Comparative genomics of Bacillus velezensis and Bacillus subtilis reveals distinction and evolution of lipopeptide antimicrobial gene clusters

Species belonging to the genus Bacillus are recognized as important biocontrol agents, especially the Bacillus subtilis and Bacillus velezensis exhibit the excellent antifungal activity, being found in a variety of habitats and demonstrating significant metabolic versatility. However, knowledge regarding the genetic diversity of different Bacillus species is limited. In this study, we employed comparative genomics to elucidate the genetic diversity and evolutionary relationships between B. velezensis and B. subtilis. Our results indicated that the antibacterial activity and colonization features, including biofilm formation and swarming, of B. velezensis strains were significantly greater than those of B. subtilis strains. We conducted a comprehensive genomic analysis of various Bacillus group strains and found that the genome size of B. velezensis was larger than that of B. subtilis, while the GC content of B. subtilis was higher than that of B. velezensis. The Average Nucleotide Identidy (ANI) value and phylogenetic analysis revealed ambiguous classifications among some Bacillus strains. Furthermore, the 20 Bacillus strains examined yielded a pangenome size of 7068 genes, with strain-specific genes ranging from 24 to 305. The core and specific genome of B. velezensis strains, annotated for secondary metabolite biosynthesis, transport and catabolism, were significantly more abundant than those of B. subtilis. The most pronounced difference between B. velezensis and B. subtilis strains was observed in the gene cluster encoding the iturin family of lipopeptides. Evolutionary analysis suggested that the iturin gene cluster of Bacillus may have been transferred from Paenibacillus spp. via horizontal gene transfer (HGT) events during the evolution. Additionally, functional analysis demonstrated that the iturin gene cluster effectively inhibits Fusarium pathogens. Collectively, these findings provide a foundation for a deep understanding of the evolution of different Bacillus strains and establish a theoretical basis for the application of Bacillus strains in agricultural production.