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.     

Natural Variation in Susceptibility of Listeria Strains to Class IIa Bacteriocins

Thirty-one Listeria strains were tested for sensitivity to four class IIa bacteriocins, namely, enterocin A, mesentericin Y105, divercin V41, and pediocin AcH, and to nisin A. Class IIa bacteriocins displayed surprisingly similar antimicrobial patterns ranging from highly susceptible to fully resistant strains, whereas nisin A showed a different pattern in which all Listeria strains were inhibited. Particularly, it was observed that the strain Listeria monocytogenes V7 could not be inhibited by any of the class IIa bacteriocins tested. These observations suggest that Listeria strains resistant to the whole range of class IIa bacteriocins may occur in natural environments, which could be of great concern with regard to the use of these peptides as food preservatives. Received: 22 October 1999 / Accepted: 15 December 1999     

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

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

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

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

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

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

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

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

Detection of Bacteriocins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of bacteriocins was investigated. A 30-s water wash of the sample on the MALDI-TOF MS probe was effective in removing contaminants of the analyte. This method was used for rapid detection of nisin, pediocin, brochocin A and B, and enterocin A and B from culture supernatants and for detection of enterocin B throughout its purification.     

Potential Applications of the Cyclic Peptide Enterocin AS-48 in the Preservation of Vegetable Foods and Beverages

Bacteriocins are antimicrobial peptides produced by bacteria. Among them, the enterococcal bacteriocin (enterocin) AS-48 stands for its peculiar characteristics and broad-spectrum antimicrobial activity. AS-48 belongs to the class of circular bacteriocins and has been studied in depth in several aspects: peptide structure, genetic determinants, and mode of action. Recently, a wealth of knowledge has accumulated on the antibacterial activity of this bacteriocin against foodborne pathogenic and spoilage bacteria in food systems, especially in vegetable foods and drinks. This work provides a general overview on the results from tests carried out with AS-48 in different vegetable food categories (such as fruit juices, ciders, sport and energy drinks, fresh fruits and vegetables, pre-cooked ready to eat foods, canned vegetables, and bakery products). Depending on the food substrate, the bacteriocin has been tested alone or as part of hurdle technology, in combination with physico-chemical treatments (such as mild heat treatments or high-intensity pulsed electric fields) and other antimicrobial substances (such as essential oils, phenolic compounds, and chemical preservatives). Since the work carried out on bacteriocins in preservation of vegetable foods and drinks is much more limited compared to meat and dairy products, the results reported for AS-48 may open new possibilities in the field of bacteriocin applications.     

Mechanistic action of pediocin and nisin: recent progress and unresolved questions

Nisin and pediocin PA-1 are examples of bacteriocins from lactic acid bacteria (LAB) that have found practical applications as food preservatives. Like other natural antimicrobial peptides, LAB bacteriocins act primarily at the cytoplasmic membranes of susceptible microorganisms. Studies with in vivo as well as in␣vitro membrane systems are directed toward understanding how bacteriocins interact with membranes so as to provide a mechanistic basis for their rational applications. The dissipation of proton motive force was identified early on as the common mechanism for the lethal activity of LAB bacteriocin. Models for nisin/membrane interactions propose that the peptide forms poration complexes in the membrane through a multi-step process of binding, insertion, and pore formation. This review focuses on the current knowledge of: (1) the mechanistic action of nisin and pediocin-like bacteriocins, (2) the requirement for a cell factor such as a membrane protein, (3) the influence of membrane potential, pH, and lipid composition on the of specificity and efficacy of bacteriocins, and (4) the roles of specific amino acids and structural domains of the bacteriocins in their action. Received: 3 April 1998 / Received last revision: 27 July 1998 / Accepted: 29 July 1998     

Trends in utilization of agro-industrial byproducts for production of bacteriocins and their biopreservative applications

Bacteriocins are proteinaceous, ribosomally synthesized bio-molecules having major roles in food preservation due to their antimicrobial action against food spoilage microorganisms. These have gained importance in the last decades because of increasing interest in natural products and their applications in the field of biopreservation, pharmaceutical, aquaculture, livestock, etc. Their production is quite expensive which includes the cost of synthetic media and downstream processing of which 30% of the total production cost relies on synthetic media and nutritional supplements used for growth of microorganisms. The low cost agro-industrial by-products, rich in nutritional supplements, can act as a good substitute for high valued synthetic media. This review provides comprehensive information on the use of cost effective, renewable agro-industrial by-products as substrates for the production of bacteriocins and their application in food as biopreservatives.     

