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ABSTRACT: The psychrophilic bacterium Moritella dasanensis strain ArB 0140 was isolated near a glacier in Kongsfjorden, Svalbard Archipelago, Norway. Here we report a 4.89-Mb draft genome sequence of Moritella dasanensis ArB 0140, which could provide comprehensive information on a psychrophilic mechanism in extreme environments.
Journal of bacteriology 10/2012; 194(19):5452-3. · 3.94 Impact Factor
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ABSTRACT: Expressed sequence tags (ESTs) from the Antarctic green algae Pyramimonas gelidicola were analyzed to obtain molecular information on cold acclimation of psychrophilic microorganisms. A total of 2,112 EST clones were sequenced, generating 222 contigs and 219 singletons, and 200 contigs and 391 singletons from control (4 degrees C) and cold-shock conditions (-2 degrees C), respectively. The complete EST sequences were deposited to the DDBJ EST database (http:// www.ddbj.nig.ac.jp/index-e.html) and the nucleotide sequences reported in this study are available in the DDBJ/EMBL/ GenBank. These EST databases of Antarctic green algae can be used in a wide range of studies on psychrophilic genes expressed by polar microorganisms.
Journal of Microbiology and Biotechnology 07/2012; 22(7):902-6. · 1.38 Impact Factor
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ABSTRACT: Antifreeze proteins (AFPs) have important functions in many freeze-tolerant organisms. The proteins non-colligatively lower the freezing point and functionally inhibit ice recrystallization in frozen solutions. In our previous studies, we found that the Arctic yeast Leucosporidium sp. produces an AFP (LeIBP), and that the protein could be successfully produced in Pichia expression system. The present study showed that recombinant LeIBP possesses the ability to reduce the damage induced to red blood cells (RBCs) by freeze thawing. In addition to 40 % glycerol, both 0.4 and 0.8 mg/ml LeIBPs significantly reduced freeze-thaw-induced hemolysis at either rapid- (45 °C) or slow-warming (22 °C) temperatures. Post-thaw cell counts of the cryopreserved RBCs were dramatically enhanced, in particular, in 0.8 mg/ml LeIBP. Interestingly, the cryopreserved cells in the presence of LeIBP showed preserved cell size distribution. These results indicate that the ability of LeIBP to inhibit ice recrystallization helps the RBCs avoid critically damaging electrolyte concentrations, which are known as solution effects. Considering all these data, LeIBP can be thought of as a key component in improving RBC cryopreservation efficiency.
Applied biochemistry and biotechnology 05/2012; 167(4):824-34. · 1.94 Impact Factor
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ABSTRACT: Previously, we reported the ice-binding protein (LeIBP) from the Arctic yeast Leucosporidium sp. AY30. In this study we provide physicochemical characterization of this IBP, which belongs to a class of IBPs that exhibited no significant similarity in primary structure to other known antifreeze proteins (AFPs). We compared native, glycosylated and non-glycosylated recombinant LeIBPs. Interestingly, size-exclusion chromatography and analytical ultracentrifugation revealed that LeIBP self-associates with a reversible dimer with K(d) values in the range 3.45-7.24×10(-6) M. Circular dichroism (CD) spectra showed that LeIBP, glycosylated or non-glycosylated, is predominantly composed of β-strand secondary structural elements (54.6%), similar to other β-helical antifreeze proteins (AFPs). In thermal hysteresis (TH) activity measurements, native LeIBP was twice more active (0.87 °C at 15 mg/mL) than that of the recombinant IBPs (0.43-0.42 °C at 10.8 mg/mL). This discrepancy is probably due to uncharacterized enhancing factors carried over during ice affinity purification, because glycosylated and non-glycosylated recombinant proteins displayed similarly low activity. Ice recrystallization inhibition (RI) activities of the native and recombinant LeIBPs were comparable. Measurements of CD, TH activity, and RI showed that glycosylation does not cause structural changes and is not required for function. An ice-etching experiment using green fluorescent protein-tagged IBP revealed that LeIBP binds, just as hyperactive AFPs, to both basal and pyramidal prism planes of the ice crystal. Taken together, our results indicate that LeIBP, structurally similar to hyperactive AFPs, is moderately active and that a reversible dimer has no effect on its activity.
