Abstract:

【Objective】The aim of this study is to clone and sequence the Bac operon responsible for synthesis of the bacillomycin L from Bacillus subtilis Bs916 and determine the structure and biological activities of the bacillomycin L. 【Method】 LA-PCR and gene walking were performed to clone the Bac operon responsible for synthesis of bacillomycin L. Analysis of the Bac operon genetic structure was made using bioinformatics. The molecular weight of the bacillomycin L homologues was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS). ESI/MS-CID was used to determine the bacillomycin L peptide moiety primary structure. 【Result】 The 39.0 kb Bac operon responsible for synthesis of bacillomycin L was identified and cloned from Bacillus subtilis Bs-916. The operon contained a promoter and four ORFs and the four ORFs designated BacD, BacA, BacB, and BacC, respectively. Although the amino acid sequences encoded by the Bac operon share high similarity with the countpart amino acid sequences encoded by itu, myc and bam operons, a low similarity region was also found and it presumed to be a novel Ser activation domain. According to the Bac sequence, the lipoepeptide presumed to belong to bacillomycin L. The molecular weights of the bacillomycin L were 1 008,1 022,1 036 and 1 050 Da. They were presumed to belong to homologues differed by a structure of -CH2. The bacillomycin L peptide moiety primary structure was [cyclo-(Asn-Tyr-Asn-Ser-Glu-Ser-Thr-β-amino fatty acid)] and it is the same as the peptide sequence of bacillomycin L reported previously. In vitro tests to pathogenic fungi indicated that bacillomycin L have a broad antifungal activities and hemolytic activities.【Conclusion】This paper reported the cloning, sequencing and characterization of a whole operon Bac which is responsible for synthesis of bacillomycin L. Through bio-information and chemical analysis, the authors also further confirmed the primary structure of bacillomycin L which has paradox reported before. The bacillomycin L synthesized by Bac has a broad antifungal activities and plays crucial part in Bs-916 bio-control activities.

Key words: Bacillus subtilis, bacillomycin L, operon, structure, biological activity