Integrated Genome Mining and Bioactivity-Guided Isolation of Antimicrobial Peptides from Bacillus amyloliquefaciens BS4

Bacterial resistance remains a critical global health challenge, driving the continuous search for novel antimicrobial agents. Bacillus amyloliquefaciens is a recognized repository of bioactive metabolites; however, its full biosynthetic potential requires integrated genomic and experimental validation. This study characterized the antimicrobial profile of B. amyloliquefaciens BS4 through a hybrid pipeline. Genome sequencing and de novo assembly revealed a 3.9 Mb chromosome with a G + C content of 46.14%. Functional annotation identified 3,887 coding sequences, including pathways for siderophore biosynthesis and a complete bacilysin biosynthetic cluster. BGC analysis using antiSMASH v7.1.0 and BAGEL4 identified 18 biosynthetic gene clusters, while similarity network analysis via BiG-SCAPE highlighted unique singleton BGCs, indicating untapped biosynthetic diversity. Although in silico screening via Macrel predicted two putative cationic antimicrobial peptides (AMPs), bioactivity-guided purification utilizing sequential RP-HPLC, and de novo sequencing revealed a distinct set of four active peptides. Notably, three of these sequences were identified as fragments derived from the BclA exosporium protein family, highlighting the structural proteome as a non-canonical source of antimicrobials. The purified fractions exhibited activity against M. luteus and E. coli, while displaying no significant hemolytic activity or cytotoxicity, even above the MIC values. Molecular docking further supported the interaction of these candidates with bacterial targets. Overall, this hybrid strategy effectively uncovers the antimicrobial complexity of BS4, revealing 'cryptic' peptide candidates with therapeutic potential.