IJSEM Papers in Press. Published December 9, 2014 as doi:10.1099/ijs.0.000018
International Journal of Systematic and Evolutionary Microbiology Flavicella marina gen. nov., sp. nov., a carotenoid-producing bacterium from surface seawater. --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00053R1
Full Title:
Flavicella marina gen. nov., sp. nov., a carotenoid-producing bacterium from surface seawater.
Short Title:
Flavicella marina gen. nov., sp. nov
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Maki Teramoto Kochi University Nankoku, JAPAN
First Author:
Maki Teramoto
Order of Authors:
Maki Teramoto Miyuki Nishijima
Manuscript Region of Origin:
JAPAN
Abstract:
A Gram-stain-negative, non-motile, mesophilic, aerobic, rod- or spherical-shaped bacterium, strain 2A-7T, was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain produced pigment(s), absorption spectrum of which closely resembled that of β-carotene. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Flavobacteriaceae and clustered distantly with the type strains of the genus Lutibacter (up to 93.9% similarity). The DNA G+C content was 34.1 mol%. The major fatty acids were iso-C15:0 2-OH and/or C16:1 ω7c, iso-C17:0 3-OH and iso-C15:0 3-OH. The major polar lipids were phosphatidylethanolamine and three unidentified lipids. Menaquinone-6 was detected as the sole isoprenoid quinone. On the basis of phenotypic, genotypic and chemotaxonomic data, strain 2A-7T represents a novel genus and species, for which the name Flavicella marina gen. nov., sp. nov. is proposed. The type strain of Flavicella marina is 2A-7T (=NBRC 110145T=KCTC 42197T).
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1
Title: Flavicella marina gen. nov., sp. nov., a carotenoid-producing bacterium from
2
surface seawater.
3 4
Author: Maki Teramoto1* and Miyuki Nishijima2
5 6
1
7
Japan.
Oceanography Section, Kochi University, Kohasu, Oko, Nankoku, Kochi 783-8505,
8 9 10
2
TechnoSuruga Laboratory Co. Ltd. 330 Nagasaki, Shimizu-ku Shizuoka 424-0065
Japan
11 12
*To whom correspondence should be addressed
13
Tel: 81-88-880-2240, Fax: 81-88-880-2177, E-mail: [email protected]
14 15 16
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of
17
strain 2A-7T is AB915171.
18
Three supplementary figures are available with the online version of this paper.
19 20 21
Category: New taxa – Bacteroidetes
22
Running title: Flavicella marina gen. nov., sp. nov.
23 24
1
1
Abstract
2
A Gram-stain-negative, non-motile, mesophilic, aerobic, rod- or spherical-shaped
3
bacterium, strain 2A-7T, was isolated from surface seawater at Muroto city, Kochi
4
prefecture, Japan. The strain produced pigment(s), absorption spectrum of which closely
5
resembled that of -carotene. Phylogenetic analyses based on 16S rRNA gene
6
sequences showed that the strain fell within the family Flavobacteriaceae and clustered
7
distantly with the type strains of the genus Lutibacter (up to 93.9% similarity). The
8
DNA G+C content was 34.1 mol%. The major fatty acids were iso-C15:0 2-OH and/or
9
C16:1 7c, iso-C17:0 3-OH and iso-C15:0 3-OH. The major polar lipids were
10
phosphatidylethanolamine and three unidentified lipids. Menaquinone-6 was detected as
11
the sole isoprenoid quinone. On the basis of phenotypic, genotypic and
12
chemotaxonomic data, strain 2A-7T represents a novel genus and species, for which the
13
name Flavicella marina gen. nov., sp. nov. is proposed. The type strain of Flavicella
14
marina is 2A-7T (=NBRC 110145T=KCTC 42197T).
15 16 17
Main Text
18
A large number of carotenoids of different molecular structures are known to be
19
biosynthesized and have been attracting great attention due to their beneficial effects on
20
human health, e.g. their potential in the prevention of diseases such as cancer and
21
cardiovascular disease (van den Berg et al., 2000). Marine bacteria of the family
22
Flavobacteriaceae usually produce carotenoids (Bernardet et al., 2002), which include
23
zeaxanthin (a bicyclic carotenoid; Asker et al., 2007) and myxol (a monocyclic
24
carotenoid; Shindo et al., 2007; Teramoto et al., 2003; Yokoyama & Miki, 1995), and
25
are thus attractive in health-related applications. The family Flavobacteriaceae,
26
belonging to the phylum Bacteroidetes (the Cytophaga–Flavobacterium–Bacteroides
27
group), currently comprises more than 110 genera (http://www.bacterio.cict.fr).
2
1
Members of this family have been isolated from terrestrial as well as marine
2
environments.
3 4
Muroto city, Kochi prefecture, Japan is one of the rare areas where deep seawater is
5
upwelling. Deep seawater is relatively rich in nitrogen and phosphorus (DeLong et al.,
6
2006; Hansman et al., 2009), which are the two main growth-limiting factors in marine
7
environments. Therefore, surface seawater around Muroto could be more nutrient-rich
8
than surface seawater in other areas, suggesting that rich microbial resources could be
9
harboured in the Muroto surface seawater. The present study describes a bacterial strain,
10
designated strain 2A-7T, isolated from the Muroto surface seawater (33˚18’ N 134˚11’ E,
11
water depth of 0.5 m) in summer 2011, which was found to be a novel member of
12
Flavobacteriaceae.
13 14
Strain 2A-7T was obtained from the seawater by direct plating on ONR7a medium
15
(artificial seawater medium; Dyksterhouse et al., 1995) supplemented with (per litre) 15
16
g agar, 0.05 g sodium acetate, 0.05 g sodium pyruvate and 0.1 g yeast extract (Bacto
17
yeast extract; BD).
18 19
Strain 2A-7T was cultivated at 28 ˚C for 48 h on a dMB1 plate, which contained (per
20
litre) 7.48 g marine broth 2216 (MB; BD), 15 g agar, 0.2 litres distilled water and 0.8
21
litres artificial seawater (ASW; Marine Art SF-1; Tomita Pharmaceutical).
22
Phase-contrast microscopy (BX50F4; Olympus) showed that the strain was not motile.
