Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication

Lactic acid bacteria (LAB) fight competing Gram-positive microorganisms by secreting anti-microbial peptides called bacteriocins. Peptide bacteriocins are usually divided into lantibiotics (class I) and non-lantibiotics (class II), the latter being the main topic of this review. During the past decade many of these bacteriocins have been isolated and characterized, and elements of the genetic mechanisms behind bacteriocin production have been unravelled. Bacteriocins often have a narrow inhibitory spectrum, and are normally most active towards closely related bacteria likely to occur in the same ecological niche. Lactic acid bacteria seem to compensate for these narrow inhibitory spectra by producing several bacteriocins belonging to different classes and having different inhibitory spectra. The latter may also help in counteracting the possible development of resistance mechanisms in target organisms. In many strains, bacteriocin production is controlled in a cell-density dependent manner, using a secreted peptide-pheromone for quorum-sensing. The sensing of its own growth, which is likely to be comparable to that of related species, enables the producing organism to switch on bacteriocin production at times when competition for nutrients is likely to become more severe. Although today a lot is known about LAB bacteriocins and the regulation of their production, several fundamental questions remain to be solved. These include questions regarding mechanisms of immunity and resistance, as well as the molecular basis of target-cell specificity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Novel bacteriocins from lactic acid bacteria (LAB): various structures and applications

Bacteriocins are heat-stable ribosomally synthesized antimicrobial peptides produced by various bacteria, including food-grade lactic acid bacteria (LAB). These antimicrobial peptides have huge potential as both food preservatives, and as next-generation antibiotics targeting the multiple-drug resistant pathogens. The increasing number of reports of new bacteriocins with unique properties indicates that there is still a lot to learn about this family of peptide antibiotics. In this review, we highlight our system of fast tracking the discovery of novel bacteriocins, belonging to different classes, and isolated from various sources. This system employs molecular mass analysis of supernatant from the candidate strain, coupled with a statistical analysis of their antimicrobial spectra that can even discriminate novel variants of known bacteriocins. This review also discusses current updates regarding the structural characterization, mode of antimicrobial action, and biosynthetic mechanisms of various novel bacteriocins. Future perspectives and potential applications of these novel bacteriocins are also discussed.     

Exploration of antimicrobial potential in LAB by genomics

A tremendous flow of information has been created through various genome sequencing projects worldwide. So far, 128 bacterial genome sequences have been completed and 391 are under way. Many of these bacteria, including several lactic acid bacteria (LAB), are used in the production and preservation of food and feed. The major antimicrobial and biopreservative substance produced by LAB is organic acid; however, some LAB produce additional antimicrobial compounds. Among these, the bacteriocins have demonstrated great potential as food preservatives. Additionally, antimicrobial compounds different from the bacteriocins have recently been identified, of which several display strong antifungal activity. The information obtained from genomics and related technologies will have great impact on the future identification and development of new antimicrobial agents. Developments will include the identification of pathways for the production of antimicrobials and genome mining for new antimicrobial peptides.     

细菌素的合成与作用机制

细菌素是由细菌产生的抗菌蛋白,可以杀死与产生菌相近的细菌。很多乳酸菌产生不同多样性的细菌素,虽然这些细菌素都是由发酵或非发酵食品中发现的乳酸菌产生的,但是迄今只有乳酸链球菌素(Nisin)作为食品防腐剂被广泛应用。和抗生素不同的是,细菌素由核糖体合成,需经翻译后修饰活化并且通过特定转运系统输到胞外才能发挥其功能,它一般通过作用于靶细胞膜来抑制靶细胞的生长,同时本身合成细菌素的细胞对其产物具有免疫性。细菌素能安全有效地抑制病原体生长,在食品行业中具有广阔的应用前景。     

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.     

