Bacteria synthesize a bacteriocin within the cell membrane

ABSTRACT Bacteria use bacteriocins to kill competitors and shape microbial communities. Many bacteriocins are ribosomally synthesized and post-translationally modified peptides (RiPPs), a diverse class of natural products and a source of new antimicrobials. The diversity of bacterial RiPPs stems from the combination of genetically encoded precursor peptides and biosynthetic proteins that post-translationally process the peptides. Characterized RiPP pathways involve soluble precursor peptides and biosynthetic proteins that modify them in the bacterial cytoplasm before secretion across the cell membrane. TMcins, a recently discovered RiPP class, challenge this paradigm by possessing a transmembrane helix in the mature product, indicating that bacteria have evolved a membrane-localized bacteriocin synthesis pathway. Here, we show that the precursor peptide TmcA is integrated into the producing cell membrane and, using an inducible TMcin biosynthesis system guided by structure prediction, is modified within the membrane, with three of four biosynthetic events mediated by intramembrane proteins. Notably, this approach revealed that a previously uncharacterized membrane protein performed an essential escort role during TMcin maturation within the cell membrane. Together, our findings reveal a membrane-localized pathway of RiPP biosynthesis, provide insights into its evolution, and establish TMcins as a model for understanding how bacteria produce peptide natural products from within the cell membrane. IMPORTANCE Bacteria have evolved numerous and diverse pathways to produce and secrete bacteriocins as weapons of bacterial warfare. These molecules are processed inside the cell and then transported across the bacterial membrane for secretion. We recently discovered the TMcins, a distinct class of bacteriocins found across gram-positive bacteria that contain a transmembrane helix and kill target bacteria by forming large pores. Using a Staphylococcus aureus strain that naturally produces TMcin, we show that TMcin production occurs in the cell membrane and reveal how bacteria process a membrane-spanning peptide in a lipid environment. This work expands our understanding of how bacteria produce antimicrobials and provides a model for discovering other membrane-localized natural products.