The three-dimensional structures in dodecylphosphocholine (DPC) micelles and in trifluoroethanol (TFE) of the pediocin-like antimicrobial peptide sakacin P and an engineered variant of sakacin P (termed sakP[N24C+44C]) have been determined by use of nuclear magnetic resonance spectroscopy. SakP[N24C+44C] has an inserted non-native activity- and structure-stabilizing C-terminal disulfide bridge that ties the C-terminus to the middle part of the peptide. In the presence of DPC, the cationic N-terminal region (residues 1-17) of both peptides has an S-shaped conformation that is reminiscent of a three-stranded antiparallel beta-sheet and that is more pronounced when the peptide was dissolved in TFE instead of DPC. The four positively charged residues located in the N-terminal part are found pointing to the same direction. For both peptides, the N-terminal region is followed by a well-defined central amphiphilic alpha-helix (residues 18-33), and this in turn is followed by the C-terminal tail (residues 34-43 for sakacin P and 34-44 for sakP[N24C+44C]) that lacks any apparent common secondary structural motif. In the presence of DPC, the C-terminal tails in both peptides fold back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal halves. The lack of long-range NOEs between the beta-sheet Nu-terminal region and the hairpin-like C-terminal half indicates that there is a flexible hinge between these regions. We discuss which implications such a structural arrangement has on the interaction with the target cell membrane.

A rapid and simple two-step procedure suitable for both small-and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.Gene-encoded, ribosomally synthesized antimicrobial peptides are widely distributed in nature, being produced by bacteria, plants, and a wide variety of animals, including humans (28,29,32,34). The peptides are often cationic and amphiphilic or hydrophobic, and many of them kill bacteria by permeabilizing the target cell membrane. The peptides may be developed into new and useful antimicrobial additives and drugs. An example of this is the antimicrobial peptide nisin, which is produced by lactic acid bacteria (LAB). This peptide is used as a food preservative (9) and has been considered for use for treatment of gastric Heliobacter infections and/or ulcers (16).There has especially been considerable interest in antimicrobial peptides (bacteriocins) produced by LAB because of the "food-grade quality" and industrial importance of these bacteria. LAB are used in food production, are part of the natural microbial flora in food that humans have consumed for centuries, and constitute a significant part of the indigenous flora of mammals, including humans. Thus, LAB and the bacteriocins that they produce may be considered safe agents for preventing growth of pathogenic and/or undesirable microorganisms.Many of the LAB bacteriocins belong to the pediocin-like family; these bacteriocins are of special interest because of their antilisterial activity. The family contains at least 15 different bacteriocins, of which pediocin PA-1 (3,19,23,30), leucocin A UAL-187 (17), mesentericin Y105 (18), sakacin P (35) and curvacin A (identical to sakacin A [20,35]) were the first to be identified. All pediocin-like bacteriocins are cationic, contain between 35 and 50 amino acid residues, permeabilize target cell membranes, and have very similar primary structures but differ markedly with respect to their target cell specificity (5-7, 10-14, 22, 24, 29, 32, 37).The use of pediocin-like bacteriocins and other antimicrobial peptides as additives or drugs requires a simple and rapid method by which large quantities may be purified to homogeneity. Present methods for purification of pediocin-like bacteriocins and other cationic bacteriocins generally include a centrifugati...

Lactococcin G (LcnG) is an antimicrobial substance (bacteriocin) consisting of two peptides, LcnG-A and LcnG-β. The structures of intact LcnG-A and LcnG-β as well as various fragments of these peptides were studied by circular dichroism (CD) under several conditions. All peptides had a non-structured conformation in aqueous solutions. In the presence of trifluoroethanol, dodecylphosphocholine micelles and (negatively charged) dioleoylglycerophosphoglycerol (Ole 2 GroPGro) liposomes, varying amounts of A-helical structure were induced. Comparisons of the various fragments showed that helicity was concentrated in those parts of LcnG-A and LcnG-β that would become amphiphilic if an A-helical structure was adopted. In the presence of zwitterionic dioleoylglycerophosphocholine (Ole 2 GroPCho) liposomes, the peptides were much less (if at all) structured, suggesting that the excess of positive charge on the antimicrobial peptides needs to be compensated by an excess of negative charge on the membrane. The structuring of LcnG-A and LcnG-β in the presence of Ole 2 GroPGro liposomes was considerably enhanced when both peptides were presented simultaneously to the membranes. Consecutive addition of the two peptides to Ole 2 GroPGro liposomes did not give this additional structuring, indicating that the individual LcnG-A and LcnG-β peptides associate with the membrane in a virtually irreversible manner that makes them inaccessible for interaction with the complementary peptide. The results suggest that upon arrival at and interaction with the target membrane, LcnG-A and LcnG-β form a complex that consists of approximately 50% amphiphilic A-helices.

