Crystal structure of the reactive intermediate/imine deaminase A homolog from the Antarctic bacterium Psychrobacter sp. PAMC 21119.

The RidA subfamily proteins catalyze the deamination reaction of enamine/imine intermediates, which are metabolites of amino acids such as threonine and serine. Numerous structural and functional studies have been conducted on RidA isolated from mesophiles and thermophiles. However, little is known about the structure of the RidA proteins isolated from psychrophiles. In the present study, we elucidated the crystal structure of RidA from the Antarctic bacterium Psychrobacter sp. PAMC 21119 (Pp-RidA) at 1.6 Å resolution to identify the structural properties contributing to cold-adaptability. Although the overall structure of Pp-RidA is similar to those of its homologues, it exhibits specific structural arrangements of a loop positioned near the active site, which is assumed to play a role in covering the active site of catalysis. In addition, the surface electrostatic potential of Pp-RidA suggested that it exhibits stronger electrostatic distribution relative to its homologues. Our results provide novel insights into the key determinants of cold-adaptability.

Crystal structure of a transcription factor, GerE (PaGerE), from spore-forming bacterium Paenisporosarcina sp. TG-14.

In cold and harsh environments such as glaciers and sediments in ice cores, microbes can survive by forming spores. Spores are composed of a thick coat protein, which protects against external factors such as heat-shock, high salinity, and nutrient deficiency. GerE is a key transcription factor involved in spore coat protein expression in the mother cell during sporulation. GerE regulates transcription during the late sporulation stage by directly binding to the promoter of cotB gene. Here, we report the crystal structure of PaGerE at 2.09 Šresolution from Paenisporosarcina sp. TG-14, which was isolated from the Taylor glacier. The PaGerE structure is composed of four α-helices and adopts a helix-turn-helix architecture with 68 amino acid residues. Based on our DNA binding analysis, the PaGerE binds to the promoter region of CotB to affect protein expression. Additionally, our structural comparison studies suggest that DNA binding by PaGerE causes a conformational change in the α4-helix region, which may strongly induce dimerization of PaGerE.

Proteomic and transcriptomic investigations on cold-responsive properties of the psychrophilic Antarctic bacterium Psychrobacter sp. PAMC 21119 at subzero temperatures.

Psychrobacter sp. PAMC 21119, isolated from Antarctic permafrost soil, grows and proliferates at subzero temperatures. However, its major mechanism of cold adaptation regulation remains poorly understood. We investigated the transcriptomic and proteomic responses of this species to cold temperatures by comparing profiles at -5°C and 20°C to understand how extreme microorganisms survive under subzero conditions. We found a total of 2,906 transcripts and 584 differentially expressed genes (≥ twofold, P <0.005) by RNA-seq. Genes for translation, ribosomal structure and biogenesis were upregulated, and lipid transport and metabolism was downregulated at low temperatures. A total of 60 protein spots (≥ 1.8 fold, P < 0.005) showed differential expression on two-dimensional gel electrophoresis and the proteins were identified by mass spectrometry. The most prominent upregulated proteins in response to cold were involved in metabolite transport, protein folding and membrane fluidity. Proteins involved in energy production and conversion, and heme protein synthesis were downregulated. Moreover, isoform exchange of cold-shock proteins was detected at both temperatures. Interestingly, pathways for acetyl-CoA metabolism, putrescine synthesis and amino acid metabolism were upregulated. This study highlights some of the strategies and different physiological states that Psychrobacter sp. PAMC 21119 has developed to adapt to the cold environment in Antarctica.

Effect of the antifreeze protein from the arctic yeast Leucosporidium sp. AY30 on cryopreservation of the marine diatom Phaeodactylum tricornutum.

