Science Advances

Supplementary Materials

This PDF file includes:

  • Section S1. Bacterial strains and plasmids used in this study
  • Section S2. Peptide synthesis
  • Section S3. Permeabilization of the E. coli cell envelope
  • Section S4. Macromolecular synthesis assays
  • Section S5. Photoaffinity labeling
  • Section S6. Thanatin-resistant E. coli mutants
  • Section S7. Production of LptA-His6, LptAm, LptD/E, and Thanatin in E. coli
  • Section S8. Binding assays with LptA by FP and thermophoresis
  • Section S9. Binding assays with LptD/E by FP and thermophoresis
  • Section S10. NMR studies and structure determination of thanatin-LptAm complex
  • Scheme S1. Synthesis of thanatin-PAL5.
  • Scheme S2. Synthesis of thanatin-BDP-FL.
  • Scheme S3. Structure of thanatin-Cy3.
  • Fig. S1. Membrane permeabilization monitored by uptake (or absence thereof) of SYTOX Green.
  • Fig. S2. Assays for release of ß-lactamase and of ß-galactosidase.
  • Fig. S3. Relative incorporations of 3H label from labeled precursor over 20 min, at 37°C, performed in triplicate.
  • Fig. S4. SDS-PAGE of purified LptD/EHis complex from E. coli.
  • Fig. S5. Binding assays with LptA by FP.
  • Fig. S6. Binding assays with LptA by thermophoresis.
  • Fig. S7. Binding assays with LptD/E.
  • Fig. S8. HSQC spectra of 15N-labeled LptA.
  • Fig. S9. HSQC spectra of 15N-labeled thanatin in free form and bound to LptAm.
  • Fig. S10. Weighted 15N,1H chemical shift changes (Δδ) between the free and thanatin-bound LptAm as a function of residue number.
  • Fig. S11. Ribbon representation of the 20 lowest energy NMR conformers in two different orientations.
  • Fig. S12. Sequence and structure comparisons of the N-terminal regions of LptA and LptD.
  • Table S1. Bacterial strains and plasmids used in this study.
  • Table S2. ThanR isolates from two independent passaging experiments, with MICs against thanatin after four generations without selection pressure, together with point mutations detected by lptA sequencing.
  • Table S3. MIC values (μg/ml) of three selected mutants (ThanR-2, ThanR-4, and ThanR-8) and WT against thanatin and seven standard antibiotics (colistin, erythromycin, gentamicin, vancomycin, rifampicin, ampicillin, and ciprofloxaxin).
  • Table S4. Genes mutated in the mutant strain compared to WT (+, indicates a mutation; −, indicates no mutation in the selected gene).
  • Table S5. Antimicrobial activities of thanatin (MIC, μg/ml) against E. coli WT strains and strains containing plasmids shown.
  • Table S6. Sequences of primers used for cloning experiments.
  • Table S7. Statistics from the NMR structure calculations for LptAm-thanatin.
  • References (3746

Download PDF

Files in this Data Supplement: