Numerous members of the third kingdom, Archaea, are extreme hyperthermophiles. Such organisms pose many interesting fundamental questions in molecular biology, since all of their life processes occur at elevated temperatures. For instance, how do regulatory proteins recognize and bind to operators at temperatures above 100 degrees C? How can the DNA replicate with high fidelity, and how do these organisms avoid excessive mutation due to heat-induced DNA lesions, and what repair strategies do they employ? We propose to develop a functional genomic analysis of one of the high temperature archaea, Pyrobaculum aerophilum, that can grow up to 104 degrees C, with an optimal growth temperature of 100 degrees C. We have sequenced and fully annotated the 2.2 mega base genome of Pyrobaculum aerophilum, and are completing the construction of microarrays containing every ORF, in collaboration with Todd Lowe at Stanford/U.C. Santa Cruz. We will carry out experiments on genome-wide gene expression in different environments, and use the data to identify coregulated genes. Through extended analyses, we hope to use the data to set up experiments to identify regulatory pathways and regulatory proteins. We will extend the development of a genetic system with the aim of being able to carry out reverse genetics in this organism. We have begun an indepth investigation of the repair systems in this organism, and we will continue to characterize DNA repair strategies and proteins.
