Improved Antimicrobial Activities of Synthetic-Hybrid Bacteriocins Designed from Enterocin E50-52 and Pediocin PA-1

Two hybrid bacteriocins, enterocin E50-52/pediocin PA-1 (EP) and pediocin PA-1/enterocin E50-52 (PE), were designed by combining the N terminus of enterocin E50-52 and the C terminus of pediocin PA-1 and by combining the C terminus of pediocin PA-1 and the N terminus of enterocin E50-52, respectively. Both hybrid bacteriocins showed reduced MICs compared to those of their natural counterparts. The MICs of hybrid PE and EP were 64- and 32-fold lower, respectively, than the MIC of pediocin PA-1 and 8- and 4-fold lower, respectively, than the MIC of enterocin E50-52. In this study, the effect of hybrid as well as wild-type (WT) bacteriocins on the transmembrane electrical potential (ΔΨ) and their ability to induce the efflux of intracellular ATP were investigated. Enterocin E50-52, pediocin PA-1, and hybrid bacteriocin PE were able to dissipate ΔΨ, but EP was unable to deplete this component. Both hybrid bacteriocins caused a loss of the intracellular concentration of ATP. EP, however, caused a faster efflux than PE and enterocin E50-52. Enterocin E50-52 and hybrids PE and EP were active against the Gram-positive and Gram-negative bacteria tested, such as Micrococcus luteus, Salmonella enterica serovar Enteritidis 20E1090, and Escherichia coli O157:H7. The hybrid bacteriocins designed and described herein are antimicrobial peptides with MICs lower those of their natural counterparts. Both hybrid peptides induce the loss of intracellular ATP and are capable of inhibiting Gram-negative bacteria, and PE dissipates the electrical potential. In this study, the MIC of hybrid bacteriocin PE decreased 64-fold compared to the MIC of its natural peptide counterpart, pediocin PA-1. Inhibition of Gram-negative pathogens confers an additional advantage for the application of these peptides in therapeutics.     

Purification and mass spectrometry based characterization of a pediocin produced by Pediococcus acidilactici 13

Bacteriocins are antimicrobial peptides produced by several bacterial species. Among the bacteriocins pediocin-like bacteriocins have a significant inhibitory activity on the foodborne pathogens especially on Listeria monocytogenes. This study aims to select a simple and usable purification method to purify/concentrate the antimicrobial peptide and characterization of the bacteriocin produced by Pediococcus acidilactici 13 by using proteomic approaches which is a recent omic technology. For purification dialysis, ultrafiltration method was used, and as a result of this study the bacteriocin activity reached 819,200 AU/mL from 102,400 AU/mL initially. Two dimensional gel electrophoresis and then matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) analysis were carried out to identify the current bacteriocin and related proteins. Obtained data revealed similarity to pediocin PA-1 transport/processing ATP-binding protein PedD (accession number: P36497), pediocin operon PedC (accession number: Q68GC4) and bacteriocin pediocin PA-1 (accession number: P29430) from UniProtKB/Swiss-Prot databank, thus the bacteriocin produced by P. acidilactici 13 is considered similar to pediocin PA-1.     

Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0.

The plasmid-encoded bacteriocin pediocin PA-1, produced by the gram-positive bacterium Pediococcus acidilactici strain PAC-1.0, was purified to homogeneity. The purified product exhibited antibacterial activity against several gram-positive bacterial strains, including the food pathogen Listeria monocytogenes. Pediocin PA-1 is a 4629-Da peptide with 44 amino acids and two disulfide bonds. The amino acid sequence and arrangement of the disulfide bonds were determined. Sequence data were used to calculate an isoelectric point of 10.0. The small and basic nature of PA-1 is comparable to several other bacteriocins produced by gram-positive bacteria. Reported sequences of other bacteriocins and of other antimicrobial peptides from diverse origins bear no resemblance to the sequence reported here.     

