Bacillus amyloliquefaciens WH1 produces multiple antibiotics with antimicrobial activity and can control bacterial wilt disease caused by Ralstonia solanacearum. Antibacterial substances produced by WH1 and the regulation mechanism are unknown. In this study, it was found that difficidin, and to a minor extent bacillibactin, exhibited antibacterial activity against R. solanacearum. Lipopeptides, macrolactin, bacillaene, and bacilysin had no antibacterial activity. Ferric iron uptake transcriptional regulator Fur bound the promoter region of the dhb gene cluster of bacillibactin biosynthesis. Mutant Δfur showed a higher bacillibactin production and its antibacterial activity increased by 27% than wild-type WH1. Difficidin inhibited R. solanacearum growth and disrupted the integrity of the cells. Lack of transcription factor Spo0A abolished difficidin biosynthesis. Spo0A bound the promoter region of the dfn gene cluster of difficidin biosynthesis. Changing phosphorylation levels of Spo0A via deletion of phosphatase gene spo0E and histidine kinases genes kinA and kinD affected the biosynthesis of difficidin. Deletion of spo0E increased the phosphorylation level of Spo0A and consequently improved the difficidin production. The antibacterial activity of mutant Δspo0E and ΔkinA increased by 12% and 19%. The antibacterial activity of mutant ΔkinD decreased by 28%. Collectively, WH1 produced difficidin to disrupt the cell of R. solanacearum and secreted siderophore bacillibactin to compete for ferric iron. Spo0A regulated difficidin biosynthesis. Spo0A regulates quorum-sensing responses and controls the biosynthesis of secondary metabolites in B. amyloliquefaciens. This study has important findings in the regulation mechanism of antibiotic synthesis and helps to improve antibiotic yield in Bacillus. IMPORTANCE Pathogen R. solanacearum causes bacterial wilt disease in many crops. There is no chemical bactericide that can control bacterial wilt disease. It is vital to find antagonistic microorganisms and antibacterial substances that can efficiently control bacterial wilt disease. B. amyloliquefaciens WH1 could inhibit the growth of R. solanacearum. Via genetic mutation, it was found that difficidin and to a minor extent bacillibactin produced by WH1 acted efficiently against R. solanacearum. The transcription factor Spo0A regulated the synthesis of difficidin. Phosphorylation of Spo0A affected the production of difficidin. Increasing the phosphorylation level of Spo0A improved the difficidin production and antibacterial activity. In-depth analysis of the regulation mechanism of antibiotic difficidin is meaningful for enhancing the control efficiency of WH1. B. amyloliquefaciens WH1 and the antibacterial substances have vast application potential in controlling bacterial wilt disease.
Keywords: Bacillus amyloliquefaciens; Ralstonia solanacearum; Spo0A; bacillibactin; difficidin.