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149. Optimal growth conditions and protein expression of psychrophilic bacteria, KOPRI-ArB0140 Moritella sp.
Abstracts / Cryobiology 53 (2006) 367–446 147. Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria. Elias Anastassopoulos, TEI of Larissa, Department of Plant Production, 411 10 Larissa, Greece An agar plate freezing assay is described based on the incorporation of fluorescein dye in the agar medium. Upon addition of fluorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g., in directed evolution experiments involving ice nucleation genes. (Conflict of interest: The described assay is the subject of a patent pending. Source of funding: The General Secretary of Research and Technology, Hellas.) doi:10.1016/j.cryobiol.2006.10.148
148. Growth and amplified fragment length polymorphism analysis of Euglena gracilis after cryopreservation by alginate encapsulation dehydration. Keith Harding a,b, Julia M} uller c, Hella Timmerc c c mann , Maike Lorenz , Thomas Friedl , John G. Day d, a Plant Conservation Group, University Abertay Dundee, Dundee DD1 1HG, UK; b DAMAR, Conservation, Environmental Science & Biotechnology, Cuparmuir, Fife, Scotland KY15 5RJ, UK; c Albrecht-von-Haller-Institut fu¨r Pflanzenwissenschaften, Universit} ut Go¨ttingen, (SAG), 37073 Go¨ttingen, Germany; d Culture Collection of Algae and Protozoa (CCAP), SAMS, Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland PA37 1QA, UK Serial subculture is the most routine maintenance method for microalgae of actively growing cell cultures under suboptimal conditions that extend the transfer intervals to reduce costs and increase resource locations. Although effective, this approach has disadvantages, often being labour intensive, without the guarantee of genetic and phenotypic stability of the strains over decades of maintenance. Cryopreservation in liquid nitrogen where metabolic activity and cellular function is considered to cease at extremely low ( 196 °C) temperatures, is often assumed to prevent genetic changes that alter phenotypic characteristics of the preserved strains. For algal culture collections, cryopreservation may well avoid the selection pressures and/or genetic drift inherent in routine serial subculture. Despite these advantages over serial subculture, the cryopreservation process may induce various stresses on the organism by the formation of intracellular ice, osmotic shock or toxicity of the cryoprotectants. Oxidative stress results in the formation of free radicals during cryoinjury, which may potentially lead to genetic alterations. Fundamental to any conservation technique is the risk of genomic alteration that cannot be excluded, but it is necessary to quantitatively determine its magnitude. Cryopreservation (or certain components of the cryostorage protocol) may allow the selection of algal subpopulations, which differ in their genotypic properties as compared to their non-frozen counterparts, but in the absence of phenotypic changes, may not represent a selective advantage. The genetic stability of plants after cryopreservation can be investigated at the phenotypic, cytological, biochemical and molecular levels. But due to the small cell size of microalgae they frequently lack distinctive morphological characters, where analysis at the molecular level has fundamental appli-
429
cations in their characterization. Although, the genetic stability of algae after cryopreservation requires investigation, there has been only one report evaluating genetic stability in the CCAP. After long-term cryostorage strains of Chlorella vulgaris showed no genomic differences between duplicate (CCAP & SAG) strains of the same isolate maintained by continuous subculture over several decades and neither within those stored for longer than 20 years. Microalgae are diverse taxonomically and morphologically different algae (e.g., flagellates, coccoids, and filamentous) display differential levels of survival using the same cryopreservation methodology, making stability assessments essential to protocol validation. Following the cryopreservation of Euglena gracilis cultures by the alginate bead encapsulation dehydration technique, we report a continuation in genetic stability studies to examine the possible genetic changes detectable by Amplified Fragment Length Polymorphism (AFLP) and alterations to the phenotype resulting in growth characteristic changes during post-thaw recovery. (Conflict of interest: None declared. Source of funding: EU 5th Framework Programme, Quality of Life and Management of Living Resources, COBRA Project QLRT-2000-01645. Carnegie Trust for the Universities of Scotland.)
doi:10.1016/j.cryobiol.2006.10.149
149. Optimal growth conditions and protein expression of psychrophilic bacteria, KOPRI-ArB0140 Moritella sp.. Soyoung Park, Jung Min Lee, Sung-Ho Kang, Polar Applied Science Division, Korea Polar Research Institute, 406-840 Incheon, South Korea More than three-quarters of the earth’s surface is occupied by cold ecosystems, including the deep oceans, and polar and alpine regions. These permanently cold environments have been successfully colonized by a class of extremophilic microorganisms known as psychrophiles. Psychrophiles are adapted not only to low temperature but frequently also to further environmental constraints. We isolated psychrophilic bacteria KOPRIArB0140 Moritella sp. (KCTC 10671BP) from sea water near a glacier of Kongsfjorden in Svalbard Island where the Korean Arctic Dasan Base is located. KOPRI-ArB0140 Moritella sp. is a gram-negative, motile, psychrophilic, rod-shaped bacterium. We tested the optimal growth conditions for Moritella sp. of temperature, salinity, media, pH. The optimal growth temperature was 9 °C and growth temperature range was from 3 to 17 °C. No growth was observed above 18 °C. The optimal salinity was 2.5% NaCl concentration and optimal growth media was tryptic soy broth. The optimal growth pH was 8.5. We measured ice activity for whether KOPRI-ArB0140 Moritella sp. secreted icebinding proteins (IBPs) and also performed protein expression on the freeze-thaw method [Raymond et al. 2001] using 2-DE. There were four major spots in the SDS–PAGE (12%) and the expressed proteins are speculated to be IBPs. The expressed proteins are totally unknown proteins so far. Identification and purification of IBPs are the next steps to understanding the ecophysiology of psychrophilic bacteria. (Conflict of interest: None declared. Source of funding: Korea Polar Research Institute (KOPRI).)
