YcsE-mediated dephosphorylation of ComP regulates surfactin and iturin synthesis in Bacillus velezensis HN-1.

Antifungal lipopeptides are crucial bioactive compounds produced by Bacillus velezensis through non-ribosomal peptide synthase (NRPS). However, the roles of phosphatases and histidine kinases in regulating lipopeptides synthesis in Bacillus species remain understudied. Here, we investigate the regulatory mechanisms of the phosphatase YcsE and the histidine kinase ComP in lipopeptides synthesis in B. velezensis. Physiological and biochemical indices, bacteriostatic activity, protein interaction and in vitro dephosphorylation were performed to study the roles of the YcsE mediated ComP dephosphorylation in B. velezensis lipopeptides synthesis. The EC and MIC assays revealed that the ycsE::TnYLB-1 and comP::TnYLB-1 exhibited only 3.48 % and 6.05 % against Colletotrichum fructicola HD-1 compared to the wild-type strain HN-1. Furthermore, inhibitory activity against Xanthomonas oryzae pv. oryzae decreased by 48.34 % and 75 %, respectively. In the ycsE::TnYLB-1 mutant, the concentrations of Bacillomycin D and Surfactin A were reduced to 0.65 mg/mL and 2.24 mg/mL, representing a decrease of 90.37 % and 62.16 %, respectively. Similarly, in the comP::TnYLB-1 mutant, Bacillomycin D and Surfactin A levels were 0.61 mg/mL and 2.82 mg/mL, corresponding to reductions of 90.96 % and 52.36 %, respectively. Notably, there were significant reductions in swimming, biofilm, oil-draining, and hemolytic activity. GST pull-down confirmed that YcsE interacts with SrfAA and Sfp, while ComP interacts with genes involved in lipopeptides synthesis. In vitro dephosphorylation experiments showed that YcsE-mediated the dephosphorylation of ComP. In summary, this study identifies a novel histidine kinase in regulating lipopeptides synthesis, through dephosphorylation by phosphatase YcsE, providing a theoretical foundation for improving high-yield B. velezensis.