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     

An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley

AIMS: The aim of this study was to perform a detailed characterization of bacteriocins produced by lactic acid bacteria (LAB) isolated from malted barley. METHODS AND RESULTS: Bacteriocin activities produced by eight LAB, isolated from various types of malted barley, were purified to homogeneity by ammonium sulphate precipitation, cation exchange, hydrophobic interaction and reverse-phase liquid chromatography. Molecular mass analysis and N-terminal amino acid sequencing of the purified bacteriocins showed that four non-identical Lactobacillus sakei strains produced sakacin P, while four Leuconostoc mesenteroides strains were shown to produce bacteriocins highly similar or identical to leucocin A, leucocin C or mesenterocin Y105. Two of these bacteriocin-producing strains, Lb. sakei 5 and Leuc. mesenteroides 6, were shown to produce more than one bacteriocin. Lactobacillus sakei 5 produced sakacin P as well as two novel bacteriocins, which were termed sakacin 5X and sakacin 5T. The inhibitory spectrum of each purified bacteriocin was analysed and demonstrated that sakacin 5X was capable of inhibiting the widest range of beer spoilage organisms. CONCLUSION: All bacteriocins purified in this study were class II bacteriocins. Two of the bacteriocins have not been described previously in the literature while the remaining purified bacteriocins have been isolated from environments other than malted barley. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a thorough analysis of bacteriocin-producing LAB from malt and demonstrates, for the first time, the variety of previously identified and novel inhibitory peptides produced by isolates from this environment. It also highlights the potential of these LAB cultures to be used as biological controlling agents in the brewing industry.     

Bacteriocins from lactic acid bacteria: production, purification, and food applications

In fermented foods, lactic acid bacteria (LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with industrial potential have been purified and characterized. The kinetics of bacteriocin production by LAB in relation to process factors have been studied in detail through mathematical modeling and positive predictive microbiology. Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness), in fermented sausage (anti-listerial effect), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level. The highly promising results of these studies underline the important role that functional, bacteriocinogenic LAB strains may play in the food industry as starter cultures, co-cultures, or bioprotective cultures, to improve food quality and safety. In addition, antimicrobial production by probiotic LAB might play a role during in vivo interactions occurring in the human gastrointestinal tract, hence contributing to gut health.     

Bacteriocins from lactic acid bacteria and their potential in the preservation of fruit products

Bacteriocins produced by lactic acid bacteria (LAB) are well-recognized for their potential as natural food preservatives. These antimicrobial peptides usually do not change the sensorial properties of food products and can be used in combination with traditional preservation methods to ensure microbial stability. In recent years, fruit products are increasingly being associated with food-borne pathogens and spoilage microorganisms, and bacteriocins are important candidates to preserve these products. Bacteriocins have been extensively studied to preserve foods of animal origin. However, little information is available for their use in vegetable products, especially in minimally processed ready-to-eat fruits. Although, many bacteriocins possess useful characteristics that can be used to preserve fruit products, to date, only nisin, enterocin AS-48, bovicin HC5, enterocin 416K1, pediocin and bificin C6165 have been tested for their activity against spoilage and pathogenic microorganisms in these products. Among these, only nisin and pediocin are approved to be commercially used as food additives, and their use in fruit products is still limited to certain countries. Considering the increasing demand for fresh-tasting fruit products and concern for public safety, the study of other bacteriocins with biochemical characteristics that make them candidates for the preservation of these products are of great interest. Efforts for their approval as food additives are also important. In this review, we discuss why the study of bacteriocins as an alternative method to preserve fruit products is important; we detail the biotechnological approaches for the use of bacteriocins in fruit products; and describe some bacteriocins that have been tested and have potential to be tested for the preservation of fruit products.     

Peptide signal molecules and bacteriocins in Gram-negative bacteria: a genome-wide in silico screening for peptides containing a double-glycine leader sequence and their cognate transporters

