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Jon Nissenmeyer - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of residues in the helical region of the class iia bacteriocin pediocin pa 1
Applied and Environmental Microbiology, 2011Co-Authors: Helen Sophie Haugen, Gunnar Fimland, Jon NissenmeyerAbstract:A 15-mer fragment that is derived from the helical region in the C-terminal half of pediocin PA-1 inhibited the activity of pediocin PA-1. Of 13 other pediocin-like (hybrid) bacteriocins, only the hybrid bacteriocin Sak/Ped was markedly inhibited by the 15-mer fragment. Sak/Ped was the only one of these bacteriocins that had a sequence (in the C-terminal helix-containing half) identical to that of the 15-mer fragment, indicating that the fragment inhibits pediocin-like bacteriocins in a sequence-dependent manner. By replacing (one at a time) all 15 residues in the fragment with Ala or Leu, five residues (K1, A2, T4, N8, and A15) were identified as being especially important for the inhibitory action of the fragment. The results suggest that the corresponding residues (K20, A21, T23, N27, and A34, respectively) in pediocin PA-1 might be involved in interactions between pediocin PA-1 and its receptor. To characterize the environment surrounding these five residues when pediocin PA-1 interacts with target cells, these residues were replaced (one at a time) with a hydrophobic large (Leu) residue, a hydrophilic charged (Asp or Arg) residue, and a small (Ala or Gly) residue. The results revealed that residues A21 and A34 are in a spatially constrained environment, since the replacement with a small (Gly) residue was the only substitution that did not markedly reduce the bacteriocin activity. The positive charge in K20 and the polar amide group in N27 appeared to interact with electronegative groups, since the replacement of these two residues with a positive (Arg) residue was well tolerated, while replacement with a negative (Asp) residue was detrimental to the bacteriocin activity. K20 was in a less constrained environment than N27, since the replacement of K20 with a large hydrophobic (Leu) residue was tolerated fairly well and to a greater extent than N27. T23 seemed to be in an environment that was not restricted with respect to size, polarity, and charge, since replacements with large (Leu) and small (Ala) hydrophobic residues and a hydrophilic negative (Asp) residue were tolerated fairly well (2- to 6-fold reduction in activity). Moreover, the replacement of T23 with a large positive (Arg) residue resulted in wild-type or better-than-wild-type activity.
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1 6 a crystal structure of enta im a bacterial immunity protein conferring immunity to the antimicrobial activity of the pediocin like bacteriocin enterocin a
Journal of Biological Chemistry, 2005Co-Authors: Line Johnsen, Bjorn Dalhus, Ingar Leiros, Jon NissenmeyerAbstract:Many Gram-positive bacteria produce ribosomally synthesized antimicrobial peptides, often termed bacteriocins. Genes encoding pediocin-like bacteriocins are generally cotranscribed with or in close vicinity to a gene encoding a cognate immunity protein that protects the bacteriocin-producer from their own bacteriocin. We present the first crystal structure of a pediocin-like immunity protein, EntA-im, conferring immunity to the bacteriocin enterocin A. Determination of the structure of this 103-amino acid protein revealed that it folds into an antiparallel four-helix bundle with a flexible C-terminal part. The fact that the immunity protein conferring immunity to carnobacteriocin B2 also consists of a four-helix bundle (Sprules, T., Kawulka, K. E., and Vederas, J. C. (2004) Biochemistry 43, 11740-11749) strongly indicates that this is a conserved structural motif in all pediocin-like immunity proteins. The C-terminal half of the immunity protein contains a region that recognizes the C-terminal half of the cognate bacteriocin, and the flexibility in the C-terminal end of the immunity protein might thus be an important characteristic that enables the immunity protein to interact with its cognate bacteriocin. By homology modeling of three other pediocin-like immunity proteins and calculation of the surface charge distribution for EntA-im and the three structure models, different charge distributions were observed. The differences in the latter part of helix 3, the beginning of helix 4, and the loop connecting these helices might also be of importance in determining the specificity.