Production of Pediocin PA-1 by Lactococcus lactis Using the Lactococcin A Secretory Apparatus

The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp. lactis bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions −1 and −2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an in-frame fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L. lactis IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting L. lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.Bacteriocins of lactic acid bacteria have received considerable attention in recent years due to their potential application in the food industry as natural preservatives. Most interest has focused on lantibiotics (class I bacteriocins), e.g., nisin, and small heat-stable non-lanthionine-containing bacteriocins (class II) (22, 23). A major subgroup of class II bacteriocins (IIa) has been given the generic name of pediocin family (28) after its most extensively studied member, pediocin PA-1. Members of this class have a number of features in common, including a very strong antimicrobial activity against Listeria species (28). The food-borne pathogen Listeria monocytogenes is a major concern in the dairy industry since it can grow in a variety of dairy products at low temperature and pH (13). Although a pediocin PA-1-producing Lactobacillus plantarum strain has recently been isolated (12), this bacteriocin is generally produced by Pediococcus acidilactici strains of meat origin (3, 16, 18, 29, 31). Because of its antilisterial activity, the expression of pediocin PA-1 in strains of dairy origin would be highly desirable.Pediocin PA-1 production, immunity, and secretion are determined by an operon containing four genes (26). The structural gene, pedA, encodes the pediocin PA-1 precursor, pedB specifies immunity, and the pedC and pedD gene products are membrane-bound proteins required for secretion of the active peptide (39). Homologs of these genes have been described for related peptides. Biosynthesis of the well-characterized class II bacteriocin, lactococcin A, produced by strains of Lactococcus lactis also involves four genes (20, 36, 40). In addition to the structural gene (lcnA) and immunity gene (lciA), there are two genes (lcnC and lcnD) whose products together form a transport system dedicated to the translocation of lactococcin through the host membrane. The LcnC protein belongs to the family of ATP-binding cassette transporter proteins (40), and LcnD acts as an accessory protein (14). These two proteins have considerable homology to PedD and PedC, respectively (39), suggesting that the latter proteins play a similar role in the transport of active pediocin. The two bacteriocins also share the double glycine-processing site found in many lactic acid bacteria class II bacteriocins, some lantibiotics, and the Escherichia coli bacteriocin, colicin V (17).Van Belkum et al. (38) have recently investigated the role of leader sequences of the class II bacteriocins in the recognition of the precursor peptide by the dedicated translocation machinery of the host organism. By constructing hybrid genes, they demonstrated that the leader peptides of leucocin A, lactococcin A, and colicin V, which are cleaved at the Gly-Gly (positions −2 and −1) site, can direct the secretion of the nonrelated bacteriocin divergicin A. Our studies have focused on the class II bacteriocins pediocin PA-1 and lactococcin A. Since these peptides have a number of features in common, it might be expected that a pediocin PA-1 precursor could be secreted and processed by using the lactococcin A translocation machinery. L. lactis IL1403 is a plasmid-free strain that does not produce bacteriocin but contains chromosomal copies of genes analogous to lcnC and lcnD (33, 40). In addition, the natural resistance of this strain to pediocin PA-1 (8) makes it an ideal candidate for a production host to investigate the expression of pediocin PA-1 in lactococci.This paper describes the development of an expression system geared to the production of heterologous peptides in L. lactis. Testing the system with pediocin PA-1 involved the construction of a vector containing an in-frame fusion between sequences encoding the lactococcin A leader and the structural part of mature pediocin PA-1. The hybrid genes were introduced into L. lactis IL1403, and the ability of these strains to produce and secrete pediocin PA-1 was investigated.     

Production of active pediocin PA-1 in Escherichia coli using a thioredoxin gene fusion expression approach: cloning, expression, purification, and characterization

Antimicrobial peptides possess cationic and amphipathic properties that allow for interactions with the membrane of living cells. Bacteriocins from lactic acid bacteria, in particular, are currently being studied for their potential use as food preservatives and for applications in health care. However, bacteriocin exploitation is often limited owing to low production yields. Gene cloning and heterologous protein or peptide production is one way to possibly achieve overexpression of bacteriocins to support biochemical studies. In this work, production of recombinant active pediocin PA-1 (PedA) was accomplished in Escherichia coli using a thioredoxin (trx) gene fusion (trx-pedA) expression approach. Trx-PedA itself did not show any biological activity, but upon cleavage by an enterokinase, biologically active pediocin PA-1 was obtained. Recombinant pediocin PA-1 characteristics (molecular mass, biological activity, physicochemical properties) were very similar to those of native pediocin PA-1. In addition, a 4- to 5-fold increase in production yield was obtained, by comparison with the PA-1 produced naturally by Pediococcus acidilactici PAC 1.0. The new production method, although not optimized, offers great potential for supporting further investigations on pediocin PA-1 and as a first-generation process for the production of pediocin PA-1 for high-value applications.     

