IJSEM Papers in Press. Published August 5, 2014 as doi:10.1099/ijs.0.066738-0
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Psychrobium conchae gen. nov, sp. nov., a psychrophilic marine bacterium isolated from
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the Iheya North hydrothermal field, Okinawa Trough, off Japan.
3 4
Yuichi Nogi1, Mariko Abe2, Shinsuke Kawagucci2 and Hisako Hirayama2
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1
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Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka
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237-0061, Japan.
Research and Development Center for Marine Biosciences, Japan Agency for
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2
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Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka
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237-0061, Japan.
Department of Subsurface Geobiological Analysis and Research, Japan Agency for
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Running title: Psychrobium conchae gen. nov, sp., nov.,
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Subject category for the content list: Proteobacteria
17 18
*Corresponding author: Yuichi Nogi
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Phone: +81-468-67-9697, Fax: +81-468-66-6364,
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E-mail: [email protected]
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1
1
Abbreviations:
2
ASW: artificial seawater
3
DPG: diphosphatidylglycerol
4
LB: Luria-Bertani
5
MA: Marine Agar
6
MB: Marine Broth
7
PE: phosphatidylethanolamine
8
PG: phosphatidylglycerol
9
PL: phospholipid
10
PME: phosphatidylmethylethanolamine
11
NJ: neighbor-joining
12
ML: maximum-likelihood
13
UPGMA: unweighted pair group method with arithmetic mean
14 15 16
The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequences of strain
17
BJ-1T is AB930131.
18 19
2
1 2
Abstract A novel psychrophilic marine bacterial strain designated BJ-1T was isolated from
3
the Iheya North hydrothermal field, Okinawa Trough off Japan. Cells were
4
Gram-negative, rod-shaped, non-spore-forming aerobic chemo-organotrophs and motile
5
by means of a single polar flagellum. Growth occurred at temperatures below 16 °C, with
6
the optimum between 9 and 12 °C. Phylogenetic analysis based on the 16S rRNA gene
7
sequence indicated that the closest relatives of strain BJ-1T were Shewanella denitrificans
8
OS-217T (93.5 %), Shewanella profunda DSM 15900T (92.9 %), Shewanella gaetbuli
9
TF-27T (92.9 %), Paraferrimonas sedimenticola Mok-106T (92.1 %) and Ferrimonas
10
kyonanensis Asr22-7T (91.7 %). The major respiratory quinone was Q-8. The
11
predominant fatty acids were C16:1ω7c and C16:0. The G + C content of the novel strain
12
was 40.5 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, it is
13
proposed that strain BJ-1T represents a novel species in a new genus for which the name
14
Psychrobium conchae gen. nov., sp. nov., is proposed. The type strain of the type species
15
is BJ-1T (=JCM 30103T =DSMZ 28701T).
16 17
3
1
The genus Shewanella, currently represented by a single member of the family
2
Shewanellaceae (Ivanova et al., 2004a), belongs to the order Alteromonadales. The
3
genus was defined by the following features: aerobic or facultatively anaerobic,
4
Gram-negative, motile, rod-shaped bacteria. Nitrate-reducing and having 14:0, 16:1ω7,
5
16:0 and 17:1ω6 as the major fatty acids. In this study, we used a polyphasic taxonomic
6
approach to investigate a psychrophilic strain that was found to represent a novel genus
7
and species belonging to the family Shewanellaceae.
8 9
Strain BJ-1T was isolated from the gill tissue of the deep-sea hydrothermal vent mussel
10
Bathymodiolus japonicus at the Iheya North hydrothermal field, Okinawa Trough, off
11
Japan (latitude: 27°47.438 N, longitude: 126°53.736 E, depth: 990 m), collected by the
12
remotely operated vehicle Hyper-Dolphin during JAMSTEC NT12-06 cruise in March
13
2012 (Kawagucci et al., 2013). The fresh gill tissue was crushed with a small amount of
14
sterilized artificial seawater (ASW; RohtoMarine, Rei-Sea Co., Tokyo) with ice-cooling
15
by using a mortar and pestle, and was made into a fine paste. The tissue paste suspended
16
in ASW was spread on a Marine Agar 2216 (MA; Difco) plates and incubated at 10 °C
17
upon isolation. After isolation of strain BJ-1T, the strain maintained on MA plates or in
18
Marine Broth 2216 (MB; Difco) were incubated aerobically for 2 or 3 days at 12 °C and
19
stored at –80 ºC. Unless otherwise indicated, the physiological tests were performed with
20
a slight modification (use of ASW) of the general procedures described by Barrow &
21
Feltham (1993) and Baumann et al. (1972). The effects of temperature, NaCl
22
concentration and pH on cell growth were determined by examining the time course of
23
optical density (temperature gradient incubator with a bio-photorecorder, model
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TVS126MA; Advantec). Growth at 4–20 °C was tested in MB. Cell growth was observed
′
′
4
1
at 16 °C or less (optimum 12 °C), but not above 18 °C. Growth at various NaCl
2
concentrations (0, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5 % w/v) was examined in Luria-Bertani
3
(LB) medium (1.0 % [w/v] tryptone [Difco], 0.5 % [w/v] yeast extract [Difco]) and
4
incubated at 12 °C. Cell growth was observed at NaCl concentrations of 2 % to 4 %
5
(optimum 3%), but not at less than 1 % or more than 5 % NaCl. Growth at various pH
6
values (5.0–10.0 in increments of 0.5 pH units) was measured in 3 % NaCl LB medium
7
and incubated at 12 °C. Cell growth was observed at pH 5.5 to 8.5 (optimum pH 6.0–6.5),
8
but not at pH 5.0 or at greater than pH 9.0.
9
Species of the genera Shewanella, Paramoritella and Ferrimonas which not grow at a 5%
10
NaCl concentration can be grown at a 1% or lower NaCl concentration, and this species
11
that cannot be grown at a 1% NaCl concentration can be grown at a 5% or higher
12
concentration (Table 1).
13
The morphology of living and non-living stained cells was determined by light
14
microscopy and transmission electron microscopy, respectively. For negative staining, 1
15
drop of a culture was placed on a copper grid coated with Pioloform and carbon and
16
stained with 1% potassium phosphotungstic acid adjusted to pH 6.5 with potassium
17
hydroxide. The negatively stained cells were observed with a model Tecnai 20
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transmission electron microscope (FEI, Oregon USA) at an accelerating voltage of 200kV.
19 20
Growth under anaerobic conditions was tested on MA 1 week after the addition of 0.1 %
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KNO3 to the MA plates with the AnaeroPak system (Mitsubishi Gas Chemical). Acid
22
production from sugars was assessed using modified oxidative-fermentative medium
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(Hugh & Leifson, 1953) containing ASW, 0.05 % (NH4)2SO4, 0.01 % yeast extract
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(Difco), 0.05 % Tris, 1 % test sugar and 0.003 % bromothymol blue (pH adjusted to 7.2 at
5
1
20 °C) with incubation at the optimum temperature. Oxidase activity was determined by
2
spreading cell pellets on oxidase test paper (Nissui Pharmaceutical). Hydrolysis of gelatin,
3
casein, starch, Tween 40 and 80, chitin and esculin was detected on MA plates using
4
substrate concentrations of 1 % (w/v). DNase activity was assessed using DNase Test
5
Agar (Difco). The susceptibility of the strain to antibiotics was tested using MA plates
6
and 6-mm sensitivity discs (Becton, Dickinson and Company) according to the
7
manufacturer’s instructions. The following antibiotics were examined: ampicillin (10 µg),
8
cefotaxime (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), clindamycin (2 µg),
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erythromycin (15 µg), gentamicin (10 µg), penicillin (10 IU), polymyxin B (300 µg),
10
rifampicin (5 µg), tetracycline (30 µg) and vancomycin (30 µg). The effects of
11
antimicrobial compounds on cell growth were assessed after 2 days at 12 °C. The
12
diameter of the inhibition zone was used to judge susceptibility according to the
13
manufacturer’s manual.
14
The cells of strain BJ-1T were rod-shaped, Gram-negative, strictly aerobic, non-spore
15
forming and motile by means of a single polar flagellum. Colonies were whitish in color,
16
smooth, circular and 1.0–2.0 mm in diameter after 3 days incubation at 12 °C on MA.
17
Detailed results from the phenotypic and biochemical tests are given in the species
18
description and shown in Table 1. Among related genera, only strain BJ-1T and some
19
species of the genus Shewanella cannot be grown under anaerobic conditions.
20
Chromosomal DNA was purified using the phenol extraction method (Saito & Miura,
21
1963). The DNA G + C content was determined using reversed-phase HPLC (Tamaoka &
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Komagata, 1984). The 16S rRNA gene was amplified using the PCR method with
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primers 27F and 1492R (Lane, 1991). The 16S rRNA gene sequence of strain BJ-1T was
24
obtained by direct sequencing of PCR-amplified DNA as described previously (Uchida et
6
1
al., 2012). The resulting 16S rRNA gene sequence (1482 nt) of strain BJ-1T was
2
compared with available 16S rRNA gene sequences from the DDBJ using the BLAST
3
program (http://blast.ddbj.nig.ac.jp/top-j.html) to determine an approximate phylogenetic
4
affiliation, and gene sequences were aligned with those of closely related species using
5
the CLUSTAL X software program (Thompson et al., 1997). In addition, sequence
6
similarity values were calculated using the GENETYX-MAC program ver. 17.0.2 (SDC
7
Software Development) between the novel strain and other related members.
8
Phylogenetic analyses were conducted in MEGA 5.2 (Tamura et al., 2011) using the NJ
9
method (Saitou & Nei, 1987). Bootstrap analysis to evaluate the stability of phylogenetic
10
trees was performed by obtaining a consensus tree based on 1000 randomly generated
11
trees
12
maximum-likelihood (ML) and unweighted pair group method with arithmetic mean
13
(UPGMA) analyses (data not shown), which confirmed the robust nature of this group of
14
organisms, the branching order and high bootstrap values. The results of the phylogenetic
15
analyses indicated that the novel organism belonged to the class Gammaproteobacteria.
16
The novel bacterium was most closely related to Shewanella denitrificans OS-217T,
17
Shewanella profunda LT13aT, Shewanella waksmanii KMM3823T and Shewanella
18
gaetbuli TF-27T, with pairwise similarity values of 93.5 %, 93.2 %, 92.9 % and 92.9 %,
19
respectively (Fig. 1). More distantly related organisms included Paraferrimonas
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sedimenticola Mok-106T (92.1 %), Moritella marina ATCC15381T (91.3 %),
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Paramoritella alkaliphila A3F-7T (91.1 %) and Ferrimonas balearica DSM 9799T
22
(90.8 %). This cluster was supported by a significant bootstrap value of 90 %. There is no
23
precise correlation between 16S rRNA gene sequence divergence and species delineation,
24
but it is generally recognized that divergence values of 3% or more are significant
(Felsenstein,
1985).