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the c terminal domain of pediocin like antimicrobial peptides class iia bacteriocins is involved in specific recognition of the c terminal part of cognate immunity proteins and in determining the antimicrobial spectrum
Journal of Biological Chemistry, 2005Co-Authors: Line Johnsen, Gunnar Fimland, Jon NissenmeyerAbstract:Abstract The pediocin-like bacteriocins contain two domains: a cationic N-terminal β-sheet domain that mediates binding of the bacteriocin to the target cell surface and a more hydrophobic C-terminal hairpin-like domain that penetrates into the hydrophobic part of the target cell membrane. The two domains are joined by a hinge, which enables movement of the domains relative to each other. In this study, 12 different hybrid bacteriocins were constructed by exchanging domains between 5 different bacteriocins. The hybrid bacteriocins were by and large highly potent (i.e. similar potencies as the parental bacteriocins) when constructed such that the recombination point was in the hinge region, indicating that the two domains function independently. The use of optimal recombination points was, however, crucial. Shifting the recombination point just one residue from the hinge could reduce the activity of the hybrid by 3–4 orders of magnitude. Most interestingly, the active hybrids displayed target cell specificities similar to those of the parental bacteriocin from which their membrane-penetrating C-terminal hairpin domain was derived. The results also indicate that the negatively charged aspartate reside in the hinge of most pediocin-like bacteriocins interacts with the C-terminal hairpin domain, perhaps by interacting with the positively charged residue that is present at one of the last three positions in the C-terminal end of most pediocin-like bacteriocins. Bacteria that produce pediocin-like bacteriocins also produce a cognate immunity protein that protects the producer from being killed by its own bacteriocin. Four different active hybrid immunity proteins constructed by exchanging regions between three different immunity proteins were tested for their ability to confer immunity to the hybrid bacteriocins. The results showed that the C-terminal half of the immunity proteins contains a region that directly or indirectly specifically recognizes the membrane-penetrating C-terminal hairpin domain of pediocin-like bacteriocins. The implications these results have on how pediocin-like bacteriocins and their immunity proteins interact with cellular specificity determinants (for instance a putative bacteriocin receptor) are discussed.
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structure function analysis of immunity proteins of pediocin like bacteriocins c terminal parts of immunity proteins are involved in specific recognition of cognate bacteriocins
Applied and Environmental Microbiology, 2004Co-Authors: Line Johnsen, Gunnar Fimland, Dimitris Mantzilas, Jon NissenmeyerAbstract:The immunity proteins of pediocin-like bacteriocins show a high degree of specificity with respect to the pediocin-like bacteriocin they recognize and confer immunity to. The aim of this study was to identify regions of the immunity proteins that are involved in this specific recognition. Six different hybrid immunity proteins were constructed from three different pediocin-like bacteriocin immunity proteins that have similar sequences but confer resistance to different bacteriocins. These hybrid immunity proteins were then tested for their ability to confer immunity to various pediocin-like bacteriocins. The specificities of the hybrid immunity proteins proved to be similar to those of the immunity proteins from which the C-terminal halves were derived, thus revealing that the C-terminal half of immunity proteins for pediocin-like bacteriocins contains a domain that is involved in specific recognition of the bacteriocins they confer immunity to. Moreover, the results also revealed that the effectiveness of an immunity protein is strain dependent and that its functionality thus depends in part on interplay with strain-dependent factors. To further investigate the structure-function relationship of these immunity proteins, the enterocin A and leucocin A immunity proteins (EntA-im and LeuA-im) were purified to homogeneity and structurally analyzed under various conditions by Circular dichroism (CD) spectroscopy. The results revealed that both immunity proteins are α-helical and well structured in an aqueous environment, the denaturing temperature being 78.5°C for EntA-im and 58.0°C for LeuA-im. The CD spectra also revealed that there was no further increase in the structuring or α-helical content when the immunity proteins were exposed to dodecylphosphocholine micelles or dioleoyl-l-α-phosphatidyl-dl-glycerol (DOPG) liposomes, indicating that the immunity proteins, in contrast to the bacteriocins, do not interact extensively with membranes. They may nevertheless be loosely associated with the membrane, possibly as peripheral membrane proteins, thus enabling them to interact with their cognate bacteriocin.
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a c terminal disulfide bridge in pediocin like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum
Journal of Bacteriology, 2000Co-Authors: Gunnar Fimland, Ingolf F Nes, Line Johnsen, Lars Axelsson, May Bente Brurberg, Vincent G H Eijsink, Jon NissenmeyerAbstract: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°C, whereas wild-type sakacin P was approximately 10 times less potent at 37°C than at 20°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.
