International Journal of Systematic and Evolutionary Microbiology (2009), 59, 2773–2777
DOI 10.1099/ijs.0.007906-0
Flavobacterium tiangeerense sp. nov., a cold-living
bacterium isolated from a glacier
Yu-Hua Xin,1 Zhi-Hong Liang,2 De-Chao Zhang,1 Hong-Can Liu,1
Jian-Li Zhang,3 Yong Yu,4 Ming-Shuang Xu,2 Pei-Jin Zhou1
and Yu-Guang Zhou1
Correspondence
Yu-Guang Zhou
[email protected]
1
China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese
Academy of Sciences, Beijing 100101, PR China
2
China Agricultural University, Beijing 100083, PR China
3
School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, PR China
4
SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136,
PR China
A novel cold-living, Gram-stain-negative, yellow-pigmented and obligately aerobic bacterium
designated strain 0563T was isolated from the China No. 1 glacier. Phylogenetic analysis showed
that strain 0563T was a member of the genus Flavobacterium, sharing the highest 16S rRNA gene
sequence similarities with Flavobacterium limicola JCM 11473T (98.5 %) and Flavobacterium
psychrolimnae NBRC 102679T (98.2 %). The genomic DNA G+C content was 34.8 mol%. Strain
0563T displayed the common phenotypic and chemotaxonomic features of the genus
Flavobacterium, containing menaquinone-6 (MK-6) as the major quinone and iso-C15 : 0, iso-C15 : 0
3-OH, summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c), C15 : 1v6c, iso-C15 : 1 G
and iso-C16 : 0 3-OH as the major fatty acids. On the basis of phenotypic characteristics,
phylogenetic analysis and DNA–DNA relatedness data, the novel species Flavobacterium
tiangeerense sp. nov. is proposed; the type strain is 0563T (5CGMCC 1.6847T 5JCM 15087T).
The genus Flavobacterium was proposed by Bergey et al.
(1923). Following the emendation of its description
(Bernardet et al., 1996), it currently accommodates 49
Gram-stain-negative, non-spore-forming, yellow-pigmented and rod-shaped bacteria, usually motile by gliding,
containing menaquinone-6 (MK-6) as the major respiratory
quinone, with a DNA G+C content range of 30–41 mol%
(Bernardet & Bowman, 2006; Qu et al., 2008). The genus
Flavobacterium belongs to the family Flavobacteriaceae
in the phylum Bacteroidetes (previously, the Cytophaga–
Flavobacterium–Bacteroides group) (Bernardet et al., 2002).
Members of the genus Flavobacterium have been isolated
from diverse habitats, such as diseased fish, freshwater and
river sediments, seawater and marine sediments, soil,
glaciers and Antarctic lakes. A number of Flavobacterium
species isolated from glaciers, sea ice and Antarctic lakes are
cold-adapted.
During the investigation of psychrotrophic organisms from
the China No.1 glacier located in the Xinjiang Uygur
Autonomous Region, north-west China, a novel psychrotrophic bacterial strain was isolated.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of Flavobacterium tiangeerense 0563T is EU036219.
007906 G 2009 IUMS
Strain 0563T was isolated from frozen soil collected from
the China No.1 glacier using previously described media
and methods (Zhu et al., 2003). The strain was routinely
grown aerobically at 23 uC on PYG agar (Zhang et al.,
2006). Flavobacterium limicola JCM 11473T and
Flavobacterium psychrolimnae NBRC 102679T were
obtained from the corresponding culture collections,
cultivated as recommended (Tamaki et al., 2003; Van
Trappen et al., 2005) and used as reference strains for fatty
acid analysis, DNA–DNA hybridization experiments and
phenotypic tests.
DNA was extracted according to the method of Marmur
(1961). For PCR amplification of the 16S rRNA gene the
following primer set was used: 27f (59-AGAGTTTGATCCTGGCTCAG-39) and 1492r (59-TACGGCTACCTTGTTACGACTT-39) (Lane, 1991). The PCR product was
sequenced using the ABI BigDye3.1 Sequencing kit
(Applied Biosystems) and an automated DNA sequencer
(model ABI 3730; Applied Biosystems). BLASTN searches
with the nearly complete (1427 bp) 16S rRNA gene
sequence of strain 0563T, performed in GenBank and
EMBL, revealed that the new isolate shared high sequence
similarity (~98.4 %) with members of the genus
Flavobacterium. Phylogenetic trees were constructed
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2773
Y.-H. Xin and others
according to the neighbour-joining (Saitou & Nei, 1987),
maximum-likelihood (Felsenstein, 1981) and maximumparsimony algorithms with Kimura’s two-parameter calculation model (Kimura, 1980), using the software package
MEGA version 3.0 (Kumar et al., 2004). The same results
were obtained using these three methods. The tree
topology and distances were determined with bootstrap
analysis based on 1000 resamplings. Analysis of the almostcomplete 16S rRNA gene sequence (Fig. 1) showed that
strain 0563T was a member of the genus Flavobacterium
and formed a distinct cluster with F. limicola JCM 11473T
(98.5 %) and F. psychrolimnae NBRC 102679T (98.2 %).