All
major
branching
7
nodes
were
subjected
to
1
(Stackebrandt & Goebel, 1994). The sequence divergence values of 6% or greater
2
displayed between the novel isolate and species within the genus Shewanella, combined
3
with physiological and chemotaxonomic criteria strongly suggest that the new isolate
4
represents a novel genus.
5
Cellular fatty acids, isoprenoid quinones and polar lipids extracted from the isolated
6
strain were cultured in MB at 12 °C for up to 2 days. Cells were washed twice with 0.7 %
7
NaCl at 4 °C. The fatty acids of these strains were obtained from cells by saponification,
8
methylation and extraction according to the Sherlock Microbial Identification System
9
(MIDI, 1999). Fatty acid compositions were determined using a Finnigan TRACE DSQ
10
GC-MS system (Thermo Fisher Scientific) equipped with a DB-5 column (J&W
11
Scientific) under a helium flow of 1.5 ml min-1 and an oven temperature program
12
increasing from 140 °C (5 min) to 280 °C (5 min) at 4 °C min-1.
13
The fatty acids making up more than 1 % of the total in strain BJ-1T were C16:1ω7c
14
(67.0 %), C16:0 (26.3 %), C14:1 (2.2 %), C18:1ω7c (1.9 %), C14:0 3-OH (1.5 %) and C14:0
15
(1.2 %).
16
Isoprenoid quinones were extracted with chloroform/methanol (2:1) from dried cells (200
17
mg), purified on TLC and analyzed using reversed-phase HPLC according to the methods
18
described previously (Miyazaki et al., 2006). Standard quinones (Q-6, Q-7, Q-9, Q-10)
19
were obtained from Sigma Chemical Co.Ubiquinone Q-8 was the only quinone detected.
20
Polar lipids were extracted using the procedures described by Minnikin et al. (1984) and
21
identified using two-dimensional TLC followed by spraying with the appropriate
22
detection reagents (Komagata & Suzuki, 1987). Polar lipid analysis of strain BJ-1T
23
showed the presence of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE),
24
phosphatidylglycerol (PG), phosphatidylmethylethanolamine (PME) and small amounts
8
1
of unidentified phospholipid (PL1) (Fig. 2).
2
Based on the distinctive genotypic, chemotaxonomic and other phenotypic characteristics,
3
strain BJ-1T is considered to represent a new genus and species within
4
Gammaproteobacteria, for which the name Psychrobium conchae gen. nov., sp. nov. is
5
proposed.
6 7
Description of Psychrobium gen. nov.
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Psychrobium gen. nov. (Psy.chro'bi.um. Gr. adj. psychros, cold; Gr. n. bios, life; N. L.
9
neutr. n. Psychrobium, a living entity coming from the cold).
10
Cells are Gram-negative, psychrophilic, chemo-organotrophic, strictly aerobic, motile by
11
means of a single polar flagellum and positive for catalase and cytochrome oxidase.
12
Nitrate is reduced to nitrite and nitrite is reduced to ammonia (assimilated). No growth
13
occurs at temperatures higher than 20 °C. No growth occurs at NaCl concentrations of
14
less than 1 % and higher than 5 %. Major fatty acids (>10 %) are C16:0 and C16:1 ω7c. The
15
isoprenoid quinone is Q-8. Polar lipids are diphosphatidylglycerol,
16
phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmethylethanolamine.
17
The DNA G+C content of the type strain of the type species is 40.5 mol%. This genus
18
belongs to the class Gammaproteobacteria and the type species is Psychrobium conchae.
19 20
Description of Psychrobium conchae sp. nov.
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Psychrobium conchae (con'chae. L. gen. n. conchae, of [from] a mussel).
22
Cells are rod shaped; cell width ranges from 0.7 to 1.5 μm, and cell length ranges from
23
3.0 to 6.0 μm. Cells are Gram-negative, strictly aerobic chemo-organotrophs and motile
24
by means of single polar flagellum. Colonies on MA are circular, smooth, convex,
9
1
slightly whitish and 1 to 2 mm in diameter after 2 days of incubation at 12 °C. The
2
temperature range for growth is less than 16 °C (optimum 12 °C). No growth occurs at
3
temperatures higher than 18 °C. Optimal growth occurs at the NaCl concentration of 3 %,
4
and cells are able to grow at a concentration of 4 % NaCl in LB medium. The optimal pH
5
is 6.0–6.5, and the pH range at which growth occurs is 5.5–8.5. The organism tests
6
positive for DNase and hydrolysates of Tween 20 and is negative for agarase, amylase,
7
chitinase, proteinase, hydrolysates of Tween 40 and 80 and esculin. Acid is formed
8
oxidatively from D-galactose, D-glucose, myo-inositol, maltose and D-trehalose. No acid
9
is produced from L-arabinose, cellobiose, D-fructose, glycerol, D-lactose, D-mannitol,
10
D-mannose, D-raffinose, L-rhamnose, D-sorbitol, sucrose and D-xylose. It is sensitive to
11
ampicillin, cefotiam, chloramphenicol, ciprofloxacin, polymyxin B, rifampicin and
12
vancomycin, but resistant to clindamycin, penicillin and tetracycline. P. conchae was
13
isolated from the gill tissue of the deep-sea hydrothermal vent mussel Bathymodiolus
14
japonicus at the Iheya North hydrothermal field, Okinawa Trough, Japan. The type strain
15
is strain BJ-1T (=JCM 30103T = DSM 28701T).
16 17
Acknowledgements
18
We would like to thank the captain and crew of the R/V Natsushima and the operation
19
team of the ROV Hyper-Dolphin for their cooperation in collecting invaluable samples.
20
We are very grateful to Professor Dr. Bernhard Schink for help with the Latin
21
nomenclature, Dr. Tomoo Watsuji for helping us to collect and transport the deep-sea
22
samples and Mr. Katsuyuki Uematsu for performing the transmission electron
23
microscopy.
24
10
1
References
2
Barrow, G. I. & Feltham, R. K. A. (1993). Cowan and Steel’s Manual for the
3
Identification of Medical Bacteria, 3rd edn. New York, NY: Cambridge University Press.
4
Baumann, L., Baumann, P., Mandel, M. & Allen, R. D. (1972). Taxonomy of aerobic
5
marine eubacteria. J Bacteriol 110, 402–429.
6
Benediktsdottir, E., Verdonck, L., Sproer, C., Helgason, S. & Swings, J. (2000).
7
Characterization of Vibrio viscosus and Vibrio wodanis isolated at different geographical
8
locations: a proposal for reclassification of Vibrio viscosus as Moritella viscosa comb.
9
nov. Int J Syst Evol Microbiol 50, 479–488.
10
Bowman, J. P. (2005). Genus XIII. Shewanella MacDonell and Colwell 1986, 355VP
11
(Effective publication: MacDonell and Colwell 1985, 180). In Bergey’s Manual of
12
Systematic Bacteriology, 2nd edn, vol. 2B, pp. 480–491. Edited by Brenner, D. J., Krieg,
13
N. R., Staley, J. T., Garrity, G. M. New York: Springer.
14
Bowman, J. P., McCammon, S. A., Nichols, D. S., Skerratt, J. H., Rea, S. M., Nichols,
15
P. D. & McMeekin, T. A. (1997). Shewanella gelidimarina sp. nov. and Shewanella
16
frigidimarina sp. nov., novel Antarctic species with the ability to produce
17
eicosapentaenoic acid (20:5 3) and grow anaerobically by dissimilatory Fe(III)
18
reduction. Int J Syst Bacteriol 47, 1040–1047.
19
Bozal, N., Montes, M. J., Miñana-Galbis, D., Manresa, A. & Mercadé, E. (2009).
20
Shewanella vesiculosa sp. nov., a psychrotolerant bacterium isolated from an Antarctic
21
coastal area. Int J Syst Evol Microbiol 59, 336–340.
22
Bozal, N., Montes, M. J., Tudela, E., Jiménez, F. & Guinea, J. (2002). Shewanella
23
frigidimarina and Shewanella livingstonensis sp. nov. isolated from Antarctic coastal
24
areas. Int J Syst Evol Microbiol 52, 195–205.
ω
11
1
Brettar, I., Christen, R. & Höfle, M. G. (2002). Shewanella denitrificans sp. nov., a
2
vigorously denitrifying bacterium isolated from the oxic–anoxic interface of the Gotland
3
Deep in the central Baltic Sea. Int J Syst Evol Microbiol 52, 2211–2217.
4
Campbell S., Harada, R. M. & Li, Q. X. (2007). Ferrimonas senticii sp. nov., a novel
5
gammaproteobacterium isolated from the mucus of a puffer fish caught in Kaneohe Bay,
6
Hawai'i. Int J Syst Evol Microbiol 57, 2670–2673.
7
Chang, H. W., Roh, S. W., Kim, K. H., Nam, Y. D., Jeon, C. O., Oh, H. M. & Bae, J.
8
W. (2008). Shewanella basaltis sp. nov., a marine bacterium isolated from black sand. Int
9
J Syst Bacteriol 58, 1907–1910.
10
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the
11
bootstrap. Evolution 39, 783–791.
12
Gao, H., Obraztova, A., Stewart, N., Popa, R., Fredrickson, J. K., Tiedje J. M.,
13
Nealson, K. H. & Zhou, J. (2006). Shewanella loihica sp. nov., isolated from iron-rich
14
microbial mats in the Pacific Ocean. Int J Syst Evol Microbiol 56, 1911–1916.
15
Hirota, K., Nodasaka, Y., Orikasa, Y., Okuyama, H. & Yumoto, I. (2005).
16
Shewanella pneumatophori sp. nov., an eicosapentaenoic acid-producing marine
17
bacterium isolated from the intestines of Pacific mackerel (Pneumatophorus japonicus).
18
Int J Syst Evol Microbiol 55, 2355–2359.
19
Hosoya S., Suzuki, S., Adachi, K., Matsuda, S. & Kasai, H. (2009). Paramoritella
20
alkaliphila gen. nov., sp. nov., a member of the family Moritellaceae isolated in the
21
Republic of Palau. Int J Syst Evol Microbiol 59, 411–416.
22
Huang, J., Sun, B. & Zhang, X. (2010). Shewanella xiamenensis sp. nov., isolated from
23
coastal sea sediment. Int J Syst Evol Microbiol 60, 1585–1589.
24
Hugh, R. & Leifson, E. (1953). The taxonomic significance of fermentative versus
12
1
oxidative metabolism of carbohydrates by various Gram negative bacteria. J Bacteriol 66,
2
22–26.
3
Ivanova E. P., Flavier S., & Christen R. (2004a). Phylogenetic relationships among
4
marine Alteromonas-like proteobacteria: emended description of the family
5
Alteromonadaceae and proposal of Pseudoalteromonadaceae fam. nov., Colwelliaceae
6
fam. nov., Shewanellaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam.
7
nov., Idiomarinaceae fam. nov. and Psychromonadaceae fam. nov. Int J Syst Evol
8
Microbiol 54, 1773–1788.
9
Ivanova, E. P., Gorshkova, N. M., Bowman, J. P., Lysenko, A. M., Zhukova, N. V.,
10
Sergeev, A. F., Mikhailov, V. V. & Nicolau, D. V. (2004b). Shewanella pacifica sp.
