Altitudin S, an Antimicrobial Peptide Produced by Bacillus altitudinis ECC22, Defines a Novel Subgroup of Circular Bacteriocins

Bacteriocins are ribosomally synthesized antimicrobial peptides with 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 purified active supernatant fractions confirmed the production of altitudin S, with a molecular mass of 8379 Da, consistent with head-to-tail cyclization. Altitudin S 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 also produced using an in vitro cell-free protein synthesis system coupled with 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 with distinctive sequence features and physicochemical properties, including an exceptionally high isoelectric point, strong cationic charge, and low hydrophobicity. Homologs were identified across diverse Bacillales species, displaying high sequence similarity, conserved structural features, and a shared physicochemical profile. Their biosynthetic gene clusters were also highly conserved and consistently encoded an M48-family metallopeptidase. Collectively, these findings support the classification of altitudin S and its homologs as representatives of a novel subgroup of circular bacteriocins.