Altitudin S from Bacillus altitudinis ECC22 defines a new subgroup of circular bacteriocins

Bacteriocins are ribosomally synthesized antimicrobial peptides exhibiting diverse structures and mechanisms of action. Bacillus altitudinis ECC22 , previously shown to produce the circular bacteriocins pumilarin and altitudin A, was found to harbor an additional biosynthetic gene cluster encoding a novel circular bacteriocin, designated altitudin S Proteomic analysis of active supernatant fractions confirmed the production of altitudin S, with a molecular mass of 8379 Da, consistent with head-to-tail cyclization. The peptide is synthesized as a 132-residue precursor comprising a 56-amino-acid leader and a 76-residue circular mature core. Structural modeling predicted a compact saposin-like fold composed of five α-helices and a strongly cationic surface (pI ≈ 11.0, net charge +13). Altitudin S was successfully produced using a cell-free protein synthesis system coupled to split-intein mediated ligation (IV-CFPS/SIML) and exhibited a narrow but reproducible antimicrobial spectrum. Comprehensive sequence, structural, and phylogenetic analyses revealed that altitudin S is a highly divergent circular bacteriocin, defined by distinctive sequence features and physicochemical properties, including an exceptionally high isoelectric point, net charge, and low hydrophobicity. Bioprospecting across sequence databases identified homologs of altitudin S in diverse Bacillales species, all showing high sequence similarity, conserved structural features and preservation of its distinctive physicochemical profile. Genomic analysis further revealed a conserved biosynthetic gene cluster among all altitudin S homologs, notably including a gene encoding a characteristic M48-family metallopeptidase. Altogether, these findings support the classification of altitudin S and its homologs as representatives of a novel subgroup of circular bacteriocins.