Development of a Fluorescent Reporter-System to Quantify Gene Expression in vivo in the Hyperthermophilic Archaeon, Thermococcus kodakarensis

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The Ohio State University

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The hyperthermophilic anaerobic archaeon Thermococcus kodakarensis has emerged as the most complete model system for genetic dissection of the unique and widely varied metabolisms encoded in archaeal genomes. The genetic toolkit currently available is substantial, but the demands for ever more complicated experimentation continue, necessitating improvements and additions to the genetic techniques that can be employed. Reporter gene systems are integral tools in molecular biology to understand gene function or regulation and many commonly used reporter genes induce visually identifiable characteristics involving fluorescent or luminescent proteins. These proteins are usually derived from mesophilic organisms and the photochemistry involved almost always depends on the presence of oxygen, which excludes their usage in hyperthermophilic anaerobic Archaea. To expand the molecular toolbox for this important group of archaeal species, we are developing a new versatile reporter gene system based on TK1971, encoding a O6-methylguanine-DNA methyltransferase (MGMT). Naturally, MGMT is involved in DNA repair, recognizing and removing methyl groups form methylated guanine residues. In this reaction, the methyl group is transferred and covalently linked to the protein. In the envisioned reporter system, we will take advantage of the enzymatic properties by using commercially available flurophore-coupled guanine analogs to covalently label MGMT. To limit endogenous background activity of the reporter system, a knock-out strain of MGMT (ΔTK1971) has been generated using a recently developed markerless deletion technique. Furthermore, several shuttle plasmids were constructed carrying PCR-mutagenized versions of a genetically engineered MGMT gene.



Archaea, Thermococcus, reporter gene, confocal scanning laser microscopy, methylguanine methyltransferase