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.     

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.     

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.     

Detection of specific bacteriocin-producing lactic acid bacteria by colony hybridization

A colony hybridization method for detecting lactic acid bacteria encoding specific bacteriocins was developed. Specific PCR-generated probes were used to detect colonies of pediocin PA-1, lactococcin A, enterocin AS-48, nisin A and lacticin 481 producing strains. The probes were shown to be sensitive and specific for sequences belonging to the structural genes of the respective bacteriocins.     

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.     

Interactions of nisin and pediocin PA-1 with closely related lactic acid bacteria that manifest over 100-fold differences in bacteriocin sensitivity.

The natural variation in the susceptibilities of gram-positive bacteria towards the bacteriocins nisin and pediocin PA-1 is considerable. This study addresses the factors associated with this variability for closely related lactic acid bacteria. We compared two sets of nonbacteriocinogenic strains for which the MICs of nisin and pediocin PA-1 differed 100- to 1,000-fold: Lactobacillus sake DSM20017 and L. sake DSM20497 and Pediococcus dextrinicus and Pediococcus pentosaccus. Strikingly, the bacteriocin-sensitive and -insensitive strains showed a similar concentration-dependent dissipation of their membrane potential (delta psi) after exposure to these bacteriocins. The bacteriocin-induced dissipation of delta psi below the MICs for the insensitive strains did not coincide with a reduction of intracellular ATP pools and glycolytic rates. This was not observed with the sensitive strains. Analysis of membrane lipid properties revealed minor differences in the phospho- and glycolipid compositions of both sets of strains. The interactions of the bacteriocins with strain-specific lipids were not significantly different in a lipid monolayer assay. Further lipid analysis revealed higher in situ membrane fluidity of the bacteriocin-sensitive Pediococcus strain compared with that for the insensitive strain, but the opposite was found for the L. sake strains. Our results provide evidence that the association of bacteriocins with the cell membrane and their subsequent insertion take place in a similar way for cells that have a high or a low natural tolerance towards bacteriocins. For insensitive strains, overall membrane constitution rather than mere membrane fluidity may preclude the formation of pores with sufficient diameters and lifetimes to ultimately cause cell death.     

Rice Hull Ash and Silicic Acid as Adsorbents for Concentration of Bacteriocins

A model procedure has been developed for the rapid extraction of five bacteriocins (nisin, pediocin RS2, leucocin BC2, lactocin GI3, and enterocin CS1) from concentrated freeze-dried crude culture supernatants by adsorption onto acid or alkaline rice hull ash (RHA) or silicic acid (SA). Bacteriocins were adsorbed onto RHA or SA by a pH-dependent method and desorbed by decreasing the pH to 2.5 or 3.0 and heating at 90°C for 5 min. The maximum adsorption and optimal pH range for different bacteriocins were as follows: nisin, 97% at pH 7.0; lactocin GI3, 94% at pH 6.0; pediocin RS2, 97% at pH 8.0 to 9.0; leucocin BC2, 88% at pH 9.0; and enterocin CS1, 94% at pH 5.0. The desorption level of lactocin GI3 or enterocin CS1 from the surfaces of both RHA and SA was 94%, while the desorption level of pediocin RS2 and leucocin BC2 was 50% or less. Nisin was desorbed readily from SA (91%) but not from RHA (50% or less). The adsorption of bacteriocins onto RHA and SA increased with the increasing concentration of bacteriocins. Analysis of the desorbed bacteriocins after dialysis and sodium dodecyl sulfate–16% polyacrylamide gel electrophoresis showed a single band that gave a single inhibition zone when overlaid with Lactobacillus plantarum for detection of lactocin GI3, enterocin CS1, and nisin. RHA appears useful for extraction, concentration, and partial purification of the five bacteriocins.     

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.     

