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

Abstract 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 structure, the biosynthetic pathway and the mode of action of a new RiPP family called bufferins. With thousands of homologous biosynthetic gene clusters throughout the eubacterial phylogenetic tree, bufferins form by far the largest family of RiPPs modified by multinuclear non-heme iron-dependent oxidases (MNIO, DUF692 family). Using Caulobacter vibrioides bufferins as a model, we showed that the conserved Cys residues of their precursors are transformed into 5-thiooxazoles, further expanding the reaction range of MNIO enzymes. This rare modification is installed in conjunction with a partner protein of the DUF2063 family. Bufferin precursors are the first examples of bacterial RiPPs found to feature an N-terminal Sec signal peptide and thus to be exported by the ubiquitous Sec pathway, a new paradigm in the RiPP field. Other original features of bufferins are their large size and protein-like fold, which blurs the line between modified peptides and proteins. We reveal that bufferins are involved in copper homeostasis, and their metal-binding propensity requires the thiooxazole heterocycles. Bufferins enhance bacterial growth under copper stress by sequestering excess metal ions in the periplasm. Our study thus describes a large family of RiPP metallophores and unveils a widespread but overlooked metal homeostasis mechanism in eubacteria likely to be relevant to One-Health strategies. Significance statement Copper is both essential and toxic in excess. Bacteria face copper in their environments, notably in phagocytes, hence they have developed several defense mechanisms. We discovered a widespread strategy of protection from copper, through the biosynthesis of natural products that we call bufferins. Bufferins are ri bosomally synthesized p ost-translationally modified p eptides (RiPPs), natural products with key roles in bacterial physiology and ecology. Bufferins enhance bacterial growth under copper stress by complexing with the metal using thiooxazole heterocycles that result from enzymatic modification of cysteine residues. With thousands of homologs throughout the eubacterial phylogenetic tree, bufferins represent a highly prevalent strategy of adaptation to metal stress. They are larger in size than most RiPPs, expanding the concept of RiPPs to modified proteins.