Development and Characterization of a Salt-Tolerant Luciferase to Investigate Genetic Regulation in Haloferax volcanii

Abstract

Haloarchaea, especially Haloferax volcanii, are of particular interest for genetics research due to the wide array of tools and procedures available. However, few reporters exist for Archaea that provide a measurable output in vivo. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesohalic proteins lack these characteristics. However, proteins with salt-stabilizing mutations can lead to proper function in haloarchaea. Using laboratory directed evolution, a salt tolerant luciferase was developed and applied for use in genetic studies involving a previously described inducible promoter and genetic translation inhibitor in H. volcanii. A luciferase assay offers several advantages over other reporter systems: capturing data is done in real time due to a high temporal fidelity; data can be captured continuously using solid or liquid media with proper equipment; and the reporter gene itself is innocuous to the organism. Lastly, the individual mutations of the newly developed luciferase were characterized in order to identify changes in the protein leading to improved haline environment luminescence.