International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3276–3280
DOI 10.1099/ijsem.0.000405
Fabibacter misakiensis sp. nov., a marine
bacterium isolated from coastal surface water
Shu-Kuan Wong,1 Sanghwa Park3,1 Jung-Sook Lee,2 Keun Chul Lee,2
Hiroshi Xavier Chiura,1 Kazuhiro Kogure1 and Koji Hamasaki1
Correspondence
Koji Hamasaki
[email protected]
1
Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa,
Chiba 277-8564, Japan
2
Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and
Biotechnology (KRIBB), 125 Gwahak-ro, Daejeon 305-806, Korea
A slightly curved-rod-shaped, pink-pigmented, Gram-stain-negative, aerobic bacterial strain with
gliding motility, designated SK-8T, was isolated from coastal surface water of Misaki, Japan.
Phylogenetic trees generated using 16S rRNA gene sequences revealed that strain SK-8T
belonged to the genus Fabibacter and showed 96.0 % sequence similarity to the type strain of
the most closely related species, Fabibacter pacificus DY53T. The novel isolate was
phenotypically and physiologically different from previously described strains. The major cellular
fatty acids were iso-C15 : 1 G, iso-C15 : 0 and iso-C17 : 0 3-OH. Major polar lipids were
phosphatidylethanolamine, two aminophospholipids and an unidentified phospholipid. The DNA
G+C content was 39.1 mol% and MK-7 was the only predominant isoprenoid quinone. On the
basis of this taxonomic study employing a polyphasic approach, it was suggested that strain
SK-8T represents a novel species of the genus Fabibacter, with the newly proposed name
Fabibacter misakiensis sp. nov. The type strain is SK-8T (5NBRC 110216T5KCTC 32969T).
The family Flammeovirgaceae in the order Cytophagales
was proposed by Yoon et al. (2011) and is made up of 19
different genera that were isolated mainly from soil and
marine environments. The genus Fabibacter, a member of
the family Flammeovirgaceae, was first proposed by Lau
et al. (2006). At the time of writing, the genus Fabibacter
comprises two species with validly published names. Fabibacter halotolerans UST030701-097T, the type strain of the
type species of the genus, was isolated from a marine
sponge in the Bahamas (Lau et al., 2006) while Fabibacter
pacificus DY53T, a slightly halophilic strain, was isolated
from a deep-sea water sample of the Pacific Ocean (Huo
et al., 2013). In this study, a novel bacterial strain assigned
as SK-8T was isolated from coastal surface seawater of
Misaki, Japan and the taxonomic position was investigated
using a polyphasic approach.
Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ,
neighbour-joining.
3Present address: Center of Molecular Biosciences, University of the
Ryukyus, Senbaru1, Nishihara, Okinawa 903-0213, Japan.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strains SK-8T is KM502320.
Two supplementary figures are available with the online Supplementary
Material.
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Surface water was collected from a depth of 20 cm near the
pier of Misaki Marine Biological Station, University of
Tokyo (358 09.59 N 1398 36.59 E), Aburatsubo Inlet,
Misaki, Kanagawa Prefecture, Japan. Aliquots (100 ml) of
seawater were plated onto 1/10-strength ZoBell agar
medium [0.5 g peptone, 0.1 g yeast extract, 15 g agar in
1 l of 80 % aged natural seawater (80 % seawater+20 %
water, aged for at least one year)] and incubated at 20 8C
for 4 days. Routine cultivation for subsequent characterizations were performed on half-strength marine agar 2216
(Difco) supplemented with 1.0 % NaCl (w/v) at 25 8C,
hereby known as 1/2 MA. The isolate was maintained at
280 8C as a suspension in half-strength marine broth
2216 (Difco) supplemented with 1.0 % NaCl (w/v), containing glycerol (20 %, w/v). Cell morphology was examined according to Børsheim et al. (1990). After staining
for 30 s with 2 % uranyl acetate, grids were examined at
675 000 magnification at an acceleration voltage of
80 kV using a JEM-1400EX transmission electron microscope (JEOL); at least 50 fields were selected for examination.