11
nov., a polyunsaturated fatty acid-producing bacterium isolated from sea water. Int J Syst
12
Evol Microbiol 54, 1083–1087.
13
Ivanova, E. P., Nedashkovskaya, O. I., Zhukova, N. V., Nicolau, D. V., Christen, R.
14
& Mikhailov, V. V. (2003a). Shewanella waksmanii sp. nov., isolated from a sipuncula
15
(Phascolosoma japonicum). Int J Syst Evol Microbiol 53, 1471–1477.
16
Ivanova, E. P., Sawabe, T., Gorshkova, N. M., Svetashev, V. I., Mikhailov, V. V.,
17
Nicolau, D. V. & Christen, R. (2001). Shewanella japonica sp. nov. Int J Syst Evol
18
Microbiol 51, 1027–1033.
19
Ivanova, E. P., Sawabe, T., Hayashi, K., Gorshkova, N. M., Zhukova, N. V.,
20
Nedashkovskaya, O. I., Mikhailov, V. V., Nicolau, D. V. & Christen, R. (2003b).
21
Shewanella fidelis sp. nov., isolated from sediments and sea water. Int J Syst Evol
22
Microbiol 53, 577–582.
13
1
Ji, S., Zhao, R., Li, Z., Li, B., Shi, X. & Zhang, X. H. (2013). Ferrimonas sediminum
2
sp. nov., isolated from coastal sediment of an amphioxus breeding zone. Int J Syst Evol
3
Microbiol 63, 977–981.
4
Katsuta, A., Adachi, K., Matsuda, S., Shizuri, Y. & Kasai, H. (2005). Ferrimonas
5
marina sp. nov. Int J Syst Evol Microbiol 55, 1851–1855.
6
Khan, S. T. & Harayama, S. (2007). Paraferrimonas sedimenticola gen. nov., sp. nov.,
7
a marine bacterium of the family Ferrimonadaceae. Int J Syst Evol Microbiol 57,
8
1493–1498.
9
Kawagucci, S., Miyazaki, J., Nakajima, R., Nozaki, T., Takaya, Y., Kato, Y.,
10
Shibuya, T., Konno, U., Nakaguchi, Y., Hatada, K., Hirayama, H., Fujikura, K.,
11
Furushima, Y., Yamamoto, H., Watsuji, T., Ishibashi, J. & Takai, K. (2013).
12
Post-drilling changes in fluid discharge pattern, mineral deposition, and fluid chemistry
13
in the Iheya North hydrothermal field, Okinawa Trough. Geochem. Geophys. Geosyst. 14,
14
4774–4790.
15
Kim, D., Baik, K. S., Kim, M. S., Jung, B.-M., Shin, T.-S., Chung, G.-H., Rhee, M. S.
16
& Seong, C. N. (2007). Shewanella haliotis sp. nov., isolated from the gut microflora of
17
abalone, Haliotis discus hannai. Int J Syst Evol Microbiol 57, 2926–2931.
18
Kim, H. J., Park, S., Lee, J. M., Park, S., Jung, W., Kang, J.-S., Joo, H. M., Seo,
19
K.-W. & Kang, S.-H. (2008). Moritella dasanensis sp. nov., a psychrophilic bacterium
20
isolated from the Arctic Ocean. Int J Syst Evol Microbiol 58, 817–820.
21
Kim, K.-K., Kim, Y.-O., Park, S., Kang, S.-J., Nam, B.-H., Kim, D. N., Oh, T.-K. &
22
Yoon, J.-H. (2011). Shewanella upenei sp. nov., a lipolytic bacterium isolated from
23
bensasi goatfish Upeneus bensasi. J Microbiol 49, 381–386.
24
Kim, S. J., Park, S. J., Oh, Y. S., Lee, S. A., Shin, K. S., Roh, D. H. & Rhee, S. K.
14
1
(2012). Shewanella arctica sp. nov., an iron-reducing bacterium isolated from Arctic
2
marine sediment. Int J Syst Evol Microbiol 62, 1128–1133.
3
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics.
4
Method Microbiol 19, 161–207.
5
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial
6
Systematics, pp. 115–148. Edited by E. Stackebrandt & M. Goodfellow. New York, NY:
7
John Wiley and Sons.
8
Lee, M.-H. & Yoon, J.-H. (2012). Shewanella litorisediminis sp. nov., a
9
gammaproteobacterium isolated from a tidal flat sediment. Antonie van Leeuwenhoek
10
102, 591–599.
11
Lee, O. O., Lau, S. C. K., Tsoi, M. M. Y., Li, X., Plakhotnikova, I., Dobretsov, S.,
12
Wu, M. C. S., Wong, P.-K., Weinbauer, M. & Qian, P.-Y. (2006). Shewanella irciniae
13
sp. nov., a novel member of the family Shewanellaceae, isolated from the marine sponge
14
Ircinia dendroides in the Bay of Villefranche, Mediterranean Sea. Int J Syst Evol
15
Microbiol 56, 2871–2877.
16
Leonardo, M. R., Moser, D. P., Barbieri, E., Branther, C. A., MacGregor, B. J.,
17
Paster, B. J., Stackebrandt, E. & Nealson, K. H. (1999). Shewanella pealeana sp. nov.,
18
a member of the microbial community associated with the accessory nidamental gland of
19
the squid Loligo pealei. Int J Syst Bacteriol 49, 1341–1351.
20
Makemson, J. C., Fulayfil, N. R., Landry, W., Van Ert, L. M., Wimpee, C. F.,
21
Widder, E. A. & Case, J. F. (1997). Shewanella woodyi sp. nov., an exclusively
22
respiratory luminous bacterium isolated from the Alboran Sea. Int J Syst Bacteriol 47,
23
1034–1039.
15
1
MIDI (1999). Sherlock, Microbial Identification System, Operating Manual, version 3.0.
2
Newark, DE: MIDI, Inc
3
Minnikin, D. E., Odonnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal,
4
A. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial
5
isoprenoid quinones and polar lipids. J Microbiol Meth 2, 233–241.
6
Miyazaki, M., Nogi, Y., Usami, R. & Horikoshi, K. (2006). Shewanella surugensis sp.
7
nov., Shewanella kaireitica sp. nov. and Shewanella abyssi sp. nov., isolated from
8
deep-sea sediments of Suruga Bay, Japan. Int J Syst Evol Microbiol 56, 1607–1613.
9
Nakagawa, T., Iino, T., Suzuki, K. & Harayama, S. (2006). Ferrimonas futtsuensis sp.
10
nov. and Ferrimonas kyonanensis sp. nov., selenate-reducing bacteria belonging to the
11
Gammaproteobacteria isolated from Tokyo Bay. Int J Syst Evol Microbiol 56,
12
2639–2645.
13
Nogi, Y. & Kato, C. (1999). Taxonomic studies of extremely barophilic bacteria isolated
14
from the Mariana Trench and description of Moritella yayanosii sp. nov., a new
15
barophilic bacterial isolate. Extremophiles 3, 71–77.
16
Nogi, Y., Kato, C. & Horikoshi, K. (1998a). Moritella japonica sp. nov., a novel
17
barophilic bacterium isolated from a Japan Trench sediment. J Gen Appl Microbiol 44,
18
289–295.
19
Nogi, Y., Kato, C. & Horikoshi, K. (1998b). Taxonomic studies of deep-sea barophilic
20
Shewanella strains and description of Shewanella violacea sp. nov. Arch Microbiol 170,
21
331–338.
22
Park, H. Y. & Jeon, C. O. (2013). Shewanella aestuarii sp. nov., a marine bacterium
23
isolated from a tidal flat. Int J Syst Evol Microbiol 63, 4683–4690.
16
1
Park, S. C., Baik, K. S., Kim, M. S., Kim, D. & Seong, C. N. (2009). Shewanella
2
marina sp. nov., isolated from seawater. Int J Syst Evol Microbiol 59, 1888–1894.
3
Rahman, M. & Cha, C.-J. (2013). Ferrimonas gelatinilytica sp. nov., isolated from tidal
4
flat sediment. Int J Syst Evol Microbiol 63, 4309–4314.
5
Rosselló-Mora, R. A., Ludwig, W., Kämpfer, P., Amann, R. & Schleifer, K.-H.
6
(1995). Ferrimonas balearica gen. nov., spec. nov., a marine facultative Fe(III)-reducing
7
bacterium. Syst Appl Microbiol 18, 196–202.
8
Saito, H. & Miura, K. (1963). Preparation of transforming deoxyribonucleic acid by
9
phenol treatment. Biochim Biophys Acta 72, 612-629.
10
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for
11
reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
12
Satomi, M., Oikawa, H. & Yano, Y. (2003). Shewanella marinintestina sp. nov.,
13
Shewanella schlegeliana sp. nov. and Shewanella sairae sp. nov., novel
14
eicosapentaenoic-acid-producing marine bacteria isolated from sea-animal intestines. Int
15
J Syst Evol Microbiol 53, 491–499.
16
Satomi, M., Vogel, B. F., Gram, L. & Venkateswaran, K. (2006). Shewanella
17
hafniensis sp. nov. and Shewanella morhuae sp. nov., isolated from marine fish of the
18
Baltic Sea. Int J Syst Evol Microbiol 56, 243–249.
19
Satomi, M., Vogel, B. F., Venkateswaran, K. & Gram, L. (2007). Description of
20
Shewanella glacialipiscicola sp. nov. and Shewanella algidipiscicola sp. nov., isolated
21
from marine fish of the Danish Baltic Sea, and proposal that Shewanella affinis is a later
22
heterotypic synonym of Shewanella colwelliana. Int J Syst Evol Microbiol 57, 347–352.
23
Shnit-Orland, M., Sivan, A. & Kushmaro, A.(2010). Shewanella corallii sp. nov., a
24
marine bacterium isolated from a Red Sea coral. Int J Syst Evol Microbiol 60, 2293–2297.
17
1
Skerratt, J. H., Bowman, J. P. & Nichols, P. D. (2002). Shewanella olleyana sp. nov., a
2
marine species isolated from a temperate estuary which produces high levels of
3
polyunsaturated fatty acids. Int J Syst Evol Microbiol 52, 2101–2106.
4
Sravan Kumar, R., Sasi Jyothsna, T. S., Sasikala, Ch., Seong, C. N., Lim, C. H.,
5
Park, S. C. & Ramana, ChV. (2010). Shewanella fodinae sp. nov., isolated from a coal
6
mine and from a marine lagoon. Int J Syst Evol Microbiol 60, 1649–1654.
7
Stackebrandt, E. & Goebel, B. M.(1994). Taxonomic note: a place for DNA–DNA
8
reassociation and 16S rDNA sequence analysis in the present species definition in
9
bacteriology. Int J Syst Bacteriol 44, 846–849.
10
Sucharita, K., Sasikala, Ch., Park, S. C., Baik, K. S., Seong, C. N. & Ramana, Ch. V.