Michael L. Chikindas - One of the best experts on this subject based on the ideXlab platform.
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improved antimicrobial activities of synthetic hybrid bacteriocins designed from enterocin e50 52 and pediocin pa 1
Applied and Environmental Microbiology, 2015Co-Authors: Katia Sutyak Noll, Santosh Kumar Tiwari, Veronica L Cavera, Michael L. ChikindasAbstract: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.
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Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832
International journal of food microbiology, 2005Co-Authors: R. Bauer, Michael L. ChikindasAbstract:Pediocin PD-1 is a ribosomally synthesized antimicrobial peptide produced by Pediococcus damnosus NCFB1832. It inhibits the growth of several food spoilage bacteria, including malolactic bacteria isolated from wine. Pediocin PD-1 is 2866.87F0.4 Da in size, has an isoelectric point (pI) of ca. 9.0 and, on amino acid composition, has partial homology to the lantibiotic plantaricin C. The highest activity of pediocin PD-1 against cells of Oenococcus oeni was observed at an external pH of 5.0 and at 25 8C. The primary mode of action of pediocin PD-1 is most probably due to pore formation, as indicated by the efflux of K + from metabolically active cells of O. oeni. In the presence of 10 mM gadolinium (Gd 3+ ), pediocin PD-1 did not affect cells of O. oeni. This suggests that the mode of action of pediocin PD-1 relies on a net negatively charged cell surface. In comparison to nisin, pediocin PD-1 is less active against non-growing cells of O. oeni. D 2004 Elsevier B.V. All rights reserved.
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functional analysis of the pediocin operon of pediococcus acidilactici pac1 0 pedb is the immunity protein and pedd is the precursor processing enzyme
Molecular Microbiology, 1995Co-Authors: K. Venema, Jan Kok, G. Venema, Aat M. Ledeboer, Joey D Marugg, Maria Yvonne Toonen, Michael L. ChikindasAbstract:The bacteriocin pediocin PA-1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA-1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N-terminal domains of PedD and a subgroup of bacteriocin ABC-transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.
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Rapid and Efficient Purification Method for Small, Hydrophobic, Cationic Bacteriocins: Purification of Lactococcin B
1995Co-Authors: Koen Venema, Alfred J. Haandrikman, Gerard Venema, Michael L. Chikindas, Jos F. M. L. Seegers, Kees J. Leenhouts, Jan KokAbstract:The bacteriocins lactococcin B and pediocin PA-1 were purified by ethanol precipitation, preparative isoelectric focusing, and ultrafiltration. The procedure reproducibly leads to high final yields in comparison to the generally low yields obtained by column chromatography. Specifically, during isoelectric focusing no loss of activity occurs. The method, in general, should be applicable to small, hydrophobic, cationic bacteriocins. The well-documented ability of lactic acid bacteria (LAB) to inhibit the growth of other bacteria (14) is of special interest to the food and feed industry, since LAB bacteriocins could po-tentially be used as food preservatives (2, 12, 16). Lactococcins and Pediocins are bacteriocins produced by Lactococcus and Pediococcus spp., respectively. Because bacteriocins are secreted into the growth medium, most approaches for purification start with a concentration step from the culture supernatant, such as salt precipitation (8, 9), acid precipitation (5), vacuum concentration, or extraction with organic solvents (18). Although these procedures are nec
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Pediocin PA-1, a Bacteriocin from Pediococcus acidilactici PAC1.0, Forms Hydrophilic Pores in the Cytoplasmic Membrane of Target Cells
Applied and environmental microbiology, 1993Co-Authors: Michael L. Chikindas, Jon Nissen-meyer, Maria J. García-garcerá, Arnold J. M. Driessen, Aat M. Ledeboer, Tjakko Abee, Wilhelmus Konings, G. VenemaAbstract: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.
Bibek Ray - One of the best experts on this subject based on the ideXlab platform.
