POWRE: Strategies for the Construction of Knock out Mutants in Hyperthermophilic archaea

University Of Maryland Biotechnology Institute, Baltimore MD

Investigators

Abstract

The long-term goals of the project are to determine the molecular mechanisms that allow hyperthermophiles to survive at temperatures near the boiling point of water. The underlying hypothesis of these studies is that growth at temperature in excess of 90oC requires the presence of co-regulated DNA repair genes. In previous studies, sequence analysis, physiological, and biochemical studies have been used to investigate DNA repair processes and maintenance of genome integrity in hyperthermophiles. Although this approach has been very successful, it is increasingly clear that new techniques must be developed to understand how hyperthermophiles prevent thermodegradation of their DNA. The goal of this work is to establish genetic methods for gene disruption in hyperthermophiles. These methods are widely used to determine gene function in Bacteria and Eukarya, but have yet to be applied in hyperthermophilic Archaea. To achieve this goal, the Principal Investigator will visit Dr. Forterre's laboratory in Paris and receive training in manipulation of archaeal vectors and transformation protocols for hyperthermophiles. The Principal Investigator will use the information obtained from comparative genome analysis to design selectable markers for shuttle vectors. Two strategies will be used to accomplish gene disruption, in vitro transposon mutagenesis and knock out mutant constructions of known genes. With the first strategy, new DNA repair mutants will be identified and characterized. The second strategy will yield information necessary to determine the functions in vivo of repair genes previously identified. Mutant phenotypes will be investigated for their sensitivity to DNA damaging agents. The location and identity of the disrupted genes will be determined, and regulation of their expression in cells exposed to mutagenizing treatments will be analyzed. This POWRE award will allow Dr. DiRuggiero to develop an independent line of research by learning new skills and methodologies.