Author response image 4. Impact of antibiotics on expression of full-length and truncated Cfr protein.

E. coli BW25113 were transformed with pZA constructs containing either CfrWT expressed using the Ptet (WT) or native Pcfr promoter (SCFS1) and plated on LB agar plates containing selection antibiotic and either 0, 0.5, 1, or 2 µg/mL of chloramphenicol (CHL). Overnight cultures were diluted into fresh media containing the respective concentration of CHL and grown to mid-log phase. (a) Cfr protein levels were assessed by immunoblotting against a C-terminal FLAG tag. Asterisks denote the truncated Cfr product. Em = empty vector control. (b) Relative protein expression of full-length Cfr (teal) and the truncation corresponding to translation initiation at Met95 (yellow) compared to CfrWT expression with no CHL. Signal was normalized to housekeeping protein RNA polymerase β-subunit. (c) Minimum inhibitory concentration of CHL required to inhibit bacterial growth of E. coli BW25113 transformants that had been pre-incubated with either 0, 0.5, 1, or 2 µg/mL of CHL.