Production of bacteriocins by lactic acid bacteria is in some cases regulated by a quorum sensing mechanism that involves a secreted bacteriocin-like peptide pheromone. In the case of Lactobacillus plantarum C11, this pheromone, the 26-mer plantaricin A (PlnA), has the interesting property of having both bacteriocin and pheromone activities. To gain insight into how PlnA functions as a pheromone and as a bacteriocin, the l- and d-enantiomers of an N-terminally truncated form of PlnA were synthesized (PlnA-22L and PlnA-22D; PlnA-22L has full biological activity). With circular dichroism, it was shown that the two peptides are unstructured in aqueous solution, but they adopt mirror-image amphiphilic helical structures in the presence of trifluoroethanol and membrane-mimicking entities such as micelles of dodecylphosphocholine and negatively charged Ole2GroPGro liposomes, but not in the presence of zwitterionic Ole2GroPCho liposomes. Thus, the negative charge on the membrane is important for structuring of the (positively charged) PlnA peptides. In terms of in vivo antimicrobial activity, PlnA-22L and PlnA-22D behaved almost identically. Likewise, the peptides dissipated the membrane potential and the transmembrane pH gradient in sensitive cells equally effectively. PlnA-22L induced bacteriocin production in L. plantarum C11 (i.e., displayed pheromone activity), the level of induction being clearly dose-dependent. PlnA-22D did not display pheromone activity, but, at high concentrations, was able to inhibit the pheromone activity of PlnA-22L. The results indicate that the antimicrobial activity of PlnA does not require chiral interactions and is mediated through the formation of a strongly amphiphilic α-helical structure. In contrast, PlnA's pheromone activity is dependent on a chiral interaction between the amphiphilic helix (PlnA-22L) and a receptor protein. One may speculate that PlnA is an evolutionary intermediate between a true bacteriocin and a pheromone.

Lactobacillus plantarum C11 produces plantaricin E/F (PlnE/F) and plantaricin J/K (PlnJ/K), two bacteriocins whose activity depends on the complementary action of two peptides (PlnE and PlnF; PlnJ and PlnK). Three of the individual Pln peptides possess some antimicrobial activity, but the highest bacteriocin activity is obtained by combining complementary peptides in about a one-to-one ratio. Circular dichroism was used to study the structure of the Pln peptides under various conditions. All four peptides were unstructured under aqueous conditions but adopted a partly alpha-helical structure in the presence of trifluoroethanol, micelles of dodecylphosphocholine, and negatively charged dioleoylphosphoglycerol (DOPG) liposomes. Far less structure was induced by zwitterionic dioleoylglycerophosphocholine liposomes, indicating that a net negative charge on the phospholipid bilayer is important for a structure-inducing interaction between (positively charged) Pln peptides and a membrane. The structuring of complementary peptides was considerably enhanced when both (PlnE and PlnF or PlnJ and PlnK) were added simultaneously to DOPG liposomes. Such additional structuring was not observed in experiments with trifluoroethanol or dodecylphosphocholine, indicating that the apparent structure-inducing interaction between complementary Pln peptides requires the presence of a phospholipid bilayer. The amino acid sequences of the Pln peptides are such that the alpha-helical structures adopted upon interaction with the membrane and each other are amphiphilic in nature, thus enabling membrane interactions.

Plantaricin EF and JK are both two-peptide bacteriocins produced by Lactobacillus plantarum C11. The mechanism of plantaricin EF and JK action was studied on L. plantarum 965 cells. Both plantaricins form pores in the membranes of target cells and dissipate the transmembrane electrical potential (⌬) and pH gradient (⌬pH). The plantaricin EF pores efficiently conduct small monovalent cations, but conductivity for anions is low or absent. Plantaricin JK pores show high conductivity for specific anions but low conductivity for cations. These data indicate that L. plantarum C11 produces bacteriocins with complementary ion selectivity, thereby ensuring efficient killing of target bacteria.

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was puri®ed to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (a and b) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known`double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the puri®ed a and b peptides in the residues serine, threonine, cysteine and alanine. Post-translational modi®cation, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are¯anked by IS946 elements, suggesting an en bloc transposition. The ®ndings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.

No abstract