International Journal of Systematic and Evolutionary Microbiology (2015), 65, 2310 – 2314
DOI 10.1099/ijs.0.000260
Draconibacterium sediminis sp. nov., isolated from
river sediment
Juan Du,3 Qiliang Lai,3 Yang Liu, Chunming Dong, Yanrong Xie and
Zongze Shao
Correspondence
Zongze Shao
[email protected]
State Key Laboratory Breeding Base of Marine Genetic Resources; Key Laboratory of Marine
Genetic Resources The Third Institute of State Oceanic Administration; Collaborative Innovation
Center of Marine Biological Resources; Key Laboratory of Marine Genetic Resources of Fujian
Province, Xiamen 361005, PR China
A Gram-reaction-negative, facultatively anaerobic and rod-shaped bacterium, designated strain
JN14CK-3T, was isolated from surface sediment of the Jiulong River of China and was
characterized phenotypically and phylogenetically. Phylogenetic analysis of the 16S rRNA gene
sequences indicated that strain JN14CK-3T belonged to the genus Draconibacterium, with the
highest sequence similarity (98.3 %) to Draconibacterium orientale FH5T. By contrast, strain
JN14CK-3T shared low 16S rRNA gene sequence similarities (,91.0 %) with other type strains.
The sole respiratory quinone was MK-7.The polar lipids were phosphatidylethanolamine and
several unidentified phospholipids and lipids. The major fatty acids were iso-C15:0, iso-C16:0,
anteiso-C15:0, C17:0 2-OH, iso-C16:0 3-OH and iso-C17:0 3-OH. The G+C content of the
genomic DNA was 40.9 mol%. The digital DNA–DNA hybridization value and average
nucleotide identity (ANI) between strain JN14CK-3T and D. orientale FH5T were 34.2¡2.5 %
and 87.1 %, respectively. The combined genotypic and phenotypic data showed that strain
JN14CK-3T represents a novel species of the genus Draconibacterium, for which the name
Draconibacterium sediminis sp. nov. is proposed, with the type strain JN14CK-3T (5MCCC
1A00734T5KCTC 42152T).
To investigate survivability of sediment bacteria of the
Jiulong River (Fujian, China) in the marine environment,
a number of strains were recovered. One of the strains,
designated JN14CK-3T, was characterized taxonomically.
16S rRNA gene sequence analysis indicated that strain
JN14CK-3T belonged to the genus Draconibacterium. The
genus Draconibacterium, which was proposed by Du et al.
(2014), belongs to the family Prolixibacteraceae (Iino
et al., 2014). The aim of this work was to determine the
exact taxonomic position of strain JN14CK-3T by using a
polyphasic approach.
Surface sediment was sampled from the Jiulong River
(24.578 N 117.538 E) of Nanjing County (Fujian, China)
3 Juan Du and Qiliang Lai contributed equally to this work.
Abbreviations: ANI, average nucleotide identity; dDDH, digital DNA–
DNA hybridization; L, unknown lipids; PE, phosphatidylethanolamine;
PL, unidentified phospholipids.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of Draconibacterium sediminis JN14CK-3T is KM255689.
The accession number for the draft genome sequence of strain
JN14CK-3T is JRHC00000000.
Four supplementary figures and one supplementary table are available
with the online Supplementary Material.
2310
in June 2013. In order to assess survivability of sediment
bacteria in the marine environment, surface sediment was
placed in a cylindrical container. Two sides of the container
were coated with 0.22 mm pore size filter membranes,
which only allow exchange of water and nutrients. Subsequently, this container was kept in coastal seawater
from Xiamen for 7 days. Finally, surface sediment was
removed from the container and used to isolate bacteria
by the standard dilution plating technique on modified
R2A medium (BD Difco) with aged seawater (2.7 %
NaCl). After incubation at 28 8C for 7 days under aerobic
conditions, individual colonies were picked off and purified by successive streaking on marine agar 2216 (MA;
BD Difco). Unless otherwise stated, MA medium was
used for routine bacterial cultivation and most of the
phenotypic tests.
Genomic DNA was extracted using the AxyPrep Bacterial
Genomic DNA Miniprep kit (Axygen Biosciences) according to the manufacturer’s instructions. The 16S rRNA gene
was amplified using previously described primers (Liu &
Shao, 2005). Sequence similarity was determined using
the EzTaxon-e database (Kim et al., 2012). Phylogenetic
trees based on 16S rRNA gene sequences were reconstructed using MEGA version 5.0 (Tamura et al., 2011).
Distance options according to the Kimura two-parameter
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Draconibacterium sediminis sp. nov.
model and clustering with the neighbour-joining (Saitou &
Nei, 1987), maximum-likelihood (Felsenstein, 1981) and
minimum-evolution (Rzhetsky & Nei, 1992) methods was
performed using MEGA version 5.0. Bootstrap analysis
based on 1000 replications was used to estimate the confidence level of tree topologies.
