Structure-function relationships of the non-lanthionine-containing peptide (class II) bacteriocins produced by gram-positive bacteria.

This review focuses on the structure and mode-of-action of non-lanthionine-containing peptide bacteriocins produced by Gram-positive bacteria. These bacteriocins may be divided into four groups: (i) the anti-listerial one-peptide pediocin-like bacteriocins that have very similar amino acid sequences, (ii) the two-peptide bacteriocins that consist of two different peptides, (iii) the cyclic bacteriocins, and (iv) the linear non-pediocin-like one-peptide bacteriocins. These bacteriocins are largely cationic, contain 20 to 70 residues, and kill cells through membrane-permeabilization. The pediocin-like bacteriocins are the ones that are best characterized. Upon contact with target membranes, their cationic N-terminal half forms a beta-sheet-like structure that binds to the target cell surface, while their more hydrophobic helical-containing C-terminal half penetrates into the hydrophobic core of target-cell membranes and apparently binds to the mannose phosphotransferase permease in a manner that results in membrane leakage. Immunity proteins that protect cells from being killed by pediocin-like bacteriocins bind to the bacteriocin-permease complex and prevent bacteriocin-induced membrane-leakage. Recent structural analyses of two-peptide bacteriocins indicate that they form a helix-helix structure that penetrates into cell membranes. Also these bacteriocins may act by binding to integrated membrane proteins. It is proposed that many membrane-active peptide bacteriocins kill target-cells through basically the same mechanism; the common theme being that a membrane-penetrating part of bacteriocins bind to a membrane embedded region of an integrated membrane protein, thereby causing conformational alterations in the protein that in turn lead to membrane-leakage and cell death.

Characterization of a bacteriocin produced by Prevotella nigrescens ATCC 25261.

AIMS: To characterize the antimicrobial activity produced by Prevotella nigrescens ATCC 25261, and to evaluate its safety on cultured gingival fibroblasts. METHODS AND RESULTS: An antimicrobial activity was obtained from purifying the culture supernatant of Pr. nigrescens ATCC 25261. Purification of the active compound was achieved with ammonium sulphate precipitation followed by anion-exchange and gel filtration chromatography. As revealed by SDS-PAGE, the active fraction was relatively homogeneous, showing a protein with an approximate molecular weight of 41 kDa. The antimicrobial compound, named nigrescin, exhibited a bactericidal mode of action against Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Actinomyces spp. Nigrescin was stable in a pH range between 6.5 and 9.5, at 100 degrees C for 10 min, and resistant to lyophilization. But its activity was lost after proteinase K treatment. Despite at very high concentrations beyond the minimum inhibitory concentration (MIC), nigrescin was not toxic to the gingival fibroblasts. CONCLUSION: Nigrescin is a novel bacteriocin produced by Pr. nigrescens ATCC 25261. It exhibits antimicrobial activity against species that are implicated in periodontal diseases. The absence of toxicity on the gingival fibroblasts suggests the possibility in using of nigrescin for an application in periodontal treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Novel evidence on nigrescin would make Pr. nigrescens ATCC 25261 attractive in biotechnological applications as an antimicrobial agent in clinical dentistry.

Engineering increased stability in the antimicrobial peptide pediocin PA-1.

Pediocin PA-1 is a food grade antimicrobial peptide that has been used as a food preservative. Upon storage at 4 degrees C or room temperature, pediocin PA-1 looses activity, and there is a concomitant 16-Da increase in the molecular mass. It is shown that the loss of activity follows first-order kinetics and that the instability can be prevented by replacing the single methionine residue (Met31) in pediocin PA-1. Replacing Met by Ala, Ile, or Leu protected the peptide from oxidation and had only minor effects on bacteriocin activity (for most indicator strains 100% activity was maintained). Replacement of Met by Asp was highly deleterious for bacteriocin activity.

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105.

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) 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 purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, 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 flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.

A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum.

Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37 degrees C, whereas wild-type sakacin P was approximately 10 times less potent at 37 degrees C than at 20 degrees C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys-->Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.

Complementary and overlapping selectivity of the two-peptide bacteriocins plantaricin EF and JK.

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 (Deltapsi) and pH gradient (DeltapH). 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.

Isolation of a low-molecular-weight growth inhibitory factor from hybridoma cell cultures.

A low-molecular-weight growth inhibitory factor was produced by hybridoma cells. The number of viable cells in hybridoma cell cultures reached a maximum of about 5 x 10(5) cells/ml when the inhibitory factor had accumulated to a critical level, after which the number of viable cells declined with a concomitant increase in the number of dead cells. The growth inhibitory factor was purified to apparent homogeneity by ultrafiltration, reverse-phase chromatography, passage through cation exchangers, and gel filtration. Analysis by reverse-phase chromatography and micellar electrokinetic chromatography using a capillary electrophoresis system indicated that the final inhibitory fraction was pure. The factor had a molecular weight of 500 or less, as judged by ultrafiltration, and its behavior upon ion-exchange chromatography indicated that it was uncharged. Its absorbance maximum at 263 nm indicated that it was not a peptide, but that it may have a conjugated system of carbon-carbon double bonds.

Membrane-mimicking entities induce structuring of the two-peptide bacteriocins plantaricin E/F and plantaricin J/K.

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.

The bactericidal activity of pediocin PA-1 is specifically inhibited by a 15-mer fragment that spans the bacteriocin from the center toward the C terminus.

A 15-mer peptide fragment derived from pediocin PA-1 (from residue 20 to residue 34) specifically inhibited the bactericidal activity of pediocin PA-1. The fragment did not inhibit the pediocin-like bacteriocins sakacin P, leucocin A, and curvacin A to nearly the same extent as it inhibited pediocin PA-1. Enterocin A, however, was also significantly inhibited by this fragment, although not as greatly as pediocin PA-1. This is consistent with the fact that enterocin A contains the longest continuous sequence identical to that of pediocin PA-1 in the region spanned by the fragment. The fragment inhibited pediocin PA-1 to a much greater extent than did the other 29 possible 15-mer fragments that span pediocin PA-1. The results suggest that the fragment-by interacting with the target cells and/or pediocin PA-1-interferes specifically with pediocin-target cell interaction.

Plantaricin A is an amphiphilic alpha-helical bacteriocin-like pheromone which exerts antimicrobial and pheromone activities through different mechanisms.

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 alpha-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.

Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A.

Six bacteriocinlike peptides (plantaricin A [PlnA], PlnE, PlnF, PlnJ, PlnK, and PlnN) produced by Lactobacillus plantarum C11 were detected by amino acid sequencing and mass spectrometry. Since purification to homogeneity was problematic, all six peptides were obtained by solid-phase peptide synthesis and were tested for bacteriocin activity. It was found that L. plantarum C11 produces two two-peptide bacteriocins (PlnEF and PlnJK); a strain-specific antagonistic activity was detected at nanomolar concentrations when PlnE and PlnF were combined and when PlnJ and PlnK were combined. Complementary peptides were at least 10(3) times more active when they were combined than when they were present individually, and optimal activity was obtained when the complementary peptides were present in approximately equal amounts. The interaction between complementary peptides was specific, since neither PlnE nor PlnF could complement PlnJ or PlnK, and none of these peptides could complement the peptides constituting the two-peptide bacteriocin lactococcin G. Interestingly, PlnA, which acts as an extracellular signal (pheromone) that triggers bacteriocin production, also possessed a strain-specific antagonistic activity. No bacteriocin activity could be detected for PlnN.

Amphiphilic alpha-helices are important structural motifs in the alpha and beta peptides that constitute the bacteriocin lactococcin G--enhancement of helix formation upon alpha-beta interaction.