11
(2009). Shewanella chilikensis sp. nov., a moderately alkaliphilic gammaproteobacterium
12
isolated from a lagoon. Int J Syst Evol Microbiol 59, 3111–3115.
13
Sung, H. R., Yoon, J. H. & Ghim, S. Y. (2012). Shewanella dokdonensis sp. nov.,
14
isolated from seawater. Int J Syst Evol Microbiol 62, 1636–1643.
15
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by
16
reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25,
17
125–128.
18
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011).
19
MEGA5: molecular evolutionary genetics analysis using maximum likelihood,
20
evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
21
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
22
(1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence
23
alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
18
1
Toffin, L., Bidault, A., Pignet, P., Tindall, B. J., Slobodkin, A., Kato, C., & Prieur, D.
2
(2004). Shewanella profunda sp. nov., isolated from deep marine sediment of the Nankai
3
Trough. Int J Syst Evol Microbiol 54, 1943–1949.
4
Uchida, H., Hamana, K., Miyazaki, M., Yoshida, T. & Nogi, Y. (2012).
5
Parasphingopyxis lamellibrachiae gen. nov., sp. nov., isolated from a marine organism in
6
Kagoshima Bay, Japan. Int J Syst Evol Microbiol 62, 2224–2228.
7
Urakawa, H., Kita-Tsukamoto, K., Steven, S. E., Ohwada, K. & Colwell, R. R.
8
(1998). A proposal to transfer Vibrio marinus (Russell 1891) to a new genus Moritella
9
gen. nov. as Moritella marina comb. nov. FEMS Microbiol Lett 165, 373–378.
10
Venkateswaran, K., Dollhopf, M. E., Aller, R., Stackebrandt, E. & Nealson, K. H.
11
(1998). Shewanella amazonensis sp. nov., a novel metal-reducing facultative anaerobe
12
from Amazonian shelf muds. Int J Syst Bacteriol 48, 965–972.
13
Venkateswaran, K., Moser, D. P., Dollhopf, M. E., Lies, D. P., Saffarini, D. A.,
14
MacGregor, B. J., Ringelberg, D. B., White, D. C., Nishijima, M. & other authors
15
(1999). Polyphasic taxonomy of the genus Shewanella and description of Shewanella
16
oneidensis sp. nov. Int J Syst Bacteriol 49, 705–724.
17
Verma, P., Pandey, P. K., Gupta, A. K., Kim, H. J., Baik, K. S., Seong, C. N., Patole,
18
M. S. & Shouche, Y. S. (2011). Shewanella indica sp. nov., isolated from sediment of
19
the Arabian Sea. Int J Syst Evol Microbiol 61, 2058–2064.
20
Xiao, X., Wang, P., Zeng, X., Bartlett, D. H. & Wang, F. (2007). Shewanella
21
psychrophila sp. nov. and Shewanella piezotolerans sp. nov., isolated from west Pacific
22
deep-sea sediment. Int J Syst Evol Microbiol 57, 60–65.
23
Xu, M., Guo, J., Cen, Y., Zhong, X., Cao, W. & Sun, G. (2005). Shewanella
24
decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a
19
1
waste-water treatment plant. Int J Syst Evol Microbiol 55, 363–368.
2
Xu, Y., Nogi, Y., Kato, C., Liang, Z., Ruger, H. J., De Kegel, D. & Glansdorff, N.
3
(2003). Moritella profunda sp. nov. and Moritella abyssi sp. nov., two
4
psychropiezophilic organisms isolated from deep Atlantic sediments. Int J Syst Evol
5
Microbiol 53, 533–538.
6
Yang, S.-H., Kwon, K. K., Lee, H.-S. & Kim, S.-J. (2006). Shewanella spongiae sp.
7
nov., isolated from a marine sponge. Int J Syst Evol Microbiol 56, 2879–2882.
8
Yang, S.-H., Lee, J.-H., Ryu, J.-S., Kato, C. & Kim, S.-J. (2007). Shewanella
9
donghaensis sp. nov., a psychrophilic, piezosensitive bacterium producing high levels of
10
polyunsaturated fatty acid, isolated from deep-sea sediments. Int J Syst Evol Microbiol 57,
11
208–212.
12
Yang, S.-H., Seo, H.-S., Lee, J.-H., Kim, S.-J. & Kwon, K. K. (2013). Paramoritella
13
sediminis sp. nov., isolated from marine sediment, and emended descriptions of the genus
14
Paramoritella Hosoya et al. 2009 and Paramoritella alkaliphila. Int J Syst Evol
15
Microbiol 63, 2265–2269.
16
Yim, K. J., Lee, M., Lee, H.-W., Kim, K.-N., Yang, H.-M., Kim, M.-J., Hyun, D.-W.,
17
Bae, J.-W., Nam, Y.-D., Yoon, C., Kim, M.-S., Roh, S. W. & Kim, D. (2013).
18
Ferrimonas pelagia sp. nov., isolated from seawater. Int J Syst Evol Microbiol 63,
19
3175–3179.
20
Yoon, J. H., Kang, K. H., Oh, T. K. & Park, Y. H. (2004a). Shewanella gaetbuli sp.
21
nov., a slight halophile isolated from a tidal flat in Korea. Int J Syst Evol Microbiol 54,
22
487–491.
23
Yoon, J.-H., Park, S., Jung, Y.-T. & Lee J.-S. (2012). Shewanella seohaensis sp. nov.,
24
isolated from a tidal flat sediment. Antonie van Leeuwenhoek 102, 149–156.
20
1
Yoon, J. H., Yeo, S. H., Kim, I. G. & Oh, T. K. (2004b). Shewanella marisflavi sp. nov.
2
and Shewanella aquimarina sp. nov., slightly halophilic organisms isolated from sea
3
water of the Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 2347–2352.
4
Zhao, J.-S., Manno, D., Beaulieu, C., Paquet, L. & Hawari, J. (2005). Shewanella
5
sediminis sp. nov., a novel Na+-requiring and
6
hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading bacterium from marine sediment. Int J
7
Syst Evol Microbiol 55, 1511–1520.
8
Zhao, J.-S., Manno, D., Leggiadro, C., O’Neil, D. & Hawari, J. (2006). Shewanella
9
halifaxensis sp. nov., a novel obligately respiratory and denitrifying psychrophile. Int J
10
Syst Evol Microbiol 56, 205–212.
11
Zhao, J. S., Manno, D., Thiboutot, S., Ampleman, G. & Hawari, J. (2007).
12
Shewanella canadensis sp. nov. and Shewanella atlantica sp. nov., manganese dioxide-
13
and hexahydro-1,3,5-trinitro-1,3,5-triazine-reducing, psychrophilic marine bacteria. Int J
14
Syst Evol Microbiol 57, 2155–2162.
15
Ziemke, F., Höfle, M. G. Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of
16
Shewanella putrefaciens Owen’s genomic group II as Shewanella baltica sp. nov. Int J
17
Syst Bacteriol 48, 179–186.
18 19
21
1 Table 1. Differential characteristics of strain BJ-1T (Psychrobium gen. nov.) and related genera Data for reference genera were taken from the following studies: Bowman, J. P. (2005), Bowman et al. (1997), Bozal et al. (2002, 2009), Brettar et al. (2002), Chang et al. (2008), Gao et al. (2006), Hirota et al. (2005), Huang et al. (2010), Ivanova et al. (2001, 2003a, b, 2004b), Kim et al. (2007, 2011, 2012), Lee et al. (2006), Lee & Yoon (2012), Leonardo et al. (1999), Makemson et al. (1997), Miyazaki et al. (2006), Nogi et al. (1998b), Park et al. (2009), Park & Jeon (2013), Satomi et al. (2003, 2006, 2007), Shnit-Orland et al. (2010), Skerratt et al. (2002), Sravan Kumar et al. (2010), Sucharita et al. (2009), Sung et al. (2012), Toffin et al. (2004), Venkateswaran et al. (1998, 1999), Verma et al. (2011), Xiao et al. (2007), Xu et al. (2005), Yang et al. (2006, 2007), Yoon et al. (2004a, b, 2012), Zhao et al. (2005, 2006, 2007), Ziemke et al. (1998) (Shewanella); Hosoya et al. (2009), Yang et al. (2013) (Paramoritella); Benediktsdóttir et al. (2000), Kim et al. (2008), Nogi & Kato (1999), Nogi et al. (1998a), Urakawa et al. (1998), Xu et al. (2003) (Moritella); Campbell et al. (2007), Ji et al. (2013), Katsuta et al. (2005), Nakagawa et al. (2006), Rahman & Cha (2013), Rosselló-Mora et al. (1995), Yim et al. (2013) (Ferrimonas); Khan & Harayama (2007) (Paraferrimonas). +, All species positive; -, all species negative; ND, no data available. (Numbers in parentheses are percentages of the species positive or negative). A, Aerobic; F, facultatively anaerobic. Characteristic
Psychrobium
Shewanella
Paramoritella
Moritella
Ferrimonas
Paraferrimonas
No. of species
1
61
2
7
8
1
Metabolism
A
F (93.3 %)
F
F
F
F
4 °C
+
+ (80.3 %)
-
+
-
-
20 °C
-
+ (95.1 %)
+
-
+
+
37 °C
-
- (59.0 %)
+
-
+ (62.5 %)
+
42 °C
-
- (83.6 %)
-
-
- (62.5 %)
-
1 % NaCl
-
+ (96.6 %)
+
ND
+ (62.5 %)
+
5 % NaCl Major fatty acids
16:1ω7c, 16:0
+ (86.9 %) iso-13:0, iso-15:0, 14:0, 15:0, 16:0, 16:1ω7c, 17:1ω8c, 18:1ω7c
+ (50 %) 16:1ω7c, 16:0, 18:1ω7c, 14:0
ND 16:1, 16:0, 14:0, 22:6ω3
+ (87.5 %) iso-15:0, 16:0, 16:1ω9c, 17:1ω8c, 18:1ω9c
iso-15:0, 18:1ω7c, 16:0, iso-13:0
Major quinone(s)
Q-8
Q-7, Q-8, MK-7
Q-8
Q-8
Q-7, Q-8, MK-7
Q-7, MK-6, MK-7
DNA G + C content (mol %)
40.5
39-54
56-57
41-47
54-60
50-51
Growth at:
2 3
22
1
Figure legends
2 3
Fig. 1. Phylogenetic tree derived from the 16S rRNA gene sequences of strain BJ-1T and
4
the type species of closely related genera within the order Alteromonadales, based on the
5
NJ algorithm. Numbers at nodes indicate bootstrap values (>50%) as calculated on the
6
basis of probabilities, expressed as percentages of 1000 replications. Escherichia coli
7
ATCC 11775T was used as an outgroup. Bar, 0.01 substitution per nucleotide position.
8 9
Fig. 2. Two-dimensional TLC after staining with ethanolic phosphomolybdic acid showing
the
total
polar
lipid
reagent
11
diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL1,
12
unidentified phospholipid; PME, phosphatidylmethylethanolamine.
13 14
23
profiles
of
strain
BJ-1T.
