E. coli ATCC 25922 was grown in MH-II broth (20 ml) to OD600nm = 1 at 37°C with 200 rpm shaking. This bacterial culture (100 μl, corresponding to ≈5 × 107 CFU) was plated onto MH-II agar plates containing thanatin (10 to 50 μg/ml) and incubated at 37°C overnight. Growing colonies were then passaged at least four times over MH-II plates without selection on thanatin, and then MIC values against thanatin were determined. Two independent experiments were performed. Of the initial 10 isolates, the mutants ThanR-2, ThanR-4, ThanR-8, ThanR-9, and ThanR-10 exhibited stable resistance (MIC of ≥64 μg/ml), and, in each, the lptA gene was sequenced, which revealed mutations Q62L or D31N in the primary sequence of the protein (table S2). The antimicrobial activity of thanatin and seven standard antibiotics against the three selected mutants (ThanR-2, ThanR-4, and ThanR-8) are shown in table S3.

The complete genomes of three selected mutants (ThanR-2, ThanR-4, and ThanR-8) and the WT (ATCC 25922) strain used in these studies were sequenced using the Illumina MiSeq platform (MiSeq Reagent Kit V2; 500 cycles). Briefly, genomic DNA (gDNA) of the WT and the selected mutants was extracted using the Sigma NA2100 1Kit. One microgram of gDNA was sheared to 500 base pairs by sonication (Covaris). DNA fragments were further processed with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S/L). Genome mapping and identification of genetic variants were performed using CLC Genomics Workbench 10.1.1 (CLC bio). The genes mutated in the resistant strains, compared to the WT, are shown in table S4. In the ThanR-8 resistant strain, only a single base pair change was found in the entire genome, in the lptA gene, corresponding to a Q62L change in the primary sequence of the protein.

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