Subpeptin JM4-A and subpeptin JM4-B are two novel antimicrobial peptides produced by Bacillus subtilis JM4. To identify putative genes involved in their production, degenerate PCR primers targeted to conserved motifs of nonribosomal peptide synthetases (NRPSs) were used. A resulting 1.2 kb PCR product had high sequence similarity to genes of NRPSs, and then a 2.8 kb DNA fragment flanking it was cloned subsequently. Gene disruption of the resulting 4 kb DNA fragment produced subpeptin-deficient mutant, suggesting that subpeptin JM4-A and subpeptin JM4-B were biosynthesized by NRPSs. Based on this result, a 48 kb gene Cluster was cloned, which consisted of nine coding sequences (CDSs) involved in antimicrobial peptide biosynthesis, regulation, and resistance. Disruption of two relatively large CDSs subA and subC led to subpeptin-deficient mutants, which supported the involvement of the cloned gene cluster in subpeptin biosynthesis. (c) 2006 Elsevier Inc. All rights reserved.