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 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. ### Competing Interest Statement The authors have declared no competing interest.