Macario The mechanisms that regulate heat-shock (stress) genes in the various groups of organisms belonging to the phylogenetic domain archaea have been elucidated. While stress genes in eubacteria and eukaryotes have not been studied for decades, those in archaea are only now attracting interest. It has become clear that study of archaeal stress genes, and their regulatory mechanisms, will provide crucial information for understanding the evolution of the transcriptional machinery involved in the expression of these genes, which are likely to have played a decisive role in positive selection over time, and are definitely keys to the survival of present-day cells. The specific objective is to construct and test a modular cassette with a reporter gene to study functions of the promoter region for the hsp70 (dnaK) heat-shock (stress) gene in the methanogenic archaeon M. mazei S-6. The long-term goal is to develop a multi-purpose system applicable to the analysis of the regulatory mechanisms of other archaeal heat-shock genes, like hsp23 (grpE) and hsp40 (dnaJ). There are virtually no means at the present time to perform in vivo analyses of promoter regions and transcription factors for stress-inducible genes in methanogenic archaea. This project will provide a genetic system that will make these functional analyses possible. This research will create tools for examining the mechanisms by which gene expression is increased by exposure to high temperature in archaebacteria. Archaebacteria are fundamentally different from the other two main kinds of organisms, eubacteria and eukaryotes, and tend to live in unusual environments. Expected practical spin-offs of this research are the generation of: a) an expression system in an archaeal host (i.e., M. mazei S-6) for large-scale production of foreign, recombinant proteins; and b) tools for insertion of useful genes in the M. mazei genome that will broaden the array of substrates this organism can metabolize, and thus multiply its applications in waste bioconversion and bioremediation.