Gliding motility was observed under light microscopy (BX60;
Olympus). Gram staining was performed according to
instructions provided in the Gram Stain kit (BD). Temperature (4 8C, 10–30 8C at 5 8C intervals, 37 8C and 45 8C) and
salt tolerance were determined using 1/2 MA with different
concentrations of NaCl (0–1 % at 0.05 % intervals, 1–8 %
at 1 % intervals and 10–15 % at 5 % intervals, w/v) and
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Fabibacter misakiensis sp. nov.
adjusted to pH 7.5 using 1 M NaOH. The pH tolerance
range (pH 5–12 at 1 pH unit intervals; adjusted to different
pH values with 1 M HCl or 1 M NaOH) was tested on cells
incubated on 1/2 MA at 25 8C for 4 days. Growth under
anaerobic conditions was determined after incubation with
AnaeroPack (Mitsubishi Gas Chemical Co.) on 1/2 MA for
4 weeks. Tests for degradation of DNA [using DNase agar
(Oxoid) and flooding the plates with 1M HCl], starch
(Atlas, 1993), chitin (Sakazaki et al., 1995) and CM-cellulose
(Bowman, 2000) were performed and evaluated after
10 days. The presence of flexirubin-type pigments was
tested using 20 % (w/v) KOH solution. Catalase activity
was determined from bubble formation in 3 % hydrogen
peroxide solution. Oxidase activity was tested using cytochrome oxidase paper (Nissui Pharmaceutical). For API
series tests and fatty acid analysis, strain SK-8T was compared to the reference strains F. halotolerans JCM 13334T
and F. pacificus JCM 18885T purchased from the Japan
Collection of Microorganisms (JCM) culture collection. Biochemical properties of strain SK-8T and the two reference
strains were determined using API ZYM, API 20E, API
20NE and API 50CH strips (bioMérieux). All suspension
media in API test strips were supplemented with 2 % (w/
v) NaCl (final concentration). API 20E, API 20NE and
API 50CH strips were incubated at 25 8C and read after
5 days and API ZYM strips were read after 2 days.
For fatty acids analysis, cells of strain SK-8T and the reference strains were grown for 5 days at 25 8C on 1/2 MA.
Fatty acid methyl esters were prepared according to the
standard protocol of the Microbial Identification System
(MIS; MIDI) and identified using Microbial Identification
(MIDI) (Sasser, 1990) with the TSBA database version 6.10.
Isoprenoid quinones were extracted with chloroform/
methanol (2 : 1, v/v) and purified using TLC on Kieselgel
60 F254 plates (20620 cm, 0.5 mm thick; Merck) with
petroleum ether/diethyl ether (9 : 1, v/v) as the solvent.
Quinones were identified using reversed-phase HPLC as
described by Shin et al. (1996). Polar lipids were identified
using two-dimensional TLC and identified according to
previously published procedures (Minnikin et al., 1977).
The spots for polar lipids were identified by spraying
with 10 % phosphomolybdic acid in ethanol (Sigma
P4869), a-naphthol and ninhydrin.
The 16S rRNA gene of strain SK-8T was amplified using the
universal primer set 27F/ 1492R (Lane, 1991) and
sequenced using an ABI 3130xl DNA sequencer. The
sequence obtained (1419 bp) was subjected to BLAST in
GenBank and EzTaxon-e databases, and sequences of
related type strains were obtained. Multiple alignments of
sequences were performed using the CLUSTAL W option in
MEGA 5.0 software (Tamura et al., 2011). Phylogenetic
and molecular evolutionary analyses were also conducted
with MEGA 5.0 software using evolutionary distances calculated from Kimura’s two-parameter model (Kimura, 1983)
and clustered using neighbour-joining (NJ; Saitou & Nei,
1987), maximum-likelihood (ML; Felsenstein, 1981) and
maximum-parsimony (MP; Fitch, 1971) methods. The
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robustness of the phylogenetic tree was assessed through
bootstrap resampling values of 1000 replicates (Felsenstein,
1985). Pairwise sequence similarities were calculated using
EzTaxon-e (Kim et al., 2012). For the measurement of
genomic DNA G+C content, genomic DNA was extracted
and purified using a Blood & Cell Culture DNA Midi kit
(Qiagen), and degraded enzymically into nucleosides. The
genomic DNA G+C content of the deoxyribonucleosides
was determined by reverse-phase HPLC (Tamaoka &
Komagata, 1984).
The 16S rRNA gene sequence of strain SK-8T showed highest similarity to Fabibacter pacificus DY53T (96.0 %) and
Fabibacter halotolerans UST030701-097T (95.6 %). Phylogenetic trees obtained by the NJ, MP and ML methods
revealed clear affiliations between the novel isolate SK-8T
and the cluster composed of the species F. pacificus and
F. halotolerans, at bootstrap confidence levels of 99 %
(NJ), 97 % (ML) and 98 % (MP) (Fig. 1).
