A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to copper stress

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products that bacteria employ in their survival strategies. Herein, we characterized the mode of action, structure, and biosynthetic pathway of bufferins, products of mysterious ‘gold-induced genes’ ( gig) -like operons. We show that bufferins form a new family of RiPPs ubiquitous in Eubacteria. Using Caulobacter vibrioides as a model organism, we elucidate the important role of bufferins in protecting bacteria from copper-induced stress. The cleavable N-terminal signal peptide guides bufferins export to the periplasm, where they efficiently sequester the copper ions. The metal-binding propensity of bufferins relies on the post-translational transformation of cysteine residues in the precursor peptide into 5-thioxazoles. This rare modification is installed by a multinuclear non-heme iron-dependent oxidative enzyme (MNIO, DUF692 family) in conjunction with its partner protein belonging to the DUF2063 family. Our study broadens the landscape of known peptide modifications installed by MNIO enzymes and unveils a widespread but overlooked bacterial copper defense strategy.