35s l methionine

Infected or uninfected cells were metabolically labelled for 1h with [³⁵S]-L-methionine (500Ci/mmol, MP Biomedical, USA) at various times p.i. as indicated in the text. After labelling, cells were lysed in disruption buffer, sonicated and heated for 5 min at 100°C and proteins were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were fixed, stained, dried, and resolved radiolabelled bands visualized by phosphorimager analysis.

Inhibition of translation by retapamulin and lefamulin was determined using metabolic labeling. All manipulations were performed at 37°C. D39 Δcps was inoculated from a starter culture (OD₆₀₀ of 1) into 6 mL and grown in CDM lacking methionine and containing 0.5% Oxyrase to an OD₆₀₀ of 0.5 at 37°C with 5% CO₂. Cells were diluted 10-fold into CDM without methionine and containing 0.5% Oxyrase and grown until the culture density reached an OD₆₀₀ of ~0.2, and three 350-μL aliquots were transferred to microcentrifuge tubes (two drug conditions and one control group). Retapamulin and lefamulin were individually added to Eppendorf tubes at a final concentration of 100× MIC. Prior to and immediately following the addition of antibiotics (0, 1, 2.5, 5, and 15 min), 28 μL of the culture was added to microcentrifuge tubes containing 0.3 μCi [³⁵S]l-methionine (specific activity of 1,175 Ci/mmol; MP Biomedicals) in 2 μL of CDM. After a 1-min incubation, 25 μL of the mixture was spotted onto Whatman 3MM paper discs prewetted with 7% trichloroacetic acid (TCA). The discs were boiled twice in 7% TCA for 5 min, soaked in 100% acetone for 2 min, and then air dried prior to being placed into a 5-mL scintillation cocktail and being read using a scintillation counter.

The inhibition of cellular protein synthesis by Api was analyzed by metabolic labeling as described previously (Meydan et al., 2019 (link)) with the following modifications. E. coli BL21 ∆tolC cells were grown overnight in MOPS medium (Teknova) lacking methionine (MOPSΔMet). Cells were diluted 1:200 into fresh MOPS-Met and grown at 37°C until the culture reached a density of A₆₆₀ of 0.2. Aliquots of the exponentially growing cells were added to tubes containing appropriately diluted Api in MOPSΔMet medium to obtain 0.75, 1.5, 3.1, 6.25, 12.5, 25, and 50 µM as final Api concentrations in the total volume of 100 µL. After 2 min incubation, 28 µL were transferred to another tube containing 2 µL MOPSΔMet medium supplemented with 0.3 µCi of L-[³⁵S]-methionine (specific activity 1,175 Ci/mmol; MP Biomedicals). Following 90 s incubation, the content was transferred onto Whatman 3 MM paper discs pre-wetted with 5% TCA and the procedure was continued as described previously (Meydan et al., 2019 (link)).
The time course of protein synthesis inhibition was performed as described above, except that Api was directly added to a final concentration of 6.25 µM to the exponentially growing culture in MOPSΔMet medium and aliquots were taken at 0, 2, 5, and 10 min.

Infected or uninfected cells that had or had not been pretreated with IFN for 12 h prior to infection were metabolically labeled for 1 h with l-[³⁵S]methionine (500 Ci/mmol; MP Biomedical, USA) at 18 h postinfection (p.i.). After labeling, cells were lysed in disruption buffer, sonicated, and heated for 5 min at 100°C and then analyzed by gel electrophoresis (SDS-PAGE). The gels were fixed, stained, and dried, and resolved bands were visualized by phosphorimager analysis. When appropriate, the same amounts of cell equivalents were run on PAGE. Furthermore, the amount of protein in each sample was monitored by staining the polyacrylamide gels (PAGs) with Coomassie brilliant blue.

Total E. coli protein was isolated from the 50 mL exponentially growing E. coli culture (strain MG1655) as described previously (53 (link)). The experimental details can be found in SI Appendix, Supplementary Materials and Methods. The radiolabeled proteins were isolated from the 60 μL exponential cultures exposed for 3 min to 10 mg/mL of KSG, then incubated with 10 µCi of L-[³⁵S]-methionine (specific activity 1,175 Ci/mmol, MP Biomedicals) and quenched after 3 min with an excess of unlabeled L-methionine.
The isolated proteins were resolved following the 2-D Electrophoresis Workflow manual (fourth edition) (BioRad). See SI Appendix, Supplementary Materials and Methods for experimental details.