International Journal of Systematic and Evolutionary Microbiology (2015), 65, 2222 – 2226
DOI 10.1099/ijs.0.000252
Dokdonia pacifica sp. nov., isolated from seawater
Zenghu Zhang,13 Xin Gao,13 Long Wang1 and Xiao-Hua Zhang1,2
Correspondence
1
Xiao-Hua Zhang
2
College of Marine Life Sciences, Ocean University of China, Qingdao, PR China
Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, PR China
[email protected]
A Gram-stain-negative, aerobic, non-flagellated, non-gliding, oxidase- and catalase-positive,
rod-shaped, yellow-pigmented bacterium, designated strain SW230T, was isolated from a
surface seawater sample collected from the South Pacific Gyre. Phylogenetic analysis based on
16S rRNA gene sequences indicated that strain SW230T shared highest similarity with
members of the genus Dokdonia (95.0–94.5 %), exhibiting 95.0 % sequence similarity to
Dokdonia genika NBRC 100811T. Optimal growth occurred in the presence of 2–3 % (w/v)
NaCl, at pH 8.0 and at 28 8C. The DNA G+C content of strain SW230T was 36 mol%.
The major fatty acids (.10 % of the total) were iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH, and
C16:1v7c and/or C16:1v6c. The major respiratory quinone was menaquinone-6. The major polar
lipids were phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids.
On the basis of data from the present polyphasic study, strain SW230T is considered to
represent a novel species of the genus Dokdonia, for which the name Dokdonia pacifica
sp. nov. is proposed. The type strain is SW230T (5CGMCC 1.12184T5JCM 18216T).
The genus Dokdonia, a member of the family Flavobacteriaceae in the phylum Bacteroidetes, was proposed by
Yoon et al. (2005) for a single species, Dokdonia donghaensis, isolated from seawater around the Korean island of
Dokdo. Subsequently, three novel species of the genus Krokinobacter, Krokinobacter genikus, Krokinobacter diaphorus
and Krokinobacter eikastus, were isolated from marine sediment samples collected from the Pacific coastline of Japan
(Khan et al., 2006). Based on high levels of sequence
similarity, K. genikus, K. diaphorus and K. eikastus were
reclassified in the genus Dokdonia as D. genika comb.
nov., D. diaphoros comb. nov. and D. eikasta comb. nov.,
respectively, by Yoon et al. (2012) and the description of
the genus Dokdonia was also emended. In this study, a
novel bacterial strain, designated SW230T, was isolated
from surface seawater of the South Pacific Gyre at station
U1371 (458 589 S 1638 119 W) during the Integrated
Ocean Drilling Program (IODP) Expedition 329. The aim
of the present study was to investigate the possibility that
strain SW230T may represent a novel species of the genus
Dokdonia. Here, we describe the phenotypic and phylogenetic characteristics of strain SW230T.
3These authors contributed equally to this work.
Abbreviation: IODP, Integrated Ocean Drilling Program
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
gene sequence of Dokdonia pacifica SW230T is KP862606.
Four supplementary figures are available with the online Supplementary
Material.
2222
Surface water of the South Pacific Gyre was collected in
November 2010 and spread on marine agar 2216 (MA;
Becton Dickinson). Strain SW230T, which formed transparent, circular (0.8–1.0 mm in diameter), convex colonies
with entire margins on MA after culturing at 28 8C for
3 days, was picked and purified by streaking three times
on MA. The culture was maintained on MA plates at
28 8C, and stocks were preserved in sterile 0.85 % (w/v)
saline supplemented with 15 % (v/v) glycerol at 280 8C.
D. genika NBRC 100811T and D. donghaensis DSM
17200T were used as reference strains, and were cultured
at the same conditions as for strain SW230T [in marine
broth 2216 (MB; BD), 28 8C], unless otherwise specified.
Gram-staining and flagellum staining were investigated using
standard methods (Beveridge et al., 2007). Cell morphology
was determined by transmission electron microscopy
(JEM-1200EX; JEOL) after cells had been negatively stained
with 1 % (w/v) phosphotungstic acid. The presence of gliding
motility and the production of flexirubin-type pigments were
investigated using the methods described by Bernardet et al.
