoautotrophic
249
growth is not possible in the presence of sulfide and thiosulfate as electron donor and
250
NaHCO3 as carbon source. Chemoorganoheterotrophic growth is not possible in presence of
251
pyruvate as carbon source under aerobic in dark conditions and fermentative growth is also
252
not
253
Photoorganoheterotrophy with various organic compounds is the preferred growth mode.
254
Substrates that are utilized as carbon source/electron donors under photoheterotrophic
255
condition include acetate, fumarate, glycolate, malate, propionate, pyruvate, succinate,
256
valerate and yeast extract but not butyrate, citrate, crotonate, fructose, glucose, glycerol,
257
methanol, sucrose, casamino acids and trehalose. Ammonium chloride, glutamine, nitrate and
258
yeast extract are utilized as nitrogen source but not nitrite. Sulfate (2 mM), thiosulfate (2
259
mM) and sulfide (1.0 mM) are utilized as sulfur sources under photoheterotrophic condition
260
but not sulfur. Ferrous iron cannot act as electron donor. Thiamine and riboflavin are required
261
for growth.
262
The type strain AK40T (= JCM 19219T = MTCC 11824T) was isolated from a sediment
263
sample collected from Coringa mangrove forest, Andhra Pradesh, India.
C, optimum 30 oC), can grow both in acidic and alkaline conditions (pH range 5.0–9.0,
observed
under
264 265
Acknowledgements
anaerobic
dark
conditions
in
the
presence
of
pyruvate.
11 266
We thank Council of Scientific and Industrial Research (CSIR), Directors of IMTECH,
267
Chandigarh and NIO, Goa and Department of Biotechnology, Government of India for
268
encouragement and financial assistance. We would like to thank Mr. Deepak for his help in
269
sequencing 16S rRNA gene and Dr. Subash and Mr. Anil Theophilus for help in transmission
270
electron microscopy. TNRS is thankful to project PSC0105 for providing facilities and
271
funding respectively. IMTECH communication number is 0158/2013.
272 273 274 275 276 277 278 279 280
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18 447 448 449 450 451 452
Figure Legends:
453
Fig. 1. Electron micrograph of negatively stained cells of strain AK40T. Bar, 0.5 µm.
454
Fig. 2. Maximum-likelihood tree based on 16S rRNA gene sequences representing the
455
phylogenetic relationships between the strain AK40T and the type species of the genera
456
belongs to phototrophic members of the family Chromatiaceae. Numbers at the nodes are
457
bootstrap values >50%. Escherichia coli ATCC 11775T (X80725) was taken as the out group
458
organism. Bar, 0.02 substitutions per nucleotide position.
459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485
19 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501
Table 1. Phenotypic features that distinguish strain AK40T with the closely related type strain Phaeochromatium fluminis JA418T Data for strains AK40T and Phaeochromatium fluminis JA418T are from the present study. Both strains were rod-shaped, Gram-negative-staining and motile, optimally grew at 30 °C and 1.5-3 % NaCl (w/v), utilize acetate, fumarate, propionate, pyruvate, and succinate as carbon sources; ammonium chloride, glutamine as nitrogen sources; sulfate, sulfide and thiosulfate as sulfur sources. Both strains cannot grow under aerobic condition, does not utilize glucose, glycerol and citrate as carbon sources. +, Substrate utilized/present; (+), weak positive; −, substrate not utilized/absent; PLA, photolithoautotrophic; PLH, photolithoheterotrophic; POH, photoorganoheterotrophic; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; AL, unidentified aminolipid; L, unidentified lipid. Organic substrate utilization was tested during photoheterotrophic growth. For testing carbon sources, minimal media supplemented with respective carbon sources (5mM) were used. For Nitrogen sources, NH4Cl was replaced by different nitrogen sources with final concentration 0.12% (w/v). For sulfur sources, MgSO4.7H2O was replaced by respective sulfur sources like sodium thiosulfate, sodium sulfide of concentration (2mM) was added in media.
