The direct regulatory effects of Bacilysin on the cellular physiology of Bacillus subtilis

Background: Bacillus subtilis antibiotics, particularly non-ribosomally synthesized peptide antibiotics, possess distinct biological functions beyond their antimicrobial activity. Bacilysin, produced by certain B. subtilis strains, is one such peptide antibiotic. To gain deeper insight into its regulatory functions, this study aimed to explore its direct regulatory effects on B. subtilis physiology. Results: Bacilysin was purified from a bacilysin-producing strain B. subtilis PY79 by RP-HPLC using a ZORBAX Eclipse XDB-C18 column, with a retention time of 6.5 min (flow rate:4 mL/min) and confirmed by UPLC-MS with an [M + H]⁺ ion at m/z 271.14. A sublethal concentration of the purified compound was applied to exponentially growing cells (OD₆₀₀ 0.8) cultured in LB medium, where endogenous bacilysin production is not induced. RNA-Seq based transcriptomic analysis was performed to assess the gene expression differences between bacilysin-treated and -untreated cells. Exposure to bacilysin led to transcriptional changes in 121 genes (60 upregulated, 61 downregulated), with 98 of them newly identified as differentially expressed in response to bacilysin. Conclusion: This study reveals a multilayered regulatory network through which bacilysin not only controls its own biosynthesis but also contributes to the modulation of various adaptation processes to changing conditions, including the utilization of secondary carbon and nitrogen sources, sporulation, swarming motility, biofilm formation, biofilm-associated toxin production, antimicrobial biosynthesis, iron and zinc homeostasis, and coping with various stress conditions. One of the key findings is the existence of an antagonistic regulatory interaction between bacilysin and both the carbon catabolite regulator-CcpA and the GTP-binding global transcriptional regulator-CodY, accompanied by a repressive effect of bacilysin on tnrA expression, encoding a pivotal nitrogen metabolism regulator. Bacilysin plays a crucial role in the temporal and spatial regulation of gene expression during spore development. Furthermore, our data suggest that bacilysin creates a positive feedback loop on its own biosynthetic operon by cooperating with its positive regulators, including the srfA operon and the phrC gene. Bacilysin also appears to fine-tune its own biosynthesis by modulating SinR-dependent regulatory circuits, thereby contributing to the dynamic regulation of biofilm development. As an additional regulatory route, bacilysin may engage in a feedback loop involving the two component regulatory system-DegS/DegU, thereby linking its activity to the control of biofilm-associated toxin production.