Sequence similarity of strain 0563T with other
Flavobacterium species was 93.6–98.5 %.
The DNA G+C content of strain 0563T was determined
using the thermal denaturation method (Marmur & Doty,
1962) with Escherichia coli K-12 as a reference. DNA–DNA
hybridization experiments were performed using the liquid
renaturation method (De Ley et al., 1970). The tests were
performed using a model Lambda 35 UV/VIS spectrometer
equipped with a temperature program controller (Perkin–
Elmer). The DNA G+C content of strain 0563T was
34.8 mol%. The DNA–DNA relatedness between strain
0563T and F. limicola JCM 11473T and F. psychrolimnae
NBRC 102679T was 40.4 and 32.2 %, respectively, demonstrating that strain 0563T could not be attributed to
these species.
The following tests were performed on strain 0563T only.
Colony morphology was observed on PYG agar after
incubation at 23 uC for 48 h. The presence of gliding
motility was determined using the hanging drop technique
(Bernardet et al., 2002). The Gram reaction was performed
according to the method described by Gerhardt et al. (1994).
Growth was assessed on laboratory-prepared nutrient,
trypticase soy and marine 2216 agars. Growth was assessed
under microaerobic and anaerobic conditions using the
Oxoid atmosphere generation system. The temperature
range for growth was assessed from 4 to 37 uC (at 1 uC
intervals) with a TN3F temperature-gradient incubator
(Advantec). Oxidase activity was tested by determining the
oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine
(Merck) and catalase activity was evaluated by determining
the production of oxygen bubbles in a 3 % (v/v) aqueous
hydrogen peroxide solution.
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationship of strain 0563T and
representative Flavobacterium species. The tree was rooted with the type strain of Leeuwenhoekiella marinoflava. Numbers at
nodes represent bootstrap values (percentages based on 1000 resampled datasets) higher than 50 %. GenBank accession
numbers of 16S rRNA gene sequences are given in parentheses. Bar, 0.01 % sequence divergence.
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Flavobacterium tiangeerense sp. nov.
The following tests were performed in parallel on strains
0563T, F. limicola JCM 11473T and F. psychrolimnae NBRC
102679T. The pH range for growth was determined in PYG
broth adjusted to pH 5.0, 6.0, 6.5, 7.0, 8.0, 9.0 and 10.0
with HCl or NaOH (15 mol l21). The pH was verified after
autoclaving. Salt tolerance was tested using PYG broth
supplemented with 0, 0.5, 1, 1.5 and 2.0 % (w/v) NaCl for
3 days at 23 uC. The hydrolysis of casein, gelatin, Tweens
20 and 80, aesculin, urea, alginate, chitin, pectin, DNA,
starch, tyrosine and carboxylmethylcellulose was investigated on PYG agar after 7 days of incubation, according to
previously described methods (Smibert & Krieg, 1981;
Reichenbach, 1992). The production of a precipitate on egg
yolk agar was assessed on PYG agar supplemented with
50 % egg yolk emulsion. Strains were inoculated and then
incubated for 7 days. Additional enzyme activities and
biochemical features were tested using the API 20NE, API
20E and API ZYM systems (bioMérieux) according to the
manufacturer’s instructions. Growth with carbon sources
was tested using Gram-negative MicroPlates (Biolog)
according to the manufacturer’s instructions. The results
of API ZYM tests were read after 6 h of incubation and the
results of API 20NE, API 20E and GN MicroPlate tests were
read after 48 h. All systems were incubated at 23 uC.
Congo-red adsorption and the production of flexirubintype pigments were assessed according to Bernardet et al.
(2002). Acid production from carbohydrates was determined as described by Hugh & Leifson (1953). The
morphological, cultural, physiological and biochemical
characteristics of strain 0563T are given in the species
description and in Table 1.
