Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.     

Antagonistic Activity of Lactobacillus plantarum C11: Two New Two-Peptide Bacteriocins, Plantaricins EF and JK, and the Induction Factor Plantaricin A

Six bacteriocinlike peptides (plantaricin A [PlnA], PlnE, PlnF, PlnJ, PlnK, and PlnN) produced by Lactobacillus plantarum C11 were detected by amino acid sequencing and mass spectrometry. Since purification to homogeneity was problematic, all six peptides were obtained by solid-phase peptide synthesis and were tested for bacteriocin activity. It was found that L. plantarum C11 produces two two-peptide bacteriocins (PlnEF and PlnJK); a strain-specific antagonistic activity was detected at nanomolar concentrations when PlnE and PlnF were combined and when PlnJ and PlnK were combined. Complementary peptides were at least 10³ times more active when they were combined than when they were present individually, and optimal activity was obtained when the complementary peptides were present in approximately equal amounts. The interaction between complementary peptides was specific, since neither PlnE nor PlnF could complement PlnJ or PlnK, and none of these peptides could complement the peptides constituting the two-peptide bacteriocin lactococcin G. Interestingly, PlnA, which acts as an extracellular signal (pheromone) that triggers bacteriocin production, also possessed a strain-specific antagonistic activity. No bacteriocin activity could be detected for PlnN.     

