Regulation of bacteriocin production in Lactobacillus plantarum depends ona conserved promoter arrangement with consensus binding sequence

Bacteriocin production in Lactobacillus plantarum? C11 is regulated by a th ree-component signal transduction system comprising a peptide pheromone (Pl nA), a histidine protein kinase (PlnB), and two homologous response regulat ors (RRs; PlnC and PlnD). Both RRs are DNA-binding proteins that bind to pr omoter-proximal elements in the pin regulon. The binding site for the two r egulators consists of two 9-bp direct repeats, that conform to the consensu s sequence 5 ' TACGTTAAT-3 '. and the repeats are separated by an interveni ng 12-bp AT-rich spacer region. In the present work, the plnA promoter was used as a model to evaluate the significance of the binding sequence and co nserved promoter arrangement. Point substitutions in the consensus sequence , particularly those in invariant positions, either abolished or significan tly reduced binding of PlnC and PlnD. Both regulators bind as homodimers to DNA fragments containing a complete set of regulatory elements, while remo val of either repeat, or alterations in the length of the spacer region, si gnificantly weakened binding of both protein dimers. DNase I footprinting d emonstrated that PlnC and PlnD both bind to, and protect, the direct repeat s. By fusing the plnA promoter region to the beta -glucuronidase (GUS) gene , it was shown that promoter activity is dependent on an intact set of accu rately organized repeats. The in vitro and in vivo results presented here c onfirm the involvement of the repeats as regulatory elements in the regulat ion of bacteriocin production.