10
DPG,
Fig. 1
1
Psychrobium conchae gen. nov, sp. nov., a psychrophilic marine bacterium isolated from
2
the Iheya North hydrothermal field, Okinawa Trough, off Japan.
3 4
Yuichi Nogi1, Mariko Abe2, Shinsuke Kawagucci2 and Hisako Hirayama2
5 6
1
7
Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka
8
237-0061, Japan.
Research and Development Center for Marine Biosciences, Japan Agency for
9 10
2
11
Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka
12
237-0061, Japan.
Department of Subsurface Geobiological Analysis and Research, Japan Agency for
13
14
Running title: Psychrobium conchae gen. nov, sp., nov.,
15 16
Subject category for the content list: Proteobacteria
17 18
*Corresponding author: Yuichi Nogi
19
Phone: +81-468-67-9697, Fax: +81-468-66-6364,
20
E-mail: [email protected]
21 22
1
1
Abbreviations:
2
ASW: artificial seawater
3
DPG: diphosphatidylglycerol
4
LB: Luria-Bertani
5
MA: Marine Agar
6
MB: Marine Broth
7
PE: phosphatidylethanolamine
8
PG: phosphatidylglycerol
9
PL: phospholipid
10
PME: phosphatidylmethylethanolamine
11
NJ: neighbor-joining
12
ML: maximum-likelihood
13
UPGMA: unweighted pair group method with arithmetic mean
14 15 16
The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequences of strain
17
BJ-1T is AB930131.
18 19
2
1 2
Abstract A novel psychrophilic marine bacterial strain designated BJ-1T was isolated from
3
the Iheya North hydrothermal field, Okinawa Trough off Japan. Cells were
4
Gram-negative, rod-shaped, non-spore-forming aerobic chemo-organotrophs and motile
5
by means of a single polar flagellum. Growth occurred at temperatures below 16 °C, with
6
the optimum between 9 and 12 °C. Phylogenetic analysis based on the 16S rRNA gene
7
sequence indicated that the closest relatives of strain BJ-1T were Shewanella denitrificans
8
OS-217T (93.5 %), Shewanella profunda DSM 15900T (92.9 %), Shewanella gaetbuli
9
TF-27T (92.9 %), Paraferrimonas sedimenticola Mok-106T (92.1 %) and Ferrimonas
10
kyonanensis Asr22-7T (91.7 %). The major respiratory quinone was Q-8. The
11
predominant fatty acids were C16:1ω7c and C16:0. The G + C content of the novel strain
12
was 40.5 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, it is
13
proposed that strain BJ-1T represents a novel species in a new genus for which the name
14
Psychrobium conchae gen. nov., sp. nov., is proposed. The type strain of the type species
15
is BJ-1T (=JCM 30103T =DSMZ 28701T).
16 17
3
1
The genus Shewanella, currently represented by a single member of the family
2
Shewanellaceae (Ivanova et al., 2004a), belongs to the order Alteromonadales. The
3
genus was defined by the following features: aerobic or facultatively anaerobic,
4
Gram-negative, motile, rod-shaped bacteria. Nitrate-reducing and having 14:0, 16:1ω7,
5
16:0 and 17:1ω6 as the major fatty acids. In this study, we used a polyphasic taxonomic
6
approach to investigate a psychrophilic strain that was found to represent a novel genus
7
and species belonging to the family Shewanellaceae.
8 9
Strain BJ-1T was isolated from the gill tissue of the deep-sea hydrothermal vent mussel
10
Bathymodiolus japonicus at the Iheya North hydrothermal field, Okinawa Trough, off
11
Japan (latitude: 27°47.438 N, longitude: 126°53.736 E, depth: 990 m), collected by the
12
remotely operated vehicle Hyper-Dolphin during JAMSTEC NT12-06 cruise in March
13
2012 (Kawagucci et al., 2013). The fresh gill tissue was crushed with a small amount of
14
sterilized artificial seawater (ASW; RohtoMarine, Rei-Sea Co., Tokyo) with ice-cooling
15
by using a mortar and pestle, and was made into a fine paste. The tissue paste suspended
16
in ASW was spread on a Marine Agar 2216 (MA; Difco) plates and incubated at 10 °C
17
upon isolation. After isolation of strain BJ-1T, the strain maintained on MA plates or in
18
Marine Broth 2216 (MB; Difco) were incubated aerobically for 2 or 3 days at 12 °C and
19
stored at –80 ºC. Unless otherwise indicated, the physiological tests were performed with
20
a slight modification (use of ASW) of the general procedures described by Barrow &
21
Feltham (1993) and Baumann et al. (1972). The effects of temperature, NaCl
22
concentration and pH on cell growth were determined by examining the time course of
23
optical density (temperature gradient incubator with a bio-photorecorder, model
24
TVS126MA; Advantec). Growth at 4–20 °C was tested in MB. Cell growth was observed
′
′
4
1
at 16 °C or less (optimum 12 °C), but not above 18 °C. Growth at various NaCl
2
concentrations (0, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5 % w/v) was examined in Luria-Bertani
3
(LB) medium (1.0 % [w/v] tryptone [Difco], 0.5 % [w/v] yeast extract [Difco]) and
4
incubated at 12 °C. Cell growth was observed at NaCl concentrations of 2 % to 4 %
5
(optimum 3%), but not at less than 1 % or more than 5 % NaCl. Growth at various pH
6
values (5.0–10.0 in increments of 0.5 pH units) was measured in 3 % NaCl LB medium
7
and incubated at 12 °C. Cell growth was observed at pH 5.5 to 8.5 (optimum pH 6.0–6.5),
8
but not at pH 5.0 or at greater than pH 9.0.
9
Species of the genera Shewanella, Paramoritella and Ferrimonas which not grow at a 5%
10
NaCl concentration can be grown at a 1% or lower NaCl concentration, and this species
11
that cannot be grown at a 1% NaCl concentration can be grown at a 5% or higher
12
concentration (Table 1).
13
The morphology of living and non-living stained cells was determined by light
14
microscopy and transmission electron microscopy, respectively. For negative staining, 1
15
drop of a culture was placed on a copper grid coated with Pioloform and carbon and
16
stained with 1% potassium phosphotungstic acid adjusted to pH 6.5 with potassium
17
hydroxide. The negatively stained cells were observed with a model Tecnai 20
18
transmission electron microscope (FEI, Oregon USA) at an accelerating voltage of 200kV.
19 20
Growth under anaerobic conditions was tested on MA 1 week after the addition of 0.1 %
21
KNO3 to the MA plates with the AnaeroPak system (Mitsubishi Gas Chemical). Acid
22
production from sugars was assessed using modified oxidative-fermentative medium
23
(Hugh & Leifson, 1953) containing ASW, 0.05 % (NH4)2SO4, 0.01 % yeast extract
24
(Difco), 0.05 % Tris, 1 % test sugar and 0.003 % bromothymol blue (pH adjusted to 7.2 at
5
1
20 °C) with incubation at the optimum temperature. Oxidase activity was determined by
2
spreading cell pellets on oxidase test paper (Nissui Pharmaceutical). Hydrolysis of gelatin,
3
casein, starch, Tween 40 and 80, chitin and esculin was detected on MA plates using
4
substrate concentrations of 1 % (w/v). DNase activity was assessed using DNase Test
5
Agar (Difco). The susceptibility of the strain to antibiotics was tested using MA plates
6
and 6-mm sensitivity discs (Becton, Dickinson and Company) according to the
7
manufacturer’s instructions. The following antibiotics were examined: ampicillin (10 µg),
8
cefotaxime (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), clindamycin (2 µg),
9
erythromycin (15 µg), gentamicin (10 µg), penicillin (10 IU), polymyxin B (300 µg),
10
rifampicin (5 µg), tetracycline (30 µg) and vancomycin (30 µg). The effects of
11
antimicrobial compounds on cell growth were assessed after 2 days at 12 °C. The
12
diameter of the inhibition zone was used to judge susceptibility according to the
13
manufacturer’s manual.
14
The cells of strain BJ-1T were rod-shaped, Gram-negative, strictly aerobic, non-spore
15
forming and motile by means of a single polar flagellum. Colonies were whitish in color,
16
smooth, circular and 1.0–2.0 mm in diameter after 3 days incubation at 12 °C on MA.
17
Detailed results from the phenotypic and biochemical tests are given in the species
18
description and shown in Table 1. Among related genera, only strain BJ-1T and some
19
species of the genus Shewanella cannot be grown under anaerobic conditions.
20
Chromosomal DNA was purified using the phenol extraction method (Saito & Miura,
21
1963). The DNA G + C content was determined using reversed-phase HPLC (Tamaoka &
22
Komagata, 1984). The 16S rRNA gene was amplified using the PCR method with
23
primers 27F and 1492R (Lane, 1991). The 16S rRNA gene sequence of strain BJ-1T was
24
obtained by direct sequencing of PCR-amplified DNA as described previously (Uchida et
6
1
al., 2012). The resulting 16S rRNA gene sequence (1482 nt) of strain BJ-1T was
2
compared with available 16S rRNA gene sequences from the DDBJ using the BLAST
3
program (http://blast.ddbj.nig.ac.jp/top-j.html) to determine an approximate phylogenetic
4
affiliation, and gene sequences were aligned with those of closely related species using
5
the CLUSTAL X software program (Thompson et al., 1997). In addition, sequence
6
similarity values were calculated using the GENETYX-MAC program ver. 17.0.2 (SDC
7
Software Development) between the novel strain and other related members.
8
Phylogenetic analyses were conducted in MEGA 5.2 (Tamura et al., 2011) using the NJ
9
method (Saitou & Nei, 1987). Bootstrap analysis to evaluate the stability of phylogenetic
10
trees was performed by obtaining a consensus tree based on 1000 randomly generated
11
trees
12
maximum-likelihood (ML) and unweighted pair group method with arithmetic mean
13
(UPGMA) analyses (data not shown), which confirmed the robust nature of this group of
14
organisms, the branching order and high bootstrap values. The results of the phylogenetic
15
analyses indicated that the novel organism belonged to the class Gammaproteobacteria.
16
The novel bacterium was most closely related to Shewanella denitrificans OS-217T,
17
Shewanella profunda LT13aT, Shewanella waksmanii KMM3823T and Shewanella
18
gaetbuli TF-27T, with pairwise similarity values of 93.5 %, 93.2 %, 92.9 % and 92.9 %,
19
respectively (Fig. 1). More distantly related organisms included Paraferrimonas
20
sedimenticola Mok-106T (92.1 %), Moritella marina ATCC15381T (91.3 %),
21
Paramoritella alkaliphila A3F-7T (91.1 %) and Ferrimonas balearica DSM 9799T
22
(90.8 %). This cluster was supported by a significant bootstrap value of 90 %. There is no
23
precise correlation between 16S rRNA gene sequence divergence and species delineation,
24
but it is generally recognized that divergence values of 3% or more are significant
(Felsenstein,
1985).