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.     

Enhanced production of pediocin PA-1 and coproduction of nisin and pediocin PA-1 by Lactococcus lactis.

The production and secretion of class II bacteriocins share a number of features that allow the interchange of genetic determinants between certain members of this group of antimicrobial peptides. Lactococcus lactis IL1403 encodes translocatory functions able to recognize and mediate secretion of lactococcin A. The ability of this strain to also produce the pediococcal bacteriocin pediocin PA-1, has been demonstrated previously by the introduction of a chimeric gene, composed of sequences encoding the leader of lactococcin A and the mature part of pediocin PA-1 (N. Horn, M. I. Martínez, J. M. Martínez, P. E. Hernández, M. J. Gasson, J. M. Rodríguez, and H. M. Dodd, Appl. Environ. Microbiol. 64:818-823, 1998). This heterologous expression system has been developed further with the introduction of the lactococcin A-dedicated translocatory function genes, lcnC and lcnD, and their effect on bacteriocin yields in various lactococcal hosts was assessed. The copy number of lcnC and lcnD influenced production levels, as did the particular strain employed as host. Highest yields were achieved with L. lactis IL1403, which generated pediocin PA-1 at a level similar to that for the parental strain, Pediococcus acidilactici 347, representing a significant improvement over previous systems. The genetic determinants required for production of pediocin PA-1 were introduced into the nisin-producing strain L. lactis FI5876, where both pediocin PA-1 and nisin A were simultaneously produced. The implications of coproduction of these two industrially relevant antimicrobial agents by a food-grade organism are discussed.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Characterization and Antilisterial Effect of Phosphatidylcholine Nanovesicles Containing the Antimicrobial Peptide Pediocin

Encapsulation may provide increased stability and antimicrobial efficiency to bacteriocins. In this work, the antilisterial peptide pediocin was encapsulated in nanovesicles prepared from partially purified soybean phosphatidylcholine. The maintenance of antimicrobial activity and properties of free and encapsulated pediocin was observed during 13 days at 4 °C, and after this period, the encapsulated pediocin retained 50 % its initial activity. The maintenance of the bioactive properties of free and encapsulated pediocin was observed against different species of Listeria, inhibiting Listeria monocytogenes, Listeria innocua and Listeria ivanovii. The size of vesicles containing pediocin was determined by dynamic light scattering as an average of 190 nm, with little change throughout the observation period. Polydispersity index values were around 0.201 and are considered satisfactory, indicating an adequate size distribution of liposomes. The efficiency of encapsulation was 80 %. Considering these results, the protocol used was appropriate for the encapsulation of this bacteriocin. Results demonstrate the production of stable nanoparticulate material. The maintenance of the properties of pediocin encapsulated in liposomes is fundamental to prospect the stability in different conditions of the food matrix.     

Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins

Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.     

Heterologous Processing and Export of the Bacteriocins Pediocin PA-1 and Lactococcin A in Lactococcus Lactis: A Study with Leader Exchange

The bacteriocins pediocin PA-1 and lactococcin A are synthesized as precursors carrying N-terminal extensions with a conserved cleavage site preceded by two glycine residues in positions -2 and -1. Each bacteriocin is translocated through the cytoplasmic membrane by an integral membrane protein of the ABC cassette superfamily which, in the case of pediocin PA-1, has been shown to possess peptidase activity responsible for proteolytic cleavage of the pre-bacteriocin. In each case, another integral membrane protein is essential for bacteriocin production. In this study, a two-step PCR approach was used to permutate the leaders of pediocin PA-1 and lactococcin A. Wild-type and chimeric pre-bacteriocins were assayed for maturation by the processing/export machinery of pediocin PA-1 and lactococcin A. The results show that pediocin PA-1 can be efficiently exported by the lactococcin machinery whether it carries the lactococcin or the pediocin leader. It can also compete with wild-type lactococcin A for the lactococcin machinery. Pediocin PA-1 carrying the lactococcin A leader or lactococcin A carrying that of pediocin PA-1 was poorly secreted when complemented with the pediocin PA-1 machinery, showing that the pediocin machinery is more specific for its bacteriocin substrate. Wild-type pre-pediocin and chimeric pre-pediocin were shown to be processed by the lactococcin machinery at or near the double-glycine cleavage site. These results show the potential of the lactococcin LcnC/LcnD machinery as a maturation system for peptides carrying double-glycine-type amino-terminal leaders.     

Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis: detection by specific peptide-directed antibodies

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536-4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available.     

Stability and Inhibitory Activity of Pediocin PA-1 Against Listeria sp. in Simulated Physiological Conditions of the Human Terminal Ileum

Listeria monocytogenes is responsible for severe foodborne infections, which can be life-threatening especially for infants and elderly populations. The emergence of antibiotic-resistant pathogens has stimulated the search for new strategies, such as the use of bacteriocins, to prevent or cure foodborne infectious diseases in the intestine. In this study, we evaluated the efficacy of the bacteriocin pediocin PA-1 from Pediococcus acidilactici UL5 to inhibit Listeria ivanovii, used as a surrogate for L. monocytogenes, under physiological conditions of the terminal ileum, simulated in a continuous in vitro fermentation model. A fecal sample from a healthy adult was immobilized and propagated for 30?days in a continuous stirred tank reactor, fed with a nutritive medium simulating the ileal chime (pH 7.5). After reaching a pseudo-steady state, the reactor was inoculated five times with L. ivanovii to reach a final concentration of 10⁷ CFU/ml within the reactor. Two spikes of L. ivanovii without adjunction of pediocin PA-1 served as control assays, and three other spikes were done to test the effects of three concentrations of pediocin PA-1 corresponding to 2, 3, and 5× the minimum inhibitory concentration (MIC) active against L. ivanovii. The concentration of L. ivanovii in the reactor was followed for 8?h using the PALCAM selective medium. The different groups of commensal bacteria were enumerated on selective medium or using fluorescence in situ hybridization. Our data showed that pediocin PA-1 is stable in the ileum conditions and that it is able to exert its inhibition activity against L. ivanovii in a dose-dependent manner. The addition of pediocin PA-1 at 5?×?MIC induced a complete disappearance of L. ivanovii (5 log reduction) within 5?h, compared to a reduction of 2 logs, corresponding to the washout phenomenon, when no pediocin PA-1 was added. Reduction of 0.8 and 1.3 logs within 8?h was also obtained with the addition of 2 and 3?×?MIC, respectively. The same experiment has shown that addition of pediocin-PA1 in the reactor had a negligible effect on the balance of commensal bacteria.     

Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2

The lactic acid bacteria (LAB) are of great interest because of their food grade quality and industrial importance. In the recent past, the pediocin PA-1 like bacteriocin was found to be synthesized in cross-species and cross-genera. Hence, the present work was carried out in order to determine the transfer of plasmid encoded pediocin PA-1 like bacteriocin among LAB. The objective of this study is to demonstrate the dissemination of bacteriocin-encoded plasmid from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Enterococcus faecalis JH2-2 under in vitro (filter mating method) and in situ (soymilk model) conditions. The fermentation of the soymilk was determined by the selected pediocin producers. E. faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the native pediocin producer P. acidilactici NCIM 5424 transferred the bacteriocin under both the methods used. The in situ method gave more transfer frequency, ranging from 10^?7 to 10^?4 transconjugants per recipient cell. No conjugal transfer by L. plantarum Acr2 was observed. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the obtained transconjugants. The results suggest the horizontal gene transfer (HGT) and the natural spread of pediocin PA-1-like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1-like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and construction of novel cultures.     

Secretion of Recombinant Pediocin PA-1 by Bifidobacterium longum, Using the Signal Sequence for Bifidobacterial α-Amylase

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.     

