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

A gene disruption system for was developed using simvastatin (HMG-CoA reductase encoding gene) for positive selection and 5-Fluoroorotic acid (5-FOA), a 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 , consistently correlated with the gene (encoding a hypoxanthine-guanine phosphoribosyl-transferase). We thus replaced by on our suicide vector and tested strain sensitivity to 6-MP before and after transformation. Wild-Type (WT) is less sensitive to 6-MP than WT , and an increase of cell resistance was achieved after deletion of the gene homologous to . Results confirmed the natural resistance of to 6-MP and show that can confer sensitivity. This new counterselection system vastly improves genetic manipulations in 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., B and B).