(2002). To test for anaerobic growth, bacterial strains were
cultured on MA with resazurin (0.02 %, w/v) as an indicator
of anaerobic conditions in an anaerobic jar filled with
nitrogen and a packet of AneroPack-Anaero (Mitsubishi
Gas Chemical) at 28 8C for 1 month. The temperature
range for growth was determined on MA by incubating
cultures at 10–37 8C (10, 16, 22, 28, 32, 35, 37 8C) for
7 days, and at 0 and 4 8C on MA for 30 days. Salinity and
pH ranges supporting growth were investigated in 96-well
microplates by measuring the optical densities (wavelength
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Dokdonia pacifica sp. nov.
590 nm). In the salinity experiment, distilled water was
used to prepare synthetic marine ZoBell broth (per litre:
5 g peptone, 1 g yeast extract and 0.1 g FePO4). NaCl
concentrations were adjusted to 0–15.0 % (w/v, at intervals
of 1.0 %). Growth in MB was evaluated at pH 2.0–10.0 at
intervals of 1 pH unit using the following buffer systems:
H3PO4/KH2PO4 (pH 2.0), sodium acetate/acetic acid
(pH 3.0–6.0), KH2PO4/NaOH (pH 7.0–8.0) and Na2CO3/
NaHCO3 (pH 9.0–10.0). Various phenotypic properties
of strain SW230T and the reference strains were tested
according to standard approaches (Tindall et al., 2007),
including catalase and oxidase and hydrolysis of starch,
casein, gelatin and Tweens 20, 40 and 80 (method 2).
DNase activity was examined by using DNase agar
(Qingdao Hope Bio-technology) according to the manufacturer’s instructions. Activities of constitutive enzymes
and other physiological properties were determined after
growth on MA at 28 8C for 2 days by using API 20E, API
20NE, API 50CH and API ZYM strips (bioMérieux) and
Gram-negative MicroPlates (Biolog) according to the
manufacturers’ instructions, except that the strips were
inoculated with a heavy bacterial suspension in artificial
seawater (Yoon et al., 2002). The morphological, physiological and biochemical characteristics of strain SW230T
are shown in Fig. S1 (available in the online Supplementary
Material), Table 1 and the species description.
For cellular fatty acid analysis, strain SW230T and the reference strains were grown on MA at 28 8C for 2–3 days when
the bacterial communities reached the late-exponential stage
of growth according to the four quadrants streak method
(Sasser, 1990). Fatty acid methyl esters were prepared and
analysed according to the standard protocol of MIDI
(Sherlock Microbial Identification System, version 6.0)
and identified using the TSBA6.0 database of the Microbial
Identification System (Sasser, 1990). Cell biomass for identification of polar lipids and respiratory quinones was
obtained by centrifugation after shaking at 28 8C in MB
for 4 days. Polar lipids were extracted according to the procedures described by Minnikin et al. (1984), and separated
by two-dimensional TLC on silica gel 60 F254 plates
(Merck) using chloroform/methanol/water (65:25:4, v/v)
for the first dimension and chloroform/methanol/acetic
acid/water (80:12:15:4, v/v) for the second dimension
(Collins & Shah, 1984). Identification of individual lipids
was performed by spraying with the appropriate detection
reagents (Komagata & Suzuki, 1987). The respiratory quinones of strain SW230T were extracted with chloroform/
methanol (2:1, v/v), separated by TLC and identified by
HPLC. DNA of strain SW230T was extracted according
to the procedure of Moore et al. (1999) and the G+C
content was determined by reversed-phase HPLC according to Mesbah & Whitman (1989).
The cellular fatty acid profiles of strain SW230T and the
reference strains are given in Table 2. The dominant fatty
acids (.10 % of the total) of strain SW230T were
iso-C15:1 G (22.4 %), iso-C15:0 (18.4 %), iso-C17:0 3-OH
(15.1 %) and summed feature 3 (comprising C16:1v7c
http://ijs.sgmjournals.org
and/or C16:1v6c, 11.7 %), which were similar to those of
the reference strains although some quantitative differences
were noted; for example, SW230T contained less iso-C15:0
than the reference strains. The polar lipid profile of strain
SW230T comprised phosphatidylethanolamine (PE), two
unidentified aminolipids (AL1 and AL2) and two unidentified lipids (L1 and L2) (Fig. S2), which was identical to
that of the two reference strains. In accordance with the
reference strains, strain SW230T contained menaquinone6 as the major respiratory quinone. The DNA G+C content of strain SW230T was 36 mol%, which falls within
the range reported for members of the genus Dokdonia
(33–39 mol%) (Yoon et al., 2012).