Dynamic relationships among type IIa bacteriocins: temperature effects on antimicrobial activity and on structure of the C-terminal amphipathic alpha helix as a receptor-binding region

Dynamic aspects of structural relationships among class IIa bacteriocins, which are antimicrobial peptides from lactic acid bacteria (LAB), have been examined by use of circular dichroism (CD), molecular dynamics (MD) simulations, and activity testing. Pediocin PA-1 is a potent class IIa bacteriocin, which contains a second C-terminal disulfide bond in addition to the highly conserved N-terminal disulfide bond. A mutant of pediocin PA-1, ped[M31Nle], wherein the replacement of methionine by norleucine (Nle) gives enhanced stability toward aerobic oxidation, was synthesized by solid-phase peptide synthesis to study the activity of the peptide in relation to its structure. The secondary structural analysis from CD spectra of ped[M31Nle], carnobacteriocin B2 (cbn B2), and leucocin A (leuA) at different temperatures suggests that the alpha-helical region of these peptides is important for target recognition and activity. Using molecular modeling and dynamic simulations, complete models of pediocin PA-1, enterocin P, sakacin P, and curvacin A in 2,2,2-trifluoroethanol (TFE) were generated to compare structural relationships among this class of bacteriocins. Their high sequence similarity allows for the use of homology modeling techniques. Starting from homology models based on solution structures of leuA (PDB code 1CW6) and cbnB2 (PDB code 1CW5), results of 2-4 ns MD simulations in TFE and water at 298 and 313 K are reported. The results indicate that these peptides have a common helical C-terminal domain in TFE but a more variable beta sheet or coiled N terminus. At elevated temperatures, pediocin PA-1 maintains its overall structure, whereas peptides without the second C-terminal disulfide bond, such as enterocin P, sakacin P, curvacin A, leuA, and cbnB2 experience partial disruption of the helical section. Pediocin PA-1 and ped[M31Nle] were found to be equally active at different temperatures, whereas the other peptides that lack the second C-terminal disulfide bond are 30-50 times less antimicrobially potent at 310 K (37 degrees C) than at 298 K (25 degrees C). These results indicate that the structural changes in the helical region observed at elevated temperatures account for the loss of activity of these peptides. The presence of C-terminal hydrophobic residues on one side of the amphipathic helix in class IIa bacteriocins is an important feature for receptor recognition and specificity toward particular organisms. This study assists in the understanding of structure-activity relationships in type IIa bacteriocins and demonstrates the importance of the conserved C-terminal amphipathic alpha helix for activity.     

PrfA activation in Listeria monocytogenes increases the sensitivity to class IIa bacteriocins despite impaired expression of the bacteriocin receptor

BackgroundThe scope of the present work was to characterize the activity of class IIa bacteriocins in Listeria (L.) monocytogenes cells that constitutively express an activated form of PrfA, the virulence master regulator, since bacteriocin sensitivity was only characterized in saprophytic cells so far. The mannose phosphotransferase system (Man-PTS) has been shown to be the class IIa bacteriocin receptor in Listeria; hence, special attention was paid to its expression in virulent bacteria.MethodsL. monocytogenes FBprfA* cells were obtained by transconjugation. Bacterial growth was studied in TSB and glucose containing-minimal medium. Sensitivity to antimicrobial peptides was assessed by killing curves. Membranes of L. monocytogenes FBprfA* cells were characterized using proteomic and lipidomic approaches.ResultsThe mannose phosphotransferase system (Man-PTS) was downregulated upon expression of PrfA*, and these cells turned out to be more sensitive to enterocin CRL35 and pediocin PA-1, while not to nisin. Proteomic and lipidomic analysis showed differences between wild type (WT) and PrfA* strains. For instance, phosphatidic acid was only detected in PrfA* cells, whereas, there was a significant decline of plasmalogen-phosphatidylglycerol in the same strain.ConclusionsOur results support a model in which Man-PTS acts just as a docking molecule that brings class IIa bacteriocins to the plasma membrane. Furthermore, our results suggest that lipids play a crucial role in the mechanism of action of bacteriocins.General significanceThis is the first demonstration of the link between L. monocytogenes virulence and the bacterial sensitivity toward pediocin-like peptides.     

Identification of a New Plasmid-Encoded sec-Dependent Bacteriocin Produced by Listeria innocua 743

Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917 into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon into Escherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeria spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.     

Fermenting Cucumber, a Potential Source for the Isolation of Pediocin-Like Bacteriocin Producers

Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin. These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic strains.