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

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

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

Characterization of Mundticin L,a Class IIa Anti-Listeria Bacteriocin from Enterococcus mundtii CUGF08

Enterococcus mundtii CUGF08, a lactic acid bacterium isolated from alfalfa sprouts, was found to produce mundticin L, a new class IIa bacteriocin that has a high level of inhibitory activity against the genus Listeria. The plasmid-associated operons containing genes for the mundticin L precursor, the ATP binding cassette (ABC) transporter, and immunity were cloned and sequenced. The fifth residue of the conservative consensus sequence YGNGX in the mature bacteriocin is leucine instead of valine in the sequences of the homologous molecules mundticin KS (ATO6) and enterocin CRL35. The primary structures of the ABC transporter and the immunity protein are homologous but unique.Bacteriocins are ribosomally synthesized proteinaceous compounds that inhibit closely related bacteria (19). Due to consumer concerns with chemical and irradiation preservation methods and due to the rising demand for minimally processed food products, alternative methods for shelf life extension and enhanced safety are needed. Bacteriocins are considered “natural” antimicrobials since many bacteriocins are produced by food grade lactic acid bacteria, which are generally recognized as safe. Bacteriocins can be divided into three main classes: the class I lanthionine-containing lantibiotics, exemplified by nisin; the class II non-lanthionine-containing bacteriocins; and the class III heat-labile, large proteins (6). Class III bacteriocins have limited application due to their thermal instability and cytolytic activity against eukaryotic cells. Class II can be further divided into class IIa containing pediocin-like bacteriocins, class IIb containing two-peptide bacteriocins, and class IIc containing other bacteriocins (8). Class IIa bacteriocins have been extensively studied since pediocin PA-1 was first discovered (12) and characterized (20). Currently, only nisin in class I has been approved by the FDA as a natural food additive. Bacteriocins belonging to class IIa are promising alternative antimicrobials since they are more stable over a broader range of heating regimens and pH conditions. In addition, these bacteriocins exhibit stronger antimicrobial activity against the genus Listeria than nisin (17) but have a narrower antimicrobial spectrum.The potential applications of class IIa bacteriocins in both meat and plant-based foods as a means to provide protection against potential food-borne pathogens and extend shelf life continue to expand. In an attempt to use biological methods for controlling food-borne pathogens on fresh sprouts, a number of food grade lactic acid bacteria were isolated from the indigenous microbiota on alfalfa sprouts. Some of these isolates were found to be bacteriocinogenic. This study describes a new class IIa bacteriocin, mundticin L produced by Enterococcus mundtii CUGF08 isolated from alfalfa sprouts.     

Applications of the bacteriocin,nisin

Nisin was first introduced commercially as a food preservative in the UK approximately 30 years ago. First established use was as a preservative in processed cheese products and since then numerous other applications in foods and beverages have been identified. It is currently recognised as a safe food preservative in approximately 50 countries. The established uses of nisin as a preservative in processed cheese, various pasteurised dairy products, and canned vegetables will be briefly reviewed. More recent applications of nisin include its use as a preservative in high moisture, hot baked flour products (crumpets) and pasteurised liquid egg. Renewed interest is evident in the use of nisin in natural cheese production. Considerable research has been carried out on the antilisterial properties of nisin in foods and a number of applications have been proposed. Uses of nisin to control spoilage lactic acid bacteria have been identified in beer, wine, alcohol production and low pH foods such as salad dressings. Further developments of nisin are likely to include synergistic action of nisin with chelators and other bacteriocins, and its use as an adjunct in novel food processing technology such as higher pressure sterilisation and electroporation. Production of highly purified nisin preparations and enhancement by chelators has led to interest in the use of nisin for human ulcer therapy, and mastitis control in cattle.     