For 16S rRNA gene sequencing, the genomic DNA of
strain SW230T was extracted and purified using standard
methods (Ausubel et al., 1995). The 16S rRNA gene
was amplified by PCR with two universal primers
(B8F:
59-AGAGTTTGATCCTGGCTCAG-39;
B1510:
59-GGTTACCTTGTTACGACTT-39) (Weisburg et al., 1991).
Table 1. Differential characteristics between strain SW230T
and reference species of the genus Dokdonia
Strains: 1, SW230T; 2, D. genika NBRC 100811T (Yoon et al., 2012; Khan
et al., 2006); 3, D. donghaensis DSM 17200T (Yoon et al., 2005). All the
data are from the present study except cell size and DNA G+C content,
which are from the original references. All the strains are yellowpigmented and rod-shaped bacteria, positive for DNase, oxidase,
catalase, alkaline phosphatase, esterase lipase (C8), leucine arylamidase
and valine arylamidase activities, and hydrolysis of casein, aesculin
and Tweens 20, 40 and 80; all are negative for gliding motility,
presence of lipase (C14), trypsin, a-galactosidase, b-galactosidase,
b-glucuronidase, a-glucosidase, b-glucosidase, a-mannosidase and
a-fucosidase activities. All have MK-6 as a major respiratory quinone.
+, Positive reaction; 2, negative reaction; W , weakly positive.
Characteristic
1
2
3
Cell size (mm)
Length
1.2–1.5 1.5–25 2.5–4.0
Width
0.3–0.4 0.3–0.6 0.5–0.7
Growth at/with:
4 8C
2
+
2
35 8C
+
+
2
1 % NaCl
+
+
2
10 % NaCl
2
2
+
Hydrolysis of:
Gelatin
2
+
2
Starch
+
2
2
API ZYM results:
W
+
Esterase
+
W
2
Cystine arylamidase
2
a-Chymotrypsin
+
2
2
Acid phosphatase
+
+
2
Naphthol-AS-BI-phosphohydrolase
+
+
2
N-Acetyl-b-glucosaminidase
+
2
2
DNA G+C content (mol%)
36
37
38
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Z. Zhang and others
Table 2. Cellular fatty acid contents (%) of strain SW230T
and reference species of the genus Dokdonia
Strains: 1, SW230T; 2, D. genika NBRC 100811T; 3, D. donghaensis
DSM 17200T. All data are from this study. TR , Trace amount
(,1 %); 2, not detected.
Fatty acid
iso-C14:0
iso-C15:0
iso-C16:0
iso-C15:1 G
anteiso-C15:1 A
anteiso-C15:0
iso-C14:0 3-OH
iso-C15:0 3-OH
iso-C16:0 3-OH
iso-C17:0 3-OH
C15:0 2-OH
C16:0 3-OH
C17:0 2-OH
C17:1v6c
Summed features*
3
9
1
2
3
2
18.4
1.9
22.4
2.5
3.3
1.8
21.1
7.9
20.8
1.6
7.5
1.2
22.4
4.0
22.5
2.6
3.3
TR
TR
TR
8.5
5.4
15.1
1.5
4.5
10.1
8.6
1.7
4.9
5.3
13.6
1.7
TR
TR
TR
2.1
3.8
1.8
TR
TR
TR
11.7
2.8
9.8
2
10.2
2
*Summed features are groups of two or three fatty acids that are treated
together for the purpose of evaluation in the MIDI system and include
both peaks with discrete equivalent chain-lengths (ECLs) as well as
those where the ECLs are not reported separately (Montero-Calasanz
et al., 2013). Summed feature 3 contains C16:1v7c and/or C16:1v6c;
summed feature 9 contains C16:0 10-methyl and/or iso-C17:1v9c.