23
Gram-staining
24
Gram-stain-negative. The strain was non-sporulating, and formed smooth lenticular
25
opaque light-yellow circular colonies with an entire edge that possessed a butyrous
26
consistency on the dMB1 plate. The strain was oxidase-positive and catalase-positive
27
(Barrow & Feltham, 1993), and negative for acid production from D-glucose on
tests
(Favor
G
kit;
Nissui)
3
showed
that
the
strain
was
1
modified Hugh-Leifson’s O-F medium (Leifson, 1963). Flexirubin-type pigments were
2
not detected by flooding 2A-7T cells with 20% (w/v) KOH (Fautz & Reichenbach,
3
1980). The API 20 NE kit (bioMerieux) was used for assessing biochemical
4
characteristics according to the manufacturer’s instructions, with the exception that the
5
cells were suspended in ASW. In addition, API ZYM tests (bioMerieux) were conducted
6
to examine enzyme activities as described in the manufacturer’s manual, with the
7
exceptions that the inoculum was prepared using ASW and the incubation time was
8
extended to 24 h at 28 ˚C. The strain was positive for aesculin hydrolase and
9
-galactosidase and negative for reduction of nitrate to nitrite, indole production from
10
tryptophan, fermentation of glucose, arginine dihydrolase, urease and gelatinase. Other
11
detailed phenotypic features of strain 2A-7T are given in the species description.
12 13
Strain 2A-7T did not glide by the method of Bowman (2000) on the quarter-strength MB
14
plate containing (per litre) 9.35 g MB, 0.25 litres distilled water and 0.75 litres ASW
15
and solidified with 1% (w/v) agar. The pigment(s) was extracted with acetone from
16
cells of strain 2A-7T by shaking vigorously for 10 min. The extract was centrifuged in
17
order to remove cell debris, and the resulting supernatant was subjected to a
18
spectrophotometer (DU 730; Beckman Coulter). The visible absorption spectrum of the
19
pigment(s) had absorption maxima at 455 and 475 nm (Fig. S1, available in the online
20
Supplementary Material), which closely resembled that of authentic -carotene reported
21
in the literature (Takaichi & Shimada, 1992), suggesting that strain 2A-7T could have
22
contained -carotene or a carotenoid structurally similar to -carotene, such as
23
zeaxanthin (dihydroxy--carotene), as a dominant pigment.
24 25
For transmission electron microscopy (H7600; Hitachi), strain 2A-7T was grown at 20
26
˚C for 7 days on a dMB plate (composition same as for the dMB1 plate except that
27
Muroto surface seawater was used instead of ASW), and negatively stained with 2%
4
1
uranyl acetate. Cells of strain 2A-7T were rod- or spherical-shaped (0.4–0.9 × 0.7–1.5
2
m) and possessed a flagellum-like structure (Fig. S2). Although strain 2A-7T had a
3
flagellum-like structure, we couldn't have observed its motility as described above.
4
Motility of strain 2A-7T was also tested at its early growth phase (grown for 16 h at 10
5
˚C and at 15 ˚C) on the quarter-strength MB plate solidified with 0.2% (w/v) agar.
6
However, the strain also did not show motility. Therefore, it was unclear whether the
7
flagellum-like structure was a flagellum or not.
8 9
Anaerobic growth was tested on dMB plates at 20 ˚C in a GasPak anaerobic jar (BD)
10
for 2 months. Strain 2A-7T did not grow under anaerobic conditions. Growth
11
temperature was tested on dMB plates at 4, 10, 15, 20, 22, 25, 28, 30, 35, 37 and 40 ˚C
12
for 2 months. Strain 2A-7T grew at 4–30 ˚C (optimally at 25–28 ˚C). NaCl requirement
13
was tested at 28 ˚C for 2 weeks on modified MB (NaCl-deficient; Sohn et al., 2004)
14
supplemented with 0, 1, 3, 5, 10, 12, 15 and 17% (w/v) NaCl. Strain 2A-7T grew with
15
3% (w/v) NaCl but not with the other concentrations of NaCl. The pH range for growth
16
(tested at pH 2–11, intervals of 1 pH unit) was determined at 20 ˚C for 1 month with
17
shaking in filter-sterilized dMB medium, which contained (per litre) 7.48 g MB, 0.2
18
litres distilled water and 0.8 litres Muroto surface seawater. Strain 2A-7T grew at pH 7–
19
8 (optimally at pH 7).
20 21
The almost full-length 16S rRNA gene sequence of strain 2A-7T (1375 bp) was
22
obtained as described previously (Teramoto et al., 2009). The most similar species to
23
strain 2A-7T was Lutibacter agarilyticus (93.9% similarity to its type strain), and strain
24
2A-7T showed 93.7% similarity to the type species of Lutibacter (Lutibacter litoralis;
25
strain CL-TF09T), suggesting that strain 2A-7T does not belong to this genus in the
26
family Flavobacteriaceae (Yarza et al., 2008; Tindal et al., 2010). On the other hand,
27
the closest GenBank relative to strain 2A-7T indicated by BLAST search (Altschul et al.,
5
1
1990) was an uncultured marine bacterium clone SeaWat_F492 (JQ198834) with the
2
similarity of 98.5%. The sequence of strain 2A-7T was aligned with related sequences of
3
members of the family Flavobacteriaceae available in public databases using
4
CLUSTAL X (ver. 2.1) (Larkin et al., 2007). The alignments were manually modified
5
where necessary, and trimming of gaps was performed. Phylogenetic trees were inferred
6
from the aligned sequences of 1351 bp using the neighbour-joining algorithm (Saitou &
7
Nei, 1987) by CLUSTAL X with default parameters (including Kimura’s correction)
8
and maximum-likelihood algorithm (Felsenstein, 1981) by MEGA 6.06 (Tamura et al.,
9
2013) and analyzed using bootstrapping (Felsenstein, 1985) based on 1000 resamplings.
10
The neighbour-joining tree is shown in Fig. 1. Strain 2A-7T fell within the family
11
Flavobacteriaceae and was most closely related with the genus Lutibacter and an
12
invalidly described genus ‘Marinitalea’. However, strain 2A-7T clustered distantly with
13
these and the other members of the family Flavobacteriaceae, which was supported by
14
high bootstrap values (95% using the neighbour-joining algorithm and 95% with the
15
maximum-likelihood algorithm). Strain 2A-7T showed 93.2% similarity in the 16S
16
rRNA gene sequence to the type strain of ‘Marinitalea’ (‘Marinitalea sucinacia’
17
JC2131T; FJ387163). Together with the low 16S rRNA gene sequence similarities of
18
strain 2A-7T to the type strains of the closely related genera, these results indicate that
19
strain 2A-7T could represent a novel genus of the family Flavobacteriaceae in the class
20
Flavobacteriia.
