9631006 Schroder The objective of this project is to study the mechanism of denitrification under extremely thermophilic conditions. The focus of the research will be the enzyme of the first step of denitrification, nitrate reductase. The hyperthermophile archaeon which has an optimal growth temperature of 100 C will be Pyrobaculum aerophilum studied as the model system. The objectives of this project include: (1) the improvement of cell culture conditions for P. aerophilum. This will be done using pressurized vials and/or a fed-batch fermentor to prevent the toxic accumulation of nitrite during nitrate respiration. It is anticipated that the cell yields can be increased once the nutritional needs of the organism are better known. We will attempt to identify additional carbon sources, electron donors, and trace elements which may improve cell yields. (2) the purification and physiological characterization of the nitrate reductase will give insight into the biochemistry of a high temperature enzyme. Besides the already existing enzyme assay using artificial electron donor, additional enzyme assays involving quinones as electron donor will be developed to characterize the membrane-bound enzyme. The enzyme will be extracted from the membrane with detergents and purified using appropriate techniques. The biochemical properties of this high temperature enzyme will be examined: Is the high temperature nitrate reductase a molybdenum containing enzyme like the known mesophilic bacterial nitrate reductases? Alternatively, does it contain tungsten as do other metallo-enzymes from thermophilic archaea? What is the nature of electron transfer to the enzyme? (3) to gain further structural, regulatory and evolutionary information, the nitrate reductase genes will be cloned and sequenced. A gene library will be prepared and the nar genes will be cloned using reverse genetics or PCR probing. The DNA sequence should reveal considerable information as to the relatedness to mesophilic counterparts. This res earch project will contribute to our understanding of the energy metabolism in extremely thermophilic organisms and widen our knowledge as to how energy metabolism has evolved. %%% This project is concerned with understanding unusual bacteria known as archae that grow at very high temperatures. Pyrobaculum aerophilum grows best at 100 C and can also grow in the absence of oxygen using nitrate instead. In this work the enzyme nitrate reductase, a key enzyme for using nitrate, will be studied. The enzyme will be purified, and its properties will be determined. This will provide new and interesting information about life at high temperatures and may lead to biotechnology applications that make use of high temperature enzymes. ***
