Evidence for dual functionality of the operon plnABCD in the regulation of bacteriocin production in Lactobacillus plantarum.

The regulatory operon (plnABCD) involved in bacteriocin production in Lactobacillus plantarum C11 encodes four different proteins: a cationic prepeptide (PlnA); a histidine protein kinase (PlnB); and two highly homologous response regulators (PlnC and PlnD; over 75% sequence similarity). The mature product of PlnA, plantaricin A, serves as an extracellular pheromone that induces bacteriocin production. The exact roles of plnBCD in bacteriocin production have not been established experimentally. A reporter system containing the gusA gene fused with the plnA promoter was used to study plnABCD. We demonstrated that the plnABCD operon codes for an autoregulatory unit capable of activating its own promoter. Deletion analyses, performed in a heterologous expression host to define the roles of the individual genes, confirmed that both the inducer gene (plnA) and the kinase gene (plnB) are required for autoactivation. Apparently, the latter gene encodes a protein that serves as a receptor for the pheromone peptide. It was also demonstrated conclusively that the two regulators PlnC and PlnD, which have been shown previously to bind specifically to the DNA regulatory repeats of the plnA promoter, possess differential activities on the plnA promoter, with PlnC being much more active than PlnD. The functions of the response regulators were investigated further in the bacteriocin producer strain C11 in order to reveal their roles in bacteriocin production. Surprisingly, the two response regulators display totally opposite functions: although overexpression of plnC activated transcription and bacteriocin production, the overexpression of plnD repressed both processes, thus strongly suggesting that PlnD plays a role in the downregulation of bacteriocin synthesis. To our knowledge, this is the first evidence for a protein involved directly in negative regulation of bacteriocin production, and also it was shown for the first time that two highly homologous response regulators, with opposite functions, are encoded by genes located on the same operon.

Regulation of bacteriocin production in Lactobacillus plantarum depends on a conserved promoter arrangement with consensus binding sequence.

Bacteriocin production in Lactobacillus plantarum C11 is regulated by a three-component signal transduction system comprising a peptide pheromone (PlnA), a histidine protein kinase (PlnB), and two homologous response regulators (RRs; PlnC and PlnD). Both RRs are DNA-binding proteins that bind to promoter-proximal elements in the pln regulon. The binding site for the two regulators consists of two 9-bp direct repeats, that conform to the consensus sequence 5'-TACGTTAAT-3', and the repeats are separated by an intervening 12-bp AT-rich spacer region. In the present work, the plhA promoter was used as a model to evaluate the significance of the binding sequence and conserved promoter arrangement. Point substitutions in the consensus sequence, particularly those in invariant positions, either abolished or significantly reduced binding of PlnC and PlnD. Both regulators bind as homodimers to DNA fragments containing a complete set of regulatory elements, while removal of either repeat, or alterations in the length of the spacer region, significantly weakened binding of both protein dimers. DNase I footprinting demonstrated that PlnC and PlnD both bind to, and protect, the direct repeats. 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 accurately organized repeats. The in vitro and in vivo results presented here confirm the involvement of the repeats as regulatory elements in the regulation of bacteriocin production.