Lactococcal bacteriocins - mode of action and immunity
TL;DR: The lactococcal bacteriocins are hydrophobic cationic peptides, which form pores in the cytoplasmic membrane of sensitive cells.
About: This article is published in Trends in Microbiology.The article was published on 1995-08-01 and is currently open access. It has received 78 citations till now. The article focuses on the topics: Bacteriocin & Antimicrobial peptides.
Summary (2 min read)
Jump to: [Lactococcal bacteriocins: mode of action and immunitv] – [Mode of action of lantlbiotics: nisin] – [REVIEWS] – [Mode of action of non-Iantibiotics Diplococcin] – [Lactostrepcin 5] – [Lactococcins A and B] – [Nisin immunity and resistance] and [Conclusions and perspectives]
Lactococcal bacteriocins: mode of action and immunitv
- It is tempting to assume that different strains of a species produce these substances to enable them to compete for the same ecological niche.
- Class I bacteriocins, or lantibiotics, are small membrane-active peptides that contain the unusual amino acids lanthionine, P-methyllanthionine, dehydroalanine and dehydrobutyrine.
- All the lactococcal bacteriocins that have been thoroughly characterized so far belong to class I or II.
Mode of action of lantlbiotics: nisin
- Nisin is the only lantibiotic produced by L. luctis for which the mode of action has been studied.
- It is active against a broad spectrum of Gram-positive bacteria; Escherichia coli and other Gram-negative bacteria are only affected when their outer membranes are weakened or disrupted by treatment with EDTA or osmotic shock21,22, which makes their inner membrane accessible to the lantibiotic.
REVIEWS
- Nisin has a dual activity against spore-forming bacteria: it inhibits the outgrowth of spores and kills cells in the vegetative state.
- The 2,3_didehydroamino acid residues in nisin are thought to act against spores by interacting with the membrane sulfhydryl groups of germinating spores 23.
- It dissipates the membrane potential of whole cells, cytoplasmic membrane vesicles and artificial membrane vesicles ( liposomes)24125, indicating that the peptide does not require a specific receptor protein for activity or for membrane insertion.
- Membrane disruption is believed to result from the incorporation of nisin into the cytoplasmic membrane to form an ion channel or pore.
- This may account for the differences in sensitivity seen be-tween bacterial species or strains, as permeabilization only occurs in liposomes that contain zwitterionic phospholipids28T2p.
Mode of action of non-Iantibiotics Diplococcin
- The effect of purified diplococcin from L. lactis subsp.
- The addition of 8 arbitrary units of diplococcin to sensitive cells completely abolishes DNA and RNA synthesis within 2 min, which may partially interrupt protein synthesis.
- Small pores allow leakage of protons and other small ions only, whereas amino acids leak through larger pores.
- No receptor is required for nisin activity.
Lactostrepcin 5
- Lactostrepcin 5 (Las.5) and other lactostrepcins have a strong and rapid bactericidal effect on sensitive cells33; only Las5 has been characterized in detail.
- It inhibits uridine uptake and causes leakage of K+ ions and ATP from cells.
- Like diplococcin, Las5 inhibits DNA, RNA and protein synthesis, probably by the inhibition of transport of precursors required for macromolecular synthesis, energy depletion of the cell and/or leakage from the cell of small solutes that are required for various metabolic activities.
- Las5 is equally active against energized and energy-depleted cells33.
Lactococcins A and B
- They belong to a group of small, cationic hydrophobic peptides (including several lantibiotics) that permeabilize membranes28934"6.
- The mode of action of purified lactococcin A has been studied using whole cells of sensitive lactococcal strains and membrane vesicles made from such cells, and also using liposomes obtained from lactococcal phospholipids3'.
- Similar studies on whole cells have also been done using partially purified lactococcin B (Ref. 38) .
- These results indicate that both lactococcins form pores in the cytoplasmic membrane in a voltage-independent manner.
- Low concentrations of lactococcin B allow leakage of protons and ions, whereas ISO-fold more bacteriocin is needed for leakage of glutamate to occur38, which indicates that pores of different sizes can exist.