For cloning and sequencing of the 16S rRNA gene, the PCR
product was purified using a TIANgel MIDI Purification kit
(TIANGEN Biotech), ligated into the pUCm-T vector
(TaKaRa) and sequenced at BGI. Pairwise similarity values
between strain SW230T and closely related type strains were
calculated using the EzTaxon-e server (http://eztaxon-e.
ezbiocloud.net/; Kim et al., 2012). The 16S rRNA gene
sequences of related strains were downloaded from the NCBI
database (http://www.ncbi.nlm.nih.gov) and aligned using
the CLUSTAL X program (Thompson et al., 1997). Phylogenetic
trees based on the neighbour-joining (Fig. 1), maximum-likelihood (Fig. S3) and maximum-parsimony (Fig. S4) algorithms were constructed by MEGA version 5.0 (Tamura et al.,
2011). The genetic distance matrices were estimated by
the Kimura two-parameter model (Kimura, 1980) for the
neighbour-joining tree. In each case, bootstrap values were
calculated based on 1000 replicates.
Pairwise alignment according to the nearly complete 16S
rRNA gene sequence (1484 bp) of strain SW230T revealed
highest sequence similarity with members of the genus
Dokdonia (95.0–94.5 %), exhibiting 95.0 % sequence
similarity to D. genika NBRC 100811T. Lower sequence
similarities (#92.4 %) were found with all other representatives of the family Flavobacteriaceae. This close relationship between strain SW230T and species of the genus
2224
Dokdonia was also evident in the neighbour-joining phylogenetic tree (Fig. 1). Strain SW230T formed a distinct
phyletic line within the radiation of the genus Dokdonia.
The monophyletic clade comprising strain SW230T and
the four recognized species of the genus Dokdonia was
consistently recovered in all tree-making methods used
(Figs. 1, S3 and S4). However, the relatively low level of
sequence similarity to the type strains of recognized species
of the genus Dokdonia implied that strain SW230T may
represent a novel species (Stackebrandt & Goebel, 1994).
The major features of strain SW230T, including the major
respiratory quinone, hydrolysis of casein and Tweens 20,
40 and 80, and the presence of DNase, catalase and oxidase
activity, are in line with the two reference strains. The predominant cellular fatty acids (.10 %) and polar lipid
profile of strain SW230T were similar to those of the two
reference strains, sufficient to classify strain SW230T as a
member of the genus Dokdonia and differentiate it from
other genera in the family Flavobacteriaceae. For example,
the major polar lipids detected in Salinimicrobium gaetbulicola BB-My20T and Salinimicrobium catena JCM 14015T
were phosphatidylethanolamine and one unidentified
lipid (Lee et al., 2012), and these possessed fewer aminolipids than strain SW230T and the two reference strains;
in addition, iso-C15:1 G was a minor cellular fatty acid in
Aquimarina agarilytica ZC1T and Aquimarina intermedia
JCM 13506T (Lin et al., 2012), but is a major component
in strain SW230T and the two reference strains. Although
strain SW230T and the two reference strains shared many
common phenotypic and chemical characteristics, they
showed differences in some features including cell morphology, hydrolysis of starch and gelatin, and numerous
enzyme activities (Table 1) and differences in the
proportion of some fatty acids (Table 2). On the basis of
phenotypic characteristics and phylogenetic inference,
strain SW230T should be classified in the genus Dokdonia
as a member of a novel species, for which the name
Dokdonia pacifica sp. nov. is proposed.
Description of Dokdonia pacifica sp. nov.
Dokdonia pacifica (pa.ci9fi.ca. L. fem. adj. pacifica peaceful,
referring to the Pacific Ocean from where the type strain
was isolated).
Cells are Gram-stain-negative, aerobic, non-gliding rods
(1.2–1.5 mm in length, 0.3–0.4 mm in width after culturing
on MA for 24 h at 28 uC) without any flagellum. Colonies
are transparent, yellow, circular (0.8–1.0 mm in diameter)
and convex on MA after 3 days at 28 uC. Growth occurs
at 10–35 uC (optimum 28 uC). The salinity range for
growth is 1–7% (w/v) NaCl (optimum 2–3%) and the
pH range is 7.0–10.0 (optimum pH 8.0). Flexirubin-type
pigments are not formed. Oxidase and catalase activities
are present. Positive for hydrolysis of DNA, starch, casein
and Tweens 20, 40 and 80, but negative for hydrolysis of
gelatin. In the API 20E and 20NE strips, positive results
are obtained for aesculin hydrolysis, acetoin production,
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Dokdonia pacifica sp. nov.