Respiratory quinones were extracted and purified according to Collins (1985) and analysed by HPLC (Wu et al.,
1989), using MK-6 from F. limicola JCM 11473T as a
reference. The cellular fatty acid compositions of strain
0563T, F. limicola JCM 11473T and F. psychrolimnae NBRC
102679T were determined from cultures grown on PYG
agar at 23 uC for 3 days. Fatty acids were extracted,
methylated and analysed using the standard MIDI
(Microbial Identification, Sherlock version 6.0) procedure
(Sasser, 1990). Cells of strain 0563T contained MK-6 as the
major respiratory quinone, in line with all members of the
family Flavobacteriaceae (Bernardet & Bowman, 2006). The
predominant cellular fatty acids of strain 0563T were isoC15 : 0 (26.9 %), iso-C15 : 0 3-OH (10.7 %), summed feature
3 (comprising C16 : 1v7c and/or C16 : 1v6c, 9.3 %), C15 : 1v6c
(9.1 %), iso-C15 : 1 G (5.4 %) and iso-C16 : 0 3-OH (5.1 %).
The detailed fatty acid compositions of strain 0563T, F.
limicola JCM 11473T and F. psychrolimnae NBRC 102679T
grown under the same conditions are compared in Table 2.
Overall, the fatty acid composition of strain 0563T
conformed to those of its closest relatives although it
contained larger proportions of summed feature 4, isoC15 : 0 3-OH and iso-C15 : 0 and smaller proportions of
C17 : 1v6c and anteiso-C15 : 0.
Based on phenotypic and molecular data, it is concluded
that strain 0563T represents a novel species of the genus
http://ijs.sgmjournals.org
Table 1. Differential characteristics between strain 0563T and
its closest phylogenetic relatives
Strains: 1, F. tiangeerense sp. nov. 0563T; 2, F. limicola JCM 11473T; 3,
F. psychrolimnae NBRC 102679T. +, Positive; 2, negative; (+)
weakly positive. All data from this study.
Characteristic
1
2
3
Growth at pH 6.0
Growth at pH 10.0
Growth with 1.0 % NaCl
Enzyme activities
N-Acetyl-b-glucosaminidase
Esterase (C4)
a-Chymotrypsin
Utilization of:
a-Cyclodextrin
Tween 40
Cellobiose
a-D-Glucose
D-Mannose
D-Psicose
Raffinose
Sucrose
Trehalose
Turanose
Acetic acid
L-Alanyl glycine
L-Asparagine
Glycyl L-aspartic acid
Glycyl L-glutamic acid
L-Threonine
D,L-a-Glycerol
a-D-Glucose 1-phosphate
D-Glucose 6-phosphate
Acid production from:
Cellobiose
Mannose
Fructose
Maltose
Inulin
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
+
2
+
+
+
+
+
+
+
+
+
+
2
+
+
+
+
2
+
+
(+)
2
2
2
2
+
+
+
2
2
2
2
2
+
Flavobacterium, for which the name Flavobacterium
tiangeerense sp. nov. is proposed.
Description of Flavobacterium tiangeerense
sp. nov.
Flavobacterium tiangeerense [ti.an.geer.en9se. N.L. neut. adj.
tiangeerense pertaining to the mountain Tiangeer (meaning
‘close to the sky’ in Mongolian language), the location of
the China No. 1 glacier, where the type strain was isolated].
Cells are Gram-stain-negative rods, non-flagellated and
0.4–0.5 mm wide and 2.5–8.5 mm long. Gliding motility is
not observed. Colonies are yellow, smooth, circular and
convex with entire margins. Growth occurs at 4–26 uC
(optimum, 22–23 uC) and at pH 6.5–9.0 (optimum,
approximately pH 7.0–8.0). Growth occurs in the presence
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Y.-H. Xin and others
Table 2. Cellular fatty acid compositions (%) of F. tiangeerense 0563T and its closest phylogenetic neighbours
Strains: 1, F. tiangeerense sp. nov. 0563T; 2, F. psychrolimnae NBRC
102679T; 3, F. limicola JCM 11473T. All data from this study. Fatty
acids amouting to ,1 % of the total fatty acids in all strains are not
shown. tr, Trace (,1 %). ND, Not detected.
