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International Journal of Systematic and Evolutionary Microbiology (2011), 61, 2338–2341
Glaciecola arctica sp. nov., isolated from Arcticmarine sediment
Yan-Jiao Zhang,1 Xi-Ying Zhang,1 Zi-Hao Mi,1 Chun-Xiao Chen,1Zhao-Ming Gao,1 Xiu-Lan Chen,1 Yong Yu,2 Bo Chen2
1The State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center,
Shandong University, Jinan 250100, PR China
2SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136,
A Gram-negative, motile, psychrotolerant, oxidase- and catalase-positive bacterium, designatedBSs20135T, was isolated from Arctic marine sediment. Cells were straight or slightly curved rodsand formed circular, convex and yellowish-brown colonies. Buds and prosthecae could beproduced. The strain grew at 4–28 6C (optimum 25 6C) and with 1–5 % (w/v) NaCl (optimum2 %) and hydrolysed aesculin and DNA, but did not reduce nitrate to nitrite. Phylogenetic analysisof 16S rRNA gene sequences indicated that strain BSs20135T belonged to the genus Glaciecolaand shared 93.6–97.7 % sequence similarity with the type strains of known species of the genusGlaciecola. The major cellular fatty acids of strain BSs20135T were summed feature 3(comprising C16 : 1v7c and/or iso-C15 : 0 2-OH), C16 : 0, C17 : 1v8c and C18 : 1v7c. The genomicDNA G+C content was 40.3 mol%. Based on 16S rRNA gene sequence analysis, DNA–DNAhybridization data and phenotypic and chemotaxonomic characterization, strain BSs20135Trepresents a novel species, for which the name Glaciecola arctica sp. nov. is proposed. The typestrain is BSs20135T (5CCTCC AB 209161T 5KACC 14537T).
The genus Glaciecola, belonging to the class Gamma-
depth of 2200 m during the second Chinese National
proteobacteria, was originally proposed by
Arctic Research Expedition cruise of the Chinese icebreaker
to accommodate two Gram-negative, psychrophilic,
Xue Long into the Canada Basin in August 2003. Samples
aerobic and seawater-requiring species that were isolated
were stored in sterilized plastic bags (250 ml) and
from sea-ice cores collected from coastal areas of eastern
transported to the laboratory at 4 uC. Bacterial strains
Antarctica, i.e. Glaciecola punicea (type species) and
were isolated from sediment samples and purified as
Glaciecola pallidula. Since then, seven more species of
this genus have been isolated from diverse marine habitats:
sterilized natural Arctic seawater was used. Strain
BSs20135T was routinely cultivated in TYS broth [contain-
ing 0.5 % tryptone (Oxoid), 0.1 % yeast extract (Oxoid),
artificial seawater (2.75 % NaCl, 0.5 % MgCl2, 0.2 %
seawater G. psychrophila from Arctic sea-
MgSO4, 0.05 % CaCl2, 0.1 % KCl, 0.0001 % FeSO4 and
agar (1.5 % agar) and was stored at 280 uC in TYS broth
with 16 % (v/v) glycerol. G. chathamensis JCM 13645T
coastal surface seawater In this study, we
(from the Japan Collection of Microorganisms, Saitama,
report on a novel bacterium, designated BSs20135T, that was
Japan), G. mesophila DSM 15026T and G. polaris DSM
isolated from Arctic marine sediment.
16457T (from DSMZ, Braunschweig, Germany) and G.
psychrophila 170T (kept in our laboratory) were used as
Surface sediment samples (0–5 cm) were collected from
reference strains in some experiments. The reference strains
the Arctic Ocean (162u 319 050 W 77u 319 400 N) at a water
were routinely cultivated on TYS agar or in TYS broth at25 uC (12 uC for G. psychrophila 170T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA genesequence of strain BSs20135T is EU365479.
Genomic DNA extraction and PCR amplification and
A supplementary table, references and figure are available with the
sequencing of the 16S rRNA gene were performed as
rRNA gene sequence of strain BSs20135T was aligned
enzyme activities and biochemical characteristics were
manually with reference sequences retrieved from GenBank
detected using API ZYM, API 20 E, API 20 NE strips
(bioMe´rieux) and GN2 MicroPlates (Biolog) according to
phylogenetic tree was generated using MEGA with the
the manufacturers’ instructions except that cells for
inoculation were suspended in artificial seawater. Growth
Evolutionary distances were calculated using the model
under anaerobic conditions was determined in marine broth
2216 (Difco) for 10 days using an anaerobic chamber
resultant tree was evaluated by bootstrap analysis with 1000
An almost-complete (1497 nt) 16S rRNA gene sequence of
content was determined by the thermal denaturation
strain BSs20135T was obtained. Analysis revealed that
strain BSs20135T was affiliated with the genus Glaciecola.
Escherichia coli K-12 genomic DNA as a control. DNA–
The isolate showed the highest 16S rRNA gene sequence
DNA hybridization experiments were carried out using the
similarity to G. psychrophila 170T (97.7 %), G. mesophila
thermal denaturation and renaturation method
KMM 241T (97.4 %) and G. polaris LMG 21857T (97.1 %);
lower 16S rRNA gene sequence similarities (93.6–96.5 %)
were observed with the other members of the genus
Cellular fatty acid analysis was performed using the
Glaciecola. In the neighbour-joining tree strain
standard Microbial Identification System (MIDI) at the
BSs20135T and G. psychrophila 170T formed a coherent
Institute of Microbiology and Epidemiology, Academy of
cluster within the genus Glaciecola, which was supported
Military Medical Sciences, Beijing, PR China. The Gram
by a high bootstrap level (99 %). DNA–DNA relatedness
reaction was examined following the non-staining method
between strain BSs20135T and G. psychrophila 170T, G.