The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G

Lactococcin G and enterocin 1071 are two homologous two-peptide bacteriocins. Expression vectors containing the gene encoding the putative lactococcin G immunity protein (lagC) or the gene encoding the enterocin 1071 immunity protein (entI) were constructed and introduced into strains sensitive to one or both of the bacteriocins. Strains that were sensitive to lactococcin G became immune to lactococcin G when expressing the putative lactococcin G immunity protein, indicating that the lagC gene in fact encodes a protein involved in lactococcin G immunity. To determine which peptide or parts of the peptide(s) of each bacteriocin that are recognized by the cognate immunity protein, combinations of wild-type peptides and hybrid peptides from the two bacteriocins were assayed against strains expressing either of the two immunity proteins. The lactococcin G immunity protein rendered the enterococcus strain but not the lactococcus strains resistant to enterocin 1071, indicating that the functionality of the immunity protein depends on a cellular component. Moreover, regions important for recognition by the immunity protein were identified in both peptides (Lcn-α and Lcn-β) constituting lactococcin G. These regions include the N-terminal end of Lcn-α (residues 1 to 13) and the C-terminal part of Lcn-β (residues 14 to 24). According to a previously proposed structural model of lactococcin G, these regions will be positioned adjacent to each other in the transmembrane helix-helix structure, and the model thus accommodates the present results.Lactic acid bacteria (LAB) produce ribosomally synthesized antimicrobial peptides, generally referred to as bacteriocins. There are two main classes of these bacteriocins (8, 22): the class I bacteriocins (often referred to as lantibiotics) that contain the modified amino acid residues lanthionine and/or β-methyllanthionine and the class II bacteriocins that lack modified residues (8). The class II bacteriocins are further divided into four subclasses, IIa, IIb, IIc, and IId (8). Class IIa contains the pediocin-like bacteriocins, which have very similar amino acid sequences, class IIc consists of the cyclic bacteriocins, and the one-peptide, noncyclic bacteriocins that show no sequence similarity to the pediocin-like bacteriocins are placed in class IId (8). The unmodified two-peptide bacteriocins are placed in class IIb. They are unique in that they consist of two different peptides, both of which must be present, in about equal amounts, to obtain optimal antimicrobial activity (25). More than 10 two-peptide bacteriocins have been isolated and characterized (see reference 25 for original references) since the first isolation of such a bacteriocin (lactococcin G) in 1992 (21). For the two-peptide bacteriocins that have been genetically characterized, the genes encoding the two bacteriocin peptides are always found next to each other in the same operon, along with the gene encoding the immunity protein that protects the bacteriocin producer from being killed by its own bacteriocin.Lactococcin G is perhaps the best-characterized two-peptide bacteriocin (12, 18, 19, 21, 24, 26, 27). It consists of the 39-residue α peptide (termed Lcn-α) and the 35-residue β peptide (termed Lcn-β) (Fig. ​(Fig.1A).1A). Like all two-peptide bacteriocins whose mode of action has been studied, lactococcin G causes cell death by rendering the membranes of target cells permeable to various ions (18, 19). Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have revealed that the two lactococcin G peptides adopt mainly α-helical structures when they are individually exposed to membrane-like entities (12, 27). Based on the NMR structures and findings from site-directed mutagenesis studies, a structural model of lactococcin G has recently been proposed (23, 26, 27). In this model, the two complementary peptides form parallel helices that span the target cell membrane. The helix-helix segment consists of the N-terminal region of Lcn-α (from about Trp-3 to Gly-22) and the C-terminal region of Lcn-β (from about Tyr-13 to Trp-32). The model also proposes that the cationic C-terminal end (residues 35 to 39, R-K-K-K-H) of Lcn-α is unstructured and forced through the target cell membrane by the membrane potential, thereby positioning the C termini of the two peptides inside the target-cell (Fig. ​(Fig.2).2). The tryptophan-rich N-terminal end of Lcn-β is also proposed to be relatively unstructured and to position itself in the outer membrane interface, thus forcing the N termini of the two peptides to remain on the outer side of the target cell membrane and the helix-helix segment to transverse the membrane (Fig. ​(Fig.2).2). This proposed structure is presumably also valid for the two-peptide bacteriocins enterocin 1071 (4, 5, 11), enterocin C (17), and lactococcin Q (32), since their sequence similarities to lactococcin G (lactococcin G has about 88 and 57% sequence identity to lactococcin Q and enterocin 1071, respectively; enterocin 1071 and enterocin C are identical except for one residue) indicate that these four bacteriocins have similar three-dimensional structures.Open in a separate windowFIG. 1.(A) Amino acid sequence alignment of enterocin 1071 (peptides Ent1071A and Ent1071B) and lactococcin G (peptides Lcn-α and Lcn-β) and the cognate immunity proteins (Ent1071im and LcnGim, respectively). Ent1071A and Lcn-α show 59% sequence identity, whereas Ent1071B and Lcn-β show 54% sequence identity. The immunity proteins consist of 110 amino acid residues each and show 38% sequence identity. Identical amino acid residues are colored in red. (B) Amino acid sequences of the two hybrid peptides. The Lcn-α-Ent1071A hybrid peptide (α-hybrid) is termed α[1-16]/A[14-39] in this study (it is designated α2-4 in reference 24). Residues are numbered according to the corresponding amino acid positions in Lcn-α (Fig. 1A). Residues in orange are derived from Lcn-α, and residues in blue are derived from Ent1071A. The overlapping region (i.e., residues 14 to 16) is marked in red, and this region consists of residues that are identical in Lcn-α and Ent1071A. The α-hybrid peptide contains an additional lysine residue in the C-terminal end derived from Lcn-α (see reference 24 for the construction of the hybrid peptide). The Lcn-β-Ent1071B hybrid peptide (β-hybrid) is termed β[1-13]/B[11-35] in this study (it is designated β1-6 in reference 24). Residues in orange are derived from Lcn-β, and residues in blue are derived from Ent1071B. The overlapping region (i.e., residues 11 to 13) is marked in red, and residues in this region are identical in Lcn-β and Ent1071B.Open in a separate windowFIG. 2.Proposed structural model of lactococcin G. The two peptides (Lcn-α and Lcn-β) form a transmembrane helix-helix structure, with the flexible tryptophan-rich N-terminal end of Lcn-β positioned in the outer membrane interface and the unstructured, highly cationic C-terminal end of Lcn-α inside the target cell membrane. The transmembrane helix-helix segment consists of the N-terminal region of Lcn-α (from about Trp-3 to Gly-22) and the C-terminal region of Lcn-β (from about Tyr-13 to Trp-32). (Adapted from reference 26 with permission of the publisher. Copyright 2008 American Chemical Society.)The sequence of the lactococcin G operon (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ938036","term_id":"238480353","term_text":"FJ938036"}}FJ938036) has been determined, and a gene (lagC) encoding the putative lactococcin G immunity protein has been identified downstream of the two genes encoding the two lactococcin G peptides. Downstream of the two genes encoding the two enterocin 1071 peptides, a gene (entI) encoding the enterocin 1071 immunity protein has been identified (4, 11). The putative lactococcin G immunity protein shows 38% amino acid sequence identity to the enterocin 1071 immunity protein (the sequence of which was obtained from Franz et al. [11]), and both proteins consist of 110 amino acid residues (Fig. ​(Fig.1A).1A). The aim of this study was to identify which peptides or which parts of the peptides of the two-peptide bacteriocins lactococcin G and enterocin 1071 are recognized by these immunity proteins. To achieve this, combinations of wild-type lactococcin G peptides, wild-type enterocin 1071 peptides, and hybrid lactococcin-enterocin peptides were assayed against sensitive strains that were transformed with an expression plasmid carrying either the lactococcin G or the enterocin 1071 immunity gene.     

