Download Glaciecola psychrophila sp. nov., a novel psychrophilic bacterium

International Journal of Systematic and Evolutionary Microbiology (2006), 56, 2867–2869
DOI 10.1099/ijs.0.64575-0
Glaciecola psychrophila sp. nov., a novel
psychrophilic bacterium isolated from the Arctic
De-Chao Zhang,1,2 Yong Yu,3 Bo Chen,3 He-Xiang Wang,1 Hong-Can Liu,2
Xiu-Zhu Dong2 and Pei-Jin Zhou2
Correspondence
Pei-Jin Zhou
[email protected]
1
State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural
University, Beijing 100094, China
2
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100080, China
3
SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136,
China
A novel bacterial strain, designated 170T, was collected from high latitude Arctic locations (776 309 N
to approximately 816 129 N), including the Canadian Basin and Greenland Sea. Phylogenetic
analysis based on 16S rRNA gene sequence comparisons showed that strain 170T was related to
members of the genus Glaciecola and had the highest 16S rRNA gene sequence similarity to
Glaciecola mesophila. Cells were Gram-negative, psychrophilic, motile rods. The temperature
range for growth was 4–15 6C, with optimum growth at 12 6C and at approximately pH 6?0–9?0.
Strain 170T contained C16 : 1v7c, C16 : 0, C12 : 1 3-OH and C18 : 1v7c as major fatty acids. The
genomic DNA G+C content was 42?9 mol%. On the basis of phenotypic characterization,
phylogenetic analysis and DNA–DNA relatedness data, strain 170T is considered to represent a
novel species of the genus Glaciecola, for which the name Glaciecola psychrophila is proposed.
The type strain is 170T (=CGMCC 1.6130T=JCM 13954T).
The genus Glaciecola was proposed by Bowman et al. (1998)
to accommodate Gram-negative, aerobic, psychrophilic,
pigmented and seawater-requiring bacteria. At the time of
writing, the genus comprised four recognized species:
Glaciecola punicea and Glaciecola pallidula (Bowman et al.,
1998), Glaciecola mesophila (Romanenko et al., 2003) and
Glaciecola polaris (Van Trappen et al., 2004). Members of
the genus Glaciecola have been isolated from sea-ice samples
collected from coastal areas of eastern Antarctica, marine
invertebrate specimens and polar seawater. On the basis of
the polyphasic evidence presented herein, a bacterial Arctic
strain, designated 170T, is considered to represent a novel
species of the genus Glaciecola.
Strain 170T was isolated from sea-ice samples collected using a
MARK II ice auger during the Second Chinese National Arctic
Research Expedition cruise of the USCGC icebreaker Xue
Long into the Canada Basin in August 2003. Sea-ice samples
were cut carefully into 10 to 20 cm sections using a sterile saw
and placed in sterile plastic bottles to be melted at 4 uC. The
meltwater was then spread onto marine agar 2216 (MA;
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain 170T is DQ007436.
A table showing the fatty acid profile of strain 170T and other
Glaciecola species is available as supplementary material in IJSEM
Online.
64575 G 2006 IUMS
Difco) plates and incubated at 4 uC for 2–6 weeks. Strain 170T
was obtained in pure culture after three successive transfers to
fresh agar medium and stored at 280 uC in 30 % (v/v)
glycerol. G. mesophila DSM 15026T was kindly provided by Dr
E. Stackebrandt for reference. Cultures of both strains were
routinely grown in marine broth 2216 (MB; Difco).
Cell morphology was examined under a light microscope
(BH-2; Olympus). Colony morphology was observed on
MA after incubation at 12 uC for 4–5 days. Growth
temperature was determined with a TN3F temperaturegradient incubator (Advantec). The pH range for growth
was determined for the culture in MB at various pH values
adjusted with HCl or NaOH (1 mol l21). General physiological tests were performed using conventional methods
(Dong & Cai, 2001). Biochemical traits were determined
using API kits (API 20 E, API 20 NE, API ZYM;
bioMérieux). Acid production from carbohydrates was
determined as described by Leifson (1963).
