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.     

Selection and fitness in bacteriocin-producing bacteria.

Bacteriocins are proteinaceous anticompetitor molecules produced by bacteria against closely related species. A number of theoretical models have been used to explain experimental data that indicate high polymorphisms among bacteriocins and a frequency-dependent nature of selection for bacteriocin-producing strains. The majority of these experimental data were, however, obtained from investigations into the colicin group of bacteriocins produced by Gram-negative bacteria. The conclusions drawn from these models have been extrapolated to other bacteriocins and allelopathic compounds in general. Examination of more recent experimental investigations into the bacteriocins of Gram-positive bacteria indicate a lower degree of polymorphism and a less frequency dependent mode of selection among these strains them among the colicin-producing strains. Here we examine these contradictions in the light of the assumptions and conclusions of the theoretical models and reported data. We show that fitness costs as indicated by decreased relative maximum growth rate associated with bacteriocin production may be much lower in many cases than is assumed in the present models. A lower fitness cost associated with bacteriocin production adequately explains the newer data from Gram-positive bacteria cited here, and indicates that extrapolation of existing models to all bacteriocins and other allelopathic compounds is not appropriate.     

Bacteriocins of lactic acid bacteria

Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. 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, inhibiting a diverse group of Gram-positive microorganisms. The following review will discuss biochemical and genetic aspects of bacteriocins that have been identified and characterized from lactic acid bacteria.     

Bacterioides fragilis: production and sensitivity to bacteriocins

Bacteriocin production and sensitivity to bacteriocins have been successfully applied as an epidemiological tool in several species of bacteria. However, little work has been carried out on the bacteriocins produced by Bacteroides fragilis, which is the most frequently isolated anaerobe species from clinical specimens. Thirty two clinical isolates of B. fragilis grown anaerobically on a 0.22 microm membrane filter spotted on an agar plate, were tested for bacteriocin production and used in a screen for bacteriocin sensitivity. Sensitivity to at least one bacteriocin was found in 94% of the strains, 62.5% were sensitive to two bacteriocins, whereas 34.4% were sensitive to three or more and finally one strain was found sensitive to 17 bacteriocins. Of the strains studied, 94% inhibited at least one strain, 66% inhibited two strains, and 30% inhibited at least three strains or more. Finally, one strain was extremely active by inhibiting the growth of 17 strains. Bacteriocin types are characterised by geographic variation, and their epidemiological investigation by a simple method could be promoted.     

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     

Developing applications for lactococcal bacteriocins

While much of the applied research carried out to date with bacteriocins has concerned nisin, lactococci produce other bacteriocins with economic potential. An example is the two component bacteriocin lacticin 3147, which is active over a wide pH range and has a broad spectrum of activity against Gram-positive bacteria. Since the genetic determinants for lacticin 3147 are encoded on a large self-transmissible plasmid, the bacteriocin genes may be conveniently transferred to different lactococcal starters. The resulting food-grade strains can then be used to make a significant impact on the safety and quality of a variety of fermented foods, through the inhibition of undesirable microflora. The bacteriocin is heat stable so it can also be used as an ingredient in a powdered form such as a spray-dried fermentate. Given the observation that lacticin 3147 is effective at physiological pH, there is also considerable potential for biomedical applications. Field trials have demonstrat ed its efficacy in the prevention of mastitis infections in dairy cows. In contrast to lacticin 3147, the lactococcin bacteriocins A, B and M have a narrow spectrum of activity limited to lactococci. Strains which produce these inhibitors can be exploited in the acceleration of cheese ripening by assisting the premature lysis of starter 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.     

Lack of Heterogeneity in Bacteriocin Production Across a Selection of Commercial Probiotic Products

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Bacteriocin production has often been mooted as a desirable probiotic trait and, in specific cases, has been shown to promote probiotic survival within the gastrointestinal tract, contribute to the control of pathogens and even influence host gene expression in the gut. However, it is not clear what proportion of probiotic strains routinely found in commercial products produces bacteriocins, and additionally, it is not known which bacteriocins are produced most frequently. To address this, we conducted a culture-based assessment of the bacteriocinogenic ability of bacterial strains found in a variety of commercially available probiotic products. We detected eight bacteriocin-producing isolates from 16 tested products. Interestingly, in all cases, the isolates were Lactobacillus acidophilus, and the bacteriocin produced was identified as the narrow spectrum class II bacteriocin, lactacin B. The apparent absence of other bacteriocin-producing strains from across these products suggests a lack of heterogeneity in bacteriocin production within probiotic products and suggests that bacteriocin production is not being optimally harnessed as a probiotic trait.     