Fatty acid
Saturated straight-chain
C16 : 0
Branched
iso-C14 : 0
iso-C15 : 0
anteiso-C15 : 0
iso-C15 : 1 G
anteiso-C15 : 1 A
iso-C16 : 0
iso-C16 : 1 H
Unsaturated
C15 : 1v6c
C17 : 1v6c
C17 : 1v8c
C18 : 1v5c
Hydroxy
C15 : 0 3-OH
iso-C15 : 0 3-OH
C16 : 0 3-OH
iso-C16 : 0 3-OH
iso-C17 : 0 3-OH
Summed feature 3*
Summed feature 4*
Summed feature 9*
1
2
3
1.3
1.4
1.6
3.1
26.9
2.8
5.4
tr
3.6
3.2
3.1
12.3
8.1
6.3
1.4
4.3
5.1
3.2
14.0
8.7
6.7
1.1
5.0
3.8
9.1
3.8
tr
tr
8.2
9.6
1.1
1.1
7.4
8.2
1.0
tr
ND
1.8
5.9
1.1
5.7
3.2
12.9
1.3
6.9
1.4
7.8
5.2
8.3
10.7
1.1
5.1
4.6
9.3
1.4
2.7
ND
ND
3.9
3.1
*Summed features are groups of two or three fatty acids that cannot
be separated by GLC with the MIDI system (Sherlock version 6.0).
Summed feature 3 comprised C16 : 1v7c and/or C16 : 1v6c; summed
feature 4 comprised iso-C17 : 1 I and/or anteiso-C17 : 1 B; summed
feature 9 comprised iso-C12 : 1v7c and/or 10-methyl C16 : 0.
of 0–0.5 % (w/v) NaCl. Good growth occurs under aerobic
conditions. Weak growth occurs under microaerobic
conditions (approximately 10 % CO2, 11 % O2). No growth
occurs under anaerobic conditions (O2,1 %, 9–13 %
CO2). Growth occurs on nutrient agar and trypticase soy
agar, but not on seawater agar. No brown pigment is
produced on tyrosine agar. No precipitate is formed on egg
yolk agar. Casein and aesculin are hydrolysed. Gelatin,
starch, tyrosine, chitin, pectin, alginate, DNA, carboxymethylcellulose and Tweens 20 and 80 are not hydrolysed.
Congo red is not absorbed by colonies and flexirubin-type
pigments are not produced. Hydrogen sulfide and indole
are not produced. Nitrate is not reduced. The methyl red
and Voges–Proskauer tests are negative. Oxidase, catalase,
naphthol-AS-BI-phosphohydrolase, acid phosphatase,
alkaline phosphatase, b-glucosidase, a-chymotrypsin, trypsin, esterase lipase (C8), esterase (C4), lipase (C14), cystine
2776
arylamidase, leucine arylamidase and valine arylamidase
activities are present. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan
deaminase, a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. Citrate is
not utilized. Acid is produced from cellobiose. Acid is
weakly produced from starch and mannose. Acid is not
produced from L-arabinose, adonitol, sorbitol, glycerol,
melibiose, sorbose, galactose, rhamnose, mannitol, melezitose, sucrose, trehalose, glucose, lactose, glycogen, raffinose, methyl a-D-glucoside, meso-erythritol, amygdalin,
xylose, inositol, dulcitol, salicin, ribose, fructose, maltose
and inulin. The following substrates are utilized as sole
carbon sources in the GN MicroPlate: a-cyclodextrin,
dextrin, glycogen, a-D-glucose, maltose, L-alaninamide,
L-alanine, L-glutamic acid, L-ornithine, L-proline, uridine,
a-D-glucose 1-phosphate and D-glucose 6-phosphate. All
other substrates in the GN MicroPlate are not utilized as
sole carbon sources. Menaquinone-6 (MK-6) is the major
respiratory quinone. The predominant cellular fatty acids
(.5 % of the total fatty acids) are iso-C15 : 0, iso-C15 : 0
3-OH, summed feature 3 (comprising C16 : 1v7c and/or
C16 : 1v6c), C15 : 1v6c, iso-C15 : 1 G and iso-C16 : 0 3-OH. The
DNA G+C content of the type strain is 34.8 mol%.
The type strain is 0563T (5CGMCC 1.6847T 5JCM
15087T), isolated from the China No. 1 glacier (Xinjiang
Uygur Autonomous Region, China).
Acknowledgements
This work was supported by the National Basic Research Program 170
of China (2004CB719601).
References
Bergey, D. H., Harrison, F. C., Breed, R. S., Hammer, B. W. &
Huntoon, F. M. (1923). Genus II. Flavobacterium gen. nov. In Bergey’s
Manual of Determinative Bacteriology, pp. 97–117. Baltimore:
Williams & Wilkins.
Bernardet, J. F. & Bowman, J. P. (2006). The genus Flavobacterium. In
The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 7,
pp. 481–531. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H.