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gene organization and sequences of pediocin ach pa 1 production operons in pediococcus and lactobacillus plasmids
Letters in Applied Microbiology, 2005Co-Authors: K W Miller, P Ray, T Steinmetz, T Hanekamp, Bibek RayAbstract:Aims: To determine the locations and sequences of pediocin AcH production genes in Pediococcus parvulus ATO77 from vegetables, Lactobacillus plantarum WHE92 from Muenster cheese, and a lactose-fermenting isolate Pediococcus pentosaceus S34 from buffalo milk. Methods and Results: Plasmid curing, Southern blot hybridization, and DNA sequence analysis indicate that pediocin AcH production genes are encoded by highly similar operons in unique plasmids designated pATO77 from P. parvulus ATO77, pS34 from P. pentosaceus S34, and pWHE92 from Lact. plantarum WHE92. Structure, immunity and secretion system genes are linked together in the operons, and the promoter sequences are the same. The amino acid sequences of the encoded proteins are highly conserved between plasmids. Conclusions: Pediocin AcH production genes are located within a plasmid-borne operon cassette in all lactic acid bacterial strains examined to date. All four genes needed for production are present within a single plasmid in each strain. Significance and Impact of the Study: This is the first demonstration that the expression of a class IIa bacteriocin is directed by a common gene cassette that has been disseminated to unique plasmids in different genera of lactic acid bacteria. These plasmids should be useful for expressing pediocin AcH in Pediococcus and Lactobacillus strains used in food production.
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hydrostatic pressure and bacteriocin triggered cell wall lysis of leuconostoc mesenteroides
Innovative Food Science and Emerging Technologies, 2002Co-Authors: N. Kalchayanand, C Frethem, P Dunne, A Sikes, Bibek RayAbstract:Exposure of cell suspensions of Leuconostoc mesenteroides Ly to either high hydrostatic pressure (HP: 345 MPa at 25 °C for 5 min) or pediocin AcH (2000 or 5000 AU/ml) produced cell viability loss over 6 log cycles and reduction in optical density (OD) of approximately 80% in 1 h at 600nm. The reduction in OD was associated with cell lysis. Time-lapse studies following treatments revealed that cell death and cell lysis are two separate but dependent events. Both HP and pediocin AcH damaged the cell wall, which then probably triggered the autolysin system of the cells to further degrade the cell walls. When cells were pressurized in the presence of pediocin AcH, cell wall degradation was faster and more extensive.
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production of active chimeric pediocin ach in escherichia coli in the absence of processing and secretion genes from the pediococcus pap operon
Applied and Environmental Microbiology, 1998Co-Authors: Kurt W Miller, Robin Schamber, Yanling Chen, Bibek RayAbstract:Minimum requirements have been determined for synthesis and secretion of the Pediococcus antimicrobial peptide, pediocin AcH, in Escherichia coli. The functional mature domain of pediocin AcH (Lys+1 to Cys+44) is targeted into the E. coli sec machinery and secreted to the periplasm in active form when fused in frame to the COOH terminus of the secretory protein maltose-binding protein (MBP). The PapC-PapD specialized secretion machinery is not required for secretion of the MBP-pediocin AcH chimeric protein, indicating that in Pediococcus, PapC and PapD probably are required for recognition and processing of the leader peptide rather than for translocation of the mature pediocin AcH domain across the cytoplasmic membrane. The chimeric protein displays bactericidal activity, suggesting that the NH2 terminus of pediocin AcH does not span the phospholipid bilayer in the membrane-interactive form of the molecule. However, the conserved Lys+1-Tyr-Tyr-Gly-Asn-Gly-Val+7-sequence at the NH2 terminus is important because deletion of this sequence abolishes activity. The secreted chimeric protein is released into the culture medium when expressed in a periplasmic leaky E. coli host. The MBP fusion-periplasmic leaky expression system should be generally advantageous for production and screening of the activity of bioactive peptides.
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Pediocins of Pediococcus Species
Bacteriocins of Lactic Acid Bacteria, 1994Co-Authors: Bibek RayAbstract:The genus Pediococcus currently includes eight species. The cells are spherical and divide into two planes to form tetrads; however, cells in pairs (but never singly) can also be present in a culture. They are Gram-positive and facultative anaerobes with optimum growth temperature ranging between 25°C and 40°C. Glucose is fermented by the Embden-Meyerhof-Parnas pathway to DL or L(+)-lactic acid. They do not produce gas, but some strains can produce diacetyl and acetoin from pyruvate. They are catalase negative, but strains can contain pseudocatalase (Garvie, 1986).