A phylogenetic approach to assessing the targets of microbial warfare

Bacteriocins are the most abundant and diverse defense systems in bacteria. As a result of the specific mechanisms of bacteriocin recognition and translocation into the target cell it is assumed that these toxins mediate intra-specific or population-level interactions. However, no published studies specifically address this question. We present here a survey of bacteriocin production in a collection of enteric bacteria isolated from wild mammals in Australia. A subset of the bacteriocin-producing strains was assayed for the ability to kill a broad range of enteric bacteria from the same bacterial collection. A novel method of estimating killing breadth was developed and used to compare the surveyed bacteriocins in terms of the phylogenetic range over which they kill. The most striking result is that although bacteriocin-producers kill members of their own species most frequently, some kill phylogenetically distant taxa more frequently than they kill closer relatives. This study calls into question the role these toxins play in natural populations. A significant number of bacteriocins are highly effective in killing inter-specific strains and thus bacteriocins may serve to mediate bacterial community interactions.     

Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae

The production of bacteriocins can be favorable for colonization of the host by eliminating other bacterial species that share the same environment. In Streptococcus pneumoniae, the pnc (blp) locus encoding putative bacteriocins, immunity, and export proteins is controlled by a two-component system similar to the comCDE system required for the induction of genetic competence. A detailed comparison of the pnc clusters of four genetically distinct isolates confirmed the great plasticity of this locus and documented several repeat sequences. Members of the multiple-antibiotic-resistant Spain23F-1 clone, one member of the Spain9V-3 clone, sensitive 23F strain 2306, and the TIGR4 strain produced bactericidal substances active against other gram-positive bacteria and in some cases against S. pneumoniae as well. However, other strains did not show activity against the indicator strains despite the presence of a bacteriocin cluster, indicating that other factors are required for bacteriocin activity. Analysis of strain 2306 and mutant derivatives of this strain confirmed that bacteriocin production was dependent on the two-component regulatory system and genes involved in bacteriocin transport and processing. At least one other bacteriocin gene, pncE, is located elsewhere on the chromosome and might contribute to the bacteriocin activity of this strain.     

Production, recovery and purification of bacteriocins from lactic acid bacteria

Bacteriocins produced by lactic acid bacteria are a heterogeneous group of peptide inhibitors which include lantibiotics (class I, e.g. nisin), small heat-stable peptides (class II, e.g. pediocin AcH/PA1) and large heat-labile proteins (class III, e.g. helveticin J). Many bacteriocins belonging to the first two groups can be successfully used to inhibit undesirable microorganisms in foods, but only nisin is produced industrially and is licensed for use as a food preservative in a partially purified form. This review focuses on the production and purification of class I and class II bacteriocins from lactic acid bacteria. Bacteriocin production is growth associated but the yield of bacteriocin per unit biomass is affected by several factors, including the producing strain, media (carbohydrate and nitrogen sources, cations, etc.) and fermentation conditions (pH, temperature, agitation, aeration and dilution rate in continuous fermentations). Continuous fermentation processes with cell recycle or immobilized cells can result in a dramatic improvement in productivity over batch fermentations. Several simple recovery processes, based on adsorbing bacteriocin on resins or silica compounds, have been developed and can be used to build integrated production processes. Received: 29 December 1998 / Received revision: 23 April 1999 / Accepted: 23 April 1999     

The cost and benefit of quorum sensing‐controlled bacteriocin production in Lactobacillus plantarum

Bacteria eliminate competitors via ‘chemical warfare’ with bacteriocins. Some species appear to adjust bacteriocin production conditionally in response to the social environment. We tested whether variation in the cost and benefit of producing bacteriocins could explain such conditional behaviour, in the bacteria Lactobacillus plantarum. We found that: (a) bacterial bacteriocin production could be upregulated by either the addition of a synthetic autoinducer peptide (PLNC8IF; signalling molecule), or by a plasmid which constitutively encodes for the production of this peptide; (b) bacteriocin production is costly, leading to reduced growth when grown in poor and, to a lesser extent, in rich media; (c) bacteriocin production provides a fitness advantage, when grown in competition with sensitive strains; and (d) the fitness benefits provided by bacteriocin production are greater at higher cell densities. These results show how the costs and benefits of upregulating bacteriocin production can depend upon abiotic and biotic conditions.     

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.     

The bacteriocins of ruminal bacteria and their potential as an alternative to antibiotics

Beef cattle have been fed ionophores and other antibiotics for more than 20 years to decrease ruminal fermentation losses (e.g methane and ammonia) and increase feed efficiency, and these improvements have been explained by an inhibition of gram-positive ruminal bacteria. Ionophores are not used to treat human disease, but there has been an increased perception that antibiotics should not be used as feed additives. Some bacteria produce small peptides (bacteriocins) that inhibit gram-positive bacteria. In vitro experiments indicated that the bacteriocin, nisin, and the ionophore, monensin, had similar effects on ruminal fermentation. However, preliminary results indicated that mixed ruminal bacteria degraded nisin, and the ruminal bacterium, Streptococcus bovis, became highly nisin-resistant. A variety of ruminal bacteria produce bacteriocins, and bacteriocin production has, in some cases, been correlated with changes in ruminal ecology. Some ruminal bacteriocins are as potent as nisin in vitro, and resistance can be circumvented. Based on these results, ruminal bacteriocins may provide an alternative to antibiotics in cattle rations.     