Cryobiology 03/2012; 64(3):286-96. · 2.06 Impact Factor
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ABSTRACT: Adenoviruses have been identified in humans and a wide range of vertebrate animals, but not previously from the polar region. Here, we report the entire 26,340-bp genome of a novel adenovirus, detected by PCR, in tissues of six of nine South Polar skuas (Catharacta maccormicki), collected in Lake King Sejong, King George Island, Antarctica, from 2007 to 2009. The DNA polymerase, penton base, hexon and fiber genes of the South Polar skua adenovirus (SPSAdV) exhibited 68.3%, 75.4%, 74.9% and 48.0% nucleotide sequence similarity with their counterparts in turkey hemorrhagic enteritis virus. Phylogenetic analysis based on the entire genome revealed that SPSAdV belonged to the genus Siadenovirus, family Adenoviridae. This is the first evidence of a novel adenovirus, SPSAdV, from a large polar seabird (family Stercorariidae) in Antarctica.
Virology 11/2011; 422(1):144-50. · 3.35 Impact Factor
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ABSTRACT: A Gram-negative, rod-shaped and motile strain, designated CL-AS9(T), was isolated from polar seawater of the Arctic. Analysis of the 16S rRNA gene sequence of the strain showed an affiliation with the genus Spongiibacter, sharing 93.9% and 93.7% sequence similarities with the type strains of Spongiibacter tropicus CL-CB221(T) and Spongiibacter marinus HAL40b(T), respectively. Phylogenetic analyses revealed that strain CL-AS9(T) formed a separate branch that was distinct from a clade comprising Spongiibacter marinus HAL40b(T), Spongiibacter tropicus CL-CB221(T) and Melitea salexigens 5IX/A01/131(T). Cells of the strain grew optimally at 20-25 °C and pH 6.6-8.0 in the presence of 3-4% (w/v) sea salts. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified aminophospholipid. The major quinone was ubiquinone 8. The major cellular fatty acids were C(16:1)ω7c and/or iso-C(15:0) 2-OH (23.1%), C(17:1)ω8c (22.1%) and C(18:1)ω7c (15.6%). The genomic DNA G+C content was 53.6 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic data presented, we propose the name Spongiibacter borealis sp. nov. with the type strain CL-AS9(T) (=KCCM 90094(T) =JCM 17304(T)) and the reclassification of Melitea salexigens as a later heterotypic synonym of Spongiibacter marinus. We also provide emended descriptions of the genus Spongiibacter and Spongiibacter marinus.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 01/2011; 61(Pt 12):2895-900. · 2.11 Impact Factor
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ABSTRACT: A psychrophilic yeast was isolated from an Arctic pond and its culture supernatant showed ice-binding activity. This isolate, identified as Leucosporidium sp. based on an analysis of the D1/D2 and ITS regions of its ribosomal DNA, produced a secretory ice-binding protein (IBP). Yeast IBP was purified from the culture medium to near homogeneity by the ice affinity method and appeared to be glycosylated with a molecular mass of approximately 26 kDa. In addition, the yeast IBP was shown to have thermal hysteresis (TH) and recrystallization inhibition (RI) activities. The full-length cDNA for yeast IBP was determined and was found to encode a 261 amino acid protein with molecular weight of 26.8 kDa that includes an N-terminal signal peptide and one potential N-glycosylation site. The deduced protein showed high sequence identity with other IBPs and hypothetical IBPs from fungi, diatoms, and bacteria, clustering with a class of ice-active proteins.
Cryobiology 04/2010; 60(2):222-8. · 2.06 Impact Factor
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ABSTRACT: The antifreeze protein gene (Cn-AFP) from the Antarctic marine diatom, Chaetoceros neogracile was cloned and characterized. The full-length Cn-AFP cDNA contained an open reading frame of 849 bp and the deduced 282 amino acid peptide chain encodes a 29.2 kDa protein, which includes a signal peptide of 30 amino acids at the N terminus. Both the Cn-AFP coding region with and without the signal sequence were cloned and expressed in Escherichia coli. Recombinant Cn-AFPs were shown to display antifreeze activities based on measuring the thermal hysteresis and modified morphology of single ice crystals. Recombinant mature Cn-AFP showed 16-fold higher thermal hysteresis activity than that of pre-mature Cn-AFP at the same concentration. The ice crystal shape changed to an elongated hexagonal shape in the presence of the recombinant mature Cn-AFP, while single ice crystal showed a circular disk shape in absence of Cn-AFP. Northern analysis demonstrated a dramatic accumulation of Cn-AFP transcripts when the cells were subjected to freezing stress. This rapid response to freeze stress, and the antifreeze activity of recombinant Cn-AFPs, indicates that Cn-AFP plays an important role in low temperature adaptation.