All
major
branching
7
nodes
were
subjected
to
1
(Stackebrandt & Goebel, 1994). The sequence divergence values of 6% or greater
2
displayed between the novel isolate and species within the genus Shewanella, combined
3
with physiological and chemotaxonomic criteria strongly suggest that the new isolate
4
represents a novel genus.
5
Cellular fatty acids, isoprenoid quinones and polar lipids extracted from the isolated
6
strain were cultured in MB at 12 °C for up to 2 days. Cells were washed twice with 0.7 %
7
NaCl at 4 °C. The fatty acids of these strains were obtained from cells by saponification,
8
methylation and extraction according to the Sherlock Microbial Identification System
9
(MIDI, 1999). Fatty acid compositions were determined using a Finnigan TRACE DSQ
10
GC-MS system (Thermo Fisher Scientific) equipped with a DB-5 column (J&W
11
Scientific) under a helium flow of 1.5 ml min-1 and an oven temperature program
12
increasing from 140 °C (5 min) to 280 °C (5 min) at 4 °C min-1.
13
The fatty acids making up more than 1 % of the total in strain BJ-1T were C16:1ω7c
14
(67.0 %), C16:0 (26.3 %), C14:1 (2.2 %), C18:1ω7c (1.9 %), C14:0 3-OH (1.5 %) and C14:0
15
(1.2 %).
16
Isoprenoid quinones were extracted with chloroform/methanol (2:1) from dried cells (200
17
mg), purified on TLC and analyzed using reversed-phase HPLC according to the methods
18
described previously (Miyazaki et al., 2006). Standard quinones (Q-6, Q-7, Q-9, Q-10)
19
were obtained from Sigma Chemical Co.Ubiquinone Q-8 was the only quinone detected.
20
Polar lipids were extracted using the procedures described by Minnikin et al. (1984) and
21
identified using two-dimensional TLC followed by spraying with the appropriate
22
detection reagents (Komagata & Suzuki, 1987). Polar lipid analysis of strain BJ-1T
23
showed the presence of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE),
24
phosphatidylglycerol (PG), phosphatidylmethylethanolamine (PME) and small amounts
8
1
of unidentified phospholipid (PL1) (Fig. 2).
2
Based on the distinctive genotypic, chemotaxonomic and other phenotypic characteristics,
3
strain BJ-1T is considered to represent a new genus and species within
4
Gammaproteobacteria, for which the name Psychrobium conchae gen. nov., sp. nov. is
5
proposed.
6 7
Description of Psychrobium gen. nov.
8
Psychrobium gen. nov. (Psy.chro'bi.um. Gr. adj. psychros, cold; Gr. n. bios, life; N. L.
9
neutr. n. Psychrobium, a living entity coming from the cold).
10
Cells are Gram-negative, psychrophilic, chemo-organotrophic, strictly aerobic, motile by
11
means of a single polar flagellum and positive for catalase and cytochrome oxidase.
12
Nitrate is reduced to nitrite and nitrite is reduced to ammonia (assimilated). No growth
13
occurs at temperatures higher than 20 °C. No growth occurs at NaCl concentrations of
14
less than 1 % and higher than 5 %. Major fatty acids (>10 %) are C16:0 and C16:1 ω7c. The
15
isoprenoid quinone is Q-8. Polar lipids are diphosphatidylglycerol,
16
phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmethylethanolamine.
17
The DNA G+C content of the type strain of the type species is 40.5 mol%. This genus
18
belongs to the class Gammaproteobacteria and the type species is Psychrobium conchae.
19 20
Description of Psychrobium conchae sp. nov.
21
Psychrobium conchae (con'chae. L. gen. n. conchae, of [from] a mussel).
22
Cells are rod shaped; cell width ranges from 0.7 to 1.5 μm, and cell length ranges from
23
3.0 to 6.0 μm. Cells are Gram-negative, strictly aerobic chemo-organotrophs and motile
24
by means of single polar flagellum. Colonies on MA are circular, smooth, convex,
9
1
slightly whitish and 1 to 2 mm in diameter after 2 days of incubation at 12 °C. The
2
temperature range for growth is less than 16 °C (optimum 12 °C). No growth occurs at
3
temperatures higher than 18 °C. Optimal growth occurs at the NaCl concentration of 3 %,
4
and cells are able to grow at a concentration of 4 % NaCl in LB medium. The optimal pH
5
is 6.0–6.5, and the pH range at which growth occurs is 5.5–8.5. The organism tests
6
positive for DNase and hydrolysates of Tween 20 and is negative for agarase, amylase,
7
chitinase, proteinase, hydrolysates of Tween 40 and 80 and esculin. Acid is formed
8
oxidatively from D-galactose, D-glucose, myo-inositol, maltose and D-trehalose. No acid
9
is produced from L-arabinose, cellobiose, D-fructose, glycerol, D-lactose, D-mannitol,
10
D-mannose, D-raffinose, L-rhamnose, D-sorbitol, sucrose and D-xylose. It is sensitive to
11
ampicillin, cefotiam, chloramphenicol, ciprofloxacin, polymyxin B, rifampicin and
12
vancomycin, but resistant to clindamycin, penicillin and tetracycline. P. conchae was
13
isolated from the gill tissue of the deep-sea hydrothermal vent mussel Bathymodiolus
14
japonicus at the Iheya North hydrothermal field, Okinawa Trough, Japan. The type strain
15
is strain BJ-1T (=JCM 30103T = DSM 28701T).
16 17
Acknowledgements
18
We would like to thank the captain and crew of the R/V Natsushima and the operation
19
team of the ROV Hyper-Dolphin for their cooperation in collecting invaluable samples.
20
We are very grateful to Professor Dr. Bernhard Schink for help with the Latin
21
nomenclature, Dr. Tomoo Watsuji for helping us to collect and transport the deep-sea
22
samples and Mr. Katsuyuki Uematsu for performing the transmission electron
23
microscopy.
24
10
1
References
2
Barrow, G. I. & Feltham, R. K. A. (1993). Cowan and Steel’s Manual for the
3
Identification of Medical Bacteria, 3rd edn. New York, NY: Cambridge University Press.
4
Baumann, L., Baumann, P., Mandel, M. & Allen, R. D. (1972). Taxonomy of aerobic
5
marine eubacteria. J Bacteriol 110, 402–429.
6
Benediktsdottir, E., Verdonck, L., Sproer, C., Helgason, S. & Swings, J. (2000).
7
Characterization of Vibrio viscosus and Vibrio wodanis isolated at different geographical
8
locations: a proposal for reclassification of Vibrio viscosus as Moritella viscosa comb.
9
nov. Int J Syst Evol Microbiol 50, 479–488.
10
Bowman, J. P. (2005). Genus XIII. Shewanella MacDonell and Colwell 1986, 355VP
11
(Effective publication: MacDonell and Colwell 1985, 180). In Bergey’s Manual of
12
Systematic Bacteriology, 2nd edn, vol. 2B, pp. 480–491. Edited by Brenner, D. J., Krieg,
13
N. R., Staley, J. T., Garrity, G. M. New York: Springer.
14
Bowman, J. P., McCammon, S. A., Nichols, D. S., Skerratt, J. H., Rea, S. M., Nichols,
15
P. D. & McMeekin, T. A. (1997). Shewanella gelidimarina sp. nov. and Shewanella
16
frigidimarina sp. nov., novel Antarctic species with the ability to produce
17
eicosapentaenoic acid (20:5 3) and grow anaerobically by dissimilatory Fe(III)
18
reduction. Int J Syst Bacteriol 47, 1040–1047.
19
Bozal, N., Montes, M. J., Miñana-Galbis, D., Manresa, A. & Mercadé, E. (2009).
20
Shewanella vesiculosa sp. nov., a psychrotolerant bacterium isolated from an Antarctic
21
coastal area. Int J Syst Evol Microbiol 59, 336–340.
22
Bozal, N., Montes, M. J., Tudela, E., Jiménez, F. & Guinea, J. (2002). Shewanella
23
frigidimarina and Shewanella livingstonensis sp. nov. isolated from Antarctic coastal
24
areas. Int J Syst Evol Microbiol 52, 195–205.
ω
11
1
Brettar, I., Christen, R. & Höfle, M. G. (2002). Shewanella denitrificans sp. nov., a
2
vigorously denitrifying bacterium isolated from the oxic–anoxic interface of the Gotland
3
Deep in the central Baltic Sea. Int J Syst Evol Microbiol 52, 2211–2217.
4
Campbell S., Harada, R. M. & Li, Q. X. (2007). Ferrimonas senticii sp. nov., a novel
5
gammaproteobacterium isolated from the mucus of a puffer fish caught in Kaneohe Bay,
6
Hawai'i. Int J Syst Evol Microbiol 57, 2670–2673.
7
Chang, H. W., Roh, S. W., Kim, K. H., Nam, Y. D., Jeon, C. O., Oh, H. M. & Bae, J.
8
W. (2008). Shewanella basaltis sp. nov., a marine bacterium isolated from black sand. Int
9
J Syst Bacteriol 58, 1907–1910.
10
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the
11
bootstrap. Evolution 39, 783–791.
12
Gao, H., Obraztova, A., Stewart, N., Popa, R., Fredrickson, J. K., Tiedje J. M.,
13
Nealson, K. H. & Zhou, J. (2006). Shewanella loihica sp. nov., isolated from iron-rich
14
microbial mats in the Pacific Ocean. Int J Syst Evol Microbiol 56, 1911–1916.
15
Hirota, K., Nodasaka, Y., Orikasa, Y., Okuyama, H. & Yumoto, I. (2005).
16
Shewanella pneumatophori sp. nov., an eicosapentaenoic acid-producing marine
17
bacterium isolated from the intestines of Pacific mackerel (Pneumatophorus japonicus).
18
Int J Syst Evol Microbiol 55, 2355–2359.
19
Hosoya S., Suzuki, S., Adachi, K., Matsuda, S. & Kasai, H. (2009). Paramoritella
20
alkaliphila gen. nov., sp. nov., a member of the family Moritellaceae isolated in the
21
Republic of Palau. Int J Syst Evol Microbiol 59, 411–416.
22
Huang, J., Sun, B. & Zhang, X. (2010). Shewanella xiamenensis sp. nov., isolated from
23
coastal sea sediment. Int J Syst Evol Microbiol 60, 1585–1589.
24
Hugh, R. & Leifson, E. (1953). The taxonomic significance of fermentative versus
12
1
oxidative metabolism of carbohydrates by various Gram negative bacteria. J Bacteriol 66,
2
22–26.
3
Ivanova E. P., Flavier S., & Christen R. (2004a). Phylogenetic relationships among
4
marine Alteromonas-like proteobacteria: emended description of the family
5
Alteromonadaceae and proposal of Pseudoalteromonadaceae fam. nov., Colwelliaceae
6
fam. nov., Shewanellaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam.
7
nov., Idiomarinaceae fam. nov. and Psychromonadaceae fam. nov. Int J Syst Evol
8
Microbiol 54, 1773–1788.
9
Ivanova, E. P., Gorshkova, N. M., Bowman, J. P., Lysenko, A. M., Zhukova, N. V.,
10
Sergeev, A. F., Mikhailov, V. V. & Nicolau, D. V. (2004b). Shewanella pacifica sp.
