Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus

Abstract

A gene disruption system for <i>Thermococcus barophilus</i> was developed using simvastatin (HMG-CoA reductase encoding gene) for positive selection and 5-Fluoroorotic acid (5-FOA), a <i>pyrF</i> gene for negative selection. Multiple gene mutants were constructed with this system, which offers the possibility of complementation in trans, but produces many false positives (<80%). To significantly reduce the rate of false positives, we used another counterselective marker, 6-methylpurine (6-MP), a toxic analog of adenine developed in <i>Thermococcus kodakarensis</i>, consistently correlated with the <i>TK0664</i> gene (encoding a hypoxanthine-guanine phosphoribosyl-transferase). We thus replaced <i>pyrF</i> by <i>TK0664</i> on our suicide vector and tested <i>T. barophilus</i> strain sensitivity to 6-MP before and after transformation. Wild-Type (WT) <i>T. barophilus</i> is less sensitive to 6-MP than WT <i>T. kodakarensis</i>, and an increase of cell resistance was achieved after deletion of the <i>T. barophilus</i><i>TERMP_00517</i> gene homologous to <i>T. kodakarensis</i><i>TK0664</i>. Results confirmed the natural resistance of <i>T. barophilus</i> to 6-MP and show that <i>TK0664</i> can confer sensitivity. This new counterselection system vastly improves genetic manipulations in <i>T. barophilus</i> MP, with a strong decrease in false positives to <15%. Using this genetic tool, we have started to investigate the functions of several genes involved in genomic maintenance (e.g., <i>pol</i>B and <i>rnh</i>B).