Quorum sensing (QS) in Gram-negative bacteria is generally assumed to be mediated by N-acyl-homoserine lactone molecules while Gram-positive bacteria make use of signaling peptides. We analyzed the occurrence in Gram-negative bacteria of peptides and transporters that are involved in quorum sensing in Gram-positive bacteria. Many class II bacteriocins and inducing factors produced by lactic acid bacteria (LAB) and competence stimulating peptides (CSPs) synthesized by streptococci are processed by their cognate ABC-transporters during their secretion. During transport, a conserved leader sequence, termed the double-glycine motif (GG-motif), is cleaved off by the N-terminal domain of the transporter, which belongs to the Peptidase C39 protein family. Several peptides containing a GG-motif were recently described in Gram-negative bacteria (Trends Microbiol 2001;9:164-8). To screen for additional putative GG-motif containing peptides, an in silico strategy based on MEME, HMMER2.2 and Wise2 was designed. Using a curated training set, a motif model of the leader peptide was built and used to screen over 120 fully sequenced bacterial genomes. The screening methodology was applied at the nucleotide level as probably many small peptide genes have not been annotated and may be absent from the non-redundant databases. It was found that 33% of the screened genomes of Gram-negative bacteria contained one or more transporters carrying a Peptidase C39 domain, compared to 44% of the genomes of Gram-positive bacteria. The transporters can be subdivided into four classes on the basis of their domain organization. Genes coding for putative peptides containing 23-142 amino acids and a GG-motif were found in close association with genes coding for Peptidase C39 domain containing proteins. These peptides show structural similarity to bacteriocins and peptide pheromones of Gram-positive bacteria. The possibility of signal transduction based on peptide signaling in Gram-negative bacteria is discussed.     

Native and heterologous production of bacteriocins from gram-positive microorganisms

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.     

The structure of pyogenecin immunity protein, a novel bacteriocin-like immunity protein from Streptococcus pyogenes

Background  

Many Gram-positive lactic acid bacteria (LAB) produce anti-bacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. These peptides fall structurally into three different classes, I, II, III, with class IIa being pediocin-like single entities and class IIb being two-peptide bacteriocins. Self-protective cognate immunity proteins are usually co-transcribed with these toxins. Several examples of cognates for IIa have already been solved structurally. Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens, so knowledge of how it competes against other LAB species is likely to prove invaluable.     

Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods

Lactic acid bacteria (LAB) isolated from different sources (dairy products, fruits, fresh and fermented vegetables, fermented cereals) were screened for antimicrobial activity against other bacteria, including potential pathogens and food spoiling bacteria. Six strains have been shown to produce bacteriocins: Lactococcus lactis 19.3, Lactobacillus plantarum 26.1, Enterococcus durans 41.2, isolated from dairy products and Lactobacillus amylolyticus P40 and P50, and Lactobacillus oris P49, isolated from bors. Among the six bacteriocins, there were both heat stable, low molecular mass polypeptides, with a broad inhibitory spectrum, probably belonging to class II bacteriocins, and heat labile, high molecular mass proteins, with a very narrow inhibitory spectrum, most probably belonging to class III bacteriocins. A synergistic effect of some bacteriocins mixtures was observed. We can conclude that fermented foods are still important sources of new functional LAB. Among the six characterized bacteriocins, there might be some novel compounds with interesting features. Moreover, the bacteriocin-producing strains isolated in our study may find applications as protective cultures.     

Bacteriocin Production and Different Strategies for Their Recovery and Purification

Bacteriocins from lactic acid bacteria (LAB) are a diverse group of antimicrobial proteins/peptides, offering potential as biopreservatives, and exhibit a broad spectrum of antimicrobial activity at low concentrations along with thermal as well as pH stability in foods. High bacteriocin production usually occurs in complex media. However, such media are expensive for an economical production process. For effective use of bacteriocins as food biopreservatives, there is a need to have heat-stable wide spectrum bacteriocins produced with high-specific activity in food-grade medium. The main hurdles concerning the application of bacteriocins as food biopreservatives is their low yield in food-grade medium and time-consuming, expensive purification processes, which are suitable at laboratory scale but not at industrial scale. So, the present review focuses on the bacteriocins production using complex and food-grade media, which mainly emphasizes on the bacteriocin producer strains, media used, different production systems used and effect of different fermentation conditions on the bacteriocin production. In addition, this review emphasizes the purification processes designed for efficient recovery of bacteriocins at small and large scale.     

The continuing story of class IIa bacteriocins.

Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.     

