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.

The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe. Based on a three-dimensional model (Y. Chen, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Microbiol. 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes. Binding of pediocin PA-1 to vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted into the hydrophobic core of the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out. Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the lipid vesicles paralleled the net positive charge in the peptide. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch. Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues. These results demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane.

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Colicin V is a ribosomally synthesized antimicrobial peptide produced by Escherichia coli. Four recently characterized genes, arranged in two convergent operons on the plasmid pCoIV-K30, are required for colicin V synthesis, export and immunity. We report the purification and N-terminal amino acid sequencing of the colicin V protein. Our results demonstrate that the colicin V primary translation product, which consists of 103 amino acids, is proteolytically processed. A leader peptide, consisting of 15 amino acid residues, is removed from the N-terminus during maturation of colicin V. This leader peptide is not related to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the Sec pathway. The molecular mass of colicin V, obtained by mass spectrometry analysis, showed that the peptide consists of only unmodified amino acids. The deduced amino acid sequence of the leader peptide was highly homologous to the N-terminal extensions found in non-lantibiotic, peptide bacteriocins produced by Gram-positive bacteria. These findings strongly indicate that colicin V belongs to a family of small peptide bacteriocins that have been found previously only among the Gram-positive lactic acid bacteria.