Surface properties of bacteria sensitive and resistant to the class IIa carnobacteriocin Cbn BM1

Aims: Class IIa bacteriocins are small antimicrobial peptides synthesized by lactic acid bacteria. The proposed mechanisms of action for class IIa bacteriocins suggest that the physicochemical properties of the target bacterial surface govern the bacteriocin antimicrobial activity. The aim of this study is to decipher the relationship between both sensitivity and resistance to a class IIa bacteriocin, carnobacteriocin BM1 and physicochemical surface properties of bacteria. Methods and Results: The study was performed on 18 strains by a microbial adhesion to solvents process and with electrophoretic mobility measurements considering bacteria as soft particles. A large variation in bacterial surface properties is observed among the bacterial populations. Electro‐hydrodynamic parameters values appear to be more homogeneous for sensitive strains than for the resistant ones, which can exhibit more extreme values. Conclusions: Physicochemical surface properties of 18 strains determined show large variations between the strains. However, no direct link between these surface properties and the resistant/sensitive phenotypes of the strains can be stated. Significance and Impact of the Study: The surface physicochemical properties tested have a low predictive power to discriminate sensitive or resistant strains when determined at the bacterial population scale.     

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides.     

Pediococcus spp.: An important genus of lactic acid bacteria and pediocin producers

Probiotics have gained increasing attention due to several health benefits related to the human digestive and immune systems. Pediococcus spp. are lactic acid bacteria (LAB) that are widely described as probiotics and characterized as coccus-shaped bacteria (arranged in tetrads), Gram-positive, non-motile, non-spore forming, catalase-negative, and facultative anaerobes. There are many Pediococcus strains that produce pediocin, an effective antilisterial bacteriocin. Pediocins are small, cationic molecules consisting of a conserved hydrophilic N-terminal portion containing the YGNGV motif and an amphiphilic or hydrophobic C-terminal variable portion. A number of studies have been developed with Pediococcus isolated from multiple biological niches to conduct fermentation processes for pediocin or Pediococcus cell production. This review gathers the most significant information about the cultivation, mode of action, and variability of bacteriocins produced by Pediococcus spp., emphasizing their applications in the areas of food and clinical practice. This updated panorama assists in delimiting the challenges that still need to be overcome for pediocin use to be approved for human consumption and the food industry.     

Cell Wall-active Bacteriocins and Their Applications Beyond Antibiotic Activity

Microorganisms synthesize several compounds with antimicrobial activity in order to compete or defend themselves against others and ensure their survival. In this line, the cell wall is a major protective barrier whose integrity is essential for many vital bacterial processes. Probably for this reason, it represents a ??hot spot?? as a target for many antibiotics and antimicrobial peptides such as bacteriocins. Bacteriocins have largely been recognized by their pore-forming ability that collapses the selective permeability of the cytoplasmic membrane. However, in the last few years, many bacteriocins have been shown to inhibit cell wall biosyntheis alone, or in a concerted action with pore formation like nisin. Examples of cell wall-active bacteriocins are found in both Gram-negative and Gram-positive bacteria and include a wide diversity of structures such as nisin-like and mersacidin-like lipid II-binding bacteriocins, two-peptide lantibiotics, and non-modified bacteriocins. In this review, we summarize the current knowledge on these antimicrobial peptides as well as the role, composition, and biosynthesis of the bacterial cell wall as their target. Moreover, even though bacteriocins have been a matter of interest as natural food antimicrobials, we propose them as suitable tools to provide new means to improve biotechnologically relevant microorganisms.     

Amylocyclicin,a Novel Circular Bacteriocin Produced by Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization–time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.     

Colicin V can be produced by lactic acid bacteria

Colicin V is a small, proteinaceous bacterial toxin, produced by many strains of Escherichia coli and other members of the Enterobacteriaceae, that fits the definition of class II bacteriocins of Gram-positive bacteria. Export of colicin V is dependent on specific ABC (ATP-binding cassette) secretion proteins which recognize a double-glycine-type leader peptide on the immature colicin V bacteriocin. Replacement of the colicin V leader peptide by a signal peptide from the signal sequence-dependent bacteriocin divergicin A allowed expression of colicin V in lactic acid bacteria. This system may serve as a model for the heterologous expression of other small bacteriocins active against Gram-negative bacteria and other antibacterial peptides from lactic acid bacteria.