IJSEM Papers in Press. Published March 26, 2014 as doi:10.1099/ijs.0.061614-0
1
Defluviimonas indica sp. nov., a new marine bacterium isolated from a deep-sea
2
hydrothermal vent environment in the Southwest Indian Ocean
3
Lijing Jiang1,2, Hongxiu Xu2, Zongze Shao2*, Minnan Long1*
4
1
School of Energy Research, Xiamen University, Xiamen 361005, PR China
5
2
State Key Laboratory Breeding Base of Marine Genetic Resources; Key Laboratory
6
of Marine Genetic Resources, Third Institute of Oceanography, SOA; Key Laboratory
7
of Marine Genetic Resources of Fujian Province, Xiamen 361005, China.
8 9
*Corresponding author:
10
Zongze Shao. Tel: +86-592-2195390. Fax: +86-592-2085376.
11
E-mail: [email protected]
12
Minnan Long. Tel: +86-592-2185731. Fax: +86-592-2188053.
13
E-mail: [email protected]
14
Subjective category: Proteobacteria
15
Running title: Defluviimonas indica sp. nov.
16
Abbreviations: MCCC, Marine Culture Collection of China
17 18
The GenBank[/EMBL/DDBJ] accession number for the nucleotide sequences
19
reported in this study is HM050420 (Defluviimonas indica 20V17T, 16S rRNA gene).
1
20
Abstract
21
A Gram-negative, strictly aerobic, chemoheterotrophic marine bacterium, designated
22
20V17T, was isolated from a deep-sea hydrothermal vent chimney collected from
23
Southwest Indian Ridge. Cells of strain 20V17T were motile short rods, 1.2-1.8 µm in
24
length and 0.5-0.7 µm in width. Growth was observed between 20℃ and 37℃
25
(optimum 25℃ and 28℃), pH 5.0 and 8.0 (optimum pH 7.0) and 0.5 and 8% (w/v)
26
NaCl (optimum 1.5 and 2.0% NaCl). The major fatty acids were C18:1 ω7c (74.4%),
27
C19:0 cyclo ω8c (11%), C18:0 (5.1%) and C18:0 3OH (2.8%), and the polar lipid profile
28
comprised
29
glycolipid and four unidentified phospholipid. Ubiquinone 10 was the major quinone.
30
The G+C content of genomic DNA was 66.3 mol%. Phylogenetic analyses based on
31
16S rRNA gene sequences showed that strain 20V17T belonged to the genus
32
Defluviimonas and shared 96.5% and 96.1% sequence similarity with Defluviimonas
33
denitrificans D9-3T and Defluviimonas aestuarii BS14T, respectively. On the basis of
34
the taxonomic data obtained in this study, strain 20V17T represents a novel species of
35
the genus Defluviimonas, for which the name Defluviimonas indica sp. nov. is
36
proposed. The type strain is 20V17T (CGMCC 1.10859T =JCM 17871T=MCCC
37
1A01802T).
diphosphatidylglycerol,
phosphatidylethanolamine,
an
unidentified
2
38
Main text:
39
The genus Defluviimonas, which classified within the family Rhodobacteraceae, was
40
firstly proposed by Foesel et al. (2011) and subsequently emended by Math et al.
41
(2013). Cells of this genus are Gram-negative rods, non-motile or motile with single
42
polar flagella, strictly or facultative aerobic, chemoorganotrophic, non-pigmented,
43
non-phototrophic, moderately halophilic, catalase and cytochrome c-oxidase positive.
44
Currently the genus contained two species, Defluviimonas denitrificans, which
45
isolated from the biofilters of a recirculating marine aquaculture system in Rehovot,
46
Israel (Cytryn et al., 2003) and Defluviimonas aestuarii, which isolated from a tidal
47
flat of South Sea in Korea (Math et al., 2013).
48 49
In our study of the microbial diversity at the deep-sea hydrothermal fields, an aerobic
50
and heterotrophic, Gram-negative bacterium, strain 20V17T, was obtained that was
51
phylogenetically related to members of the genus Defluviimonas (Foesel et al., 2011).
52
On the basis of its chemotaxonomic, physiological and phylogenetic characteristics,
53
we propose the isolate as a novel species within the genus Defluviimonas. Here the
54
characterization and classification of strain 20V17T based on a polyphasic approach
55
are described.