Sensitivity to the two‐peptide bacteriocin lactococcin G is dependent on UppP,an enzyme involved in cell‐wall synthesis

Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor‐mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two‐peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G‐resistant mutants and performed whole‐genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non‐sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.     

Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in G?ttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the alpha and beta peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.     

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.     

Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6.

On the bacteriocin plasmid p9B4-6 of Lactococcus lactis subsp. cremoris 9B4, a third bacteriocin determinant was identified. The genes encoding bacteriocin production and immunity resided on a 1.2-kb CelII-ScaI fragment and were located adjacent to one of two previously identified bacteriocin operons (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). The fragment was sequenced and analyzed by deletion and mutation analyses. The bacteriocin determinant consisted of two genes which were transcribed as an operon. The first gene (lcnB), containing 68 codons, was involved in bacteriocin activity. The second gene (lciB) contained 91 codons and was responsible for immunity. The specificity of this novel bacteriocin, designated lactococcin B, was different from that of the other two bacteriocins specified by p9B4-6. Part of the nucleotide sequence of the lactococcin B operon was similar to a nucleotide sequence also found in the two other bacteriocin operons of p9B4-6. This conserved region encompassed a nucleotide sequence upstream of the bacteriocin gene and the 5' part of the gene. When the lactococcin B operon was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter, antagonistic activity could be detected.     

Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6.

On the bacteriocin plasmid p9B4-6 of Lactococcus lactis subsp. cremoris 9B4, a third bacteriocin determinant was identified. The genes encoding bacteriocin production and immunity resided on a 1.2-kb CelII-ScaI fragment and were located adjacent to one of two previously identified bacteriocin operons (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). The fragment was sequenced and analyzed by deletion and mutation analyses. The bacteriocin determinant consisted of two genes which were transcribed as an operon. The first gene (lcnB), containing 68 codons, was involved in bacteriocin activity. The second gene (lciB) contained 91 codons and was responsible for immunity. The specificity of this novel bacteriocin, designated lactococcin B, was different from that of the other two bacteriocins specified by p9B4-6. Part of the nucleotide sequence of the lactococcin B operon was similar to a nucleotide sequence also found in the two other bacteriocin operons of p9B4-6. This conserved region encompassed a nucleotide sequence upstream of the bacteriocin gene and the 5' part of the gene. When the lactococcin B operon was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter, antagonistic activity could be detected.     

Lactococcin Q, a novel two-peptide bacteriocin produced by Lactococcus lactis QU 4

A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, alpha and beta, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qalpha plus Qbeta) and lactococcin G (Galpha plus Gbeta) clarified that hybrid combinations (Qalpha plus Gbeta and Galpha plus Qbeta) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qalpha plus Gbeta was 32 times higher than that of Qalpha plus Qbeta, suggesting that the difference in beta peptides was important for the intensity of antibacterial activity.     

Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.     

An ‘Upp'‐turn in bacteriocin receptor identification

Bacteriocins are gene encoded, bacterially produced antimicrobial peptides that have been the focus of considerable scientific interest but which are relatively underutilized by the food, veterinary and medical industries. One means via which the latter issue can be overcome is through a better understanding of how these peptides work or, more specifically, the identification of bacteriocin receptors and the subsequent application of such information to enhance the potency, and commercial value, of bacteriocins. For a time since the identification of lipid II and subunits of the mannose phosphotransferase system as receptors for several class I (modified) and class II (unmodified) bacteriocins, respectively, there were relatively few developments in this area. However, a number of recent studies have addressed this issue, resulting in the identification of a maltose ABC transporter and metallopeptidase as the targets for the garvicin ML (class IIc) and LsbB (class IId) bacteriocins, respectively, and, most recently, the identification of UppP as the receptor for lactococcin G and enterocin 1071 (both class IIb). In addition to these exciting discoveries, the development, and further application, of new strategies to facilitate receptor identification has the potential to lead to even further breakthroughs in bacteriocin research.     

