Generation of Mini-Tn10 transposon insertion mutant library of Bacillus thuringiensis for the investigation of genes required for its bacteriocin production

Abstract

Bacillus thuringiensis strain BUPM4 is known for its ability to produce a bacteriocin, called Bacthuricin F4 (BF4), which inhibits the growth of several Gram-positive bacteria and particularly Bacillaceae. This study aimed to use the insertional transposon mutagenesis approach for disrupting and thus identifying genes associated with BF4 synthesis. Here, the mini-Tn10 transposon was used to generate a library of B. thuringiensis mutants. Twenty thousand clones were screened for the search of mutants with affected bacteriocin synthesis. By molecular hybridization, it was demonstrated that the mini-Tn10 transposition occurred in different sites. Clone MB1, containing a mini-Tn10 single-copy insertion, lost the BF4 synthesis, but maintained its immunity to BF4. The flanking sequences surrounding the mini-Tn10 insertion were cloned and sequenced. Homology searches of the surrounding ORFs revealed a strong similarity to a phage tail component, which allowed us to postulate that BUPM4 bacteriocin could be a phage tail-like one.