Genome analysis of Bacillus subtilis JCL16 and the synergistic relationship among its metabolites reveal its potential for biocontrol of Nocardia seriolae

Nocardia seriolae is one of the major causative agents of fish nocardiosis, responsible for large-scale fish die-offs and economic losses in the aquaculture industry worldwide. Therefore, research efforts towards developing efficacious alternatives to aquatic antibiotics, such as vaccines, immunomodulatory additives including aquatic probiotics, to control this disease are of high importance. Recently, we isolated a soil-derived strain of Bacillus subtilis JCL16, and the 21-day challenge tests results showed that the addition of B. subtilis JCL16 culture to the basal control diet significantly increased the survival rate of largemouth bass with nocardiosis from 21.67% to 68.3%. To reveal the putative functional factors, we determined the genome sequence of B. subtilis JCL16 using PacBio RS II and Illumina HiSeq 4000 platform, and predicted its secondary metabolites using antiSMASH, which showed that it contains 11 functional gene clusters, three of which, srf, bac and sbo, have previously been verified to have antagonistic bacterial functions. Subsequently, mutants Δsrf, Δbac, Δsbo were constructed using homologous recombination techniques, and HPLC results further elucidated that the three gene clusters are responsible for the synthesis of surfactin, bacilysin and subtilosin A, respectively. To our surprise, a significant reduction in the inhibition of N. seriolae occurred in all three mutants compared to the wild type, so we speculated that surfactin, bacilysin and subtilosin A might have adopted a synergistic strategy. Next, we prepared their pure products and further confirmed our inference by Oxford Cup and MIC assays. In summary, the good biocontrol effect of B. subtilis JCL16 against fish Nocardia is likely due to the synergistic strategy among surfactin, bacilysin and subtilosin A. Here we propose a synergistic mechanism among them, which provides some insight into the powerful biocontrol mechanisms of Bacillus.