Lactococcin Q, a Novel Two-Peptide Bacteriocin Produced by Lactococcus lactis QU 4

A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, α and β, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qα plus Qβ) and lactococcin G (Gα plus Gβ) clarified that hybrid combinations (Qα plus Gβ and Gα plus Qβ) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qα plus Gβ was 32 times higher than that of Qα plus Qβ, suggesting that the difference in β peptides was important for the intensity of antibacterial activity.     

Biochemical and genetic characterization of the two-peptide bacteriocin enterocin 1071 produced by Enterococcus faecalis FAIR-E 309

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.     

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.     

Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5

Lactobacillus sakei 5, isolated from malted barley, produces three bacteriocins. Genetic and functional analysis of the purified bacteriocins showed that this strain produces a plasmid-encoded bacteriocin that is identical to sakacin P, as well as two novel, chromosomally encoded bacteriocins, which were designated sakacin T and sakacin X. The structural genes specifying sakacin T and sakacin X are part of the sakacin TX locus, which consists of two adjacent but divergently oriented gene clusters. The first gene cluster includes stxP, stxR, stxK, and stxT, which, based on functional and comparative sequence analysis, are believed to encode an inducing peptide and proteins involved in regulation and secretion of these bacteriocins. The second gene cluster includes the structural and immunity genes for sakacin T, a class IIb two-peptide bacteriocin composed of SakTalpha and SakTbeta, and sakacin X, a class IIa bacteriocin. Interestingly, a so-called transport accessory protein was absent from the locus, and based on our results it appears that a dedicated accessory protein is not required for processing and transport of sakacin T and sakacin X.     

Biochemical and Genetic Characterization of the Two-Peptide Bacteriocin Enterocin 1071 Produced by Enterococcus faecalis FAIR-E 309

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.     

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.     

Purification and partial amino acid sequence of plantaricin 1.25ααα and 1.25βββ, two bacteriocins produced by Lactobacillus plantarum TMW1.25

Two bacteriocins produced by Lactobacillus plantarum TMW1.25 have been purified by a four-step purification procedure, including ammonium sulphate precipitation and cation-exchange chromatography followed by hydrophobic-interaction chromatography on octyl sepharose. The final purification was performed by repeated reversed-phase chromatography steps which yielded two bacteriocin fractions designated plantaricin 1.25 alpha and plantaricin 1.25 beta. The molecular masses of the peptides in these fractions were 5979 and 5203 Da, respectively. Combination of the fractions did not have any synergistic effects on bacteriocin activity, indicating that they each contain a one-peptide bacteriocin. The major peptide in the alpha fraction was blocked at its N-terminus, and a partial sequence (25 residues) could only be obtained after cleavage with CNBr. This sequence did not show clear homologies with known bacteriocins. The beta peptide has been sequenced almost completely and consists, presumably, of 53 residues. This peptide displayed strong homology to the known N-terminal part of brevicin 27 produced by Lactobacillus brevis SB27. The results showed that the beta peptide contains as many as six consecutive lysine residues at the N-terminus.     

Complementary and Overlapping Selectivity of the Two-Peptide Bacteriocins Plantaricin EF and JK

Plantaricin EF and JK are both two-peptide bacteriocins produced by Lactobacillus plantarum C11. The mechanism of plantaricin EF and JK action was studied on L. plantarum 965 cells. Both plantaricins form pores in the membranes of target cells and dissipate the transmembrane electrical potential (Deltapsi) and pH gradient (DeltapH). The plantaricin EF pores efficiently conduct small monovalent cations, but conductivity for anions is low or absent. Plantaricin JK pores show high conductivity for specific anions but low conductivity for cations. These data indicate that L. plantarum C11 produces bacteriocins with complementary ion selectivity, thereby ensuring efficient killing of target bacteria.