Marine Biotechnology 12/2009; 12(6):630-9. · 3.43 Impact Factor
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ABSTRACT: Two Gram-negative, strictly aerobic bacterial strains, designated CL-AP6(T) and CL-AP22, were isolated from a culture of a green alga, Pyramimonas gelidicola, established from the Antarctic. Cells of the strains were straight rods and motile by means of a single polar flagellum. The strains grew with 0.5-8 % (w/v) NaCl (optimum, 1-2 %) and at 4-33 degrees C (optimum, 25 degrees C) and pH 6.5-9.1 (optimum, pH 7.5-8.1). The two strains shared 98.8 % 16S rRNA gene sequence similarity. Analysis of the 16S rRNA gene sequences of strains CL-AP6(T) and CL-AP22 revealed that they were members of the genus Pseudomonas and were most closely related to Pseudomonas xiamenensis C10-2(T) (95.5-95.8 % sequence similarity) and next to Pseudomonas pertucinogena NBRC 14163(T) (95.1-95.5 % sequence similarity) and to other members of the genus Pseudomonas (<95.2 % sequence similarity). Phylogenetic analyses based on the 16S rRNA gene sequences showed that strain CL-AP6(T) formed a robust clade with strain CL-AP22, and that this clade clustered tightly with the nearest clade containing P. xiamenensis and P. pertucinogena. The major isoprenoid quinone of strains CL-AP6(T) and CL-AP22 was Q-9 and the major cellular fatty acids were C(18 : 1)omega7c (40.2-41.6 %), summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH; 26.3-26.8 %), C(16 : 0) (13.7-13.9 %) and C(12 : 0) (5.8-6.2 %). The genomic DNA G+C contents of strains CL-AP6(T) and CL-AP22 were 59.1 and 57.2 mol%, respectively. DNA-DNA hybridization experiments revealed high relatedness values (98.5+/-0.5 %; mean+/-sem, n=2) between strains CL-AP6(T) and CL-AP22, indicating that the two strains constituted a single species. However, the two strains differed phenotypically from P. xiamenensis by their inability to grow without NaCl, temperature range for growth, hydrolysis of starch and production of certain enzymes. The phylogenetic analysis and physiological and biochemical data showed that strains CL-AP6(T) and CL-AP22 should be classified as representing a novel species in the genus Pseudomonas, for which the name Pseudomonas pelagia sp. nov. is proposed. The type strain is CL-AP6(T) (=KCCM 90073(T)=JCM 15562(T)).
International journal of systematic and evolutionary microbiology 07/2009; 59(Pt 12):3019-24. · 2.27 Impact Factor
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ABSTRACT: A psychrophilic, Gram-negative, dark orange-pigmented bacterium, designated CL-AP4(T), was isolated from a culture of the green alga Pyramimonas gelidicola obtained from the Southern Ocean. Strain CL-AP4(T) grew optimally at 10 degrees C, in the presence of 3-4 % sea salts and at pH 8. 16S rRNA gene sequence analysis revealed that strain CL-AP4(T) belonged to the family Flavobacteriaceae, with Maribacter arcticus KOPRI 20941(T) as its closest relative (97.2 % similarity). A number of chemotaxonomic characteristics supported affiliation of strain CL-AP4(T) with the genus Maribacter, i.e. iso-C(15 : 0) (17.2 %), iso-C(15 : 1) (16.8 %) and iso-C(17 : 0) 3-OH (14.9 %) were the dominant fatty acids, MK-6 was the major menaquinone and the DNA G+C content was 37.1 mol%. DNA-DNA relatedness between CL-AP4(T) and M. arcticus KOPRI 20941(T) was only 10 %, suggesting that they are genomically distinct species. In addition, strain CL-AP4(T) differed phenotypically from M. arcticus in its optimum growth temperature, its ability to hydrolyse starch, Tween 40 and Tween 80, and production of certain enzymes. On the basis of the results of the polyphasic analysis, strain CL-AP4(T) was classified in the genus Maribacter as belonging to a novel species, for which the name Maribacter antarcticus sp. nov. is proposed; the type strain is CL-AP4(T) (=KCCM 90069(T)=JCM 15445(T)).