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

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

Combined Effect of Enterocin and Lipase From Enterococcus faecium NCIM5363 Against Food Borne Pathogens: Mode of Action Studies

Food borne diseases have a major impact on public health whose epidemiology is rapidly changing. The whole cells of pathogens involved or their toxins/metabolites affect the human health apart from spoiling sensory properties of the food products finally affecting the food industry as well as consumer health. With pathogens developing mechanisms of antibiotic resistance, there has been an increased need to replace antibiotics as well as chemical additives with naturally occurring bacteriocins. Bacteriocins are known to act mainly against Gram-positive pathogens and with little or no effect towards Gram-negative enteric bacteria. In the present study, combination effect of lipase and bacteriocin produced by Enterococcus faecium NCIM5363, a highly lipolytic lactic acid bacterium against various food pathogens was assessed. The lipase in combination with enterocin exhibited a lethal effect against Gram-negative pathogens. Scanning electron microscopy studies carried out to ascertain the constitutive mode of action of lipase and enterocin revealed that the lipase degrades the cell wall of Gram-negative bacteria and creates a pore through which enterocin enters thereby resulting in cell death. The novelty of this work is the fact that this is the first report revealing the synergistic effect of lipase with enterocin against Gram-negative bacteria.     

Lantibiotics, class I bacteriocins from the genus Bacillus

Antimicrobial peptides exhibit high levels of antimicrobial activity against a broad range of spoilage and pathogenic microorganisms. Compared with bacteriocins produced by lactic acid bacteria, antimicrobial peptides from the genus Bacillus have been relatively less recognized despite their broad antimicrobial spectra. These peptides can be classified into two different groups based on whether they are ribosomally (bacteriocins) or nonribosomally (polymyxins and iturins) synthesized. Because of their broad spectra and high activity, antimicrobial peptides from Bacillus spp. may have great potential for applications in the food, agricultural, and pharmaceutical industries to prevent or control spoilage and pathogenic microorganisms. In this review, we introduce ribosomally synthesized antimicrobial peptides, the lantibiotic bacteriocins produced by members of Bacillus. In addition, the biosynthesis, genetic organization, mode of action, and regulation of subtilin, a well-investigated lantibiotic from Bacillus subtilis, are discussed.     

Gassericin A: a circular bacteriocin produced by Lactic acid bacteria Lactobacillus gasseri

During the recent years extensive efforts have been made to find out bacteriocins from lactic acid bacteria (LAB) active against various food spoilage and pathogenic bacteria, and superior stabilities against heat treatments and pH variations. Bacteriocins isolated from LAB have been grouped into four classes. Circular bacteriocins which were earlier grouped among the four groups of bacteriocins, have recently been proposed to be classified into a different class, making it class V bacteriocins. Circular bacteriocins are special molecules, whose precursors must be post translationally modified to join the N to C termini with a head-to-tail peptide bond. Cyclization appears to make them less susceptible to proteolytic cleavage, high temperature and pH, and, therefore, provides enhanced stability as compared to linear bacteriocins. The advantages of circularization are also reflected by the fact that a significant number of macrocyclic natural products have found pharmaceutical applications. Circular bacteriocins were unknown two decades ago, and even to date, only a few circular bacteriocins from a diverse group of Gram positive organisms have been reported. The first example of a circular bacteriocin was enterocin AS-48, produced by Enterococcus faecalis AS-48. Gassereccin A, produced by Lactobacillus gasseri LA39, Reutericin 6 produced by Lactobacillus reuteri LA6 and Circularin A, produced by Clostridium beijerinickii ATCC 25,752, are further examples of this group of antimicrobial peptides. In the present scenario, Gassericin A can be an important tool in the food preservation owing to its properties of high pH and temperature tolerance and the fact that it is produced by LAB L. gasseri, whose many strains are proven probiotic.     

Antimicrobial activity of Enterococcus faecium against Listeria spp.

Listeria spp. have been isolated from vegetation, silage, the intestinal tracts of animals and foods such as milk and cheese. Lisleria spp. are taxonomically related to lactobacilli (Seeliger & Jones 1986) and some bacteriocins produced by lactic acid bacteria will inhibit growth of Listeria spp. Bacteriocins such as nisin from Lactococcus lactis and pediocin A from Pediococcus pentosoreus, are active against microorganisms from several Gram-positive genera, and will inhibit L. monocytogenes. Bacteriocins (e.g. helveticin J and lactacin F) which only inhibit strains closely related to the producing micro-organism are not effective against L. monocytogenes     

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.