Schleifer & E. Stackebrandt. New York: Springer.
Bernardet, J.-F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K.
& Vandamme, P. (1996). Cutting a Gordian knot: emended
classification and description of the genus Flavobacterium, emended
description of the family Flavobacteriaceae, and proposal of
Flavobacterium hydatis nom. nov. (basonym, Cytophaga aquatilis
Strohl and Tait 1978). Int J Syst Bacteriol 46, 128–148.
Bernardet, J. F., Nakagawa, Y. & Holmes, B. (2002). Proposed
minimal standards for describing new taxa of the family
Flavobacteriaceae and emended description of the family. Int J Syst
Evol Microbiol 52, 1049–1070.
Collins, M. D. (1985). Isoprenoid quinone analysis in classification
and identification. In Chemical Methods in Bacterial Systematics, pp.
267–287. Edited by M. Goodfellow & D. E. Minnikin. London:
Academic Press.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 59
IP: 88.99.165.207
On: Fri, 16 Jun 2017 04:33:46
Flavobacterium tiangeerense sp. nov.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative
Reichenbach, H. (1992). The order Cytophagales. In The Prokaryotes,
measurement of DNA hybridization from renaturation rates. Eur J
Biochem 12, 133–142.
2nd edn, vol. 4, pp. 3631–3675. Edited by A. Balows, H. G. Trüper, M.
Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors)
(1994). Methods for General and Molecular Bacteriology. Washington,
Sasser, M. (1990). Identification of bacteria by gas chromatography of
DC: American Society for Microbiology.
Smibert, R. M. & Krieg, N. R. (1981). General characterization. In
Hugh, R. & Leifson, E. (1953). The taxonomic significance of
Manual of Methods for General Microbiology, pp. 409–443. Edited by
P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A.
Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American
Society for Microbiology.
fermentative versus oxidative metabolism of carbohydrates by various
Gram-negative bacteria. J Bacteriol 66, 24–26.
Kimura, M. (1980). A simple method for estimating evolutionary rates
of base substitutions through comparative studies of nucleotide
sequences. J Mol Evol 16, 111–120.
integrated software
for molecular evolutionary genetics analysis and sequence alignment.
Brief Bioinform 5, 150–163.
Kumar, S., Tamura, K. & Nei, M. (2004).
MEGA3:
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid
Techniques in Bacterial Systematics, pp. 115–175. Edited by E.
Stackebrandt & M. Goodfellow. Chichester: Wiley.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic
acid from microorganisms. J Mol Biol 3, 208–218.
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
Tamaki, H., Hanada, S., Kamagata, Y., Nakamura, K., Nomura, N.,
Nakano, K. & Matsumura, M. (2003). Flavobacterium limicola sp. nov.,
a psychrophilic, organic-polymer-degrading bacterium isolated from
freshwater sediments. Int J Syst Evol Microbiol 53, 519–526.
Van Trappen, S., Vandecandelaere, I., Mergaert, J. & Swings, J.
(2005). Flavobacterium fryxellicola sp. nov. and Flavobacterium
psychrolimnae sp. nov., novel psychrophilic bacteria isolated from
microbial mats in Antarctic lakes. Int J Syst Evol Microbiol 55, 769–772.
Wu, C., Lu, X., Qin, M., Wang, Y. & Ruan, J. (1989). Analysis of
menaquinone compound in microbial cells by HPLC. Microbiology
[English translation of Microbiology (Beijing)] 16, 176–178.
Marmur, J. & Doty, P. (1962). Determination of the base composition
of deoxyribonucleic acid from its thermal denaturation temperature.
J Mol Biol 5, 109–118.
Zhang, D.-C., Wang, H.-X., Liu, H.-C., Dong, X.-Z. & Zhou, P.-J. (2006).
Qu, J.-H., Li, H.-F., Yang, J.-S. & Yuan, H.-L. (2008). Flavobacterium
Zhu, F., Wang, S. & Zhou, P. (2003). Flavobacterium xinjiangense sp.
nov. and Flavobacterium omnivorum sp. nov., novel psychrophiles
from the China No. 1 glacier. Int J Syst Evol Microbiol 53, 853–857.
cheniae sp. nov., isolated from sediment of a eutrophic reservoir. Int J
Syst Evol Microbiol 58, 2186–2190.
http://ijs.sgmjournals.org
Flavobacterium glaciei sp. nov., a novel psychrophilic bacterium isolated
from the China No.1 glacier. Int J Syst Evol Microbiol 56, 2921–2925.
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