Antifreeze proteins are a group of proteins that allow organisms to survive in subzero environments. These proteins possess thermal hysteresis and ice recrystallization inhibition activities. In the present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Arctic yeast Leucosporidium sp. AY30, LeIBP, in cryopreservation of the marine diatom Phaeodactylum tricornutum, which is one of the classical model diatoms and has most widely been studied with regard to its ecology, physiology, biochemistry, and molecular biology. P. tricornutum cells were frozen by either a fast or two-step freezing method in freezing medium containing 10 % dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol, respectively, with or without LeIBP supplement. When cells were frozen using the two-step freezing method, cell survival was significantly increased and statistically the same as that of unfrozen native cells in the presence of 0.1 mg/ml LeIBP in 10 % propylene glycol or 10 % ethylene glycol at day 11 of post-thaw culture. In the presence of LeIBP, the concentration of chlorophyll a was dramatically increased to 14-, 48-, 1.6-, and 8.8-fold when cells were frozen in freezing medium containing dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. Scanning electron microscopy observations demonstrated that the cells were also successfully preserved and epitheca or hypotheca were not deformed. These results demonstrate that LeIBP was successfully applied to improve cryopreservation of the marine diatom P. tricornutum.

Draft Genome Sequence of Pseudomonas pelagia CL-AP6, a Psychrotolerant Bacterium Isolated from Culture of Antarctic Green Alga Pyramimonas gelidicola.

Pseudomonas pelagia CL-AP6, isolated from a culture of the Antarctic green alga Pyramimonas gelidicola, is a psychrotolerant bacterium. Here, we report the draft genome sequence of this strain, which may provide insights into the mutualistic interaction between microalgae and bacteria in sea ice, as well as the cold adaptation mechanisms of bacteria.

Antifreeze peptides and glycopeptides, and their derivatives: potential uses in biotechnology.

Antifreeze proteins (AFPs) and glycoproteins (AFGPs), collectively called AF(G)Ps, constitute a diverse class of proteins found in various Arctic and Antarctic fish, as well as in amphibians, plants, and insects. These compounds possess the ability to inhibit the formation of ice and are therefore essential to the survival of many marine teleost fishes that routinely encounter sub-zero temperatures. Owing to this property, AF(G)Ps have potential applications in many areas such as storage of cells or tissues at low temperature, ice slurries for refrigeration systems, and food storage. In contrast to AFGPs, which are composed of repeated tripeptide units (Ala-Ala-Thr)n with minor sequence variations, AFPs possess very different primary, secondary, and tertiary structures. The isolation and purification of AFGPs is laborious, costly, and often results in mixtures, making characterization difficult. Recent structural investigations into the mechanism by which linear and cyclic AFGPs inhibit ice crystallization have led to significant progress toward the synthesis and assessment of several synthetic mimics of AFGPs. This review article will summarize synthetic AFGP mimics as well as current challenges in designing compounds capable of mimicking AFGPs. It will also cover our recent efforts in exploring whether peptoid mimics can serve as structural and functional mimics of native AFGPs.

Draft genome sequence of Paenisporosarcina sp. strain TG-20, a psychrophilic bacterium isolated from the basal ice of Taylor Glacier.

We report the draft genome sequence of Paenisporosarcina sp. strain TG-20, which is 4.12 Mb in size and consists of 4,071 protein-coding genes and 76 RNA genes. The genome sequence of Paenisporosarcina sp. TG-20 may provide useful information about molecular adaptations that enhance survival in icy subsurface environments.

Draft genome sequence of Paenisporosarcina sp. strain TG-14, a psychrophilic bacterium isolated from sediment-laden stratified basal ice from Taylor Glacier, McMurdo Dry Valleys, Antarctica.

The psychrophilic bacterium Paenisporosarcina sp. TG-14 was isolated from sediment-laden stratified basal ice from Taylor Glacier, McMurdo Dry Valleys, Antarctica. Here we report the draft genome sequence of this strain, which may provide useful information on the cold adaptation mechanism in extremely variable environments.

Draft genome sequence of Moritella dasanensis strain ArB 0140, a psychrophilic bacterium isolated from the Arctic Ocean.

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.

Cryopreservative effects of the recombinant ice-binding protein from the arctic yeast Leucosporidium sp. on red blood cells.

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.

Ramalin, a novel nontoxic antioxidant compound from the Antarctic lichen Ramalina terebrata.