Mechanism of Nisin, Pediocin 34, and Enterocin FH99 Resistance in Listeria monocytogenes

Nisin-, pediocin 34-, and enterocin FH99-resistant variants of Listeria monocytogenes ATCC 53135 were developed. In an attempt to clarify the possible mechanisms underlying bacteriocin resistance in L. monocytogenes ATCC 53135, sensitivity of the resistant strains of L. monocytogenes ATCC 53135 to nisin, pediocin 34, and enterocin FH99 in the absence and presence of different divalent cations was assessed, and the results showed that the addition of divalent cations significantly reduced the inhibitory activity of nisin, pediocin 34, and enterocin FH99 against resistant variants of L. monocytogenes ATCC 53135. The addition of EDTA, however, restored this activity suggesting that the divalent cations seem to affect the initial electrostatic interaction between the positively charged bacteriocin and the negatively charged phospholipids of the membrane. Nisin-, pediocin 34-, and enterocin-resistant variants of L. monocytogenes ATCC 53135 were more resistant to lysozyme as compared to the wild-type strain both in the presence as well as absence of nisin, pediocin 34, and enterocin FH99. Ultra structural profiles of bacteriocin-sensitive L. monocytogenes and its bacteriocin-resistant counterparts revealed that the cells of wild-type strain of L. monocytogenes were maximally in pairs or short chains, whereas, its nisin-, pediocin 34-, and enterocin FH99-resistant variants tend to form aggregates. Results indicated that without a cell wall, the acquired nisin, pediocin 34, and enterocin FH99 resistance of the variants was lost. Although the bacteriocin-resistant variants appeared to lose their acquired resistance toward nisin, pediocin 34, and enterocin FH99, the protoplasts of the resistant variants appeared to be more resistant to bacteriocins than the protoplasts of their wild-type counterparts.     

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.     

Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum.

Enterocin P is a new bacteriocin produced by Enterococcus faecium P13 isolated from a Spanish dry-fermented sausage. Enterocin P inhibited most of tested spoilage and food-borne gram-positive pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum. Enterocin P is produced during growth in MRS broth from 16 to 45 degrees C; it is heat resistant (60 min at 100 degrees C; 15 min at 121 degrees C) and can withstand exposure to pH between 2.0 and 11.0, freeze-thawing, lyophilization, and long-term storage at 4 and -20 degrees C. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation, gel filtration, cation-exchange, hydrophobic-interaction, and reverse-phase liquid chromatography. The sequence of 43 amino acids of the N terminus was obtained by Edman degradation. DNA sequencing analysis of a 755-bp region revealed the presence of two consecutive open reading frames (ORFs). The first ORF encodes a 71-amino-acid protein containing a hydrophobic N-terminal sec-dependent leader sequence of 27 amino acids followed by the amino acid sequence corresponding to the purified and sequenced enterocin P. The bacteriocin is apparently synthesized as a prepeptide that is cleaved immediately after the Val-Asp-Ala residues (positions -3 to -1), resulting in the mature bacteriocin consisting of 44 amino acids, and with a theoretical molecular weight of 4,493. A second ORF, encoding a putative immunity protein composed of 88 amino acids with a calculated molecular weight of 9,886, was found immediately downstream of the enterocin P structural gene. Enterocin P shows a strong antilisterial activity and has the consensus sequence found in the pediocin-like bacteriocins; however, enterocin P is processed and secreted by the sec-dependent pathway.     

In vitro inhibition activity of nisin A, nisin Z, pediocin PA-1 and antibiotics against common intestinal bacteria

AIMS: To evaluate the sensitivity of 21 common intestinal bacteria to six antibiotics and three broad-spectrum bacteriocins (nisins Z and A and pediocin PA-1). METHODS AND RESULTS: Neutralized cell-free culture supernatants containing active bacteriocins, and antibiotics were tested with the agar diffusion test and the disc-diffusion method, respectively. The tested intestinal strains showed high sensitivity to most antibiotics except for streptomycin and oxacillin. Nisins A and Z (8 mug per well) had similar activity spectra and inhibited all Gram-positive intestinal bacteria at different levels (except Streptococcus salivarius), with bifidobacteria (except Bifidobacterium breve and Bif. catenulatum), Collinsella aerofaciens and Eubacterium biforme being the most sensitive strains, but they were not active against Gram-negative bacteria. Surprisingly, none of the tested strains were inhibited by pediocin PA-1 (16 mug per well). CONCLUSION: Pediocin PA-1 which is very active against Listeria spp. and other food pathogens did not inhibit major intestinal species in the human intestine in contrast to both nisins A and Z. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that pediocin PA-1 has potential to inhibit Listeria within the intestinal microbiota without altering commensal bacteria.     