11
nov., a polyunsaturated fatty acid-producing bacterium isolated from sea water. Int J Syst
12
Evol Microbiol 54, 1083–1087.
13
Ivanova, E. P., Nedashkovskaya, O. I., Zhukova, N. V., Nicolau, D. V., Christen, R.
14
& Mikhailov, V. V. (2003a). Shewanella waksmanii sp. nov., isolated from a sipuncula
15
(Phascolosoma japonicum). Int J Syst Evol Microbiol 53, 1471–1477.
16
Ivanova, E. P., Sawabe, T., Gorshkova, N. M., Svetashev, V. I., Mikhailov, V. V.,
17
Nicolau, D. V. & Christen, R. (2001). Shewanella japonica sp. nov. Int J Syst Evol
18
Microbiol 51, 1027–1033.
19
Ivanova, E. P., Sawabe, T., Hayashi, K., Gorshkova, N. M., Zhukova, N. V.,
20
Nedashkovskaya, O. I., Mikhailov, V. V., Nicolau, D. V. & Christen, R. (2003b).
21
Shewanella fidelis sp. nov., isolated from sediments and sea water. Int J Syst Evol
22
Microbiol 53, 577–582.
13
1
Ji, S., Zhao, R., Li, Z., Li, B., Shi, X. & Zhang, X. H. (2013). Ferrimonas sediminum
2
sp. nov., isolated from coastal sediment of an amphioxus breeding zone. Int J Syst Evol
3
Microbiol 63, 977–981.
4
Katsuta, A., Adachi, K., Matsuda, S., Shizuri, Y. & Kasai, H. (2005). Ferrimonas
5
marina sp. nov. Int J Syst Evol Microbiol 55, 1851–1855.
6
Khan, S. T. & Harayama, S. (2007). Paraferrimonas sedimenticola gen. nov., sp. nov.,
7
a marine bacterium of the family Ferrimonadaceae. Int J Syst Evol Microbiol 57,
8
1493–1498.
9
Kawagucci, S., Miyazaki, J., Nakajima, R., Nozaki, T., Takaya, Y., Kato, Y.,
10
Shibuya, T., Konno, U., Nakaguchi, Y., Hatada, K., Hirayama, H., Fujikura, K.,
11
Furushima, Y., Yamamoto, H., Watsuji, T., Ishibashi, J. & Takai, K. (2013).
12
Post-drilling changes in fluid discharge pattern, mineral deposition, and fluid chemistry
13
in the Iheya North hydrothermal field, Okinawa Trough. Geochem. Geophys. Geosyst. 14,
14
4774–4790.
15
Kim, D., Baik, K. S., Kim, M. S., Jung, B.-M., Shin, T.-S., Chung, G.-H., Rhee, M. S.
16
& Seong, C. N. (2007). Shewanella haliotis sp. nov., isolated from the gut microflora of
17
abalone, Haliotis discus hannai. Int J Syst Evol Microbiol 57, 2926–2931.
18
Kim, H. J., Park, S., Lee, J. M., Park, S., Jung, W., Kang, J.-S., Joo, H. M., Seo,
19
K.-W. & Kang, S.-H. (2008). Moritella dasanensis sp. nov., a psychrophilic bacterium
20
isolated from the Arctic Ocean. Int J Syst Evol Microbiol 58, 817–820.
21
Kim, K.-K., Kim, Y.-O., Park, S., Kang, S.-J., Nam, B.-H., Kim, D. N., Oh, T.-K. &
22
Yoon, J.-H. (2011). Shewanella upenei sp. nov., a lipolytic bacterium isolated from
23
bensasi goatfish Upeneus bensasi. J Microbiol 49, 381–386.
24
Kim, S. J., Park, S. J., Oh, Y. S., Lee, S. A., Shin, K. S., Roh, D. H. & Rhee, S. K.
14
1
(2012). Shewanella arctica sp. nov., an iron-reducing bacterium isolated from Arctic
2
marine sediment. Int J Syst Evol Microbiol 62, 1128–1133.
3
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics.
4
Method Microbiol 19, 161–207.
5
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial
6
Systematics, pp. 115–148. Edited by E. Stackebrandt & M. Goodfellow. New York, NY:
7
John Wiley and Sons.
8
Lee, M.-H. & Yoon, J.-H. (2012). Shewanella litorisediminis sp. nov., a
9
gammaproteobacterium isolated from a tidal flat sediment. Antonie van Leeuwenhoek
10
102, 591–599.
11
Lee, O. O., Lau, S. C. K., Tsoi, M. M. Y., Li, X., Plakhotnikova, I., Dobretsov, S.,
12
Wu, M. C. S., Wong, P.-K., Weinbauer, M. & Qian, P.-Y. (2006). Shewanella irciniae
13
sp. nov., a novel member of the family Shewanellaceae, isolated from the marine sponge
14
Ircinia dendroides in the Bay of Villefranche, Mediterranean Sea. Int J Syst Evol
15
Microbiol 56, 2871–2877.
16
Leonardo, M. R., Moser, D. P., Barbieri, E., Branther, C. A., MacGregor, B. J.,
17
Paster, B. J., Stackebrandt, E. & Nealson, K. H. (1999). Shewanella pealeana sp. nov.,
18
a member of the microbial community associated with the accessory nidamental gland of
19
the squid Loligo pealei. Int J Syst Bacteriol 49, 1341–1351.
20
Makemson, J. C., Fulayfil, N. R., Landry, W., Van Ert, L. M., Wimpee, C. F.,
21
Widder, E. A. & Case, J. F. (1997). Shewanella woodyi sp. nov., an exclusively
22
respiratory luminous bacterium isolated from the Alboran Sea. Int J Syst Bacteriol 47,
23
1034–1039.
15
1
MIDI (1999). Sherlock, Microbial Identification System, Operating Manual, version 3.0.
2
Newark, DE: MIDI, Inc
3
Minnikin, D. E., Odonnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal,
4
A. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial
5
isoprenoid quinones and polar lipids. J Microbiol Meth 2, 233–241.
6
Miyazaki, M., Nogi, Y., Usami, R. & Horikoshi, K. (2006). Shewanella surugensis sp.
7
nov., Shewanella kaireitica sp. nov. and Shewanella abyssi sp. nov., isolated from
8
deep-sea sediments of Suruga Bay, Japan. Int J Syst Evol Microbiol 56, 1607–1613.
9
Nakagawa, T., Iino, T., Suzuki, K. & Harayama, S. (2006). Ferrimonas futtsuensis sp.
10
nov. and Ferrimonas kyonanensis sp. nov., selenate-reducing bacteria belonging to the
11
Gammaproteobacteria isolated from Tokyo Bay. Int J Syst Evol Microbiol 56,
12
2639–2645.
13
Nogi, Y. & Kato, C. (1999). Taxonomic studies of extremely barophilic bacteria isolated
14
from the Mariana Trench and description of Moritella yayanosii sp. nov., a new
15
barophilic bacterial isolate. Extremophiles 3, 71–77.
16
Nogi, Y., Kato, C. & Horikoshi, K. (1998a). Moritella japonica sp. nov., a novel
17
barophilic bacterium isolated from a Japan Trench sediment. J Gen Appl Microbiol 44,
18
289–295.
19
Nogi, Y., Kato, C. & Horikoshi, K. (1998b). Taxonomic studies of deep-sea barophilic
20
Shewanella strains and description of Shewanella violacea sp. nov. Arch Microbiol 170,
21
331–338.
22
Park, H. Y. & Jeon, C. O. (2013). Shewanella aestuarii sp. nov., a marine bacterium
23
isolated from a tidal flat. Int J Syst Evol Microbiol 63, 4683–4690.
16
1
Park, S. C., Baik, K. S., Kim, M. S., Kim, D. & Seong, C. N. (2009). Shewanella
2
marina sp. nov., isolated from seawater. Int J Syst Evol Microbiol 59, 1888–1894.
3
Rahman, M. & Cha, C.-J. (2013). Ferrimonas gelatinilytica sp. nov., isolated from tidal
4
flat sediment. Int J Syst Evol Microbiol 63, 4309–4314.
5
Rosselló-Mora, R. A., Ludwig, W., Kämpfer, P., Amann, R. & Schleifer, K.-H.
6
(1995). Ferrimonas balearica gen. nov., spec. nov., a marine facultative Fe(III)-reducing
7
bacterium. Syst Appl Microbiol 18, 196–202.
8
Saito, H. & Miura, K. (1963). Preparation of transforming deoxyribonucleic acid by
9
phenol treatment. Biochim Biophys Acta 72, 612-629.
10
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for
11
reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
12
Satomi, M., Oikawa, H. & Yano, Y. (2003). Shewanella marinintestina sp. nov.,
13
Shewanella schlegeliana sp. nov. and Shewanella sairae sp. nov., novel
14
eicosapentaenoic-acid-producing marine bacteria isolated from sea-animal intestines. Int
15
J Syst Evol Microbiol 53, 491–499.
16
Satomi, M., Vogel, B. F., Gram, L. & Venkateswaran, K. (2006). Shewanella
17
hafniensis sp. nov. and Shewanella morhuae sp. nov., isolated from marine fish of the
18
Baltic Sea. Int J Syst Evol Microbiol 56, 243–249.
19
Satomi, M., Vogel, B. F., Venkateswaran, K. & Gram, L. (2007). Description of
20
Shewanella glacialipiscicola sp. nov. and Shewanella algidipiscicola sp. nov., isolated
21
from marine fish of the Danish Baltic Sea, and proposal that Shewanella affinis is a later
22
heterotypic synonym of Shewanella colwelliana. Int J Syst Evol Microbiol 57, 347–352.
23
Shnit-Orland, M., Sivan, A. & Kushmaro, A.(2010). Shewanella corallii sp. nov., a
24
marine bacterium isolated from a Red Sea coral. Int J Syst Evol Microbiol 60, 2293–2297.
17
1
Skerratt, J. H., Bowman, J. P. & Nichols, P. D. (2002). Shewanella olleyana sp. nov., a
2
marine species isolated from a temperate estuary which produces high levels of
3
polyunsaturated fatty acids. Int J Syst Evol Microbiol 52, 2101–2106.
4
Sravan Kumar, R., Sasi Jyothsna, T. S., Sasikala, Ch., Seong, C. N., Lim, C. H.,
5
Park, S. C. & Ramana, ChV. (2010). Shewanella fodinae sp. nov., isolated from a coal
6
mine and from a marine lagoon. Int J Syst Evol Microbiol 60, 1649–1654.
7
Stackebrandt, E. & Goebel, B. M.(1994). Taxonomic note: a place for DNA–DNA
8
reassociation and 16S rDNA sequence analysis in the present species definition in
9
bacteriology. Int J Syst Bacteriol 44, 846–849.
10
Sucharita, K., Sasikala, Ch., Park, S. C., Baik, K. S., Seong, C. N. & Ramana, Ch. V.