Nisin immunity and resistance
- There are several mechanisms by which bacteria protect themselves against nisin.
- Nisin resistance (Nis') is not genetically linked to nisin production.
- These results have been united in a model for LciA topology (Fig. 3b ).
- Residues 29-47 are considered to span the cytoplasmic membrane as an amphiphilic a helix by interacting with another transmembrane protein, possibly the lactococcin A receptor.
Conclusions and perspectives
- The past few years have seen significant progress in their understanding of nisin and the lactococcins.
- The structural and immunity genes and the genes encoding the secretion and post-translational modification machinery have been cloned, and the authors are now beginning to understand the modes of action of nisin and the lactococcins A and B, and the way in which the lactococcin A immunity protein LciA works.
- This knowledge, combined with structure-function studies of the bacteriocins, should allow the construction of molecules with enhanced or altered activities and broader specificities for use as, for example, food preservatives.
- A tropism for the mucous membranes of the human respiratory tract; indeed, the upper respiratory tract of humans is virtually the sole reservoir for this organism.
- Infection by H. in/kenzae illustrates the complex interplay that can occur between the host and the pathogen, in a relationship that does not always culminate in disease4.
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TL;DR: Bacteriocin produced by lactic acid bacteria (lactobacillus lactis) isolated from fresh cow milk, showed broad spectrum of antibacterial activity against grampositive bacteria (Staphylococcus aureus and gramnegative bacteria Escherichia coli, Salmonella typhimurium).
Abstract: Bacteriocin produced by lactic acid bacteria (lactobacillus lactis) isolated from fresh cow milk, showed broad spectrum of antibacterial activity against grampositive bacteria (Staphylococcus aureus and gramnegative bacteria Escherichia coli, Salmonella typhimurium). Maximum Bacteriocin production was observed at 35c, pH 5.5 and 20h of incubation period. Introduction: Bacteriocin form lactic acid bacteria are natural antimicrobial peptides. These contain between 30 and 60 residues. They usually have a basic character, and parts of their sequence show amphiphilicity when projected on to a helical wheel. Some bacteriocins undergo posttranslational modification and are called antibiotics (1 , 2). Lactic acid bacteria produce a number of antimicrobial substances such as organic acids, ammonia reuterin, diacetyl, hydrogen peroxide. Bacteriocins have the capacity to inhibit the growth of food spoilage and pathogenic organisms (3 , 4 , 5). Most of bacteriocins produced by grampositive bacteria are from lactic acid bacteria (6). Antimicrobial activity of bacteriocin produced by a local isolate of ... 84 Lactobacillus bacteriocins are found with in each of the four major classes of antimicrobial proteins produced by lactic acid bacteria. Class I , bacteriocins (antibiotics) were discovered in the lactobacillaceae(7). These bacteriocins are small membrane active peptides (< 5KDg) containing an unusual amino acids, kanthionine. The class II , bacteriocins are small,. heat-stable non lanthionine. containing membrane – active peptides (< 10 KDa), The class III , bacteriocins have been found in lactobacillus, include heat labile proteins of large molecular mass. The class IV , bacteriocins are a group of complex proteins, associated with other lipid or carbohydrate to be required for activity, they are relatively, hydrophobic and heat stable (8 , 9). Bacteriocins have considerable potential for food preservations, as well as for human therapy as potential supplements or replacements for currently used antibiotics (10 , 11). In the present work we describe the antibacterial activity of bacteriocin produced by a local isolate of Lactobacillus lactic. Materials and methods Isolation and culture