Ramalin (γ-glutamyl-N'-(2-hydroxyphenyl)hydrazide), a novel compound, was isolated from the methanol-water extract of the Antarctic lichen Ramalina terebrata by several chromatographic methods. The molecular structure of ramalin was determined by spectroscopic analysis. The experimental data showed that ramalin was five times more potent than commercial butylated hydroxyanisole (BHA) in scavenging 1-diphenyl-2-picryl-hydazil (DPPH) free radicals, 27 times more potent in scavenging 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid free radicals (ABTS(+)) than the vitamin E analogue, trolox, and 2.5 times more potent than BHT in reducing Fe(3+) to Fe(2+) ions. Similarly, ramalin was 1.2 times more potent than ascorbic acid in scavenging superoxide radicals and 1.25 times more potent than commercial kojic acid in inhibiting tyrosinase enzyme activity, which ultimately leads to whitening of skin cells. Ramalin showed no or very little cytotoxicity in human keratinocyte and fibroblast cells at its antioxidant concentration. Furthermore, ramalin was assessed to determine its antioxidant activity in vivo. One microgram per milliliter ramalin significantly reduced the released nitric oxide (NO) and 0.125 µg/ml ramalin reduced the produced hydrogen peroxide (H(2)O(2)) in LPS (lipopolysaccharide)-stimulated murine macrophage Raw264.7 cells. Considering all the data together, ramalin can be a strong therapeutic candidate for controlling oxidative stress in cells.

Expression of recombinant endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 in Pichia pastoris by codon optimization.

An endochitinase was previously purified and the gene was cloned from the psychrophilic Antarctic bacterium, Sanguibacter antarcticus (KCTC 13143). In the present study, recombinant endochitinase, rChi21702, was expressed using a yeast expression system (Pichia pastoris) and codon optimization. The expressed rChi21702 was purified by Phenyl-Sepharose column chromatography. Optimal expression yielded 1-mg purified enzyme from 1-L bioreactor culture. When p-NP-(GlcNAc)(2) was used as a substrate, the specific activity of the enzyme was determined to be 20U/mg. In vitro assays and thin-layer chromatography demonstrated that the recombinant enzyme has endochitinase activity that produces diacetyl-chitobiose as a dominant end product when chitooligomers, colloidal chitin, and the chromogenic p-NP-(GlcNAc)(2) are used as substrates. Optimal activity for rChi21702 was observed at 37 degrees C and a pH of 7.6. Interestingly, rChi21702 exhibited 63% of optimal activity at 10 degrees C and 44% activity at 0 degrees C. Taken together, the results indicate that rChi21702 has psychrotolerant endochitinase activity even after recombinant expression in yeast cells.

Colwellia asteriadis sp. nov., a marine bacterium isolated from the starfish Asterias amurensis.

A marine bacterial strain, KMD 002T, was isolated from an Amur starfish, Asterias amurensis, collected in the East Sea of Korea. Strain KMD 002T was a Gram-negative, beige-pigmented, rod-shaped bacterium. The strain was capable of growth at relatively low temperatures (4-25 degrees C) and over a broad pH range (pH 4.0-10.0). The major fatty acids were C16:1omega7c and/or iso-C15:0 2-OH and C16:0 and the predominant isoprenoid quinone was Q-8. The DNA G+C content of strain KMD 002T was 40.3 mol%. Phylogenetic analysis using 16S rRNA gene sequences revealed that strain KMD 002T belonged to the genus Colwellia. However, various phenotypic properties as well as low 16S rRNA gene sequence similarities to members of the genus Colwellia (94.1-96.7%) suggested that strain KMD 002T is a representative of a novel species, for which the name Colwellia asteriadis sp. nov. is proposed. The type strain is KMD 002T (=KCCM 90077T =JCM 15608T).

Human dermal fibroblast proliferation activity of usimine-C from Antarctic lichen Ramalina terebrata.

Type I collagen is the major structural protein in dermis and its presence is used to monitor skin cell proliferation and aging. Recently, novel usimine compounds have been found in the Antarctic lichen Ramalina terebrata. In the present study, usimine-C induced cell proliferation of human dermal fibroblast, CCD-986SK, up to 1.6-fold after treating with 90 microg/ml for 48 h. Type I procollagen synthesis was significantly increased 1.3-fold, 3-fold, and 5-fold after treating with 0.14, 0.72, and 3.6 microg usimine-C/ml for 24 h, respectively, whereas no significant increase in type I procollagen was observed after treating with usimine-A or -B. Usimines are usnic acid derivatives. Considering that the difference among the derivatives is a side chain, the proliferation activity may be related to this side chain, triggering an internal signal for type I procollagen expression. Further studies still remain to clarify the signaling pathways for the type I procollagen induction, which is activated by usimine-C.