Figure 3.

Changes in aa-tRNA levels upon CdiA–CT^EC869 induction in vivo. (A) Growth inhibition by CdiA–CT^EC869 induction. Overnight cultures of E. coli MG1655 transformed with pBAD33 (control) and pBAD33_CdiA–CT^EC869 were serially diluted, and the dilutions were spotted on LB agar plates containing 50 μg/ml chloramphenicol and supplemented with 1% (w/v) arabinose (lower panel) or 1% (w/v) glucose (upper panel). (B) Growth curves of E. coli MG1655 transformed with pBAD33 and pBAD33_CdiA–CT^EC869. Induction of CdiA–CT^EC869 suppresses the growth of E. coli harboring pBAD33_CdiA–CT^EC869 in LB containing 50 μg/ml chloramphenicol. When the OD₆₆₀ reached ∼0.3, arabinose was added (final concentration 0.02%) to the medium, and the culture was continued at 37°C. The arrow in the graph indicates the point of CdiA–CT^EC869 induction by arabinose addition. (C) Schematic diagram of quantification and comparison of relative amounts of each aa-tRNA by LC/MS. (D) LC/MS analysis of RNase I–digested fragments of Ac-aa-tRNAs (or formyl-methionyl-tRNA_f^Met, fMet-A76) prepared from aa-tRNAs from E. coli with (pBAD33_CdiA–CT) or without (pBAD33, control) induction of CdiA–CT^EC869. The amount of each Ac-aa-A76 derived from aa-tRNA prepared from cells with or without CdiA–CT^EC869 induction is expressed as the amount relative to Ac-Ala-A76 in cells with or without induction of CdiA–CT^EC869, respectively. (E) Change of relative amounts of each aa-tRNA (or fMet-tRNA_f^Met) in E. coli after CdiA–CT^EC869 induction. The relative amounts of each aa-tRNA in E. coli with CdiA–CT^EC869 induction were normalized against the amount of the corresponding aa-tRNA in E. coli without induction of CdiA–CT^EC869. Error bars represent SDs of more than three independent experiments, and the data are presented as mean values ± SD. (F) Cleavage of each tRNA in E. coli was also evaluated by northern blotting of tRNAs (0.125 μg) prepared from E. coli with (+) or without (-) induction of CdiA–CT^EC869.