21 22
The cellular fatty acid composition of strain 2A-7T was analyzed by using cells grown
23
on Marine Agar 2216 (BD) at 30 ˚C for 3 days when growth reached its maximum (in
24
the early stationary growth phase). Cellular fatty acid methyl esters were prepared and
25
analyzed by GC (7890A GC system; Agilent Technologies) according to the
26
instructions given for the Microbial Identification System version 6.0 and were
27
compared using the database TSBA40 (MIDI). The results are given in Table 1 with
6
1
those of the type strains of the closest genera Lutibacter and ‘Marinitalea’. The major
2
cellular fatty acids of strain 2A-7T were iso-C15:0 2-OH and/or C16:1 7c (26.9% of the
3
total), iso-C17:0 3-OH (16.8%) and iso-C15:0 3-OH (10.6%). Differences in the
4
composition were present between strain 2A-7T and the type strains of Lutibacter
5
especially in iso-C15:0 2-OH and/or C16:1 7c (26.9% versus 0.6–6.0%) and iso-C16:0
6
3-OH (1.2% versus 7.1–13.4%). The differences were also observed between strain
7
2A-7T and the type strain of ‘Marinitalea’ especially in iso-C14:0 (not detected versus
8
7.1%), anteiso-C17:19c (not detected versus 5.1%) and iso-C15:0 2-OH and/or C16:17c
9
(26.9% versus 4.1%).
10 11
Isoprenoid quinones were extracted from cells of strain 2A-7T grown in MB with
12
shaking at 28 ˚C for 2 days (to early stationary phase) as described previously
13
(Nishijima et al., 1997) and were analyzed by HPLC (Waters 600; Nihon Waters).
14
Menaquinone-6 (MK-6) was detected from strain 2A-7T as the sole isoprenoid quinone.
15
Members of the family Flavobacteriaceae generally exhibit MK-6 as their only or
16
major respiratory quinone (Bernardet et al., 2002).
17 18
For polar lipid analysis, cells of strain 2A-7T were grown in MB with shaking at 28 ˚C
19
for 2 days. Polar lipids were extracted, separated and revealed as described previously
20
(Teramoto et al., 2011) except that the total lipid content was revealed by spraying with
21
10% (w/v) molybdophosphoric acid in 2-propanol and incubating at 180 ˚C for 20 min.
22
The reagents used to reveal specific functional groups were ninhydrin (for free amino
23
groups), Dittmer and Lester reagent (for phosphate groups), anisaldehyde/sulfuric
24
acid/ethanol (1 : 1 : 18, by vol.) (for glycolipids) and periodic acid–Schiff stain (for
25
vicinal hydroxyl groups) (Teramoto et al., 2011). The polar lipid profile is shown in Fig.
26
S3. The major polar lipids were phosphatidylethanolamine (PE) and three unidentified
27
lipids; several unidentified lipids were found as minor components.
7
1 2
To determine the G+C content, genomic DNA was extracted and purified from cells of
3
strain 2A-7T grown in MB with shaking at 28 ˚C for 3 days based on the protocol of
4
Marmur (1961). During the protocol, DNA was first separated from protein by shaking
5
with phenol as described by Saito & Miura (1963) before with chloroform and isoamyl
6
alcohol. The genomic DNA was digested to nucleotides with nuclease P1 using a
7
DNA-GC kit (Seikagaku Kogyo) according to the procedures described by
8
Katayama-Fujimura et al. (1984). The G+C content of the DNA was determined by
9
HPLC (LC-10; Shimadzu) with an RP Aqueous column (4.6 x 250 mm; Nomura
10
Chemical) and a UV-visual spectrophotometric detector (SPD-10AV; Shimadzu) at 270
11
nm. The DNA G+C content of strain 2A-7T was 34.1 mol%.
12 13
The phylogenetic tree showed that strain 2A-7T was related most closely to members of
14
the genera Lutibacter and ‘Marinitalea’ but clustered distantly with these and the other
15
members of the family Flavobacteriaceae, which was supported by high bootstrap
16
values (Fig. 1). Also, strain 2A-7T showed low 16S rRNA gene sequence similarity to
17
the type strains of Lutibacter (up to 93.9%) and to the type strain of ‘Marinitalea’
18
(93.2%). In addition, strain 2A-7T differed from the type strains of Lutibacter and
19
‘Marinitalea’ based on fatty acid compositions (Tables 1 and 2) and oxidase activity
20
(Table 2). DNA G+C content and major polar lipids of strain 2A-7T were also different
21
from those of the type strain of ‘Marinitalea’ (Table 2). On the basis of genotypic (Fig.
22
1) and chemotaxonomic (Tables 1 and 2) data as well as phenotypic characteristics
23
(Table 2), we propose that strain 2A-7T represents a novel genus and species, for which
24
the name Flavicella marina gen. nov., sp. nov. is proposed.
25 26
Description of Flavicella gen. nov.
27
Flavicella (Fla.vi.cel'la. L. adj. flavus yellow; L. fem. n. cella, a cell; N.L. fem. n.
8
1
Flavicella a yellow cell.)
2 3
According to 16S rRNA gene sequence analysis, belongs to the Flavobacteriaceae.
4
Cells are Gram-stain-negative, non-motile, non-gliding, aerobic and rod- or
5
spherical-shaped. Catalase- and oxidase-positive. Carotenoids are contained while
6
flexirubin-type pigments are not. Predominant cellular fatty acids are iso-C15:0 2-OH
7
and/or C16:1 7c, iso-C17:0 3-OH and iso-C15:0 3-OH. MK-6 is detected as the sole
8
isoprenoid quinone. Major polar lipids are PE and three unidentified lipids. The DNA
9
G+C content of a known strain of the type species is 34.1 mol%. The type species is
10
Flavicella marina.
11 12
Description of Flavicella marina sp. nov.
13
Flavicella marina (ma.ri'na. L. fem. adj. marina of the sea, marine).
14 15
Displays the following properties in addition to those given in the genus description.