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chapter 8 Pediocins
Bacteriocins of Lactic Acid Bacteria, 1993Co-Authors: Bibek RayAbstract:Publisher Summary This chapter provides an overview on Pediocins. Pediococcus is a group of nonmotile, nonencapsulated, homofermentative, facultative anaerobes whose spherical cells form pairs or tetrads. Bacteriocin biosynthesis in Pediococcus has been demonstrated to be often associated with plasmids, and the antagonistic effect is easily detected using agar plates. The chapter also discusses other potential applications for Pediocins. Pediococci are commonly part of the natural flora of ensiled plant products. Pediococci are potential candidates for probiotic therapy because in addition to producing bacteriocins, they can colonize the GI tract, producing hydrogen peroxide and organic acids, while possibly promoting deconjugation of bile acids and salts to yield more inhibitory free bile acids.
Juan M. Rodríguez - One of the best experts on this subject based on the ideXlab platform.
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Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other wine bacteria.
Food microbiology, 2012Co-Authors: Lorena Díez, Juan M. Rodríguez, Beatriz Rojo-bezares, Myriam Zarazaga, Carmen Torres, Fernanda Ruiz-larreaAbstract:Abstract Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficiently well characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditional antimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage. In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide and ethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; in addition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grape must is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocin PA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted more sensitive to pediocin PA-1 (IC 50 = 19 ng/ml) than the other LAB species (IC 50 = 312 ng/ml). Cooperative inhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth. Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce the bacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to 80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative in winemaking.
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Production of pediocin PA-1, and coproduction of nisin A and pediocin PA-1, by wild Lactococcus lactis strains of dairy origin
International Dairy Journal, 2005Co-Authors: C. Reviriego, Nikki Horn, A. H. Fernandez, Eva Rodríguez, M.l. Marín, Leonides Fernández, Juan M. RodríguezAbstract:Abstract Heterologous production of pediocin PA-1 in nisin and non-nisin-producing Lactococcus lactis strains, which had been previously selected because of their technological properties for cheese making, was investigated. Plasmid pFI2160, which contains a hybrid gene ( L-pedA ) encoding the fusion between the lactococcin A leader and propediocin PA-1, and also the genes lcnC and lcnD , that encode the lactococcin A secretion apparatus, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis + ). The pediocin production level of their respective transformants, L. lactis CL1 and L. lactis CL2 (Nis + ), was approximately 600 and 400 ng mL −1 , respectively, which represents a 30% and a 20% of the quantity produced by the natural pediocin PA-1 producer Pediococcus acidilactici 347. Transformation of L. lactis ESI 515 with pFI2160 did not affect its ability to produce nisin. Pediocin bioassays showed the stability of pFI2160 in both heterologous hosts under selective and non-selective conditions.
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antimicrobial activity of pediocin producing lactococcus lactis on listeria monocytogenes staphylococcus aureus and escherichia coli o157 h7 in cheese
International Dairy Journal, 2005Co-Authors: Eva Rodríguez, Juan M. Rodríguez, Javier Calzada, Juan L Arques, M Nunez, Margarita MedinaAbstract:Abstract The antimicrobial activity of two pediocin-producing transformants obtained from wild strains of Lactococcus lactis on the survival of Listeria monocytogenes , Staphylococcus aureus and Escherichia coli O157:H7 during cheese ripening was investigated. Cheeses were manufactured from milk inoculated with the three pathogens, each at approximately 6 log cfu mL −1 . Pediococcus acidilactici 347 (Ped + ), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis + ) and their respective pediocin-producing transformants Lc. lactis CL1 (Ped + ) and Lc. lactis CL2 (Nis + , Ped + ) were added at 1% as adjuncts to the starter culture. After 30 d, L. monocytogenes , S. aureus and E. coli O157:H7 counts were 5.30, 5.16 and 4.14 log cfu g −1 in control cheese made without adjunct culture. On day 30, pediocin-producing derivatives Lc. lactis CL1 and Lc. lactis CL2 lowered L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157:H7 by 0.84 and 1.69 log units with respect to control cheese. All cheeses made with nisin-producing LAB exhibited bacteriocin activity throughout ripening. Pediocin activity was only detected throughout the whole ripening period in cheese with Lc. lactis CL1. Because of the antimicrobial activity of pediocin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.