conditions Samples of fresh cow milk were collected for isolation of Lactobacillus lactis, were put in sterile vials, then inoculated on brain heart infusion broth (BHI) and incubated at 37C for 48h.anerobically by using candle jar. Identification of Lactobacillus species was performed according to Bergey's manual of systematic bacteriology Identification of L. lactis The identification of L. lactis was made on the basis of colony morphology and biochemical test, results as Gram positive, rod, Creamy, sticky and smooth round colonies, Uniform, turbidity of Growth in (BHI) Broth, Fermentation positive of Glucose, sucrose, Lactose, mannitol and Positive of H2S production. Test Bacteria Staphylococcus aureus, E.coli, Salmonella typhimurium, were used to demonstrate the antibacterial activity of bacteriocin. These bacteria were obtained from College of Science Department of biology/ university of Mosul . Assay for antibacterial activity The isolated bacterium (L. lactis) was grown in (BHI) broth, pH 5.5 at 37c for 48h, incubated anerobically, after that the culture was centrifuged at 10.000 rpm for 15 min , at 4c and then the supernatant was adjusted to pH 6.5 – 7.0 with 1N NaOH (11 , 14). The antibacterial activity of the supernatant was determined using the well diffusion method (15). The supernatant from a 48h culture of L. lactis was filtered, sterilized by filtration using membrane filter size (0.45mm). Reem Zuhair Shinashal & Ena'am Jasim AL.hamdani 85 Aliquots (30 l) of the sterile supernatant were placed in 4-mm diameter wells that had been made in nutrient agar plates previously seeded with the test bacteria. S. aureus, E.coli, S. typhimurium. After 24h of incubation, the diameters of the zones of growth inhibition surrounding the agar wells, were measured. Optimization of culture conditions The local isolate of L. lactis was subjected to different culture conditions to derive the optimum conditions for bacteriocin production. The growth and bacteriocin production were estimated at various incubation temperature (30, 35, 40, and 45) pH (4.5, 5.5, 6.5, and 7.5) and incubation period (5, 10, 20 and 40 h), then the antibacterial activity was determined (16 , 17). Results and Discussion: Antibacterial activity: Only (8) isolates of L. lactis were observed to have bacteriocin, effect against (Three) types of bacteria, as shown in the table (1), figure (1). Table (1): Sensitivity of S. aureus, E. coli, S. typhimurium to bacteriocin produced by L. lactis isolate. Bacteriocin activity ■Sensitivity Test Bacteria No. of test Bacteria Zone of inhibition (mm) ●Resistant S.aureus 3 2 12 (mm) 1 E. coli 3 3 18 (mm) 0 S. typhimurium 3 2 17 (mm) 1 6 2 Total isolates of L. lactis were observed to have bacteriocin, effect against (Three) types of bacteria 8 ■ No of test Bacteria sensitive to Bacteriocin and Measured by (m.m). ● No of test Bacteria Resistant to Bacteriocin. Figure (1): Zone of inhibition of L. lactis against: A: S. aureus. B: E. coli . C: S. typhimurium. Antimicrobial activity of bacteriocin produced by a local isolate of ... 86 The present study was primarly aimed for determining cultural conditions for obtaining better and stable, bacteriocin production. L. lactis was able to produce bacteriocin, which had a wide inhibitory spectrum towards pathogenic bacteria. It inhibited all test bacteria S. aureus. E. coli, S. typhimurium, these agree with other published data (18 , 19 , 20). possession of bacteriocin by L. lactis is an indication that this bacteria can be used as probiotic and biopreservative (19). Incubation temperature, pH of medium and incubation period. palyed important role in growth as well as bacteriocin production. The bacteriocin activity was tested with different temperature (30, 35, 40 and 45C). furthermore, the maximum level was obtained at 35C, while minimum level was recorded at 30C. Regarding pH the maximum level was obtained at PH (5.5),and minimum level was at pH (4.5). Regarding incubation period tested, the