A versatile system for the expression of nonmodified bacteriocins in Escherichia coli

AIMS: To develop a method and plasmid vectors suitable for expression of class II bacteriocins from Escherichia coli. METHODS AND RESULTS: The expression vector pSuV1 was constructed by inserting the PelB secretion signal coding sequence and a number of restriction endonuclease sites for cloning, into pTYB1. Codon optimized genes encoding the active mature region of each bacteriocin were constructed and inserted into pSuV1. Transfer of these constructs to a host expressing T7 RNA polymerase allowed for expression of secreted mature or fusion forms of the bacteriocins. Generation of the fusion, to the adjacent intein-chitin-binding domain gene, was achieved by removal of a small intervening BseRI fragment. The bacteriocins BacR1, divercin V41, enterocin P, pediocin PA-1 and piscicolin 126 were expressed from this system. For piscicolin 126, expression levels of 200 microg l(-1) in the mature form and 1100 microg l(-1) when cleaved from the fusion partner were achieved. All expressed bacteriocins displayed antimicrobial activity. CONCLUSIONS: Several class II bacteriocins have been expressed in E. coli using purpose designed plasmid vectors described here. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides a common expression system capable of producing a range of different class II bacteriocins. It allows researchers to study class II bacteriocins without access to the original producer strain, the native bacteriocin gene, or highly specific heterologous producing strains. Resulting expression levels are as high or higher than those previously reported for related bacteriocins.     

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.     

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms.Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACD^Cgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.     

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.     

Differences in susceptibility of Listeria monocytogenes strains to sakacin P,sakacin A,pediocin PA-1, and nisin

Two hundred strains of Listeria monocytogenes collected from food and the food industry were analyzed for susceptibility to the class IIa bacteriocins sakacin P, sakacin A, and pediocin PA-1 and the class I bacteriocin nisin. The individual 50% inhibitory concentrations (IC(50)) were determined in a microtiter assay and expressed in nanograms per milliliter. The IC(50) of sakacin P ranged from 0.01 to 0.61 ng ml(-1). The corresponding values for pediocin PA-1, sakacin A, and nisin were 0.10 to 7.34, 0.16 to 44.2, and 2.2 to 781 ng ml(-1), respectively. The use of a large number of strains and the accuracy of the IC(50) determination revealed patterns not previously described, and for the first time it was shown that the IC(50) of sakacin P divided the L. monocytogenes strains into two distinct groups. Ten strains from each group were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified fragment length polymorphism. The results from these studies essentially confirmed the grouping based on the IC(50) of sakacin P. A high correlation was found between the IC(50) of sakacin P and that of pediocin PA-1 for the 200 strains. Surprisingly, the correlation between the IC(50) of the two class IIa bacteriocins sakacin A and sakacin P was lower than the correlation between the IC(50) of sakacin A and the class I bacteriocin nisin.     

Use of Synthetic Genes for Cloning,Production and Functional Expression of the Bacteriocins Enterocin A and Bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis

The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.     

Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin

Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modified by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. The O-linked N-acetylglucosamine moieties are essential for the antimicrobial activity of enterocin F4-9. Further analysis of the enterocin F4-9 gene cluster identified enfC, which has high sequence similarity to a glycosyltransferase. The antimicrobial activity of enterocin F4-9 covered a limited range of bacteria, including, interestingly, a Gram-negative strain, Escherichia coli JM109. Enterocin F4-9 is sensitive to protease, active at a wide pH range, and moderately resistant to heat.     

Vegetable-Associated Pediococcus parvulus Produces Pediocin PA-1

Two bacteriocin-producing strains of Pediococcus parvulus were isolated from minimally processed vegetables. Recombinant DNA techniques revealed the presence of the pediocin PA-1 gene in both strains. Biochemical analysis confirmed the production of pediocin PA-1 and excluded the presence of other bacteriocins.