Figure 1. Evolved variants of Cfr exhibit improved resistance to PhLOPS_A ribosome antibiotics.

(a) Evolution of Cfr under selection by the PTC-targeting antibiotic tiamulin. (b) Cfr homology model based on RlmN generated by I-TASSER server (Yang and Zhang, 2015) with mutagenic hotspots in red. N-terminal domain (NTD) is labeled. Active site denoted by S-adenosylmethionine (SAM, gray) and [4Fe-4S] cluster (orange). (c) Evolved variants containing Cfr mutations were selected for further study. Ptet* indicates alterations to promoter sequence. (d) Promoter architecture of CfrV6 and CfrV7 where pPtet designates a partial Ptet promoter sequence and Ins designates a variable insertion sequence. (e) Fold improvement in MIC resistance value for PhLOPS_A antibiotics and trimethoprim compared to empty pZA vector control determined from three biological replicates by microbroth dilution method. Trimethoprim is a negative control antibiotic that does not target the ribosome. LZD testing was performed against Escherichia coli BW25113 lacking efflux pump, acrB. Numerical MIC values are displayed in Figure 1—source data 1.

Figure 1—figure supplement 1. Cfr variants confer increased resistance to hygromycin A.

(a) Antibiotic susceptibility testing of hygromycin A (HygA) against Escherichia coli BW25113 lacking efflux pump acrB transformed with pZA plasmid encoding Cfr variants. Results were determined from three biological replicates by microbroth dilution method. (b) Approximate fold improvement in MIC resistance value for HygA compared to empty pZA vector control derived from source data presented in panel (a).

Figure 1—figure supplement 2. CfrV7 does not require an inducer for resistance or expression.

(a) Antibiotic susceptibility testing of Escherichia coli transformed with pZA plasmid encoding Cfr variants. Dose-dependent susceptibility testing toward tiamulin (TIA) with plates containing ampicillin (Amp, 100 µg/ml) and anhydrotetracycline (AHT, 20 ng/ml) to induce Cfr expression. (b) Dose-dependent susceptibility testing toward TIA with plates containing Amp but lacking AHT. Neg designates empty pZA plasmid. WT and 1–7 designate pZA encoding CfrWT-His₆ or Cfr variants 1–7. For panels (a) and (b), results are representative of three biological replicates. (c) Protein expression of full-length CfrV7 without AHT inducer compared to CfrWT with AHT inducer detected by immunoblotting against a C-terminal FLAG tag. Em = empty vector control. Original uncropped blot images are shown in Figure 1—source data 2.