FIG. 4.

(A) Results of LC-MS-MS analysis of E. coli tRNA extracted from different strains. Results for WT (W6) are in upper panels; results for VDC5211 (ΔfolE::Kan^r) are in lower panels; the UV traces (left) at 254 nm and the extraction ion chromatograms (right) for 410 m/z are shown. (B) Complementation of the Q⁻ phenotype analyzed by LC-MS-MS. The ratios of Q/Ψ in tRNA extracted from the E. coli ΔfolE::Kan^r strains complemented with E. coli folE (VDC7049), B. subtilis folE2 (VDC5196), or M. maripaludis folE2 (VDC5198) are shown by the filled bars. To control for the amount of tRNA, the Ψ content in the complemented strains was compared to the Ψ content in the WT control (white bars). The result of a typical experiment is presented. (C) Upper panel shows results for complementation of the ΔfolE::Kan^r strain dT auxotrophy by the archaeal folE genes on LB supplemented with Amp and dT (left plate), LB supplemented with Amp (middle plate), and LB supplemented with Amp and 0.02% arabinose (right plate). The control is ΔfolE::Kan^r transformed by pBAD24. The plates were incubated at 37^°C for 48 h. Lower panel shows results of PCR amplifications to check for the presence of the ΔfolE::Kan^r allele and to check for the presence of an insert of the expected size in the pBAD derivatives (performed as described in Materials and Methods on the ΔfolE::Kan^r strain transformed with plasmids expressing M. maripaludis folE2 or S. solfataricus folE or with the pBAD24 control). The expected size of the PCR product from detecting ΔfolE::Kan^r is about 1.5 kb, while the same primers amplify a 1.2-kb product in the WT strain.