Motility was examined by light microscopy
mesophila DSM 15026T and G. polaris DSM 16457T was
(CX21; Olympus) using wet mounts. Cell morphology was
39.3, 37.5 and 28.1 %, respectively. These values were below
observed by transmission electron microscopy (JEM-
the 70 % cut-off for species discrimination, which indi-
100CX II; JEOL) after cells were negatively stained with
cated that the isolate represented a novel species in the
2 % phosphotungstic acid for 5 and 10 s. Colony morpho-
logy was observed after incubation on TYS agar at 25 uC for
7–15 days. Growth at 4, 10, 15, 20, 25, 28, 30 and 37 uC was
The DNA G+C content of strain BSs20135T was
measured in TYS broth. Growth with 0, 1, 2, 3, 4, 5, 6, 8 and
40.3 mol%, which was within the range of G+C contents
10 % (w/v) NaCl was measured in modified TYS broth.
reported for the genus Glaciecola (40–45 mol%). The
Oxidase activity was determined using commercial oxidase
major fatty acids of strain BSs20135T were summed feature
test strips (Merck). Catalase activity was detected by bubble
production in a 3 % (v/v) hydrogen peroxide solution.
16 : 1v7c and/or iso-C15 : 0 2-OH; 40.7 %),
DNase activity was tested using DNase test agar (Oxoid)
16 : 0 (24.6 %), C17 : 1v8c (10.0 %) and C18 : 1v7c (7.5 %),
which were similar to those of known members of the
prepared with artificial seawater. Hydrolysis of casein, starch
genus Glaciecola (Supplementary Table S1, available in
and Tween 80 was tested on TYS agar supplemented with
1 % (w/v) skimmed milk, 0.2 % (w/v) soluble starch or 1 %(v/v) Tween 80. Susceptibility to antibiotics was tested using
Cells of strain BSs20135T were Gram-negative, straight or
commercial filter paper discs with different antibiotics
slightly curved rods and could produce buds and
(Tianhe Micro-organism Reagent Co) on TYS agar. Other
prosthecae (Supplementary Fig. S1). The phenotypic
Fig. 1. Neighbour-joining phylogenetic treebased on 16S rRNA gene sequences showingthe phylogenetic position of strain BSs20135Tamong members of the genus Glaciecola andsome other related genera. Bootstrap values(.50 %) based on 1000 replications areshown at branch nodes. Bar, 0.01 substitu-tions per nucleotide position.
characteristics of strain BSs20135T are given in the species
Description of Glaciecola arctica sp. nov.
description and Strain BSs20135T shared somephenotypic characteristics with other members of the
Glaciecola arctica (arc9ti.ca. L. fem. adj. arctica northern,
genus Glaciecola, such as the presence of oxidase and
catalase, no growth at or above 37 uC, the requirement of
Cells are Gram-negative, straight or slightly curved rods
sodium ions for growth and the ability to produce buds
(0.4–0.861.3–4.8 mm). Motile by a single polar flagellum.
and prosthecae. However, strain BSs20135T could be
Buds and prosthecae can be formed. Colonies on TYS agar
phenotypically differentiated from other members of the
are circular, convex and smooth with entire edges. In TYS
genus Glaciecola by a combination of phenotypic char-
broth, cell aggregates are yellowish-brown. Oxidase- and
acteristics, such as range and optimal temperature for
catalase-positive. Grows at 4–28 uC (optimum 25 uC) and
growth, growth with 6 % (w/v) NaCl, nitrate reduction,
with 1–5 % (w/v) NaCl (optimum 2 % NaCl); NaCl is
hydrolysis of different substrates and carbon-source
required for growth. Hydrolyses aesculin and DNA, but
does not hydrolyse casein, Tween 80 or starch (API 20 NE).
In total, phylogenetic analysis of 16S rRNA gene sequences,
Does not reduce nitrate to nitrite (API 20 NE). Does not
phenotypic and chemotaxonomic characteristics and
produce indole, acetoin (Voges–Proskauer reaction) or
DNA–DNA relatedness indicate that strain BSs20135T
H2S (API 20 E). Produces alkaline phosphatase, leucine
should be assigned to the genus Glaciecola as a represent-
ative of a novel species, for which the name Glaciecola
naphthol-AS-BI-phosphohydrolase, esterase (C4) (weak),
esterase lipase (C8) (weak), cystine arylamidase (weak) and
Table 1. Differential characteristics of strain BSs20135T and members of the genus Glaciecola
Strains: 1, Glaciecola arctica sp. nov. BSs20135T; 2, G. psychrophila 170T; 3, G. mesophila DSM 15026T; 4, G. polaris DSM 16457T (data for columns1–4 were taken from this study); 5, G. chathamensis JCM 13645T 6, G. agarilytica LMG 23762T 7, G.
lipolytica JCM 15139T 8, G. nitratireducens FR 1064T 9, G. pallidula ACAM 615T 10, G.
punicea ACAM 611T +, Positive; 2, negative; ND, no data available.
*Data from DData from dData from this study.
International Journal of Systematic and Evolutionary Microbiology 61
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