DNA was extracted and purified as described by Sambrook
et al. (1989). The gene encoding 16S rRNA was amplified by
PCR with forward primer 59-AGAGTTTGATCCTGGCTCAG-39 and reverse primer 59-AAGGAGGTGATCCAGCCGCA-39 (Liu et al., 2000). The purified PCR product
was ligated to the plasmid pMD 18-T (TaKaRa) and cloned
according to the manufacturer’s instructions. Sequencing
reactions were carried out using an ABI BigDye 3.1 sequencing
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D.-C. Zhang and others
kit (Applied Biosystems) and an automated DNA sequencer
(model ABI3730; Applied Biosystems). The 16S rRNA gene
sequence of strain 170T was submitted to GenBank and EMBL
to search for similar sequences revealed using the BLAST
algorithm. A phylogenetic tree was constructed using
Kimura’s two-parameter and pairwise-deletion model analysis implemented in the MEGA version 3.0 program (Kumar
et al., 2004). The resultant tree topologies were evaluated by
bootstrap analysis based on 1000 replicates.
The G+C content of the genomic DNA was determined by
thermal denaturation with Escherichia coli K-12 as the
reference strain. DNA–DNA hybridization experiments
were carried out by using the liquid renaturation method
(De Ley et al., 1970) as modified by Huß et al. (1983). Both
experiments were carried out using a DU800 spectrophotometer (Beckman).
Cellular fatty acids were determined for a culture grown on
MA at 12 uC for 4 days and were extracted, methylated and
analysed using the standard MIDI (Microbial Identification)
system (Sasser, 1990).
formed a distinct cluster with G. mesophila DSM 15026T
(96?3 % sequence similarity) and G. polaris LMG 21857T
(96?0 %). The level of DNA–DNA relatedness between strain
170T and G. mesophila DSM 15026T was 44?9 %. The DNA
G+C content of strain 170T was 42?9 mol%.
The predominant cellular fatty acids of strain 170T were
C16 : 1v7c (38?31 %), C16 : 0 (13?59 %), C12 : 1 3-OH (6?47 %),
C18 : 1v7c (6?23 %), C14 : 0 (5?48 %) and C17 : 1v8c (2?73 %).
The fatty acid profile resembled those determined for other
Glaciecola species (Bowman et al., 1998; Romanenko et al.,
2003; Van Trappen et al., 2004) except that C12 : 1 3-OH has
not been found in the other Glaciecola species. The fatty acid
profiles of strain 170T and other Glaciecola species are given
in Supplementary Table S1 in IJSEM Online.
Strain 170T could be distinguished from the type strains of
other Glaciecola species by a combination of physiological
and biochemical properties (Table 1). Based on these
results, it is concluded that strain 170T represents a novel
species of the genus Glaciecola, for which the name
Glaciecola psychrophila sp. nov. is proposed.
Cells of strain 170T were rod-shaped, Gram-negative and
motile. Colonies on MA were white, smooth, circular and
convex with entire margins. Strain 170T grew aerobically;
the optimal growth temperature was 12 uC and growth
occurred at 4–15 uC.
Glaciecola psychrophila (psy.chro9phi.la. Gr. adj. psychros
cold; Gr. adj. philus loving; N.L. fem. adj. psychrophila cold
loving).
The nearly complete 16S rRNA gene of strain 170T
(1509 bp) was PCR amplified and sequenced. Phylogenetic analysis (Fig. 1) based on a consensus 1340-bp
length of 16S rRNA gene sequences showed that strain 170T
was grouped with members of the genus Glaciecola and
Cells are Gram-negative, psychrophilic, motile rods,
0?5–0?8 mm by 1?2–4?5 mm in size. Colonies are nonpigmented, convex, circular and smooth with entire edges.
Grows aerobically and produces catalase and cytochrome
oxidase. Growth occurs at 4–15 uC and pH 5?0–10?0, with
Description of Glaciecola psychrophila sp. nov.
Fig. 1. Phylogenetic tree showing the position of strain 170T and related species based on 16S rRNA gene sequence
analysis. The tree was constructed by using the neighbour-joining method. Numbers at nodes represent percentage bootstrap
support based on a neighbour-joining analysis of 1000 resampled datasets. GenBank accession numbers are given in
parentheses. Bar, 1 % sequence divergence.
