IJSEM Papers in Press. Published December 11, 2014 as doi:10.1099/ijs.0.066027-0
1
Marivirga lumbricoides sp. nov., a marine bacterium isolated
2
from the South China Sea Yongle Xu, Rui Zhang, Qipei Li, Keshao Liu, Nianzhi Jiao
3 4
State Key Laboratory of Marine Environmental Science, Institute of Marine Microbes and
5
Ecospheres, Xiamen University, Xiamen 361005, PR China
6
Author for correspondence: Nianzhi Jiao; Rui Zhang
7
State Key Laboratory of Marine Environmental Science
8
Xiamen University
9
Xiamen 361005, P. R. China
10
Tel: +86-592-2187869,
Email: [email protected]; [email protected]
11
Running Title: Marivirga lumbricoides sp. nov.
12
Keywords: Marivirga lumbricoides sp. nov., polyphasic taxonomy, seawater
13
Subject category: New Taxa (Bacteroidetes)
14
Sequence deposited: The GenBank accession number for the 16S rRNA gene sequence of strain
15
JLT2000T is HQ638976
16
-------------------------------------------------------------------------------------
17
Abstract:
18
A novel, aerobic, heterotrophic, orange-pigmented, Gram-staining-negative, rod-shaped, gliding
19
bacterium strain designated JLT2000T was isolated from the surface seawater of South China Sea.
20
The strain was oxidase and catalase positive. The major cellular fatty acids of strain JLT2000T
21
were C12:0, iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH, Summed feature 3 (comprising C16:1 ω 7c and/or
22
C16:1 ω 6c), C16:0, C18:0 and iso-C17:0 3-OH. MK-7 is the major respiratory quinone. The major
23
polar lipids of strain JLT2000T are phosphatidylcholine, phosphatidylethanolamine. The genomic
24
DNA G+C content of JLT2000T is 37.8 mol%. Phylogenetic analysis based on 16S rRNA gene
25
sequences showed strain JLT2000T formed a branch within genus Marivirga but were clearly
26
separated from the two established species Marivirga tractuosa and Marivirga sericea. The
27
sequence similarity with the type strains of the two species are 95.8% and 96.1% respectively.
28
Strain JLT2000T has shorter cell length and wider growth range in different temperatures and
29
salinities than that of M. tractuosa NBRC 15989 T and strain M. sericea NBRC 15983T. In addition,
30
strain JLT2000T can utilize more kinds of carbon source, and hydrolyse more kinds of polymers
31
than strain M. tractuosa NBRC 15989T and strain M. sericea NBRC 15983 T. Based on these
32
polyphasic analysis, strain JLT2000T represents a novel species of the genus Marivirga, for which
33
the name Marivirga lumbricoides sp. nov. is proposed. The type strain is JLT2000T (= JCM
34
18012T = CGMCC 1.10832T).
35 36
The genus Mariviga was established by Nedashkovskaya et al. (2010), and belongs to the family
37
Flammeovirgaceae of the phylum Bacteroidetes (Ludwig et al., 2008). The genus Mariviga
38
contains two species: M. sericea and M. tractuosa, with M. tractuosa as the type species. These
39
two species once belonged to the genus Microscilla (Lewin, 1969). [Microscilla] tractuosa was
40
then transferred to the genus Flexibacter by Leadbetter (1974). In 1989, Reichenbach reclassified
41
the marine representatives of this species back into the genus Microscilla (Reichenbach, 1989).
42
Due to the considerable phylogenetic heterogeneity between members of genera Flexibacter and
43
Microscilla based on the 16S rRNA gene sequences, it was suggested to restrict these genera to
44
their respective type species, and to reclassify the other species (Nakagawa et al., 2002).
45
According to the extensive taxonomic studies, [Flexibacter] tractuosus NRBC 2958T and
46
[Microscilla] sericea LMG 13021T were reclassified into the novel genus Mariviga as two distinct
47
species (Nedashkovskaya et al., 2010).
48
The genus Mariviga was proposed as rod-shaped, motile by gliding, Gram-staining-negative,
49
aerobic bacterium with the lowest DNA G+C content in the family Flammeovirgaceae
50
(Nedashkovskaya et al., 2010). At the time of writing, the genus is most closely related to the
51
genera Cesiribacter and Nafulsella based on the 16S rRNA gene phylogeny.
52
Strain JLT2000T was isolated from the surface water of South China Sea at 22°3'36" N,
53
118°26'24" E by direct plating of 200 µl sea water using modified rich organic (RO) medium
54
(Yurkov et al., 1999). After primary incubation and purification, JLT2000T was preserved in RO
55
medium supplemented with 20% (v/v) glycerol at -80 ºC and was routinely maintained using RO
56
medium at 28 ºC.