16
Smooth lenticular opaque light-yellow circular colonies with an entire edge are formed
17
on dMB1 plate. Cells are 0.4–0.9 × 0.7–1.5 m. Growth occurs at 4–30 ˚C (optimally at
18
25–28 ˚C) at pH 7–8 (optimally at pH 7) and with 3% (w/v) NaCl. In API 20 NE tests,
19
positive for aesculin hydrolase and -galactosidase, but negative for reduction of nitrate
20
to nitrite, indole production from tryptophan, fermentation of glucose, arginine
21
dihydrolase, urease, gelatinase and assimilation of glucose, L-arabinose, D-mannose,
22
D-mannitol, N-acetyl-D-glucosamine, maltose, potassium gluconate, n-caprate, adipate,
23
DL-malate, sodium citrate and phenylacetate. In API ZYM tests, positive for alkaline
24
phosphatase,
25
naphthol-AS-BI-phosphohydrolase, but negative for esterase (C4), esterase lipase (C8),
26
lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin,
27
-galactosidase, -galactosidase, -glucuronidase,
leucine
arylamidase,
9
acid
phosphatase
-glucosidase,
and
-glucosidase,
1
N-acetyl--glucosaminidase, -mannosidase and -fucosidase.
2 3
The type strain is 2A-7T (=NBRC 110145T=KCTC 42197T), isolated from surface
4
seawater at Muroto city, Kochi prefecture, Japan.
5 6
Acknowledgements
7
We thank Yasuyoshi Nakagawa for discussion on gliding motility, Ayumi Komatsu for
8
technical assistance, Satoru Ibuki & Takahiro Tsushima for providing Muroto seawater.
9
This study was performed through Program to Disseminate Tenure Tracking System of
10
the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
11
10
1
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reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
13
Shindo, K., Kikuta, K., Suzuki, A., Katsuta, A., Kasai, H., Yasumoto-Hirose, M.,
14
Matsuo, Y., Misawa, N. & Takaichi, S. (2007). Rare carotenoids, (3R)-saproxanthin
15
and (3R,2'S)-myxol, isolated from novel marine bacteria (Flavobacteriaceae) and their
16
antioxidative activities. Appl Microbiol Biotechnol 74, 1350–1357.
17
Sohn, J. H., Kwon, K. K., Kang, J. H., Jung, H. B. & Kim, S. J. (2004).
18
Novosphingobium pentaromativorans sp. nov., a high-molecular-mass polycyclic
19
aromatic hydrocarbon-degrading bacterium isolated from estuarine sediment. Int J Syst
20
Evol Microbiol 54, 1483–1487.
21
Takaichi, S. & Shimada, K. (1992). Characterization of carotenoids in photosynthetic
22
bacteria. Methods Enzymol 213, 374–385.
23
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6:
24
Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30, 2725–2729.
25
Teramoto, M., Ohuchi, M., Hatmanti, A., Darmayati, Y., Widyastuti, Y.,
26
Harayama, S. & Fukunaga, Y. (2011). Oleibacter marinus gen. nov., sp. nov., a
13
1
bacterium that degrades petroleum aliphatic hydrocarbons in a tropical marine
2
environment. Int J Syst Evol Microbiol 61, 375–380.
3
Teramoto, M., Suzuki, M., Okazaki, F., Hatmanti, A. & Harayama, S. (2009).
4
Oceanobacter-related bacteria are important for the degradation of petroleum aliphatic
5
hydrocarbons in the tropical marine environment. Microbiology 155, 3362–3370.
6
Teramoto, M., Takaichi, S., Inomata, Y., Ikenaga, H. & Misawa, N. (2003).
7
Structural and functional analysis of a lycopene beta-monocyclase gene isolated from a
8
unique marine bacterium that produces myxol. FEBS Lett 545, 120–126.
9
Tindall, B. J., Rosselló-Móra, R., Busse, H.-J., Ludwig,W. & Kämpfer, P. (2010).
10
Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst
11
Evol Microbiol 60, 249–266.
12
van den Berg, H., Faulks, R., Granado, H. F., Hirschberg, J., Olmedilla, B.,
13
Sandmann, G., Southon, S. & Stahl, W. (2000). The potential for the improvement of
14
carotenoid levels in foods and the likely systemic effects. J Sci Food Agric 80, 880–912.
15
Yarza, P., Richter, M., Peplies, J., Euzéby, J., Amann, R., Schleifer, K. H., Ludwig,
16
W., Glöckner, F. O. & Rosselló-Móra, R. (2008). The All-Species Living Tree
17
project: a 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst Appl
18
Microbiol 31, 241–250.
19
Yokoyama, A. & Miki, W. (1995). Isolation of myxol from a marine bacterium
20
Flavobacterium sp. associated with a marine sponge. Fish Sci 61, 684–686.
21 22
Figure legends
23
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences (1351 bp) showing
24
the phylogenetic position of strain 2A-7T in the family Flavobacteriaceae. Type strains
25
of the closest genera are shown. Bootstrap values (above 50%) based on 1000
26
resamplings are shown at branch nodes. Filled circles indicate that the corresponding
27
nodes were recovered in a tree generated with the maximum-likelihood method with
14
1
bootstrap values >60%. Cryomorpha ignava LMG21436T (AF170738) was used as an
2
outgroup (not shown). Bar, 0.01 substitutions per nucleotide position.
3 4
Supplementary Fig. S1. Absorption spectrum of pigment(s) produced in cells of
5
Flavicella marina gen. nov., sp. nov. 2A-7T. The spectrum of pigment(s) dissolved in
6
acetone is shown.
7 8
Supplementary Fig. S2. Transmission electron micrographs of negatively stained cells
9
of strain 2A-7T. Bars, 200 nm. The strain possessed a flagellum-like structure (indicated
10
by arrows).
11 12
Supplementary Fig. S3. A two-dimensional thin-layer chromatogram of polar lipids
13
extracted from strain 2A-7T, stained with molybdophosphoric acid to detect the total
14
lipids. PE, phosphatidylethanolamine; and L1−10, unidentified lipids.
15 16
15
1
Table 1. Cellular fatty acid composition of strain 2A-7T and type strains of the closest
2
genera Lutibacter and ‘Marinitalea’.
3
Strains: 1, 2A-7T (grown on MA at 30 ˚C for 3 days); 2, Lutibacter litoralis CL-TF09T
4
(grown on MA at 30 ˚C for 1 day; data from Choi & Cho, 2006); 3, L. maritimus S7-2T
5
(grown on MA at 30 ˚C for 2 days; Park et al., 2010); 4, L. aestuarii MA-My1T (grown
6
on MA at 30 ˚C for 2 days; Lee et al., 2012); 5, L. flavus IMCC1507T (grown on MA at
7
30 ˚C for up to 3 days; Choi et al., 2013); 6, L. agarilyticus KYW566T (grown on MA
8
at 25 ˚C for 2 days; Park et al., 2013); 7, ‘Marinitalea sucinacia’ JC2131T (grown on
9
MA at 28 ˚C for 2-7 days; Kim et al., 2011). Values are percentages of the total fatty
10
acids and are shown when present at ≥0.5% in strain 2A-7T or S7-2T or ≥1.0% in strain
11
CL-TF09T, MA-My1T, IMCC1507T, KYW566T or JC2131T. –, Not detected or no data
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
available. Fatty acid C11:0 C14:0 C15:0 C15:0 2-OH C15:0 3-OH C16:0 C16:0 3-OH C17:0 2-OH C18:0 iso-C13:0 anteiso-C13:0 iso-C14:0 iso-C14:0 3-OH iso-C15:0 anteiso-C15:0 iso-C15:0 3-OH iso-C15:1 G anteiso-C15:1 A iso-C16:0 iso-C16:0 3-OH iso-C16:1 H iso-C17:0 3-OH anteiso-C17:19c C13:1 at 12–13 C15:16c C17:16c C18:17c C18:19c C20:46,9,12,15c Unknown C11.543 Unknown C13.565
1
2
– 0.6 2.2 1.6 0.6 1.8 4.2 2.0 – 3.9 0.5 – – 8.8 2.6 10.6 5.0 – 0.2 1.2 – 16.8 – 0.5 1.1 0.3 – – 1.6 0.5 2.4
– – 1.7 2.1 1.5 – – 3.6 1.3 – 1.4 2.2 – 16.7 15.1 17.4 4.2 1.6 – 13.4 1.3 3.9 – – 1.5 1.0 – – – 2.3 –
3 – – 7.2 – 2.3 – – 0.6 –
International Journal of Systematic and Evolutionary Microbiology Flavicella marina gen. nov., sp. nov., a carotenoid-producing bacterium from surface seawater. --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00053R1
Full Title:
Flavicella marina gen. nov., sp. nov., a carotenoid-producing bacterium from surface seawater.
Short Title:
Flavicella marina gen. nov., sp. nov
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Maki Teramoto Kochi University Nankoku, JAPAN
First Author:
Maki Teramoto
Order of Authors:
Maki Teramoto Miyuki Nishijima
Manuscript Region of Origin:
JAPAN
Abstract:
A Gram-stain-negative, non-motile, mesophilic, aerobic, rod- or spherical-shaped bacterium, strain 2A-7T, was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain produced pigment(s), absorption spectrum of which closely resembled that of β-carotene. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Flavobacteriaceae and clustered distantly with the type strains of the genus Lutibacter (up to 93.9% similarity). The DNA G+C content was 34.1 mol%. The major fatty acids were iso-C15:0 2-OH and/or C16:1 ω7c, iso-C17:0 3-OH and iso-C15:0 3-OH. The major polar lipids were phosphatidylethanolamine and three unidentified lipids. Menaquinone-6 was detected as the sole isoprenoid quinone. On the basis of phenotypic, genotypic and chemotaxonomic data, strain 2A-7T represents a novel genus and species, for which the name Flavicella marina gen. nov., sp. nov. is proposed. The type strain of Flavicella marina is 2A-7T (=NBRC 110145T=KCTC 42197T).
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1
Title: Flavicella marina gen. nov., sp. nov., a carotenoid-producing bacterium from
2
surface seawater.
3 4
Author: Maki Teramoto1* and Miyuki Nishijima2
5 6
1
7
Japan.
Oceanography Section, Kochi University, Kohasu, Oko, Nankoku, Kochi 783-8505,
8 9 10
2
TechnoSuruga Laboratory Co. Ltd. 330 Nagasaki, Shimizu-ku Shizuoka 424-0065
Japan
11 12
*To whom correspondence should be addressed
13
Tel: 81-88-880-2240, Fax: 81-88-880-2177, E-mail: [email protected]
14 15 16
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of
17
strain 2A-7T is AB915171.
18
Three supplementary figures are available with the online version of this paper.
19 20 21
Category: New taxa – Bacteroidetes
22
Running title: Flavicella marina gen. nov., sp. nov.
23 24
1
1
Abstract
2
A Gram-stain-negative, non-motile, mesophilic, aerobic, rod- or spherical-shaped
3
bacterium, strain 2A-7T, was isolated from surface seawater at Muroto city, Kochi
4
prefecture, Japan. The strain produced pigment(s), absorption spectrum of which closely
5
resembled that of -carotene. Phylogenetic analyses based on 16S rRNA gene
6
sequences showed that the strain fell within the family Flavobacteriaceae and clustered
7
distantly with the type strains of the genus Lutibacter (up to 93.9% similarity). The
8
DNA G+C content was 34.1 mol%. The major fatty acids were iso-C15:0 2-OH and/or
9
C16:1 7c, iso-C17:0 3-OH and iso-C15:0 3-OH. The major polar lipids were
10
phosphatidylethanolamine and three unidentified lipids. Menaquinone-6 was detected as
11
the sole isoprenoid quinone. On the basis of phenotypic, genotypic and
12
chemotaxonomic data, strain 2A-7T represents a novel genus and species, for which the
13
name Flavicella marina gen. nov., sp. nov. is proposed. The type strain of Flavicella
14
marina is 2A-7T (=NBRC 110145T=KCTC 42197T).
15 16 17
Main Text
18
A large number of carotenoids of different molecular structures are known to be
19
biosynthesized and have been attracting great attention due to their beneficial effects on
20
human health, e.g. their potential in the prevention of diseases such as cancer and
21
cardiovascular disease (van den Berg et al., 2000). Marine bacteria of the family
22
Flavobacteriaceae usually produce carotenoids (Bernardet et al., 2002), which include
23
zeaxanthin (a bicyclic carotenoid; Asker et al., 2007) and myxol (a monocyclic
24
carotenoid; Shindo et al., 2007; Teramoto et al., 2003; Yokoyama & Miki, 1995), and
25
are thus attractive in health-related applications. The family Flavobacteriaceae,
26
belonging to the phylum Bacteroidetes (the Cytophaga–Flavobacterium–Bacteroides
27
group), currently comprises more than 110 genera (http://www.bacterio.cict.fr).
2
1
Members of this family have been isolated from terrestrial as well as marine
2
environments.