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Glaciecola psychrophila sp. nov.
Table 1. Phenotypic characteristics that differentiate strain
170T from other Glaciecola species
Taxa: 1, strain 170T; 2, G. mesophila (data from Romanenko et al.,
2003); 3, G. polaris (Van Trappen et al., 2004); 4, G. punicea
(Bowman et al., 1998); 5, G. pallidula (Bowman et al., 1998). +,
Positive; 2, negative; W, weakly positive.
Characteristic
1
Pigmentation
2
Growth at 0–4 uC
+
Growth at 18 uC
2
Growth in 10 % NaCl
2
Hydrolysis of:
Aesculin
2
Egg yolk
2
Starch
+
Tween 80
+
Urea
2
Utilization of:
Acetate
2
Cellobiose
2
Fructose
2
Galactose
+
Glucose
2
Glutamate
W
Glycerol
2
Lactate
2
Maltose
+
Mannitol
2
D-Mannose
2
Propionate
2
Sucrose
+
Trehalose
2
Enzyme activity (API ZYM):
a-Galactosidase
2
b-Galactosidase
+
DNA G+C content (mol%) 42?9
2
3
2
2
+
2
2 Pink–red Pale pink
2
+
+
+
+
+
+
2
2
W
2
+
+
2
+
+
+
+
2
+
2
2
2
W
2
2
+
+
2
2
+
+
+
+
+
2
2
+
+
+
2
+
+
+
+
+
+
+
+
2
2
+
+
+
+
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
2
2
2
+
+
+
2
2
2
2
2
2
+ W
+ +
44 44
4
+
+
44–46
5
+
2
40
optimum growth at 12 uC and at approximately pH 6?0–9?0.
Growth occurs in the presence of 1–6 % (w/v) NaCl and no
growth occurs in the absence of NaCl. Indole production
and Voges–Proskauer reaction are negative. Negative results
for the reduction of nitrate and production of hydrogen
sulfide. Negative in tests for arginine dihydrolase, lysine
decarboxylase, ornithine decarboxylase, urease, gelatinase,
caseinase, agarase, chitinase, lecithinase, esterase (C4),
esterase lipase (C8), acid phosphatase, b-glucuronidase,
b-glucosidase, a-glucosidase, N-acetyl-b-glucosaminidase,
a-mannosidase and a-fucosidase. Weak enzymic activity is
observed for trypsin, lipase (C14) and cystine arylamidase.
Tests for naphthol-AS-BI-phosphohydrolase, a-chymotrypsin, amylase, alkaline phosphatase, leucine arylamidase,
valine arylamidase and b-galactosidase are positive. The
following substrates are utilized as sole carbon sources:
sucrose, maltose, lactose, galactose, gluconate and pyruvate.
Acid is weakly produced from maltose and galactose. The
following substrates are not utilized as sole carbon sources:
http://ijs.sgmjournals.org
glucose, D-mannose, mannitol, fructose, cellobiose, Larabinose, xylose, L-rhamnose, D-melibiose, raffinose, Dsorbitol, sorbinose, glycerol, melezitose, ribose, galactitol,
inositol, erythritol, salicin, inulin, valine, glycine, cysteine,
arginine, histidine, lysine, methionine, N-acetylglucosamine,
acetate, fumarate, hippurate, lactate, malate, citrate, succinate, tartrate, alginate, capric acid, adipic acid, phenylacetic
acid and uric acid. The predominant cellular fatty acids are
C16 : 1v7c (38?31 %), C16 : 0 (13?59 %), C12 : 1 3-OH (6?47 %),
C18 : 1v7c (6?23 %), C14 : 0 (5?48 %) and C17 : 1v8c (2?73 %).
The G+C content of the DNA is 42?9 mol%.
The type strain, 170T (=CGMCC 1.6130T=JCM 13954T),
was collected from high latitude Arctic locations (77u 309 N
to approximately 81u 129 N), including the Canadian Basin
and Greenland Sea.
Acknowledgements
We are grateful to Dr Erko Stackebrandt for providing the type strain of
G. mesophila. This work was supported by the National Basic Research
Program of China (2004CB719601) and the National Natural Science
Foundation of China (30500001).
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