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.