Identification of the bacteriocin subtilosin A and loss of purL results in its high-level production in Bacillus amyloliquefaciens.

A mini-Tn10 transposon mutagenesis library of strain Bacillus amyloliquefaciens BAhja NK10 was constructed and one mutant, L4, exhibiting markedly enhanced antagonistic activity against Micrococcus luteus, was screened and possessed an inactivated purL gene. A similar antibacterial spectrum was observed for both L4 and wild-type strains, but L4 displayed a markedly strengthened antimicrobial effect. Extracts of the corresponding culture media were separated by means of liquid chromatography and subjected to antimicrobial assay wherein the active molecule was further purified. Mass spectrometry and genetic analysis data revealed that the bacteriocin subtilosin A was produced and was responsible for the antibacterial activity of B. amyloliquefaciens. Expression of the subtilosin-A-encoding gene sboA was determined to occur earlier in the purL mutant, while the transcription level of the negative regulator AbrB declined by 3.3-fold. Correspondingly, an increase was observed in the mRNA levels of two early sporulation genes spo0A and sigH, which are implicated in repressing abrB expression and regulating subtilosin A production. Moreover, RT-PCR showed that the expression of three Kin family kinases was coordinately upregulated. We speculated that purL mutation could result in induced expression of Kin kinases and accelerated accumulation of phosphorylated Spo0A, with the latter repressing transcription of abrB which ultimately led to earlier and increased production of the bacteriocin subtilosin A.