Fig. 3.

AK_Tn complementation of PQN1. (A) Single colonies of PQN1 and HB8 cells grown at 65°C on EvTM-agar plates were used to inoculate 5 ml EvTM cultures. After an overnight incubation at 60°C while shaking at 150 rpm, 1 ml of each culture was used to inoculate fresh 50 ml EvTM cultures. These cultures were grown at the indicated temperatures, the change in optical density at 600 nm was measured until the stationary phase was reached (≥12 h) and the growth rate was calculated. For each strain and temperature, the mean and standard error were calculated from three independent measurements. (B) AK_Tn complementation of PQN1 on solid medium. Colonies of HB8, PQN1 and PQN1 transformed with a plasmid expressing full-length AK_Tn streaked on EvTM-Gelrite plates after incubation at 65, 78 and 81°C for 48 h. (C) PQN1 complementation by AK_Tn fragments. Colonies of PQN1 transformed with plasmids that coexpress TnN and TnC, TnN and TnC^C133A and TnN and TnC^C156A streaked on EvTM-Gelrite plates after incubation for 48 h at 65, 78 and 81°C. Representative streaks are shown from selections that were performed in triplicate.