FIGURE 5.

Plasmid stability in the Thermus. thermophilus mutants. (A) Comparison of plasmid loss frequencies using a replicative plasmid conferring Hyg resistance (pMotH1103A) in HB27pyrEK control strain and in ppol:kat and addAB:kat insertion mutant strains. Transformed cells were re-streaked twice on selective plates and grown on selective media liquid for 24 h. After that, cells were further incubated for another 48 h in the absence of antibiotic to allow plasmid loss to occur. We finally plated cells on both non-selective and selective plates and the plasmid loss frequency was measured as the CFUs on selective media divided by CFUs on non-selective plates (total cells). (B) Comparison of plasmid loss frequencies using a replicative plasmid conferring Kn resistance (pMotK1103A) in HB27 control strain and its derivatives ppol:lox72, ppol_cat, ppol_comp, addAB:lox72 and addAB_ppol mutants. Each data point is an average of 3 independent experiments. Error bars correspond to the standard deviation of the means. Asterisks indicate significant statistical differences (**P-value <0.0002 when comparing the mean of addAB:lox72 with the mean of addAB_ppol).