Mutation sites in AT14. (A) Deletion of Gln143 in TTC0795 (upper panel) and comparison of heat stability between TTC0795 proteins from the wild-type strain and AT14 (lower panel). Lane 1, not heated; 2, 31.0°C; 3, 34.1°C; 4, 36.1°C; 5, 39,0°C; 6, 41.0°C; 7, 42.4°C; 8, 45.2°C; 9, 50.2°C; 10, 56.0°C; 11, 60.8°C; 12, 63.5°C; 13, 65°C. (B) Deletion of Leu175 in TTC0969 (upper panel) and comparison of heat stability between TTC0969 proteins from wild-type strain and AT14 (lower panel). Lanes are the same as those for panel A. (C) Growth of AT14 in MM at permissive and nonpermissive temperatures. Lanes 1 and 2, cultured at 70°C; lanes 3 and 4, cultured at 50°C. Lanes 1 and 3, culture in the absence of 500 μM AEC; lanes 2 and 4, culture in the presence of 500 μM AEC. (D) TTC0966-TTC0970 region in wild-type T. thermophilus HB27. (E) TTC0793-TTC0807 region in wild-type T. thermophilus HB27. Periplasmic substrate-binding protein is abbreviated as SBP.