ACR3 as a Key Regulator Antimicrobial Lipopeptide Synthesis and Efflux in Bacillus velezensis HN-1

Bacillus velezensis is known for producing and secreting antimicrobial lipopeptides, yet the regulatory mechanisms behind these processes remain unclear. In this study, we constructed arsenite transporter protein gene (acr3)-deficient strains (HN-1ΔQ acr3) and complementary strains (HN-1ΔC acr3) to elucidate the roles of ACR3 in antimicrobial lipopeptide synthesis and efflux. Our findings demonstrate that ACR3 dysfunction compromised swarming and biofilm formation, completely abolishing the antifungal capabilities of the HN-1ΔQ acr3 strains. Transcriptomic analysis and reverse transcription-quantitative PCR revealed significant downregulation of lipopeptide biosynthesis genes, including sfp, fenD and bmyC, in HN-1ΔQ acr3. Notably, the addition of purified ACR3 protein restored the expression of lipopeptide biosynthesis genes and partially rescued antifungal activities in the HN-1ΔQ acr3 strains. Furthermore, yeast two-hybrid assays indicated an interaction between ACR3 and proteins involved in cell survival, substrate uptake and antifungal activities, such as ribose ABC transporter substrate-binding protein (RbsB), fengycin synthetases and phosphopantetheine transferases. In parallel, our findings indicate that ACR3 interacts with the ABC transporter pathway to tune antifungal lipopeptide transport in Bacillus velezensis. Collectively, these results suggest that ACR3 functions as a crucial regulator, mediating both the synthesis and efflux of lipopeptides in B. velezensis. These research establish an experimental foundation for elucidating the mechanisms of antimicrobial compound synthesis and secretion in B. velezensis and provide a theoretical basis for the optimisation and engineering of high-yield biocontrol strains.