high level of baceriocin production was recorded at 20h., while low level was recorded as shown in table (2). Table (2): Optimization of culture conditions (incubation temperature, pH, and incubation period). No. of producing isolate of bacteriocin Incubation temperature (C) pH Incubation period (h.) 1, 3 , 5 , 8 35 5.5 20 2 , 6 30 4.5 5 Bacteriocin production was strongly dependent on incubation temperature, pH and incubation period as claimed by (21). Various physiochemical factor seemed to affect bacteriocin production as well it’s activity, similar results were observed (19 , 22 , 23). In conclusion, therefore, the peculiar antimicrobial characteristics of L. lactis can positively have impact on their use as starter cultures for traditional fermented foods, with a view to improving the hygiene and safety of the food products. References 1) Venema, K. G. venema and T. kok, (1995). Lactococcal bacteriocin: mode of action and immunity. Trends Microbiol., 3: 299304. 2) Vingent G. H. Eijlsink, May B. Brurbery, p. Hans Middle Lhoven and Ingolf. F. Nes , (1996) Induction of Bacteriocin production in Lactobacillus Sake by a secreted peptide T. of Bac., Apr., p.22322237. 3) Hol Zapfel, W. H., P. Habere, R. Geisen, J. Bjorkroth and S. Vlrich, (2001) Taxonomy and important features of probiotic micro organisims in food and nutrition. Am. J. clin. Nutr., 73: 365373. Reem Zuhair Shinashal & Ena'am Jasim AL.hamdani 87 4) Lealsa'nchez, M.V. Jime' ne zDaiz, R., Maldonado – Barraga'n, A., Garrido Ferna' dez, A., Ruiz. Barba, J. L. (2002) Optimization of Bacteriocin production by Batch Fermentation of Lactobacillus plantarum , Lpco 10. Appl , And, Envin. Microbiol., 68(9) : 44654474. 5) Hirano, J. T. Yoshida, T. sugiyama, N. Koid, I. Mori and T. Yokochi, (2003) the effect of Lactobcillus rhamnosus on enterohemorrhagic Escherichia Coli infection of human intestinal cells in vitro., Microbiol Immunol., 47: 405409. 6) Garneau, S., N. I. Martin and J. c. Vederas, (2002) Tow-peptide bacteriocin produce by lactic acid bacteria, J. Biochem., 84: 577592. 7) Sengul Alpay Karaoglu, Faruk Aydin, S. Sirri Kilic, Ali O. kilic, (2003). Antimcrobial activity and characteristic of bacteriocin produced by vaginal Lactobacilli, Turk J. Med. Sci., 33: 7-13. 8) Twomey D., Ross R. p, Ryan M., Meaney B, Hill c., Lantibiotics produced by Lactic acid bacteria structure, function and applications, Antonie van Leeuwenhook (2002), 82: 165: 185. 9) Eunahar, S. ,T. Sashihara K. Sonomoto and A. Ishizaki, (2000) class IIa bacteriocin biosynthesis, structure and activity, FEMS microbiol. Rew., 24: 85106. 10) Fricourt Bv, Barefoot, S. F. Testin RF. Hayaka SS (1994). Detection and activity of plantaricin F. an antibacteria, substance from Lactobacillus palntarum Bf 001, isolated from processed channel catifich. J. food, prot. 37 (8): 698708. 11) Ogunbanwo S. T., Sanni, A. l. and Onilude A.A. (2003) Influence of cultural conditions on the production of bacteriocin by Lactobacillus brevis OG1, African J. of biotech, vol. 2. (7), pp. 179184 . july. 12) Klander O. and Regular W. N. (1986) Nonsporing gram-positive rods. In: Bergey's manual of systematic bacteriology (Edited by sneath, PHA, Mair NS, Sharpe ME, Holt. JG, pp. 1208-1298, 34 Williams and wilkins Baltimore. 13) Holt, J. G, N. R. , Kriy, J.T. Staley and S.T. Williams, (1994) Gram positive cocci. Bergey's, manual of determinative Bacteriology, 9 Edu., prestons street, Baltimore, Maryland, 21202 USA., pp: 528540. 14) Ammar, S., G.Tauveron, E. Dufour, and I. chevallier (2006) Antibacterial activity of Lactic acid bacteria against spolage and pathogenic bacteria isolated from the same meat small scale facility: 1 screening and characterization of the antibacterial compounds Food control : 17
11 Jul 2018
TL;DR: The presence of indigenous lactabacilus in meat is an additional factor that ensures the flow of desired biochemical processes and the safety of raw smoked sausages and the risk of production of unsafe products is reduced.
