a, Darobactin and polymyxin B MIC studies against E. coli MG1655 were performed in the presence of LPS. Addition of LPS antagonized polymyxin activity, but not darobactin activity. Data are mean ± s.d. of triplicate experiments. b, Groups of five mice were infected intraperitoneally with 107 E. coli ATCC 25922, and subsequently euthanized at 24 h (if not already dead), after which the livers and spleens collected, homogenized and plated for c.f.u. analysis. Wild-type E. coli caused 60% death and showed high c.f.u. burdens in liver and spleen. All three darobactin-resistant bamA mutant strains had reduced virulence, with 100% survival in all groups at 24 h. The burden of bacteria of the triple bamA mutant was close to the limit of detection in organs, the G429R-expressing mutant was found at low but detectable levels, whereas the G429V-expressing mutant was found at relatively high loads in the organs. n = 5. Data are mean ± s.d. c, Schematic of the BAM activity assay in which BAM (BamA–E) was first inserted into lipid nanodiscs. Unfolded OmpT, along with the periplasmic chaperone SurA, was then mixed with the BAM–nanodiscs, and BAM folds OmpT into the nanodisc. OmpT, a protease, cleaves an internally quenched peptide, which produces a fluorescent signal. d, BAM–nanodisc assays performed in the presence of increasing concentrations of darobactin (left). The results show that darobactin is able to specifically inhibit BAM–nanodisc activity in a dose-dependent manner. These data were then normalized against the ‘no darobactin’ sample and the highest concentration of darobactin and plotted, and an IC 50 was calculated using the online IC 50 calculator tool (AAT Bioquest) (right). ND, nanodisc. n = 3 biologically independent experiments. Data are mean ± s.d. e, As a control to the BAM–nanodisc assays, we prepared OmpT–nanodiscs and assayed OmpT–nanodisc activity in the presence of increasing concentrations of darobactin. To prepare the OmpT–nanodiscs, we first expressed OmpT as inclusion bodies and then refolded the protein using previously reported methods55,56. We then incorporated OmpT into nanodiscs using the same methods as described for BAM. The assays were performed using 0.4 μM of OmpT–nanodiscs. The results show that darobactin has almost no effect on OmpT–nanodisc activity, thus confirming that darobactin does not affect OmpT activity itself or disrupting the nanodiscs themselves. A representative plot is shown from a triplicate experiment. f, The WNWSKSF peptide does not inhibit BAM–nanodiscs. As a control to darobactin, the BAM–nanodisc assays were performed in the presence of increasing concentrations of a linear peptide WNWSKSF. The results show that the WNWSKSF peptide has only minimal effects on BAM–nanodisc activity, even at the highest concentrations. A representative plot is shown from a triplicate experiment. g, h, Specific binding of darobactin to BamA/BAM. Mole ratio is the protein:ligand ratio. g, Plot of ITC experiments of wild-type BAM titrated with darobactin. K d = 1.2 μM, N = 0.52, ΔH = –25 kcal mol−1 and ΔS = −56 cal mol−1 K−1. The experiment was repeated independently twice with similar results. h, Plot of ITC experiments of wild-type BAM titrated with the peptide WNWSKSF shows that there is no binding within the same concentration range as was used for darobactin. The experiment was repeated independently twice with similar results. i, j, Two-dimensional [15N, 1H]-TROSY spectra of 250 μM BamA-β in 0.1% w/v LDAO. i, BamA-β in the absence (left) and in the presence of darobactin with a molar ratio of 1:0.5 (middle) and 1:1 (right) of BamA-β:darobactin. The red dashed line outlines an example spectral region that shows substantial spectral changes during the titration. The experiment was repeated independently twice with similar results. j, An overlay of apo BamA-β (black) (250 μM) on BamA-β and a scrambled linear peptide WNKWSFS (green) (230 μM). The experiment was performed once. Source data