3 4
Muroto city, Kochi prefecture, Japan is one of the rare areas where deep seawater is
5
upwelling. Deep seawater is relatively rich in nitrogen and phosphorus (DeLong et al.,
6
2006; Hansman et al., 2009), which are the two main growth-limiting factors in marine
7
environments. Therefore, surface seawater around Muroto could be more nutrient-rich
8
than surface seawater in other areas, suggesting that rich microbial resources could be
9
harboured in the Muroto surface seawater. The present study describes a bacterial strain,
10
designated strain 2A-7T, isolated from the Muroto surface seawater (33˚18’ N 134˚11’ E,
11
water depth of 0.5 m) in summer 2011, which was found to be a novel member of
12
Flavobacteriaceae.
13 14
Strain 2A-7T was obtained from the seawater by direct plating on ONR7a medium
15
(artificial seawater medium; Dyksterhouse et al., 1995) supplemented with (per litre) 15
16
g agar, 0.05 g sodium acetate, 0.05 g sodium pyruvate and 0.1 g yeast extract (Bacto
17
yeast extract; BD).
18 19
Strain 2A-7T was cultivated at 28 ˚C for 48 h on a dMB1 plate, which contained (per
20
litre) 7.48 g marine broth 2216 (MB; BD), 15 g agar, 0.2 litres distilled water and 0.8
21
litres artificial seawater (ASW; Marine Art SF-1; Tomita Pharmaceutical).
22
Phase-contrast microscopy (BX50F4; Olympus) showed that the strain was not motile.
23
Gram-staining
24
Gram-stain-negative. The strain was non-sporulating, and formed smooth lenticular
25
opaque light-yellow circular colonies with an entire edge that possessed a butyrous
26
consistency on the dMB1 plate. The strain was oxidase-positive and catalase-positive
27
(Barrow & Feltham, 1993), and negative for acid production from D-glucose on
tests
(Favor
G
kit;
Nissui)
3
showed
that
the
strain
was
1
modified Hugh-Leifson’s O-F medium (Leifson, 1963). Flexirubin-type pigments were
2
not detected by flooding 2A-7T cells with 20% (w/v) KOH (Fautz & Reichenbach,
3
1980). The API 20 NE kit (bioMerieux) was used for assessing biochemical
4
characteristics according to the manufacturer’s instructions, with the exception that the
5
cells were suspended in ASW. In addition, API ZYM tests (bioMerieux) were conducted
6
to examine enzyme activities as described in the manufacturer’s manual, with the
7
exceptions that the inoculum was prepared using ASW and the incubation time was
8
extended to 24 h at 28 ˚C. The strain was positive for aesculin hydrolase and
9
-galactosidase and negative for reduction of nitrate to nitrite, indole production from
10
tryptophan, fermentation of glucose, arginine dihydrolase, urease and gelatinase. Other
11
detailed phenotypic features of strain 2A-7T are given in the species description.
12 13
Strain 2A-7T did not glide by the method of Bowman (2000) on the quarter-strength MB
14
plate containing (per litre) 9.35 g MB, 0.25 litres distilled water and 0.75 litres ASW
15
and solidified with 1% (w/v) agar. The pigment(s) was extracted with acetone from
16
cells of strain 2A-7T by shaking vigorously for 10 min. The extract was centrifuged in
17
order to remove cell debris, and the resulting supernatant was subjected to a
18
spectrophotometer (DU 730; Beckman Coulter). The visible absorption spectrum of the
19
pigment(s) had absorption maxima at 455 and 475 nm (Fig. S1, available in the online
20
Supplementary Material), which closely resembled that of authentic -carotene reported
21
in the literature (Takaichi & Shimada, 1992), suggesting that strain 2A-7T could have
22
contained -carotene or a carotenoid structurally similar to -carotene, such as
23
zeaxanthin (dihydroxy--carotene), as a dominant pigment.
24 25
For transmission electron microscopy (H7600; Hitachi), strain 2A-7T was grown at 20
26
˚C for 7 days on a dMB plate (composition same as for the dMB1 plate except that
27
Muroto surface seawater was used instead of ASW), and negatively stained with 2%
4
1
uranyl acetate. Cells of strain 2A-7T were rod- or spherical-shaped (0.4–0.9 × 0.7–1.5
2
m) and possessed a flagellum-like structure (Fig. S2). Although strain 2A-7T had a
3
flagellum-like structure, we couldn't have observed its motility as described above.
4
Motility of strain 2A-7T was also tested at its early growth phase (grown for 16 h at 10
5
˚C and at 15 ˚C) on the quarter-strength MB plate solidified with 0.2% (w/v) agar.
6
However, the strain also did not show motility. Therefore, it was unclear whether the
7
flagellum-like structure was a flagellum or not.
8 9
Anaerobic growth was tested on dMB plates at 20 ˚C in a GasPak anaerobic jar (BD)
10
for 2 months. Strain 2A-7T did not grow under anaerobic conditions. Growth
11
temperature was tested on dMB plates at 4, 10, 15, 20, 22, 25, 28, 30, 35, 37 and 40 ˚C
12
for 2 months. Strain 2A-7T grew at 4–30 ˚C (optimally at 25–28 ˚C). NaCl requirement
13
was tested at 28 ˚C for 2 weeks on modified MB (NaCl-deficient; Sohn et al., 2004)
14
supplemented with 0, 1, 3, 5, 10, 12, 15 and 17% (w/v) NaCl. Strain 2A-7T grew with
15
3% (w/v) NaCl but not with the other concentrations of NaCl. The pH range for growth
16
(tested at pH 2–11, intervals of 1 pH unit) was determined at 20 ˚C for 1 month with
17
shaking in filter-sterilized dMB medium, which contained (per litre) 7.48 g MB, 0.2
18
litres distilled water and 0.8 litres Muroto surface seawater. Strain 2A-7T grew at pH 7–
19
8 (optimally at pH 7).
20 21
The almost full-length 16S rRNA gene sequence of strain 2A-7T (1375 bp) was
22
obtained as described previously (Teramoto et al., 2009). The most similar species to
23
strain 2A-7T was Lutibacter agarilyticus (93.9% similarity to its type strain), and strain
24
2A-7T showed 93.7% similarity to the type species of Lutibacter (Lutibacter litoralis;
25
strain CL-TF09T), suggesting that strain 2A-7T does not belong to this genus in the
26
family Flavobacteriaceae (Yarza et al., 2008; Tindal et al., 2010). On the other hand,
27
the closest GenBank relative to strain 2A-7T indicated by BLAST search (Altschul et al.,
5
1
1990) was an uncultured marine bacterium clone SeaWat_F492 (JQ198834) with the
2
similarity of 98.5%. The sequence of strain 2A-7T was aligned with related sequences of
3
members of the family Flavobacteriaceae available in public databases using
4
CLUSTAL X (ver. 2.1) (Larkin et al., 2007). The alignments were manually modified
5
where necessary, and trimming of gaps was performed. Phylogenetic trees were inferred
6
from the aligned sequences of 1351 bp using the neighbour-joining algorithm (Saitou &
7
Nei, 1987) by CLUSTAL X with default parameters (including Kimura’s correction)
8
and maximum-likelihood algorithm (Felsenstein, 1981) by MEGA 6.06 (Tamura et al.,
9
2013) and analyzed using bootstrapping (Felsenstein, 1985) based on 1000 resamplings.