Lactococcin G (LcnG) is an antimicrobial substance (bacteriocin) consisting of two peptides, LcnG-alpha and LcnG-beta. The structures of intact LcnG-alpha and LcnG-beta 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 (Ole2GroPGro) liposomes, varying amounts of alpha-helical structure were induced. Comparisons of the various fragments showed that helicity was concentrated in those parts of LcnG-alpha and LcnG-beta that would become amphiphilic if an alpha-helical structure was adopted. In the presence of zwitterionic dioleoylglycerophosphocholine (Ole2GroPCho) 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-alpha and LcnG-beta in the presence of Ole2GroPGro liposomes was considerably enhanced when both peptides were presented simultaneously to the membranes. Consecutive addition of the two peptides to Ole2GroPGro liposomes did not give this additional structuring, indicating that the individual LcnG-alpha and LcnG-beta 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-alpha and LcnG-beta form a complex that consists of approximately 50% amphiphilic alpha-helices.

Mechanistic properties of the two-component bacteriocin lactococcin G.

Lactococcin G is a bacteriocin whose activity depends on the complementary action of two peptides, termed alpha and beta. Biologically active, synthetic lactococcin G was used to study the mode of action on sensitive cells of Lactococcus lactis. The alpha and beta peptides can bind independently to the target cell surface, but activity requires the complementary peptide. Once bound to the cell surface, the peptides cannot be displaced to the surfaces of other cells. A complex of alpha and beta peptides forms a transmembrane pore that conducts monovalent cations but not protons. Efflux of potassium ions is observed only above pH 5.0, and the rate of efflux increases steeply with the pH. The consequences of cation fluxes for the viability of the target cells are discussed.

Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action.

Ribosomally synthesized peptides with antimicrobial activity are produced by prokaryotes, plants, and a wide variety of animals, both vertebrates and invertebrates. These peptides represent an important defense against micro-organisms. Although the peptides differ greatly in primary structures, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of these peptides kill their target cells by permeabilizing the cell membrane. Moreover, many of these peptides may roughly be placed into one of three groups: (1) those that have a high content of one (or two) amino acid(s), often proline, (2) those that contain intramolecular disulfide bonds, often stabilizing a predominantly beta-sheet structure, and (3) those with amphiphilic regions if they assume an alpha-helical structure. Most known ribosomally synthesized antimicrobial peptides have been identified and characterized during the past 15 years. As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as innate immunity, membrane-protein interactions, and protein modification and secretion. Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives and drugs. This review presents a broad overview of the main types of ribosomally synthesized antimicrobial peptides produced by eukaryotes and prokaryotes.

New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity.

The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.

Lactococcin G is a potassium ion-conducting, two-component bacteriocin.

Lactococcin G is a novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides, termed alpha and beta. Peptide synthesis of the alpha and beta peptides yielded biologically active lactococcin G, which was used in mode-of-action studies on sensitive cells of Lactococcus lactis. Approximately equivalent amounts of both peptides were required for optimal bactericidal effect. No effect was observed with either the alpha or beta peptide in the absence of the complementary peptide. The combination of alpha and beta peptides (lactococcin G) dissipates the membrane potential (delta omega), and as a consequence cells release alpha-aminoisobutyrate, a non-metabolizable alanine analog that is accumulated through a proton motive-force dependent mechanism. In addition, the cellular ATP level is dramatically reduced, which results in a drastic decrease of the ATP-driven glutamate uptake. Lactococcin G does not form a proton-conducting pore, as it has no effect on the transmembrane pH gradient. Dissipation of the membrane potential by uncouplers causes a slow release of potassium (rubidium) ions. However, rapid release of potassium was observed in the presence of lactococcin G. These data suggest that the bactericidal effect of lactococcin G is due to the formation of potassium-selective channels by the alpha and beta peptides in the target bacterial membrane.

Production and pH-Dependent Bactericidal Activity of Lactocin S, a Lantibiotic from Lactobacillus sake L45.