Antibacterial metabolites of lactic acid bacteria: their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Potential of bacteriocin-producing lactic acid bacteria for improvements in food safety and quality

Lactic acid bacteria (LAB) have been used for centuries in the fermentation of a variety of dairy products. The preservative ability of LAB in foods is attributed to the production of anti-microbial metabolites including organic acids and bacteriocins. Bacteriocins generally exert their anti-microbial action by interfering with the cell wall or the membrane of target organisms, either by inhibiting cell wall biosynthesis or causing pore formation, subsequently resulting in death. The incorporation of bacteriocins as a biopreservative ingredient into model food systems has been studied extensively and has been shown to be effective in the control of pathogenic and spoilage microorganisms. However, a more practical and economic option of incorporating bacteriocins into foods can be the direct addition of bacteriocin-producing cultures into food. This paper presents an overview of the potential for using bacteriocin-producing LAB in foods for the improvement of the safety and quality of the final product. It describes the different genera of LAB with potential as biopreservatives, and presents an up-to-date classification system for the bacteriocins they produce. While the problems associated with the use of some bacteriocin-producing cultures in certain foods are elucidated, so also are the situations in which incorporation of the bacteriocin-producer into model food systems have been shown to be very effective.     

Isolation and characterization of lactobacilli from some traditional fermented foods and evaluation of the bacteriocins

Lactic acid bacteria (LAB) commonly used in food as starter cultures are known to produce antimicrobial substances such as bacteriocins and have great potential as food biopreservatives. LAB isolated from traditional fermented foods (appam batter and pickles) were screened for bacteriocin production. Two lactobacilli, LABB and LABP (one from each source) producing bacteriocins were characterized. Both the bacilli were homo-fermentative, catalase negative and micro-aerophilic in nature. LABB was found to be a thermobacterium growing at 45 degrees C while LABP was a streptobacterium growing at 15 degrees C. Both were able to grow at pH 4.5-8.6 but were intolerant to high salt concentration. They failed to produce gas from glucose as well as ammonia from arginine. Among the sugars examined they could not ferment arabinose, raffinose, rhamnose or xylose. Additionally, LABB could not ferment esculin, gluconate or mannose. LABB is identified as Lactobacillus acidophilus while LABP as Lb. casei. Their bacteriocins showed a broad inhibitory spectrum against the indicator organisms tested. They were active below pH 8.0 and after autoclaving as well. There was a complete loss of activity when treated with proteolytic enzymes such as trypsin indicating the proteinaceous nature of the active molecules. SDS-PAGE of partially purified bacteriocins indicated the molecular mass of the bacteriocin as 3.8 and 4.5 kDa for LABB and LABP respectively.     

Antibacterial metabolites of lactic acid bacteria: Their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Strong synergy between a eukaryotic antimicrobial peptide and bacteriocins from lactic acid bacteria

The antimicrobial effect obtained upon combining the prokaryotic antimicrobial peptides (AMPs; more commonly referred to as bacteriocins) pediocin PA-1, sakacin P, and curvacin A (all produced by lactic acid bacteria [LAB]) with the eukaryotic AMP pleurocidin (from fish) has been investigated. The three LAB AMPs alone were active against gram-positive Listeria ivanovii bacteria at nanomolar concentrations, whereas they were inactive against gram-negative Escherichia coli bacteria. Pleurocidin alone was active against both of these types of bacteria at micromolar concentrations. Little if any synergy between the LAB AMPs and pleurocidin against the gram-positive L. ivanovii strain was obtained. In contrast, the LAB AMPs and pleurocidin acted highly synergistically against the gram-negative E. coli strain. Nanomolar concentrations of LAB AMPs increased the growth inhibitory potency of pleurocidin by about fourfold. When micromolar concentrations of LAB AMPs were combined with 2 micro g of pleurocidin/ml, 100% growth inhibition was attained, whereas pleurocidin alone at a concentration of 2 micro g/ml gave no growth inhibition. Most noteworthy, when high concentrations (128 micro g/ml) of pleurocidin in the absence of LAB AMPs were used over a long period of incubation (1 week), some growth of E. coli was observed, whereas 16 micro g of pleurocidin/ml completely abolished growth in the presence of 64 to 128 ng of LAB AMPs/ml over the same period of time. The results clearly demonstrate that combining eukaryotic and prokaryotic AMPs can greatly increase the specific activity and broaden the target-cell range of these peptides.