10
The neighbour-joining tree is shown in Fig. 1. Strain 2A-7T fell within the family
11
Flavobacteriaceae and was most closely related with the genus Lutibacter and an
12
invalidly described genus ‘Marinitalea’. However, strain 2A-7T clustered distantly with
13
these and the other members of the family Flavobacteriaceae, which was supported by
14
high bootstrap values (95% using the neighbour-joining algorithm and 95% with the
15
maximum-likelihood algorithm). Strain 2A-7T showed 93.2% similarity in the 16S
16
rRNA gene sequence to the type strain of ‘Marinitalea’ (‘Marinitalea sucinacia’
17
JC2131T; FJ387163). Together with the low 16S rRNA gene sequence similarities of
18
strain 2A-7T to the type strains of the closely related genera, these results indicate that
19
strain 2A-7T could represent a novel genus of the family Flavobacteriaceae in the class
20
Flavobacteriia.
21 22
The cellular fatty acid composition of strain 2A-7T was analyzed by using cells grown
23
on Marine Agar 2216 (BD) at 30 ˚C for 3 days when growth reached its maximum (in
24
the early stationary growth phase). Cellular fatty acid methyl esters were prepared and
25
analyzed by GC (7890A GC system; Agilent Technologies) according to the
26
instructions given for the Microbial Identification System version 6.0 and were
27
compared using the database TSBA40 (MIDI). The results are given in Table 1 with
6
1
those of the type strains of the closest genera Lutibacter and ‘Marinitalea’. The major
2
cellular fatty acids of strain 2A-7T were iso-C15:0 2-OH and/or C16:1 7c (26.9% of the
3
total), iso-C17:0 3-OH (16.8%) and iso-C15:0 3-OH (10.6%). Differences in the
4
composition were present between strain 2A-7T and the type strains of Lutibacter
5
especially in iso-C15:0 2-OH and/or C16:1 7c (26.9% versus 0.6–6.0%) and iso-C16:0
6
3-OH (1.2% versus 7.1–13.4%). The differences were also observed between strain
7
2A-7T and the type strain of ‘Marinitalea’ especially in iso-C14:0 (not detected versus
8
7.1%), anteiso-C17:19c (not detected versus 5.1%) and iso-C15:0 2-OH and/or C16:17c
9
(26.9% versus 4.1%).
10 11
Isoprenoid quinones were extracted from cells of strain 2A-7T grown in MB with
12
shaking at 28 ˚C for 2 days (to early stationary phase) as described previously
13
(Nishijima et al., 1997) and were analyzed by HPLC (Waters 600; Nihon Waters).
14
Menaquinone-6 (MK-6) was detected from strain 2A-7T as the sole isoprenoid quinone.
15
Members of the family Flavobacteriaceae generally exhibit MK-6 as their only or
16
major respiratory quinone (Bernardet et al., 2002).
17 18
For polar lipid analysis, cells of strain 2A-7T were grown in MB with shaking at 28 ˚C
19
for 2 days. Polar lipids were extracted, separated and revealed as described previously
20
(Teramoto et al., 2011) except that the total lipid content was revealed by spraying with
21
10% (w/v) molybdophosphoric acid in 2-propanol and incubating at 180 ˚C for 20 min.
22
The reagents used to reveal specific functional groups were ninhydrin (for free amino
23
groups), Dittmer and Lester reagent (for phosphate groups), anisaldehyde/sulfuric
24
acid/ethanol (1 : 1 : 18, by vol.) (for glycolipids) and periodic acid–Schiff stain (for
25
vicinal hydroxyl groups) (Teramoto et al., 2011). The polar lipid profile is shown in Fig.
26
S3. The major polar lipids were phosphatidylethanolamine (PE) and three unidentified
27
lipids; several unidentified lipids were found as minor components.
7
1 2
To determine the G+C content, genomic DNA was extracted and purified from cells of
3
strain 2A-7T grown in MB with shaking at 28 ˚C for 3 days based on the protocol of
4
Marmur (1961). During the protocol, DNA was first separated from protein by shaking
5
with phenol as described by Saito & Miura (1963) before with chloroform and isoamyl
6
alcohol. The genomic DNA was digested to nucleotides with nuclease P1 using a
7
DNA-GC kit (Seikagaku Kogyo) according to the procedures described by
8
Katayama-Fujimura et al. (1984). The G+C content of the DNA was determined by
9
HPLC (LC-10; Shimadzu) with an RP Aqueous column (4.6 x 250 mm; Nomura
10
Chemical) and a UV-visual spectrophotometric detector (SPD-10AV; Shimadzu) at 270
11
nm. The DNA G+C content of strain 2A-7T was 34.1 mol%.
12 13
The phylogenetic tree showed that strain 2A-7T was related most closely to members of
14
the genera Lutibacter and ‘Marinitalea’ but clustered distantly with these and the other
15
members of the family Flavobacteriaceae, which was supported by high bootstrap
16
values (Fig. 1). Also, strain 2A-7T showed low 16S rRNA gene sequence similarity to
17
the type strains of Lutibacter (up to 93.9%) and to the type strain of ‘Marinitalea’
18
(93.2%). In addition, strain 2A-7T differed from the type strains of Lutibacter and
19
‘Marinitalea’ based on fatty acid compositions (Tables 1 and 2) and oxidase activity
20
(Table 2). DNA G+C content and major polar lipids of strain 2A-7T were also different
21
from those of the type strain of ‘Marinitalea’ (Table 2). On the basis of genotypic (Fig.
22
1) and chemotaxonomic (Tables 1 and 2) data as well as phenotypic characteristics
23
(Table 2), we propose that strain 2A-7T represents a novel genus and species, for which
24
the name Flavicella marina gen. nov., sp. nov. is proposed.
25 26
Description of Flavicella gen. nov.
27
Flavicella (Fla.vi.cel'la. L. adj. flavus yellow; L. fem. n. cella, a cell; N.L. fem. n.