11
(2009). Shewanella chilikensis sp. nov., a moderately alkaliphilic gammaproteobacterium
12
isolated from a lagoon. Int J Syst Evol Microbiol 59, 3111–3115.
13
Sung, H. R., Yoon, J. H. & Ghim, S. Y. (2012). Shewanella dokdonensis sp. nov.,
14
isolated from seawater. Int J Syst Evol Microbiol 62, 1636–1643.
15
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by
16
reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25,
17
125–128.
18
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011).
19
MEGA5: molecular evolutionary genetics analysis using maximum likelihood,
20
evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
21
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
22
(1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence
23
alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
18
1
Toffin, L., Bidault, A., Pignet, P., Tindall, B. J., Slobodkin, A., Kato, C., & Prieur, D.
2
(2004). Shewanella profunda sp. nov., isolated from deep marine sediment of the Nankai
3
Trough. Int J Syst Evol Microbiol 54, 1943–1949.
4
Uchida, H., Hamana, K., Miyazaki, M., Yoshida, T. & Nogi, Y. (2012).
5
Parasphingopyxis lamellibrachiae gen. nov., sp. nov., isolated from a marine organism in
6
Kagoshima Bay, Japan. Int J Syst Evol Microbiol 62, 2224–2228.
7
Urakawa, H., Kita-Tsukamoto, K., Steven, S. E., Ohwada, K. & Colwell, R. R.
8
(1998). A proposal to transfer Vibrio marinus (Russell 1891) to a new genus Moritella
9
gen. nov. as Moritella marina comb. nov. FEMS Microbiol Lett 165, 373–378.
10
Venkateswaran, K., Dollhopf, M. E., Aller, R., Stackebrandt, E. & Nealson, K. H.
11
(1998). Shewanella amazonensis sp. nov., a novel metal-reducing facultative anaerobe
12
from Amazonian shelf muds. Int J Syst Bacteriol 48, 965–972.
13
Venkateswaran, K., Moser, D. P., Dollhopf, M. E., Lies, D. P., Saffarini, D. A.,
14
MacGregor, B. J., Ringelberg, D. B., White, D. C., Nishijima, M. & other authors
15
(1999). Polyphasic taxonomy of the genus Shewanella and description of Shewanella
16
oneidensis sp. nov. Int J Syst Bacteriol 49, 705–724.
17
Verma, P., Pandey, P. K., Gupta, A. K., Kim, H. J., Baik, K. S., Seong, C. N., Patole,
18
M. S. & Shouche, Y. S. (2011). Shewanella indica sp. nov., isolated from sediment of
19
the Arabian Sea. Int J Syst Evol Microbiol 61, 2058–2064.
20
Xiao, X., Wang, P., Zeng, X., Bartlett, D. H. & Wang, F. (2007). Shewanella
21
psychrophila sp. nov. and Shewanella piezotolerans sp. nov., isolated from west Pacific
22
deep-sea sediment. Int J Syst Evol Microbiol 57, 60–65.
23
Xu, M., Guo, J., Cen, Y., Zhong, X., Cao, W. & Sun, G. (2005). Shewanella
24
decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a
19
1
waste-water treatment plant. Int J Syst Evol Microbiol 55, 363–368.
2
Xu, Y., Nogi, Y., Kato, C., Liang, Z., Ruger, H. J., De Kegel, D. & Glansdorff, N.
3
(2003). Moritella profunda sp. nov. and Moritella abyssi sp. nov., two
4
psychropiezophilic organisms isolated from deep Atlantic sediments. Int J Syst Evol
5
Microbiol 53, 533–538.
6
Yang, S.-H., Kwon, K. K., Lee, H.-S. & Kim, S.-J. (2006). Shewanella spongiae sp.
7
nov., isolated from a marine sponge. Int J Syst Evol Microbiol 56, 2879–2882.
8
Yang, S.-H., Lee, J.-H., Ryu, J.-S., Kato, C. & Kim, S.-J. (2007). Shewanella
9
donghaensis sp. nov., a psychrophilic, piezosensitive bacterium producing high levels of
10
polyunsaturated fatty acid, isolated from deep-sea sediments. Int J Syst Evol Microbiol 57,
11
208–212.
12
Yang, S.-H., Seo, H.-S., Lee, J.-H., Kim, S.-J. & Kwon, K. K. (2013). Paramoritella
13
sediminis sp. nov., isolated from marine sediment, and emended descriptions of the genus
14
Paramoritella Hosoya et al. 2009 and Paramoritella alkaliphila. Int J Syst Evol
15
Microbiol 63, 2265–2269.
16
Yim, K. J., Lee, M., Lee, H.-W., Kim, K.-N., Yang, H.-M., Kim, M.-J., Hyun, D.-W.,
17
Bae, J.-W., Nam, Y.-D., Yoon, C., Kim, M.-S., Roh, S. W. & Kim, D. (2013).
18
Ferrimonas pelagia sp. nov., isolated from seawater. Int J Syst Evol Microbiol 63,
19
3175–3179.
20
Yoon, J. H., Kang, K. H., Oh, T. K. & Park, Y. H. (2004a). Shewanella gaetbuli sp.
21
nov., a slight halophile isolated from a tidal flat in Korea. Int J Syst Evol Microbiol 54,
22
487–491.
23
Yoon, J.-H., Park, S., Jung, Y.-T. & Lee J.-S. (2012). Shewanella seohaensis sp. nov.,
24
isolated from a tidal flat sediment. Antonie van Leeuwenhoek 102, 149–156.
20
1
Yoon, J. H., Yeo, S. H., Kim, I. G. & Oh, T. K. (2004b). Shewanella marisflavi sp. nov.
2
and Shewanella aquimarina sp. nov., slightly halophilic organisms isolated from sea
3
water of the Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 2347–2352.
4
Zhao, J.-S., Manno, D., Beaulieu, C., Paquet, L. & Hawari, J. (2005). Shewanella
5
sediminis sp. nov., a novel Na+-requiring and
6
hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading bacterium from marine sediment. Int J
7
Syst Evol Microbiol 55, 1511–1520.
8
Zhao, J.-S., Manno, D., Leggiadro, C., O’Neil, D. & Hawari, J. (2006). Shewanella
9
halifaxensis sp. nov., a novel obligately respiratory and denitrifying psychrophile. Int J
10
Syst Evol Microbiol 56, 205–212.
11
Zhao, J. S., Manno, D., Thiboutot, S., Ampleman, G. & Hawari, J. (2007).
12
Shewanella canadensis sp. nov. and Shewanella atlantica sp. nov., manganese dioxide-
13
and hexahydro-1,3,5-trinitro-1,3,5-triazine-reducing, psychrophilic marine bacteria. Int J
14
Syst Evol Microbiol 57, 2155–2162.
15
Ziemke, F., Höfle, M. G. Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of
16
Shewanella putrefaciens Owen’s genomic group II as Shewanella baltica sp. nov. Int J
17
Syst Bacteriol 48, 179–186.
18 19
21
1 Table 1. Differential characteristics of strain BJ-1T (Psychrobium gen. nov.) and related genera Data for reference genera were taken from the following studies: Bowman, J. P. (2005), Bowman et al. (1997), Bozal et al. (2002, 2009), Brettar et al. (2002), Chang et al. (2008), Gao et al. (2006), Hirota et al. (2005), Huang et al. (2010), Ivanova et al. (2001, 2003a, b, 2004b), Kim et al. (2007, 2011, 2012), Lee et al. (2006), Lee & Yoon (2012), Leonardo et al. (1999), Makemson et al. (1997), Miyazaki et al. (2006), Nogi et al. (1998b), Park et al. (2009), Park & Jeon (2013), Satomi et al. (2003, 2006, 2007), Shnit-Orland et al. (2010), Skerratt et al. (2002), Sravan Kumar et al. (2010), Sucharita et al. (2009), Sung et al. (2012), Toffin et al. (2004), Venkateswaran et al. (1998, 1999), Verma et al. (2011), Xiao et al. (2007), Xu et al. (2005), Yang et al. (2006, 2007), Yoon et al. (2004a, b, 2012), Zhao et al. (2005, 2006, 2007), Ziemke et al. (1998) (Shewanella); Hosoya et al. (2009), Yang et al. (2013) (Paramoritella); Benediktsdóttir et al. (2000), Kim et al. (2008), Nogi & Kato (1999), Nogi et al. (1998a), Urakawa et al. (1998), Xu et al. (2003) (Moritella); Campbell et al. (2007), Ji et al. (2013), Katsuta et al. (2005), Nakagawa et al. (2006), Rahman & Cha (2013), Rosselló-Mora et al. (1995), Yim et al. (2013) (Ferrimonas); Khan & Harayama (2007) (Paraferrimonas). +, All species positive; -, all species negative; ND, no data available. (Numbers in parentheses are percentages of the species positive or negative). A, Aerobic; F, facultatively anaerobic. Characteristic
Psychrobium
Shewanella
Paramoritella
Moritella
Ferrimonas
Paraferrimonas
No. of species
1
61
2
7
8
1
Metabolism
A
F (93.3 %)
F
F
F
F
4 °C
+
+ (80.3 %)
-
+
-
-
20 °C
-
+ (95.1 %)
+
-
+
+
37 °C
-
- (59.0 %)
+
-
+ (62.5 %)
+
42 °C
-
- (83.6 %)
-
-
- (62.5 %)
-
1 % NaCl
-
+ (96.6 %)
+
ND
+ (62.5 %)
+
5 % NaCl Major fatty acids
16:1ω7c, 16:0
+ (86.9 %) iso-13:0, iso-15:0, 14:0, 15:0, 16:0, 16:1ω7c, 17:1ω8c, 18:1ω7c
+ (50 %) 16:1ω7c, 16:0, 18:1ω7c, 14:0
ND 16:1, 16:0, 14:0, 22:6ω3
+ (87.5 %) iso-15:0, 16:0, 16:1ω9c, 17:1ω8c, 18:1ω9c
iso-15:0, 18:1ω7c, 16:0, iso-13:0
Major quinone(s)
Q-8
Q-7, Q-8, MK-7
Q-8
Q-8
Q-7, Q-8, MK-7
Q-7, MK-6, MK-7
DNA G + C content (mol %)
40.5
39-54
56-57
41-47
54-60
50-51
Growth at:
2 3
22
1
Figure legends
2 3
Fig. 1. Phylogenetic tree derived from the 16S rRNA gene sequences of strain BJ-1T and
4
the type species of closely related genera within the order Alteromonadales, based on the
5
NJ algorithm. Numbers at nodes indicate bootstrap values (>50%) as calculated on the
6
basis of probabilities, expressed as percentages of 1000 replications. Escherichia coli
7
ATCC 11775T was used as an outgroup. Bar, 0.01 substitution per nucleotide position.
8 9
Fig. 2. Two-dimensional TLC after staining with ethanolic phosphomolybdic acid showing
the
total
polar
lipid
reagent
11
diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL1,
12
unidentified phospholipid; PME, phosphatidylmethylethanolamine.
13 14
23
profiles
of
strain
BJ-1T.
10
DPG,
Fig. 1