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pediocin pa 1 a wide spectrum bacteriocin from lactic acid bacteria
Critical Reviews in Food Science and Nutrition, 2002Co-Authors: Juan M. Rodríguez, María I. MartínezAbstract:Pediocin PA-1 is a broad-spectrum lactic acid bacteria bacteriocin that shows a particularly strong activity against Listeria monocytogenes, a foodborne pathogen of special concern among the food industries. This antimicrobial peptide is the most extensively studied class IIa (or pediocin family) bacteriocin, and it has been sufficiently well characterized to be used as a food biopreservative. This review focuses on the progress that have been made in the elucidation of its structure, mode of action, and biosynthesis, and includes an overview of its applications in food systems. The aspects that need further research are also addressed. In the future, protein engineering, genetic engineering and/or chemical synthesis may lead to the development of new antimicrobial peptides with improved properties, based on some features of the pediocin PA-1 molecule.
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Enhanced Production of Pediocin PA-1 and Coproduction of Nisin and Pediocin PA-1 by Lactococcus lactis
Applied and environmental microbiology, 1999Co-Authors: Nikki Horn, Michael J. Gasson, Juan M. Rodríguez, María I. Martínez, J. M. Martínez, Pablo E. Hernández, Helen M. DoddAbstract: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. Martinez, J. M. Martinez, P. E. Hernandez, M. J. Gasson, J. M. Rodriguez, 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.
Gunnar Fimland - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of residues in the helical region of the class iia bacteriocin pediocin pa 1
Applied and Environmental Microbiology, 2011Co-Authors: Helen Sophie Haugen, Gunnar Fimland, Jon NissenmeyerAbstract:A 15-mer fragment that is derived from the helical region in the C-terminal half of pediocin PA-1 inhibited the activity of pediocin PA-1. Of 13 other pediocin-like (hybrid) bacteriocins, only the hybrid bacteriocin Sak/Ped was markedly inhibited by the 15-mer fragment. Sak/Ped was the only one of these bacteriocins that had a sequence (in the C-terminal helix-containing half) identical to that of the 15-mer fragment, indicating that the fragment inhibits pediocin-like bacteriocins in a sequence-dependent manner. By replacing (one at a time) all 15 residues in the fragment with Ala or Leu, five residues (K1, A2, T4, N8, and A15) were identified as being especially important for the inhibitory action of the fragment. The results suggest that the corresponding residues (K20, A21, T23, N27, and A34, respectively) in pediocin PA-1 might be involved in interactions between pediocin PA-1 and its receptor. To characterize the environment surrounding these five residues when pediocin PA-1 interacts with target cells, these residues were replaced (one at a time) with a hydrophobic large (Leu) residue, a hydrophilic charged (Asp or Arg) residue, and a small (Ala or Gly) residue. The results revealed that residues A21 and A34 are in a spatially constrained environment, since the replacement with a small (Gly) residue was the only substitution that did not markedly reduce the bacteriocin activity. The positive charge in K20 and the polar amide group in N27 appeared to interact with electronegative groups, since the replacement of these two residues with a positive (Arg) residue was well tolerated, while replacement with a negative (Asp) residue was detrimental to the bacteriocin activity. K20 was in a less constrained environment than N27, since the replacement of K20 with a large hydrophobic (Leu) residue was tolerated fairly well and to a greater extent than N27. T23 seemed to be in an environment that was not restricted with respect to size, polarity, and charge, since replacements with large (Leu) and small (Ala) hydrophobic residues and a hydrophilic negative (Asp) residue were tolerated fairly well (2- to 6-fold reduction in activity). Moreover, the replacement of T23 with a large positive (Arg) residue resulted in wild-type or better-than-wild-type activity.
