Due to their capacity to produce antimicrobial peptides that can prevent the growth of diseases, many Bacillus spp. are beneficial to plants. In this study, we looked into the antagonistic activity of the B. pumilus 3-19 strain and its derivatives following targeted genome editing. Two peptide genes with antibacterial action, bacilysin (bac) and bacteriocin (bact), and the sigF gene, which encodes the sigma factor of sporulation, were specifically inactivated using the CRISPR-Cas9 system in the genome of B. pumilus 3-19. Antibacterial activity against B. cereus and Pantoea brenneri decreased as a result of the inactivation of target genes in the B. pumilus 3-19 genome, with a noticeable effect against bacilysin. The growth dynamics of the culture changed when the bac, bact, and sigF genes were inactivated, and the altered strains had less proteolytic activity. An asporogenic mutant of B. pumilus 3-19 was obtained by inactivating the sigF gene. It has been proven that bacilysin plays a unique part in the development of B. pumilus 3-19’s antagonistic action against soil microorganisms.

The Bacillus pumilus 3-19 strain exhibits increased secretion of various hydrolases, including proteases, and is a promising plant growth-promoting (PGP) agent. A unique secreted minor metalloproteinase, MprBp, which has no homologs among prokaryotic enzymes, has been identified in its genome. The role of the enzyme in B. pumilus cells is poorly understood. Using CRISPR/Cas9 genome editing, a deletion mutant of B. pumilus 3-19 with an inactivated mprBp gene was obtained. Comparative analysis of the mutant and native strains by quantitative reverse transcription PCR (RT-qPCR) revealed a significant decrease in the expression of other minor proteases. The mutant strain also demonstrated a two-fold increase in biofilm formation, suggesting that MprBp acts as a negative regulator of this process. Additionally, seed treatment with B. pumilus ΔmprBp led to an increase in the morphometric parameters of barley seedlings (Hordeum vulgare L.).