A nearly complete 16S rRNA gene sequence of strain
JN14CK-3T was determined (1481 bp). A neighbourjoining phylogenetic tree of the 16S rRNA gene sequences
of all members of seven genera within the family Prolixibacteraceae was reconstructed. As shown in Fig. 1, strain
JN14CK-3T and Draconibacterium orientale FH5T formed
an independent monophyletic cluster with a high level of
similarity, 98.3 %. Sequence similarity values between
strain JN14CK-3T and other type strains were less than
91.0 %. A similar topology was retrieved in the maximum-likelihood tree and the minimum-evolution tree
(Figs S1 and S2, available in the online Supplementary
Material). The high similarity of the 16S rRNA gene
sequence to that of D. orientale FH5T confirmed that
strain JN14CK-3T belonged to the genus Draconibacterium.
The draft genome sequence of strain JN14CK-3T was
sequenced at Shanghai Majorbio Bio-pharm Technology
(Shanghai, China) using Solexa paired-end (500 bp library)
sequencing technology. The draft genome sequence of
strain JN14CK-3T was deposited into NCBI and assigned
accession number JRHC00000000. The complete genome
sequence of D. orientale FH5T was obtained from NCBI
(CP007451.1). The genomic DNA G+C content of strain
JN14CK-3T was 40.9 mol% according to its draft genome
sequence, which was similar to that of D. orientale FH5T
(41.3 mol%, according to its complete genome sequence).
The digital DNA–DNA hybridization (dDDH) value was
calculated using the genome-to-genome distance calculator
57
100
98
53
Gram reaction, catalase, oxidase and lipase (Tween 80)
activities, hydrolysis of DNA, protein, starch and casein
and other cell morphology properties were determined
according to previously established procedures (Lai et al.,
2009). Cell morphology, size and presence of flagella were
determined using transmission electron microscopy
(TEM-1230, JEOL). For transmission electron microscopy,
exponential-phase cells were harvested, suspended and
absorbed on a Formvar/carbon-coated grid, then negatively
stained with 1 % (w/v) phosphotungstic acid (Tindall et al.,
2007). Motility was observed by using the hanging-drop
method described by Skerman (1967). Growth under
anaerobic conditions was performed in an anaerobic
chamber (Anoxomat Mark II, Mart Microbiology BV;
N2:H2:O2, 89.9:9.9:0.2) on MA at 30 8C for 2 weeks. The
temperature ranges for growth were determined in
marine broth 2216 (MB; Difco) incubated at 25–45 8C
for 7 days and at 4–20 8C for 3 weeks. Tolerance to NaCl
was evaluated in an artificial marine broth medium according to the formula of MB except for the modification of
NaCl concentration to be 0, 0.5, 1, 2, 3, 5, 7, 9, 12, 15 or
18 % (w/v) (Sambrook & Russell, 2001). The pH ranges
for growth was evaluated in MB adjusted to pH 2.0–11.0,
at 1 pH unit intervals, with citrate/phosphate (pH 2.0–
‘Sunxiuqinia dokdonensis’ DH1 (KF646787)
99
0.02
(GGDC 2.0) (Meier-Kolthoff et al., 2013) and average
nucleotide identity (ANI) was calculated using the
algorithm of Goris et al. (2007) using the EzGenome web
service. The dDDH and ANI values between strain
JN14CK-3T and D. orientale FH5T were 34.2¡2.5 % and
87.1 %, respectively. Both values were far below the cutoff values recommended for bacterial species delineation
(Thompson et al., 2013; Wayne et al., 1987). These results
confirmed that strain JN14CK-3T was a representative of a
novel species of the genus Draconibacterium.
Sunxiuqinia faeciviva JAM-BA0302T (AB362263)
Sunxiuginia rutila HG677T (AB851476)
Sunxiuqinia elliptica DQHS4T (GQ200190)
Mangrovibacterium diazotrophicum SCSIO NO430T (JX983191)
Meniscus glaucopis ATCC 29398T (GU269545)
52
Prolixibacter bellariivorans JCM 13498T (BALJ01000075)
100
Draconibacterium sediminis JN14CK-3T (KIVI255689)
Draconibacterium orientate FH5T (CP007451)
Mariniphaga anaerophila Full-5T (AB921558)
100
98
Tangfeifania diversioriginum G22T (JQ683777)
Cytophaga xylanolytica DSM 6779T (FR733683)
Fig. 1. Neighbour-joining phylogenetic tree of 16S rRNA gene sequences showing the position of strain JN14CK-3T with all
members of seven genera within the family Prolixibacteraceae. Nodes indicated by solid circles were recovered by three
algorithms (neighbour-joining, minimum-evolution and maximum-likelihood methods). Cytophaga xylanolytica DSM 6779T
(FR733683) was used as the outgroup. Bootstrap confidence values (.50%) based on 1000 replicates are shown at branch
nodes. Bar, 0.02 nucleotide substitution rate (Knuc) units.