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the c terminal domain of pediocin like antimicrobial peptides class iia bacteriocins is involved in specific recognition of the c terminal part of cognate immunity proteins and in determining the antimicrobial spectrum
Journal of Biological Chemistry, 2005Co-Authors: Line Johnsen, Gunnar Fimland, Jon NissenmeyerAbstract:Abstract The pediocin-like bacteriocins contain two domains: a cationic N-terminal β-sheet domain that mediates binding of the bacteriocin to the target cell surface and a more hydrophobic C-terminal hairpin-like domain that penetrates into the hydrophobic part of the target cell membrane. The two domains are joined by a hinge, which enables movement of the domains relative to each other. In this study, 12 different hybrid bacteriocins were constructed by exchanging domains between 5 different bacteriocins. The hybrid bacteriocins were by and large highly potent (i.e. similar potencies as the parental bacteriocins) when constructed such that the recombination point was in the hinge region, indicating that the two domains function independently. The use of optimal recombination points was, however, crucial. Shifting the recombination point just one residue from the hinge could reduce the activity of the hybrid by 3–4 orders of magnitude. Most interestingly, the active hybrids displayed target cell specificities similar to those of the parental bacteriocin from which their membrane-penetrating C-terminal hairpin domain was derived. The results also indicate that the negatively charged aspartate reside in the hinge of most pediocin-like bacteriocins interacts with the C-terminal hairpin domain, perhaps by interacting with the positively charged residue that is present at one of the last three positions in the C-terminal end of most pediocin-like bacteriocins. Bacteria that produce pediocin-like bacteriocins also produce a cognate immunity protein that protects the producer from being killed by its own bacteriocin. Four different active hybrid immunity proteins constructed by exchanging regions between three different immunity proteins were tested for their ability to confer immunity to the hybrid bacteriocins. The results showed that the C-terminal half of the immunity proteins contains a region that directly or indirectly specifically recognizes the membrane-penetrating C-terminal hairpin domain of pediocin-like bacteriocins. The implications these results have on how pediocin-like bacteriocins and their immunity proteins interact with cellular specificity determinants (for instance a putative bacteriocin receptor) are discussed.
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structure function analysis of immunity proteins of pediocin like bacteriocins c terminal parts of immunity proteins are involved in specific recognition of cognate bacteriocins
Applied and Environmental Microbiology, 2004Co-Authors: Line Johnsen, Gunnar Fimland, Dimitris Mantzilas, Jon NissenmeyerAbstract:The immunity proteins of pediocin-like bacteriocins show a high degree of specificity with respect to the pediocin-like bacteriocin they recognize and confer immunity to. The aim of this study was to identify regions of the immunity proteins that are involved in this specific recognition. Six different hybrid immunity proteins were constructed from three different pediocin-like bacteriocin immunity proteins that have similar sequences but confer resistance to different bacteriocins. These hybrid immunity proteins were then tested for their ability to confer immunity to various pediocin-like bacteriocins. The specificities of the hybrid immunity proteins proved to be similar to those of the immunity proteins from which the C-terminal halves were derived, thus revealing that the C-terminal half of immunity proteins for pediocin-like bacteriocins contains a domain that is involved in specific recognition of the bacteriocins they confer immunity to. Moreover, the results also revealed that the effectiveness of an immunity protein is strain dependent and that its functionality thus depends in part on interplay with strain-dependent factors. To further investigate the structure-function relationship of these immunity proteins, the enterocin A and leucocin A immunity proteins (EntA-im and LeuA-im) were purified to homogeneity and structurally analyzed under various conditions by Circular dichroism (CD) spectroscopy. The results revealed that both immunity proteins are α-helical and well structured in an aqueous environment, the denaturing temperature being 78.5°C for EntA-im and 58.0°C for LeuA-im. The CD spectra also revealed that there was no further increase in the structuring or α-helical content when the immunity proteins were exposed to dodecylphosphocholine micelles or dioleoyl-l-α-phosphatidyl-dl-glycerol (DOPG) liposomes, indicating that the immunity proteins, in contrast to the bacteriocins, do not interact extensively with membranes. They may nevertheless be loosely associated with the membrane, possibly as peripheral membrane proteins, thus enabling them to interact with their cognate bacteriocin.
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a c terminal disulfide bridge in pediocin like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum
Journal of Bacteriology, 2000Co-Authors: Gunnar Fimland, Ingolf F Nes, Line Johnsen, Lars Axelsson, May Bente Brurberg, Vincent G H Eijsink, Jon NissenmeyerAbstract: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°C, whereas wild-type sakacin P was approximately 10 times less potent at 37°C than at 20°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.
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new biologically active hybrid bacteriocins constructed by combining regions from various pediocin like bacteriocins the c terminal region is important for determining specificity
Applied and Environmental Microbiology, 1996Co-Authors: Gunnar Fimland, Knut Sletten, O R Blingsmo, Gunther Jung, I F Nes, Jon NissenmeyerAbstract:The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.