Abstract: Cattle carcasses of ritual slaughter (Halal) from the lateral and medial side were investigated in order to identify the indigenous strains of lactic acid microorganisms capable of producing bacteriocins. Only 14 strains, from 36 strains of microorganisms isolated from washings taken from carcasses, were used for further research and identified as lactabacilus. In the study of their bacteriocinogenicity by the method of two-layer agars of modified composition, high inhibitory efficiency was proved with respect to the growth of the pathogen Salmonella typhimurium, only 6 isolated strains of lactabacilus. The use of this method of evaluation will not only reveal the presence of the ability of the lactabacilus to produce bacteriocins, but also to study their antagonistic activity against many of the studied microorganisms, which are indicators of compliance with various modes of technological processes. The presence of such microorganisms in the meat will increase the shelf life of meat, due to the suppression of the growth of closely related lactabacilus, some of which are spoilage microorganisms, as well as the growth of opportunistic and pathogenic microflora. In addition, the use of lactabacilus with bacteriocinogenicity in the production technology of raw sausages will reduce the risk of production of unsafe products. Thus, the presence of indigenous lactabacilus in meat is an additional factor that ensures the flow of desired biochemical processes and the safety of raw smoked sausages. Contribution All authors bear responsibility for the work and presented data. All authors made an equal contribution to the work. Dagmara S. Bataeva developed scientific and methodological approaches to work, determined the scope of research, analyzed the data obtained, performed the narrative and corrected it after submitting to the editorial office. Olga V. Sokolova conducted a review and analysis of the literature, carried out the descriptive part Elena V. Zaiko selected research objects, carried out microbiological analysis. Victoria V. Pashkova carried out preparatory work and microbiological analysis. The authors were equally involved in writing the manuscript and bear the equal responsibility for plagiarism. Conflict of interest The authors declare no conflict of interest.
01 Jan 2012
TL;DR: Bacteriocin-like substance produced by Enterococcus strains may find application as biopreservatives in food products, and the antimicrobial activity was altered after treatment with trypsin, α-chymotryps in, papain which confirms the proteinaceous nature of the inhibition.
Abstract: Some lactic acid bacteria and particularly species belonging to the genus Enetrococcus are known to produce bacteriocin like inhibitory substance (BLIS). Usually they are small cationic peptide with bactericidal activity. The antimicrobial peptide produced by bacteria that deserve considerable interest for their use as natural and non-toxic food preservatives. The use of bacteriocin is among the new approaches as it has major potential in preservatives. Broad spectrum activities against prominent pathogens make it an issue of medical interest. The ability to produce such a biocompound may play role in providing an ecological advantage on non-bacteriocin producer species. 34 strains of Enterococci were isolated from different sources. These strains were identified to species: E. faecalis and E. faecium. Direct antimicrobial activity against indicator strain S. aureus was detected in 34 of the tested isolates. From these, only 7 displayed strong inhibitory activity against this indicator strain. The antimicrobial activity was altered after treatment with trypsin, α-chymotrypsin, papain which confirms the proteinaceous nature of the inhibition. This fact suggests that bacteriocin-like substance produced by Enterococcus strains may find application as biopreservatives in food products. Hence, the focus here is put on bacteriocin like substance screened by Enterococcus species isolated from different sources
Cites background from "Lactococcal bacteriocins - mode of ..."
...These peptides are often cationic and amphiphilic or hydrophobic, and many of them kill bacteria by permeabilising the target cell membrane (Venema et al., 1995)....
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TL;DR: The biochemical and genetic characteristics of these antimicrobial proteins are reviewed and common elements are discussed between the different classes of bacteriocins produced by these Gram-positive bacteria.
Abstract: Lactic acid bacteria produce a variety of bacteriocins that have recently come under detailed investigation. The biochemical and genetic characteristics of these antimicrobial proteins are reviewed and common elements are discussed between the different classes of bacteriocins produced by these Gram-positive bacteria.
2,013 citations
TL;DR: The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow, inhibiting only those strains that are closely related to the producer organism, or wide, inhibited a diverse group of Gram-positive microorganisms as mentioned in this paper.
1,754 citations
TL;DR: It is demonstrated that nisin is bactericidal to Salmonella species and that the observed inactivation can be demonstrated in other gram-negative bacteria.
