Project description:Dihydrodipicolinate synthase (DHDPS) is an oligomeric enzyme that catalyzes the first committed step of the lysine-biosynthesis pathway in plants and bacteria, which yields essential building blocks for cell-wall and protein synthesis. DHDPS is therefore of interest to drug-discovery research as well as to studies that probe the importance of quaternary structure to protein function, stability and dynamics. Accordingly, DHDPS from the psychrophilic (cold-dwelling) organism Shewanella benthica (Sb-DHDPS) was cloned, expressed, purified and crystallized. The best crystals of Sb-DHDPS were grown in 200?mM ammonium sulfate, 100?mM bis-tris pH 5.0-6.0, 23-26%(w/v) PEG 3350, 0.02%(w/v) sodium azide and diffracted to beyond 2.5?Å resolution. Processing of diffraction data to 2.5?Å resolution resulted in a unit cell with space group P2(1)2(1)2(1) and dimensions a = 73.1, b = 84.0, c = 143.7?Å. These studies of the first DHDPS enzyme to be characterized from a bacterial psychrophile will provide insight into the molecular evolution of enzyme structure and dynamics.
Project description:Organisms living in deep seas such as the Mariana Trench must be adapted to the extremely high pressure environment. For example, the 3-isopropylmalate dehydrogenase from the obligate piezophile Shewanella benthica DB21MT-2 (SbIPMDH) remains active in extreme conditions under which that from the land bacterium S. oneidensis MR-1 (SoIPMDH) becomes inactivated. In order to unravel the differences between these two IPMDHs, their structures were determined at ~1.5 Å resolution. Comparison of the structures of the two enzymes shows that SbIPMDH is in a more open form and has a larger internal cavity volume than SoIPMDH at atmospheric pressure. This loosely packed structure of SbIPMDH could help it to avoid pressure-induced distortion of the native structure and to remain active at higher pressures than SoIPMDH.
Project description:Physiological and gene expression studies of deep-sea bacteria under pressure conditions similar to those experienced in their natural habitat are critical to understand growth kinetics and metabolic adaptations to in situ conditions. The Epslilonproteobacterium, Nautilia sp. strain PV1, was isolated from hydrothermal fluids released from an active deep-sea hydrothermal vent at 9°N on the East Pacific Rise. Using a high pressure/high temperature continuous culture system we established that strain PV-1 has the shortest generation time of all known piezophilic microorganisms and we investigated its protein expression pattern in response to different hydrostatic pressures. Proteomic analyses of strain PV-1 grown at 200 Bars and 5 Bars showed that pressure adaptation is not restricted only to stress response or homeoviscous adaptation, but that it is more diversified and protein specific, with a fine and variegated regulation of enzymes involved even in the same metabolic pathway. As previously reported, proteins synthesis, motility, transport and energy metabolism are all affected by pressure, although to different extents. In strain PV-1, low pressure condition seems to activate the synthesis of phage-related proteins and an overexpression of enzymes involved in central carbon metabolism.
Project description:The goal of this study was to identify the key functions of the six main symbionts that are hosted in gills of the marine bivalve, Idas modiolaeformis, which lives at deep-sea hydrocarbon seeps and wood falls in the Eastern Atlantic Ocean and the Mediterranean Sea. These symbionts include the main autotrophic methane- and sulfur-oxidizing lineages (Methyloprofundus, Thioglobus, Thiodubillierella), as well as a Methylophagaceae methylotrophic autotroph, a flavobacterial degrader of complex polysaccharides Urechidicola and a Nitrincolaceae heterotroph that specializes in degradation of nitrogen-rich compounds such as peptides and nucleosides. Four I. modiolaeformis individuals were preserved in RNAlater following retrieval from a brine pool habitat in the Eastern Mediterranean at 1,150 m water depth (32° 13.4' N 34° 10.7' E), using a remotely-operated vehicle. RNAlater was discarded after 24 hours, and the specimens were kept at -80°C until DNA/RNA/protein co-extraction using the AllPrep DNA/RNA/Protein Mini Kit (Cat. No. 80004, Qiagen).