57
The Gram reaction was determined according to the method described by Gerhardt et al. (1994).
58
Cell morphology was examined by TEM (JEM 2100 HC) using cells growing on marine agar for
59
12 h at 28 ºC. Gliding motility was tested using method described by Bowman (2000).
60
Growth at various NaCl concentrations (0%, 2%, 3%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, 16%,
61
and 18%) was assessed by RO medium at 28 ºC. Growth range of pH was tested by adjusting the
62
final pH of RO medium to values of 3, 4, 5, 6, 7, 8, 9, 10, 11 using 1 N H2SO4 (3-5), 10 mM
63
HEPES-NaOH (6-8), and 0.1 M NaHCO3/Na2CO 3 (9-11) with a final Na+ concentration of 0.6 M
64
(Harrison Jr, 1984; Sorokin et al, 2003). Growth at different temperatures was determined by
65
incubation at temperatures from 4 ºC to 45 ºC (4 ºC, 10 ºC, 15 ºC, 25 ºC, 30 ºC, 37 ºC, 40 ºC, 45
66
ºC). The susceptibility to antibiotics was tested using the disc-diffusion plate (Kirby–Bauer)
67
method according to Fraser & Jorgensen (1997) and Andrews (2008). Discs containing
68
tetracycline (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), medemycin (30 µg),
69
ofloxacin (5 µg), ciprofloxacin (5 µg), clarithromycin (15 µg), clindamycin (2 µg), ampicillin (10
70
µg), streptomycin (10 µg), kanamycin (30 µg), gentamicin (10 µg), aztreonam (30 µg), penicillin
71
(10 µg), cefotaxime (30 µg), cefoperazone (75 µg), polymyxin B (30 µg), cefalothin (30 µg) were
72
used in this test.
73
API 20E, API 20NE, API ZYM galleries (BioMérieux) were used to investigate the biochemical
74
properties. Oxidase activity was determined using method described by Smibert & Krieg (1994).
75
The activity of catalase was assessed by dropping 3% H2O2 to the overnight colony collected from
76
the marine agar (Dong & Cai, 2001). Presence of flexirubin pigments was investigated by using
77
the bathochromic shift test with a 20% (w/v) KOH solution (Fautz & Reichenbach 1980;
78
Bernardet et al., 2002).
79
Carbon source utilization was tested according to the methods described by Nedashkovskaya et al.
80
(2010), using both API 20NE strips and a medium containing 0.2 g NaNO3, 0.2 g NH4Cl, 0.05 g
81
yeast extract and 0.4% (w/v) carbon source (glucose, sucrose, inositol, sorbitol, maltose, citrate,
82
cellobiose, arabinose, D-galactose, malic acid, α-lactose, mannitol, pyruvate, gluconate) per liter
83
of artificial seawater as described by Suzuki et al. (2001). Hydrolysis of DNA, cellulose (filter
84
paper), agar, starch, chitin, and casein was examined according to the methods described by Dong
85
& Cai (2001) using RO medium without organic matter. The oxidative or fermentative utilization
86
of glucose was determined according to the method described by Dong & Cai (2001) using RO
87
medium without organic matter. Acid production from the carbohydrates was tested according to
88
the method described by Cappuccino & Sherman (2002).
89
The cellular fatty acids were extracted from cells grown on marine agar at 28 ºC for 48 h
90
according to the method described by Komagata & Suzuki (1987) and analyzed on Microbial
91
Identification System (MIDI) Sherlock version 6.0 and the TSBA6 6.00 library. Isoprenoid
92
quinones were extracted from freeze-dried cells using the two-stage method as described by
93
Tindall (1990a, b) and analysed using reverse-phase HPLC. Polar lipids were extracted using a
94
chloroform / methanol system and analysed using one- & two-dimensional TLC, as described
95
previously (Kates, 1986).
96
The genomic DNA was extracted from cells grown in RO medium after 24 h at 28 ºC using
97
TIANamp Bacteria DNA kit (Tiangen) according to the instruction of the manufacture. The
98
genomic DNA G + C content of strain JLT2000T was determined by a HPLC method according to
99
Mesbah et al. (1989). The 16S rRNA gene was amplified using universal bacterial primers 27F
100
and 1492R (Lane, 1991). The PCR product was cloned and sequenced from the both sides and
101
identified using the BLAST search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the EzTaxon
102
server (http://www.ezbiocloud.net/eztaxon; Kim et al., 2012). The 16S rRNA gene sequences of
103
the representatives of the genus Mariviga, closely related and other more distantly related genera
104
of family Flammeovirgaceae were retrieved from the GenBank (http://www.ncbi.nlm.nih.gov).