The amount of lactocin S activity in a growing culture depends on the growth stage of the bacteria, the pH of the medium, the presence of ethanol, and the aeration of the culture. We observed the highest levels of bacteriocin activity in the early stationary growth phase of cultures at 30 deg C. When Lactobacillus sake L45 was grown in a fermentor at pH 5, it produced 2,000 to 3,000 bacteriocin units per ml, which represented an 8- to 10-fold increase in bacteriocin production compared with production during batch culture fermentation. Less than 10% of this level of bacteriocin activity was observed during fermentation at pH 6.0. When 1% ethanol was included in the growth medium, a two- to fourfold increase in the bacteriocin yield was observed. Aerating the culture during growth almost completely eliminated the production of active bacteriocin. Our results also showed that lactocin S-mediated killing of target cells depended on the pH of the culture. The pH had to be less than 6 in order to obtain a bactericidal effect with lactocin S-sensitive cells. At pH values greater than 6, lactocin S had no apparent effect on sensitive cells.

In vivo conversion of L-serine to D-alanine in a ribosomally synthesized polypeptide.

In the course of characterizing the bacteriocin lactocin S and its encoding gene, we discovered three alanine-for-serine substitutions which, apparently, is a violation of the genetic code. Subsequent chiral analysis of lactocin S hydrolysates revealed a correlation between D-alanine content and the three substitutions, implying a conversion of L-serine to D-alanine in lactocin S maturation. In order to explain this observation, we suggest a sequence of events initiated by the dehydration of serine, which is common in the biosynthesis of the lanthionine-containing polycyclic lantibiotics (Schnell, N., Entian, K.-D., Schneider, U., Götz, F., Zähner, H., Kellner, R. & Jung, G. (1988) Nature 333, 276-278; Jung, G. (1991) Angew, Chem. Int. Ed. Engl. 30, 1051-1068; Bierbaum, G. & Sahl, H.-G. (1993) Zentralbl. Bakteriol. 278, 1-22) and completed by the stereospecific reduction of dehydroalanine residues. The occurrence of non-lanthionine alpha-carbon stereoinversion in lactocin S maturation substantiates the hypothetical alpha-epimerization scheme originally put forward by Bycroft (Bycroft, B. W. (1969) Nature 224, 595-597), and we propose a revision of this model to accommodate the lactocin S-type stereoinversion. Lactocin S is the first prokaryotic exception to the rule that only L-amino acids are included in ribosomally synthesized peptides.

The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system.

Purification and amino acid sequencing of plantaricin A, a bacteriocin from Lactobacillus plantarum C11, revealed that maximum bacteriocin activity is associated with the complementary action of two almost-identical peptides, alpha and beta (J. Nissen-Meyer, A. G. Larsen, K. Sletten, M. Daeschel, and I. F. Nes, J. Gen. Microbiol. 139:1973-1978, 1993). A 5-kb chromosomal HindIII restriction fragment containing the structural gene of plantaricin A was cloned and sequenced. Only one gene encoding plantaricin A was found. The gene, termed plnA, encodes a 48-amino-acid precursor peptide, of which the 22 and 23 C-terminal amino acids correspond to the purified peptides. Northern (RNA) blot analysis demonstrated that a probe complementary to the coding strand of the plantaricin A gene hybridized to a 3.3-kb mRNA transcript. Further analysis of the 3.3-kb transcript demonstrated that it contains three additional open reading frames (plnB, plnC and plnD) downstream of plnA. The DNA sequences of plnB, plnC, and plnD revealed that their products closely resemble members of bacterial two-component signal transduction systems. The strongest homology was found to the accessory gene regulatory (agr) system, which controls expression of exoproteins during post-exponential growth in Staphylococcus aureus. The finding that plnABCD are transcribed from a common promoter suggests that the biological role played by the bacteriocin is somehow related to the regulatory function of the two-component system located on the same operon.