Purification, Identification And Machanism Of Action Of Lipopeptide Antibiotic Bacillomycin L Produced By Bacillus Amyloliquefaciens

In order to provided technical support and theoretical basis for the development and application of lipopeptide antibiotics as a biological control agent, the purification, identification, physical and chemical characteristics, mechanism of action along with application in biocontrol of bacillomycin L produced by Bacillus amyloliquefaciens K103, isolated from a lemon sample and suppressing Rhizoctonia solani Kiihn, were investigated in detail.(1)The organism of interest was isolated from the surface of a lemon sample, identified as a B. amyloliquefaciens strain, and designated as B.amyloliquefaciens K103using biochemical, physiological, and16S ribosomal DNA sequencing.(2)Two antifungal compounds were purified from the culture broth using acid precipitation, gel permeation chromatography, and reversed-phase high-performance liquid chromatography. Based on its amino acid composition and MS/MS spectrometry analysis, they were successfully identified as bacillomycin L and its derivatives, varying only in terms of the length of the fatty acid aliphatic chains from C14to C15.(3)Results obtained from charateristics analysis of bacillomycin L shows it has a broad spectrum, high stability to heat, enzyme, and ultra-vioket radiation,which indicated it has a potentail application in biological control and food conservation.(4)Various fluorescent techniques, electron microscopy and model membrane assay show that the surface of cells was rough with more wrinkles and vesicles, the membranes show deformation and materials within the cytoplasm seem to be shrunken an unevenly dispersed, the fluidity of membrane was increased and the integrity and permeabilization of membrane was disrupted.(5)Gel retardation experiments and absorption titrations assay showed that bacillomycin L could bind to R. solani hyphal cells DNA in vitro,indicating that bacillomycin L was likely to interaction with the intracellular targets.(6) The protein bands of R. solani hyphal cells detected by SDS-PAGE were fleeted but not disappeared. Native-PAGE showed that non-denaturation protein bands of R. solani hyphal cells were diffenrent markedly with some bands fleeted and disappeared.(7)As compared with the untreated sample, there were48spots whose abundence varied significantly (more than2-fold),43spots significantly increased and5spots decreased after treated by bacillomycin L. matrixassisted laser desorption/ionization time-of-flight tandem mass spectrometry(MALDI-TOF/TOF-MS) and function classification analysis indicated these protein were involved in carbohydrate metabolism, nucleotide metabolism, amino acids metabolism, transmembrane transport, stress response,transcription, nucleotide binding, protein degradation, and cellular component organization. Real-time PCR and ATPase activity analysis of the hyphal proteins differentially expressed indicated that these differentially expression were consistented with corresponding gene expression roughly. (8)A pot experiment was carried out to investigate the effects of bacillomycin L and Bacillus amyloliquefaciens K103on the growth of cucumber seedlings, absorption and utilization of nutrient and the suppression of the seedlings damping-off disease. The results indicated that the application of bacillomycin L and Bacillus amyloliquefaciens K103could effectively promote growth, nutrition absorption of banana seedlings, and reduce the occurrence of seedlings damping-off.