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J. Du and others
Table 1. Differential physiological characteristics of strain JN14CK-3T and D. orientale FH5T
Tests for hydrolysis of CM-cellulose, starch, casein, gelatin and DNA, tolerance of temperature, NaCl and pH, and those using the API 20E, API
20NE and API ZYM strips were performed in this study. Both strains were Gram-reaction-negative, facultatively anaerobic, non-motile and positive
for hydrolysis of aesculin, but negative for H2S production, and hydrolysis of CM-cellulose, starch, casein, gelatin and DNA. The temperature range
for growth of both strains was 15–408C. In API ZYM tests, the two strains were positive for alkaline phosphatase, esterase (C4), leucine arylamidase,
trypsin, acid phosphatase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase and a-fucosidase; weakly positive for naphthol-AS-BI-phosphoamidase; negative for b-glucuronidase and a-mannosidase. +, Positive; W , weakly positive; 2, negative; PE, phosphatidylethanolamine; PL, unidentified phospholipid; AL, aminolipid; L, unknown lipid.
Characteristic
Cell morphology
Temperature optimum (8C)
NaCl range for growth (%, w/v)
NaCl optimum (%, w/v)
pH range for growth
pH optimum
Nitrate reduced
Acid production from D -glucose
Oxidase
Catalase
API ZYM results
Esterase lipase (C8)
Lipase (C14)
Valine arylamidase
Cystine arylamidase
a-Chymotrypsin
Polar lipids
DNA G+C content (mol%)
JN14CK-3T
FH5T
Slightly curved rods
30–35
2.0–7.0
3.0–5.0
5.0–8.0
7.0
+
Straight to slightly curved rods
28–32
2.0–5.0
2.0–3.0
5.0–9.0
7.0–7.5
2
+
+
+
W
W
W
+
+
W
W
PE, PL, L
40.9
7.0), Tris/HCl (pH 8.0–9.0) or sodium carbonate/sodium
bicarbonate (pH 10.0–11.0) buffers. Other biochemical
tests were carried out using API 20E, API 20NE and API
ZYM systems (bioMérieux) according to the manufacturer’s instructions, with the single modification of adjusting the NaCl concentration to 3.0 % in all tests. Type strain
D. orientale FH5T was a generous gift from Dr Zongjun Du
and was used as a reference strain in this study. Cells of
strain JN14CK-3T were Gram-reaction-negative, nonmotile, non-pigmented, slightly curved rods, 0.6–0.8 mm
wide, 2.9–3.4 mm long (Fig. S3). The physiological and biochemical characteristics of strain JN14CK-3T are given in
Table 1 and the species description.
For analysis of fatty acids, respiratory quinones and polar
lipids, cell mass of strains JN14CK-3T and FH5T were harvested after incubation in MB at 30 8C for 48 h. Fatty acids
of whole cells were saponified, methylated, extracted and
analysed according to the standard protocol described in
the MIDI (Microbial Identification) system (Sasser,
1990). The fatty acid profile of strain JN14CK-3T and
D. orientale FH5T are given in Table S1. The major fatty
acids (.5 % of total fatty acids) of strain JN14CK-3T
were iso-C15:0, iso-C16:0, anteiso-C15:0, C17:0 2-OH,
iso-C16:0 3-OH and iso-C17:0 3-OH. The amounts of anteiso-C15:0 and iso-C15:0 in strain JN14CK-3T were 14.96 %
and 25.66 %; but in D. orientale FH5T these fatty acids
2312
W
W
2
2
2
2
PE, PL, AL, L
41.3
represented 28.27 % and 20.86 %, respectively. As the
fatty acid profiles of strain JN14CK-3T and D. orientale
FH5T were determined under the same conditions, strain
JN14CK-3T could be distinguished from D. orientale
FH5T on the basis of the differences in proportion.
Determination of respiratory quinone was performed by
the identification service of the Zhejiang University according to the methods described by Collins (1985). The novel
isolate contained menaquinone MK-7 (100 %) as the only
quinone, which was in accordance with D. orientale FH5T.
Determination of the polar lipids was performed by the
identification service of the Jiangsu University according to
the methods described by Kates (1972). Strain JN14CK-3T
contained phosphatidylethanolamine (PE), several unidentified phospholipids (PL) and two unknown lipids (L)
(Fig. S4). This profile was similar to that of D. orientale
FH5T in the presence of PE, PL and L. In addition,
strain JN14CK-3T could be obviously distinguished from
D. orientale FH5T by the absence of aminolipid.
The high similarity of the 16S rRNA gene sequences
between strain JN14CK-3T and D. orientale FH5T
confirmed that strain JN14CK-3T belonged to the genus
Draconibacterium. However, strain JN14CK-3T could be
differentiated from D. orientale FH5T by low values of
dDDH and ANI. The differential physiological, biochemical
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Draconibacterium sediminis sp. nov.
and chemotaxonomic characteristics between strain JN14CK3T and D. orientale FH5T are given in Table 1. On the basis of
the data described above, strain JN14CK-3T should be placed
in a novel species of the genus Draconibacterium, for which the
name Draconibacterium sediminis sp. nov. is proposed.
Dr Zongjun Du for kindly providing the type strain Draconibacterium
orientale FH5T.
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This work was supported financially by the High-Tech Research and
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