56 57
Strain 20V17T was isolated from the hydrothermal sulfide chimney sample collected
58
from 2783 m depth on the Southwest Indian Ridge (E 50.64°, S 37.78°) during the
59
cruise of “Da-Yang Yi-Hao” in 2008. The chimney sample was diluted and spread on
60
Luria Bertani (LB) agar medium (10 g tryptone L-1, 5 g yeast extract L-1, 30.0 g NaCl 3
61
L-1 and 15g agar L-1), which was then incubated at 28°C. Strain 20V17T was isolated
62
and subsequently purified on LB agar plate at 28 °C three times. Unless stated
63
otherwise, the strain was grown routinely on LB at 28°C for characterization studies.
64 65
Genomic DNA was extracted by using the method of Ausubel et al. (1995) and the
66
16S rRNA gene was PCR amplified according to the previous study (Liu & Shao,
67
2005). The almost complete 16S rRNA gene sequence (1431bp) of strain 20V17T was
68
obtained and analyzed using BLAST Search against the GenBank and EzTaxon
69
databases (Altschul et al., 1997; Chun et al., 2007). The 16S rRNA gene sequences of
70
closely related taxa obtained from the GenBank database were aligned using SINA
71
sequence alignment program (http://www.arb-silva.de/aligner/; Pruesse et al., 2012)
72
based on the secondary structures. Phylogenetic analysis was performed using the
73
program MEGA 6.0 (Tamura et al., 2013). Distance matrices were calculated
74
according to the Kimura two-parameter model (Kimura, 1980). Phylogenetic trees
75
were inferred using the neighbor-joining (Saitou & Nei, 1987), minimum-evolution
76
(Rzhetsky & Nei, 1992, 1993) and maximum-likelihood methods (Felsenstein 1981).
77
Bootstrap values were determined based on 1000 replications. The neighbor-joining
78
tree was shown in Fig.1. Clustering results with the minimum-evolution and
79
maximum-likelihood approaches were similar to that obtained using neighbor-joining
80
method, which shown in the supplementary Fig.S3 and Fig.S4.
81 82
Phylogenetic analysis based on the almost complete 16S rRNA gene sequence of
4
83
20V17T placed the novel isolate in the family Rhodobacteraceae. Strain 20V17 T was
84
related closely to Defluviimonas denitrificans D9-3T and Defluviimonas aestuarii
85
BS14T with 96.5% and 96.1% 16S rRNA gene sequence similarity, respectively,
86
exhibited sequence similarity of
1
Defluviimonas indica sp. nov., a new marine bacterium isolated from a deep-sea
2
hydrothermal vent environment in the Southwest Indian Ocean
3
Lijing Jiang1,2, Hongxiu Xu2, Zongze Shao2*, Minnan Long1*
4
1
School of Energy Research, Xiamen University, Xiamen 361005, PR China
5
2
State Key Laboratory Breeding Base of Marine Genetic Resources; Key Laboratory
6
of Marine Genetic Resources, Third Institute of Oceanography, SOA; Key Laboratory
7
of Marine Genetic Resources of Fujian Province, Xiamen 361005, China.
8 9
*Corresponding author:
10
Zongze Shao. Tel: +86-592-2195390. Fax: +86-592-2085376.
11
E-mail: [email protected]
12
Minnan Long. Tel: +86-592-2185731. Fax: +86-592-2188053.
13
E-mail: [email protected]
14
Subjective category: Proteobacteria
15
Running title: Defluviimonas indica sp. nov.
16
Abbreviations: MCCC, Marine Culture Collection of China
17 18
The GenBank[/EMBL/DDBJ] accession number for the nucleotide sequences
19
reported in this study is HM050420 (Defluviimonas indica 20V17T, 16S rRNA gene).
1
20
Abstract
21
A Gram-negative, strictly aerobic, chemoheterotrophic marine bacterium, designated
22
20V17T, was isolated from a deep-sea hydrothermal vent chimney collected from
23
Southwest Indian Ridge. Cells of strain 20V17T were motile short rods, 1.2-1.8 µm in
24
length and 0.5-0.7 µm in width. Growth was observed between 20℃ and 37℃
25
(optimum 25℃ and 28℃), pH 5.0 and 8.0 (optimum pH 7.0) and 0.5 and 8% (w/v)
26
NaCl (optimum 1.5 and 2.0% NaCl). The major fatty acids were C18:1 ω7c (74.4%),
27
C19:0 cyclo ω8c (11%), C18:0 (5.1%) and C18:0 3OH (2.8%), and the polar lipid profile
28
comprised
29
glycolipid and four unidentified phospholipid. Ubiquinone 10 was the major quinone.