Characteristic Cell diameter (μm) Sulfur granules per cell (n) NaCl range (%) pH range pH optimum Temperature range (°C) Growth modes Colour of cell suspension Carotenoids Vitamin requirement Other growth factor requirement Sulfide tolerance (mM) Carbon/electron donors Oxoglutarate Methanol Fructose Valerate Malate Sucrose Nitrogen sources Nitrate Chemotaxonomy Major fatty acids
Phaeobacterium nitratireducens AK40T 2-4 x 0.9-1.3 No 0-7 5-9 7.5 22-37 PLH, POH Brown Spirilloxanthin group Thiamine and riboflavin None 1
Phaeochromatium fluminis JA418T 4-6 x 0.4-0.5 1-3 0.5-6.5 6.5-10 8-8.5 25-30 PLH, PLA, POH Reddish-brown Spirilloxanthin group None Yeast extract 4
+ (+) -
(+) (+) + (+)
+
-
C12:0, C16:0, C18:1 7c and C16:1 7c/iso-C15:0 2OH Polar lipids DPG, PE, PG, L1-8 DNA G + C content (mol%) 65.5 502 *Data taken from Shivali et al., 2012.
C16 : 0, C16 : 1ω7c/ C16 : 1 ω6c and C18 : 1 ω7c *DPG, PE, PG, AL1-2 *71.4
20 503 504 505 506 507 508 509 510 511
Table 2. Phenotypic features that distinguish strain AK40T with the type species of the genera, Chromatiaceae 1. Strain AK40T; 2. Allochromatium (Imhoff, 2005b); 3. Halochromatium (Imhoff & Caumette, 2005a); 4. Isochromatium (Imhoff, 2005c); 5. Lamprocystis (Imhoff, 2001); 6. Marichromatium (Imhoff, 2005d); 7. Nitrosococcus (Koops & Roser, 2005); 8. Phaeochromatium (Sucharita et al., 2010 & Shivali et al., 2012); 9. Rhabdochromatium (Imhoff, 2005e), 10. Rheinheimera (Brettar et al., 2002); 11. Thermochromatium (Imhoff & Madigan, 2005); 12. Thioalkalicoccus (Imhoff, 2005f); 13. Thiobaca (Rees et al., 2002); 14. Thiocapsa (Imhoff & Caumette, 2005b); 15. Thiococcus (Imhoff, 2005g); 16. Thiocystis (Imhoff, 2005h); 17. Thiodictyon (Imhoff, 2005i); 18. Thioflavicoccus (Imhoff & Pfennig, 2001); 19.Thiohalocapsa (Imhoff & Caumette, 2005c); 20. Thiolamprovum (Guyoneaud et al., 1998); 21. Thiopedia (Imhoff, 2005j); 22. Thiophaeococcus (Anil Kumar et al., 2008b); 23. Thiorhodococcus (Guyoneaud et al., 1997); 24. Thiorhodovibrio (Overmann et al., 1992); 25. Thiospirillum (Imhoff, 2005k).
Characteristics Cell shape Cell size (µm)
1 R 2-4
2 R 1-4
3 RO 2-4
5 SO 2-3.5
6 R 1-1.7
+ -
4 R 3.54.5 + -
Motility Slime capsule Gas vesicles Aggregate formation
+ -
+ -
Internal photosynthetic Membranes Colour of cell suspension Bacteriochlorophyll Carotenoid group
V
+ + + B
+ Spc
V
V
V
V
V
Rb
Ob
Pr
Pv
a RN
a RA
a SP
PvPuv a RA
pH optimum
7.5
6.5-7.6
Temperature range (oC) Salt requirement Growth mode(s)
2237 POH
25-35
7.27.6 20-35
7.07.3 20-30
PLA,
+ PLA, CLA, COH
6.57.6 2530 + PLA
Sulfate assimilation Vitamin B12 requirement DNA G+C content (mol%)
-
+ ±
+ ±
65.5
55.166.3
64.666.3
512 513 514 515 516 517 518
7 SE 1.52.5 + NA NA Si pi EI
8 R 0.40.5 + NA NA -
9 R 1.51.7 + NA NA -
10 RC 0.51.5 + NA NA -
11 R 1-2 + NA -
12 SO 1.31.8 -
V
V
-
V
T
Br Pur a SP Ok 6.57.6 25-37
NA
Db
BOB
NA
NA
a RN
a LY
DblLbl NA
NA
7.0
NA
7.0
NA
6.57.5 NA
4-30
PLA, CLA
+ PLA, CLA, COH
+ CLH
+ PLH
2035 + PLA
+ +
NA NA
+ -
NA NA
NA -
62.262.8
63.464.1
68.970.4
50.5
68-69
a RA
13 R 1.6
14 S 1.2-3
15 S 1.2-1.5
16 R, SO 2.5-3
17 R 1.5-2
+ NA Si/ Pi V