Cultural, physiological and biochemical characteristics of
strain SK-8T were compared with related species, F. pacificus
and F. halotolerans (Table 1). Cells of strain SK-8T were
curved rods ranging from 2.4–3.5 mm in length (Fig. S1, available in the online Supplementary material). The pH tolerance
range for strain SK-8T (optimum pH 7.0–9.0) was almost
similar to the reference strains from the genus Fabibacter.
However, strain SK-8T had lower salt (1–5 % NaCl) and
lower temperature (10–30 8C) tolerance ranges. Another
differential feature of the novel strain from the reference
strains was its inability to hydrolyse starch. Strain SK-8T and
the two reference strains were positive for catalase, oxidase,
leucine arylamidase, valine arylamidase, cystine arylamidase,
trypsin, a-chymotrypsin, acid phosphate, naphthol-AS-BIphosphohydrolase, b-glucosidase and b-galactosidase but
the enzyme activity for alkaline phosphate, esterase lipase
(C8), lipase (C14), a-galactosidase and N-acetyl-b-glucosaminidase were different among the three strains. Mannitol and
gluconate assimilation were positive only for strain SK-8T.
The DNA G+C content of strain SK-8T was 39.1 mol%.
The major fatty acids were iso-C15 : 1 G (47.6 %), iso-C15 : 0
(18.1 %) and iso-C17 : 0 3-OH (9.8 %) (Table 2). The major
fatty acids of strain SK-8T, F. halotolerans JCM 13334T and
F. pacificus JCM 18885T were similar but the absence of
summed feature 3 (comprising C16 : 1v6c and/or C16 : 1v7c)
and the higher portion of iso-C15 : 1 G distinguished strain
SK-8T from the two reference strains. The polar lipids of
strain SK-8T comprised phosphatidylethanolamine, two aminophospholipids and an unidentified phospholipid (Fig. S2).
Similarities and differences in phylogenetic, chemotaxonomic and phenotypic evidences mentioned above suggested
that strain SK-8T belongs to the genus Fabibacter but can be
differentiated from other members within the same genus.
It is concluded that strain SK-8T represents a novel species
of the genus Fabibacter with the newly proposed name Fabibacter misakiensis sp. nov.
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3277
S.-K. Wong and others
Fabibacter pacificus DY53T (KC005305)
(82/81/90)
Fabibacter halotolerans UST030701-097T (DQ080995)
(99/97/98)
0.02
Fabibacter misakiensis SK-8T (KM502320)
(100/100/99)
Roseivirga spongicola UST030701-084T (DQ080996)
(74/64/56)
Roseivirga echinicomitans KMM 6058T (AY753206)
(71/68/68)
(100/100/100)
Roseivirga ehrenbergii KMM 6017T (AY608410)
Marinoscillum pacificum MRN461T (DQ660388)
(83/71/61)
Marinoscillum luteum SJP7T (NR108992)
(100/100/100)
Marinoscillum furvescens NBRC 15994T (AB078079)
(-/-/-)
Fulvivirga imtechensis AK7T (FR687203)
(100/99/100)
Fulvivirga kasyanovii KMM 6220T (DQ836305)
(82/82/74)
Reichenbachiella agariperforans KMM 3525T (AB058919)
(98/96/96)
Reichenbachiella faecimaris PCP11T (GU143096)
(100/100/100)
Cesiribacter andamanensis AMV16T (FN396961)
(100/100/99)
Cesiribacter roseus 311T (HM775387)
(68/71/-)
Marivirga sericea IFO 15983T (AB078081)
(91/85/59)
Marivirga tractuosa DSM 4126T (CP002349)
(100/100/100)
Aureibacter tunicatorum A5Q-118T (AB572584)
(61/-/-)
Persicobacter diffluens NBRC 15940T (AB260929)
(99/98/-)
(100/100/100)
Persicobacter psychrovividus NBRC 101262T (AB260934)
Flexithrix dorotheae IFO 15987T (AB078077)
(100/100/99)
Rapidithrix thailandica TISTR 1750T (AB265192)
Sediminitomix flava Mok-1-85T (AB255370)
(83/87/-)
Limibacter armeniacum YM11-185T (AB359907)
(72/84/-)
Perexilibacter aurantiacus Shu-F-UV2-2T (AB276355)
(66/51/-)
Flammeovirga pacifica WPAGA1T (HQ412594)
(100/100/100)
(88/92/78)
Flammeovirga yaeyamensis NBRC 100898T (AB247554)
Flammeovirga kamogawensis YS10T (AB251933)
(100/100/100)
(93/88/96)
(97/75/93)
Flammeovirga aprica JL-4T (AB247553)
Flammeovirga arenaria NBRC 15982T (AB078078)
Thermonema rossianum NR-27T (Y08956)
(100/100/100)
Thermonema lapsum DSM 5718T (HE582775)
Flavobacterium aquatile DSM 1132T (AM230485)
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic position of strain SK-8T. Numbers at nodes are bootstrap values based on 1000 replications (NJ/ML/MP); 2 indicates branch with bootstrap values
,50 %. Bar, 2.0 % sequence divergence.
