Global regulatory systems operating in Bacilysin biosynthesis in Bacillus subtilis.

In Bacillus subtilis, bacilysin is a nonribosomally synthesized dipeptide antibiotic composed of L-alanine and L-anticapsin. The biosynthesis of bacilysin depends on the bacABCDEywfG operon (bac operon)and the adjacent ywfH gene. To elucidate the effects of global regulatory genes on the expression of bac operon, we used the combination of lacZ fusion analysis and the gel mobility shift assays. The cell density-dependent transition state induction of the bac operon was clearly shown. The basal expression level of the bac operon as well as transition state induction of bac is directly ComA dependent. Three Phr peptides, PhrC, PhrF and PhrK, are required for full-level expression of ComA-dependent bac operon expression, but the most important role seemed to be played by PhrC in stimulating bac expression through a RapC-independent manner. Spo0A is another positive regulator which participates in the transition state induction of bac both directly by interacting with the bac promoter and indirectly by repressing abrB expression. AbrB and CodY proteins do not only directly repress the bac promoter, but they also mutually stimulate the transition state induction of bac indirectly, most likely by antagonizing their repressive effects without preventing each other's binding since both proteins can bind to the bac promoter simultaneously.

The novel gene yvfI in Bacillus subtilis is essential for bacilysin biosynthesis.

Using transposon mutagenesis in Bacillus subtilis PY79, three independent mutants defective in production of bacilysin were isolated. To identify the genes in these mutant loci affecting bacilysin biosynthesis, the inserted transposon and its flanking regions were cloned and sequenced from each mutant. Transposon insertions in these three mutants were found to be in the yvfI gene which encodes an unknown protein similar to GntR family transcriptional regulators. For further confirmation, deletion mutants were constructed in which nucleotides 196-314 of the yvfI gene were removed. All resulting yvfI (Delta196-314)::spc deletion mutants exhibited bacilysin-negative phenotypes, as in the case of the yvfI::Tn10::spc insertional mutants. The lacR gene, encoding a transcriptional regulator, resides immediately downstream from the yvfI gene. Therefore, an insertion mutation was created in the lacR gene to demonstrate that the bacilysin negative phenotype is actually due to the mutation in the yvfI gene and not a polar effect of yvfI mutation on the downstream gene. As expected, all resulting lacR mutant derivatives of PY79 still produced bacilysin.