International journal of systematic and evolutionary microbiology 07/2009; 59(Pt 6):1455-9. · 2.27 Impact Factor
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ABSTRACT: Statistical experimental designs; involving (i) a fractional factorial design (FFD) and (ii) a central composite design (CCD) were applied to optimize the culture medium constituents for production of a unique antifreeze protein by the Antartic microalgae Chaetoceros neogracile. The results of the FFD suggested that NaCl, KCl, MgCl2, and Na2SiO3 were significant variables that highly influenced the growth rate and biomass production. The optimum culture medium for the production of an antifreeze protein from C. neogracile was found to be Kalleampersandrsquor;s artificial seawater, pH of 7.0ampersandplusmn;0.5, consisting of 28.566 g/l of NaCl, 3.887 g/l of MgCl2, 1.787 g/l of MgSO4, 1.308 g/l of CaSO4, 0.832 g/l of K2SO4, 0.124 g/l of CaCO3, 0.103 g/l of KBr, 0.0288 g/l of SrSO4, and 0.0282 g/l of H3BO3. The antifreeze activity significantly increased after cells were treated with cold shock (at -5oC) for 14 h. To the best of our knowledge, this is the first report demonstrating an antifreeze-like protein of C. neogracile.
Journal of Microbiology and Biotechnology 01/2009; 18(12):1919-26. · 1.38 Impact Factor
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ABSTRACT: An aerobic, motile, Gram-negative, ice-active substance-producing, rod-shaped psychrophile, designated strain ArB 0140T, was isolated from seawater collected from near a glacier in Kongsfjorden, Svalbard Archipelago, Norway. Phylogenetic analysis using 16S rRNA gene sequences indicated that strain ArB 0140T showed a distinct phyletic line within the genus Moritella. Characteristic chemotaxonomic data [predominant isoprenoid quinone, Q8; major fatty acids, C14 : 0, C14 : 1, C16 : 0, C16 : 1 and C22 : 6 (docosahexaenoic acid; DHA)] also corroborated the affiliation of strain ArB 0140T to the genus Moritella. The maximal growth rate of the novel strain was observed at 9 degrees C, with a maximum temperature for growth of 18 degrees C. The genomic DNA G+C content was 46.9 mol%. Based on the data obtained from this polyphasic study, including DNA-DNA relatedness, physiological and biochemical tests and ice-controlling activity, strain ArB 0140T was found to be genetically and phenotypically different from other recognized species of the genus Moritella. Therefore strain ArB 0140T represents a novel species, for which the name Moritella dasanensis sp. nov. is proposed. The type strain is ArB 0140T (=KCTC 10814T=KCCM 42845T=JCM 14759T).
International journal of systematic and evolutionary microbiology 05/2008; 58(Pt 4):817-20. · 2.27 Impact Factor
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ABSTRACT: 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly (CTD(GG)) and gly-ser-ser-gly (CTD(GSSG)) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), 15N-HSQC, and 31P-NMR revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.
Journal of Microbiology and Biotechnology 09/2007; 17(8):1385-9. · 1.38 Impact Factor
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ABSTRACT: To better understand the gene expression of the cold-adapted polar diatom, we conducted a survey of the Chaetoceros neogracile transcriptome by cDNA sequencing and expression of interested cDNAs from the Antarctic diatom. A non-normalized cDNA library was constructed from the C. neogracile, and a total of 2,500 cDNAs were sequenced to generate 1,881 high-quality expressed sequence tags (ESTs) (accession numbers EL620615-EL622495). Based on their clustering, we identified 154 unique clusters comprising 342 ESTs. The remaining 1,540 ESTs did not cluster. The number of unique genes identified in the data set is thus estimated to be 1,694. Taking advantage of various tools and databases, putative functions were assigned to 939 (55.4%) of these genes. Of the remaining 540 (31.9%) unknown sequences, 215 (12.7%) appeared to be C. neogracile-specific since they lacked any significant sequence similarity to any sequence available in the public databases. C. neogracile consisted of a relatively high percentage of genes involved in metabolism, genetic information processing, cellular processes, defense or stress resistance, photosynthesis, structure, and signal transduction. From the ESTs, the expression of these putative C. neogracile genes was investigated: fucoxanthin chlorophyll (chl) a,c-binding protein (FCP), ascorbate peroxidase (ASP), and heat-shock protein 90 (HSP90). The abundance of ASP and HSP90 changed substantially in response to different culture conditions, indicating the possible regulation of these genes in C. neogracile.
Journal of Microbiology and Biotechnology 09/2007; 17(8):1330-7. · 1.38 Impact Factor
