(A) Growth of trmD-deg and trmD-cont G78 cells on an LB plate. An overnight culture of trmD-deg and trmD-cont cells in LB + Glc was serially diluted, spotted on an LB + Ara plate to turn on clpXP expression, and incubated at 37°C overnight. (B) Representative growth of trmD-deg and trmD-cont G78 cells in a liquid LB culture. An overnight culture of trmD-deg and trmD-cont G78 cells in LB + Glc was grown in LB for 1–2 hr, then diluted into LB + Ara at OD600 of 0.1 and grown to OD600 of 0.3 at 37°C. The cycle of dilution and re-growth was repeated three times to observe a growth defect of the trmD-deg strain. (C) Western blot analysis of TrmD protein. An overnight culture of trmD-deg and trmD-cont cells in LB + Glc was diluted to LB + Ara at T = 0, and was sampled at the indicated time points. Cell lysates were separated on a 12% SDS-PAGE and TrmD protein was detected by primary antibody against E. coli TrmD and a secondary antibody against rabbit IgG. (D) Primer-extension inhibition analysis. An overnight culture of trmD-deg and trmD-cont cells in LB + Glc was diluted to LB + Ara at T = 0 and the fresh culture was taken through three cycles of dilution and re-growth. Total RNA was extracted over the time course, probed with a 5'-[32P]-labeled DNA primer targeting E. coli tRNALeu/CAG, and analyzed by a 12% PAGE/7 M urea gel and phosphorimaging. Primer extension would terminate in the control at 1 nt downstream of m1G37, generating a 15 nt fragment, whereas primer extension would continue to the 5'-end in TrmD deficiency, generating a 53 nt fragment. The fraction of m1G37 in each sample is calculated based on all primer-extension products as shown in the source file, not including the primer. Because the same amount of cell culture was used for extraction of RNA, an increased primer-extension stop at the 15 nt band relative to the primer position was observed in trmD-cont cells, reflecting an increased cell density 0–3 hr and increased synthesis of m1G37-tRNA. In all samples collected for trmD-cont cells, no synthesis of the read-through 53 nt band was observed, indicating 100% methylation. At the time of cell harvest, the m1G37 level was 100% for trmD-cont cells at T = 3 hr, but was below 25% for trmD-deg cells at T = 4 hr.