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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
Correspondence
Pei-Jin Zhou
zhou{at}sun.im.ac.cn
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ABSTRACT |
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7c, C16 : 0, C12 : 1 3-OH and C18 : 17c 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).
A table showing the fatty acid profile of strain 170T and other Glaciecola species is available as supplementary material in IJSEM Online.
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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 °C. The meltwater was then spread onto marine agar 2216 (MA; Difco) plates and incubated at 4 °C for 2–6 weeks. Strain 170T was obtained in pure culture after three successive transfers to fresh agar medium and stored at –80 °C 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 °C for 4–5 days. Growth temperature was determined with a TN3F temperature-gradient 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 l–1). 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 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse primer 5'-AAGGAGGTGATCCAGCCGCA-3' (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 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 °C for 4 days and were extracted, methylated and analysed using the standard MIDI (Microbial Identification) system (Sasser, 1990).
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 °C and growth occurred at 4–15 °C.
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 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%.
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7c (38.31 %), C16 : 0 (13.59 %), C12 : 1 3-OH (6.47 %), C18 : 17c (6.23 %), C14 : 0 (5.48 %) and C17 : 18c (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.
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Cells are Gram-negative, psychrophilic, motile rods, 0.5–0.8 µm by 1.2–4.5 µm in size. Colonies are non-pigmented, convex, circular and smooth with entire edges. Grows aerobically and produces catalase and cytochrome oxidase. Growth occurs at 4–15 °C and pH 5.0–10.0, with optimum growth at 12 °C 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, 
-glucuronidase, -glucosidase, 
-glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Weak enzymic activity is observed for trypsin, lipase (C14) and cystine arylamidase. Tests for naphthol-AS-BI-phosphohydrolase, -chymotrypsin, amylase, alkaline phosphatase, leucine arylamidase, valine arylamidase and -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: glucose, D-mannose, mannitol, fructose, cellobiose, L-arabinose, xylose, L-rhamnose, D-melibiose, raffinose, D-sorbitol, 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 : 17c (38.31 %), C16 : 0 (13.59 %), C12 : 1 3-OH (6.47 %), C18 : 17c (6.23 %), C14 : 0 (5.48 %) and C17 : 18c (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 (77° 30' N to approximately 81° 12' N), including the Canadian Basin and Greenland Sea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Dong, X.-Z. & Cai, M.-Y. (editors) (2001). Determination of biochemical properties. In Manual for the Systematic Identification of General Bacteria. pp. 370–398. Beijing: Science Press (in Chinese).
Huß, V. A. R., Festl, H. & Schleifer, K.-H. (1983). Studies on the spectrometric determination of DNA hybridisation from renaturation rates. Syst Appl Microbiol 4, 184–192.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. J Bacteriol 85, 1183–1184.
Liu, H., Xu, Y., Ma, Y. & Zhou, P. (2000). Characterization of Micrococcus antarcticus sp. nov., a psychrophilic bacterium from Antarctica. Int J Syst Evol Microbiol 50, 715–719.[Abstract]
Romanenko, L. A., Zhukova, N. V., Rohde, M., Lysenko, A. M., Mikhailov, V. V. & Stackebrandt, E. (2003). Glaciecola mesophila sp. nov., a novel marine agar-digesting bacterium. Int J Syst Evol Microbiol 53, 647–651.
Sambrook, J., Frisch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Sasser, M. (1990). Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. Technical Note 101. Newark, DE: MIDI Inc.
Van Trappen, S., Tan, T.-L., Yang, J., Mergaert, J. & Swings, J. (2004). Glaciecola polaris sp. nov., a novel budding and prosthecate bacterium from the Arctic Ocean, and emended description of the genus Glaciecola. Int J Syst Evol Microbiol 54, 1765–1771.
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