105
Multiple alignments of the 16S rRNA gene sequences were performed using ClustalX2 program
106
(Larkin et al., 2007). Phylogenetic trees were constructed assuming Neighbor-Joining,
107
maximum-likelihood and maximum-parsimony methods using MEGA 5.0 software package
108
(Tamura et al., 2011).
109
The predominant fatty acids (>4%) of strain JLT2000T are C12:0 (4.47), iso-C15:1 G (22%), iso-C15:0
110
(14.6%), iso-C17:0 3-OH (7.4%), Summed feature 3 (comprising C16:1
111
(19.9%), C16:0 (7.3%), C18:0 (6.4%) and iso-C17:0 3-OH (7.4%) (Table 1). JLT2000T shares similar
112
fatty acid constituents with M. sericea NBRC 15983T and M. tractuosa NBRC 15989 T, but has
113
remarkably lower proportion of iso-C15:0, and higher proportion of C16:1 ω 7c and/or C16:1 ω 6c
114
compared to the other two strains (Table 1). MK-7 is the major respiratory lipoquinone of strain
115
JLT2000T. The major polar lipids of strain JLT2000T, M. sericea NBRC 15983T and M. tractuosa
116
NBRC 15989T are all phosphatidylcholine, phosphatidylethanolamine. The polar lipid profile of
117
strain JLT2000T consists of phosphatidylcholine, phosphatidylethanolamine, four unknown
118
phospholipid and one unknown aminolipid (Fig. S1). The DNA G+C content of strain JLT2000T is
119
37.9 mol%. Nearly complete 16S rRNA gene sequence (1443 bp) was gained. The phylogenetic
120
analysis shows strain JLT2000T is closely related to the two species in genus Marivirga (Fig. 1):
121
M. sericea NBRC 15983T (96.05% similarity) and M. tractuosa NBRC 15989T (95.77%), and
122
formed a distinct cluster with the two species. Members of the genera Cesiribacter, Fabibacter
123
Fulvivirga, Marinoscillum, Nafulsella and Roseivirga were the next closest relatives of the new
124
isolate, with sequence similarities
1
Marivirga lumbricoides sp. nov., a marine bacterium isolated
2
from the South China Sea Yongle Xu, Rui Zhang, Qipei Li, Keshao Liu, Nianzhi Jiao
3 4
State Key Laboratory of Marine Environmental Science, Institute of Marine Microbes and
5
Ecospheres, Xiamen University, Xiamen 361005, PR China
6
Author for correspondence: Nianzhi Jiao; Rui Zhang
7
State Key Laboratory of Marine Environmental Science
8
Xiamen University
9
Xiamen 361005, P. R. China
10
Tel: +86-592-2187869,
Email: [email protected]; [email protected]
11
Running Title: Marivirga lumbricoides sp. nov.
12
Keywords: Marivirga lumbricoides sp. nov., polyphasic taxonomy, seawater
13
Subject category: New Taxa (Bacteroidetes)
14
Sequence deposited: The GenBank accession number for the 16S rRNA gene sequence of strain
15
JLT2000T is HQ638976
16
-------------------------------------------------------------------------------------
17
Abstract:
18
A novel, aerobic, heterotrophic, orange-pigmented, Gram-staining-negative, rod-shaped, gliding
19
bacterium strain designated JLT2000T was isolated from the surface seawater of South China Sea.
20
The strain was oxidase and catalase positive. The major cellular fatty acids of strain JLT2000T
21
were C12:0, iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH, Summed feature 3 (comprising C16:1 ω 7c and/or
22
C16:1 ω 6c), C16:0, C18:0 and iso-C17:0 3-OH. MK-7 is the major respiratory quinone. The major
23
polar lipids of strain JLT2000T are phosphatidylcholine, phosphatidylethanolamine. The genomic
24
DNA G+C content of JLT2000T is 37.8 mol%. Phylogenetic analysis based on 16S rRNA gene
25
sequences showed strain JLT2000T formed a branch within genus Marivirga but were clearly
26
separated from the two established species Marivirga tractuosa and Marivirga sericea. The
27
sequence similarity with the type strains of the two species are 95.8% and 96.1% respectively.
