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)     

Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0

The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.     

Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0.

The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.     

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.     

Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells.

Pediocin PA-1 is a bacteriocin which is produced by Pediococcus acidilactici PAC1.0. We demonstrate that pediocin PA-1 kills sensitive Pediococcus cells and acts on the cytoplasmic membrane. In contrast to its lack of impact on immune cells, pediocin PA-1 dissipates the transmembrane electrical potential and inhibits amino acid transport in sensitive cells. Pediocin interferes with the uptake of amino acids by cytoplasmic membrane vesicles derived from sensitive cells, while it is less effective with membranes derived from immune cells. In liposomes fused with membrane vesicles derived from both sensitive and immune cells, pediocin PA-1 elicits an efflux of small ions and, at higher concentrations, an efflux of molecules having molecular weights of up to 9,400. Our data suggest that pediocin PA-1 functions in a voltage-independent manner but requires a specific protein in the target membrane.     

Complete nucleotide sequence of pSMB 74, a plasmid encoding the production of pediocin AcH in Pediococcus acidilactici

Several Pediococcus acidilactici strains produce a plasmid-encoded bacteriocin, pediocin AcH. Previous studies have shown that this plasmid, designated as pSMB 74, encodes genes associated with the production of prepediocin, its post-translation processing to pediocin AcH, transmembrane translocation of these molecules, and immunity of producer cells against pediocin AcH. We report here the complete nucleotide sequence of pSMB 74. The plasmid has a total of 8877 bp. Four genes have been located on pSMB 74. The genes are arranged in a gene cluster of 3500 bp and share a common promoter and rho-independent stem-loop terminator. The four genes, each with independent ribosome binding sites (rbs), initiation and termination codons and spacer sequences in between, were designated as pap A, pap B, pap C and pap D and encode respectively for proteins of 62, 112, 174 and 724 amino acids. The results of this study can be useful either to introduce a suitable marker at a unique restriction site in pSMB 74 and use it as a vector or to clone the pap gene cluster in a suitable plasmid and transform desirable strains for pediocin AcH production. The gene sequence has been submitted to Gene Bank (Acc. No. U02482).     

Cloning, expression, and nucleotide sequence of genes involved in production of lactococcin DR, a bacteriocin from lactococcus lactis subsp. lactis.

The partial nucleotide sequence of a Lactococcus lactis subsp. lactis ADRIA 85LO30 bacteriocin-producing operon was determined. The first two open reading frames of the operon are necessary to get bacteriocin expression in L. lactis IL1403R.     

Factors influencing immunity and resistance of Pediococcus acidilactici to the bacteriocin, pediocin AcH

In pediocin AcH producing Pediococcus acidilactici strains the genes for both the production of pediocin and immunity against it are encoded in an 8.9 kb plasmid pSMB74. Following loss of this plasmid, the variants lost the ability to produce pediocin AcH, but some retained the resistance against it. This resistance was a transient trait, acquired while nonproducing cells grew in the presence of pediocin AcH but lost when the cells were grown in the absence of it.     

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.     

Monoclonal antibody-colony immunoblot method specific for isolation of Pediococcus acidilactici from foods and correlation with pediocin (bacteriocin) production.

BALB/c mice were immunized with broken, heat-killed cells of Pediococcus acidilactici H. After murine cell fusions, one monoclonal antibody (MAb), Ped-2B2, was selected on the basis of its positive reaction with seven of seven strains tested in an enzyme-linked immunosorbent assay with whole cells of P. acidilactici. The MAb Ped-2B2 did not show any cross-reactions with other lactic-acid bacteria or other gram-positive or gram-negative organisms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of surface proteins of P. acidilactici indicated that Ped-2B2 reacted with a protein of 116 kDa. MAb Ped-2B2 was used as a probe to isolate Pediococcus species from fermented-meat products by colony immunoblotting. A total of 18 Ped-2B2-reactive Pediococcus spp. isolates were isolated from eight food samples and assayed for bacteriocin production. All of the isolates produced bacteriocins which were heat stable, proteinaceous, and inhibitory to Lactobacillus plantarum NCDO 955. Biochemical characterization of these isolates indicated that they were all P. acidilactici.     

Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum.

Lactic acid bacteria were isolated from Spanish dry-fermented sausages and screened for bacteriocin production. About 10% of the isolates produced antimicrobial substances when grown on solid media, but only 2% produced detectable activity in liquid media. Strain L50, identified as Pediococcus acidilactici, showed the strongest inhibitory activity and was active against members of all of the gram-positive genera tested. The strain produced a heat-stable bacteriocin when grown at 8 to 32 degrees C but not at 45 degrees C. The bacteriocin was purified to homogeneity. Its mass was determined to be 5,250.11 +/- 0.30 by electrospray mass spectrometry. The N terminus of the bacteriocin was blocked for sequencing by Edman degradation, but a partial sequence of 42 amino acids was obtained after cleavage of the peptide by cyanogen bromide. The sequence showed no similarity to those of other bacteriocins. Pediocin L50 appears to contain modified amino acids but not lanthionine or methyl-lanthionine.     