30
The G+C content of genomic DNA was 66.3 mol%. Phylogenetic analyses based on
31
16S rRNA gene sequences showed that strain 20V17T belonged to the genus
32
Defluviimonas and shared 96.5% and 96.1% sequence similarity with Defluviimonas
33
denitrificans D9-3T and Defluviimonas aestuarii BS14T, respectively. On the basis of
34
the taxonomic data obtained in this study, strain 20V17T represents a novel species of
35
the genus Defluviimonas, for which the name Defluviimonas indica sp. nov. is
36
proposed. The type strain is 20V17T (CGMCC 1.10859T =JCM 17871T=MCCC
37
1A01802T).
diphosphatidylglycerol,
phosphatidylethanolamine,
an
unidentified
2
38
Main text:
39
The genus Defluviimonas, which classified within the family Rhodobacteraceae, was
40
firstly proposed by Foesel et al. (2011) and subsequently emended by Math et al.
41
(2013). Cells of this genus are Gram-negative rods, non-motile or motile with single
42
polar flagella, strictly or facultative aerobic, chemoorganotrophic, non-pigmented,
43
non-phototrophic, moderately halophilic, catalase and cytochrome c-oxidase positive.
44
Currently the genus contained two species, Defluviimonas denitrificans, which
45
isolated from the biofilters of a recirculating marine aquaculture system in Rehovot,
46
Israel (Cytryn et al., 2003) and Defluviimonas aestuarii, which isolated from a tidal
47
flat of South Sea in Korea (Math et al., 2013).
48 49
In our study of the microbial diversity at the deep-sea hydrothermal fields, an aerobic
50
and heterotrophic, Gram-negative bacterium, strain 20V17T, was obtained that was
51
phylogenetically related to members of the genus Defluviimonas (Foesel et al., 2011).
52
On the basis of its chemotaxonomic, physiological and phylogenetic characteristics,
53
we propose the isolate as a novel species within the genus Defluviimonas. Here the
54
characterization and classification of strain 20V17T based on a polyphasic approach
55
are described.
56 57
Strain 20V17T was isolated from the hydrothermal sulfide chimney sample collected
58
from 2783 m depth on the Southwest Indian Ridge (E 50.64°, S 37.78°) during the
59
cruise of “Da-Yang Yi-Hao” in 2008. The chimney sample was diluted and spread on
60
Luria Bertani (LB) agar medium (10 g tryptone L-1, 5 g yeast extract L-1, 30.0 g NaCl 3
61
L-1 and 15g agar L-1), which was then incubated at 28°C. Strain 20V17T was isolated
62
and subsequently purified on LB agar plate at 28 °C three times. Unless stated
63
otherwise, the strain was grown routinely on LB at 28°C for characterization studies.
64 65
Genomic DNA was extracted by using the method of Ausubel et al. (1995) and the
66
16S rRNA gene was PCR amplified according to the previous study (Liu & Shao,
67
2005). The almost complete 16S rRNA gene sequence (1431bp) of strain 20V17T was
68
obtained and analyzed using BLAST Search against the GenBank and EzTaxon
69
databases (Altschul et al., 1997; Chun et al., 2007). The 16S rRNA gene sequences of
70
closely related taxa obtained from the GenBank database were aligned using SINA
71
sequence alignment program (http://www.arb-silva.de/aligner/; Pruesse et al., 2012)
72
based on the secondary structures. Phylogenetic analysis was performed using the
73
program MEGA 6.0 (Tamura et al., 2013). Distance matrices were calculated
74
according to the Kimura two-parameter model (Kimura, 1980). Phylogenetic trees
75
were inferred using the neighbor-joining (Saitou & Nei, 1987), minimum-evolution
76
(Rzhetsky & Nei, 1992, 1993) and maximum-likelihood methods (Felsenstein 1981).
77
Bootstrap values were determined based on 1000 replications. The neighbor-joining
78
tree was shown in Fig.1. Clustering results with the minimum-evolution and
79
maximum-likelihood approaches were similar to that obtained using neighbor-joining
80
method, which shown in the supplementary Fig.S3 and Fig.S4.
81 82
Phylogenetic analysis based on the almost complete 16S rRNA gene sequence of
4
83
20V17T placed the novel isolate in the family Rhodobacteraceae. Strain 20V17 T was
84
related closely to Defluviimonas denitrificans D9-3T and Defluviimonas aestuarii
85
BS14T with 96.5% and 96.1% 16S rRNA gene sequence similarity, respectively,
86
exhibited sequence similarity of