28
Strain JLT2000T has shorter cell length and wider growth range in different temperatures and
29
salinities than that of M. tractuosa NBRC 15989 T and strain M. sericea NBRC 15983T. In addition,
30
strain JLT2000T can utilize more kinds of carbon source, and hydrolyse more kinds of polymers
31
than strain M. tractuosa NBRC 15989T and strain M. sericea NBRC 15983 T. Based on these
32
polyphasic analysis, strain JLT2000T represents a novel species of the genus Marivirga, for which
33
the name Marivirga lumbricoides sp. nov. is proposed. The type strain is JLT2000T (= JCM
34
18012T = CGMCC 1.10832T).
35 36
The genus Mariviga was established by Nedashkovskaya et al. (2010), and belongs to the family
37
Flammeovirgaceae of the phylum Bacteroidetes (Ludwig et al., 2008). The genus Mariviga
38
contains two species: M. sericea and M. tractuosa, with M. tractuosa as the type species. These
39
two species once belonged to the genus Microscilla (Lewin, 1969). [Microscilla] tractuosa was
40
then transferred to the genus Flexibacter by Leadbetter (1974). In 1989, Reichenbach reclassified
41
the marine representatives of this species back into the genus Microscilla (Reichenbach, 1989).
42
Due to the considerable phylogenetic heterogeneity between members of genera Flexibacter and
43
Microscilla based on the 16S rRNA gene sequences, it was suggested to restrict these genera to
44
their respective type species, and to reclassify the other species (Nakagawa et al., 2002).
45
According to the extensive taxonomic studies, [Flexibacter] tractuosus NRBC 2958T and
46
[Microscilla] sericea LMG 13021T were reclassified into the novel genus Mariviga as two distinct
47
species (Nedashkovskaya et al., 2010).
48
The genus Mariviga was proposed as rod-shaped, motile by gliding, Gram-staining-negative,
49
aerobic bacterium with the lowest DNA G+C content in the family Flammeovirgaceae
50
(Nedashkovskaya et al., 2010). At the time of writing, the genus is most closely related to the
51
genera Cesiribacter and Nafulsella based on the 16S rRNA gene phylogeny.
52
Strain JLT2000T was isolated from the surface water of South China Sea at 22°3'36" N,
53
118°26'24" E by direct plating of 200 µl sea water using modified rich organic (RO) medium
54
(Yurkov et al., 1999). After primary incubation and purification, JLT2000T was preserved in RO
55
medium supplemented with 20% (v/v) glycerol at -80 ºC and was routinely maintained using RO
56
medium at 28 ºC.
57
The Gram reaction was determined according to the method described by Gerhardt et al. (1994).
58
Cell morphology was examined by TEM (JEM 2100 HC) using cells growing on marine agar for
59
12 h at 28 ºC. Gliding motility was tested using method described by Bowman (2000).
60
Growth at various NaCl concentrations (0%, 2%, 3%, 5%, 6%, 8%, 10%, 12%, 14%, 15%, 16%,
61
and 18%) was assessed by RO medium at 28 ºC. Growth range of pH was tested by adjusting the
62
final pH of RO medium to values of 3, 4, 5, 6, 7, 8, 9, 10, 11 using 1 N H2SO4 (3-5), 10 mM
63
HEPES-NaOH (6-8), and 0.1 M NaHCO3/Na2CO 3 (9-11) with a final Na+ concentration of 0.6 M
64
(Harrison Jr, 1984; Sorokin et al, 2003). Growth at different temperatures was determined by
65
incubation at temperatures from 4 ºC to 45 ºC (4 ºC, 10 ºC, 15 ºC, 25 ºC, 30 ºC, 37 ºC, 40 ºC, 45
66
ºC). The susceptibility to antibiotics was tested using the disc-diffusion plate (Kirby–Bauer)
67
method according to Fraser & Jorgensen (1997) and Andrews (2008). Discs containing
68
tetracycline (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), medemycin (30 µg),
69
ofloxacin (5 µg), ciprofloxacin (5 µg), clarithromycin (15 µg), clindamycin (2 µg), ampicillin (10
70
µg), streptomycin (10 µg), kanamycin (30 µg), gentamicin (10 µg), aztreonam (30 µg), penicillin
71
(10 µg), cefotaxime (30 µg), cefoperazone (75 µg), polymyxin B (30 µg), cefalothin (30 µg) were
72
used in this test.
73
API 20E, API 20NE, API ZYM galleries (BioMérieux) were used to investigate the biochemical
74
properties. Oxidase activity was determined using method described by Smibert & Krieg (1994).
75
The activity of catalase was assessed by dropping 3% H2O2 to the overnight colony collected from
76
the marine agar (Dong & Cai, 2001). Presence of flexirubin pigments was investigated by using
77
the bathochromic shift test with a 20% (w/v) KOH solution (Fautz & Reichenbach 1980;
78
Bernardet et al., 2002).
