Project description:Experimentally mapped transcriptome structure of Pyrococcus furiosus DSM 3638 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 16 nt).
Project description:Experimentally mapped transcriptome structure of Pyrococcus furiosus DSM 3638 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 16 nt). Pyrococcus furiosus DSM 3638 growth curve experiments were conducted in batch culture. Reference samples were cultured at mid-log phase (OD600 = 0.096). Seven samples were collected that spanned the key phases of the growth curve. Total RNA from samples of growth curve and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. Dye-flip experiments were done for each sample. Log ratios were calculated for each probe (growth curve sample/reference). Transcriptome browser is available at http://baliga.systemsbiology.net/enigma/.
Project description:In recent years, there has been a large increase in the amount of experimental evidence for diverse archaeal organisms, and these findings allow for a comprehensive analysis of archaeal genetic organization. However, studies about regulatory mechanisms in this cellular domain are still limited. In this context, we identified a repertoire of 86 DNA-binding transcription factors (TFs) in the archaeon Pyrococcus furiosus DSM 3638, that are clustered into 32 evolutionary families. In structural terms, 45% of these proteins are composed of one structural domain, 41% have two domains, and 14% have three structural domains. The most abundant DNA-binding domain corresponds to the winged helix-turn-helix domain; with few alternative DNA-binding domains. We also identified seven regulons, which represent 13.5% (279 genes) of the total genes in this archaeon. These analyses increase our knowledge about gene regulation in P. furiosus DSM 3638 and provide additional clues for comprehensive modeling of transcriptional regulatory networks in the Archaea cellular domain.
Project description:Glycerophosphodiester phosphodiesterases (GDPD) are enzymes which degrade various glycerophosphodiesters to produce glycerol-3-phosphate and the corresponding alcohol moiety. Apart from this, a very interesting finding is that this enzyme could be used in the degradation of toxic organophosphorus esters, which has resulted in much attention on the biochemical and application research of GDPDs. In the present study, a novel GDPD from Pyrococcus furiosus DSM 3638 (pfGDPD) was successfully expressed in Escherichia coli and biochemically characterized. This enzyme hydrolyzed bis(p-nitrophenyl) phosphate, one substrate analogue of organophosphorus diester, with an optimal reaction temperature 55 °C and pH 8.5. The activity of pfGDPD was strongly dependent on existing of bivalent cations. It was strongly stimulated by Mn(2+) ions, next was Co(2+) and Ni(2+) ions. Further investigations were conducted on its substrate selectivity towards different phospholipids. The results indicated that except of glycerophosphorylcholine (GPC), this enzyme also possessed lysophospholipase D activity toward both sn1-lysophosphatidylcholine (1-LPC) and sn2-lysophosphatidylcholine (2-LPC). Higher activity was found for 1-LPC than 2-LPC; however, no hydrolytic activity was found for phosphatidylcholine (PC). Molecular docking based on the 3D-modeled structure of pfGDPD was conducted in order to provide a structural foundation for the substrate selectivity.
