Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica

M.K. and P.N.G. designed the work; T.N.C. performed physiological studies; M.K., M.F., Y.A.-R., A.B., N.L.-C., M.E.G., O.R.K., T.Y.N., S.K., I.L., O.V.G., M.M.Y. R.R. and P.N.G. were associated with genome annotation; H.J.H. performed lipids and FAME analysis; M.F., M-l.F., S.J., S.C. and J.P.A performed chaperonin anti-proteome analysis; A.-x. S., O.K., O.E., P.A.P., P.S. and Y.K. were associated with structural proteomics; A.T. and R.F. were associated with functional proteomics; H.L. performed electron microscopy; R.D. performed real-time PCR; M.M.-G. and M.F. performed DIGE proteome analysis; M.G. was involved in siderophore production; O.N.R. performed genomic islands’ analysis; H.T. performed storage lipid compounds’ analysis; P.N.G. coordinated manuscript writing.Accession Codes: The genome sequence of Oleispira antarctica RB-8 has been deposited in GenBank under accession core FO203512. Protein structures have deposited in PDB under accession codes 3QVM (a/b hydrolase, OLEAN_C08020), 3QVQ (phosphodiesterase, OLEAN_C20330), 3M16 (transaldolase, OLEAN_C18160), 3LQY (isochorismatase, OLEAN_C07660), 3LNP (amidohydrolase, OLEAN_C13880), 3V77/3L53 (fumarylacetoacetate isomerase/hydrolase, OLEAN_C35840), 3VCR/3LAB (2-keto-3-deoxy-6-phosphogluconate aldolase, OLEAN_C25130), 3IRU (phoshonoacetaldehyde hydrolase, OLEAN_C33610), 3I4Q (inorganic pyrophosphatase, OLEAN_C30460), 3LMB (protein with unknown function, OLEAN_C10530).Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis—the paradigm of mesophilic hydrocarbonoclastic bacteria—O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.We acknowledge the funding from the EU Framework Program 7 to support Projects MAMBA (226977), ULIXES (266473), MAGIC PAH (245226) and MICROB3 (287589) This work received the support of the Government of Canada through Genome Canada and the Ontario Genomics Institute (grant 2009-OGI-ABC-1405 to A.F.Y. and A.S.), and the U.S. Government National Institutes of Health (grants GM074942 and GM094585 (to A.S. through Midwest Center for Structural Genomics). The study was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft through project KU 2679/2-1 and BU 890/21-1. We thank the sequencing team of the AG Reinhardt for technical assistance and Alfred Beck for computational support. The skilful work of electron microscopic sample preparation by Mrs. Ingeborg Kristen (Dept. VAM, HZI Braunschweig) is gratefully acknowledged. Authors thank Professor Ken Timmis for his critical reading the manuscript and useful comments.http://www.nature.com/naturecommunicationsam201

Immunocytochemical localization of CDW60 antigens on human peripheral T-cells

About 25 - 35% of human T cells display the CDw60 ganglioside (9-O-acetyl-GD3) antigen at the cell surface [E.P. Rieber, in W. Knapp, B. Dörken, W.R. Gilks, E.P. Rieber, R.E. Schmidt, H. Stein, A.E.G.K. von dem Borne (Eds.), Leucocyte Typing IV, Oxford University, Oxford, 1989, p. 361.]. Other leucocytes do not express this antigen on the cell surface. This led us to investigate its presence by flow cytometry and immunoelectron microscopy (IEM). Flow cytometric analysis of isolated peripheral T cells showed 26% of the cell population to have the CDw60 antigen expressed on the cell surface whereas 74% did not. Similarly, IEM analysis of 262 random T cells by the preembedding immunogold labeling technique revealed CDw60 surface expression to be tetrapartite: (a) the majority of 63.7% of the T cells did not show any surface associated gold label; (b) 19.5% were of low CDw60 surface exposition, corresponding to a linear density of 0.05–2.0 gold markers per ?m; (c) about 13.4% showed a medium surface exposition with a linear density of 2.1–4.5 gold markers per ?m; and (d) a high exposition, ranging from 4.6 to 9.0 gold markers per ?m, was seen at 3.4% of the T cells. From postembedding label experiments, which additionally make access to the antigen localized within the cytoplasm, it was found that nearly all T cells contained low levels of intracellular CDw60. Most of it was found to be associated with the cytoplasmic membrane or vesicles, derived from the Golgi. Immunogold conjugates associated with the cytoplasmic membrane showed a linear density up to 0.6 gold markers per µm. The asymmetric expression of the CDw60 antigen on human T cells and its occurrence in nearly all T cells suggests that its surface presentation is tightly regulated

Thiobaca trueperi gen. nov., sp nov., a phototrophic purple sulfur bacterium isolated from freshwater lake sediment

Two strains of a novel species of phototrophic micro-organism were isolated from the sediments of a shallow, freshwater, eutrophic lake. Both strains grew photolithoheterotrophically with sulfide as an electron donor, transiently accumulating intracellular sulfur globules. Photolithoautotrophic growth was not observed. One strain was designated BCHT (the type strain) and was studied in most detail. Cells contained bacteriochlorophyll a, and the dominant carotenoid was lycopene. Cell suspensions were brown. The photosynthetic membranes had a vesicular arrangement. Acetate, propionate, pyruvate, succinate and fumarate were each used as electron donors and carbon sources in the presence of sulfide and bicarbonate. In the presence of light, growth did not occur with hydrogen, thiosulfate or iron(II). The optimum temperature for growth was between 25 and 30 degreesC, the maximum being 36 degreesC. The G+C content of the genomic DNA of strain BCHT was 63 mol %. Analysis of the 16S RNA genes showed that both strains belonged to the gamma-subclass of the Proteobacteria but were phylogenetically distinct from any described phototrophic organisms within the Chromatiaceae. On the basis of phylogenetic and physiological differences from other phototrophic microorganisms, strain BCHT is described as a novel species of a new genus, Thiobaca trueperi gen. nov., sp. nov