79
Carbon source utilization was tested according to the methods described by Nedashkovskaya et al.
80
(2010), using both API 20NE strips and a medium containing 0.2 g NaNO3, 0.2 g NH4Cl, 0.05 g
81
yeast extract and 0.4% (w/v) carbon source (glucose, sucrose, inositol, sorbitol, maltose, citrate,
82
cellobiose, arabinose, D-galactose, malic acid, α-lactose, mannitol, pyruvate, gluconate) per liter
83
of artificial seawater as described by Suzuki et al. (2001). Hydrolysis of DNA, cellulose (filter
84
paper), agar, starch, chitin, and casein was examined according to the methods described by Dong
85
& Cai (2001) using RO medium without organic matter. The oxidative or fermentative utilization
86
of glucose was determined according to the method described by Dong & Cai (2001) using RO
87
medium without organic matter. Acid production from the carbohydrates was tested according to
88
the method described by Cappuccino & Sherman (2002).
89
The cellular fatty acids were extracted from cells grown on marine agar at 28 ºC for 48 h
90
according to the method described by Komagata & Suzuki (1987) and analyzed on Microbial
91
Identification System (MIDI) Sherlock version 6.0 and the TSBA6 6.00 library. Isoprenoid
92
quinones were extracted from freeze-dried cells using the two-stage method as described by
93
Tindall (1990a, b) and analysed using reverse-phase HPLC. Polar lipids were extracted using a
94
chloroform / methanol system and analysed using one- & two-dimensional TLC, as described
95
previously (Kates, 1986).
96
The genomic DNA was extracted from cells grown in RO medium after 24 h at 28 ºC using
97
TIANamp Bacteria DNA kit (Tiangen) according to the instruction of the manufacture. The
98
genomic DNA G + C content of strain JLT2000T was determined by a HPLC method according to
99
Mesbah et al. (1989). The 16S rRNA gene was amplified using universal bacterial primers 27F
100
and 1492R (Lane, 1991). The PCR product was cloned and sequenced from the both sides and
101
identified using the BLAST search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the EzTaxon
102
server (http://www.ezbiocloud.net/eztaxon; Kim et al., 2012). The 16S rRNA gene sequences of
103
the representatives of the genus Mariviga, closely related and other more distantly related genera
104
of family Flammeovirgaceae were retrieved from the GenBank (http://www.ncbi.nlm.nih.gov).
105
Multiple alignments of the 16S rRNA gene sequences were performed using ClustalX2 program
106
(Larkin et al., 2007). Phylogenetic trees were constructed assuming Neighbor-Joining,
107
maximum-likelihood and maximum-parsimony methods using MEGA 5.0 software package
108
(Tamura et al., 2011).
109
The predominant fatty acids (>4%) of strain JLT2000T are C12:0 (4.47), iso-C15:1 G (22%), iso-C15:0
110
(14.6%), iso-C17:0 3-OH (7.4%), Summed feature 3 (comprising C16:1
111
(19.9%), C16:0 (7.3%), C18:0 (6.4%) and iso-C17:0 3-OH (7.4%) (Table 1). JLT2000T shares similar
112
fatty acid constituents with M. sericea NBRC 15983T and M. tractuosa NBRC 15989 T, but has
113
remarkably lower proportion of iso-C15:0, and higher proportion of C16:1 ω 7c and/or C16:1 ω 6c
114
compared to the other two strains (Table 1). MK-7 is the major respiratory lipoquinone of strain
115
JLT2000T. The major polar lipids of strain JLT2000T, M. sericea NBRC 15983T and M. tractuosa
116
NBRC 15989T are all phosphatidylcholine, phosphatidylethanolamine. The polar lipid profile of
117
strain JLT2000T consists of phosphatidylcholine, phosphatidylethanolamine, four unknown
118
phospholipid and one unknown aminolipid (Fig. S1). The DNA G+C content of strain JLT2000T is
119
37.9 mol%. Nearly complete 16S rRNA gene sequence (1443 bp) was gained. The phylogenetic
120
analysis shows strain JLT2000T is closely related to the two species in genus Marivirga (Fig. 1):
121
M. sericea NBRC 15983T (96.05% similarity) and M. tractuosa NBRC 15989T (95.77%), and
122
formed a distinct cluster with the two species. Members of the genera Cesiribacter, Fabibacter
123
Fulvivirga, Marinoscillum, Nafulsella and Roseivirga were the next closest relatives of the new
124
isolate, with sequence similarities