Abstract: Nisin, produced by Lactococcus lactis subsp. lactis, has a broad spectrum of activity against gram-positive bacteria and is generally recognized as safe in the United States for use in selected pasteurized cheese spreads to control the outgrowth and toxin production of Clostridium botulinum. This study evaluated the inhibitory activity of nisin in combination with a chelating agent, disodium EDTA, against several Salmonella species and other selected gram-negative bacteria. After a 1-h exposure to 50 micrograms of nisin per ml and 20 mM disodium EDTA at 37 degrees C, a 3.2- to 6.9-log-cycle reduction in population was observed with the species tested. Treatment with disodium EDTA or nisin alone produced no significant inhibition (less than 1-log-cycle reduction) of the Salmonella and other gram-negative species tested. These results demonstrated that nisin is bactericidal to Salmonella species and that the observed inactivation can be demonstrated in other gram-negative bacteria. Applications involving the simultaneous treatment with nisin and chelating agents that alter the outer membrane may be of value in controlling food-borne salmonellae and other gram-negative bacteria.
586 citations
TL;DR: Transcription analyses of several L. lactis strains indicated that an expression product of the nisA gene, together with NisR, is required for the activation of nisinA transcription, indicating that NisI plays a role in the immunity mechanism.
Abstract: The nisin gene cluster nisABTCIPR of Lactococcus lactis, located on a 10-kbp DNA fragment of the nisin-sucrose transposon Tn5276, was characterized. This fragment was previously shown to direct nisin-A biosynthesis and to contain the nisP and nisR genes, encoding a nisin leader peptidase and a positive regulator, respectively [van der Meer, J. R., Polman, J., Beerthuyzen, M. M., Siezen, R. J., Kuipers, O. P. & de Vos, W. M. (1993) J. Bacteriol. 175, 2578–2588]. Further sequence analysis revealed the presence of four open-reading frames, nisB, nisT, nisC and nisI, downstream of the structural gene nisA. The nisT, nisC and nisI genes were subcloned and expressed individually in Escherichia coli, using the T7-RNA-polymerase system. This resulted in the production of radio-labelled proteins with sizes of 45 kDa (NisC) and 32 kDa (NisI). The nisT gene product was not detected, possibly because of protein instability. The deduced amino acid sequence of NisI contained a consensus Iipoprotein signal sequence, suggesting that this protein is a lipid-modified extracellular membrane-anchored protein. Expression of nisI in L. Iactis provided the cells with a significant level of protection against exogeneously added nisin, indicating that NisI plays a role in the immunity mechanism. In EDTA-treated E. coli cells, expression of nisI conferred up to a 170-fold increase in immunity against nisin A compared to controls. Moreover, a lactococcal strain deficient in nisin-A production, designated NZ9800, was created by gene replacement of nisA by a truncated nisA gene and was 10-fold less resistant to nisin A than the wild-type strain. A wild-type immunity level to nisin and production of nisin was obtained in strain NZ9800 harboring complementing nisA and nisZ plasmids. Transcription analyses of several L. IIactis strains indicated that an expression product of the nisA gene, together with NisR, is required for the activation of nisA transcription.
564 citations
TL;DR: The data suggest that the cytoplasmic membrane is the primary target and that membrane disruption accounts for the bactericidal action of nisin.
Abstract: The peptide antibiotic nisin was shown to cause a rapid efflux of amino acids and Rb+ from the cytoplasm of gram-positive bacteria (Staphylococcus cohnii 22, Bacillus subtilis W 23, Micrococcus luteus ATCC 4698, and Streptococcus zymogenes 24). It strongly decreased the membrane potential of cells as judged by the distribution of the lipophilic tetraphenylphosphonium cation. Ascorbate-phenazine methosulfate-driven transport of L-proline by cytoplasmic membrane vesicles was blocked after addition of nisin, and accumulated amino acids were released from the vesicles. Soybean phospholipid (asolectin) vesicles were not affected by nisin. The data suggest that the cytoplasmic membrane is the primary target and that membrane disruption accounts for the bactericidal action of nisin.
379 citations