8
1
Flavicella a yellow cell.)
2 3
According to 16S rRNA gene sequence analysis, belongs to the Flavobacteriaceae.
4
Cells are Gram-stain-negative, non-motile, non-gliding, aerobic and rod- or
5
spherical-shaped. Catalase- and oxidase-positive. Carotenoids are contained while
6
flexirubin-type pigments are not. Predominant cellular fatty acids are iso-C15:0 2-OH
7
and/or C16:1 7c, iso-C17:0 3-OH and iso-C15:0 3-OH. MK-6 is detected as the sole
8
isoprenoid quinone. Major polar lipids are PE and three unidentified lipids. The DNA
9
G+C content of a known strain of the type species is 34.1 mol%. The type species is
10
Flavicella marina.
11 12
Description of Flavicella marina sp. nov.
13
Flavicella marina (ma.ri'na. L. fem. adj. marina of the sea, marine).
14 15
Displays the following properties in addition to those given in the genus description.
16
Smooth lenticular opaque light-yellow circular colonies with an entire edge are formed
17
on dMB1 plate. Cells are 0.4–0.9 × 0.7–1.5 m. Growth occurs at 4–30 ˚C (optimally at
18
25–28 ˚C) at pH 7–8 (optimally at pH 7) and with 3% (w/v) NaCl. In API 20 NE tests,
19
positive for aesculin hydrolase and -galactosidase, but negative for reduction of nitrate
20
to nitrite, indole production from tryptophan, fermentation of glucose, arginine
21
dihydrolase, urease, gelatinase and assimilation of glucose, L-arabinose, D-mannose,
22
D-mannitol, N-acetyl-D-glucosamine, maltose, potassium gluconate, n-caprate, adipate,
23
DL-malate, sodium citrate and phenylacetate. In API ZYM tests, positive for alkaline
24
phosphatase,
25
naphthol-AS-BI-phosphohydrolase, but negative for esterase (C4), esterase lipase (C8),
26
lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin,
27
-galactosidase, -galactosidase, -glucuronidase,
leucine
arylamidase,
9
acid
phosphatase
-glucosidase,
and
-glucosidase,
1
N-acetyl--glucosaminidase, -mannosidase and -fucosidase.
2 3
The type strain is 2A-7T (=NBRC 110145T=KCTC 42197T), isolated from surface
4
seawater at Muroto city, Kochi prefecture, Japan.
5 6
Acknowledgements
7
We thank Yasuyoshi Nakagawa for discussion on gliding motility, Ayumi Komatsu for
8
technical assistance, Satoru Ibuki & Takahiro Tsushima for providing Muroto seawater.
9
This study was performed through Program to Disseminate Tenure Tracking System of
10
the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
11
10
1
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21 22
Figure legends
23
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences (1351 bp) showing
24
the phylogenetic position of strain 2A-7T in the family Flavobacteriaceae. Type strains
25
of the closest genera are shown. Bootstrap values (above 50%) based on 1000
26
resamplings are shown at branch nodes. Filled circles indicate that the corresponding
27
nodes were recovered in a tree generated with the maximum-likelihood method with
14
1
bootstrap values >60%. Cryomorpha ignava LMG21436T (AF170738) was used as an
2
outgroup (not shown). Bar, 0.01 substitutions per nucleotide position.
3 4
Supplementary Fig. S1. Absorption spectrum of pigment(s) produced in cells of
5
Flavicella marina gen. nov., sp. nov. 2A-7T. The spectrum of pigment(s) dissolved in
6
acetone is shown.
7 8
Supplementary Fig. S2. Transmission electron micrographs of negatively stained cells
9
of strain 2A-7T. Bars, 200 nm. The strain possessed a flagellum-like structure (indicated
10
by arrows).
11 12
Supplementary Fig. S3. A two-dimensional thin-layer chromatogram of polar lipids
13
extracted from strain 2A-7T, stained with molybdophosphoric acid to detect the total
14
lipids. PE, phosphatidylethanolamine; and L1−10, unidentified lipids.
15 16
15
1
Table 1. Cellular fatty acid composition of strain 2A-7T and type strains of the closest
2
genera Lutibacter and ‘Marinitalea’.
3
Strains: 1, 2A-7T (grown on MA at 30 ˚C for 3 days); 2, Lutibacter litoralis CL-TF09T
4
(grown on MA at 30 ˚C for 1 day; data from Choi & Cho, 2006); 3, L. maritimus S7-2T
5
(grown on MA at 30 ˚C for 2 days; Park et al., 2010); 4, L. aestuarii MA-My1T (grown
6
on MA at 30 ˚C for 2 days; Lee et al., 2012); 5, L. flavus IMCC1507T (grown on MA at
7
30 ˚C for up to 3 days; Choi et al., 2013); 6, L. agarilyticus KYW566T (grown on MA
8
at 25 ˚C for 2 days; Park et al., 2013); 7, ‘Marinitalea sucinacia’ JC2131T (grown on
9
MA at 28 ˚C for 2-7 days; Kim et al., 2011). Values are percentages of the total fatty
10
acids and are shown when present at ≥0.5% in strain 2A-7T or S7-2T or ≥1.0% in strain
11
CL-TF09T, MA-My1T, IMCC1507T, KYW566T or JC2131T. –, Not detected or no data
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
available. Fatty acid C11:0 C14:0 C15:0 C15:0 2-OH C15:0 3-OH C16:0 C16:0 3-OH C17:0 2-OH C18:0 iso-C13:0 anteiso-C13:0 iso-C14:0 iso-C14:0 3-OH iso-C15:0 anteiso-C15:0 iso-C15:0 3-OH iso-C15:1 G anteiso-C15:1 A iso-C16:0 iso-C16:0 3-OH iso-C16:1 H iso-C17:0 3-OH anteiso-C17:19c C13:1 at 12–13 C15:16c C17:16c C18:17c C18:19c C20:46,9,12,15c Unknown C11.543 Unknown C13.565
1
2
– 0.6 2.2 1.6 0.6 1.8 4.2 2.0 – 3.9 0.5 – – 8.8 2.6 10.6 5.0 – 0.2 1.2 – 16.8 – 0.5 1.1 0.3 – – 1.6 0.5 2.4
– – 1.7 2.1 1.5 – – 3.6 1.3 – 1.4 2.2 – 16.7 15.1 17.4 4.2 1.6 – 13.4 1.3 3.9 – – 1.5 1.0 – – – 2.3 –
3 – – 7.2 – 2.3 – – 0.6 –