± tet
-
+ + Irr
V
T
Yb,O b b THS
Br
P, Rr
a LY
48-50
8.89.2 25
COH
PLA
+ PLA
+ NA
NA
+ -
NA -
60.4
47.848.9
60-63
63.664.8
a SP
19 S 1.52.5 + -
20 SO 2
21 SO 2-2.5
22 S 2-2.5
23 SO 1-2.0
NA + Irr
18 Co 0.8 -1 + -
25 RS 2.5-4
+ Si,Pi,irr
24 VS 1.21.6 + NA NA -
+ Plt
+ Rec
+ -
V
V
T
V
V
V
V
V
V
V
Ob
Puv
Puv
Pur
P, Rr
a SP
b THS
a RA
a RA
YbOb b THS
Pur
S-B
Bo
P
Y-Ob
a Ok
a SP
a OK
a LY
a SP
a SP
6.57.5 20-30
7.4-7.6
7.37.5 20
7.5
7.0-7.2
20-35
30-35
7.07.4 14-37
a LY, RN 7.0
NA
7.3
6.5 -7.5
2530 PL H, PO H NA
20-35
20-35
6.5 7.6 25-35
6.77.3 20-30
6.57.5 20-30
-§ PLA, POH, CLA, COH + -
± PLA
PLA, CLA, COH
PLA
+ PLA
+ PLA, POH, CLA, COH +
PLA, CLA
PLA
+ PLA
PLA CLA
+ PLA
PLA
-
± -
NA NA
+ -
-
-
-
-
+ -
+ +
63
63.366.3
69.469.9
61.367.9
65.366.3
66.5
65.966.6
64.566.5
62.563.5
68.5
66.8-67.0
61.0
45.5
37
Co, coccoid; R, rod; S, sphere; RO, rod or ovoid to spherical; SO, spherical to oval; RC, Rod to coccoid; SE, spherical to ellipsoidal; VS,vibroid to spirilloid, RS, rod to spiral; B, branching; Irr, Irregular; Plt, platelet; Rec, rectangular; Spc, Sin pairs clumps; Si, sin; pi, pairs; tet, tetrad; V, vesicular; T, Tubular; EI- extensive intracytoplasmic membrane systems arranged centrally in the protoplasm; Ob, orange brownish; Pv, pinkishviolet; Dbl, dark blue; Lbl, light blue; Yb, yellowish brown; Puv, purple-violet; Yb, yellow-beige; Pr, pinkish-red; Pur, purple-red; S-B, sandy brown-Baker’s chocolate; Bo, brown-orange; Rb, reddish-brown; Db, dark brown; Br, brownish; BOB, beige to orange-brown; P, pinkish; Rr, rose-red; RA, rhodopinal; SP, spirilloxanthin; RN, rhodopin; RA, rhodopinal; THS, tetrahydrospirilloxanthin; Ok, okenone; LY, lycopenal; §, marine strains may tolerate low concentrations of NaCl; PLA, photolithoautotrophic; PLH, photolithoheterotrophic; POH, photoorganoheterotrophic; CLA, chemolithoautotrophic; CLH, chemolithoheterotrophic; COH, chemoorganoheterotrophic; +, present; −, absent; ±, variable; NA, no data available.
+ NA NA -
20-25
Figure 1 Click here to download Figure: Figure 1.tiff
Figure 2 Click here to download Figure: Figure 2.pptx
Thiophaeococcus mangrovi JA304T (AM748925) Thiorhodococcus minor CE2203T (Y11316) Thiocapsa roseopersicina 1711T (AF113000)
Thiobaca trueperi BCHT (AJ404006) Lamprocystis roseopersicina DSM 229T (AJ006063)
50
Thiodictyon elegans DSM 232T (EF999973) Chromatium okenii DSM 169T (AJ223234)
59
Thiocystis violacea DSMZ 207T (Y11315) Thermochromatium tepidum DSM 3771T (M59150)
86
Allochromatium vinosum DSM 180T (FM178268)
Isochromatium buderi DSM 176T (AJ224430) Thiolamprovum pedioforme DSM 3802T (Y12297) Thioalkalicoccus limnaeus A26T (AJ277023)
64
Thioflavicoccus mobilis 8320 (AJ010125)
Phaeobacterium nitratireducens AK40T (HF968497) Phaeochromatium fluminis JA418T (FM210274) Marichromatium gracile BN 5210T (X93473)
Halochromatium salexigens 6310T (X98597)
95
Thiohalocapsa halophila 4270T (AJ002796)
53
87
Rhabdochromatium marinum DSM 5261T (X84316) Thiorhodovibrio winogradskyi MBIC2776 (AB016986) Escherichia coli ATCC 11775T (X80725)
0.02
Fig. 2
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