[Nature, 26 March 2025]

Lasso peptides (biologically active molecules with a distinct structurally constrained knotted fold) are natural products that belong to the class of ribosomally synthesized and post-translationally modified peptides. Lasso peptides act on several bacterial targets, but none have been reported to inhibit the ribosome, one of the main targets of antibiotics in the bacterial cell. Here we report the identification and characterization of the lasso peptide antibiotic lariocidin and its internally cyclized derivative lariocidin B, produced by Paenibacillus sp. M2, which has broad-spectrum activity against a range of bacterial pathogens. We show that lariocidins inhibit bacterial growth by binding to the ribosome and interfering with protein synthesis. Structural, genetic and biochemical data show that lariocidins bind at a unique site in the small ribosomal subunit, where they interact with the 16S ribosomal RNA and aminoacyl-tRNA, inhibiting translocation and inducing miscoding. Lariocidin is unaffected by common resistance mechanisms, has a low propensity for generating spontaneous resistance, shows no toxicity to human cells, and has potent in vivo activity in a mouse model of Acinetobacter baumannii infection. Our identification of ribosome-targeting lasso peptides uncovers new routes towards the discovery of alternative protein-synthesis inhibitors and offers a novel chemical scaffold for the development of much-needed antibacterial drugs