Transcription Factor Spo0A Regulates the Biosynthesis of Difficidin in Bacillus amyloliquefaciens.

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.

The cellular redox state in Bacillus amyloliquefaciens WH1 affects biofilm formation indirectly in a surfactant direct manner.

Surfactin is a signal to trigger biofilm formation against harsh environments. Generally, harsh environments can result in change of the cellular redox state to induce biofilm formation, but we know little about whether the cellular redox state influences biofilm formation via surfactin. Here, the reductant glucose could reduce surfactin and enhance biofilm formation by a surfactin-indirect way. The oxidant H2 O2 led to a decrease of surfactin accompanying with weakened biofilm formation. Spx and PerR were both necessary for surfactin production and biofilm formation. H2 O2 improved surfactin production but inhibited biofilm formation by a surfactin-indirect manner in Δspx, while it reduced surfactin production without obvious influence on biofilm formation in ΔperR. The ability against H2 O2 stress was enhanced in Δspx, but weakened in ΔperR. Thereby, PerR was favorable for resisting oxidative stress, while Spx played a negative role in this action. Knockout and compensation of rex also supported that the cells could form biofilm by a surfactin-indirect way. Collectively, surfactin is not a unique signal to trigger biofilm formation, and the cellular redox state can influence biofilm formation by a surfactin-direct or -indirect way in Bacillus amyloliquefaciens WH1.