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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.
§Sea-salt concentration.
||Data from International Journal of Systematic and Evolutionary Microbiology 61 trypsin (weak), but not lipase (C14), a-chymotrypsin, a- or Felsenstein, J. (1985). Confidence limits on phylogenies: an approach b-galactosidase, b-glucuronidase, a- or b-glucosidase, N- using the bootstrap. Evolution 39, 783–791.
acetyl-b-glucosaminidase, a-mannosidase or b-fucosidase Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the (API ZYM). Positive for acid production from glucose, but spectrophotometric determination of DNA hybridization from negative for arginine dihydrolase, lysine decarboxylase, renaturation rates. Syst Appl Microbiol 4, 184–192.
ornithine decarboxylase, urease, tryptophan deaminase, Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In gelatinase, utilization of citrate and acid production from Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro.
mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose (API 20 E). Does not assimilate Marmur, J. & Doty, P. (1962). Determination of the base composition glucose, arabinose, mannose, mannitol, N-acetylglucosa- of deoxyribonucleic acid from its thermal denaturation temperature.
J Mol Biol 5, 109–118.
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& Yumoto, I. (2006). Glaciecola chathamensis sp. nov., a novel marine are observed with GN2 MicroPlates after 7 days at 25 uC.
polysaccharide-producing bacterium. Int J Syst Evol Microbiol 56, Resistant to tetracycline (30 mg per disc); sensitive to (mg per disc) gentamicin (10), chloromycetin (30), erythromy- Romanenko, L. A., Zhukova, N. V., Rohde, M., Lysenko, A. M., cin (15), kanamycin (30), streptomycin (10), carbenicillin Mikhailov, V. V. & Stackebrandt, E. (2003). Glaciecola mesophila (100), amikacin (30), polymyxin (30) and vancomycin sp. nov., a novel marine agar-digesting bacterium. Int J Syst Evol (30). The major cellular fatty acids are summed feature 3 (comprising C16 : 1v7c and/or iso-C15 : 0 2-OH), C16 : 0, Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new C17 : 1v8c and C18 : 1v7c. The DNA G+C content of the method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– The type strain, BSs20135T (5CCTCC AB 209161T Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. InMethods for General and Molecular Bacteriology, pp. 607–654. Edited 5KACC 14537T), was isolated from Arctic sediment.
by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg.
Washington, DC: American Society for Microbiology.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place forDNA–DNA reassociation and 16S rRNA sequence analysis in the We especially thank Dr Xue-Wei Xu and Dr Fan-Xu Meng from the present species definition in bacteriology. Int J Syst Bacteriol 44, 846– Second Institute of Oceanography, State Oceanic Administration, for their help with the DNA–DNA hybridization experiments. This study Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular was supported financially by the Hi-Tech Research and Development Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Program of China (grant 2007AA091903 and 2007AA021306), the National Natural Science Foundation of China (grants 40706001 and30500001) and the COMRA Program (grant no. DYXM-115-02-2-6).
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