Nucleotide and amino acid sequence of pap-gene (pediocin AcH production) in Pediococcus acidilactici H

N-terminal analysis of purified pediocin AcH produced a partial sequence of 23 amino acids. This sequence matched perfectly with a segment of 23 amino acids in a 62 amino acid molecule generated from the 186 nucleotide sequence open reading frame in a Hind III fragment in pSMB74 encoding pap-gene (pediocin AcH production). It is suggested that the molecule is translated as inactive prepediocin AcH of 62 amino acids. Then through enzymatic modifications the leader segment of 18 amino acids is removed from the NH2-terminal. The remaining segment of 44 amino acids is active pediocin AcH of 4628 M(r).     

Monoclonal antibody-colony immunoblot method specific for isolation of Pediococcus acidilactici from foods and correlation with pediocin (bacteriocin) production.

BALB/c mice were immunized with broken, heat-killed cells of Pediococcus acidilactici H. After murine cell fusions, one monoclonal antibody (MAb), Ped-2B2, was selected on the basis of its positive reaction with seven of seven strains tested in an enzyme-linked immunosorbent assay with whole cells of P. acidilactici. The MAb Ped-2B2 did not show any cross-reactions with other lactic-acid bacteria or other gram-positive or gram-negative organisms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of surface proteins of P. acidilactici indicated that Ped-2B2 reacted with a protein of 116 kDa. MAb Ped-2B2 was used as a probe to isolate Pediococcus species from fermented-meat products by colony immunoblotting. A total of 18 Ped-2B2-reactive Pediococcus spp. isolates were isolated from eight food samples and assayed for bacteriocin production. All of the isolates produced bacteriocins which were heat stable, proteinaceous, and inhibitory to Lactobacillus plantarum NCDO 955. Biochemical characterization of these isolates indicated that they were all P. acidilactici.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

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.     

Continuous production of pediocin by immobilized Pediococcus acidilactici PO2 in a packed-bed bioreactor

 A continuous bioreactor packed with a fibrous matrix was set up. Cells of Pediococcus acidilactici PO2 were inoculated and MRS broth was fed gradually until cell growth and immobilization were achieved. Kinetics of fermentation and production of bacteriocin were investigated at dilution rates ranging from 0.63 day^-1 to 1.58 day^-1 and at pH values that varied between 4.0 and 5.5. A maximum bacteriocin activity of 6400 AU/ml was detected when the medium was fermented at dilution rates of at least 1.19 day^-1 and the pH controlled at 4.5. The maximum bacteriocin productivity was 1.0×10⁷ AUl^-1 day^-1 at a dilution rate of 1.58 day^-1 and pH 4.5. At this high dilution rate, 1.21 g cells/l medium was produced, 95.9% of the glucose in MRS broth was utilized, and 15.1 g lactic acid/l accumulated in the bioreactor effluent. The bioreactor was operated continuously for 3 months without encountering any clogging, degeneration, or contamination problems, indicating good long-term stability of the bioreactor for bacteriocin production. About 94% of the cells in the bioreactor were immobilized, and the remainder were suspended in the medium. According to scanning electron microscopic observations, cell immobilization in the fibrous matrix was attained by natural attachment to fiber surfaces and entrapment in the void volume within the fibrous matrix. In conclusion, conditions for the optimum continuous production of pediocin were defined; this may facilitate the development of large-scale industrial processes for production of this bacteriocin. Received: 25 September 1995/Received revision: 30 November 1995/Accepted: January 1996     

Effect of physical factors on the production of bacteriocin from Pediococcus acidilactici ITV 26

The effect of pH, temperature and agitation on growth and bacteriocin production by Pediococcus acidilactici ITV 126 was investigated. Experiments were made in flasks containing MRS medium at 30 to 40°C, pH 5 to 7 and agitation 0 to 200 rpm. Factor levels were arranged in a 2³ factorial design with central and axial points. Anova and Tukey paired comparison tests showed that a temperature of 35°C favored bacteriocin production, whereas 40°C was best for cell growth. A statistical interaction of temperature and agitation was observed affecting microbial growth. pH 5 favored both cell growth and bacteriocin production. Journal of Industrial Microbiology & Biotechnology (2001) 26, 191–195. Received 30 October 1999/ Accepted in revised form 31 January 2001     

Mapping of pSMB74, a plasmid-encoding bacteriocin, pediocin AcH, production (Pap+) by Pediococcus acidilactici H

Plasmids encoding bacteriocin production phenotype in four Pediococcus acidilactici strains and their derivatives were examined for restriction enzyme cleavage patterns and found to produce similar fragments. A restriction map of this plasmid, pSMB74, has been constructed.