Multi-locus pairing expands the functional repertoire of two-peptide bacteriocins

Two-peptide bacteriocins are generally viewed as self-contained systems whose peptides are encoded at the same biosynthetic locus and co-expressed to form an active antimicrobial pair. Here, we show that functional pairing extends beyond this organization. Comparative genomic analysis revealed frequent co-occurrence of putative two-peptide bacteriocins encoded at distinct chromosomal loci in streptococci. Testing pairwise combinations of 47 peptides produced by cell-free protein synthesis identified 54 combinations with enhanced antimicrobial activity, with inter-locus combinations more frequent than intra-locus combinations. In Streptococcus mitis strain C22, four dispersed bacteriocin loci are coordinately expressed during exponential growth despite regulation by partially independent signaling pathways, providing an opportunity for inter-locus pairing. Split-luciferase complementation assays showed that active peptide pairs associate and that these interactions depend predominantly on amino acid sequence. Using a bacteriocin-free S. mitis background, we further show that components produced by distinct bacterial cells can act together to increase producer competitiveness in co-culture with S. pneumoniae. These findings reveal a modular organization in which peptides encoded at different loci can form functional combinations, including when produced by separate cells. This flexibility expands the antimicrobial repertoire available to individual strains and provides a mechanism for bacteriocin diversification while retaining functional compatibility.