Project description:The 28,000-year-old remains of a woolly mammoth, named ‘Yuka’, were found in Siberian permafrost. We performed proteomic analyses of muscle and bone marrow samples obtained from the remains to gain information about the repertoire and modifications of proteins.
Project description:Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.
Project description:Permafrost soils are extreme environments that exert low-temperature, desiccation and starvation stress on bacteria over thousands to millions of years. To understand how Psychrobacter arcticus 273-4 survived for > 20,000 years in permafrost, transcriptome analysis was performed during growth at 22°C, 17°C, 0°C, and -6°C using a mixed effects ANOVA model. Genes for transcription, translation, energy production and most biosynthetic pathways were down-regulated at low temperatures. Evidence of isozyme exchange was detected over temperature for D-alanyl-D-alanine carboxypeptidases (dac1 and dac2), DEAD-box RNA helicases (csdA and Psyc_0943) and energy efficient substrate incorporation pathways for ammonium and acetate. Specific functions were compensated by up-regulation at low temperature including genes for the biosynthesis of proline, tryptophan, methionine, and histidine. RNases and peptidases were generally up-regulated at low temperatures. Changes in energy metabolism, amino acid metabolism, and RNase gene expression were consistent with induction of the stringent response by relA activity. In contrast to results observed in other psychrophiles and mesophiles, only clpB and hsp33 were up-regulated at low temperature with no up-regulation of other chaperones and peptidyl-prolyl isomerases. Knockout mutants of relA, csdA, and dac2 were all deficient in low temperature growth, but a mutant in dac1 was deficient in growth at 17°C. The combined data suggest that the basal biological machinery including translation, transcription and energy metabolism are well adapted to function across the -6°C to 22°C growth range of P. arcticus and temperature compensation by gene expression was employed to address specific challenges to low-temperature growth.
Project description:Haloalkane dehalogenases are hydrolytic enzymes with a broad range of potential practical applications such as biodegradation, biosensing, biocatalysis and cellular imaging. Two newly isolated psychrophilic haloalkane dehalogenases exhibiting interesting catalytic properties, DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17, were purified and used for crystallization experiments. After the optimization of crystallization conditions, crystals of diffraction quality were obtained. Diffraction data sets were collected for native enzymes and complexes with selected ligands such as 1-bromohexane and 1,2-dichloroethane to resolutions ranging from 1.05 to 2.49 Å.
Project description:Permafrost soils are extreme environments that exert low-temperature, desiccation and starvation stress on bacteria over thousands to millions of years. To understand how Psychrobacter arcticus 273-4 survived for > 20,000 years in permafrost, transcriptome analysis was performed during growth at 22°C, 17°C, 0°C, and -6°C using a mixed effects ANOVA model. Genes for transcription, translation, energy production and most biosynthetic pathways were down-regulated at low temperatures. Evidence of isozyme exchange was detected over temperature for D-alanyl-D-alanine carboxypeptidases (dac1 and dac2), DEAD-box RNA helicases (csdA and Psyc_0943) and energy efficient substrate incorporation pathways for ammonium and acetate. Specific functions were compensated by up-regulation at low temperature including genes for the biosynthesis of proline, tryptophan, methionine, and histidine. RNases and peptidases were generally up-regulated at low temperatures. Changes in energy metabolism, amino acid metabolism, and RNase gene expression were consistent with induction of the stringent response by relA activity. In contrast to results observed in other psychrophiles and mesophiles, only clpB and hsp33 were up-regulated at low temperature with no up-regulation of other chaperones and peptidyl-prolyl isomerases. Knockout mutants of relA, csdA, and dac2 were all deficient in low temperature growth, but a mutant in dac1 was deficient in growth at 17°C. The combined data suggest that the basal biological machinery including translation, transcription and energy metabolism are well adapted to function across the -6°C to 22°C growth range of P. arcticus and temperature compensation by gene expression was employed to address specific challenges to low-temperature growth. Cy-dye labelled cDNA populations were generated from total RNA extracted from mid-exponential growth phase cultures of Psychrobacter arcticus 273-4 grown at 22°C, 17°C (optimal growth rate), 0°C, and -6°C. All pairwise temperature comparisons were performed in this 4-level single factor experiment with dye swapping nested within biological replicate. Five biological replicates (20 samples totalled over all temperatures) were included in each comparison for a total of 30 hybridizations. Data were analyzed using R/MAANOVA with an ANOVA model which included gene, dye, biological replicate, slide, and growth temperature terms (with interactions). Spike-in mRNAs were included immediate following the cell lysis stage in order to serve as monitors for RNA degradation from lysis through hybridization.