Surfactin, bacillibactin and bacilysin are the main antibacterial substances of Bacillus subtilis JSHY-K3 that inhibited the growth of Vp AHPND (the main pathogen of acute hepatopancreatic necrosis disease in shrimp)

Acute hepatopancreatic necrosis disease (AHPND), which is caused by a particular strain of Vibrio that carries the pirA and pirB virulence protein gene, has caused major economic losses in shrimp farming. Bacillus subtilis JSHY-K3, a bacterial strain apparently antagonistic to Vp AHPND, was screened by our laboratory to better control AHPND. Our previous whole-genome sequencing analysis and untargeted metabolomics-LCMS-EMDB analysis of strain JSHY-K3 revealed that its antimicrobial material could be surfactin, aurantinin B/C/D, fengycin, sublancin 168, bacillibactin, subtilosin A, bacilysin, caffeic acid, fosfomycin, minocycline and so on. However, the specific bacteriostatic substances remain unclear and need to be further investigated. In this study, based on the whole genome sequence information obtained previously, we used CRISPR/Cas9 technology to edit the genome of B. subtilis JSHY-K3 and constructed 6 strains of gene knockout mutants, among which the bacterial solution and cell-free culture medium of the mutants Δ srfAB-AC, Δ dhbC and Δ bacA showed a significant decrease in antagonism to Vp AHPND. Therefore, we suspected that the main antibacterial substances of B. subtilis JSHY-K3 may be surfactin, bacillibactin and bacilysin. We then carried out high-resolution mass spectrometry and three-quadrupole quantitative analysis on the fermentation supernatants of strains JSHY-K3, Δ srfAB-AC, Δ dhbC and Δ bacA, and confirmed that these three gene clusters are involved in the synthesis of surfactin, bacillibactin and bacilysin, respectively. It was also confirmed that the deletion of the srfAB-AC, dhbC, bacA genes led to a reduction in the synthesis of surfactin, bacillibactin, bacilysin, which in turn led to a decrease in the inhibitory ability of the three mutant strains against Vp AHPND. In conclusion, surfactin, bacillibactin and bacilysin are the main inhibitory substances of B. subtilis JSHY-K3 to inhibit the growth of Vp AHPND.