Expression of lactococcin A and pediocin PA-1 in heterologous hosts

Pediocin PA-1 production, immunity and secretion are specified by a cluster of four genes in Pediococcus acidilactici PAC1.0. The production by, secretion of, and immunity to lactococcin A of Lactococcus lactis are also determined by four genes. Here, expression of the pediocin operon in Lactococcus lactis is reported, which could only be achieved by placing it under control of a lactococcal promoter. Expression of the lactococcin A operon in Pediococcus is also described: recombinant clones of Pediococcus were obtained that produced and secreted both active pediocin PA-1 and lactococcin A.     

Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832

Pediocin PD-1, produced by Pediococcus damnosus NCFB 1832, is inhibitory to several food spoilage bacteria and food-borne pathogens. However, pediocin PD-1 is not active against other Pediococcus spp. and differs in this respect to other pediocins produced by Pediococcus acidilactici and Pediococcus pentosaceus. Production of pediocin PD-1 starts during early growth and reaches a plateau at the end of exponential growth. Pediocin PD-1 was partially purified and its size was determined by tricine-SDS-PAGE as ≈ 3·5 kDa. The isoelectric point (pI) of pediocin PD-1 is ≈ 3·5, as determined with the Rotofor electrofocusing cell (BioRad). Pediocin PD-1 is heat-resistant (10 min at 121°C) and remains active after 30 min of incubation at pH 2–10. Pediocin PD-1 is resistant to treatment with pepsin, papain, α-chemotrypsin and trypsin, but not Proteinase K. Pediocin PD-1 is bactericidal against sensitive cells of Oenococcus oeni (previously Leuconostoc oenos ).     

Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici

An antimicrobial peptide designated pediocin AcH was isolated from Pediococcus acidilactici strain H. The pediocin AcH was purified by ion exchange chromatography. The molecular weight of pediocin AcH was determined by SDS-PAGE to be about 2700 daltons. Pediocin AcH was sensitive to proteolytic enzymes, resistant to heat and organic solvents, and active over a wide range of pH. Pediocin AcH exhibited inhibition against several food spoilage bacteria and foodborne pathogens including Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes. It was bactericidal to sensitive cells and acted very rapidly. The bactericidal effect was not produced by either cell lysis or apparent loss of membrane permeability.     

Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici

An antimicrobial peptide designated pediocin AcH was isolated from Pediococcus acidilactici strain H. The pediocin AcH was purified by ion exchange chromatography. The molecular weight of pediocin AcH was determined by SDS-PAGE to be about 2700 daltons. Pediocin AcH was sensitive to proteolytic enzymes resistant to heat and organic solvents, and active over a wide range of pH. Pediocin AcH exhibited inhibition against several food spoilage bacteria and foodborne pathogens including Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes. It was bactericidal to sensitive cells and acted very rapidly. The bactericidal effect was not produced by either cell lysis or apparent loss of membrane permeability.     

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.     

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.     

Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0.

The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0.

The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material

Expression of the pedA gene from Pediococcus acidilactici, coding for mature bacteriocin Pediocin PA-1, was investigated using the yeast Pichia pastoris to obtain larger quantities of pediocin to support additional studies, including structure-function research. Following various cloning strategies, a KM71H (Mut(s)) strain was selected. A significant concentration (74 microg/ml) of extracellular recombinant pediocin was obtained but the pediocin showed no biological activity. Supernatant fluids from P. pastoris cultures, harboring or not pedA, inhibited the biological activity of natural pediocin PA-1. The recombinant pediocin appeared as a mixture of three main fractions (7-8, 11, 20 kDa vs. 4.6 kDa for natural pediocin PA-1). The recombinant pediocin was also less hydrophobic and behaved differently when subjected to isoelectric focusing. Strong evidence indicated that some "collagen-like" material was tightly associated, most probably via covalent binding, to the recombinant pediocin. The "collagen-like" material was most probably responsible for the lack of biological activity of the recombinant pediocin and for the differences observed regarding some of the physico-chemical properties. Both the recombinant pediocin and natural pediocin were sensitive to collagenase, suggesting that pediocin PA-1 may possess a somewhat "collagen-like" nature. Interestingly, recombinant pediocin preparations showed the ability to assemble into fibrils.     

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.     

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.     

Interaction of the bacteriocin pediocin SJ-1 with the cytoplasmic membrane of sensitive bacterial cells as detected by ANS fluorescence

The l-anilino-8-naphthalenesulphonic acid (ANS) fluorescent probe was used to monitor alterations in the cytoplasmic membrane of sensitive Lactobacillus plantarurm cells, caused by pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici. The addition of pediocin SJ-1 to the sensitive cells caused an increase in fluorescence intensity of ANS and a blue shift in its emission maximum from 520 to 475 nm. None of these spectral changes could be detected when pediocin SJ-1 was added to cells of a Lact. plantarum variant resistant to pediocin SJ-1. Upon the addition of pediocin SJ-1, dose-dependent energy transfer took place between tryptophanyl residues in the cytoplasmic membrane of sensitive cells and ANS. Similar ANS-fluorescence changes were observed with the bacteriocin nisin. The concentrations of pediocin SJ-1 needed to effect changes in the fluorescence spectrum of ANS were of the same magnitude as those required for a bactericidal effect and the release of u.v.-absorbing material. A hypothesis on the mode of action of pediocin SJ-1 is proposed.     

Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain

Aims:  The aim of this study was to purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a strain previously isolated from human gut.
Methods and Results:  Purification of the bacteriocin was performed by cationic exchange chromatography followed by a reverse phase step. Biochemical and mass spectrometry analysis showed homology with pediocin PA-1. To verify if P. acidilactici MM33 carried the pediocin PA-1 gene, total DNA was used to amplify the pediocin gene. The PCR product obtained was then sequenced and the nucleotide sequence revealed to be identical to that of pediocin PA-1. Treatment of P. acidilactici MM33 with novobiocin resulted in a plasmid-cured strain without bacteriocin-producing capacity. Antimicrobial assay and molecular analysis demonstrated that this strain was ped ⁻ suggesting that the ped cluster is plasmid encoded. Antimicrobial assay revealed that pediocin was bactericidal against Listeria monocytogenes , showing a minimal inhibitory concentration (MIC) of 200 AU ml⁻¹.
Conclusions:  A two-step purification procedure was elaborated in this study. The bacteriocin secreted by the human strain P. acidilactici MM33 is carried on a plasmid and the amino acid sequence is identical to pediocin PA-1.
Significance and Impact of the Study:  Pediococcus acidilactici MM33 is the first human pediocin-producing strain reported and could be used as probiotic to prevent enteric pathogen colonization.     

The bacteriocin lactococcin A specifically increases permeability of lactococcal cytoplasmic membranes in a voltage-independent, protein-mediated manner.

Lactococcin A is a bacteriocin produced by Lactococcus lactis. Its structural gene has recently been cloned and sequenced (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). Purified lactococcin A increased the permeability of the cytoplasmic membrane of L. lactis and dissipated the membrane potential. A significantly higher concentration of lactococcin A was needed to dissipate the membrane potential in an immune strain of L. lactis. Lactococcin A at low concentrations (0.029 microgram/mg of protein) inhibited secondary and phosphate-bond driven transport of amino acids in sensitive cells and caused efflux of preaccumulated amino acids. Accumulation of amino acids by immune cells was not affected by this concentration of lactococcin A. Lactococcin A also inhibited proton motive force-driven leucine uptake and leucine counterflow in membrane vesicles of the sensitive strain but not in membrane vesicles of the immune strain. These observations indicate that lactococcin A makes the membrane permeable for leucine in the presence or absence of a proton motive force and that the immunity factor(s) is membrane linked. Membrane vesicles of Clostridium acetobutylicum, Bacillus subtilis, and Escherichia coli were not affected by lactococcin A, nor were liposomes derived from phospholipids of L. lactis. These results indicate that lactococcin A acts on the cytoplasmic membrane and is very specific towards lactococci. The combined results obtained with cells, vesicles, and liposomes suggest that the specificity of lactococcin A may be mediated by a receptor protein associated with the cytoplasmic membrane.     

Interaction of the bacteriocin produced by Pediococcus acidilactici SJ-1 with the cell envelope of Lactobacillus spp.

In spite of differences in producing strains and their plasmid profiles, amino acid sequence analysis indicates that the bacteriocin produced by Pediococcus acidilactici SJ-1 is identical to that produced by PAC 1.0 and H. Protoplasts prepared from cells of pediocin-resistant strains of Lactobacillus plantarum and Lact. fermentum were lysed by exposure to the pediocin. The interaction of the pediocin with sensitive Lact. plantarum cells did not alter the fluidity of the cell membrane.     

Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles.

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

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.     

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.     

Purification, partial characterization and plasmid-linkage of pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici

Pediococcus acidilactici SJ-1, isolated from a naturally-fermented meat product, produced an antibacterial agent active against selected strains of Lactobacillus spp., Clostridium perfringens and Listeria monocytogenes. The agent was bactericidal against sensitive indicators, and sensitive to proteolytic enzymes; it was identified as a bacteriocin, and was designated as pediocin SJ-1. It was stable over a wide pH range (3–9), and apparently most stable in the lower part of that range. At pH 3.6, pediocin SJ-1 was stable at heat-processing temperatures within the range 65–121°C; its activity decreased significantly, however, when it was heated at pH 7.0. The activity of pediocin SJ-1 on sensitive indicator cells was lost in the presence of α-amylase, suggesting that it contains a glyco moiety, necessary for its antibacterial action.
Native pediocin SJ-1 exists in the form of monomers and aggregates (with molecular weights in the range 80–150 kDa). Pediocin SJ-1 was purified 262-fold by direct application of cell-free supernatant fluids to a cation-exchange chromatography column, and was resolved by SDS-PAGE as a single peptide band with a MW of ca 4 kDa. The original pediocin SJ-1-producing strain (bac⁺) harbours three plasmids of 4.6, 23.5, and 45.7 MDa. Production of pediocin SJ-1, but not immunity to SJ-1, is associated with the 4.6 MDa plasmid.     

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.