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Abstract
The objective of this research was to understand the response of the hyperthermophilic archaeon, Pyrococcus furiosus to two environmental stresses, oxidative and cold stress. The first aim was to purify and determine the biochemical and kinetic properties of the protein rubrerythrin (Rr), and determine its role in the organisms response to oxidative stress. The native protein from P. furiosus contains three iron atoms per subunit and reduces hydrogen peroxide using Rd as the electron donor. The gene encoding Rr (PF1283) was cloned and expressed in Escherichia coli growing on various media. These recombinant Rr proteins had reduced iron contents and lower Rd peroxidase activities. A second protein involved in oxidative stress, NADPH:rubredoxin (Rd) oxidoreductase (NROR) was studied to produce an active, recombinant form of the enzyme by a reconstitution protocol using FAD and heat treatment. In addition to studying oxidative stress in P. furiosus, the cold shock response was also studied using cellular, transcriptional, translational and enzymatic approaches. Growth studies were performed to determine the cellular response to cold shock and to continuous growth at low otemperature (72 C). Whole genome DNA microarray analyses compared these transcriptional oprofiles with those of cultures grown at 95 C. The cellular response to a cold shock was a 5 hour acclimation phase, after which growth resumed at a four times lower rate than that at 95 oC.The microarray data revealed three separate types of responses, involving the up-regulation (> 2.5 fold, P < 0.01) of 55, 30 and 59 ORFs, respectively.