Description of Fabibacter misakiensis sp. nov.
Fabibacter misakiensis (mi.sa.ki9en.sis N.L. masc. adj. misakiensis pertaining to Misaki, the city of which the type
strain was isolated).
Colonies grown on 1/2 MA for 4 days are circular, shiny
with entire edges and pink-pigmented. Cells are curvedrod-shaped, 1.12+ 0.13 mm in length and 0.39+ 0.05 mm
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wide. Strictly aerobic, Gram-stain-negative, and lacking flagella but has gliding motility. Flexirubin-type pigments are
absent. The temperature range for growth is 10–30 uC.
Requires 1.0–5.0 % NaCl and pH 6–10 for growth. Does
not reduce nitrite or nitrate, nor produce acetoin or
indole. Catalase- and oxidase-positive. DNA and aesculin
are hydrolysed but starch, chitin, gelatin and urea are
not. In API ZYM tests, leucine arylamidase, valine
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Fabibacter misakiensis sp. nov.
Table 1. Differential characteristics between strain SK-8T and related members of the genus Fabibacter
Strains: 1, SK-8T; 2, F. halotolerans JCM 13334T; 3, F. pacificus JCM 18885T. All data were obtained from this study unless otherwise stated. All
strains were negative for hydrolysis of chitin, gelatin and urea. All strains were positive for catalase, oxidase, leucine arylamidase, valine arylamidase,
cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase (API ZYM). ND , Not determined; +, positive; 2, negative; w, weakly positive.
Characteristic
Isolation source
Cell morphology
Cell length6width (mm)
Gliding motility
NaCl range for growth (%)
pH range for growth (optimum)
Temperature range for growth (optimum) (8C)
Hydrolysis of:
Starch
Aesculin
DNA
Enzyme activities (API ZYM)
Alkaline phosphatase
Esterase lipase (C8)
Lipase (C14)
a-Galactosidase
N-Acetyl-b-glucosaminidase
Assimilation of (API 20NE):
Mannitol
L -Arginine
Mannose
Maltose
Gluconate
Malate
DNA G+C content (mol%)
1
2
3
Coastal seawater
Curved rod
0.92–1.3560.35–0.50
+
1–5
6–10 (7–9)
10–30 (20–25)
Marine sponge*a
Curved roda
1.560.5a
+a
0–12a
5–10 (ND )a
12–36 (28–30)a
Deep seawaterb
Rod-shapedb
1.5–5.060.5–0.7b
2
+
+
+
2
+a
+
+
+
2
2
2
2
2
+
+
2
+
+
+
+
+
+
+
+
2
2
2
w
2
39.1
2
+
+
+
2
+
42.5
2
+
+
+
2
+
40.8
ND
b
0.5–15b
6.5–8.5 (7.5)b
15–40 (35–37)b
*Data from: a, Lau et al. (2006); b, Huo et al. (2013).
Table 2. Comparison of fatty acid contents of strain SK-8T
and other closely related taxa of the genus Fabibacter
Strains: 1, SK-8T; 2, F. halotolerans JCM 13334T; 3, F. pacificus JCM
18885T. All data were obtained from this study. ND , Not detected.
Fatty acid
Branched
iso-C15 : 0
iso-C15 : 1 G
iso-C16 : 1 G
anteiso-C15 : 0
Hydroxy
C16 : 0 3-OH
iso-C15 : 0 3-OH
iso-C16 : 0 3-OH
iso-C17 : 0 3-OH
Summed feature 3*
1
2
3
18.1
47.6
1.8
2.9
30.5
21.7
2.1
7.5
19.0
32.8
2.1
5.3
1.3
6.2
4.7
9.8
2.6
4.0
2.8
10.5
8.0
1.7
5.0
3.8
17.2
5.7
ND
*Summed features are groups of two or three fatty acids that cannot
be separated by GLC with the MIDI system. Summed feature 3 contains C16 : 1v6c and/or C16 : 1v7c.