T
100 Dokdonia eikasta PMA-26 (AB198088)
Dokdonia diaphoros MSKK-32T (AB198089)
100
0.02
Dokdonia donghaensis DSM 17200T (DQ003276)
99
Dokdonia genika NBRC 100811T (AB198086)
98
Dokdonia pacifica SW230T (KP862606)
73
Aquimarina aganlytica ZC1T (JH621312)
Aquimarina gracilis PSC32T (HM998909)
100
Aquimarina intermedia KMM 6258T (AM113977)
96
Aquimarina latercula ATCC 23177T (M58769)
Salinimicrobium catena HY1T (DQ640642)
100
Salinimicrobium gaetbulicola BB-My20T (JF340052)
Leeuwenhoekiella palythoae KMM 6264T (FJ405187)
Leeuwenhoekiella blandensis MED 217T (AANC01000011)
100
Nonlabens tegetincola UST030701-324T (AY987349)
100
Nonlabens sediminis CKA-5T (AB206954)
Gilvibacter sediminis Mok-1-36T (AB255368)
Algibacter aestuarii KYW371T (HQ405792)
73
96
Jejuia pallidilutea EM39T (EU443204)
Bacteroides fragilis ATCC 25285T (X83935)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic positions
of strain SW230T and other closely related species of the genus Dokdonia. Bootstrap values above 70 % (1000 replicates)
are shown at branch nodes. Bacteroides fragilis ATCC 25285T (GenBank accession no. X83935) was used as an
outgroup. Bar, 0.02 substitutions per nucleotide position.
nitrite reduction, urea hydrolysis, and assimilation of
L -arabinose, D -mannose, maltose, potassium gluconate,
malate and adipic acid; negative results are obtained for
lysine decarboxylase, ornithine decarboxylase, tryptophan
deaminase and arginine dihydrolase activities, citrate utilization, indole and H2S production. In API 50CH strips, acid
is produced from arabinose, galactose, glucose, mannose,
rhamnose, sorbitol, amygdalin, aesculin, melibiose and
sucrose, but not from other substrates. In API ZYM strips,
alkaline phosphatase, esterase (C4), esterase lipase (C8),
leucine arylamidase, valine arylamidase, a-chymotrypsin,
acid phosphatase and N-acetyl-b-glucosaminidase activities
are present; lipase (C14), cystine arylamidase, trypsin,
a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, naphthol-AS-BI-phosphohydrolase,
a-mannosidase and a-fucosidase activities are absent.
There are positive reactions in the Biolog GN2 MicroPlate
system for a-cyclodextrin, dextrin, cellobiose, D -fructose,
D -galactose, gentiobiose, a-D -glucose, maltose, D -mannose,
melibiose, sucrose, trehalose, turanose, methyl pyruvate,
monomethyl succinate, acetic acid, a-ketobutyric acid,
a-ketovaleric acid, propionic acid, succinic acid, L -alaninamide, L -alanine, L -glutamic acid, glycyl L -glutamic acid,
http://ijs.sgmjournals.org
hydroxy-L -proline, L -ornithine, L -proline, L -serine, L -threonine, uridine, DL -a-glycerol phosphate, glucose 1-phosphate
and glucose 6-phosphate. The dominant fatty acids (w10%
of the total) are iso-C15:1 G, iso-C15:0, iso-C17:0 3-OH and
summed feature 3 (comprising C16:1v7c and/or C16:1v6c).
The major respiratory quinone is MK-6. The major polar
lipids are phosphatidylethanolamine, two unidentified
aminolipids and two unidentified lipids.
The type strain, SW230T (5CGMCC 1.12184T5JCM
18216T), was isolated from a seawater sample collected
from the South Pacific Gyre (45u 589 S 163u 119 W). The
DNA G+C content of the type strain is 36 mol%.
Acknowledgements
This research used samples provided by the IODP. We thank all of the
crew and technical staff on the JOIDES Resolution during IODP
Expedition 329. This work was supported by projects from the
National Natural Science Foundation of China (no. 41276141), the
China Ocean Mineral Resources Research & Development Association (COMRA, no. DY125-15-R-03) and the National High Technology Research & Development Program of China (863 Programs,
no. 2012AA091605).
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Z. Zhang and others
& Camacho, M. (2013). Chryseobacterium hispalense sp. nov., a
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