A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B-cereus haemolysin II

Haemolysin ll, Hlyll, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyll gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyllR, which is located immediately downstream of hlyll and regulates hlyll expression, is reported. The deduced amino acid sequence of HIyllR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyll promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E coli, hlyll transcription decreased in the presence of hlyll/R. Recombinant HlyllR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyll promoter transcription initiation point. In vitro transcription studies showed that HlyllR inhibits transcription from the hlyll promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.