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arylamidase, cystine arylamidase, trypsin, a-chymotrypsin,
acid phosphatase, naphthol-AS-BI-phosphohydrolase,
b-glucosidase and b-galactosidase activities are positive
but alkaline phosphatase, esterase (C4), esterase lipase
(C8), lipase (C14), a-galactosidase, N-acetyl-b-glucosaminidase, a-glucosidase, a-mannosidase, a-fucosidase and
b-glucuronidase activities are negative. Acid is produced
from glucose, sorbose, methyl a-D -glucoside, amygdalin,
arbutin, aesculin, salicin, cellobiose, maltose, sucrose, trehalose, glycogen, gentiobiose and turanose in API 50CH tests.
Assimilates gluconate and mannitol in API 20NE tests. The
only major menaquinone is MK-7. The polar lipids are phosphatidylethanolamine, two aminophospholipids and an unidentified phospholipid.
The type strain is SK-8T (5NBRC 110216T5KCTC 32969T),
which was isolated from the surface water (20 cm depth) of
Aburatsubo Inlet in Misaki, Kanagawa Prefecture, Japan.
The genomic DNA G+C content of the type strain is
39.1 mol%.
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S.-K. Wong and others
Acknowledgements
Kimura, M. (1983). The Neutral Theory of Molecular Evolution.
Cambridge: Cambridge University Press.
This work was supported in part by a grant from JSPS Postdoctoral
Fellowship for the Foreign Researcher (P-12404) from the Japan
Society for the Promotion of Science and the KRIBB Research Initiative Program.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid
References
Atlas, R. M. (1993). Handbook of Microbiological Media. Edited by
L. C. Parks. Boca Raton, FL: CRC Press.
Børsheim, K. Y., Bratbak, G. & Heldal, M. (1990). Enumeration and
biomass estimation of planktonic bacteria and viruses by transmission
electron microscopy. Appl Environ Microbiol 56, 352–356.
Bowman, J. P. (2000). Description of Cellulophaga algicola sp. nov.,
isolated from the surfaces of Antarctic algae, and reclassification of
Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as
Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50,
1861–1868.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences:
a maximum likelihood approach. J Mol Evol 17, 368–376.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39, 783–791.
Fitch, W. M. (1971). Toward defining the course of evolution:
minimum change for a specific tree topology. Syst Zool 20, 406–416.
Huo, Y. Y., Xu, L., Wang, C. S., Yang, J. Y., You, H., Oren, A. & Xu, X. W.
(2013). Fabibacter pacificus sp. nov., a moderately halophilic
bacterium isolated from seawater. Int J Syst Evol Microbiol 63,
3710–3714.
Techniques in Bacterial Systematics, pp. 115–175. Edited by
E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
Lau, S. C. K., Tsoi, M. M. Y., Li, X., Plakhotnikova, I., Dobretsov, S., Wu,
M., Wong, P. K., Pawlik, J. R. & Qian, P. Y. (2006). Description of
Fabibacter halotolerans gen. nov., sp. nov. and Roseivirga spongicola
sp. nov., and reclassification of [Marinicola ] seohaensis as Roseivirga
seohaensis comb. nov. Int J Syst Evol Microbiol 56, 1059–1065.
Minnikin, D. E., Patel, P. V., Alshamaony, L. & Goodfellow, M. (1977).
Polar lipid composition in the classification of Nocardia and related
bacteria. Int J Syst Bacteriol 27, 104–117.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4,
406–425.
Sakazaki, R., Miki, K. & Yoshizaki, E. (1995). New Bacterial Culture
Media, 2nd I. Tokyo, Japan: Kindai Shuppan.
Sasser, M. (1990). Identification of bacteria by gas chromatography of
cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
Shin, Y. K., Lee, J.-S., Chun, C. O., Kim, H.-J. & Park, Y.-H. (1996).
Isoprenoid quinone profiles of Leclercia adecarboxylata KCTC
1036T. J Microbiol Biotechnol 6, 68–69.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA
base composition by reversed-phase high-performance liquid
chromatography. FEMS Microbiol Lett 25, 125–128.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar,
S. (2011). MEGA 5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and
parsimony methods. Mol Biol Evol 28, 2731–2739.
maximum
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C.,
Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing
Yoon, J., Adachi, K., Park, S., Kasai, H. & Yokota, A. (2011). Aureibacter
EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with
phylotypes that represent uncultured species. Int J Syst Evol Microbiol
62, 716–721.
tunicatorum gen. nov., sp. nov., a marine bacterium isolated from a
coral reef sea squirt, and description of Flammeovirgaceae fam. nov.
Int J Syst Evol Microbiol 61, 2342–2347.
3280
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