Characterization of a gene regulating antibiotic production in Bacillus subtilis BSD-2

Abstract

Bacillus subtilis BSD-2 produces small molecule antibiotics that effectively control grey mould. To isolate the genes responsible for these antimicrobial activities, a B. subtilis BSD-2 mutant library was constructed utilizing the pHV1249 plasmid with a mini-Tn10 transposon. The antimicrobial potential of the generated strains was evaluated in the presence of cucumber grey mould and the mutant sequences were cloned by chromosome walking. Among the generated mutants, four mutants showed enhanced inhibitory effects in the presence of grey mould, and one mutant lost its inhibitory effect. The negative 1–19 mutant strains showed 98% sequence homology of the yodF gene, which encodes a Na+/metabolite permease. Next, we constructed a yodF knockout vector (pB-y-k) and used it to transform B. subtilis. A mutant yodF strain lost its inhibitory effect on grey mould, whereas reversion of the mutation restored the fungistatic activity.

Keywords

• Bacillus subtilis
• gene mapping
• chromosome walking