doi:10.1016/j.cryobiol.2006.10.150
148. Growth and amplified fragment length polymorphism analysis of Euglena gracilis after cryopreservation by alginate encapsulation dehydration. Keith Harding a,b, Julia M} uller c, Hella Timmerc c c mann , Maike Lorenz , Thomas Friedl , John G. Day d, a Plant Conservation Group, University Abertay Dundee, Dundee DD1 1HG, UK; b DAMAR, Conservation, Environmental Science & Biotechnology, Cuparmuir, Fife, Scotland KY15 5RJ, UK; c Albrecht-von-Haller-Institut fu¨r Pflanzenwissenschaften, Universit} ut Go¨ttingen, (SAG), 37073 Go¨ttingen, Germany; d Culture Collection of Algae and Protozoa (CCAP), SAMS, Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland PA37 1QA, UK Serial subculture is the most routine maintenance method for microalgae of actively growing cell cultures under suboptimal conditions that extend the transfer intervals to reduce costs and increase resource locations. Although effective, this approach has disadvantages, often being labour intensive, without the guarantee of genetic and phenotypic stability of the strains over decades of maintenance. Cryopreservation in liquid nitrogen where metabolic activity and cellular function is considered to cease at extremely low ( 196 °C) temperatures, is often assumed to prevent genetic changes that alter phenotypic characteristics of the preserved strains. For algal culture collections, cryopreservation may well avoid the selection pressures and/or genetic drift inherent in routine serial subculture. Despite these advantages over serial subculture, the cryopreservation process may induce various stresses on the organism by the formation of intracellular ice, osmotic shock or toxicity of the cryoprotectants. Oxidative stress results in the formation of free radicals during cryoinjury, which may potentially lead to genetic alterations. Fundamental to any conservation technique is the risk of genomic alteration that cannot be excluded, but it is necessary to quantitatively determine its magnitude. Cryopreservation (or certain components of the cryostorage protocol) may allow the selection of algal subpopulations, which differ in their genotypic properties as compared to their non-frozen counterparts, but in the absence of phenotypic changes, may not represent a selective advantage. The genetic stability of plants after cryopreservation can be investigated at the phenotypic, cytological, biochemical and molecular levels. But due to the small cell size of microalgae they frequently lack distinctive morphological characters, where analysis at the molecular level has fundamental appli-
429
cations in their characterization. Although, the genetic stability of algae after cryopreservation requires investigation, there has been only one report evaluating genetic stability in the CCAP. After long-term cryostorage strains of Chlorella vulgaris showed no genomic differences between duplicate (CCAP & SAG) strains of the same isolate maintained by continuous subculture over several decades and neither within those stored for longer than 20 years. Microalgae are diverse taxonomically and morphologically different algae (e.g., flagellates, coccoids, and filamentous) display differential levels of survival using the same cryopreservation methodology, making stability assessments essential to protocol validation. Following the cryopreservation of Euglena gracilis cultures by the alginate bead encapsulation dehydration technique, we report a continuation in genetic stability studies to examine the possible genetic changes detectable by Amplified Fragment Length Polymorphism (AFLP) and alterations to the phenotype resulting in growth characteristic changes during post-thaw recovery. (Conflict of interest: None declared. Source of funding: EU 5th Framework Programme, Quality of Life and Management of Living Resources, COBRA Project QLRT-2000-01645. Carnegie Trust for the Universities of Scotland.)
doi:10.1016/j.cryobiol.2006.10.149
149. Optimal growth conditions and protein expression of psychrophilic bacteria, KOPRI-ArB0140 Moritella sp.. Soyoung Park, Jung Min Lee, Sung-Ho Kang, Polar Applied Science Division, Korea Polar Research Institute, 406-840 Incheon, South Korea More than three-quarters of the earth’s surface is occupied by cold ecosystems, including the deep oceans, and polar and alpine regions. These permanently cold environments have been successfully colonized by a class of extremophilic microorganisms known as psychrophiles. Psychrophiles are adapted not only to low temperature but frequently also to further environmental constraints. We isolated psychrophilic bacteria KOPRIArB0140 Moritella sp. (KCTC 10671BP) from sea water near a glacier of Kongsfjorden in Svalbard Island where the Korean Arctic Dasan Base is located. KOPRI-ArB0140 Moritella sp. is a gram-negative, motile, psychrophilic, rod-shaped bacterium. We tested the optimal growth conditions for Moritella sp. of temperature, salinity, media, pH. The optimal growth temperature was 9 °C and growth temperature range was from 3 to 17 °C. No growth was observed above 18 °C. The optimal salinity was 2.5% NaCl concentration and optimal growth media was tryptic soy broth. The optimal growth pH was 8.5. We measured ice activity for whether KOPRI-ArB0140 Moritella sp. secreted icebinding proteins (IBPs) and also performed protein expression on the freeze-thaw method [Raymond et al. 2001] using 2-DE. There were four major spots in the SDS–PAGE (12%) and the expressed proteins are speculated to be IBPs. The expressed proteins are totally unknown proteins so far. Identification and purification of IBPs are the next steps to understanding the ecophysiology of psychrophilic bacteria. (Conflict of interest: None declared. Source of funding: Korea Polar Research Institute (KOPRI).)
doi:10.1016/j.cryobiol.2006.10.150