Abstract
BACKGROUND: Bacillus subtilis 916 has been identified as an effective biocontrol agent against Rhizoctonia solani, the causal pathogen of rice sheath blight, under greenhouse and field conditions. HPLC analysis showed that surfactin, a member of the lipopeptide family produced by B. subtilis, was the major antimicrobial substance. RESULTS: Previously, we obtained a mutant strain of B. subtilis 916, Bs-H74, which produced significantly more surfactin than the wild type and presented 10% stronger inhibitory activity against R. solani. To explore the molecular mechanism underlying the higher surfactin productivity in the mutant, high-throughput proteomic analysis was carried out to analyze the differential protein expression. Our results showed that several differentially expressed proteins are involved in OppA, DegU and Carbon Catabolite Repression (CCR) regulatory pathways, which could be positively or negatively associated with surfactin biosynthesis. At both transcriptional and translational levels, we suggested that OppA may play a key role in surfactin synthesis regulation. Based on the above findings, we proposed the hypothesis that a point mutation in the oppA gene may lead to changes in oligopeptides acquisition in B. subtilis, and then the changed oligopeptides may activate or suppress the global regulatory protein, CcpA in the CCR pathway, and ComA and DegU may indirectly regulate surfactin synthesis in Bs-H74. To further explore the regulatory mechanisms in Bs-H74, metabolomics analysis was performed in this study. Interestingly, only 16 metabolites showed changes in abundance in Bs-H74 compared to Bs-916. Neohesperidin, a type of natural flavanone glycosides from citrus with a range of biological activities, increased by 18 times over the wild type Bs-916. This result implied exciting findings in regulatory mechanisms by OppA protein. CONCLUSIONS: In summary, this study has revealed the mechanisms underlying the improved antagonistic property with increased surfactin production in Bs-H74 at the gene, protein and metabolic levels, which may help to comprehend the map of the regulatory networks in B. subtilis. Findings from our work have provided a solid physical and theoretical basis for practically applying metabolic and genetic engineering to achieve improved and high-yielding biocontrol strains.