Initiation-Factor EIF-4E of Saccharomyces-Cerevisiae - Distribution Within the Cell, Binding to Messenger- RNA, and Consequences of its Overprodutction

The eukaryotic translational initiation factor 4E (eIF-4E) is an essential protein that binds the 5' cap structure with high specificity and affinity. Yeast eIF-4E is homologous to eIF-4E of higher eukaryotes, but interacts with a different set of cap-binding complex proteins. In the present study the distribution of yeast eIF-4E in Saccharomyces cerevisiae was found to be similar to that observed in higher cells, whereby the yeast factor was more concentrated in the nucleus than in the cytoplasm. Overexpression of yeast eIF-4E in S. cerevisiae exerted at most a minimal effect on growth in liquid minimal medium and was not found to influence the translation of reporter gene mRNAs bearing secondary structure in their leader regions. In a new method to study mRNA-protein interactions, biotinylated mRNAs were synthesized in vitro for use in studies of the binding of eIF-4E in yeast extracts. Streptavidin was used to adsorb the biotinylated mRNAs plus bound initiation factors. Stem-loop structures in the leader region did not influence the binding of eIF-4E or, in comparative experiments, of eIF-4A Thus yeast eIF-4E shows both similarities and differences with respect to the distribution and function of its counterparts in higher eukaryotes

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers

Werner A, Duvar S, Müthing J, et al. Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers. BIOTECHNOLOGY AND BIOENGINEERING. 2000;68(1):59-70.Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 mu g/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 mu g/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary meta-belie functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system. (C) 2000 John Wiley & Sons, Inc

Thiobaca trueperi gen. nov., sp nov., a phototrophic purple sulfur bacterium isolated from freshwater lake sediment

Two strains of a novel species of phototrophic micro-organism were isolated from the sediments of a shallow, freshwater, eutrophic lake. Both strains grew photolithoheterotrophically with sulfide as an electron donor, transiently accumulating intracellular sulfur globules. Photolithoautotrophic growth was not observed. One strain was designated BCHT (the type strain) and was studied in most detail. Cells contained bacteriochlorophyll a, and the dominant carotenoid was lycopene. Cell suspensions were brown. The photosynthetic membranes had a vesicular arrangement. Acetate, propionate, pyruvate, succinate and fumarate were each used as electron donors and carbon sources in the presence of sulfide and bicarbonate. In the presence of light, growth did not occur with hydrogen, thiosulfate or iron(II). The optimum temperature for growth was between 25 and 30 degreesC, the maximum being 36 degreesC. The G+C content of the genomic DNA of strain BCHT was 63 mol %. Analysis of the 16S RNA genes showed that both strains belonged to the gamma-subclass of the Proteobacteria but were phylogenetically distinct from any described phototrophic organisms within the Chromatiaceae. On the basis of phylogenetic and physiological differences from other phototrophic microorganisms, strain BCHT is described as a novel species of a new genus, Thiobaca trueperi gen. nov., sp. nov

Immobilization of uranium in biofilm microorganisms exposed to groundwater seeps over granitic rock tunnel walls in Olkiluoto, Finland

In an underground rock characterization facility, the ONKALO tunnel in Finland, massive 5-10-mm thick biofilms were observed attached to tunnel walls where groundwater was seeping from bedrock fractures at a depth of 70 m. In laboratory experiments performed in a flow cell with detached biofilms to study the effect of uranium on the biofilm, uranium was added to the circulating groundwater (CGW) obtained from the fracture feeding the biofilm. The final uranium concentration in the CGW was adjusted to 4.25 x 10(-5) M, in the range expected from a leaking spent nuclear fuel (SNF) canister in a future underground repository. The effects were investigated using microelectrodes to measure pH and E-h, time-resolved laser fluorescence spectroscopy (TRLFS), energy-filtered transmission electron microscopy (EF-TEM), and electron energy-loss spectroscopy (EELS) studies and thermodynamic calculations were utilized as well. The results indicated that the studied biofilms constituted their own microenvironments, which differed significantly from that of the CGW. A pH of 5.37 was recorded inside the biofilm, approximately 3.5 units lower than the pH observed in the CGW, due to sulfide oxidation to sulfuric acid in the biofilm. Similarly, the E-h of +73 mV inside the biofilm was approximately 420 mV lower than the E-h measured in the CGW. Adding uranium increased the pH in the biofilm to 7.27 and reduced the E-h to -164 mV. The changes of E-h and pH influenced the bioavailability of uranium, since microbial metabolic processes are sensitive to metals and their speciation. EF-TEM investigations indicated that uranium in the biofilm was immobilized intracellularly in microorganisms by the formation of metabolically mediated uranyl phosphate, similar to needle-shaped autunite (Ca[UO2](2)[PO4](2)center dot 2-6H(2)O) or meta-autunite (Ca[UO2](2)[PO4](2)center dot 10-12H(2)O). In contrast, TRLFS studies of the contaminated CGW identified aqueous uranium carbonate species, likely (Ca2UO2[CO3](3)), formed due to the high concentration of carbonate in the CGW. The results agreed with thermodynamic calculations of the theoretically predominant field of uranium species, formed in the uranium-contaminated CGW at the measured geochemical parameters. This investigation clearly demonstrated that biological systems must be considered as a part of natural systems that can significantly influence radionuclide behavior. The results improve our understanding of the mechanisms of biofilm response to radionuclides in relation to safety assessments of SNF repositories. (C) 2012 Elsevier Ltd. All rights reserved