Identification and characterization of pumicyclin A, a novel circular bacteriocin from <i>Bacillus pumilus</i> with a dispensable leader peptide

ABSTRACT Foodborne pathogens such as Listeria monocytogenes and Bacillus cereus continue to pose serious threats to global food safety, driving demand for natural, consumer-friendly preservation strategies. Circular bacteriocins, a class of ribosomally synthesized antimicrobial peptides characterized by having covalently linked N- and C-termini, are promising candidates due to their superior stability and broad-spectrum activity. Here we describe pumicyclin A, a novel circular bacteriocin produced by two nearly identical but independently isolated Bacillus pumilus strains. Pumicyclin A exhibits a broad inhibitory spectrum against gram-positive bacteria and is particularly potent against Listeria species and B. cereus . Genomic analysis revealed a six-gene biosynthetic cluster, with the precursor peptide comprising a 32-amino-acid leader and a 64-amino-acid core, yielding a mature peptide of approximately 6,218 Da. A second downstream gene, encoding a smaller bacteriocin-like peptide, was found to be non-essential for pumicyclin A production. Notably, deletion mutagenesis of the leader peptide to a single methionine residue did not abolish activity, whereas specific substitutions near the circularization site were associated with loss of detectable activity, consistent with impaired biosynthesis (e.g., disrupted circularization, secretion, stability), and indicating key residues important for bacteriocin production. These results offer mechanistic insights into the production of long-leader circular bacteriocins and support pumicyclin A as a promising agent for food biopreservation. IMPORTANCE There is an urgent need for safe and effective antimicrobial solutions to control foodborne pathogens and reduce reliance on conventional chemical preservatives. Circular bacteriocins represent a relatively unexplored class of antimicrobials with desirable features, such as high stability and potent antibacterial activity. This study identifies and characterizes pumicyclin A, a new circular bacteriocin from Bacillus pumilus , with potent anti- Listeria activity and unique biosynthetic characteristics. By dissecting the roles of the leader peptide and conserved residues near the circularization site, we provide novel insights into the structure-function relationships governing circular bacteriocin maturation. These findings lay the groundwork for future (circular) peptide engineering efforts and support the potential application of pumicyclin A as a natural and effective biopreservative to enhance food safety and prolong shelf life.